discussions and conclusions in research

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• How to Write Discussions and Conclusions

The discussion section contains the results and outcomes of a study. An effective discussion informs readers what can be learned from your experiment and provides context for the results.

What makes an effective discussion?

When you’re ready to write your discussion, you’ve already introduced the purpose of your study and provided an in-depth description of the methodology. The discussion informs readers about the larger implications of your study based on the results. Highlighting these implications while not overstating the findings can be challenging, especially when you’re submitting to a journal that selects articles based on novelty or potential impact. Regardless of what journal you are submitting to, the discussion section always serves the same purpose: concluding what your study results actually mean.

A successful discussion section puts your findings in context. It should include:

• the results of your research,
• a discussion of related research, and
• a comparison between your results and initial hypothesis.

Tip: Not all journals share the same naming conventions.

You can apply the advice in this article to the conclusion, results or discussion sections of your manuscript.

Our Early Career Researcher community tells us that the conclusion is often considered the most difficult aspect of a manuscript to write. To help, this guide provides questions to ask yourself, a basic structure to model your discussion off of and examples from published manuscripts. 

Questions to ask yourself:

• Was my hypothesis correct?
• If my hypothesis is partially correct or entirely different, what can be learned from the results? 
• How do the conclusions reshape or add onto the existing knowledge in the field? What does previous research say about the topic? 
• Why are the results important or relevant to your audience? Do they add further evidence to a scientific consensus or disprove prior studies? 
• How can future research build on these observations? What are the key experiments that must be done? 
• What is the “take-home” message you want your reader to leave with?

How to structure a discussion

Trying to fit a complete discussion into a single paragraph can add unnecessary stress to the writing process. If possible, you’ll want to give yourself two or three paragraphs to give the reader a comprehensive understanding of your study as a whole. Here’s one way to structure an effective discussion:

Writing Tips

While the above sections can help you brainstorm and structure your discussion, there are many common mistakes that writers revert to when having difficulties with their paper. Writing a discussion can be a delicate balance between summarizing your results, providing proper context for your research and avoiding introducing new information. Remember that your paper should be both confident and honest about the results! 

• Read the journal’s guidelines on the discussion and conclusion sections. If possible, learn about the guidelines before writing the discussion to ensure you’re writing to meet their expectations. 
• Begin with a clear statement of the principal findings. This will reinforce the main take-away for the reader and set up the rest of the discussion. 
• Explain why the outcomes of your study are important to the reader. Discuss the implications of your findings realistically based on previous literature, highlighting both the strengths and limitations of the research. 
• State whether the results prove or disprove your hypothesis. If your hypothesis was disproved, what might be the reasons? 
• Introduce new or expanded ways to think about the research question. Indicate what next steps can be taken to further pursue any unresolved questions. 
• If dealing with a contemporary or ongoing problem, such as climate change, discuss possible consequences if the problem is avoided. 
• Be concise. Adding unnecessary detail can distract from the main findings. 

Don’t

• Rewrite your abstract. Statements with “we investigated” or “we studied” generally do not belong in the discussion. 
• Include new arguments or evidence not previously discussed. Necessary information and evidence should be introduced in the main body of the paper. 
• Apologize. Even if your research contains significant limitations, don’t undermine your authority by including statements that doubt your methodology or execution. 
• Shy away from speaking on limitations or negative results. Including limitations and negative results will give readers a complete understanding of the presented research. Potential limitations include sources of potential bias, threats to internal or external validity, barriers to implementing an intervention and other issues inherent to the study design. 
• Overstate the importance of your findings. Making grand statements about how a study will fully resolve large questions can lead readers to doubt the success of the research. 

Snippets of Effective Discussions:

Consumer-based actions to reduce plastic pollution in rivers: A multi-criteria decision analysis approach

Identifying reliable indicators of fitness in polar bears

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Discussion and Conclusions

Your Discussion and Conclusions sections should answer the question: What do your results mean?

In other words, the majority of the Discussion and Conclusions sections should be an interpretation of your results. You should:

• Discuss your conclusions in order of  most to least important.
• Compare  your results with those from other studies: Are they consistent? If not, discuss possible reasons for the difference.
• Mention any  inconclusive results  and explain them as best you can. You may suggest additional experiments needed to clarify your results.
• Briefly describe the  limitations  of your study to show reviewers and readers that you have considered your experiment’s weaknesses. Many researchers are hesitant to do this as they feel it highlights the weaknesses in their research to the editor and reviewer. However doing this actually makes a positive impression of your paper as it makes it clear that you have an in depth understanding of your topic and can think objectively of your research.
• Discuss  what your results may mean  for researchers in the same field as you, researchers in other fields, and the general public. How could your findings be applied?
• State how your results  extend the findings  of previous studies.
• If your findings are preliminary, suggest  future studies  that need to be carried out.
• At the end of your Discussion and Conclusions sections,  state your main conclusions once again .

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Discussion vs Conclusion: Researcher's Compact Guide

Table of Contents

If you are a researcher or a student, understanding the difference between a discussion and a conclusion is crucial especially when you are working on your academic projects. These two sections play distinct roles in your paper, and knowing how to approach each one can greatly improve the quality of your work.

This guide will delve into the nuances of both sections, providing a comprehensive overview of their purposes, structures, and writing strategies.

Understanding the discussion section of a research paper

The discussion section of a research paper is where you interpret and explain your research findings. It's a section for you to explore the implications of your results, compare them to previous research, and address any limitations in your study.

One of the main purposes of the discussion section is to answer the research question. You should provide a detailed explanation of the results and how they relate to your hypothesis or research question. This part of the paper is your opportunity to show that you have made a valuable contribution to your field of study.

Structure of the Discussion Section

The structure of the discussion section can vary depending on the nature of the research and the guidelines of the publication. However, a typical structure might include the following elements:

• Restatement of the research problem
• Summary of the main findings
• Interpretation of the results
• Comparison with previous research
• Explanation of any unexpected findings or discrepancies
• Discussion of the implications of the results
• Identification of limitations and suggestions for prospective research

Tips for Writing Discussion Section

When writing the discussion section, it's important to stay focused on your research question and avoid talking about unrelated areas. Be sure to interpret your findings in the context of the research question and the existing literature in your field.

It's also crucial to be honest about the limitations of your study. Acknowledging these limitations not only enhances the credibility of your research but also provides valuable information for budding researchers.

Understanding the Conclusion Section

The conclusion section of a research paper is where you summarize your research and its implications. Unlike the discussion section, the conclusion is not the place for a detailed analysis of your results. Instead, it's where you wrap up your argument and leave the reader with a clear understanding of your research and its significance.

The conclusion should provide a succinct summary of your research question, methods, results, and main findings. It should also discuss the broader implications of your research and suggest areas for future study.

Structure of the Conclusion Section

The structure of the conclusion section is typically more straightforward than that of the discussion section. A typical conclusion might include the following elements:

• Restatement of the research question
• Discussion of the implications of the research
• Suggestions for future research

Tips for Writing Conclusion Section

When writing the conclusion section, it's important to be concise and to the point. Try not to introduce new information or arguments in the conclusion section. Instead, simply focus on summarizing your research and highlighting its significance.

It's also important to make your conclusion engaging and indelible. Consider ending with a strong statement that emphasizes the importance of your research and leaves a lasting impression on the reader.

Discussion Vs. Conclusion: Key Differences

While the discussion and conclusion sections of a research paper have some similarities, they serve different purposes and should be approached differently. The discussion section is where you interpret and analyze your results, while the conclusion is where you summarize your research and highlight its significance.

Another key difference is the level of detail. The discussion section typically includes a detailed analysis of the results, while the conclusion provides a concise summary of the research. Let’s take a look at the differences between the discussion and conclusion sections in various aspects

Whether you're writing a discussion section or a conclusion, it's important to choose the right approach for your research. Consider the nature of your study, the guidelines of the publication, and the expectations of your audience when deciding how to structure and write these sections.

Remember, the goal of both sections is to communicate your research effectively and make a meaningful contribution to your field. By understanding the differences between a discussion and a conclusion, you can ensure that your research paper is clear, coherent, and impactful.

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The conclusion is intended to help the reader understand why your research should matter to them after they have finished reading the paper. A conclusion is not merely a summary of the main topics covered or a re-statement of your research problem, but a synthesis of key points derived from the findings of your study and, if applicable, where you recommend new areas for future research. For most college-level research papers, two or three well-developed paragraphs is sufficient for a conclusion, although in some cases, more paragraphs may be required in describing the key findings and their significance.

Conclusions. The Writing Center. University of North Carolina; Conclusions. The Writing Lab and The OWL. Purdue University.

Importance of a Good Conclusion

A well-written conclusion provides you with important opportunities to demonstrate to the reader your understanding of the research problem. These include:

• Presenting the last word on the issues you raised in your paper . Just as the introduction gives a first impression to your reader, the conclusion offers a chance to leave a lasting impression. Do this, for example, by highlighting key findings in your analysis that advance new understanding about the research problem, that are unusual or unexpected, or that have important implications applied to practice.
• Summarizing your thoughts and conveying the larger significance of your study . The conclusion is an opportunity to succinctly re-emphasize  your answer to the "So What?" question by placing the study within the context of how your research advances past research about the topic.
• Identifying how a gap in the literature has been addressed . The conclusion can be where you describe how a previously identified gap in the literature [first identified in your literature review section] has been addressed by your research and why this contribution is significant.
• Demonstrating the importance of your ideas . Don't be shy. The conclusion offers an opportunity to elaborate on the impact and significance of your findings. This is particularly important if your study approached examining the research problem from an unusual or innovative perspective.
• Introducing possible new or expanded ways of thinking about the research problem . This does not refer to introducing new information [which should be avoided], but to offer new insight and creative approaches for framing or contextualizing the research problem based on the results of your study.

Bunton, David. “The Structure of PhD Conclusion Chapters.” Journal of English for Academic Purposes 4 (July 2005): 207–224; Conclusions. The Writing Center. University of North Carolina; Kretchmer, Paul. Twelve Steps to Writing an Effective Conclusion. San Francisco Edit, 2003-2008; Conclusions. The Writing Lab and The OWL. Purdue University; Assan, Joseph. "Writing the Conclusion Chapter: The Good, the Bad and the Missing." Liverpool: Development Studies Association (2009): 1-8.

Structure and Writing Style

I.  General Rules

The general function of your paper's conclusion is to restate the main argument . It reminds the reader of the strengths of your main argument(s) and reiterates the most important evidence supporting those argument(s). Do this by clearly summarizing the context, background, and necessity of pursuing the research problem you investigated in relation to an issue, controversy, or a gap found in the literature. However, make sure that your conclusion is not simply a repetitive summary of the findings. This reduces the impact of the argument(s) you have developed in your paper.

When writing the conclusion to your paper, follow these general rules:

• Present your conclusions in clear, concise language. Re-state the purpose of your study, then describe how your findings differ or support those of other studies and why [i.e., what were the unique, new, or crucial contributions your study made to the overall research about your topic?].
• Do not simply reiterate your findings or the discussion of your results. Provide a synthesis of arguments presented in the paper to show how these converge to address the research problem and the overall objectives of your study.
• Indicate opportunities for future research if you haven't already done so in the discussion section of your paper. Highlighting the need for further research provides the reader with evidence that you have an in-depth awareness of the research problem but that further investigations should take place beyond the scope of your investigation.

Consider the following points to help ensure your conclusion is presented well:

• If the argument or purpose of your paper is complex, you may need to summarize the argument for your reader.
• If, prior to your conclusion, you have not yet explained the significance of your findings or if you are proceeding inductively, use the end of your paper to describe your main points and explain their significance.
• Move from a detailed to a general level of consideration that returns the topic to the context provided by the introduction or within a new context that emerges from the data [this is opposite of the introduction, which begins with general discussion of the context and ends with a detailed description of the research problem]. 

The conclusion also provides a place for you to persuasively and succinctly restate the research problem, given that the reader has now been presented with all the information about the topic . Depending on the discipline you are writing in, the concluding paragraph may contain your reflections on the evidence presented. However, the nature of being introspective about the research you have conducted will depend on the topic and whether your professor wants you to express your observations in this way. If asked to think introspectively about the topics, do not delve into idle speculation. Being introspective means looking within yourself as an author to try and understand an issue more deeply, not to guess at possible outcomes or make up scenarios not supported by the evidence.

II.  Developing a Compelling Conclusion

Although an effective conclusion needs to be clear and succinct, it does not need to be written passively or lack a compelling narrative. Strategies to help you move beyond merely summarizing the key points of your research paper may include any of the following:

• If your essay deals with a critical, contemporary problem, warn readers of the possible consequences of not attending to the problem proactively.
• Recommend a specific course or courses of action that, if adopted, could address a specific problem in practice or in the development of new knowledge leading to positive change.
• Cite a relevant quotation or expert opinion already noted in your paper in order to lend authority and support to the conclusion(s) you have reached [a good source would be from your literature review].
• Explain the consequences of your research in a way that elicits action or demonstrates urgency in seeking change.
• Restate a key statistic, fact, or visual image to emphasize the most important finding of your paper.
• If your discipline encourages personal reflection, illustrate your concluding point by drawing from your own life experiences.
• Return to an anecdote, an example, or a quotation that you presented in your introduction, but add further insight derived from the findings of your study; use your interpretation of results from your study to recast it in new or important ways.
• Provide a "take-home" message in the form of a succinct, declarative statement that you want the reader to remember about your study.

III. Problems to Avoid

Failure to be concise Your conclusion section should be concise and to the point. Conclusions that are too lengthy often have unnecessary information in them. The conclusion is not the place for details about your methodology or results. Although you should give a summary of what was learned from your research, this summary should be relatively brief, since the emphasis in the conclusion is on the implications, evaluations, insights, and other forms of analysis that you make. Strategies for writing concisely can be found here .

Failure to comment on larger, more significant issues In the introduction, your task was to move from the general [the field of study] to the specific [the research problem]. However, in the conclusion, your task is to move from a specific discussion [your research problem] back to a general discussion framed around the implications and significance of your findings [i.e., how your research contributes new understanding or fills an important gap in the literature]. In short, the conclusion is where you should place your research within a larger context [visualize your paper as an hourglass--start with a broad introduction and review of the literature, move to the specific analysis and discussion, conclude with a broad summary of the study's implications and significance].

Failure to reveal problems and negative results Negative aspects of the research process should never be ignored. These are problems, deficiencies, or challenges encountered during your study. They should be summarized as a way of qualifying your overall conclusions. If you encountered negative or unintended results [i.e., findings that are validated outside the research context in which they were generated], you must report them in the results section and discuss their implications in the discussion section of your paper. In the conclusion, use negative results as an opportunity to explain their possible significance and/or how they may form the basis for future research.

Failure to provide a clear summary of what was learned In order to be able to discuss how your research fits within your field of study [and possibly the world at large], you need to summarize briefly and succinctly how it contributes to new knowledge or a new understanding about the research problem. This element of your conclusion may be only a few sentences long.

Failure to match the objectives of your research Often research objectives in the social and behavioral sciences change while the research is being carried out. This is not a problem unless you forget to go back and refine the original objectives in your introduction. As these changes emerge they must be documented so that they accurately reflect what you were trying to accomplish in your research [not what you thought you might accomplish when you began].

Resist the urge to apologize If you've immersed yourself in studying the research problem, you presumably should know a good deal about it [perhaps even more than your professor!]. Nevertheless, by the time you have finished writing, you may be having some doubts about what you have produced. Repress those doubts! Don't undermine your authority as a researcher by saying something like, "This is just one approach to examining this problem; there may be other, much better approaches that...." The overall tone of your conclusion should convey confidence to the reader about the study's validity and realiability.

Assan, Joseph. "Writing the Conclusion Chapter: The Good, the Bad and the Missing." Liverpool: Development Studies Association (2009): 1-8; Concluding Paragraphs. College Writing Center at Meramec. St. Louis Community College; Conclusions. The Writing Center. University of North Carolina; Conclusions. The Writing Lab and The OWL. Purdue University; Freedman, Leora  and Jerry Plotnick. Introductions and Conclusions. The Lab Report. University College Writing Centre. University of Toronto; Leibensperger, Summer. Draft Your Conclusion. Academic Center, the University of Houston-Victoria, 2003; Make Your Last Words Count. The Writer’s Handbook. Writing Center. University of Wisconsin Madison; Miquel, Fuster-Marquez and Carmen Gregori-Signes. “Chapter Six: ‘Last but Not Least:’ Writing the Conclusion of Your Paper.” In Writing an Applied Linguistics Thesis or Dissertation: A Guide to Presenting Empirical Research . John Bitchener, editor. (Basingstoke,UK: Palgrave Macmillan, 2010), pp. 93-105; Tips for Writing a Good Conclusion. Writing@CSU. Colorado State University; Kretchmer, Paul. Twelve Steps to Writing an Effective Conclusion. San Francisco Edit, 2003-2008; Writing Conclusions. Writing Tutorial Services, Center for Innovative Teaching and Learning. Indiana University; Writing: Considering Structure and Organization. Institute for Writing Rhetoric. Dartmouth College.

Writing Tip

Don't Belabor the Obvious!

Avoid phrases like "in conclusion...," "in summary...," or "in closing...." These phrases can be useful, even welcome, in oral presentations. But readers can see by the tell-tale section heading and number of pages remaining that they are reaching the end of your paper. You'll irritate your readers if you belabor the obvious.

Assan, Joseph. "Writing the Conclusion Chapter: The Good, the Bad and the Missing." Liverpool: Development Studies Association (2009): 1-8.

Another Writing Tip

New Insight, Not New Information!

Don't surprise the reader with new information in your conclusion that was never referenced anywhere else in the paper. This why the conclusion rarely has citations to sources. If you have new information to present, add it to the discussion or other appropriate section of the paper. Note that, although no new information is introduced, the conclusion, along with the discussion section, is where you offer your most "original" contributions in the paper; the conclusion is where you describe the value of your research, demonstrate that you understand the material that you’ve presented, and position your findings within the larger context of scholarship on the topic, including describing how your research contributes new insights to that scholarship.

Assan, Joseph. "Writing the Conclusion Chapter: The Good, the Bad and the Missing." Liverpool: Development Studies Association (2009): 1-8; Conclusions. The Writing Center. University of North Carolina.

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Discussion and conclusions

Your discussion and conclusions sections should answer the question:.

What do your results mean?

In other words, the majority of the Discussion and Conclusions sections should be an interpretation of your results. You should:

Discuss your conclusions in order of most to least important. Compare your results with those from other studies: Are they consistent? If not, discuss possible reasons for the difference. Mention any inconclusive results and explain them as best you can. You may suggest additional experiments needed to clarify your results. Briefly describe the limitations of your study to show reviewers and readers that you have considered your study's weaknesses. Discuss what your results may mean for researchers in the same field as you, researchers in other fields, and the general public. How could your findings be applied? State how your results extend the findings of previous studies. If your findings are preliminary, suggest future studies that need to be carried out. At the end of your discussion and conclusions, state your main conclusions once again.

Example Source Feilong Wang, Wenzhi Pan, Shuming Pan, Shuyun Wang, Qinmin Ge and Junbo Ge Usefulness of N-terminal pro-brain natriuretic peptide and C-reactive protein to predict ICU mortality in unselected medical ICU patients: a prospective, observational study. Critical Care 2011;15(1):R42. 

Discussion In this large scale study of 576 unselected medical ICU patients, we found that NT-proBNP and CRP independently predicted ICU mortality even after adjustment for the APACHE II score and multiple potential confounders including eGFR, age...

...In the present study, we also used a more sensitive test of improvement in model discrimination [27]. We found that the addition of NT-proBNP to the APACHE II score significantly increased the ability to predict ICU mortality as demonstrated by the IDI (6.6%, P = 0.003) and NRI (16.6%, P = 0.007) indices. NT-proBNP was not an independent predictor of ICU mortality in the non-cardiac subgroup after adjustment for APACHE II score and CRP. Kotanidou et al. [13] found that NT-proBNP predicted mortality independently after the adjusted APACHE II score and some inflammatory cytokines levels in non-cardiac ICU patients. But they used TNF-a, IL-6, and IL-10 rather than CRP and enrolled many surgical and multiple trauma cases. In the cardiac subgroup, NT-proBNP independently predicted ICU mortality while the AUC of the APACHE II score was not different from that of NT-proBNP (0.81 ± 0.03 vs 0.77 ± 0.04; P > 0.05). The addition of NT-proBNP to the APACHE-II score can obviously increase predictive ability (IDI = 10.2%, P = 0.018; NRI = 18.5%, P = 0.028). Therefore, although NT-proBNP could predict ICU mortality in unselected medical patents, it appeared to be more useful in cardiac patients than in non-cardiac patients.

...One previous study showed no predictive value of CRP for in-hospital mortality, even in univariate analysis [21]. The scope of the study was rather small (N = 103) and, thus, the statistical power was less than that of our study. Moreover, the endpoint of the previous study was in-hospital mortality but not ICU mortality. The present study revealed that CRP was also an independent predictor of ICU mortality in unselected patients or non-cardiac patients...

Several limitations of our study should be mentioned. First, neither echocardiography was performed nor cardiac function assessed in the present study. The division of subgroups was according to primary admission cause. Thus patients in the non-cardiac group may also have cardiac disease and cardiac dysfunction. However, patients with cardiac diseases as the primary principal diagnosis leading to ICU admission must have cardiac diseases. The statistical conclusion drawn from the cardiac group was appropriate. Second, this was a single-center study, and participants did not include surgery and trauma patients. The value for NT-proBNP in prediction of adverse outcome would be a bit different if the population was different. At last, a limitation of the net reclassification improvement and other reclassification measures is that they depend on the particular categories used [26]. We had used < 10%, 10% to 30%, and 30% to 50%, and > 50% for the risk of ICU death as risk categories. But there are still no well-recognized risk categories now. If the risk categories used had been different, the NRI would be a bit different.

Conclusions In this large-scale study of unselected ICU patients, we confirmed that NT-proBNP and CRP can serve as moderate independent predictors of ICU mortality. Although the predictive ability was lower compared with the APACHE II score, but the addition of CRP or NT-proBNP or both to the APACHE II score could significantly improve the ability to predict ICU mortality, as demonstrated by IDI and NRI indices. NT-proBNP appeared to be more useful for predicting ICU outcomes in cardiac patients.

How to Write the Discussion Section of a Research Paper

The discussion section of a research paper analyzes and interprets the findings, provides context, compares them with previous studies, identifies limitations, and suggests future research directions.

Updated on September 15, 2023

Structure your discussion section right, and you’ll be cited more often while doing a greater service to the scientific community. So, what actually goes into the discussion section? And how do you write it?

The discussion section of your research paper is where you let the reader know how your study is positioned in the literature, what to take away from your paper, and how your work helps them. It can also include your conclusions and suggestions for future studies.

First, we’ll define all the parts of your discussion paper, and then look into how to write a strong, effective discussion section for your paper or manuscript.

Discussion section: what is it, what it does

The discussion section comes later in your paper, following the introduction, methods, and results. The discussion sets up your study’s conclusions. Its main goals are to present, interpret, and provide a context for your results.

What is it?

The discussion section provides an analysis and interpretation of the findings, compares them with previous studies, identifies limitations, and suggests future directions for research.

This section combines information from the preceding parts of your paper into a coherent story. By this point, the reader already knows why you did your study (introduction), how you did it (methods), and what happened (results). In the discussion, you’ll help the reader connect the ideas from these sections.

Why is it necessary?

The discussion provides context and interpretations for the results. It also answers the questions posed in the introduction. While the results section describes your findings, the discussion explains what they say. This is also where you can describe the impact or implications of your research.

Adds context for your results

Most research studies aim to answer a question, replicate a finding, or address limitations in the literature. These goals are first described in the introduction. However, in the discussion section, the author can refer back to them to explain how the study's objective was achieved. 

Shows what your results actually mean and real-world implications

The discussion can also describe the effect of your findings on research or practice. How are your results significant for readers, other researchers, or policymakers?

What to include in your discussion (in the correct order)

A complete and effective discussion section should at least touch on the points described below.

Summary of key findings

The discussion should begin with a brief factual summary of the results. Concisely overview the main results you obtained.

Begin with key findings with supporting evidence

Your results section described a list of findings, but what message do they send when you look at them all together?

Your findings were detailed in the results section, so there’s no need to repeat them here, but do provide at least a few highlights. This will help refresh the reader’s memory and help them focus on the big picture.

Read the first paragraph of the discussion section in this article (PDF) for an example of how to start this part of your paper. Notice how the authors break down their results and follow each description sentence with an explanation of why each finding is relevant. 

State clearly and concisely

Following a clear and direct writing style is especially important in the discussion section. After all, this is where you will make some of the most impactful points in your paper. While the results section often contains technical vocabulary, such as statistical terms, the discussion section lets you describe your findings more clearly. 

Interpretation of results

Once you’ve given your reader an overview of your results, you need to interpret those results. In other words, what do your results mean? Discuss the findings’ implications and significance in relation to your research question or hypothesis.

Analyze and interpret your findings

Look into your findings and explore what’s behind them or what may have caused them. If your introduction cited theories or studies that could explain your findings, use these sources as a basis to discuss your results.

For example, look at the second paragraph in the discussion section of this article on waggling honey bees. Here, the authors explore their results based on information from the literature.

Unexpected or contradictory results

Sometimes, your findings are not what you expect. Here’s where you describe this and try to find a reason for it. Could it be because of the method you used? Does it have something to do with the variables analyzed? Comparing your methods with those of other similar studies can help with this task.

Context and comparison with previous work

Refer to related studies to place your research in a larger context and the literature. Compare and contrast your findings with existing literature, highlighting similarities, differences, and/or contradictions.

How your work compares or contrasts with previous work

Studies with similar findings to yours can be cited to show the strength of your findings. Information from these studies can also be used to help explain your results. Differences between your findings and others in the literature can also be discussed here. 

How to divide this section into subsections

If you have more than one objective in your study or many key findings, you can dedicate a separate section to each of these. Here’s an example of this approach. You can see that the discussion section is divided into topics and even has a separate heading for each of them. 

Limitations

Many journals require you to include the limitations of your study in the discussion. Even if they don’t, there are good reasons to mention these in your paper.

Why limitations don’t have a negative connotation

A study’s limitations are points to be improved upon in future research. While some of these may be flaws in your method, many may be due to factors you couldn’t predict.

Examples include time constraints or small sample sizes. Pointing this out will help future researchers avoid or address these issues. This part of the discussion can also include any attempts you have made to reduce the impact of these limitations, as in this study .

How limitations add to a researcher's credibility

Pointing out the limitations of your study demonstrates transparency. It also shows that you know your methods well and can conduct a critical assessment of them.  

Implications and significance

The final paragraph of the discussion section should contain the take-home messages for your study. It can also cite the “strong points” of your study, to contrast with the limitations section.

Restate your hypothesis

Remind the reader what your hypothesis was before you conducted the study. 

How was it proven or disproven?

Identify your main findings and describe how they relate to your hypothesis.

How your results contribute to the literature

Were you able to answer your research question? Or address a gap in the literature?

Future implications of your research

Describe the impact that your results may have on the topic of study. Your results may show, for instance, that there are still limitations in the literature for future studies to address. There may be a need for studies that extend your findings in a specific way. You also may need additional research to corroborate your findings. 

Sample discussion section

This fictitious example covers all the aspects discussed above. Your actual discussion section will probably be much longer, but you can read this to get an idea of everything your discussion should cover.

Our results showed that the presence of cats in a household is associated with higher levels of perceived happiness by its human occupants. These findings support our hypothesis and demonstrate the association between pet ownership and well-being. 

The present findings align with those of Bao and Schreer (2016) and Hardie et al. (2023), who observed greater life satisfaction in pet owners relative to non-owners. Although the present study did not directly evaluate life satisfaction, this factor may explain the association between happiness and cat ownership observed in our sample.

Our findings must be interpreted in light of some limitations, such as the focus on cat ownership only rather than pets as a whole. This may limit the generalizability of our results.

Nevertheless, this study had several strengths. These include its strict exclusion criteria and use of a standardized assessment instrument to investigate the relationships between pets and owners. These attributes bolster the accuracy of our results and reduce the influence of confounding factors, increasing the strength of our conclusions. Future studies may examine the factors that mediate the association between pet ownership and happiness to better comprehend this phenomenon.

This brief discussion begins with a quick summary of the results and hypothesis. The next paragraph cites previous research and compares its findings to those of this study. Information from previous studies is also used to help interpret the findings. After discussing the results of the study, some limitations are pointed out. The paper also explains why these limitations may influence the interpretation of results. Then, final conclusions are drawn based on the study, and directions for future research are suggested.

How to make your discussion flow naturally

If you find writing in scientific English challenging, the discussion and conclusions are often the hardest parts of the paper to write. That’s because you’re not just listing up studies, methods, and outcomes. You’re actually expressing your thoughts and interpretations in words.

• How formal should it be?
• What words should you use, or not use?
• How do you meet strict word limits, or make it longer and more informative?

Always give it your best, but sometimes a helping hand can, well, help. Getting a professional edit can help clarify your work’s importance while improving the English used to explain it. When readers know the value of your work, they’ll cite it. We’ll assign your study to an expert editor knowledgeable in your area of research. Their work will clarify your discussion, helping it to tell your story. Find out more about AJE Editing.

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Discussion Vs. Conclusion: Know the Difference Before Drafting Manuscripts

The discussion section of your manuscript can be one of the hardest to write as it requires you to think about the meaning of the research you have done. An effective discussion section tells the reader what your study means and why it is important. In this article, we will cover some pointers for writing clear/well-organized discussion and conclusion sections and discuss what should NOT be a part of these sections.

What Should be in the Discussion Section?

Your discussion is, in short, the answer to the question “what do my results mean?” The discussion section of the manuscript should come after the methods and results section and before the conclusion. It should relate back directly to the questions posed in your introduction, and contextualize your results within the literature you have covered in your literature review . In order to make your discussion section engaging, you should include the following information:

• The major findings of your study
• The meaning of those findings
• How these findings relate to what others have done
• Limitations of your findings
• An explanation for any surprising, unexpected, or inconclusive results
• Suggestions for further research

Your discussion should NOT include any of the following information:

• New results or data not presented previously in the paper
• Unwarranted speculation
• Tangential issues
• Conclusions not supported by your data

Related: Avoid outright rejection with a well-structured manuscript. Check out these resources and improve your manuscript now!

How to Make the Discussion Section Effective?

There are several ways to make the discussion section of your manuscript effective, interesting, and relevant. Hear from one of our experts on how to structure your discussion section and distinguish it from the results section:

Now that we have listened to how to approach writing a discussion section, let’s delve deeper into some essential tips with a few examples:

• Most writing guides recommend listing the findings of your study in decreasing order of their importance. You would not want your reader to lose sight of the key results that you found. Therefore, put the most important finding front and center. Example: Imagine that you conduct a study aimed at evaluating the effectiveness of stent placement in patients with partially blocked arteries. You find that despite this being a common first-line treatment, stents are not effective for patients with partially blocked arteries. The study also discovers that patients treated with a stent tend to develop asthma at slightly higher rates than those who receive no such treatment.

Which sentence would you choose to begin your discussion? Our findings suggest that patients who had partially blocked arteries and were treated with a stent as the first line of intervention had no better outcomes than patients who were not given any surgical treatments.   Our findings noted that patients who received stents demonstrated slightly higher rates of asthma than those who did not. In addition, the placement of a stent did not impact their rates of cardiac events in a statistically significant way.

If you chose the first example, you are correct!

• If you are not sure which results are the most important, go back to your research question and start from there. The most important result is the one that answers your research question.
• It is also necessary to contextualize the meaning of your findings for the reader. What does previous literature say, and do your results agree? Do your results elaborate on previous findings, or differ significantly?
• In our stent example, if previous literature found that stents were an effective line of treatment for patients with partially blocked arteries, you should explore why your interpretation seems different in the discussion section. Did your methodology differ? Was your study broader in scope and larger in scale than the previous studies? Were there any limitations to previous studies that your study overcame? Alternatively, is it possible that your own study could be incorrect because of some difficulties you had in carrying it out? The discussion section should narrate a coherent story to the target audience.
• Finally, remember not to introduce new ideas/data, or speculate wildly on the possible future implications of your study in the discussion section. However, considering alternative explanations for your results is encouraged.

Avoiding Confusion in your Conclusion!

Many writers confuse the information they should include in their discussion with the information they should place in their conclusion. One easy way to avoid this confusion is to think of your conclusion as a summary of everything that you have said thus far. In the conclusion section, you remind the reader of what they have just read. Your conclusion should:

• Restate your hypothesis or research question
• Restate your major findings
• Tell the reader what contribution your study has made to the existing literature
• Highlight any limitations of your study
• State future directions for research/recommendations

Your conclusion should NOT:

• Introduce new arguments
• Introduce new data
• Fail to include your research question
• Fail to state your major results

An appropriate conclusion to our hypothetical stent study might read as follows:

In this study, we examined the effectiveness of stent placement. We compared the patients with partially blocked arteries to those with non-surgical interventions. After examining the five-year medical outcomes of 19,457 patients in the Greater Dallas area, our statistical analysis concluded that the placement of a stent resulted in outcomes that were no better than non-surgical interventions such as diet and exercise. Although previous findings indicated that stent placement improved patient outcomes, our study followed a greater number of patients than those in major studies conducted previously. It is possible that outcomes would vary if measured over a ten or fifteen year period. Future researchers should consider investigating the impact of stent placement in these patients over a longer period (five years or longer). Regardless, our results point to the need for medical practitioners to reconsider the placement of a stent as the first line of treatment as non-surgical interventions may have equally positive outcomes for patients.

Did you find the tips in this article relevant? What is the most challenging portion of a research paper for you to write? Let us know in the comments section below!

This is the most stunning and self-instructional site I have come across. Thank you so much for your updates! I will help me work on my dissertation.

Thank you so much!! It helps a lot!

very helpful, thank you

thanks a lot …

this is one of a kind! awesome, straight to the point and easy to understand! Thanks a lot

Thank you so much for this, I never comment on these types of sites but I just had too here as I’ve never seen an article that has answered everyone of the questions I wanted when I searched on Google. Certainly not to the extent and clear clarity that you have presented. Thanks so much for this it has put my mind to ease a bit with my terrible dissertation haha.

Have a nice day.

Helped massively with writing a good conclusion!

Extremely well explained all details in simple and applicable manner, Thank you very much for outstanding article. It really made life easy. Ravi, India.

Thanks a lot for such a nicely explained difference of discussion and conclusion. now got some basic idea to write what.

Thanks for clearing the great confusion. It gave real clarity to me!

Clarified my confusion. Thank you for this article

This website certainly has all of the information I wanted concerning this subject and didn’t know who to ask.

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6 Writing the Discussion and Conclusion Sections

6.1 Discussion

This section discusses your results, presenting the “so what,” or “why should the reader care” about your research. This is where you explain what you think the results show. Tell the reader the significance of your document by discussing how the results fit with what is already known as you discussed in your introduction, how the results compare with what is expected, or why are there unexpected results. Here are some words to get you thinking about this section: evaluate, interpret, examine, qualify, etc.

Start by either summarizing the the information in this section or by stating the validity of the hypothesis. This allows readers to see upfront your interpretation of the data. End the discussion by summarizing why the results matter.

Writing tips:

• Summarize the most important findings at the beginning (1-3 sentences)
• Describe patterns and relationships shown in your results
• Explain how results relate to expectations and literature cited in Introduction
• Explain contradictions and exceptions
• Describe need for future research (if no Conclusion section)
• Overgeneralize, use specific supported statements
• Ignore unexpected results or deviations from your data
• Speculate conclusions that cannot be tested in the foreseeable future.

6.2 Conclusion

The Discussion usually serves as the conclusion. If there is a separate conclusion section then it should be brief, only one or two paragraphs. In the conclusion typically authors offer either recommendations or future perspectives for the research. Figs. 2.9 and 2.10 show the Discussion and Conclusion sections from the sample paper.

Technical Writing @ SLCC Copyright © 2020 by Department of English, Linguistics, and Writing Studies at SLCC is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Results, discussion, and conclusion, results/findings.

The Results (or Findings) section follows the Methods and precedes the Discussion section. This is where the authors provide the data collected during their study. That data can sometimes be difficult to understand because it is often quite technical. Do not let this intimidate you; you will discover the significance of the results next.

The Discussion section follows the Results and precedes the Conclusions and Recommendations section. It is here that the authors indicate the significance of their results. They answer the question, “Why did we get the results we did?” This section provides logical explanations for the results from the study. Those explanations are often reached by comparing and contrasting the results to prior studies’ findings, so citations to the studies discussed in the Literature Review generally reappear here. This section also usually discusses the limitations of the study and speculates on what the results say about the problem(s) identified in the research question(s). This section is very important because it is finally moving towards an argument. Since the researchers interpret their results according to theoretical underpinnings in this section, there is more room for difference of opinion. The way the authors interpret their results may be quite different from the way you would interpret them or the way another researcher would interpret them.

Note: Some articles collapse the Discussion and Conclusion sections together under a single heading (usually “Conclusion”). If you don’t see a separate Discussion section, don’t worry.  Instead, look in the nearby sections for the types of information described in the paragraph above.

When you first skim an article, it may be useful to go straight to the Conclusion and see if you can figure out what the thesis is since it is usually in this final section. The research gap identified in the introduction indicates what the researchers wanted to look at; what did they claim, ultimately, when they completed their research? What did it show them—and what are they showing us—about the topic? Did they get the results they expected? Why or why not? The thesis is not a sweeping proclamation; rather, it is likely a very reasonable and conditional claim.

Nearly every research article ends by inviting other scholars to continue the work by saying that more research needs to be done on the matter. However, do not mistake this directive for the thesis; it’s a convention. Often, the authors provide specific details about future possible studies that could or should be conducted in order to make more sense of their own study’s conclusions.

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Hanney S, Boaz A, Jones T, et al. Engagement in research: an innovative three-stage review of the benefits for health-care performance. Southampton (UK): NIHR Journals Library; 2013 Oct. (Health Services and Delivery Research, No. 1.8.)

Engagement in research: an innovative three-stage review of the benefits for health-care performance.

Chapter 7 discussion and conclusions.

• Developing appropriate methods for data collection and analysis

This review took an innovative form because of the research topic (largely unexplored, but potentially important) and the disparate nature of the evidence that was identified. A review by Clarke and Loudon, 31 published after our proposal had been submitted, was also influential because it suggested that the evidence base to support the widely held assumption that research engagement improves health-care performance was less strong than some had thought. We therefore felt it important to see if (as our initial scoping exercise indicated) we could identify, and analyse, a larger body of evidence through conducting a focused review that would involve a more recent search than Clarke and Loudon's in 2009, 31 and would have a somewhat wider scope.

By itself, however, such a focused review seemed unlikely to enable us fully to address the request in the ITT for an evidence synthesis ‘which maps and explores the likely or plausible mechanisms through which research engagement might bear on wider performance at clinician, team, service or organisational levels’. 1 For example, both our original scoping exercise and advice from an expert suggested we should consider the way in which some health-care organisations support research engagement as part of an overall strategy to improve the quality of the health care they provide, and it was clear that important evidence on such issues came from descriptive articles that would not meet the inclusion criteria for the focused review.

We responded to these challenges by developing what we have called an hourglass review. This consisted of three stages:

• A broad scoping or mapping stage in which we explored a large number of bodies of literature that we thought might be relevant to the review topic.
• A focused (or formal) review on the narrower (though still large) core question of whether or not research engagement improves health care. For this review we searched extensively, but applied quite tight inclusion criteria.
• Finally, a wider (but less systematic) review of relevant papers that we had identified during the two earlier stages (plus continuing snowballing). We therefore included papers that did not meet our inclusion criteria for the focused review, but nevertheless had something important to say, in particular about the mechanisms through which research engagement might improve performance and about the role of organisations.

The hourglass shape refers to the scope of the analysis at each stage, and to the number of papers considered in detail; in terms of the sheer volume of titles and abstracts processed, the throughput of the review was greatest in the second (or middle) stage.

It became clear at the mapping stage that no neat patterns were emerging from the literature. Therefore, we developed a matrix to help us characterise the circumstances in which research engagement might improve health-care performance and the mechanisms that might be at work.

We identified two main dimensions along which to categorise our research on research studies: the degree of intentionality of the link between research engagement and performance and the scope of the impact on health care . (These are explained in detail in Chapter 2 and illustrated in Table 1 .)

The three overlapping categories of intentionality we used in our analysis were:

• least intentionality – where the improved health-care performance resulting from engagement in research arises as a by-product of a trial or other research which has been conducted to test a specific therapy or approach
• a middle category – where structures such as research networks promote the conduct of research on specific therapies in a particular field but can also have the wider remit of informing network members about research more generally and addressing their identified research needs
• greatest intentionality – where various interventions such as collaborative approaches, participatory and action research, and organisational approaches explicitly seek to produce improvements in health-care performance as a direct consequence of research engagement.

The two categories related to the scope of the impact were:

• broader impact – those who have engaged in research becoming more willing and/or able (because of individual or unit capacities) to provide evidence-based care that is not related to the specific findings of the research on which they were engaged
• specific impact – those who have engaged in research becoming more willing and/or able (because of increased knowledge/understanding of the findings and/or infrastructure related to the procedures, etc.) to provide evidence-based care that is related to the specific findings or processes of the research on which they were engaged.
• Emerging themes about the role of research engagement
• There is still a relatively small number of papers on this topic. The previous review by Clarke and Loudon, 31 plus the 2011 analyses by Selby 93 in Annals of Oncology , both called for further studies to be conducted.
• We identified a somewhat more substantial body of evidence than Clarke and Loudon 31 because the scope of our review was wider (e.g. not limited to trials) and we conducted it at a later date. We finished with 33 papers 97 – 129 in our focused review, including 12 97 – 102 , 104 , 105 , 108 – 111 of the 13 that Clarke and Loudon had identified.
• Twenty-eight papers 97 , 99 , 101 – 105 , 107 , 108 , 110 – 126 , 128 , 129 were positive (of which six 105 , 112 , 115 , 120 , 126 , 129 were positive/mixed) in relation to the key question of whether or not research engagement improves health-care performance. Just five papers 98 , 100 , 106 , 109 , 127 were negative (of which two 100 , 127 were mixed/negative).
• The pattern of our findings is similar to that of Clarke and Loudon. 31 Only a minority of the positive cases (seven 102 , 105 , 107 , 110 , 114 , 116 , 129 out of 28) reported some improvement in health outcomes. For the rest, the improved care took the form of improved (usually more evidence-based) processes of care.
• We identified 21 papers 97 – 117 in our ‘by-product’ category, including all 12 97 – 102 , 104 , 105 , 108 – 111 of the papers from Clarke and Loudon 31 that we included in our review. Of these we classified 17 97 , 99 , 101 – 105 , 107 , 108 , 110 – 117 as positive, or positive/mixed. By adding papers that we classified as being in the research network and the intervention categories, we increased the total number of papers by a further 12. 118 – 129 The eight included network papers 118 – 125 were classified as being positive, or positive/mixed; as were three 126 , 128 , 129 of the four 126 – 129 intervention studies. These papers are of considerable importance in enabling us to go further than previous evidence in suggesting research evidence does improve health-care performance.
• Of the 28 97 , 99 , 101 – 105 , 107 , 108 , 110 – 126 , 128 , 129 positive or mixed/positive studies, 13 99 , 102 , 103 , 108 , 110 , 113 – 119 , 122 describe broader impact and 15 97 , 101 , 104 , 105 , 107 , 111 , 112 , 120 , 121 , 123 – 126 , 128 , 129 describe specific impact. Within this overall balance 10 99 , 102 , 103 , 108 , 110 , 113 – 117 out of 17 97 , 99 , 101 – 105 , 107 , 108 , 110 – 117 of the by-product studies showed broader impact (as would fit with the absorptive capacity model), and all the intervention studies showed specific impact (as would fit a key element of the collaborative and action research theories).
• We also considered the levels of research engagement. Clarke and Loudon 31 had examined two levels: practitioners and institutions. In practice we found it very difficult to be more discerning than this because there is little consensus about the reporting terms used and we could not readily apply the separate categories of team, service and organisation. Therefore, we followed Clarke and Loudon 31 in using a twofold distinction between clinicians and institutions. Twenty-two studies 97 – 99 , 102 – 105 , 107 , 109 , 110 , 114 – 116 , 118 – 122 , 126 – 129 were at the institutional level, and 11 100 , 101 , 106 , 108 , 111 – 113 , 117 , 123 – 125 at the clinician level. Of the 28 97 , 99 , 101 – 105 , 107 , 108 , 110 – 126 , 128 , 129 positive and mixed/positive papers, 19 97 , 99 , 102 – 105 , 107 , 110 , 114 – 116 , 118 – 122 , 126 , 128 , 129 were institutional and nine 101 , 108 , 111 – 113 , 117 , 123 – 125 clinician level. Of the five 98 , 100 , 106 , 109 , 127 negative and mixed/negative papers, three 98 , 109 , 127 were institutional and two 100 , 106 clinician level.
• For this analysis we drew on the focused review and the wider review, and applied the matrix.
• The positive papers in the focused review describe a diverse variety of mechanisms through which research engagement might improve health care. We have more evidence than previously collated (and the wider review added even more), but we have also shown that there are many circumstances and mechanisms at work, that more than one mechanism is often operative, and that the evidence available for each one is limited.
• The wider review allowed a fuller data collection, and the matrix facilitated a more nuanced analysis of the various roles of research engagement.
• Using the by-product and network categories from the matrix we can reasonably suggest that when clinicians and health-care organisations engage in research there is the likelihood of a positive impact on the health-care performance, even when that has not been the primary aim of the research. This finding is further supported by evidence from the wider review, which provides examples of studies that illustrate some progress along the pathway from research engagement to improved health care. However, the wider review also indicated situations in which the impacts seemed less likely to arise, and the operation of networks proved not always easy to sustain.
• There were only four papers in the intervention category in the focused review, but a much higher number in the wider review. The latter demonstrate how collaborative or action research can encourage some progress along the pathway from research engagement towards improved health-care performance. However, various studies also demonstrated that there can be considerable obstacles to overcome, particularly with one-off initiatives.
• Health-care organisations and systems provide the context within which research engagement operates at other levels. And organisations in which the research function is fully integrated into the organisational structure can out-perform other organisations that pay less formal heed to research and its outputs. 76 , 189 – 193 However, at the organisational level, as at other levels, engagement in research operates through a variety of mechanisms and is only one of many influences on performance. Disaggregating how these mechanisms operate in complex systems is not straightforward.
• Widespread engagement in research by all the key groups of relevant stakeholders often influences the type of research pursued. What is sought in these circumstances is research that answers specific concerns, and this (deliberately) results in a programme of research driven by the needs of the health-care system and its users.
• In the USA, these initiatives include the 2003 Roadmap for Medical Research from the NIH 149 in which resources have been devoted to boost CBPPR through the Clinical and Translation Science Awards. The NIH has funded a large-scale project aimed at identifying how this initiative might best operate. 131 , 147 , 148
• In the UK, the NHS Commissioning Board has a duty to promote research and innovation: ‘The NHS Commissioning Board's objective is to ensure that the new commissioning system promotes and supports participation by NHS organisations and NHS patients in research funded by both commercial and non-commercial organisations, most importantly to improve patient outcomes, but also to contribute to economic growth’. 204 In addition, the goal of the Academic Health Science Networks is to improve patient and population health outcomes by translating research into practice and developing and implementing integrated health-care systems. 205
• Various studies have indicated that new structures such as research networks and schemes aimed at involving individual health-care professionals more fully in research can face difficulties in making progress, particularly if there are not changes at the organisational level to support the initiatives. 127 , 169
• The moves towards trials and other well-found research taking place within networks, and as part of wider interventions, means that increasingly research engagement leading to improved health-care performance shifts from being a by-product to an intended outcome.
• Pulling together the evidence related to mechanisms through which health care might be improved in research-active settings

The majority of the papers in the focused review were by-product papers, and the majority of these were positive about the relation between research engagement and improved performance. Various potential mechanisms were suggested, including:

• a specific impact, at individual level, through the increased awareness and understanding of specific findings arising from research participation
• a specific impact, at institutional level, through an increased use of the protocol and processes associated with a specific research study
• a broader impact, at individual level, through changes in attitudes to research and resulting behaviour
• a broader impact, at institutional level, through infrastructure development associated with research participation.

We also saw that the mechanisms associated with research networks (the second largest group of papers in the focused review) represent a partial formalisation and use on a regular basis (through the provision of more effective collaboration and more supportive contexts) of some of these potential mechanisms. Finally, we saw how this partial formalisation had been taken still further in interventions deliberately designed to integrate the research function into organisational structures (as described in the intervention papers in the focused review and more extensively covered in the wider review).

Building on these findings, on the theoretical approaches discussed in Chapter 2 , and on previous work on potential mechanisms by Krzyzanowska et al. , 95 we can start to develop a taxonomy of the various mechanisms and submechanisms through which outcomes may be improved in research-active settings ( Box 4 ).

Mechanisms in research-active settings through which health care improved 1. Absorptive capacity: this is most relevant for wider adoption of research in institutions:

• Limitations and robustness of the conclusions

This evidence synthesis could be described as research on research on research: it is an evidence synthesis of studies that assess the impact of research engagement. Conducting reviews of this nature is a complex task and it is not surprising that there are various potential limitations to this study. The first relates to locating the relevant literature. At the outset we anticipated that there would be many bodies of literature addressing the review question. We used the initial mapping stage to explore the potential locations of relevant literature and our findings informed the search strategy for the focused review. The search terms remained quite broad in order to cast the net as wide as possible and catch papers published in different fields, journals and countries. As a consequence, a significant amount of time within the review process was spent on mapping, refining the question and developing search terms. Owing to the nature of the research question, the search terms included a number of generic and widely used terms including ‘research’ and ‘engagement’. We sought expert advice and worked with an information scientist to develop the search strategy and conduct a search that was sufficiently sensitive to identify relevant papers. Despite the difficulty of the task, we were reassured that we identified nine of the 13 papers in the previous review by Clarke and Loudon 31 using the search terms (and the others were added by snowballing).

In order to make the search feasible, the review focused on engagement in research, rather than using the wider definition of research engagement that would have included engagement with research. The focused review shares the limitations of many systematic reviews. In pursuing a narrow question, we inevitably excluded large volumes of potentially interesting, relevant research that did not meet the inclusion criteria. As with other reviews, we have also had to exclude a number of papers owing to the lack of information provided by the authors about key elements of the study (such as design and outcomes). We have tried to address these limitations by conducting a wider additional synthesis (the wider review) to help explain the findings of the focused review and give the final review more explanatory power.

In Chapter 4 , we discussed concerns that arose in our analysis of two studies in the focused review about the extent to which engagement in small individual studies could improve performance, 100 , 106 compared with relying on reviews of all the studies in a field. We suggest this depends on the context, in this case the existing knowledge base, which provides the basis for judgements about whether or not a study is well-found and whether its findings are supported or challenged by other studies in the same field. One of the limitations of our review is that it was not always possible to make these judgements.

Finally, another common limitation reflects the reliance of reviewers on what is already published in the literature. This meant that the review was only able to capture very recent developments (such as the outcomes of UK research networks) through an abstract. The section of the focused review on networks draws exclusively on US studies of research networks. This reflects the more established nature of research networks in the USA, and also an approach to evaluation that is consistent with the rigorous inclusion criteria used for the focused review. Linked to this is another challenge common to other systematic reviews: the impact of publication bias. As with other fields, there is likely to be a bias towards the publication of studies with positive results to share with the academic community. We have sought to address this by searching the grey literature, conducting a web search and writing to some key authors in the field to identify unpublished literature. We have also kept this potential limitation in mind in conducting the analysis. Furthermore, in at least some of the studies it is complicated to interpret the findings in terms of the issues we were considering.

• Methodological developments

Given the topic and our interest in both realist synthesis and narrative synthesis we sought expert advice from the advisory group. We were advised that our review question was likely to contain too many diverse aspects to conduct a meaningful realist synthesis. We drew on narrative synthesis methods at various stages, including the mapping stage, but felt the need to develop our own approach to adequately address the review question.

Despite the limitations described above, we found the hourglass review approach had various advantages. It allowed us the opportunity to start broadly, which was useful when it was difficult to locate the examples of the phenomenon we were seeking. The hourglass approach was also useful because the detailed mapping allowed us to explore issues from many angles. It also meant that we could then conduct a more focused, or formal, review on the central question of whether or not research engagement improves health care. This was particularly desirable in the context of the review published after the submission of our proposal 31 which concluded that the evidence for suggesting research engagement improves health-care performance was not as strong as seemed to be widely assumed.

Finally, the wider review allowed us to collate a wider range of evidence and more fully address a range of issues related to the mechanisms through which research engagement might improve health-care performance. In relation to networks, it allowed us to bring in examples from beyond the USA, and also brought in some examples of problems with networks to counteract the examples in the focused review, which were all positive. In relation to issues of collaborative research and the organisational dimension, the wider review allowed us to consider a wide range of papers that did not meet the inclusion criteria adopted for the focused review.

One of the main reasons for developing the hourglass review methodology was to allow for a final, wider review stage and, as is demonstrated above, it was this less formal approach that enabled us to draw on a more diverse range of literature.

So, the approach we developed allowed considerable progress to be made, but was very resource intensive.

• Discussing future research needs and the scope for developing performance indicators

Clarke and Loudon 31 recommend that RCTs should be conducted in this area in an attempt to generate stronger evidence, but acknowledged the considerable difficulties involved. Pater et al. 94 analyse in considerable detail how clinical trials and other well-found research studies in this field could be designed, but the focus of their proposed question was narrower than the one we addressed in our review. They were asking whether or not patients who receive care in a research environment derive benefits that are attributable to that general research activity, but not simply because these patients were exposed to a successful intervention as patients in a research project. They state: ‘this question has been deliberately phrased to indicate our concern with benefits that occur contemporaneously with the research activity’ (p. vii58).

In conducting our review, we identified several areas where there appeared to be less evidence available than we might have expected to find. These included empirical studies related to the role of medical academics. In relation to research networks there seemed to be fewer studies from the UK than from the USA on how the benefits of research engagement might arise. There might be some problems in replicating exactly the type of studies identified in the focused review because there has now been such a growth of research networks in the UK; nevertheless, that only serves to highlight the importance of this area.

As explained in Chapter 1 , our review focused primarily on the issues of performance in relation to the quality agenda. We did, however, identify one study that undertook a data envelopment analysis comparing the performance of academic medical centres with other hospitals in Germany, and identified an ‘emphasis on research’ as a factor that increased efficiency. 206 This study compared the level of research undertaken with other factors, but failed to meet the inclusion criteria for our focused review because the data on health-care performance related to the volume of work and not the quality of care. Nevertheless, this is an approach on which it might be worth conducting a scoping exercise in the UK.

The issue of research engagement as a factor in performance improvement has, largely, not previously been systematically researched as a topic in its own right. The studies we identified have mainly been one-off studies and/or considerations of the issue as part of a wider study. Therefore, we believe there would be scope for deliberately considering aspects such as the potentially negative impacts of research engagement alongside benefits. Organisation-wide interventions designed to promote research engagement also require further research. However, there are significant methodological challenges in conducting evaluations of these complex interventions and there is a need for methodological development to improve evaluations of how different mechanisms operate in complex systems (as we suggest in Chapters 5 and 6 ). There is also a role for social theory in developing and understanding the role of research engagement in promoting health-care improvement. Therefore, overall we believe there would be scope for a programme of work in this field that identified the various roles that could be played by a range of different methods that would be appropriate for the various topics identified. Such methods could include observational studies of research engagement within organisations, applications of social theory, and the use of large data sets.

Through the Quality Accounts, NHS trusts have reported on the number of patients recruited annually to participate in research approved by local research ethics committees. This is a useful development, but given the importance that is now being attached to research there could be value in monitoring key issues such as the relevance of the research conducted, and establishing whether or not this research activity is producing beneficial change. Experience suggests, however, that it is difficult to capture such items, especially through the small number of indicators that it is likely to be practical to introduce. This is an issue that requires careful analysis. Furthermore, one of the key elements of the VA's use of research engagement was to identify the most appropriate performance indicators (PIs) to apply within the organisation to show improvements in the processes of care.

Overall, a key role for PIs could be to encourage research participation and a culture of maximising the benefits from such engagement. Therefore, some illustrative examples of the approach towards indicators that might be used by NHS trusts and Clinical Commissioning Groups are provided below.

• An indicator for research activity approved by local research ethics committees could continue along the lines already collected through the Quality Accounts.
• changes in care settings (e.g. additional accommodation, equipment or personnel brought in as part of a research project or programme and remaining in place afterwards)
• changes in human capital (e.g. training or updating staff through research engagement), or changes in team or individual behaviour (including more rapid uptake of evidence-based treatments)
• changes in processes of care (e.g. improvements in following clinical guidelines, improvements in monitoring and support, early access to new technologies).
• An indicator of the percentage of eligible staff who have participated in research studies involving NHS/NIHR-wide research networks might be useful.
• There might also be scope for an indicator to demonstrate the extent to which the Trust or Commissioning Group has arrangements in place to work with staff and patients to identify specific problems within a health-care system/organisation and generate and implement research-based solutions, thinking strategically as well as in the short term.
• Conclusions

The focused review identified more evidence (33 papers 97 – 129 ) than has previously been collated on the question of whether or not research engagement improves health-care performance. This evidence was broadly positive, with 28 papers 97 , 99 , 101 – 105 , 107 , 108 , 110 – 126 , 128 , 129 drawing positive conclusions about the impact of research engagement (with six 105 , 112 , 115 , 120 , 126 , 129 of these being positive/mixed). Just five papers 98 , 100 , 106 , 109 , 127 were negative (of which two 100 , 127 were negative/mixed). Drawing on the focused review (especially the by-product and network categories from the matrix) we can reasonably suggest, therefore, that when clinicians and health-care organisations engage in research there is the likelihood of a positive impact on health-care performance, but this is more likely to be on improved health-care processes than improved patient outcomes.

The wider review added additional evidence. In particular, although there were only four papers from the focused review in the intervention category of the matrix (covering interventions such as collaborative and action research), a much higher number from the wider review contribute to our analysis of the mechanisms and, for example, demonstrate how research engagement can encourage some progress along the pathway towards improved health-care performance.

Overall, we have also seen that there are many circumstances and mechanisms at work, that more than one mechanism is often operative, and that the evidence available for each one is limited. This limits the immediate implications for practice. The large-scale initiatives now being developed to encourage research engagement as a means of improving performance may also highlight the need for more research on research engagement and for adequate PIs of research engagement and of research quality in NHS trusts, building on what has previously been sought through the Quality Accounts and on the suggestions set out above. However, our findings do tell us that the mechanisms through which research engagement might improve performance overlap and rarely act in isolation, and that their effectiveness often depends on the context in which they operate. Generally, at lower levels of intentionality (where improved health-care performance is a by-product of a research study) we identified a series of one-off studies in which a diversity of detailed mechanisms were applied. At higher levels of intentionality (e.g. networks and collaborations) there had been more formalisation of potential mechanisms and research processes themselves had become an increasingly important means through which research engagement can improve health-care performance.

The number of research networks is growing, and these new structures continue to develop and evolve. The contribution of collaborative approaches to research is also developing. At an organisational level, the mechanisms through which research engagement promotes performance improvement are often only one facet within a wider, multipronged change strategy. Organisations that have deliberately integrated the research function into organisational structures demonstrate how research engagement can, among other factors, contribute to improved health-care performance.

• Recommendations for research
• Further explorations of how particular mechanisms promote research engagement. Evaluations of research networks and of existing schemes to promote the engagement of clinicians and managers in research would be particularly valuable.
• Detailed observational research focusing on research engagement within organisations, to build understanding of mechanisms, and to explore potentially negative impacts of research engagement alongside benefits.
• Analysis of organisation-wide interventions designed to promote research engagement also requires further research. There are significant methodological challenges in conducting evaluations of these complex interventions and a need for methodological development to improve evaluations of how different mechanisms operate in complex systems.
• Scoping exercises to identify possibilities of using large databases of research production and hospital performance.
• The application of social theory in developing and understanding the role of research engagement in promoting health care improvement.

Included under terms of UK Non-commercial Government License .

• Cite this Page Hanney S, Boaz A, Jones T, et al. Engagement in research: an innovative three-stage review of the benefits for health-care performance. Southampton (UK): NIHR Journals Library; 2013 Oct. (Health Services and Delivery Research, No. 1.8.) Chapter 7, Discussion and conclusions.
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• Published: 01 May 2024

Addressing loneliness and social isolation in 52 countries: a scoping review of National policies

• Nina Goldman   ORCID: orcid.org/0000-0002-3058-1251 1 , 2   na1 ,
• Devi Khanna   ORCID: orcid.org/0000-0002-9254-0869 1   na1 ,
• Marie Line El Asmar   ORCID: orcid.org/0000-0002-0733-3911 3 ,
• Pamela Qualter   ORCID: orcid.org/0000-0001-6114-3820 1 &
• Austen El-Osta   ORCID: orcid.org/0000-0002-8772-4938 2  

BMC Public Health volume  24 , Article number:  1207 ( 2024 ) Cite this article

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Even prior to the advent of the COVID-19 pandemic, there was ample evidence that loneliness and social isolation negatively impacted physical and mental health, employability, and are a financial burden on the state. In response, there has been significant policy-level attention on tackling loneliness. The objective of this scoping review was to conduct a loneliness policy landscape analysis across 52 countries of the UN European country groups. Our policy analysis sought to highlight commonalities and differences between the different national approaches to manage loneliness, with the goal to provide actionable recommendations for the consideration of policymakers wishing to develop, expand or review existing loneliness policies.

We searched governmental websites using the Google search engine for publicly available documents related to loneliness and social isolation. Seventy-eight documents were identified in total, from which 23 documents were retained. Exclusion of documents was based on predetermined criteria. A structured content analysis approach was used to capture key information from the policy documents. Contextual data were captured in a configuration matrix to highlight common and unique themes.

We could show that most policies describe loneliness as a phenomenon that was addressed to varying degrees in different domains such as social, health, geographical, economic and political. Limited evidence was found regarding funding for suggested interventions. We synthesised actionable recommendations for the consideration of policy makers focusing on the use of language, prioritisation of interventions, revisiting previous campaigns, sharing best practice across borders, setting out a vision, evaluating interventions, and the need for the rapid and sustainable scalability of interventions.

Conclusions

Our study provides the first overview of the national loneliness policy landscape, highlighting the increasing prioritisation of loneliness and social isolation as a major public health and societal issue. Our findings suggest that policymakers can sustain this momentum and strengthen their strategies by incorporating rigorous, evidence-based intervention evaluations and fostering international collaborations for knowledge sharing. We believe that policymakers can more effectively address loneliness by directing funds to develop and implement interventions that impact the individual, the community and society.

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Introduction

The significant increase in research on loneliness and social isolation over the last decade, and especially following the advent of the COVID-19 pandemic [ 1 , 2 , 3 ] highlighted the detrimental consequences of loneliness to individuals, society and governments worldwide. For older adults, the pandemic led to feelings of loneliness due to a lack of companionship and connections, which can negatively impact cognition, and mental health [ 4 ]. The paradox of social distancing, intended to protect older adults, further isolated them and exacerbated the negative effects of loneliness [ 5 ]. A longitudinal study on adolescents showed that they also experienced social isolation from peers, and that resulted in increases of loneliness due to COVID-related school closures [ 6 ]. Evidence shows that a lack of social connection impacts physical and mental health [ 7 ], employability opportunities [ 8 ], and how it is related to social disparities [ 1 , 9 ]. In response, there has been significant policy-level attention on loneliness, with, for example, the United Kingdom of Great Britain and Northern Ireland (GB) [ 10 ] and Japan [ 11 ] both appointing a Minister for Loneliness in 2018 (GB) and 2021 (Japan) respectively. In a joint press statement, both an EU Commissioner and the Japanese Loneliness Minister agreed that “loneliness and social isolation pose crucial challenges to the cohesion, economy and mental and physical health in 21st century societies across the world” [ 12 ]. In November 2023, the World Health Organization highlighted the importance of social connection, recognising the significant and often underestimated impact of loneliness and isolation on our health and well-being. This recognition led to the launch of its Commission on Social Connection (2024–2026), which aims to address this issue as a public health concern [ 13 ]. However, little is known about the extent that loneliness is currently included in national strategies and policies across the world.

Loneliness is often defined in psychological terms as an unpleasant feeling that people experience when they perceive their social relationships to be qualitatively or quantitatively inadequate [ 14 ]. The quality, rather than the quantity, of social relationships plays a greater role in loneliness [ 15 ]. While temporary loneliness is a natural human experience, chronic loneliness has serious negative consequences for health and life expectancy. There are three main types of loneliness: intimate (also known as emotional) loneliness, relational (also known as social) loneliness and collective loneliness, first identified by McWhirter (1990) [ 16 ], and empirically validated by Hawkley et al. (2005) [ 17 ] and Panayiotou et al. (2023) [ 18 ]. Loneliness is distinct from social isolation, which Nicholson Jr. (2009) [ 19 ] defines as “a state in which the individual lacks a sense of belonging socially, lacks engagement with others, has a minimal number of social contacts, and they are deficient in fulfilling and quality relationships” (p. 1346). This does not mean that socially isolated individuals necessarily feel lonely and vice versa.

There are different scales to measure loneliness and social isolation. The most commonly used instruments for measuring loneliness are the indirect measures from De Jong Gierveld Loneliness Scale [ 20 ] and the full UCLA Loneliness Scale [ 21 ], as well as the direct measure from the UK Office for National Statistics [ 22 ]. However, what these definitions fail to measure is the “intensity, frequency and duration of loneliness. Loneliness can be acute (i.e., transient) or chronic (i.e., enduring), and it can be mild to severe in its intensity” [ 23 , p.2]. There are also a variety of scales to measure social isolation, but there is no consensus on which should be used [ 24 ]. Some common scales include the Lubben Social Network Scale [ 25 ], the Cudjoe social isolation typology [ 26 ] or a social isolation index used by Shankar et al. [ 27 ].

Our study contributes to existing literature by presenting an overview of current governmental documents that address loneliness and social isolation. Our intention is that the scoping review would be used by federal agencies or local communities who want to develop their own strategies to address loneliness and social isolation, or by researchers to gain an overview of the policy landscape.

The aim of this study was to characterise the policy landscape relevant to tackling loneliness and social isolation across the UN European country groups to identify commonalities and differences between national approaches to loneliness. A secondary aim was to provide actionable recommendations including their implications based on the scoping review for the consideration of policy makers to help promote the rapid and widescale adoption and diffusion of sustainable, scalable and evidence-based interventions to manage loneliness.

We conducted a scoping review based on Mak and Thomas’ recommendations (2022) [ 28 ] to identify (i) how loneliness and social isolation are defined, (ii) the common characteristics between loneliness policies across countries, (iii) which population groups were targeted, and (iv) whether there was an identifiable commitment to action and funding. We contextualised findings using five domains (geographic, social, health, economic, political) that all affect or are affected by experiences of loneliness and social isolation. We have taken every step to make the scoping review as clear and reproducible as possible, following the PRISMA-ScR guidelines [ 29 ] [see file: Supplementary Material _PRISMA-ScR-Checklist].

Eligibility criteria

A multi-method review approach inspired by Schnable et al. (2021) [ 30 ], including a qualitative policy analysis, was used to identify and describe the characteristics of a collection of national-level government documents with reference to loneliness and social isolation. As national policy documents and commissioned governmental strategies and action plans are not available on a central database, a systematic review was not feasible.

We retrieved and reviewed policy documents that address loneliness or social isolation from a total of 52 countries from the UN European Country Groups: Albania (AL), Andorra (AD), Armenia (AM), Australia (AU), Austria (AT), Azerbaijan (AZ), Belarus (BY), Belgium (BE), Bosnia and Herzegovina (BA), Bulgaria (BG), Canada (CA), Croatia (HR), Czechia (CZ), Denmark (DK), Estonia (EE), Finland (FI), France (FR), Georgia (GE), Germany (DE), Greece (GR), Hungary (HU), Iceland (IS), Ireland (IE), Israel (IL), Italy (IT), Latvia (LV), Liechtenstein (LI), Lithuania (LT), Luxembourg (LU), Malta (MT), Monaco (MC), Montenegro (ME), Netherlands (NL), New Zealand (NZ), North Macedonia (MK), Norway (NO), Poland (PL), Portugal (PT), Republic of Moldova (MD), Romania (RO), Russian Federation (RU), San Marino (SM), Serbia (RS), Slovakia (SK), Slovenia (SI), Spain (ES), Sweden (SE), Switzerland (CH), Türkiye (TR), Ukraine (UA), United Kingdom of Great Britain and Northern Ireland (GB), and United States of America (US). We chose this geographic focus of Europe because the European Union was the first supranational union of states to put loneliness on its agenda with a policy brief published in 2018 [ 31 ]. To ensure comprehensive coverage of European nations, we chose the UN European country groups, recognising that they include some members beyond the continent’s geographical borders.

Articles including policies, reports, strategies and policy briefs were included in the analysis if they were (i) from the two UN country groups under study, (ii) officially published or commissioned by a national government, (iii) publicly available, (iv) published between 1 January 2003 and 1 July 2023, (v) related directly to loneliness and social isolation or indirectly by using other language such as social connection, (vi) published in any language.

Information sources

The main information sources were governmental websites of relevant ministries and departments of the 52 selected countries. Additionally, we used the Google search engine for all publicly available national policies related to loneliness and social isolation.

We conducted desktop research using the key terms “loneliness” and “social isolation” for all publicly available national policies, including a review of government websites to generate an asset map of key policy documents and white papers from each country. Online searches were conducted between 1st February 2023 and 1st July 2023.

Internet searches, using the Google search engine, included the following keywords: [(“loneliness” OR “social isolation” OR “social connection”)] and [(“policy” OR “strategy” OR “actions” OR “reports”) and “Country”]. If this did not yield any results for a specific country, we searched for the government website of that country using primary (loneliness and social isolation) and secondary (strategy/policy) terms to determine if governments published documents on loneliness and social isolation. The Google website translator was used to navigate non-English governmental websites.

Selection of sources of evidence

The documents were not limited to policies, but also included national strategies, technical reports, brochures and webpages published by government agencies, studies commissioned by a government agency, governmental press releases, and parliamentary enquiries from politicians to federal ministers or councillor regarding data on loneliness in their respective countries. If multiple strategies/policies from the same government were found, the most recently published one was included. We focused on national level documents only (excluding any regional strategies).

Where documents retrieved were not in English, they were translated into English using a paid (subscription) version of DeepL Pro, a powerful and sophisticated online translator. For reasons of pragmatism, no attempt was made to quality assure the translation with native speakers.

We excluded 40 documents after a first round of reviews where there was no disagreement between the researchers. For 20 documents there was no consensus, so a third researcher reviewed the documents. After reviewing each document, consensus was reached to exclude 16 of the 20 documents. Documents were excluded for the following reasons: (i) loneliness and social isolation were only mentioned in passing and did not elaborated on the issue of loneliness, or loneliness was not part of a proposed intervention, (ii) highlighted or acknowledged loneliness as a problem but we could not identify any detail or strategies or commitments on how to address it, (iii) short news piece or press releases that did not specifically touch on loneliness or social isolation, (iv) documented queries raised by political representatives addressed to parliament, (v) research articles not commissioned by the government, (vi) local focus, not national, (vii) NGO reports not commissioned by a government and (viii) older versions of included documents.

Data charting process

The principal investigator (NG) developed a coding matrix using Excel based on the study objectives and considerations from Braun and Clarke (2006) [ 32 ]. This matrix was first tested on the British documents (NG, DK, MLEA), as we knew these to be extensively detailed. In an iterative process this matrix was reviewed and adapted after testing it on a random selection of five sources of evidence (NG, DK, MLEA, PQ). After a final round of reviewing and adapting, all authors agreed by consensus that they have captured all desired variables needed to address the study objectives. Each policy document was coded independently by at least two investigators (NG, DK, MLEA) to minimise human error in information extraction.

The configuration matrix was completed for all sources of evidence containing information on: (i) document overview (title, publisher, year of publication, original language of publication), (ii) recommended measurement tool for loneliness, (iii) definitions for loneliness, social isolation and other language around social connection, (iv) target group of policy, (v) proposed or suggested actions by government (raising awareness, funding pledge, call for a development of a loneliness measure, proposed interventions or actions, type of evidence cited, commitment to work with specific charities), and (vi) five key domains (geographic, social, health, economic, political) that affect or are affected by experiences of loneliness and social isolation. We also coded whether the documents referred to five domains (geographic, social, health, economic, political) that have been shown to affect or are affected by experiences of loneliness and social isolation.

Synthesis of results

The data of the configuration matrix were consolidated and are presented as Table  1 , Supplementary Table A [see file: Supplementary Material_Table   A ], and within the text where a presentation in table format was not deemed useful (for data items 3–5 as detailed above). We used the document analysis as proposed by [ 33 ] to analyse all the included documents. This approach is based on an iterative processes of qualitative content analysis [ 34 ], with a specific thematic analysis [ 32 ]. The configuration matrix captured all extracted data from which the authors (NG, DK, MLEA) could identify emerging sub-themes within these broad pre-defined domains of loneliness (geographic, social, health, economic and political domain) using thematic analysis [ 32 ]. To create recommendations, two authors (NG, PQ) reviewed the extracted data, with the team revisiting the sources of evidence where needed.

Our scoping review identified 79 sources of evidence that discussed loneliness and social isolation from across 32 countries in both UN European country groups. We excluded a total of 56 documents after two review rounds for reasons shown in the PRISMA flowchart Fig.  1 . This yielded a subset of 23 documents that were included in our final analysis.

PRISMA flow chart based on [ 24 ]

Wider awareness of loneliness and social isolation in our study area

Here, we delve into the sources of evidence that were excluded from our study, but which are nonetheless noteworthy because they illustrate the momentum of the international conversations around loneliness. In some countries (AT, CH), we found parliamentary enquiries asking about data on loneliness in their respective countries, and whether there were any strategies in place to alleviate loneliness. DE does not have a loneliness strategy, but the governmental Committee for Family Affairs, Senior Citizens, Women and Youth has partially funded the organisation (the Competence Network on Loneliness (KNE)) which looks at the causes and consequences of loneliness and promotes the development and exchange of possible prevention and intervention measures in DE. NZ is a good example where there was no specific policy, despite there being great public awareness. They have an established nationwide trust called “Loneliness New Zealand Charitable Trust”. While some countries had excellent resources targeted at policy makers (e.g. CA), they have not yet been translated into a nationwide policy to address loneliness and social isolation. In countries where there was no national strategy, some cities have designed their own regional strategies or organisations, e.g. Barcelona [ 57 ], Helsinki [ 58 ], or Vancouver [ 59 ]. A map highlighting the loneliness policy development landscape across 52 countries of the UN European Country Groups is shown in Fig.  2 .

Current state of the loneliness policy landscape across the study area. Map created with [ 28 ]

It is important to note that for many countries in the study area we could not identify any resources that met the inclusion criteria. It is difficult to assess why loneliness and social isolation are not on the policy agenda of more national governments. Connel and t’ Hart [ 60 ] have developed a typology of policy inaction. Three of the five types may apply to our context: Type I: Calculated inaction. Governments may make a strategic decision not to act, or not to act now, because they believe that the costs of action outweigh the perceived benefits, or because they want to see a stronger evidence base on an issue. Type II: Ideological inaction. Government inaction as a product of ideology, where governments rely on non-governmental and not-for-profit organisations to address the issue of loneliness. The strong third or social-economy sector in the European Union [ 61 ], which includes more volunteers than paid employees, could give the impression that loneliness and social isolation can be managed without government policies. Type IV: Reluctant inaction. Governments do not act because they perceive an absolute or relative lack of resources to fund loneliness and social isolation policies. This may be the case for the less economically strong countries in our study area that do not have policies in place.

Characteristics of sources of evidence

Table  1 gives an overview of the 23 documents that we included in our analysis. Half the documents were published after 2020. Seven documents had to be translated into English. Certain countries released documents in conjunction with one another. For instance, Denmark published a National Strategy and an Action Plan simultaneously in 2023 that were complementary. Similarly, GB’s 2021 Action Plan builds on the GB Loneliness Strategy published in 2018.

Results of individual sources of evidence

For each of the included sources of evidence, we extracted information with our configuration matrix presented in the section Data items . We believe that presenting the results this way will better suit our study objectives, i.e., to highlight common and unique themes.

Target group of policies

Eight documents (from AL, CA, IT, MT, US) were targeted specifically at the older adult population, often classified as age 65 + years. Definitions, causes and proposed interventions for loneliness and social isolation in those documents were contextualised within the framework of old age. The other documents addressed the general population, often highlighting that there are specific groups that are more vulnerable to becoming lonely or socially isolated. Five of the documents identified target groups at increased risk of loneliness (AU, IE, CH, GB, DK). For instance, children (IE), young adults ages 18–25 years (AU, DK, IE, GB), older adults ages 65 + years (AU, CH, IE, GB), people with disabilities & special needs (AU, DK), people suffering from mental illness (CH), those with long-term illness (GB), migrants and refugees (AU, CH, GB), lower income households (AU), and people living alone (AU, CH), people with lower levels of schooling (CH), single parents (CH), young single men (CH), care leaver (GB), victims of domestic violence (US), LGBTQ + individuals (US) and minorities (DK, US).

Defining loneliness and social isolation

Of the 23 documents included in the review, 11 documents from seven countries (AU, AT, CA, DE, NL, GB, US) provided specific definitions of loneliness and social isolation. Those definitions were based on academic sources, explicitly referenced and cited, except for AT which based their definition on general “experts” rather than a specific source. Peplau and Perlman (1982)’s widely used framework is drawn upon in multiple documents, and some countries (AU, DE, NL, GB) go further in their definitions to distinguish between different types of loneliness, (e.g., social, emotional, and existential loneliness in the NL document).

The 11 documents that used a specific definition of loneliness used the Peplau and Perlman (1982) definition that highlights differences between loneliness and social isolation. Documents noted social isolation as an objective lack of social relationships, while loneliness is considered to be the subjective feelings as a result of that social isolation.

Across all the documents included in our review, both with and without specific definitions of loneliness, other language used around social connection can be classified as follows:

Inclusion in wider society, which includes the terms social inclusion (CZ, DK, IE, MT), social integration (CA) and social participation (DE, NL, CH).

Connecting with others, which includes the terms social networks (CA, DK, DE), social support (CH, US), social connection (AU, US), and social contacts (AT, DE, NL, GB).

Existing resources, which includes the terms social resources (CH), social capital (CH, CA), and social skills (CA, NL).

Covering a deficit, which includes the terms social exclusion (AL, CZ, CA), social vulnerability (IT, CA) and social recovery (AU).

Relationship between loneliness and mental health, which includes the term social wellbeing (GB), and discussions of social prescribing (GB) and the contribution of loneliness to poor mental health (IE).

Mental health, which includes the term social wellbeing (GB), and discussions of social prescribing (GB) and the contribution of loneliness to poor mental health (IE).

Funding pledges

Despite the governmental strategies and action plans to reduce loneliness and social isolation, we found little evidence of a commitment to funding. We identified concrete funding pledges or already provided funding for AL (0.75 m USD for 5 years), DK (145 m USD for 2014–2025), GB (24.8 m USD in 2018; 44.5 m USD in 2020), and NL (10.7 m USD per year for 2022–2025; 5.5 m USD 2018–2022) governments. DK provided a detailed overview of initiatives that can be achieved within the already approved budget, initiatives that could be delivered within existing financial frameworks and over 80 initiatives that should be advanced but required additional funding. The Australian government has not yet made a funding pledge but has received a specific budget and initiative proposal for funding from an alliance of three different national organisations. Other government strategies either stated that different ministries are to ensure the necessary financial and human resources for initiatives that fall under their respective jurisdiction (MT) or did not specify funding pledges, merely stating that adequate funding needs to be identified (IT). We identified that some governments (DE, SE) are (partially) funding research on loneliness to gather scientific evidence to help them build their own policy.

Interventions and partnerships

Strategies, policies and action plans proposed a variety of interventions, while technical reports focused on reviewing existing evidence. We have provided many intervention examples across various domains in the policy landscape analysis section below. Of those countries and documents included in our analysis, only AU and GB have committed to work with specific charities, organisations or initiatives to address loneliness and social isolation. Other governments (CA, IE, IT, MT) stated their intention to work with NGOs and local services, but did not mention any specific organisations.

Development of a loneliness and social isolation measure

None of the documents called for the development of new tools to measure loneliness or social isolation. US, DK and GB reviewed existing measures of loneliness for use in possible interventions and strategies. Notably, GB described its own use of a consistent and direct measure of loneliness, developed by the Office of National Statistics (ONS) in 2018. The Direct Measure of Loneliness is a single item measure developed by the ONS that should be used in conjunction with three questions from the University of California Los Angeles (UCLA) Loneliness Scale. A US documents considered multiple ways in which loneliness and social isolation should be measured in research and recommended the appropriate choice of measures in targeted interventions and in major health strategies. The US did not call for the creation of a new measure, but rather recommended the use of existing validated tools tailored to the purpose of proposed interventions. DK’s national strategy considered the applicability of adult measures to adolescents and children.

Policy landscape analysis

This section highlights the wider policy context of the loneliness debate. All 14 countries that have published documents on loneliness are aware that loneliness touches many different dimensions (geographical, health, social, economic, and political; see Table  2 for a brief overview). In 91% ( n  = 21) of the analysed documents, the social and health dimension was most prominent, highlighting the impact of loneliness on various aspects of people’s lives and across age groups, as well as the health implications. However, not all dimensions were addressed with the same level of detail. An extensive overview of the different dimensions touched upon in every document can be found in the Supplementary Table A . For each of the five dimensions, we have identified themes that recur across the documents. We have also added some intervention examples to show how loneliness could be addressed in this dimension from a policy perspective.

Geographic dimension

Most documents (74%, n  = 17) touched on various geographic dimensions that influence or are influenced by loneliness. Four governments observed geographical variation in loneliness prevalence within their country (AU, CA, DE, GB). Only one document suggested reforming the digital environment (US). Within the geographic dimension the following themes were most often mentioned as being influential regarding loneliness and social isolation in the context of geography: (i) place or residence and housing, (ii) public transport, (iii) community services, and (iv) urban planning.

Place of residence and housing

Four governments (AU, CA, DE, GB) reported that the place of residence (urban or rural) significantly influences loneliness. Loneliness levels were also considered to vary due to population changes (AT, DE) but acknowledged that regional distribution was complex and cannot be solely attributed to urban-rural differences. Relocating to a new place was also reported to lead to feelings of being disconnected from familiar social networks and support systems. Additionally, insufficient affordable and suitable housing contributed to social isolation. Living conditions were mostly mentioned in connection with older adults where the effect of the type of housing was mentioned to affect social interactions and feelings of loneliness (CA, DK). Intervention examples to manage loneliness as a result of a change in residence, or loss of housing include working within local municipal authorities’ strategies on housing policies and reform plans (IT, DK, NL), creating models of apartments that foster community life (AL, DK), creating flexible housing solutions to support life transitions, e.g. homes that can be adjusted in size or adapted to changing needs (DK).

Public transport

The impact of public transport, especially access and affordability, was mentioned as a key issue for social integration, especially for older people (AL, CA). The place of residence (especially if rural) was recognised as a barrier to public transport use. Intervention examples that were put in place to address this issue include an increase of public transport access for the poorer older adults by subsidising the costs locally (AL, DK), and further strengthening accessible transport for communities in residential areas specifically (DK, GB).

Community services

Limited awareness of or access to community services contributed to loneliness. Financial support and grants for rural projects are needed to promote social inclusion. GB, DK and NL documents highlight the importance of the central government working together with local authorities, as the latter play a key role in actively supporting local transport, voluntary groups and initiatives that promote social cohesion and reduce isolation. Intervention examples included subsidies for community work to promote social inclusion specifically in rural areas (CZ), expanding the services in and of community centres (AL), and promoting the use of tailored community-based services (US).

Urban planning

There was general awareness that the physical environment can pose challenges to social participation, especially for the more vulnerable groups, e.g. older adults (CA), in terms of access to public toilets or walkability. Intervention examples included cultivating a sense of belonging that should be considered by urban planners (CA, IT), ensuring proximity to public services (IT), access to public toilets (CA), establishment of healthy and active movement paths (IT) aimed at encouraging walking groups (IT, CH), maximising the use of underutilised community spaces (GB), and use of participatory design in the development of child-friendly neighbourhoods in local environments (CH).

Social dimension

Most documents (90.9%, n  = 20) highlighted a range of interrelated social factors associated with loneliness; the social determinants covered various aspects of people’s lives that shape experiences of loneliness across age groups. Throughout these documents were notes on groups more vulnerable to loneliness as well as everyday life transitions and triggers. Some risk factors for loneliness such as lacking contact with family and friends, the negative impact of unemployment, and inadequate income support were also prominently highlighted.

Groups vulnerable to loneliness

Many governments identified groups more vulnerable to loneliness and social isolation, in line with research findings (AL, CA, IT, MT, NL, CH, GB, US). The following groups were identified as more vulnerable to becoming lonely or socially isolated: single parents, widows, newly retirees, single households, those living in changing family structures, immigrants with language barriers or low socioeconomic status, individuals dealing with addiction, those from the LGBTIQ + community, young adults (around 18 to 29), older adults (above 80), individuals that experience bullying or harassment, and individuals with criminal records. The importance of cultivating inclusive communities and establishing safe spaces for individuals, particularly for groups like migrants, single parents, and older adults was emphasized. Interventions were often tailored to specific groups. For example, community-led interventions targeted older adults who were homebound or in residential long-term care (MT). Others strengthened the resources of older people caring for relatives (CH), invested in a Carers Action Plan (GB), levelled up the volunteering infrastructure through collaboration of the voluntary sector and the government especially for those out-of-work (GB), developed social prescribing pilots and peer support groups (GB, US), facilitated befriending and socializing (AU), and linked vulnerable groups of people in the form of self-help and enabled them to help each other (CH). Here are some examples of targeted interventions for specific groups:

Women: language classes for women who do not speak the local language with crèche facilities alongside the classes (GB), Mitigate the risks of lifelong gender inequalities that result in female old-age poverty and gender pension gaps by ensuring adequate levels of income security for older women (MT).

Men: increase offers for older (single) men such as Men’s Meeting Places or Men’s Communities (DK), active aging centres to mitigate against the tendency of older men to experience difficulties in seeking help and talking about loneliness (MT).

Young people: Strengthen detection of loneliness in day care, primary schools and educational institutions (DK), provide education courses as a source of mitigating loneliness among children (DK), create more binding communities for young people without education and jobs (DK).

Older adults and low-income households: offer free local cultural and leisure activities (CH), increase public transport access (AL), guaranteeing the living minimum and gradual improvement of lowest pensions (AL), activation of computer literacy paths (IT).

Everyday life

The impact of events like the pandemic on individuals and communities was noted, with reference to mental well-being and social interactions, including potential changes in post-pandemic work patterns that might limit personal engagement. The absence of support or opportunities within society, communities, and workplaces is discussed as hindering social integration and fosters loneliness. The role of technology and social media as both a potential mitigating and exacerbating factor was recognized. Intervention examples include enhancement of popular traditions by developing new forms of technologically-oriented interactions, while still including cultural heritage (IT), expansion of existing community interventions (MT) including specific funding allocated to national, local, and community levels (AU), development of national and community awareness or anti-stigma campaigns (AU, CA, DK, DE, IE, NL, GB, US), and awareness spreading specifically towards politicians, administrations, managers, health care providers and others who work on loneliness (DK, US).

Health dimension

The health dimension of loneliness was very prominent in most documents (91%, n  = 21), often noting that socially isolated individuals faced an increased risk of engaging in negative health behaviours. The evidence of interconnection between chronic illnesses, mental health and social isolation was also highlighted. Overlapping with recommendations identified in the social domain, the need for policy development to prioritize social function among older individuals, aiming to enhance their overall health and well-being, was mentioned by (AT, DE, IE).

Institutional intervention examples included the development of an integrated health and social system on a community basis (AL, DK), national training for health practitioners and community care services to systematically identify, monitor and direct people experiencing loneliness (AU, DK, MT, US), linking healthcare practitioners with researchers to further evaluate and use loneliness assessment tools in clinal settings (US), and the inclusion of loneliness and social isolation in electronic health records (AU, US).

Physical health

Documents noted the evidence that individuals with higher levels of chronic diseases, geriatric syndromes, reduced mobility, chronic pain, frailty, hearing and sight impairment, urinary incontinence, or other health issues necessitating long-term care were more susceptible to loneliness. Governments acknowledged these links, often targeting interventions to support disabled people. Intervention examples included the provision of sensory impairment guides for those whose social lives are impacted by a change in their senses due to accidents or disabilities (GB), strengthening bridge-building for civil society and other actors was recommended in the context of in-system transitions and among high-risk groups (DK), the establishment of mobility centres to help people stay mobile or provide information on alternative modes of transport (GB), increased focus on digital inclusion of older and disabled to reduce loneliness as they face reduced mobility (GB), and the advancement of physical activity interventions, especially promising for improving the health outcomes of older adults (US).

Mental health

The policy documents showed empirical evidence that individuals experiencing depression, mental health problems and addiction were at risk of social exclusion. Depression and anxiety are specifically mentioned as significant factors in the context of loneliness; the consequences of loneliness are also discussed, with reference to the increased risk of depression, suicide, anxiety disorders, dementia, and reduced cognitive abilities. Intervention examples included the introduction of community care for people with mental health problems (CZ), while others focused attention on cognitive behavioural therapy, interpersonal psychotherapy and mindfulness (US). The reduction of addictive substances in populations at risk of social exclusion was targeted (CZ); mental health literacy programs were also discussed (DE, IE), specifically in reference to school education initiatives such as social emotional learning programs for use in preschool, school, and youth settings (IE); mental health literacy campaigns were also highlighted (DE, IE).

Economic dimension

Economic factors relating to loneliness were also addressed most documents (74%, n  = 17). In line with research evidence, documents noted that unemployment, receiving income support, and dissatisfaction with financial situation contribute to loneliness. The need for allocating more resources to combat poverty and address the loneliness experienced by older individuals was emphasised, with reference to the fact that it plays a crucial role in enhancing their overall well-being and quality of life. The following themes were prominent within this dimension.

Economic poverty stemming from insufficient income was identified as a key concern for the older adult population. Notably, social exclusion and family poverty were found to be directly linked, posing a risk to children as well. One document (AU) noted that men ages 25–44 years with high incomes and women of all ages with low incomes have been to be more susceptible to loneliness, revealing a discrepancy based on gender. The economic burden of loneliness extended to health service utilization costs, especially for mental health services. Intervention examples included allocating more resources to combat poverty and address the loneliness of older people specifically (IT), guaranteeing dignified living conditions through the adoption of the minimum pension and the gradual improvement of the lowest pensions by offering sustainable support for the poorer elderly was also suggested within the economic domain (AL), early support interventions for children from disadvantaged families, including support for their parents (CH), and more widely to reduce risk of social exclusion due to over-indebtedness (CZ).

Unemployment

Lack of affordable and suitable housing and care options was noted as being linked to social isolation. Loneliness and lack of social support could lead to reduced community participation, hindering employment prospects and workplace progress. This can result in reduced productivity, lower job satisfaction, increased absenteeism, and longer recovery times due to stress and health issues, which in turn negatively affects the economy. Intervention examples included facilitation of the integration of vulnerable individuals into the workforce (CZ, DK), prevention of loneliness among the unemployed through volunteerism and community initiatives (GB, DK), focus on ensuring a smooth transition from work to retirement (DK), working in collaboration with job centres (GB), and creating a cultural shift in work environments for employees at risk of social exclusion (CZ).

Political dimension

Political factors pertaining to loneliness and social isolation were only identified in few documents (30%, n  = 7), indicating less governmental awareness of the political implications of loneliness. Instances of elderly individuals being denied many rights were observed to be associated with loneliness (AL). Additionally, the effects of COVID-19 lockdown policies were connected to the loneliness because of social isolation. DE mentioned the political relevance of loneliness as it correlates with decreased political engagement of individuals. Thus, it was stated that implementing political measures at the federal level is imperative to effectively foster a more socially connected society (DE). One of the documents mentioned the need for the government to establish a comprehensive national strategy targeting loneliness, accompanied by the allocation of sufficient funding, with active engagement from regions and municipalities, especially when it comes to implementation (DK). Furthermore, the same document underscored the contribution of various other key stakeholders, including research institutions, foundations, employers, and civil society, in combating loneliness (DK). Multiple countries acknowledged the relevance of working across government bodies and levels in combatting loneliness (AU, DE, NL, GB). One document highlighted the need for a “connection-in-all-Policies” [ 62 , p.49] approach as social connection, an antidote to loneliness and social isolation, is relevant in all sectors (US).

To our knowledge, this is the first study to characterise the loneliness policy landscape across the UN European country groups (52 countries). The scoping review provided comprehensive coverage of how countries address loneliness and social isolation on a national level, allowing for a much clearer understanding of the diversity in country-level strategies and better coordination across countries in tackling loneliness. This is particularly important because loneliness and social isolation have been increasingly identified as a public health concern [ 63 , 64 ]. The findings of this review can be used by a wide range of stakeholders including federal agencies and local community groups who want to develop their own strategies to address loneliness and social isolation, or by researchers to gain an overview of the policy landscape.

Summary of principal findings

While not all governments (14 of 52 countries; 27%) had official documents that addressed loneliness, the vast number of documents we identified (79 documents) highlight the growing momentum in the loneliness discourse in the study area. The inclusion of research findings in the vision and strategy documents from different nations suggests widespread evidence-to-policy across the world and calls for a cross-disciplinary approach to addressing loneliness, including efforts to leverage asset-based community development and place-based approaches to tackling loneliness [ 65 ].

All 14 countries that published documents on loneliness demonstrated an awareness that loneliness impacted various dimensions including geography (through place of residence and housing, public transport, community services, urban planning), social (some groups are more vulnerable to loneliness than others, social support, technology), health (physical and mental), economics (income, unemployment) or politics (effects of COVID policies, political engagement, working across sectors to address loneliness). Notably, none of the documents reviewed acknowledged that (i) most research on physical health and loneliness is cross-sectional, where the researcher measures both the outcome and the exposures of the study participants at the same time, and thus, the findings of these studies cannot be used to make causal inferences, and (ii) such work does not control for other predictors of health, including, for example, socioeconomic status and actual health conditions. These are important considerations because (a) we cannot be certain that healthy individuals are more likely to get sick if they experience loneliness compared to other healthy individuals who do not experience loneliness, and (b) whether the link between loneliness and health is actually driven by structural inequalities that determine our physical and social environments. We have also found that the documents rarely mention the transient nature of loneliness and the discourse often seems to frame loneliness like an illness that can be treated. The documents also did not address the cultural context (i.e. beliefs, values, religion) that can shape expectations of relationships and the welfare regime.

Policy targets proposed in the documents

Most countries in our sample showed some attempt at raising public awareness about loneliness (AL, AU, CA, DK, DE, IE, IT, MT, NL, CH, UK, US). Such policies are often informative, but there appeared to be a lack of deadlines and appropriate funding. That means the strategy cannot be evaluated. Another point of concern is the perception that loneliness is something that only affects older adults. Some documents lacked information about how to address loneliness, probably because here is limited evidence of what works and for whom. Also absent was a commitment to evaluation of interventions, which is crucial to verify the effects of any intervention and any risks related to action.

Recommendations for policy makers

Despite the adoption of an evidence-to-policy approach to loneliness, given the issues noted above, we encourage policymakers to be cautious in making claims in relation to loneliness, and to ensure that part of their strategy includes the funding of research that fill the gaps in knowledge. Policymakers should also ensure that the work they quote includes study populations that are well-represented in all relevant demographics and that the research is able to make causal claims about how loneliness impacts health. The World Health Organisation (WHO) and the European Union have identified the limits of their own knowledge and skills in this field, commissioning experts to write evidence gap reports [ 66 , 67 ] or GB and DK for example have had loneliness researchers help write their vision and strategy.

Policymakers should also adopt a similar approach in relation to interventions that address loneliness. A recent meta-analytic review [ 68 ] suggested that in order for interventions designed to reduce loneliness to be effective, matching the intervention to the loneliness type is essential, whereas a one size fits all will not be effective. For example, social support interventions and social and emotional skills training are all promising interventions for reducing loneliness, albeit they are usually only appropriate for loneliness that is linked to the perceived absence of a close friend or partner and perceived lack social encounters and acquaintances respectively. Such an understanding of the nuances surrounding loneliness interventions is absent from the documents we evaluated, and policymakers will want to fill that gap in their knowledge so that appropriate decisions about intervention work, and suitable funding, can be provided. The effects of current interventions have been shown to be only moderate, highlighting the need for funding for rigorous and systematically developed interventions that are also appropriately evaluated.

Based on our scoping review and underlying evidence we propose a list of actionable recommendations for national and regional governments wishing to establish or incorporate loneliness into their policy documents (Table  3 ). In sum, we believe that revisiting previous national and local campaigns to identify connection points for loneliness interventions is an effective way to include loneliness into the policy agenda. For example, a walkability campaign that focuses on making cities more pedestrian friendly will benefit individuals in terms of physical health and mental health but it also increases the likelihood of social encounters when walkability is higher [ 69 ]. We also believe that sharing best-practice approaches internationally and accessible to everyone ensures the development of a strong knowledge base. The EU has taken the lead as the first supranational union to address loneliness amongst its member states by recently organizing various roundtables and conferences around loneliness [ 70 ]. Globally, WHO has recently published an evidence gap report on in-person interventions for reducing social isolation and loneliness [ 67 ]. Lastly, we argue that policies would be meaningless if there are no concrete funding streams allocated towards evidence generation, intervention design and implementation and the evaluation thereof. Because our review could not identify clear funding streams for all countries, we strongly encourage policy makers to make the funding streams transparent within their loneliness policies.

Limitations

The primary limitation of our scoping review was concerned with identifying documents from countries that did not provide information in English. That limitation was partially overcome by the use of Google’s website translator. Another limitation is the reliance on machine translation for the identified documents. Documents were translated into English from German, Danish, Finnish, French, Dutch and Norwegian using DeepL. For German and French, the quality of the translation was checked by the author team and considered sufficient to meet our study aim. The cross-sectional design of our scoping review also does not account for how a country’s policy may have changed over time. This is a general issue in policy evaluation. That limitation can be overcome by conducting this review every two to four years. Another challenge with our study is that the data reflect the existence of policies and not the effectiveness of their implementation. Further, only funding that was explicitly allocated to reducing loneliness and social isolation was considered. We acknowledge that other initiatives that received governmental funding pledges, such as establishing community centres for older adults, might also reduce feelings of loneliness. However, it is beyond the scope of the current paper to identify which initiatives specifically reduce loneliness and how much funding has been allocated to them, especially as evidence on which interventions have proven successful are scarce. Additionally, there may be other funding streams we are not aware of or that might have been part of other documents (e.g., state budgets) not included in this analysis.

More work is needed to assess if the various proposed interventions are implemented and successful. Evaluating interventions is crucial if we want to effectively use the pledged funding, to identify what tools (online or other) are being developed to promote loneliness interventions on national and regional levels and to map out the role of the emerging national loneliness networks.

Our study provides the first comprehensive overview of the national loneliness policy landscape across 52 countries, highlighting the increasing prioritisation of loneliness and social isolation as significant public health and societal issues. While the momentum in addressing loneliness is evident, with most policies being informed by scientific evidence, gaps remain, particularly around intervention strategies and their effectiveness. Our findings urge policymakers to not only sustain this momentum but to also strengthen their strategies by incorporating rigorous, evidence-based intervention evaluations and fostering international collaborations for knowledge sharing. This approach can enhance the understanding and addressing of loneliness, ensuring interventions are well-targeted, effective, and scalable. By addressing these issues, policymakers can more effectively manage loneliness by directing funds to develop and implement interventions that impact the individual (e.g. through therapy or befriending services, thereby improving public health outcomes) and the community and society by making them genuinely inclusive, thereby increasing social cohesion.

Availability of data and materials

The references to the documents supporting the conclusions of this article are provided in Table  1 . Should a link have expired, contact the corresponding author for a pdf version of the translated and original document in question.

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Acknowledgements

The authors thank the following students for assisting with compiling the dataset of government documents: Selma Akbas and Laura Baldini. The authors also thank the following students for assisting with the first round of document coding: Kim Aleppo, Izma Ahmed, Angela Benson, Emma Marchong, Sathana Sivanantham, Keyi Le, Yaxuan Shi, Ruifeng Ding and Yiming Bi. The authors would also like to thank Mahmoud M M Al Ammouri for creating the map displayed as Fig.  2 . The lead author also thanks Claudia Kessler from Public Health Services based in Switzerland for insightful discussions on the Danish and Dutch national loneliness policies.

This research was unfunded. Nina Goldman is supported by the Swiss National Science Foundation (SNSF), Bern (Grant #: 214225). Austen El-Osta is supported by the National Institute for Health and Care Research (NIHR) Applied Research Collaboration (ARC) Northwest London. The views expressed are those of the authors and not necessarily those of the SNSF, NHS, NIHR or the Department of Health and Social Care. AEO is the guarantor.

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Nina Goldman, Devi Khanna and Marie Line El Asmar contributed equally to this work.

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Manchester Institute of Education, University of Manchester, Ellen Wilkinson Building, Devas Street, Manchester, M13 9PL, United Kingdom

Nina Goldman, Devi Khanna & Pamela Qualter

School of Public Heath, Faculty of Medicine, Imperial College London, Charing Cross Hospital, Reynolds Building, St Dunstan’s Road, London, W6 8RF, United Kingdom

Nina Goldman & Austen El-Osta

North Hampshire Hospital, Hampshire Hospitals NHS Foundation Trust, Basingstoke, United Kingdom

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All authors contributed substantially to this study: Conception (N.G., A.EO.) and design of the work (N.G., A.EO., P.Q.); Data collection (N.G.); Data analysis and interpretation (N.G., D.K., M.L.EA.); Drafting the article (N.G., D.K., M.L.EA.); Critical revision of the article (P.Q., A.EO.); Final approval of the version to be submitted (N.G., D.K., M.L.EA., A.EO., P.Q.)

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The authors span multiple nationalities and levels of seniority. All authors are based at three UK institutions (University of Manchester, Imperial College London and Hampshire Hospitals NHS Foundation Trust). The lead author is a human geographer researching loneliness from a spatial perspective, the second author has a background in international social and public policy, the third author is a medical doctor conducting mixed methods research in the area of public health, the fourth author is the UK's leading scientific expert on child and adolescent loneliness and the last author is a mixed methods public health researcher and is principal investigator of the Measuring Loneliness in the UK (INTERACT) study.

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Correspondence to Nina Goldman or Devi Khanna .

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Goldman, N., Khanna, D., El Asmar, M.L. et al. Addressing loneliness and social isolation in 52 countries: a scoping review of National policies. BMC Public Health 24 , 1207 (2024). https://doi.org/10.1186/s12889-024-18370-8

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Jeanine Mazak-Kahne is associate professor at the Indiana University of Pennsylvania History Department, email: [email protected] .

Theresa McDevitt is government information and outreach librarian at Indiana University of Pennsylvania, email: [email protected] .

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Jeanine Mazak-Kahne, Theresa McDevitt, and Lorilie Blose

Academic Libraries and Public Art

Engaging Students in a Timely Discussion

Jeanine Mazak-Kahne is associate professor at the Indiana University of Pennsylvania History Department, email: [email protected] . Theresa McDevitt is government information and outreach librarian at Indiana University of Pennsylvania, email: [email protected] . Lorilie Blose is currently a student in the Clarion University Department of Information and Library Science, email: [email protected] .

© 2024 Jeanine Mazak-Kahne, Theresa McDevitt, and Lorilie Blose

A cademic libraries are dynamic institutions that are constantly changing in response to the needs and wants of their users. At the same time, they are also the offices that house the institution’s archives and are responsible for preserving the records that document the institution’s past. While they strive to adopt best practices consistent with student needs, they also work to protect and preserve the past. Such roles can be at odds.

What do libraries do when current student and administrative priorities, tastes, and institutional missions clash with their interest in preserving the past? Are libraries’ users best served by exclusively supporting current taste and initiatives, or does preservation of the past serve students and institutions more? Can libraries use existing historic places to share significant historical stories about their institution and still be perceived as current and attractive? These questions are ones that librarians might struggle with, but they do not have to struggle alone.

Academic libraries exist in colleges and universities that are staffed by discipline-specific experts and the students who are training in these disciplines. Librarians, who work daily with faculty and students, have a ready opportunity to seek advice from both seasoned and budding experts and include them in discussions on authentic dilemmas that libraries face. Collaboration may result in more informed decision-making, as well as provide educational and professional benefits for students as they build work skills, develop more impressive vitas, and become more deeply engaged in their learning.

Public history programs provide students with civically engaged instruction, often incorporating practical projects into traditional classroom learning. Public history fields include librarianship and/or encourage strong relationships with libraries and librarians not only as a source of research, but also as tied to their nature as being public service–oriented. Both have a concern for ensuring information is preserved and shared, and shape community and identity, in shared, as well as unique ways.

During the 2022–2023 academic year, an Indiana University of Pennsylvania Libraries issue related to historic public art was brought to faculty and students with an expertise in public history when seeking a practical solution to a real-world problem. This article will describe the problem, the collaboration, and the manner in which the service-learning activity at once benefited the library, faculty, and students.

Mural tracing the evolution of communications, located in a stairwell of the IUP Stabley Library. The mural was installed c. 1960s by student and artist Wayne Hawxhurst. Photo by Rhonda Yeager.

The People and the Issue

Indiana University of Pennsylvania (IUP) is a mid-sized public university in Western Pennsylvania that began as a Normal School in 1875 to prepare teachers for common schools. Over the years, it developed into a university with an R2 or High Research Activity classification. In the summer of 2022, IUP Libraries were renovated to support student success by physically uniting a number of campus support services and creating a Learning Commons in the library facility. In the course of the renovations, a stairway previously available only to staff was again opened to the public and a mural painted in the late 1960s was rediscovered. The mural was painted as part of a course assignment by a graduate student who loved the library. It illustrated the evolution of information storage and sharing from the beginning of time to the 1960s. Although it survived for more than 50 years, in the fall of 2022 the new renovations and resulting altered traffic patterns drew attention to the mural and led some to question whether it should be preserved or painted over. Interesting in its time, and related to the library’s current and historical mission, it was not consistent with the more minimalist and standardized style of the renovated building. The question arose: should it be painted over, or should it be preserved?

Public History Instruction and Libraries

Theresa McDevitt is a librarian who has a PhD in history. Over the years she has developed several history-based initiatives in the library and university communities and long served as the embedded librarian for the History Department. In this role, McDevitt has worked closely with Jeanine Mazak-Kahne, a public history professor, to develop authentic public history assignments based upon existing local history records. Mazak-Kahne considers this relationship as essential in providing critical instruction to public history students, from information literacy to research practices in local history.

Instruction in public history is anchored in understanding and fostering the preservation of cultural heritage, whether it be on the local level or a national scale. Course materials and faculty instruction involve a historical understanding of community and memory, especially that fostered by cultural and heritage objects created (such as public art or memorials) for a given purpose and examining how purpose and meaning change over time. In contemporary discussions, the weight has fallen on monuments and memorials. Discussion is often lively and reflective; however, for many, these issues are abstract as students are removed by space and time from the objects and the surrounding debates.

Also critical to public history instruction is the understanding of community and memory. While typical case studies examined in the classroom are often part of larger debates over a national identity, in reality, peoples’ lives are most often rooted in the local identity, community, and memory. As students are part of a university community, public history professors recognize that student learning and engagement with material deepens when an understanding of place, identity, and memory is drawn from their immediate surroundings. This leads to the development of connections to the community in which they are immersed in new ways. Through projects that stem from authentic local issues, students apply what they have learned in the classroom to their immediate, discuss identity, memory, and community values and how they have changed over time.

Students in the IUP public history program comprise multiple disciplines from history and anthropology to art and English. Students continue to work in a variety of fields, including historic sites and museums, but also in archives and libraries, and they benefit from developing an understanding of how information is preserved and used in these different organizations. Therefore, it is an ideal situation to work with the embedded librarian and university library to develop authentic applications of theory at every opportunity possible.

The Assignment

Discussion of whether the library mural should be painted over came just at the time when instruction in the public history course shifted to the meaning of memory and identity as preserved and expressed in public monuments, memorials, and art. In this course, students are encouraged to think about how the meaning of public objects change over time and how evolving meaning and understanding of our past causes us to reinterpret these objects. They also seek to explore how objects have different meanings within the community and how public historians must navigate and negotiate these meanings among different groups and provide professional insight when called upon.

McDevitt and Mazak-Kahne felt that the discussion of whether the mural should be preserved or painted over would be a good authentic assignment for students. Students in two public history classes, Introduction to Public History and History Museums and Historic Sites, visited the library and read about the mural’s history in an alumni newsletter article. They were then asked to call upon what they had learned so far to provide advice to the libraries on the disposition of the mural. The mission of the libraries was shared, and the historical value of the mural and possible significance of its having been painted by an alumnus was considered. They were able to recommend that it be preserved or painted over. If they chose the latter, they were asked to suggest ways that the historical information intrinsic to the artifact could be preserved. Discussion prompts included those related to historical significance, historical content accuracy, artistic quality, and impact of ease of navigation when the space might be used for navigational signs.

While considering this real-world dilemma, they were asked to consider what the historical significance of the content of the mural was, and if it was accurate, and if that mattered. The students participated actively in the discussion, clearly drawing upon their training, and raised unanticipated issues that led to a deeper consideration of not just the mural, but of all public art in the library.

Some of the students in the classes were so intrigued with the mural that they volunteered to look more into its history. They wrote up their findings for library administration and prepared background documents that will be preserved in the Special Collections Department. They also provided wording for a plaque for the mural should it be preserved.

In reflecting on the assignment, Mazak-Kahne felt that the mural lesson enhanced student learning, immersing them in the immediate relationship between public art, public engagement, and institutional memory. She feels that it is important for students to learn that life is messy and that no solution is perfect, while giving them practice in making decisions for the common good, while not dismissing the past. She argues that engagement with this real-world problem helps students gain experience that may help them assess the complicated circumstances that professionals encounter in their field.

Based on this success, Mazak-Kahne intends to build upon this project in the future. IUP’s public history program spans multiple courses, and it is often the case that a project started in the Intro class is carried into the archives, museums, oral history, digital history, and family and local history courses. The documentation created during this project will, at the very least, be used to create a digital exhibit that will be housed on the program’s website, currently under construction.

Students who worked on the assignment echoed Mazak-Kahne’s sentiments. They reported that the mural project gave them insight and hands-on experience that corresponded to their field of interest, public history, and offered them a glimpse into the work often found within museums and archival sites.

They also agreed that the investigation of the mural led them to deepen their understanding of historical research by conducting thorough research into various primary and secondary sources in a manner that an actual public historian would. Although it was challenging, they felt that translating discovered information into an accurate and unique plaque strengthened their ability to communicate historical information effectively and efficiently.

Lorilie Blose, a student in the class, wrote, “Throughout the project, I learned about the meticulous research required to understand art and artifacts, the careful decision of what to preserve, and the significance of accurately documenting and contextualizing historical objects and art. . . . This hands-on experience helped me grasp the intricate workings of these institutions, preparing me for potential future roles in public history.” She continued that it “enhanced my academic experience but also gave me invaluable insights into the operational aspects of public history. Such projects help to bridge the gap between theory and practice, preparing students for careers in history and other related subjects.”

For the immediate future, the mural will continue to grace the walls of the stairway in the older part of the library building, and the students’ plaque text will be mounted near it in the stairway. Based on this experience, the curator of the University Museum was inspired to ask interns working at the University Museum the following semester to write descriptions for more of the art items in the library.

The decision of what to do with the historical mural presented a challenge to library personnel. Like most challenges though, it offered opportunities for learning and growth. By including history professors and students as advisors in the discussion-making process, librarian learning was extended, and students and faculty benefited as well.

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Original research article, a community health worker led approach to cardiovascular disease prevention in the uk—spices-sussex (scaling-up packages of interventions for cardiovascular disease prevention in selected sites in europe and sub-saharan africa): an implementation research project.

• 1 Department of Primary Care and Public Health, Brighton and Sussex Medical School, Brighton, United Kingdom
• 2 Department of Disease Control and Environmental Health, Makerere University, Kampala, Central Region, Uganda
• 3 Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium

Background: This paper describes a UK-based study, SPICES-Sussex, which aimed to co-produce and implement a community-based cardiovascular disease (CVD) risk assessment and reduction intervention to support under-served populations at moderate risk of CVD. The objectives were to enhance stakeholder engagement; to implement the intervention in four research sites and to evaluate the use of Voluntary and Community and Social Enterprises (VCSE) and Community Health Worker (CHW) partnerships in health interventions.

Methods: A type three hybrid implementation study design was used with mixed methods data. This paper represents the process evaluation of the implementation of the SPICES-Sussex Project. The evaluation was conducted using the RE-AIM framework.

Results: Reach: 381 individuals took part in the risk profiling questionnaire and forty-one women, and five men participated in the coaching intervention. Effectiveness: quantitative results from intervention participants showed significant improvements in CVD behavioural risk factors across several measures. Qualitative data indicated high acceptability, with the holistic, personalised, and person-centred approach being valued by participants. Adoption: 50% of VCSEs approached took part in the SPICES programme, The CHWs felt empowered to deliver high-quality and mutually beneficial coaching within a strong project infrastructure that made use of VCSE partnerships. Implementation: Co-design meetings resulted in local adaptations being made to the intervention. 29 (63%) of participants completed the intervention. Practical issues concerned how to embed CHWs in a health service context, how to keep engaging participants, and tensions between research integrity and the needs and expectations of those in the voluntary sector. Maintenance: Several VCSEs expressed an interest in continuing the intervention after the end of the SPICES programme.

Conclusion: Community-engagement approaches have the potential to have positively impact the health and wellbeing of certain groups. Furthermore, VCSEs and CHWs represent a significant untapped resource in the UK. However, more work needs to be done to understand how links between the sectors can be bridged to deliver evidence-based effective alternative preventative healthcare. Reaching vulnerable populations remains a challenge despite partnerships with VCSEs which are embedded in the community. By showing what went well and what did not, this project can guide future work in community engagement for health.

1 Introduction

Cardiovascular disease (CVD) is among the most prevalent, costly to treat, and deadly medical issues in the world ( 1 ). As part of the continual effort to combat CVD, greater emphasis is being placed on prevention. This often takes the form of behavioural or lifestyle change, focusing on the reduction of risk factors (e.g., hypertension, poor diet, obesity). Reducing these risk factors using evidence-based interventions not only works to lower rates of CVD, but also impacts rates of a variety of other medical issues, including susceptibility to severe COVID-19 infection ( 2 ), many common Noncommunicable Diseases (NCDs) including Type 2 diabetes and a wide range of cancers ( 3 ). Furthermore these preventative interventions are less expensive than reactionary care and can lower the treatment burden on strained medical systems ( 4 ).

Community-Based Participatory Research (CBPR) and Community Engagement (CE) have grown increasingly popular as potential methods to engender sustainable, long-term change in communities—particularly those communities under-served by existing medical systems and/or those at heightened risk of CVD ( 5 ). One's behaviour is influenced by their environment and the community they live in, meaning that tapping into a community's resources can be effective in changing lifestyle behaviour as well as having impacts on the wider community ( 6 , 7 ). The use of community-based practices fits within the growing South-North collaboration that this project joins as part of an international collaboration known as “Scaling-up Packages of Interventions for Cardiovascular disease prevention in selected sites in Europe and Sub-Saharan Africa: An implementation research project” (SPICES). In the Low- and Middle-Income countries (LMIC) there is evidence for the successful implementation of evidence-based community-based interventions in increasing knowledge of, and changing behaviour related to, CVD ( 8 ) however their use in the Global North is less well tested or understood ( 9 ).

In the UK, the flagship intervention to address preventative health issues is the National Health Service's (NHS) Health Check initiative, which is free to individuals ages 40–76 and which assesses risk for long term health conditions including CVD ( 10 ). Following initial assessment by a health professional, patients are advised on a course of action which often includes some degree of preventative prescribing to address behavioural risk factors ( 11 ). Just under half of eligible individuals accepts a first health-checks appointment (44.2%)—it is associated with increased detection of CVD risk, but uptake is skewed by several demographic factors (principally, age, gender, and socio-demographics), and it has struggled to create change in underserved groups ( 12 , 13 ). Marginalised coastal communities in Sussex face overall below-average healthy-life expectancy ( 14 ). This, alongside heightening inequality and the impact of COVID-19, has left some communities in Sussex significantly deprived in terms of access and engagement with health services ( 15 ). People in these communities experience transgenerational poverty, precarity, and lifestyle behaviours ingrained into the communities that lead many to be at higher risk for CVD. CBPR and CE models have the potential to lead to improved health and health behaviours among disadvantaged populations if designed properly and implemented through effective community consultation and participation ( 16 ).

CBPR and CE offer the chance to bring lessons from effective programmes in the Global South and apply them to programmes in the Global North. Community-based strategies to promote evidence-based preventative health interventions using Community Health Workers (CHWs) are often more established in the Global South where more tightly knit communities and established community health programmes fulfil a range of public health needs ( 17 , 18 ). CHWs interventions are a form of “task-sharing” intervention in which responsibility and power is shared between professional health workers and communities which have been proposed to effectively manage non-communicable disease risk ( 19 ). Lay Community Health Workers are individuals who are trained to perform of health-related functions but lack a formal professional health education. They can provide links between local communities and health care institutions thereby building and on and developing the social capital that already exists in communities ( 20 ). Although there is plenty of evidence communicating the importance and usefulness of these methods (the “what”), there remains a lack of attention given to how to do it . This article joins the work and voices attempting to begin filling that lacuna.

Within the literature on CBPR and CE, a handful of common themes emerge. The first is a push for human-centred research design ( 21 , 22 ). Yardley et al. ( 23 ) focused on this idea in their “person-based” approach to digital health interventions, where they recommended a “focus on understanding and accommodating the perspectives of the people who will use the intervention” ( 24 ). Hopkins and Rippon's ( 25 ) “asset-based” approach to CE interventions recommends recognising and adapting to the need, wants, and strengths already present in the community. Particularly the strengths, or “assets” already present in the community provide an opportunity for projects to use those assets. Such an implementation approach requires flexibility and adaptability, as well as deep involvement with the community. The second theme builds on the first, with the idea that not only should project design be person-centred, but those participants and other stakeholders in the community should be involved at every level of project planning through co-design. Yardley et al. ( 23 ) included this as a key element of their paper, writing that people from the target population should be involved in project development as well as at every stage of the intervention. Similarly, Berrera et al. ( 26 ) emphasise the need to adapt all projects to the cultural context of the community. This insight speaks to the third theme, continuous evaluation ( 27 ). As the needs of the community will be ever-shifting, so must the project adapt to those needs continually. Instead of designated periods of evaluation, a shift to continual processes of qualitative evaluation is called for to identify and adjust to the needs of the community. These processes require elevated levels of trust and participation from the community, which has its own challenges. Trust especially takes significant time and resources to develop and is an under-studied area of community engagement ( 28 ).

The SPICES-Sussex project was carried out from January 2019 and aimed to answer the following overarching research question: How can Community Health Workers (CHW) CVD prevention interventions, that have been used in the Global South, be developed, and implemented in a Global North setting and what barriers and enablers exist to their implementation? The project began with a situational analysis which included an exploration of the views and experiences of the local community with regards to CVD health and Community Health Workers and early stakeholder mapping of the research sites which was carried out between 2019 and 2020 ( 29 , 30 ).

The primary aim of the current paper is to provide a comprehensive examination of the project's implementation including complementary mixed methods analyses according to the Reach Effectiveness-Adoption Implementation and Maintenance (RE-AIM) framework ( 31 ). The secondary aims of the project are to inform future CE projects what worked (and did not work) for our project and to tie insights from our project to broader discussions in the discipline. The project is based on a protocol published in 2020 prior to the onset of COVID and was conducted through the period of the COVID-19 pandemic ( 29 ). Subsequently, several aspects of the original protocol were adapted to make implementation feasible within the constraints of this period (see Supplementary Appendix 2 ).

2.1 Study design

The project uses a type 3 hybrid implementation design ( 29 ) meaning that the primary aim of the research was to determine utility of an implementation intervention/strategy whilst the secondary aim was to assess clinical outcomes associated with the implementation trial. This means that we focused on understanding what barriers and enablers existed for the project's implementation and the context within which it operated. Effectiveness of the intervention remained important, however we were primarily interested in how and why it did (or did not) work. The project was carried out at four geographic research sites within Sussex (see Section 2.3 ) and implementation was conducted on an iterative basis from research site to research site broadly following the Medical Research Council's (MRC) framework for the development and implementation of complex interventions ( 32 ). The research team developed and then began delivering the intervention at each site before moving onto the next. At each site the following stages were carried out: (1) Development: this included stakeholder mapping, formation of implementation partners, and codesign/local adaptation of the intervention [covered in the study's pre-implementation paper ( 30 )]; (2) Implementation: this included the delivery of the CHW intervention at the research sites and collection of mixed method data pertaining to effectiveness and stakeholder experiences, and (3) Evaluation: this included the analysis of the mixed method data in line with the MRC guidance on analysis complex interventions.

2.2 Research site and voluntary and community sector enterprise partner selection

Four study sites were selected across East Sussex by identifying Middle Layer Super Output Area (MSOA) postcodes with high levels of deprivation according to the Indices of Multiple Deprivation (IMD) ( 33 ). Selection of the research sites was based on the pre-implementation community mapping phase of the project ( 30 ). Following on from CBPR practices, VCSEs and Volunteer Coordinators (VCs) were recruited to co-design and deliver the implementation strategy at each of the research sites.

VCSEs organisations were recruited as partners at each research site. The intervention was primarily run through these organisations and a paid staff member was recruited at each organisation. Their responsibilities included, CHW management, and participant recruitment. They also had a role in local adaptation activities. VCSE organisations were eligible to take part in the organisations if they were based in the research site, if they had interests and existing activities that aligned with the project's goals (CVD risk reduction and community development), and if they had existing experience of volunteer recruitment and management.

2.3 Community health worker recruitment and training

The aim was for each site to recruit a pool of five to eight CHWs. As part of this each site was asked for input into local CHW recruitment flyers, which were shared on VCSE websites and social media pages and shared on social media via existing CHWs at the VCSEs. CHWs were recruited through intermediary organisation recruitment via the VCSE partner organisation. The project was also advertised at a Virtual Volunteer Fair. Local contacts and existing volunteer pools at the VCSEs meant that the target number of CHWs was rapidly recruited at each site. CHWs were eligible to take part in the intervention if they were over 18 years of age, if they lived within the research site (determined by postcode), and if they had some kind of pre-existing relationship with the VCSE partner organisation (i.e., as a volunteer).

Potential CHWs who expressed an interest in the project were invited to attend an induction to the project, and then the local adaptation co-design meeting. Those who decided they would like to become a CHW then went on to receive five online, group training sessions (each of which lasted for 2 h, 10 h in total): an introductory session, a session covering project policies, heart health and the structure of the intervention, and three sessions on behaviour change techniques. These training sessions were developed and delivered by an external organisation (National Centre for Behaviour Change) specifically for the project after a consultation and planning process with the research team. Before the onset of the intervention at each site CHWs made various recommendations in the local adaptation meetings on the design of the training programme. These included providing information on listening techniques, engaging, and managing resistance, providing simple health information, using accessible language, using different starting points depending on the CHW's background knowledge and experience, training on conducting coaching virtually, and providing a training handbook. A Volunteer coordinator (VC) was recruited at each site. This VC was a trained and experience health coach (KFS) and provided training support and guidance through monthly group training support sessions in addition to the initial 10 h training block the received prior to the intervention onset. These monthly training and support sessions were organised into specific themes and agendas that were set with the CHW participants.

2.4 Local adaptation

Elements of the evidence-based intervention were tailored to the individuals and their community in the stakeholder-mapping phase using qualitative interviews, workshops, and focus groups with a range of stakeholders across the study site ( 30 ). Further rounds of local adaptation were carried out with VCs and CHWs at each of the research sites to tailor to individuals and their community context through iterative co-design workshops ( 34 ). CHWs and VCSE also agreed on a “volunteer charter” during the co-design session. This was a list of principles, behaviours, and practices upon which guided interactions between research staff, CHWs, VCSE and participants. The charter was designed to ensure that the practices of the project aligned with the principles of the CHW and partner organisation.

2.5 Participant recruitment and screening questionnaire

Participants (who received coaching) were eligible to take part in the eligibility screening if they lived in, or adjacent to, the study site's postcode and if they were aged eighteen or older. Participant recruitment was also based on intermediary organisation recruitment, community outreach, paid social media advertisement (through Meta™), gatekeeper and snowball sampling. Gatekeeper recruitment was conducted when interacting with a relevant statutory or non-statutory service provider (i.e., a fitness/weight loss group leader) and involved asking them to recommend the intervention to their members or to recommend participants who may be interested in taking part. Snowball sampling involved asking participants who participated in the study to sending email invitations to their social group. A social media recruitment strategy was undertaken to recruit people from the local area to the risk profiling survey to supplement the community-based recruitment through the VCSE partners. Social media was conducted on Facebook via paid advertisement in four waves of recruitment which took place over 1–2 weeks at each site. The advert targeted people who were 35+ and over and to people with 5 km of each research site. Messages were changed regularly from a list of recruitment messages drafted with CHWs during co-design sessions. Additionally, CHWs and VCSE participants were asked to send recruitment emails to any social or professional networks they thought would be interested in taking part. We did not record where participants were recruited.

Screening and risk profiling for the CVD coaching was carried out using the validated non-laboratory based INTERHEART questionnaire, presented online, for all participants that expressed an interest in the study ( 35 ). This questionnaire assessed modifiable and non-modifiable CVD risk factors and categorised participants as either “Low,” “Moderate.” Or “High” risk. See the protocol paper for further information on the INTERHEART risk profiling; for more information on the screening questionnaire, see the study protocol paper ( 29 , 35 ). Questionnaire data were collected and managed using Research Electronic Data Capture (REDCap) electronic data capture tools hosted at the University of Antwerp ( 36 ). Participants were considered to be eligible for the intervention if they were aged eighteen or over, if they lived within the research site (determined by postcode), and if they were categorised as “Moderate” risk of CVD according to the INTERHEART questionnaire. High risk participants were not included as their needs were considered to be too high for a pilot study involving CHWs. Eligible participants were then emailed by the research team with an invitation to take part in the CVD coaching intervention. After recruitment for the intervention was closed for each site, an online questionnaire survey was sent to eligible participants to gather information the reasons for not accepting the invitation to the intervention. Open response questions were used which the research team later categorised into codes.

2.6 The CVD prevention coaching intervention

The coaching intervention was based on motivational interviewing techniques which are promoted by the European commission on cardiovascular disease prevention in clinical practice ( 37 ) and which include techniques such as Open questions, Affirmation, Reflective listening and Summary reflections (OARS) ( 38 , 39 ). The use of these Behaviour Change Techniques (BCTs) used during the intervention were based on five target behaviours highlighted by the World Health Organisation including: reduce/cease smoking, increase moderate physical activity, reduce the fat, salt, and sugar content of the diet, increase fibre, oily fish (or alternatives), fruit, and vegetable content of the diet, reduce sedentary hours. The intervention involved six, one-hour long coaching sessions between participants and CHWs which were delivered every two weeks. Participants were also considered to have completed the intervention if they only completed three sessions and then notified the team of their withdrawal from the intervention.

The study team included two participant co-ordinators (PCs) who managed the participant journey through the intervention, sending welcome emails, questionnaires, and invitations to post-intervention interviews, and co-ordination between participants and CHWs to book coaching sessions. Reminders of appointments were also sent to CHWs and participants one week and two days before the session. Participants and CHWs were matched, based on gender preference and availability, and supported throughout the coaching intervention the PCs. CHWs were provided with guidance, resources, and signposting information throughout the intervention but were also given the flexibility to deliver the coaching in a way that suited them and their participant(s). Initially, counselling and goalsetting were based on their individual item INTERHEART assessment scores. Participants and CHWs were then encouraged to create an action plan with appropriate goal setting for the behaviours they wanted to change (e.g., diet, exercise habits). The goals were set in relation to when, where, and how they would undertake the behaviour, e.g., when the physical activity will be performed, where it will be performed, how often it will be performed (i.e., in a group or using specific equipment). CHWs helped participants to analyse any factors which might influence their ability to achieve the goals and to generate strategies which could help them overcome these barriers using problem solving. Full details of the participant journey through the intervention are given in Supplementary Appendix 2 in the Supplementary Material . All coaching was conducted virtually using Zoom™ to host and monitor coaching sessions and Microsoft OneDrive to store, recruit, and communicate written and visual resources with CHWs and participants. Monitoring in Zoom calls was called out by the PCs who checked whether both the participant began and ended the coaching session. If either the participants or CHW did not join, the PC could join the call to help the attendee. Feedback was obtained from the participant about the coaching session through emails after the session and by inviting participants to a follow-up interview after the intervention (see qualitative evaluation).

2.7 Evaluation

The evaluation was underpinned by the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework ( 31 ) which allows for an understanding of the multifaceted and interactive effects of personal, social, and environmental factors that determine behaviour; and for identifying behavioural and organisational leverage points and intermediaries for health promotion within organisations and communities. RE-AIM has been used to evaluate programs and setting in public health and community settings and is thought to be particularly useful when evaluate interventions in “real-world” settings ( 40 , 41 ). It has also been used to evaluate public health interventions which make use of community health workers in community-based setting ( 42 – 44 ). Results are made up of quantitative measures from the participant questionnaires, qualitative interviews with the participants, the CHWs, VCSE partners, and the research team. Primary quantitative outcome measures included implementation measures such as uptake and engagement and the pre/post changes to the self-report CVD behavioural questions which included the following three questionnaires: (1) the INTERHEART CVD risk questionnaire collected during the screening process was used as the baseline and collected again after completion of the intervention. (2) Physical activity levels were measured using the International Physical Activity Questionnaire (IPAQ) ( 45 ). The IPAQ is an internationally validated instrument to capture information about weekly physical activity habits, behaviours, and routines. (3) Diet was assessed using a 20-item questionnaire based on a modified version of the UK Diet and Diabetes Questionnaire ( 46 ), a brief food frequency questionnaire designed to assess conformity to healthy eating guidelines, and to assist in the setting of dietary goals. It was used to estimate the number of portions eaten daily or weekly of fruit and vegetables, oily fish (or alternative), and foods high in fat, salt, and sugar, what proportion of the time wholegrain cereal products were chosen, weekly units of alcohol consumed and the frequency of binge drinking. Due to the small sample sizes and non-parametric data used in this study, Wilcoxon Sign test was used to evaluate for differences in continuous variables whilst McNemar's test was used for binary categorical data. The pre-intervention assessment of the primary outcome measures was sent to participants before they participated in the intervention (no participant could begin the intervention without completing the baseline measures). Post intervention primary outcome measures were collected after their participant in the intervention was completed.

Focus groups and one-to-one interviews were conducted with four groups of stakeholders: (1) VCSE partners; (2) CHWs; (3) members of the research team, (4) participants in the intervention. Individual interviews were conducted with VCs, members of the research team, and participants, while data from the CHWs was collected in focus groups. Discussion guides for VCs, CHWs and members of the research team all included questions on the respondents' role within the project, the process of community engagement, barriers, and facilitators the implementation process, recommendations for the future and sustainability. Discussion guides for participant interviews included questions on how and why participants became involved in the project, their experience of the health coaching, and their views on the impact and usefulness of the project. Interviews and focus groups were conducted online using Zoom or MS Teams. The analysis was conducted by TGJ, IR, and RD and using qualitative framework analysis based on the components of the RE-AIM framework. Following data collection interviews and focus groups were transcribed by a professional transcription service and TGJ, IR, and RD familiarised themselves with the full set of data. They then undertook line-by-line coding of the data in NVivo using descriptive primary codes which were then interlinked with secondary codes. These secondary codes were then organised under the five elements of the RE-AIM framework (Reach, Effectiveness, Adoption, Implementation, Maintenance). The analysis was interpreted, findings were synthesised with reference to the stakeholder group and theme descriptions were produced with supplementary illustrative quotes.

The Reach of the intervention was assessed through recruitment rates for the VCSE partners, CHWs and Intervention participants and qualitative data collected from the VCSE partners, and the research team was used to understand barriers and facilitators to recruitment. Effectiveness was assessed during the primary outcome measures and barriers and facilitators to effectiveness were assessed through qualitative interviews with the participants and CHWs. Adoption was at the setting level was determined through assessment of the retention of VCSE partners and qualitatively through interviews with VCSE partners and the research team. At the individual level, Adoption was assessed through CHW retention rates and qualitatively assessed through interviews with the research team and the CHWs. Implementation was assessed qualitatively through interviews with the intervention participants focusing on intervention fidelity. Maintenance was assessed at the setting level qualitatively through interviews with VCSE partners and the research team and through a report of the status of the intervention after 6 months. No individual level maintenance data is reported. A description of the data sources which contributed to each component of the RE-AIM framework is listed in Table 1 .

Table 1 . Description of data source used to evaluate the SPICES-Sussex intervention for each of the RE-AIM components.

Ethical approval for this research was obtained from Brighton and Sussex Medical School's Research Governance and Ethics Committee (R-GEC) (application reference: ER/DG241/17BSMS9E3G/1). This ethics review covered the methods described herein, key research materials, and recruitment and consent protocols for both intervention participants and staff/CHW interviews. Due to the changes imposed on the project by COVID-19 (see Supplementary Appendix 2 ) and because of minor adaptations from research site to research sites; several minor amendments were made (final application reference: ER/BSMS9E3G/6).

Informed consent was obtained in three ways from study participants depending on the nature of their participation. (1) Online screening questionnaire: these participants were presented with an approved information sheet on the first page of the online screening questionnaire, they were then provided with an Informed Consent Form (ICF) which they had to sign with a digital signature. (2) Intervention participants: just prior to participation and data collection participants met with a research staff member to review the information sheet and to sign the ICF if they agreed to participate, consent was sought again for those intervention participants who took part in a post-intervention interview. (3) CHW and research staff members: participants were sent the information sheet and consent form several days before their interview and were asked to sign and return the ICF prior to their interview appointment.

3.1 Participant characteristics

Risk profiling data was collected from 381 participants (Females: 310, Males: 71; mean (SD) age = 58 (12.39) years. Forty-Six participants began the intervention (39 Females, 7 Males; age = 58 (11.94) years. Sixteen participants took part in one-to-one interviews at the end of the intervention (thirteen females and two males, aged 32–67 years). Seven members of the research team (6 females, 1 male), and four VCSE partners (3 females and 1 male) took part in the research team interviews. Four focus groups with a total of thirteen participants (10 females and 3 males) were conducted with CHWs from each of the research sites. Thirteen participants (no gender data collected) took part in the post-intervention questionnaire for non-participants.

3.2 Analytical framework

The remainder of these findings are organised into RE-AIM dimensions with various quantitative and qualitative methods used to evidence each dimension, see Table 1 for a description of each of the data sources. Table 2 summarises concordance and discordance with expectations of the intervention [as described in the study protocol ( 29 )] in line with the RE-AIM framework. Supplementary Appendix 3 summarises changes to the study design from the published study protocol . Throughout this section participant codes are used to attribute quotations and references to specific terminology to a respondent. The codes identify the respondent as either a member of the Research team (RT), VCSE partners (VCSE), Community Health Worker (CHW) or Participants (PP). For VCSEs, CHWs and PPs references to their sites are also made (EB, HA, NH, HG). All codes refer to gender and (F/M), and their number within each respondent category.

Table 2 . Description of concordance/discordance with our pre-implementation expectations of the SPICES intervention for each of the RE-AIM components ( 29 ).

4.1 Recruitment of voluntary and community sector enterprise partners

A community-mapping exercise was carried out during the pre-implementation phase of the project ( 30 ) in which three partner organisations were identified across three research sites in East Sussex (Hastings, East Brighton, and Newhaven). All these organisations were volunteer based community organisations with a focus on local community development and improving health, with the Hastings organisations being focused on health and wellbeing. During the intervention set up phase, the East Brighton organisations dropped out of the study due to the impact of Covid-19 whilst the Hastings, and Newhaven organisations were carried forward to deliver the intervention. The East Brighton organisations helped the research team to develop links with a health and wellbeing organisation that was associated with a local General Practice (GP) clinic in East Brighton. Finally, a fourth research site was identified in West Hove and a final VCSE partner was identified. This organisation was a local community development organisation for the area. In total four VCSE organisations were partnered with across four research sites. In each site a VC was recruited from the partner organisation to deliver the intervention with the research team.

4.2 Community health worker recruitment

The research team and VCSE partners recruited 38 individuals who attended the introductory CHW meetings (Gender: 27 females and 11 males, NH n  = 7, EB n  = 13, HG n  = 10, HA n  = 8). Twenty-seven of these individuals completed the full training for CHWs (20 females and 7 males; NH n  = 5, EB n  = 9, HG n  = 7, HA n  = 6).

4.3 Participant recruitment

Social media recruitment had a wider reach to potential participants compared with gatekeeper recruitment, however, several participants did not complete the REDCap screening questions, had a poor understanding of the study, or were not part of the study's target population. VCSE gatekeepers yielded poor recruitment results apart from when a newsletter with a particularly large reach was used. Social media was the primary strategy for recruiting participants to the study. In total the messages reached 13,086 individuals across four waves of recruitment and of these 472 (3.6%) engaged with post by clicking on the survey link. Of those who clicked on the link 80% were female and 20% were male.

The INTERHEART screening data is shown in Figure 1 and Supplementary Appendix 1 for all those who completed the screening questionnaire ( N  = 381), participants who started the intervention and then withdrew ( N  = 17), and participants who completed the intervention and on whom we have full data ( n  = 27). Of the CVD risk factors measured by the INTERHEART screening tool, the two most prevalent were stress (reported by 61% of those screened, 56% of those who started the intervention, and 78% of those who went on to complete), and physical inactivity (reported by 55%, 81% and 64% respectively).

Figure 1 . Primary outcome measures for the “Reach” and “Effectiveness” components of the SPICES-Sussex intervention. ( A ) The proportion of “Low”, “Medium”, and “High” risk participants identified during the Interheart risk profiling questionnaire; ( B ) the mean Interheart score pre and post intervention for those who completed the intervention, p value from paired t -tests; ( C ) shows the % change regularly of dietary behaviours from pre/post intervention UKDDQ score, within-group t -tests; ( D ) the change in the % of intervention participants classified as having either low or medium/high activity levels pre and post intervention, p value from McNemar's test.

Forty-six participants took part in the CVD coaching intervention across the four research sites, all of whom completed the pre-intervention quantitative questionnaires. Sixty-three percent completed the full coaching intervention, and one participant withdrew from the project after three months. We had full data for twenty-seven of twenty-nine participants who completed the full 6-month coaching intervention (note: these participants have been removed from Supplementary Appendix 1 , n  = 2), Participants' characteristics are summarised in Table 3 . Several participants withdrew (37%), reasons given for withdrawing were: ill health/poor mental health/ill health in the family (13%); the intervention was considered a poor fit for the participant/did not meet their expectations/they did not need the intervention (9%); other commitments got in the way/they were too busy with their normal lives (7%); repeated non-attendance at planned coaching sessions from the CHW (4%); did not get on well with CHW (2%), language issues (2%).

Table 3 . Facilitators and barriers to reach and recruitment.

Due to low initial recruitment rates, the recruitment areas were expanded and included more affluent adjacent areas. The proportion of those completing the screening questionnaire and of those who went on to start the intervention who were in the target population (i.e., had an address with a postcode and IMD in the most deprived three deciles) was 30% in both cases. Despite recruitment being gender neutral and without gender/sex related parameters on social media our risk profiling questionnaire recruited far more women than men (77% female, 23% male, see Supplementary Appendix 1 ). This issue was carried forward to the main intervention in which only five of the forty-six who initially took part in the study were male.

4.4 Reasons for non-participation

Reasons given for not participating included missing or not receiving an invitation to take part ( n  = 4), lack of time due to responsibilities and commitments ( n  = 4), not feeling like the intervention was a good fit for them and their circumstances ( n  = 2), not being happy with the CHW allocated to them ( n  = 2), being reluctant to take part in online activities due to a lack of privacy at home ( n  = 1). When asked what would have made them more likely to participate the most common response was more clarity/detail on what was involved ( n  = 3).

4.5 Facilitators and barriers to reach

Intervention participants referred to several intervention components that functioned as facilitators or barriers to the reach of the intervention. These barriers and facilitators were organised into themes which include: (1) Experience of CHW recruitment; (2) The value of community partnerships; (3) The experience of the risk profiling questionnaire; (4) Impacts of COVID-19. These barriers and facilitators are described in more detail in Table 3 and illustrative quotes are provided.

5 Effectiveness

5.1 primary outcomes measures.

For those participants who completed the intervention, the before and after measures of cardiovascular risk, diet, physical activity, and readiness to change were compared (see Figure 1 and Supplementary Appendix 1 ). Mean INTERHEART score fell significantly from 11.7 to 9.9, taking the mean to within the low-risk range. There were also significant improvements in the self-reported dietary measures including: an increase in the proportion of time wholegrain foods were chosen, and the daily portions of fruit and vegetables eaten, and decreases in the consumption of fatty, salty, and sugary food. No changes were observed in the consumption of oily fish. Self-reported levels of physical inactivity also dropped over the course of the intervention with the proportion of those classified in the “low” physical activity category falling from 40% to 7%. Additionally, the self-reported levels of participants' “readiness to change” during the intervention increased from 3.6 to 4.5, which indicates increased levels of motivation as a result of the intervention.

5.2 Participant reported facilitators and barriers to the effectiveness

Intervention participants referred to several intervention components that functioned as facilitators or barriers to the effectiveness of the intervention. These barriers and facilitators were organised into themes which include: (1) accountability—the ways CHWs kept participants accountable about their health behaviours; (2) connection and community—the importance of making human connections with the CHWs and feelings of community togetherness; (3) judgement-free—the importance of a judgement-free intervention experience; (4) motivation and support—the coaching role that the intervention took in the lives of participants; (5) personalisation—the feeling that the intervention was adapted to their own needs and experiences; (6) reflection—the value of reflecting on experiences during the coaching intervention; (7) self-efficacy—the ways in which CHWs made participants feel in control of their health behaviours; (8) gradual or modest impact—the feeling that the intervention largely lead to modest impacts (9) generic or inappropriate advice—the feeling that the information provided during the coaching was too generic, obvious, or inappropriate to their needs. These barriers and facilitators are described in more detail in Table 4 and illustrative quotes are provided.

Table 4 . Facilitators and barriers to intervention effectiveness.

6.1 Retention of voluntary and community sector enterprise partnerships (setting level)

Of the six VCSE organisation engaged with during the pre-implementation phase of the project, four went on to be VCSE partner organisations during the implementation phase. Disruption and staff pressures resulting from the COVID-19 pandemic were a significant barrier to recruiting partner VCSEs, with two organisations who had been involved in initial discussions deciding not to proceed for this reason. Furthermore, interruptions to communication caused by COVID-19 and research team changes led to a loss of trust and engagement in some cases. One organisation which had a group of people ready to volunteer at the beginning of the project later withdrew as this group had fragmented due to COVID-19-related delays and substantial staffing changes that took place just prior to the implementation phase between 2019 and 2020. Other factors impacting on VCSE recruitment included the availability of funding, and issues with recruiting staff to the VC role. After one of the VCSE partners dropped out of the study just prior to the implementation phase, the same organisation linked the research team with another organisation who eventually functioned as VCSE partners for the implementation phase. The need to develop trust, and having the time to achieve this, was stated by several members of the research team as being key to recruiting partner VCSEs. Quality of communication was also felt to be especially important.

6.2 Facilitators of voluntary and community sector enterprise partnerships (setting level)

VCSES and research team members referred to several intervention components that functioned as facilitators or barriers to setting level adoption. These barriers and facilitators were organised into themes which include: (1) Trust—the importance of developing trust with community- partners; (2) Local Knowledge—the value of local knowledge and to delivering appropriate community care; (3) Local Skills—the value of the skills and experiences in local communities to delivering the intervention. These barriers and facilitators are described in more detail in Table 5 and illustrative quotes are provided.

Table 5 . Facilitators and barriers to intervention setting level adoption.

6.3 Retention of community health workers (individual level)

Of the twenty-seven CHWs who were recruited and trained to be a part of the intervention, twenty-one went on to deliver one or more session as an active CHW (Gender: 15 females and 6 males NH n  = 5, EB n  = 6 HG n  = 5, HA n  = 5). Each of these CHWs completed the intervention with at least one participant and the maximum number of participants who completed the intervention with one CHW was three.

6.4 Community health worker training needs feedback (individual level)

After training sessions in our first site, a short questionnaire was conducted with CHWs who attended the training in the formof one-to-one discussions with the training coordinator and the research team. Questions were asked about the anticipated barriers that CHWs thought they would face during the coaching as well as key training needs. Anticipated barriers and challenges during the project included: a sense of mistrust amongst participants, issues of poverty and deprivation, triggers, and sensitivities to the experiences of participants (i.e., trauma or addition triggers). The key training needs identified included: the sharing of personal stories to empower participants, how to set achievable health goals, preparing CHWs with tools to challenge the participant in a supportive way, improving CHW confidence, and advice on how to communicate CVD risk to participants in a straightforward way.

6.5 Community health worker facilitators and barriers to adoption (individual level)

CHWs referred to several intervention components that functioned as facilitators or barriers to the adoption of the intervention at the individual CHW level. These barriers and facilitators were organised into themes which include: (1) Local adaptation and Codesign Sessions; (2) CHW motivation for participating; (3) CHW experiences of the training; (4) CHW experience of the support provided to them. These barriers and facilitators are described in more detail in Table 6 and illustrative quotes are provided.

Table 6 . Facilitators and barriers to intervention individual level adoption.

7 Implementation

7.1 participant retention and fidelity.

Overall, 48% ( n  = 51) of those eligible ( n  = 106) to take part in the intervention agreed to do so and provided consent, of those 90% ( n  = 46) attended their first CHW coaching session and completed the baseline questionnaire. Of those who completed their first session 63% ( n  = 29) completed three sessions and 45% completed six sessions. For the 46 participants that began the intervention there were 276 planned program contacts of which 183 (66%) were completed. Retention and attendance data are summarised in Supplementary Appendix 2 . No data was collected on the amount of time each participant spent in their coaching session.

7.2 Participant facilitators and barriers to implementation

Intervention participants referred to several intervention components that functioned as facilitators or barriers to the implementation of the intervention. These barriers and facilitators were organised into themes which include: (1) expectations of the coaching intervention, (2) the virtual coaching sessions; (3) holistic and flexible, (4) length of the coaching session, (5) administrative support, (6) past experiences, (6) mental health. These barriers and facilitators are described in more detail in Table 7 and illustrative quotes are provided.

Table 7 . Facilitators and barriers to intervention implementation .

8 Maintenance

8.1 status of the intervention after six months.

Six months after the intervention's funding period ended the program was being continued at two of the sites. One of the sites continued it as volunteer opportunity and peer support program which was covered by their existing funding for peer support programs. A second site was awarded funding from the National Health Service to continue the intervention. The latter's findings will be reported as a program evaluation in the future.

8.2 Facilitators and barriers to maintenance

Interviewees referred to several intervention components that functioned as facilitators or barriers to the maintenance of the intervention at the setting level. These barriers and facilitators were organised into themes which include: (1) continuity of the intervention; (2) funding; (3) infrastructure These barriers and facilitators are described in more detail in Table 8 and illustrative quotes are provided.

Table 8 . Facilitators and barriers to maintenance.

9 Discussion

SPICES Sussex developed strategies to implement effective community-based CVD risk reduction interventions based on behaviour change coaching with CHWs by partnering with and leveraging the experience and influence of VCSE in four underserved communities in East Sussex, UK. Despite issues with recruitment and challenges associated with COVID-19 as well as other logistic, management, and research design challenges, the project showed clear markers of success. Participants experienced the interventions positively and many made gradual, and sometimes substantial, lifestyle changes. The quantitative results showed significant reduction in participants' CVD risk after taking part in the interventions. We think these successes were due to implementing our interventions in a flexible, personalised, and holistic way, which empowered CHWs to use their skills and experiences to aid participants. These results demonstrate how CHWs-led and community-based preventative CVD interventions could be implemented, such as those seen widely across the Global South ( 17 , 18 ). They also support a “person-based” and “asset-based” approach to community-based implementation design ( 23 , 25 ) in which the strengths and assets of communities and their members are used to promote health and wellbeing.

9.1 Intervention design

The SPICES-Sussex project used community-engagement and community health worker approaches to improve CVD health that are based on practices developed and tested in Kampala, Uganda ( 47 ). As part of the SPICES consortium these practices were adapted to several global north (UK, France, Belgium) and global south settings (South Africa). In the global south social public health approaches have long advocated for the decentralisation of healthcare to community partners and for a greater focus on prevention ( 48 ). Community-based public health practices such as task-sharing are often utilised in low-resource health systems in low-and middle income countries by recruiting and training community health workers to deliver low-intensity health intervention such as health coaching and signposting ( 49 ). In global south SPICES settings, there was greater buy-in to community-based interventions from governments and much of the trust building, and infrastructure for community health workers already exists ( 50 ). These settings, including the SPICES sites that influenced the Sussex site, often rely on voluntary or unpaid volunteers to conduct public health work in order to lower cost and to make use of existing social networks.

In resource-rich global north settings, healthcare is far more institutionalised and focused on secondary care and the infrastructure for community-based and participatory interventions is far less well developed. In the UK, most health interventions must adhere to the institutional demands of the National Health Service which presents a range of resource intensive training, recruitment, safeguarding, and management practices. There is much less history of CHWs in the UK; the role of these workers is not well understood or well defined outside of the third/voluntary sector despite recent calls for their use during the COVID-19 pandemic ( 51 ). This squeezed landscape for community-based intervention and the lack of familiarity with the role makes the development and implementation of these interventions challenging. In the global north there are increasing challenges to the volunteer nature of CHWs with researchers calling for compensation, capacity building, or payment of members of the public involved in intervention delivery of research and health interventions on moral and efficacy grounds ( 52 , 53 ). In our study, the decision not to pay CHW was made as a result of us following the SPICES approach developed in the Global South ( 17 ) and because the VCSE organisations we partnered with all had existing unpaid community volunteer programs. In our post-intervention qualitative evaluation interviews, participants and CHWs both discussed the value of paying CHWs. Furthermore, the drop in CHWs and the small number of participants they were able to take on implies that the lack of payment impacted the degree to which CHWs were able to engage with the project and therefore impacted the intervention's effectiveness and sustainability. In the UK, the NIHR now recommends that members of the public who are involved in research are properly reimbursed for their involvement and provide frequently updated guidelines on how to do so ( 54 ). In the future we argue that public health intervention that make use of CHWs should reimburse and pay them in some way for their involvement.

Community volunteers with low levels of training (10 h core training plus ongoing support), such as those used during this study, are not well-suited to complex cases or acute needs that required specialised support. In our findings, participants complained of generic, inappropriate, or obvious advice from the CHWs. Participants did not seem to prioritize the knowledge or expertise of the CHWs and instead valued the personalised, holistic, and supportive relationships that were offered by the CHWs. Participants in the intervention reported having good knowledge of what they needed to do to improve their health but struggled to do it in practice. Therefore, this kind of intervention may be well-suited to providing emotional and social support to people at risk of CVD who know what they “should” do but need a support and judgement free support mechanism to make changes.

Interviews with participants revealed a tension in the study linked to the use of an individualistic lifestyle change intervention situated within a community-based and participatory study. The study design did not address community-level, socio-economic, or environmental issues known to be vital when addressing CVD health ( 55 ). Tengland ( 56 ) argues that an individualistic lens of behavioural change can limit understandings of a person's CVD health. The result can be too narrow, as the “secondary” effects of their wider environmental conditions (i.e., powerlessness, lack of control, or lack of hope), are not considered. They further suggest that interventions should focus more on the attainment of instrumental goals, such as increased real opportunities in life. For community-based projects to grow further, they should seek to become multi-faceted by combining individualistic interventions with environment/community activities such as community education ( 57 ).

The frequency with which mental health issues were raised in discussions was notable. Those who took part in the screening reported high levels of stress and depression, and rates were even higher amongst participants taking part in the programme, furthermore, participants reported lower levels of stress and depression at the end. This may show that this type of intervention is particularly well suited to people with mental health concerns for whom talking to someone can make a real difference. This was also observed in the SPICES consortium partner sites including Brest (France) ( 58 ), and Antwerp (Belgium) ( 59 ). Most non-specialist or non-clinical people do not think of their health siloed into CVD, mental health, digestive health etc ( 60 ). Instead, one's health is perceived holistically, and mental health is often the most prominent barrier and facilitator to behaviour change.

9.2 Implementation strategy

We adopted a type 3 hybrid implementation study which focused primarily on implementation factors rather than evaluation, dropping the randomisation approach and embracing flexible more emergent iterative development and growth perspective, co-design, and contextual/place-based factors. A rigid evaluation linear approach as required for a type 1/2 design, which was initially planned, caused tensions with the community-based, participatory, and “emergent” aspects of the project and (2) the pressures imposed on our voluntary sector partners by the pandemic meant that adhering to a rigid randomisation approach was less realistic ( 7 ). The planned approach placed power in the hands of the research team which negatively affected our stakeholder relationships, and a rigid adherence to study protocols would have meant we could not effectively adapt strategies or interventions to context.

Instead we adopted a type 3 approach, which has been used to assess a wide range of preventative health and eHealth interventions which operate in communities based on participatory principles ( 61 ). In their systematic review of such strategies to implement interventions, Haldane et al. ( 62 ) highlight the importance of building mutually beneficial and trust-based relationships particularly with marginalised stakeholders, and stress the importance of developing strategies and interventions contextually whilst reporting and acting on lessons learnt throughout the project. Wildman et al. ( 63 ) argue that successful community-based projects require extensive community input, learning and adaptation captured from existing programmes to facilitate the replicability of programmes in other community contexts. With the more flexible type three approach we were able to make local adaptation to meet the need and priorities of the local community and local VCSE partner organisation thereby listening to the voices of those who are involved. This iterative approach to intervention design is similar to the “scaling-out” approach suggested by Aaron et al. ( 64 ) which advocates iterative roll-out and local adaptation in place of simply “copy and pasting” interventions across context. In reality, during SPICES-Sussex the local adaption became less flexible as the intervention became more well-developed as the internal factors became more institutionalised within the research team. However, the principle of meeting the needs and priorities of the local VCSE organisation were maintained from site to site and the team sought input from local organisations where possible.

We do not know whether the changes observed will be maintained due to the short follow up period, both at an individual level or a setting level ( 65 ), and the research lacks an economic appraisal. The short follow-up period was forced on the research team because of delays to the project caused by COVID-19 which meant our funding period was not long enough to conduct a follow up assessment. An economic appraisal was not considered appropriate because the development approach taken during the study meant that any economic appraisal was not likely to reflect real-world roll-out. In the future we would advocate for greater scaling-out to include a larger sample and an economic appraisal.

9.3 Recruitment and retention

The impacts of the restrictions placed on the people, organisations, and communities involved in this research due to COVID-19 were extensive and wide-ranging. The per-implementation phase of the research began in January of 2020 with the recruitment of an implementation team and participant recruitment was due to begin in April 2020. Following the outbreak of COVID-19 in the UK, recruitment was stopped from 16 March 2020 to 1 October 2020. By June 2020, a decision was made to fully move to remote delivery of the coaching intervention using video conferencing services.

Research recruitment and retention were near constant challenges, and all activities were significantly impacted by the Covid restrictions. We believe that the use of the INTERHEART tool, presented on the REDCap platform, acted as a barrier to recruitment as evidenced through the follow sources: (1) Over 650 participants attempted to begin the screening questionnaire and our records show those who did not complete it stopped towards the beginning or mid-way through the questions, particularly when they were asked to measure their waist/hip circumference, (2) of the 380 participants who completed the survey only approximately 100 were eligible for the intervention meaning we were selecting from a very limited pool of participants, (3) many of the participants in the per implementation interviews mentioned finding the screening tool to be “clunky” or “annoying” to use. Its overly “medical” focus, as a basis for lifestyle discussions may not have been engaging for the target audience.

Our initial recruitment strategy was to rely heavily on our VCSE partners to act as gatekeepers for recruitment, a practice commonly seen in participatory research methods ( 66 ). Whilst the VCSE partners were adept in the recruitment and management of CHWs and in the development of practices and policies, they did not seem to have the reach or access for the recruitment of large numbers of potential participants. Our experience aligns with that of Williams ( 67 ), who states that VCSE and end users' relationships are often smaller in number but deep, based on trust and protection, and covered by a range of risk related policies. Instead, we relied heavily on the use of paid for social media adverts for recruitment due to our ethics restrictions. Much like the experience of other researchers who used these tools, we found that they were low cost and reached large numbers of people but engagement with the screening and risk profiling and participant recruitment was low ( 68 ). In future studies, it may be more suitable to use social media as an adjunct to mixed recruitment strategies which make use of community outreach, primary care recruitment, and media outreach ( 69 , 70 ).

The study sample was heavily skewed towards middle-aged females and much of the sample was not considered to be from vulnerable or low socio-economic groups. Furthermore, males are under-represented in both the risk profiling and intervention samples which represents a divergence with our planned recruitment targets in which we aimed for a more representative sample. The difficulties in recruiting men and vulnerable and other “seldom heard” populations to life style interventions are well-recognised ( 71 , 72 ). Recommended strategies to improve male participation in community-based interventions include engaging with male-friendly spaces, workplace-based interventions, and incorporating activity-based programming, social-support, and group activities ( 73 , 74 ). Some of these elements were suggested during the planning phase of SPICES but were not feasible due to COVID restrictions ( 30 ).

9.4 Project infrastructure

We made the key decision to bring VCSE organisations into the research team with paid roles to foster stronger community/research partnerships as promoted by CBPR researchers ( 75 ) and the NHS's PPIE (Public and Patient Involvement and Engagement) initiatives ( 76 ). Our research shows that the VCSE sector is an untapped resource within primary and community care that has a great deal of expertise, compassion, and enthusiasm to offer health provision ( 77 ). To facilitate this community-based project, we focused on the concept of trust building throughout the intervention as described by Christopher et al. ( 78 ).

VCSE partnerships brought knowledge and expertise of their local communities, policies/practices of volunteer management and, critically, perspectives of the motivations and drivers for CHWs and communities. CHWs were empowered to bring their own skills and abilities to the intervention through an asset based and flexible project development which included them in the co-design of the project ( 79 ). The strategies we used to implement the interventions were not prescriptive and did not force CHWs to follow a set of strict guidelines. This led to a highly personalised, flexible, and reflective experience for CHWs. However, our experience highlights potential problems with relying on unpaid volunteers to deliver complex interventions, including issues with volunteer commitment, attendance and drop out.

Our research highlights the importance of infrastructure when managing CHWs and partnering with VCSE sector organisations. We developed a bespoke behaviour change training course for CHWs, a range of CHW risk appraisal and mitigation policies with our VCSE partners, and a dedicated team of participant and CHW support and management coordinators. Clear protocols were developed and followed for the recruitment, onboarding, matching, and hosting of participant coaching sessions whilst CHWs were provided with multiple channels of regular communication and continuous training and feedback opportunities. We support calls for project managers, VCSEs, primary care providers, and community members to be more explicitly involved in the design and development of interventions which affect and include communities ( 80 ).

In this study, the research team also experienced issues of positionality throughout the project whereby the lines between implementor, community worker, and evaluator were blurred. Coulter et al. ( 81 ) have pointed out that research that includes CHWs in the design and delivery of interventions commonly experience a tension between fidelity of the intervention protocol and community expectations, needs, and norms. We also experienced differing goals between academic and community partners (including CHWs), where academic partners prioritized data collection and community partners prioritized funding, sustainability, and policy. This can be likened to the experience of Furman et al. ( 82 ) who discussed how community partners were hesitant to endorse their research due to conflicts with on-the-ground realities of the community members they served.

9.5 Recommendations

During this project the research team, VCSE partners, and CHWs constantly learnt lessons and were quick to make adaptations to their approach based on feedback from a range of stakeholders and capturing all of these in this paper would be an impossible task. However, several key insights can be drawn from our collective experience and evaluation of the project. They include:

1. Environmental issues are larger and more complex than any coaching intervention based on individualistic changes can hope to remedy.

2. The voluntary and community sector has a range of strengths and assets based on local experience and knowledge developed over significant periods of time that can be used for CVD prevention. However, the sector is highly under-resourced and spread thinly across a wide range of priorities. Individual VCSE partner organisations do not always have enough reach to facilitate recruitment.

3. Community engagement works best if it is built into a project early on through co-design and resources and time should be allocated to this activity.

4. CHWs bring significant advantages during the delivery of community-based interventions. They are trusted peers, they bring their own skills and experience, and they can benefit from the intervention alongside the participants.

5. Strategies to encourage the participation of men should be specifically considered during the planning phase.

6. Virtual coaching interventions are acceptable to participants, and in many cases preferable to participants, due to their flexibility and ease of use.

7. The issue of mental health must be addressed even when working with unrelated health public conditions.

8. A strong project infrastructure, made up of well-trained support/administrative staff, is essential when delivering community-based interventions.

9. CHWs should be paid or reimbursed for their involvement in research and public health interventions. Falling to do so is looked down on my stakeholders and has impacts on sustainability and effectiveness.

10. The Global North can look to innovations in the Global South for examples of success for community-based interventions, however, proper contextual or situational analyses must be conducted to understand the needs and priorities of target communities.

9.6 Conclusion

This study demonstrates the feasibility of a CHW-led preventative health interventions could be implemented with overseen and unheard communities in the UK. It highlights the wealth of untapped resources that exist with VCSE and CHWs and suggests how a beneficial community-based service could be set up to run alongside and support NHS Health Checks, to reduce the incidence of CVD. The aim was to empower CHWs to discuss health with people in their communities based on behaviour change principles. We have set out what worked well and what did not, to facilitate development of future community-based interventions in the Global North. We believe that the community-based approach need not be restricted to CVD risk reduction, and that it could easily be applied to low level mental health conditions, diabetes, or other preventable NCDs. If CHWs are confident, well supported, and well-trained, they will have the skills and ability to contribute to improving the health and wellbeing of people in their communities. The benefits do not only extend to patients but also to CHWs and to the VCSE partners involved. We believe our project shows how these interventions can become a supplementary tool that links primary care services with the VCSE sector.

Data availability statement

The datasets presented in this study can be found in the University of Sussex's data repository through the following link: https://sussex.figshare.com/ ; (doi: 10.25377/sussex.25569084).

Ethics statement

The studies involving humans were approved by Brighton and Sussex Medical School Research Governance and Ethics Committee (RGEC). The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

Author contributions

First Author (First Authorship): Thomas Grice-Jackson Second Authors (Equal Contribution): Imogen Rogers, Elizabeth Ford, Robert Dickson Third Authors (Equal Contribution): Kat-Frere Smith, Katie Goddard, Linda Silver, Catherine Topham Fourth Author (Equal Contribution): Papreen Nahar, Geofrey Musinguzi, Hilde Bastiens. Senior Author (Senior Authorship): Harm Van Marwijk. All authors contributed to the article and approved the submitted version.

This project was funded as part of an EU Commission Horizon. CORDIS (The Community Research and Development Information Service (CORDIS) Grant agreement number: 733356.

Acknowledgments

We thank the following voluntary and community sector organisations for their partnerships whilst designing and delivering this project: Active Hastings, Wellsbourne Healthcare Community Interest Company, Sussex Community Development Association, the Crew Club, and the Hangleton and Knoll project. We thank the National Centre for Behaviour Change for their contribution to the development and delivery of the Community Health Workers training. We thank all members of the SPICES consortium and European Commission who provide consultation and advice throughout the project. Finally, we thank all our Community Health Workers for giving up their time for this project. They were central to every part of this work and their contribution is greatly appreciated. We would also like to thank the editorial and reviewer team assigned to this manuscript. Their contributions improved the quality of our manuscript presentation, structure, and discussion.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/frhs.2024.1152410/full#supplementary-material

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Keywords: community based participatory research, implementation research, RE-AIM (reach, effectiveness, adoption, implementation and maintenance), cardiovascular disease, community health workers (CHW)

Citation: Grice-Jackson T, Rogers I, Ford E, Dickinson R, Frere-Smith K, Goddard K, Silver L, Topham C, Nahar P, Musinguzi G, Bastiaens H and Van Marwijk H (2024) A community health worker led approach to cardiovascular disease prevention in the UK—SPICES-Sussex (scaling-up packages of interventions for cardiovascular disease prevention in selected sites in Europe and Sub-saharan Africa): an implementation research project. Front. Health Serv. 4:1152410. doi: 10.3389/frhs.2024.1152410

Received: 27 January 2023; Accepted: 20 March 2024; Published: 7 May 2024.

Reviewed by:

© 2024 Grice-Jackson, Rogers, Ford, Dickinson, Frere-Smith, Goddard, Silver, Topham, Nahar, Musinguzi, Bastiaens and Van Marwijk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Thomas Grice-Jackson [email protected]

This article is part of the Research Topic

Hybrid Effectiveness-Implementation Trial Designs: Critical Assessments, Innovative Applications, and Proposed Advancements

Reducing carbon emissions in prefabricated buildings supply chains: a focus on component manufacturing processes

• Research Article
• Published: 06 May 2024

Cite this article

• Feng Guo 1 ,
• Junwu Wang 2 &
• Yinghui Song   ORCID: orcid.org/0000-0003-3872-7486 2  

The construction sector accounts for 23% of \({\mathrm{CO}}_{2}\) emissions from global economic activity, with China responsible for nearly 41%. Although China has vigorously promoted the development of prefabricated buildings (PBS) in pursuit of cleaner production, the carbon emissions from prefabricated component factories (PCF) should not be underestimated. So, the focus of this research was on how to promote the decarbonization of PCF. Based on the carbon trading market mechanism, the carbon emissions trading tax and revenue tax collection, the authors established a differential game model consisting of the local government and the PCF, studied the equilibrium solutions under different decision models, and analyzed the roles of the two tax systems, carbon trading revenue, and market preferences. The results are as follows: (1) The PCF’s low-carbon technology (LCT) innovation efforts can be directly affected by the carbon price, component price, and tax rate and indirectly affected by influencing the local government’s efforts. Besides, the local government and the PCF strategies can be changed through the central government’s regulation of carbon prices and tax rates. (2) PCF should set reasonable prices for components, improving economic efficiency and the LCT stocks. (3) Cost-sharing contracts can encourage PCF to increase their LCT innovation, which is conducive to increasing the optimal benefits of the PCF. (4) The local government cannot be motivated by cost-sharing contracts. They can increase their optimal benefits only if the cost-sharing coefficient is below a threshold or if the environmental benefits from low-carbon production are above a specific value.

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Acknowledgements

We would like to thank the editors and anonymous reviewers for their valuable comments.

This study was supported by National key R&D projects, grant number ( 2018YFC0704301 ), Science and Technology Project of Wuhan Urban and Rural Construction Bureau, China ( 201943 ), Research on theory and application of prefabricated building construction management ( 20201h0439 ), Wuhan Mo Dou construction consulting Co., Ltd. ( 20201h0414 ), and Preliminary Study on the Preparation of the 14th Five-Year Plan for Housing and Urban–Rural Development in Hubei Province ( 2020 2s0002 ).

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School of Civil and Hydraulic Engineering, Huazhong University of Science and Technology, Wuhan City, 430070, Hubei Province, China

School of Civil Engineering and Architecture, Wuhan Univ. of Technology, Wuhan City, 430070, Hubei Province , China

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Feng Guo: conceptualization, investigation, software, and methodology. Yinghui Song: investigation, resources, validation, and writing review. Junwu Wang: supervision.

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Correspondence to Yinghui Song .

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According to the relevant theory, the optimal value function of the PCF is as follows:

where  \({V}_{pcf}^{N}\left(P\right)=\max \limits_{{E}_{pcf}}{\int }_{t}^{\infty }{e}^{-p\left(r-t\right)}\left[\left(1-{k}_{1}\right)D\left({E}_{pcf,}{E}_{G}\right){W}_{pcf}+\left(1-{k}_{2}\right)\pi \left(P\right)-{C}_{pcf}\left({E}_{pcf}\right)+{R}_{pcf}\left(P\right)\right]dr\)   

And, \({V}_{pcf}^{N}\left(P\right)\) should satisfy the following HJB equation at \({\mathrm{P}}\ge {0}\) :

According to Pontrya-gin’s maximization principle, by taking the partial derivative of \({\mathrm{E}}_{\mathrm{pcf}}\) in the HJB equation, the optimal LCT innovation effort for the PCF is as follows:

Similarly, we can get the optimal value function of the local government factor as

where \({V}_{G}^{N}\left(P\right)=\max \limits_{{E}_{G}}{\int }_{t}^{\infty }{e}^{-p\left(r-t\right)}\left[{k}_{1}D\left({E}_{pcf,}{E}_{G}\right){W}_{pcf}+{k}_{2}\pi \left(P\right)-{C}_{G}\left({E}_{G}\right)+{R}_{G}\left(P\right)\right]dr\)   

And, \({V}_{G}^{N}\left(P\right)\) should satisfy the following HJB equation at \(P\ge 0\) :

According to relevant theory, by taking the partial derivative of \({\mathrm{E}}_{\mathrm{G}}\) in the HJB equation, the government's optimal participation in environmental governance efforts can be expressed as follows:

\({\mathrm{V}}_{\mathrm{pcf}}^{\mathrm{N}}\mathrm{(P)}\) and \({\mathrm{V}}_{\mathrm{G}}^{\mathrm{N}}\mathrm{(P}\mathrm{)}\) , the linear expression for P , can be expressed as follows:

where \({u}_{1}^{N}\) , \({{u}_{2}}^{N}\) , \({v}_{1}^{N}\) , and \({{v}_{2}}^{N}\) are the parameters to be determined. Substituting equation ( 28 ) and equation ( 37 ) into equation ( 27 ) and equation ( 32 ) and equation ( 33 ) into equation ( 35 ), the following equation can be obtained:

By solving equation ( 39 ), we can solve for \(u_1^N\) , \(u_2^N\) , \(v_1^N\) , and \(v_2^N\) as follows.

Substituting \({u}_{1}^{N}\) , \(u_2^N\) , \({v}_{1}^{N}\) , and \({{v}_{2}}^{N}\) into equations ( 28 ) and ( 31 ), the optimal equilibrium strategy of the PCF and the local government can be obtained, and substituting the obtained solution into the carbon reduction technology in the state equation, when \(P\left(0\right)= {P}_{0}\ge 0\) , we can get the optimal PCF's stocks of LCT. Substituting ( 32 ), ( 33 ) into ( 26 ), ( 29 ) yields the optimal cumulative benefits for the PCF, the government, respectively.

As mentioned above, we can get the optimal value function of the PCF as follows:

where, \({V}_{pcf}^{Y}\left(P\right)=\max \limits_{{E}_{pcf}}{\int }_{t}^{\infty }{e}^{-p\left(r-t\right)}\left[\left(1-{k}_{1}\right){DW}_{pcf}+\left(1-{k}_{2}\right)\pi \left(P\right)-\left(1-\beta \right){C}_{pcf}\left({E}_{pcf}\right)+{R}_{pcf}\left(P\right)\right]dr,\)   

According to relevant theory, the optimal LCT innovation effort of the PCF can be expressed by taking the partial derivative of \(E_{pcf}\) in the HJB equation as follows:

Similarly, we can get the optimal value function of the local government as follows:

where \({V}_{G}^{Y}\left(P\right)={{\mathrm{max}}}_{{E}_{G}}{\int }_{t}^{\infty }{e}^{-p\left(r-t\right)}\left[{k}_{1}D\left(t\right){W}_{pcf}+{k}_{2}\pi \left(P\left(t\right)\right)-{C}_{G}\left({E}_{G}\left(t\right)\right)-{\beta C}_{pcf}\left({E}_{pcf}\right)+{R}_{G}\left(t\right)\right]dr\)

And, \({V}_{G}^{Y}(P)\) should satisfy the following HJB equation at \(P\ge 0\) :

According to Pontrya-gin’s maximization principle, by taking the partial derivative of \({\mathrm{E}}_{\mathrm{G}}\) in the HJB equation, the government’s optimal participation in environmental governance efforts is as follows:

\({V}_{pcf}^{N}\left(P\right)\) and \({V}_{G}^{N}(P)\) , the linear expression for P , can be expressed as follows:

where \({u}_{1}^{Y},{u}_{2}^{Y}, {v}_{1}^{Y},\) and \({v}_{2}^{Y}\) are the parameters to be determined. Substituting equation ( 38 ) and equation ( 42 ) into equation ( 37 ) and equation ( 41 ) and equation ( 43 ) into equation ( 40 ), the following equation is obtained.

By solving equation ( 34 ), we can solve for \({u}_{1}^{Y}\) , \({u}_{2}^{Y}\) , \({v}_{1}^{Y}\) , and \({v}_{2}^{Y}\) as follows:

Substituting \({u}_{1}^{Y}\) , \({u}_{2}^{Y}\) , \({v}_{1}^{Y}\) , and \({v}_{2}^{Y}\) into equations (383) and (416), the optimal equilibrium strategy for the government and the PCF can be obtained. The resulting solution is substituted into the carbon reduction technology in Assumption 2 the state equation, when \(P(0) = {P}_{0}\ge 0\) , we can get the optimal PCF’s stocks of LCT.

Substituting ( 42 ), ( 43 ) into ( 36 ), ( 39 ) yields the optimal cumulative benefits for the PCF, the government, respectively.

As mentioned above, the optimal area value function is as follows:

where \({V}_{Gpcf}^{C}\left(P\right)=\max \limits_{{E}_{pcf,}{E}_{G}}{\int }_{0}^{\infty }{e}^{-pt}\left[D\left({E}_{pcf,}{E}_{G}\right){W}_{pcf}+\pi \left(P\right)-{C}_{G}\left({E}_{G}\right)-{C}_{pcf}\left({E}_{pcf}\right)+{R}_{pcf}\left(P\right)+{R}_{G}\right]dt\)   

And, \({V}_{Gpcf}^{C}\left(P\right)\) should satisfy the following HJB equation at \(P \ge 0\) :

According to Pontrya-gin’s maximization principle, taking the partial derivatives of \({\mathrm{E}}_{\mathrm{pcf}}\) and \({\mathrm{E}}_{\mathrm{G}}\) in the HJB equation above, the optimal LCT innovation effort of the PCF, the optimal government participation effort, can be expressed as follows:

\({V}_{Gpcf}^{C}\left(P\right)\) , a linear expression for P , can be expressed as follows:

where \({\mathrm{m}}_{1}^{\mathrm{C}}\) and \({{\mathrm{m}}_{2}}^{\mathrm{C}}\) are the parameters to be determined and substituting equation ( 48 ) and equation ( 49 ) into equation ( 47 ) yields the following equation.

By solving the above equation, one can find \({\mathrm{m}}_{1}^{\mathrm{C}}\) and \({\mathrm{m}}_{2}^{\mathrm{C}}\) as follows:

By substituting \({\mathrm{m}}_{1}^{\mathrm{C}}\) and \({\mathrm{m}}_{2}^{\mathrm{C}}\) into equation ( 48 ), the optimal equilibrium strategy for the PCF and the government can be obtained. Substituting this equilibrium strategy into the state equation of carbon abatement technology in Assumption 2, the stocks of LCT in the optimal region can be obtained when \(P\left(0\right)={P}_{0}\ge 0\) . Substituting equation ( 49 ) into equation ( 46 ), the best cumulative benefit for the region can be get.

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Guo, F., Wang, J. & Song, Y. Reducing carbon emissions in prefabricated buildings supply chains: a focus on component manufacturing processes. Environ Sci Pollut Res (2024). https://doi.org/10.1007/s11356-024-33169-1

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Stable, fluorescent markers for tracking synthetic communities and assembly dynamics

• Beatriz Jorrin   ORCID: orcid.org/0000-0002-6198-0925 1 ,
• Timothy L. Haskett   ORCID: orcid.org/0000-0003-4675-6009 1 ,
• Hayley E. Knights   ORCID: orcid.org/0000-0003-2002-4141 1 ,
• Anna Martyn   ORCID: orcid.org/0000-0002-6853-0206 1 ,
• Thomas J Underwood   ORCID: orcid.org/0000-0002-3208-7230 1 ,
• Jessic Dolliver   ORCID: orcid.org/0000-0002-1167-2713 1 ,
• Raphael Ledermann   ORCID: orcid.org/0000-0003-4612-1708 1 &
• Philip S. Poole   ORCID: orcid.org/0000-0001-5087-6455 1  

Microbiome volume  12 , Article number:  81 ( 2024 ) Cite this article

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After two decades of extensive microbiome research, the current forefront of scientific exploration involves moving beyond description and classification to uncovering the intricate mechanisms underlying the coalescence of microbial communities. Deciphering microbiome assembly has been technically challenging due to their vast microbial diversity but establishing a synthetic community (SynCom) serves as a key strategy in unravelling this process. Achieving absolute quantification is crucial for establishing causality in assembly dynamics. However, existing approaches are primarily designed to differentiate a specific group of microorganisms within a particular SynCom.

To address this issue, we have developed the differential fluorescent marking (DFM) strategy, employing three distinguishable fluorescent proteins in single and double combinations. Building on the mini-Tn 7 transposon, DFM capitalises on enhanced stability and broad applicability across diverse Proteobacteria species. The various DFM constructions are built using the pTn7-SCOUT plasmid family, enabling modular assembly, and facilitating the interchangeability of expression and antibiotic cassettes in a single reaction. DFM has no detrimental effects on fitness or community assembly dynamics, and through the application of flow cytometry, we successfully differentiated, quantified, and tracked a diverse six-member SynCom under various complex conditions like root rhizosphere showing a different colonisation assembly dynamic between pea and barley roots.

Conclusions

DFM represents a powerful resource that eliminates dependence on sequencing and/or culturing, thereby opening new avenues for studying microbiome assembly.

Video Abstract

Plant roots are colonised by a vast diversity of microorganism, with Proteobacteria and Actinobacteria amongst the most abundant groups [ 1 , 2 , 3 ]. These soil microorganisms are recruited in different root niches, including rhizosphere (few mm from root), rhizoplane (root surface) and endosphere (microbes between root cells) [ 4 ]. Furthermore, plants exude up to 20% of their fixed carbon into the rhizosphere thereby shaping their root microbiome, which in turn influences plant growth [ 5 , 6 , 7 ]. This two-way dialogue alters plant fitness, is crucial in nutrient cycling, promotes plant growth, primes plant defences and controls pathogens [ 3 , 8 , 9 , 10 ].

The last two decades have seen an explosion in microbiome research on plants, animals and humans. Most plant studies have analysed microbiome composition by amplicon or genome sequencing under multiple conditions, including species and soil type [ 11 , 12 , 13 ]. More recently, use of synthetic DNA spikes enables absolute quantification of microbiome members directly in environmental samples [ 14 ]. The cutting-edge challenge is to now move beyond describing and classifying microbiomes, to understand the mechanisms of microbiome assembly. However, due to the vast diversity of microbes, this has proved to be technically challenging.

A key strategy to understand microbiome assembly is to establish a simpler representative/synthetic community (SynCom) to study and fine tune plant–microbe interactions. One of the pivotal decisions to make when designing a SynCom is the choice of size, which mainly depends on the objective of the study to perform. Vorholt et al. [ 15 ] defined that a high-complexity SynCom (more than 100 members) aims to represent the original microbiome by maintaining the diversity and thereby reducing the risk of losing keystone species and essential microbe-microbe interactions. On the other hand, in a low-complexity SynCom (less than ten members), the stochasticity is reduced, which increases experimental reproducibility and therefore it can establish a more accurate causality [ 15 ]. Most SynComs are an attempt to produce a microbial culture collection with minimal strains representative of the original phylogenetic diversity [ 16 ]. The profile, represented by the relative abundance of each strain in the assembled SynCom, is used as a phenotype under different conditions. An example is how the absence of coumarin, or the lack of cuticle biosynthesis, shifted the SynCom composition in Arabidopsis thaliana [ 17 , 18 ]. A 185-member SynCom was used to interrogate the capacity of root growth inhibition (RGI), showing that Variovorax and related species within the SynCom have the capability to suppress RGI by manipulating plant hormone levels through auxin degradation [ 19 ]. SynComs can improve plant yield, as shown by the 22-member sugarcane community which displaced 54% of the natural rhizosphere microbiota and increased sugarcane fresh weight 3.4-fold compared to non-inoculated plants [ 20 ]. Whilst relative abundance quantification provides valuable insights, the power of absolute quantification reveals that specific microbial groups can maintain steady or increasing absolute abundance, even in scenarios where their relative abundances may decrease [ 14 ]. Absolute quantification emerges as a superior approach, offering a more accurate understanding of microbiome assembly dynamics and mitigating potential biases inherent in relative measurements. Nui et al. [ 21 ] measured the absolute abundance of each bacterial strain within a seven-member maize SynCom by complex culturing, including testing of 288 growth media and antibiotics combinations. The seven-membered community was stable on roots, where Enterobacter cloacae AA4 was a keystone species, as its absence led to collapse of the SynCom. This research highlights that one of the principal challenges in studying microbiome assembly is the identification and quantification of different bacteria during colonisation. Most SynCom studies rely on 16S RNA sequencing to describe assembly of the community, which only reveals relative microbial abundance on the roots. In contrast, differential culturing as used by Niu et al. [ 21 ] allows for experimental intervention and establishes causality in microbiome assembly, although it is labour-intensive and limited to the specific organisms for which it was developed.

Bacterial communities can be visualised and differentiate in situ by applying techniques based on the hybridisation of fluorescently labelled antisense 16S rRNA probes (FISH), which can be designed for broad groups (e.g. Actinobacteria or Betaproteobacteria), or for specific strains [ 2 , 3 , 22 , 23 ]. FISH was applied to a seven-member SynCom in which each strain-specific probe was labelled to a particular fluorescent protein which can be distinguished by image deconvolution [ 24 ]. However, FISH has limitations such as cell loss during sample fixation and low accuracy due to an imperfect probe coverage or reduced bacterial membrane permeability [ 25 , 26 ]. In small SynComs, fluorescent proteins can be expressed in bacteria; however, the limitation is the number of distinguishable ones used at the same time. Whitaker et al. [ 27 ] developed a technique with six unique fluorescent signatures by utilising two fluorescent proteins (GFP and mCherry) with different ribosome binding site (RBS)s to provide varied expression levels. When applied to a Bacteroides six-member SynCom colonising the guts of mice, each strain could be differentiated by linear deconvolution. Whilst this works well with strains of the same species, interspecies differentiation based on fluorescence intensity of a single fluorescent protein would require laborious tuning of expression.

The aforementioned limitations led us to develop a remarkably simple differential fluorescent marking (DFM) method using three fluorescent proteins (mTagBFP, sYFP2 and mCherry) with distinct excitation and emission spectra, allowing simultaneous detection by flow cytometry or fluorescence microscopy. Using the DFM strategy, we generate and distinguish six fluorescence patterns, i.e. three single fluorescent proteins and three combinations of two. Plasmid-based protein expression can lead to issues such as gene dosage-dependent toxicity, as well as plasmid stability and host-range. Therefore, we adapted a mini-Tn 7 delivery system [ 28 ] to generate the plasmid Tn7 suicidal low COPY for universal transfer (pTn7-SCOUT) family, enabling integration of transgenes downstream of the highly conserved chromosomal glmS gene in bacteria [ 29 ]. This approach is compatible with our modular and hierarchical cloning system, BEVA [ 30 ]. We tested DFM in Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) and applied it to a six-member synthetic community (OxCom6), consisting of Alpha-, Beta- and Gammaproteobacteria. Using flow cytometry, we both differentiated and quantified the assembly of individual members of OxCom6 in nutrient-rich media and during colonisation of pea and barley roots. Our results demonstrate that DFM is an outstanding resource for tracking and distinguishing bacterial communities both in vitro, but more importantly, in diverse and complex environmental settings.

Material and methods

Primer and plasmids.

Primer and plasmids used in this study are shown in Table S 1 and Table S 2 , respectively. All pTn7-SCOUT plasmids are available in Addgene, see Table S 3 for codes.

Bacterial media and growth conditions

Bacterial strains used in this work are listed in Table S 4 . Escherichia coli strains were grown in LB [ 31 ] at 37 °C, supplemented with antibiotics at the following concentrations: ampicillin (Ap) 100 µg·mL −1 , gentamicin (Gm) 10 µg·mL −1 , kanamycin (Km) 20 µg·mL −1 , tetracycline (Tc) 10 µg·mL −1 and, spectinomycin (Sp) 50 µg·mL −1 . The remaining strains were grown in rich Tryptone Yeast (TY) media [ 32 ] supplemented with 20 mM succinate at 28 °C, unless specified otherwise. The following antibiotic concentrations were used: Rhizobium leguminosarum bv. viciae (Rlv3841) Gm 20 µg·mL −1 , neomycin 40 µg·mL −1 , Tc 5, Sp 100 µg mL −1 ; Ochrobactrum pituitosum AA2 and Pseudomonas fluorescens SBW25 Gm 20 µg·mL −1 ; Enterobacter cloacae AA4 Km 20 µg·mL −1 . Achromobacter xylosoxidans AT1 Km 100 µg·mL −1 and Azoarcus olearius DQS-4 Sp 200 µg·mL −1 . For the assessment of Rlv3841 labelled with DFM (Rlv3841 DFM ), mean generation time (MGT) strains were grown in UMS [ 33 ] supplemented with 10 mM glucose and 10 mM NH 4 Cl.

Plasmids were transformed into chemically competent E . coli strains DH5 \(\mathrm{\alpha }\) , Transformax™ EC100D™ pir + (Lucigen) and Transformax™ EC100D™ pir-116 (Lucigen). Except for the pTn7-SCOUT plasmids which were introduced into recipient bacteria by triparental conjugation with E . coli DH5 \(\mathrm{\alpha }\) as plasmid donor and E . coli HB101 with the helper plasmid pRK2013 [ 34 ]. The pTn7-SCOUT plasmids were conjugated by tetraparental conjugation using E . coli Transformax™ EC100D™ pir + as plasmid donor, E . coli S17-1 containing pTNS3 as transposase and E . coli pRK2013 as helper. Nitrofurantoin 10 µg·mL −1 was used to counter select against E . coli strains.

Construction of pTn7-SCOUT plasmids

The pUC18R6KT-mini-Tn 7 T-Km [ 28 ] was obtained from Addgene (catalogue no. 64969) and used as a scaffold to generate the Golden Gate level 1 master plasmid pTn7-SCOUT10. BsaI and Esp3I restrictions sites (RS) were removed, and two cloning sites added: a Golden Gate level 1 cloning site and an Esp3I cloning site to allow addition of antibiotic markers (Fig.  1 ). Five different fragments were generated by PCR and assembled by Golden Gate using BpiI. The first fragment was amplified using oxp3349-oxp3350 from the pUC18R6KT-mini-Tn 7 T-Km multicloning site (MCS) to the BsaI RS located in the ampicillin resistance marker (Ap R ), changing a nucleotide in a serine codon (748A > G). The second fragment was amplified with oxp3351-oxp3352 from the BsaI RS located in Ap R to two Esp3I RS located in the plasmid backbone between the Ap R and R-Tn 7 . The third fragment was amplified with oxp3353-oxp3354 from the Esp3I RS in the backbone plasmid to a region between the flippase recognition site (FRT) site and 3′-end of the Km R . The fourth fragment was amplified with oxp3355-oxp3356 from the region between 5′-end of Km R and FRT site to the mini-Tn 7 MCS. The fifth fragment was amplified with oxp2980-oxp2981 from pOGG093 plasmid [ 30 ], which amplifies the Golden Gate level 1 cloning site containing the P lac :: lacZ \(\mathrm{\alpha }\) -T0 region. Fragments were amplified with DNA polymerase Q5 (NEB), cleaned (GeneJet PCR purification kit, Thermo Fisher), assembled by Golden Gate with BpiI as described by Geddes et al. [ 30 ], cloned in Transformax™ EC100D™ pir-116 (Lucigen), miniprepped, and Sanger sequenced. pTn7-SCOUT10 has BsaI RS compatible with Golden Gate level 1 assembly and lacZ \(\mathrm{\alpha }\) as cloning marker, resulting in blue/white colony colour selection when plated on media supplemented with X-gal 50 µg·mL −1 .

To generate the Golden Gate level 2 master plasmid pTn7-SCOUT20, a new selection marker was constructed. The chromogenic gene tsPurple expression cassette was amplified from pOPS1522 with oxp4051-oxp4052, cloned into pTn7-SCOUT10 by Golden Gate using BsaI, transformed in Transformax™ EC100D™ pir-116 (Lucigen), miniprepped and Sanger sequenced. pTn7-SCOUT20 has BpiI RS compatible with Golden Gate level 2 assembly and tsPurple as cloning marker, resulting in purple/white colony colour selection.

The antibiotic resistance cassettes within the mini-Tn 7 were cloned in pTn7-SCOUT10 and pTn7-SCOUT20 by a Golden Gate reaction using Esp3I. The pLVC-P2 modules of the gentamicin resistance marker (Gm R , pOGG009), tetracycline resistance marker (Tc R , pOGG042) and kanamycin resistance marker (Km R , pOGG008) were used [ 30 ]. The spectinomycin resistance marker (Sp R ) was amplified with oxp3357-oxp3358 from pUC18T-mini-Tn 7 T- aad9 [ 35 ] and cloned by Golden Gate reaction with Esp3I. A family of pTn7-SCOUT plasmids was generated: level 1 pTn7-SCOUT11 (Gm R ), pTn7-SCOUT12 (Km R ), pTn7-SCOUT13 (Tc R ), pTn7-SCOUT14 (Sp R ); and level 2 pTn7-SCOUT21 (Gm R ), pTn7-SCOUT22 (Km R ), pTn7-SCOUT23 (Tc R ), pTn7-SCOUT24 (Sp R ) (see Table  1 , Table S 2 ).

Development of compatible flippase plasmids

The antibiotic marker within the mini-Tn 7 is flanked by FRT sites, allowing its excision from the chromosome by yeast recombinase flippase (Flp) following mini-Tn 7 insertion [ 28 ]. The pFLP2 plasmid [ 36 ] with Amp R was obtained from Herbert P. Schweizer. The sacB - flp - cI genes were amplified with oxp3417-oxp3418, purified and assembly by Golden Gate with BsaI into the destination vectors pOGG024 (Gm R ), pOGG023 (Km R ) and pOGG277 (Tc R ). Three new pFLP2 plasmids were generated pFlp-Km (pOPS1466; flp-cl-sacB-pL1V-Lv1-neo-pBBR1-ELT3), pFlp-Gm (pOPS1467; flp-cl-sacB-pL1V-Lv1-gent-pBBR1-ELT3) and pFlp-Tc (pOPS1468; flp-cl-sacB-pL1V-Lv1-TetAR-pBBR1-ELT3), (Table  1 , Table S 2 ).

Assembly of Golden Gate plasmids

Assembly of plasmids was done by Golden Gate as described by Geddes et al. [ 30 ]. Esp3I was used for the assembly of level 1 cloning plasmids (pL1V-Lv1), BsaI for the assembly of the expression cassette into level 1 plasmids and BpiI for assembly of level 1 modules into level 2 plasmids. Specific details about each plasmid construction are described in Supplementary Methods .

att amplification, sequencing and analysis

DNA extraction from each DFM strain was achieved by alkaline lysis (0.05 M NaOH, 0.25% SDS) [ 37 ], and used as a template to amplify by PCR the region from the 3′-end of glmS to Tn 7 -R. Primer PTn 7 R [ 28 ] on Tn 7 -R was used as a reverse primer and a specific forward primer was designed for each strain (see Table S 1 ). Amplification was carried out in a 50 µL PCR reaction containing 5–10 ng of isolated DNA and 2 U of Q5 DNA polymerase (NEB). PCR products were visualised on 1% agarose gels, purified (Monarch® PCR & DNA Cleanup kit, NEB) and Sanger sequenced (Eurofins). Alignment of sequences was performed using MUSCLE [ 38 ] implemented in MEGA X software [ 39 ]. The alignment consensus was calculated in Jalview [ 40 ].

Development and assessment of landing pad introduction into strains

To construct the Sinorhizobium meliloti CL150 containing the landing pad ( Sm LP), we followed the same procedure as described by Haskett et al. [ 41 ]. Firstly, a 282 bp fragment containing the Tn7 attB site using oxp3192 and oxp3193 primers was PCR-amplified from Rlv3841 chromosome (Table S 1 ). Secondly, 1 kb DNA fragments of two flanking regions of the harbour site [ 42 ] of S . meliloti CL150 were amplified using primer pairs oxp3190-oxp3191 and oxp3194-oxp3195. These three fragments were assembled by HiFi (NEB) with pK19mobSacB digested with SmaI, resulting in plasmid pOPS1246. Plasmid pOPS1246 was introduced into S . meliloti CL150, and sucrose selection [ 43 ] was used to stably integrate the Tn7 attB site of Rlv3841 (landing pad) into a harbour site in the chromosome by homologous recombination, resulting in Sm LP strain.

To test mini-Tn 7 integration specificity into the landing in Azorhizobium caulinodans ORS571 containing landing pad (AcLP) and Sm LP, two sets of primer pairs were used to PCR-amplify the 5′-end of the Rlv3841 attB -containing site fragment to Tn7-R (oxp2986 and oxp1390) and the Tn 7 -L to the 3′-end of the Rlv3841 attB -containing site fragment (PTn7L and oxp5053).

Counterselection for Flp-containing plasmids

Rlv3841 containing a mini-Tn7-Gm-sfGFP (Rlv3841 G-Gm ) was conjugated with pOPS1468 (flp-cl-sacB-pL1V-Lv1-TetAR-pBBR1-ELT3), and colonies selected on TY containing Tc. Transconjugants were pooled and plated on TY supplemented with sucrose (12%). Fifty colonies were patched on TY media with and without Tc. Strains unable to grow on Tc were PCR-tested with primers oxp3878 and oxp3879, which bind between T0 and T1 and on FRT sequence. Two bands of 272 bp and 1240 bp were present in Rlv3841 G-Gm , but only the 272 bp band in the Rlv3841 G , which confirms excision of the Gm cassette.

Microscopy images

Microscopy images were taken of cultures of Rlv3841 DFM strains growing on TY agar plates using a Leica M165FC. Detection of fluorescent proteins was as follows: mTagBFP with ET BFP filter (10,450,329, excitation: 405/20 nm, barrier: 460/40 nm) and exposure time 0.7 s; sYFP2 with ET YFP filter (10,447,410, excitation: 500/20 nm, barrier: 535/30 nm) and exposure time 1 s; and mCherry with ET mCherry filter (10,450,195, excitation: 560/40 nm, emission: 630/74 nm) and exposure time 0.2 s. Gain was set at 1 × , saturation at 1.0 and gamma at 1.01 for all images.

A mix containing equal amounts of cultures of Rlv3841 DFM and unlabelled were imaged with a Zeiss LSM 880 Airy Scan confocal microscope and analysed with ZEN Black v 3.6 software. To visualise fluorescent tags, mCherry was excited with a 561 nm wavelength laser and detected between 598 and 649 nm, sYFP2 was excited with a 488 nm wavelength laser and emission detected between 498 and 562 nm and mTag was excited with a 405 nm wavelength laser and emission detected between 440 and 490 nm. Two channels were used for the overlapping excitation and emission of sYFP2 and mTag. Channel one excited and detected mCherry and mTag, channel two excited and detected sYFP2.

• Flow cytometry

An Amnis® Cellstream® (Luminex Ltd.) flow cytometer with autosampler, equipped with 405 nm, 488 nm and 561 nm to excite TagBFP, sfGFP/sYFP2 and mCherry respectively, was used. Flow rates were set to low speed/high sensitivity (3.66 µL·min −1 ) and 5000–20,000 events defined by our gating parameters as Bacteria population were counted for each sample. Using Cellstream® Analysis 1.3.384 software, the Bacteria population was defined as the concentrated events area when plotting size (FSC) and granularity (SSC). The bacteria population was afterwards gated based on FSC (threshold > 0) and the aspect-ratio of SSC (threshold > 0.4) defining the Singlets population. Then Singlets events were gated based on their fluorescence emission, generating three colour populations: Red, Yellow and Blue for each fluorescent protein, mCherry, sYFP2 and TagBFP, respectively . The Red population are singlets events detected in the 561–611/31 channel above 550 FI units. The Yellow population are singlets events detected 488–528/46 channel above 500 FI units. The Blue population are singlets events detected in the 405–456/51 channel above 450 FI units (Fig. S 1 ). Afterwards, we created six Combined populations defined as presence absence of Red, Yellow and Blue colour populations. R population (exclusively Red), Y (exclusively Yellow), B (exclusively Blue), RY (exclusively Red and Yellow), RB (exclusively Red and Blue) and YB (exclusively Yellow and Blue). For instance, an event will be assigned to the R population if belongs to the Red population whilst not belonging to either the Yellow or Blue population. This implies that only signals for mCherry detection were observed. The number of events·mL −1 (emL) was recorded for each Combined population in each sample and transformed into events·g root −1 (egr). All flow cytometer data is available at http://flowrepository.org , experiment codes are shown in Table S 5 .

Growth curves to assess growth fitness

To calculate the MGT of each Rlv3841, strain labelled with DFM was grown in minimum media (UMS, [ 33 ]). A single colony of bacteria was streaked onto 10 mL UMS agar slopes supplemented with 10 mM glucose and 10 mM NH 4 Cl and incubated for 2 days. Cultures were resuspended in 4 mL of UMS supplemented with 10 mM glucose and 10 mM NH 4 Cl and washed three times. The OD 600nm was measured and 400 µL of 10 7 cells·mL −1 were inoculated into 24-well plates (Vision Plate™, 4titude) and incubated in a plate reader (FLUOstar Omega, BMG Labtech) for 72 h, 700 rpm, 28 °C. MGT was calculated as the number of h it takes the population to double whilst in exponential growth phase [ 44 ].

Inoculum preparation for pea root colonisation

A single colony of bacteria was streaked in 10 mL of TY supplemented with 20 mM succinate agar slopes in 30 mL universal tubes. For E . cloacae AA4, O . pituitosum AA2 and P . fluorescens SBW25 cultures were incubated overnight. A . xylosoxidans AT1 cultures were incubated for 1 day and A . olearius DQS-4 and Rlv3841 for 2 days. Once grown, cultures were resuspended in 4 mL of sterile 0.9% NaCl. OD 600 nm was measured and cultures were set at 10 9 cells·mL −1 . For competition and community experiments, cultures were mixed in equal ratios at 10 9 cells·mL −1 . Inocula were diluted to 10 5 cells·mL −1 and 1 mL was added to each plant.

Root colonisation experiment

Pea seeds were sterilised in ethanol 70% for 1 min, followed by 5 min in 3% NaClO. Barley seeds were sterilised in ethanol 70% for 1 min, followed by 5 min in 7% NaClO plus 0.1% Tween20 (Sigma-Aldrich). Seeds were washed with sterile distilled water. Pea seeds were pregerminated on agar-water 0.8% for 3 days at 23 °C in the dark, and after 3 days were transferred into sterilised boiling tubes containing fine vermiculite and 25 mL of root nutrient solution [ 45 ]. Sterilised barley seeds were transfer into boiling tubes containing fine vermiculite and 25 mL of root nutrient solution [ 45 ]. At 7 days after sterilisation, each seed was inoculated with a total of 10 5 cells. At 7 days post-inoculation (dpi) (1 to 14 dpi for assembly dynamics experiment), plants were harvested by inverting and shaking the tubes. Roots were dipped in sterilised water to remove loosely attached vermiculite, separated from seed, and shoot by cutting the root below the seed, weighed, and transferred to 50-mL Falcon tubes. Then, 25 mL harvest solution (0.9% NaCl, 0.02% Silwet L-77) was added and vortexed at maximum speed for 1 min. Further, 1 mL was passed through 40 µm filters (FLOWMI™ cell strainers) and 100 µL of each sample was transferred to 96-well u-bottom plates for single cell quantification using Amnis® Cellstream® (Luminex Ltd.) flow cytometer.

Quantification of background from plant roots

Uninoculated pea and barley plants were grown for 14 days, and samples were treated as described above. For each DFM population, emL was recorded and converted into egr. The values obtained were defined as root background and subtracted from total egr obtained from samples with bacterial inoculation (Table S 6 ).

Statistical analysis

Statistical analyses were performed on Prism 10 v10.02.

Nitrogenase activity

Nitrogenase activity of A . olearius DQS-4 and A. olearius DQS-4 labelled with sYFP2 (AoDQS-4 Y ) on barley plants was assessed as described by Haskett et al. [ 41 ].

Development of pTn7-SCOUT plasmids

Genomic integration of fluorescent markers is crucial for gene stability when studying bacteria in complex environments, due to the absence of plasmid-associated antibiotic selection [ 46 ]. However, fluorescent protein expression must be tuned to ensure sufficient levels of protein required for detection by microscopy and flow cytometry, whilst also avoiding toxicity due to overexpression. To overcome this challenge, we generated the pTn7-SCOUT (plasmid Tn7 Suicidal low COpy for Universal Transfer) as a family of mini-Tn 7 delivery plasmids that are compatible with BEVA modular Golden Gate cloning, and which only replicate in strains containing the pir genes [ 30 , 47 ]. The pTn7-SCOUT plasmid family facilitates the chromosomal integration of multiple expression cassettes in a diverse group of Proteobacteria. This can be applied, as shown in this work, for tracking bacterial community through the quantification of fluorescent protein.

To develop the master pTn7-SCOUT10 (Fig.  1 ), we used the pUC18R6K-mini-Tn 7 T-Km developed by Choi et al. [ 28 ] as a scaffold. First, BsaI and Esp3I RS present in the pUC18R6K-mini-Tn 7 T-Km plasmid were mutated since BsaI and Esp3I sites are used for level 1 and antibiotic marker cloning, respectively. Secondly, the Km R located in the mini-Tn 7 between the FRT sites was replaced with an Esp3I cloning site to allow for addition of different selection markers. Lastly, the MCS located in the mini-Tn 7 was substituted with a level 1 Golden Gate cloning site ( lacZ \(\mathrm{\alpha }\) ) for blue to white selection, which facilitates the assembly of one expression cassette by using BsaI. To enable the assembly of multiple expression cassettes, we generated the level 2 master plasmid pTn7-SCOUT20 by replacing the pTn7-SCOUT10 cloning site with a level 2 ( tsPurple ) for purple to white selection. Finally, we independently cloned the antibiotic markers, gentamicin (Gm R ), kanamycin (Km R ), tetracycline (Tc R ) and spectinomycin (Sp R ) by Golden Gate reaction into the Esp3I cloning site, generating the pTn7-SCOUT family (Table  1 ).

The pTn7-SCOUT plasmid family. Engineering the plasmid pUC18R6K-miniTn 7 T-Km for BEVA plasmid assembly compatibility. The plasmid was altered by removing three restriction sites (RS) and introducing two new cloning site—a designated antibiotic site and a Level 1/Level 2 site. The schematic representation includes key elements: a yellow line with a dot above indicating the Esp3I RS and blue line with a dot above indicating BsaI RS. A black inverted triangle represents the original plasmid’s multi-cloning site. Inverted blue and purple triangles depict new BsaI and BpiI sites, respectively, facilitating Golden Gate level 1 and level 2 plasmid construction. An inverted yellow triangle designates the Esp3I RS for cloning antibiotic resistance markers, located between the FRT sites (flippase recognition site, green circle). pL0M corresponds to Level 0 modules used to assemble expression cassettes in pTn7-SCOUT10. The promoter module (pL0M-P) is represented as an arrow, the ribosome binding site module (RBS, pL0M-U) as a semicircle, the gene module (pL0M-SC) as a horizontal black arrow and the terminator module (pL0M-T) represented as a “T”. pL1M corresponds to level 1 modules, representing assembled expression cassettes (promoter-RBS-gene-terminator), for constructing multiple expression cassettes in pTn7-SCOUT20. The right (Tn 7 -R) and left (Tn 7 -L) sites of the mini-Tn 7 transposon are depicted in pink and blue, respectively. The cloning marker for pTn7-SCOUT10 cloning ( lacZ \(\mathrm{\alpha }\) ) and for pTn7-SCOUT20 cloning ( tsPurple ) are indicated in blue and purple, respectively

The existence of a FRT site on either side of the antibiotic expression cassette on mini-Tn 7 means that, following integration, the antibiotic marker can be removed using the Flp. To facilitate this, we also developed new antibiotic versions of the pFLP2 plasmid ( flp , cI , sacB Ap R [ 36 ]) (Table  1 ) to ensure compatibility with the strains used in this study. The Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) containing the mini-Tn 7 -Gm-sfGFP (Rlv3841 G−Gm ) was conjugated with pOPS1468 ( flp-Ic-sacB -Tc-pBBR) to excise the Gm R from the integrated mini-Tn 7 . After sucrose selection, 100% of the strains were sensitive to Gm and the lack of a Gm R was confirmed by PCR.

Analysis of mini-Tn7 integration delivered by pTn7-SCOUT

In the model bacteria Escherichia coli , integration of the Tn 7 transposon occurs downstream of the glmS gene [ 48 ]. Different strains of Alpha-, Beta- and Gammaproteobacteria were tested for mini-Tn 7 integration delivered by pTn7-SCOUT and its integration site was assessed. The region from the 3′ end of glmS gene to the upstream end of the mini-Tn 7 (Tn 7 -R) was PCR amplified and sequenced (see Table S 1 for primers). Nucleotide alignment of the Tn 7 integration site for these strains revealed that, as previously observed in E . coli K12 and Pseudomonas aeruginosa PAO1 [ 28 , 48 ], Tn 7 integration occurs 25 bp from the glmS stop codon (Fig.  2 ). However, in P . protegens Pf-5 and Achromobacter xylosoxidans AT1, integration occurs 24 bp downstream of glmS , and in Azoarcus olearius DQS-4 and Enterobacter cloacae AA4 at 26 bp. Whilst 90% of the time the Tn 7 transposon integrates 25 bp downstream glmS in E . coli K12, it has been shown to integrate at a lower frequency, at either 24 bp or 26 bp downstream [ 29 , 48 ]. Therefore, the different integration locations ( attB ) identified among the strains tested could be related to the nature of Tn 7 integration itself rather than a strain-specific effect. Upon Tn 7 integration there is a duplication of 5 bp immediately upstream to attB site [ 29 ]. Our results show that there is no conservation in this 5 bp sequence, suggesting that Tn 7 does not require a specific recognition sequence for integration, but rather integrates at a specific distance from the glmS gene (Fig.  2 ).

Alignment of the att Tn 7 - attB region. Nucleotide alignment of the 3′-region of the glmS gene— attB site across various tested Proteobacteria. The highlighted area in the alignment shows the differences in the distance from the stop codon of the glmS gene to the Tn 7 integration site ( attB ). Coordinate 0 corresponds to the central nucleotide of the 5 bp sequence that duplicates after Tn 7 integration (green rectangle). The 3′-end of the glmS gene is denoted by the blue arrow, whilst the att Tn 7 sequence is represented by the yellow rectangle. The pink rectangle signifies Tn 7 -R from the mini-Tn 7 transposon

Whilst we have demonstrated that Tn 7 integration occurs 25 ± 1 bp from the glmS stop codon in diverse species, we found that some bacteria such Azorhizobium caulinodans ORS571 and Sinorhizobium meliloti CL150 encode a gene in this region that appear to be lethally disrupted by mini-Tn 7 insertion. We have previously overcome this issue by introducing a Tn 7 landing pad derived from the Rlv3841 Tn 7 attB site into a neutral region of the A . caulinodans ORS571 ( Ac LP) chromosome by double homologous recombination. This landing pad provides an alternative, non-lethal site which permits integration by Tn 7 [ 41 ]. Here, we use the same strategy to integrate the landing pad into S . meliloti CL150 chromosome at the same neutral site previously used to harbour a recombinase attB [ 42 ], creating strain Sm LP. We tested the specificity of integration into these sites for Ac LP and Sm LP with three independent conjugation experiments and were able to isolate mini-Tn 7 exconjugants of each strain harbouring the landing pad, but not for their corresponding wild-type strains, indicating the landing pads were being utilised for integration. Ten of each Ac LP and Sm LP colonies putatively harbouring mini-Tn 7 from each of the three conjugation experiments were screened by PCR using bridging across the left Tn 7 attB site and chromosomal landing pad, confirming integration at the desired site in at least 90% for Ac LP (9/10, 10/10 and 9/10 colonies produced bands of the correct size) and 100% for Sm LP (10/10, 10/10, and 10/10 colonies produced bands of the correct size). One amplicon generated from each independent experiment was sequenced and successfully aligned to the predicted in silico sequences to further confirm this conclusion. Clearly this landing pad strategy is robust and can be applied to most strains recalcitrant to Tn 7 insertion at the native glmS position.

Expression of single and dual fluorescent markers permits differentiation of up to six bacteria

The use of single fluorescent proteins to track bacteria is widely used in plant–microbe interaction studies [ 49 , 50 ], but is restricted to availability of fluorophores and an ability to detect them. Our differential fluorescent marking (DFM) strategy couples use of three distinguishable fluorescent proteins, mCherry, sYFP2 and TagBFP (Fig. S 2 ) and mini-Tn 7 stable chromosomal specific integration delivered by pTn7-SCOUT plasmids. DFM uses the aforementioned fluorescent proteins in single and double combinations to generate six unique patterns. The three single constructs are formed by cloning either, mCherry (R), sYFP2 (Y) and TagBFP (B), whilst the three doubles makers were constructed by cloning the fluorescent proteins in pairs, mCherry and sYFP2 (RY), mCherry and TagBFP (RB) and sYFP2 and TagBFP (YB).

To test our DFM strategy Rlv3841 was labelled with each DFM construction (Rlv3841 R , Rlv3841 Y , Rlv3841 B , Rlv3841 RY , Rlv3841 RB and Rlv3841 YB ) (Table  2 ), spotted on agar and after two days the fluorescence of each spot was detected using a fluorescent stereomicroscope, confirming the differentiation among the six DFM patterns which are not present in the unlabelled strain (Rlv3841 U ) (Fig.  3 A). We expanded our investigation by combining Rlv3841 U and each Rlv3841 DFM in equal proportions. The resulting mixture was visualised using a Zeiss LSM 880 Airy Scan confocal microscope, confirming differentiation at the single-cell level among the six distinct DFM patters and unlabelled strain (Fig. S 3 ).

Differential Fluorescent Marking (DFM) of bacteria. Illustration of Rhizobium leguminosarum bv. viciae 3841 strains, unlabelled (Rlv3841 U ) and with distinct DFM combinations: Rlv3841 R (mCherry), Rlv3841 Y (sYFP2), Rlv3841 B (mTagBFP), Rlv3841 RY (mCherry and sYFP2), Rlv3841 RB (mCherry and mTagBFP) and Rlv3841. YB (sYFP2 and mTagBFP). A Stereomicroscope images of Rlv3841 spots growing on rich media. The first column shows bright field images, the second 560/40–630/74 channel capturing mCherry expression, the third column presents the 500/20–535/30 channel capturing sYFP2 expression and the fourth column demonstrates the 405/20–460/40 channel capturing mTagBFP expression. Scale bars indicate 500 µm. B Flow cytometry graphs, with the y-axis showing time in seconds and x-axis displaying fluorescence intensity (FI) units on a logarithmic scale. In the first column, events are plotted in 561–611/31 channel for detecting mCherry expression (above 550 Fi units), in the second column, events are shown in the 488–528/46 channel for detecting sYFP2 expression (above 500 Fi units), and in the third column, events are depicted in the 405–456/51 channel for detecting mTagBFP expression (above 450 FI units)

Subsequently, we ran these Rlv3841 DFM strains and Rlv3841 U independently through a flow cytometer and used Cellstream® Analysis software to distinguish the six strains based on the presence or absence of the three fluorescent proteins (Fig.  3 B). First, the bacteria population was defined as the concentrated area based on size (FSC) and granularity (SSC), followed by the definition of the Singlets population based on FSC and the aspect-ration of SSC (Fig. S 1 A and B). Our gating strategy is followed by the delineation of three different colour population for each fluorescent marker as follows; for mCherry expression, the Red population as events detected 561–611/31 channel above 550 FI units; for sYPF2 expression the Yellow population, events detected 488–528/46 channel above 500 FI units; and for mTagBFP expression the Blue population as the events detected in the 405–456/51 channel above 450 FI units (Fig. S 1 C). Afterwards, we assigned six Combined populations defined as presence or absence of the Colour populations Red, Yellow and Blue: R population (exclusively Red), Y (exclusively Yellow), B (exclusively Blue), RY (exclusively Red and Yellow), RB (exclusively Red and Blue) and YB (exclusively Yellow and Blue) (Fig. S 1 D). The graphs in Fig.  3 B show the detection by flow cytometry of each colour population (column) for each Rlv3841 DFM strain (rows), which confirms the six unique DFM patters observed with the stereomicroscopy (Fig.  3 A). Next, we calculated the accuracy of our flow cytometry gating strategy to assign each Rlv3841 DFM strain to its corresponding colour population, showing that more than 90% events were determined correctly, whereas Rlv3841 U showed less than 1.7% of Singlets events belonging to any of these colour population (Table  3 ). This 1.7% misassignment of events corresponds to events detected in the Blue colour population. The accuracy of our flow cytometry gating strategy for detecting each DFM pattern was assessed by calculating the percentage of each combined population (R, Y, B, RY, RB and YB) for each Rlv3841 DFM strain (Rlv3841 R , Rlv3841 Y , Rlv3841 B , Rlv3841 RY , Rlv3841 RB and Rlv3841 YB ). The results showed an accuracy of more than 95% in assigning the correct combined population to the corresponding DFM strain with almost complete accuracy for Rlv3841 B (Table  4 ). In this case, 99.9% of the events detected when running Rlv3841 B in the flow cytometer by itself were assigned as the corresponded B Combined population (Table  4 ). Next, we evaluated the precision of our gating strategy in discriminating each Rlv3841 DFM strain when present in a mixed sample, with an equal number of each strain. The number of events for each Combined population was calculated revealing that 1/6 of the total number of events were assigned to each Rlv3841 DFM version (Table  4 ).

To assess if the presence of any DFM combination had a growth effect in Rlv3841, the MGT on minimum media was calculated and compared to Rlv3841 U . No differences were observed for any of the Rlv3841 DFM strains, neither for each antibiotic version with a sfGFP expression cassette, nor for different colour combinations (Table  5 ). This is consistent with previous studies showing that the fluorescent protein has no effect on the fitness when integrated in single copy using mini-Tn 7 [ 28 ].

To validate the use of DFM combined with flow cytometry to assess bacterial colonisation on plant roots, we inoculated Rlv3841 R onto pea and quantified colonisation 7 dpi by colony counts and flow cytometry. The number of Rlv3841 R counted with flow cytometry was 6 · 10 5  ± 4 · 10 5 egr and by colony count 1.1 · 10 6  ± 8.6 · 10 5  CFU·g root −1 , showing no significant differences ( p value = 0.4375, Wilcoxon test), demonstrating that flow cytometry gives comparable numbers to CFU, as shown for Herbaspirillum colonising rice roots [ 51 ]. Subsequently, we tested the capacity of each Rlv3841 DFM strain to grow on pea roots in single inoculation and in competition with Rlv3841 U . No significant differences were observed confirming that DFM does not affect the competitive colonisation ability of the strain (Table  6 ). Finally, we examined the capacity to differentiate each Rlv3841 DFM strain when inoculated in equal amounts on pea roots. At 7 dpi, no significant differences were observed among the Rlv3841 DFM strains (Table  7 ).

These results confirm that DFM combined with flow cytometry can be used to simultaneously differentiate and quantify up to six bacterial strains from both liquid culture and plant samples with no deleterious effects on bacterial fitness.

Since one member of OxCom6 is capable of nitrogen fixation, we tested if the presence of mini-Tn 7 affects the capacity of A . olearius DQS-4 to fix nitrogen on barley roots. The nitrogenase activity of A . olearius DQS-4 wild-type strain was 208.1 ± 44.6 nmol ethylene·plant −1  h −1 , and in A. olearius DQS-4 integrated with mini-Tn 7 was 176.6 ± 24 nmol ethylene·plant −1  h −1 . t Test showed no significance differences between strains ( p value = 0.25).

Tracking bacteria in synthetic communities using differential fluorescent markers

To test the accuracy of DFM to discriminate, track and quantify individual members in a bacterial community, a model SynCom (OxCom6) was assembled with well-characterised root-colonising strains, all of which are amenable to genetic modification. These belong to Alphaproteobacteria ( Ochrobactrum pituitosum AA2, R. leguminosarum bv. viciae 3841), Betaproteobacteria ( A. xylosoxidans AT1, A. olearius DQS-4) and Gammaproteobacteria ( E . cloacae AA4 and P . fluorescens SBW25) [ 11 , 21 , 52 , 53 , 54 ]. Each member of the OxCom6 community was labelled with a specific DFM combination: O . pituitosum AA2 was labelled with mCherry (OpAA2 R ), R . leguminosarum bv. viciae 3841 mCherry and mTagBFP (Rlv3841 RB ), A . olearius DQS-4 sYFP2 (AoDQS-4 Y ), A . xylosoxidans AT1 sYFP2 and mTagBFP (AxAT1 YB ), E . cloacae AA4 mCherry and sYFP2 (EcAA4 RY ) and P . fluorescens SBW25 mTagBFP (PfSBW25 B ) (Table  2 , and Table S 4 for details on the strains used). The labelling of OxCom6 strain with each DFM pattern does not have any effect on fitness (Table S 7 ) or competitive colonisation (Table S 8 ). Similar to the observations for Rlv3841, when comparing CFU·mL −1 and events·mL −1 for each OxCom6 strains labelled with DFM, no differences were observed (Table S 9 ). Subsequently, we monitored the assembly of OxCom6 in nutrient-rich media over a span of 96 h and on pea and barley roots for a duration 14 days (Fig.  4 ).

Tracking bacterial synthetic communities using DFM. Absolute quantification of each OxCom6 member: A growing in nutrient-rich media over 96 h, B colonising pea roots over 14 days and C colonising barley roots over 14 days. Solid dots represent the average, the shaded regions depict plus/minus the standard error of the mean (± SEM), with solid lines connecting the dots. In the graphs, members of OxCom6 are colour-coded as follows: Ochrobactrum pituitosum AA2 R (red), Rhizobium leguminosarum bv. viciae 3841 RB (purple), Azoarcus olearius DQS-4 Y (yellow), Achromobacter xylosoxidans AT1 YB (green), Enterobacter cloacae AA4 RY (orange), Pseudomonas fluorescens SBW25 B (blue). egr: events·gram root −1 . emL: (events·mL − . 1 )

The results from the OxCom6 assembly in nutrient-rich media (Fig.  4 A) revealed that EcAA4 RY exhibited a robust and sustained growth, reaching a maximum count of 1.5·10 9 events·mL −1 (emL) within 24 h. In contrast, the other members of the OxCom6 reached a growth plateau at 61 h. OpAA2 R and PfSBW25 B attained peak counts of 2·10 8 and 1.7·10 8 emL respectively. Similarly, AxAT1 YB and Rlv3841 RB achieved comparable plateau levels, recording 8.4·10 6 and 8.8·10 6 emL correspondingly. Meanwhile, AoDQS-4 Y reached a maximum growth of 4.7·10 6 emL. Notably, among the strains, EcAA4 RY demonstrated the fastest growth rate, establishing itself as the most prolific member during the OxCom6 assembly in nutrient-rich media and therefore most abundant strain when OxCom6 assembled in rich media.

Subsequently, the assembly dynamics of OxCom6 was tracked on pea roots over 14 days (Fig.  4 B). At 1 dpi, EcAA4 RY emerged as the predominant coloniser, accounting for 10 6 egr. However, by 2 dpi, PfSBW25 B displayed higher counts than EcAA4 RY , recording figures of 3.7·10 6  ± 3.2·10 5 and 1.4·10 6  ± 7.3·10 5 egr respectively. This disparity became significant from 3 dpi with colonisation counts of 4.2·10 6  ± 2.4·10 6 egr for EcAA4 RY and 1.7·10 7  ± 7.9·10 6 egr for PfSBW25 B (paired t test p value = 0.001). Both strains achieved and sustained a plateau from 3 dpi onward, with counts circa 1.7–4.3·10 7 and 1.8–4.2·10 6 egr respectively. Starting at 10 dpi, a consistent rise was observed in the accounts of Rlv3841 RB and OpAA2 R . Rlv3841 RB exhibited an increase from 6 to 14 dpi, rising from 1.4·10 6 to 4.1·10 6 egr, aligning its values with those of EcAA4 RY . A similar pattern was evident for OpAA2 R , which displayed growth from 4.9·10 5 to 1.7·10 6 egr between 9 and 14 dpi. This growth correlated positively with Rlv3841 RB colonisation (Pearson r = 0.91, R 2  = 0.83, p value < 10 −4 ). Despite early events of colonisation, the Betaproteobacteria AoDQS-4 Y and AxAT1 YB were not consistently detected within the OxCom6 assembly on pea roots. At 2 dpi, AoDQS-4 Y achieved a peak colonisation of 1.4·10 6 egr. However, its counts swiftly decreased to 3.5·10 5 egr by 3 dpi, concurring with the increase of PfSWB25 B . This phenomenon finds support in a significant negative correlation between the two strains (Pearson r = -0.62, R 2  = 0.38, p value = 0.03) indicating a potential displacement of AoDQS-4 Y by PfSWB25 B . AxAT1 YB attained a maximum value of 1·10 6 egr root at 3 dpi, followed by a fluctuating pattern until 14 dpi, with counts ranging between values of 10 5 ·10 6 egr.

Finally, OxCom6 assembly dynamics were tracked on barley roots over 14 days (Fig.  4 C). EcAA4 RY emerged as the primary coloniser from 2 dpi onward, achieving a plateau of 2–3·10 7 egr by 5 dpi. The colonisation counts of PfSBW25 B at 1 dpi (1.8·10 6  ± 2.1·10 6 egr) did not significantly differ from those observed for EcAA4 RY (4.1·10 6  ± 4.4·10 6 egr), as indicated by the paired t test ( p value = 0.08). However, PfSBW25 B displayed a noteworthy decrease at 2 dpi (3.6·10 5  ± 8.1·10 4 egr), which differed significantly from the account at 1 dpi ( t test p value = 0.005). Subsequently, the count of PfSBW25 B ’s rebounded to 5–6·10 6 egr by 11 dpi when it reached plateau. Despite the early competitive events, a robust positive correlation exists between EcAA4 RY and PfSBW25 B (Pearson r = 0.81, R 2  = 0.66, p value = 0.0004). OpAA2 R was initially detected at 3 dpi and maintained a consistent count between 5·10 5 and 10 6 egr up to 14 dpi. Similarly, AxAT1 YB exhibited a steady colonisation on barley roots, ranging 2–5·10 5 egr. Rlv3841 RB was only detected at 11 and 13 dpi in one and two plants respectively, indicating that its colonisation on barley roots lacked stability in the presence of other OxCom6 members. Likewise, AoDQS-4 Y was detected during initial stages of colonisation (1–3 dpi) within the 5·10 5 to 10 6 egr range. Subsequently, it was detected at 9 and 13 dpi with roughly the same counts as before. The colonisation of AoDQS-4 Y at 3 dpi and 9 dpi was quantified in only one plant, and at 13 dpi in two plants. AoDQS-4 Y colonisation in barley roots appeared to exhibit stability during the initial colonisation events (1–3 dpi) but was subsequently outcompeted by other OxCom6 members.

Mini-Tn 7 is an excellent delivery system to use when working with a wide range of bacterial species in a non-selective environment since it is 100% stable for 100 generations in the absence of antibiotic selection [ 28 , 55 , 56 ]. Mini-Tn 7 is broad-range as demonstrated by successful delivery into multiple strains within Proteobacteria [ 57 , 58 ]. Moreover, mini-Tn 7 is highly efficient and integrates in single copy into bacterial chromosomes, site- and orientation-specifically at attB Tn 7 , located downstream of the 3′-end of the highly conserved glmS gene [ 28 ]. In contrast to plasmids, mini-Tn 7 is replicated within the chromosome, therefore it does not have a fitness cost due to copy number or replication mechanism, and it is compatible with any other cloning system [ 59 , 60 , 61 ].

Here, we developed the pTn7-SCOUT, a new family of mini-Tn 7 plasmids compatible with the Golden Gate modular cloning system BEVA [ 30 ], which allowed us to rapidly tune the expression of the different fluorescent markers used in the DFM strategy. The pTn7-SCOUT family uses the suicidal R6K as origin of replication, which only replicates in the presence of pir genes supplied in trans [ 47 ]. Moreover, in pir + E . coli strains the R6K copy number is less than 15, which reduces the toxic effect of highly-expressed cassettes [ 62 ]. We replaced the MCS for either a level 1 or level 2 compatible Golden Gate cloning site, to allow the addition of single or multiple expression cassettes respectively. These Golden Gate cloning sites have blue/purple ( lacZ \(\mathrm{\alpha }\)  / tsPurple ) to white markers to facilitate the identification of positive transformants. The presence of a Golden Gate cloning site enables the use of a vast diversity of compatible Golden Gate modules available to construct the desired fluorescent cassette [ 30 , 63 ]. Nevertheless, the pTn7-SCOUT family is not restricted to Golden Gate assembly, as the level 1 and level 2 plasmids can be digested with BsaI and BpiI respectively to become entry plasmids for classic cloning such as digestion-ligation or DNA fragment assembly methods like Gibson or HiFi (NEB). Moreover, the lacZ \(\mathrm{\alpha }\)  within the level 1 cloning site contains a polylinker for traditional cloning [ 30 ]. The pTn7-SCOUT plasmid family has an Esp3I site within the mini-Tn 7 to clone any selection marker such as antibiotic resistance genes. We successfully cloned Gm R , Tc R and Km R resistance markers using the BEVA modules [ 30 ]. However, as shown with Sp versions, any other selection marker can be cloned; by simply PCR-amplifying them with compatible overhangs, followed by cloning into pTn7-SCOUT digested with Esp3I. The level 2 Golden Gate and the antibiotic cassette cloning sites increase the modularity of the already available mini-Tn 7 delivery plasmids [ 28 , 64 ]. We expanded the pTn7-SCOUT family with new antibiotic versions of Flippase-containing plasmids to enable excision of the antibiotic resistance cassette, which are compatible with the strains used in the study, since only ampicillin (Ap R ) and Tc R version were available [ 28 , 36 ].

Characterization of the attB site has enabled us to predict the success of mini-Tn 7 integration if the host genome sequence is known. In some strains, mini-Tn 7 integration would disrupt a gene; however, we have overcome this issue by integrating a new landing pad [ 41 ], providing a new attB site where mini-Tn 7 is able to integrate (with an efficiency over 90% in the strains tested). This tool removes a bottleneck in mini-Tn 7 use.

The DMF tool combines single chromosomal integration with multi-fluorescence labelling to discriminate up to six different strains in a bacterial community when growing in nutrient-rich media or colonising plant roots (Fig.  4 ). Our flow cytometry protocol is able to discriminate with more than 95% efficiency each DFM-labelled strain (Fig.  3 , Fig. S 3 , Table  3 , Table  4 , Table  7 ), which is as efficient as the tool developed by Whitaker et al. [ 27 ] where they combined GFP and RFP with different RBS strengths to differentiate six Bacteroides strains with a 6% error. The main source of misassignment detected was with the Blue Colour population (Table  3 , Fig.  3 B). This can be partially related to autofluorescence of aromatic amino acids, thiamine and riboflavin, detected in the 405–456/61 channel [ 65 , 66 ]. However, this blue autofluorescence represents less 2% of the events in Rlv3841 U strain (Table  3 , Fig.  3 B). In addition, plant roots can show blue autofluorescence, mainly related to lignin and suberin compounds of the cell wall [ 67 ], as shown in the non-inoculated pea roots (Table S 6 ). To overcome this issue, we quantified the background on non-inoculated pea roots for each combined population and subtracted this from the colonisation values.

High expression of fluorescent proteins can affect growth, decrease fitness, and generate toxicity due to protein aggregation and solubilisation [ 68 , 69 ]. The fluorescent proteins chosen for DFM (mCherry, sYFP2 and mTagBFP) are engineered monomers with increased brightness, protein folding, extinction coefficient and maturation, which reduce deleterious effects compared to their predecessors [ 70 , 71 , 72 ]. Moreover, DFM is assembled in low-copy number plasmids and then integrated as a single copy into the bacterial chromosome, which reduces overall expression levels of the fluorescent proteins, and thereby any related toxicity. Furthermore, our results showed no deleterious effect of any DFM combinations during growth in liquid culture or colonisation of plants (Table  5 , Table  6 , Table S 7 , Table S 8 ).

We successfully applied DFM to the OxCom6, a model SynCom of Proteobacteria root colonisers. Assembly of OxCom6 showed differences between nutrient-rich media, pea and barley roots (Fig.  4 ), indicating that the findings in planta can be associated with rhizosphere adaptation, as has been proven for plant microbiome [ 4 , 73 , 74 ].

The most marked difference was the one observed between OxCom6 assembly on pea and barley roots, where each of them have a distinct dominant strain, PfSBW25 B and EcAA4 RY , respectively, and their colonisation was determined in the early stages of root occupancy (1–3 dpi) (Fig.  4 B and C). P. fluorescens SWB25, a well-known root coloniser isolated from sugar beet [ 54 ], is recognised to enhance plant growth through a combination of factors such as competing with other microorganisms, producing antimicrobial compounds and stimulating systemic resistance [ 75 ]. P . fluorescens SWB25 has the capability to generate furanomycin, which displays a potent inhibitory effect on the growth of Pseudomonas , Bacillus , Erwinia and Dickeya strains as observed in agar diffusion assay [ 76 ]. On the other hand, E . cloacae AA4 is part of a 7-member SynCom isolated from maize roots, and the absence of E . cloacae AA4 results in the collapse of the root colonisation by the SynCom. Whilst E . cloacae AA4 exhibits antifungal and nematocidal properties, it has not been shown to have any antibacterial activity [ 21 , 77 ]. The intrinsic antibiotic capabilities of both OxCom6 Gammaproteobacteria alone do not explain the distinctive OxCom6 assembly phenotype. This suggests that there may be a rhizosphere adaptation to pea in the case of PfSBW25 B and to barley in the case of EcAA4 RY , likely influenced by root exudates. The pea and barley root exudate profile have not been extensively characterised to date, but there are some studies that have provided partial descriptions of these exudates’ components. In the case of barley, a study by Calvo et al. [ 78 ] reported the presence of sugars such as sucrose, fructose and glucose at concentrations between 1 and 1.5 mg g root dry weight −1 at 71 days. On the other hand, the use of metabolite reporters on pea roots showed that at 4 dpi, the greater proportion of metabolites detected was sugars (xylose, fructose and myo-inositol), di-carboxylic acids (malonate and tartrate) and hesperetin; whereas, other sugars like sucrose were barely detected at this time point [ 79 ]. This suggests the different nature of pea and barley rhizosphere secretions, and therefore a different metabolic profile which the OxCom6 members can catabolise during the early stages of establishment, may be crucial in colonisation. In pea roots Rlv3841 RB can achieve similar levels of colonisation as EcAA4 RY , with both reaching counts of 4.1·10 6 egr at 14 dpi (Fig.  4 B). Rlv3841 is a root symbiont of pea plants known for its unique affinity for colonising pea roots and inducing formation of nitrogen-fixing nodules [ 52 ]. Therefore, colonisation of Rlv3841 RB may be associated with specific niches, such as infection threads and nodules, as evident from the presence of prominent nodules formed by Rlv3841 RB at 13 and 14 dpi, as shown in Fig. S 4 . Rlv3841 RB root colonisation numbers on pea in OxCom6 are lower compared to those in single culture at 7dpi, 1.7·10 6  ± 1.4·10 6 and 4.7·10 6  ± 1.5·10 6 egr respectively ( t test p value = 0.006) (Fig.  4 B and Table  6 ). This suggests the potential use of OxCom6 as a controlled environment to investigate competitive colonisation of legume endosymbionts, which is critical for the competitiveness of inoculants in the field [ 80 ]. On the other hand, Rlv3841 RB was not detected in the barley rhizosphere (Fig.  4 C), which reveals adaptation of this pea endosymbiont to its host rhizosphere [ 52 ]. Although A. olearius DQS-4 is capable of fixing nitrogen under free-living conditions and on barley roots, as well as promoting plant growth in rice and Setaria viridis [ 41 , 81 ], it was not able to effectively colonise pea and barley roots in the presence of other members of OxCom6. Whilst it can colonise the root intercellular spaces of rice and S . viridis , it is not a strong competitor for pea and barley root colonisation, perhaps because it was isolated from oil-contaminated soil [ 53 ]. O . pituitosum AA2, like E . cloacae AA4, is one of the seven members of the maize SynCom and a significant contributor to that community at 14 dpi [ 21 ]. OpAA2 R has a strong positive correlation with the colonisation/root infection of Rlv3841 RB on pea roots. This could be partially facilitated by Nod factor produced by rhizobia, as legume mutants with impaired Nod factor perception have been shown to have a less abundant and altered microbiome [ 82 , 83 ]. However, OpAA2 R showed similar root colonisation counts between pea and barley since a positive correlation was observed between both plants (Pearson r = 0.62, R 2  = 0.39, p value = 0.03), which suggests a good adaptation to both plant rhizospheres, and only this strain out of the six showed any significant correlation between both plants. Therefore, the correlation with Rlv3841 RB on pea cannot be attributed to Rlv3841 host specificity. A . xylosoxidans AT1 was isolated from the rhizosphere of Medicago truncatula and it promotes growth of A . thaliana , M . truncatula and Brachypodium distachyon [ 11 ]. The fluctuating colonisation of AxAT1 YB on pea roots, as shown in Fig.  4 B, may be influenced by stochastic availability of specific resources for bacteria in the pea rhizosphere, which can result in oscillation in bacterial growth [ 84 , 85 ]. However, this is not the case on barley roots, where AxAT1 YB colonises in a steadier way, suggesting a better adaptation to this rhizosphere. A . xylosoxidans AT1 was isolated from M . truncatula by Tkacz et al. [ 11 ]; however, OTUs of Achromobacter spp. were among the most abundant in the three rhizospheres studied: M . truncatula , A . thaliana and B . dystachium . This suggests that the isolation from M . truncatula may be somewhat stochastic and does not necessarily imply that A. xylosoxidans AT1 is better adapted to this plant.

These results suggest that the distinct nature of the rhizosphere resources in pea and barley can result in different metabolic profiles encountered by OxCom6 members during colonisation [ 78 , 79 ]. The availability of these resources in both plants would be just one aspect of the equation. Similarly, the catabolic capabilities of OxCom6 members in these rhizospheres could play a significant role in determining the assembly profile in each plant root based on their preference for catabolic sources [ 17 , 86 , 87 ]. However, catabolic capability alone may not be the sole determinant of this phenotype; competitive exclusion also could play a crucial role [ 88 ]. The speed at which bacteria utilise these resources could define their adaptation, and consequently, their abundance. Factors like chemotaxis and motility are pivotal in these processes [ 89 , 90 ], since once a bacterium can detect a resource and effectively access and utilise it, it would gain an advantage over others, and this would lead to more rapid increase in numbers.

The combination of DFM with flow cytometry allowed us to perform absolute quantification of bacterial root colonisation quickly and easily. This is crucial when assessing root colonisation dynamics, as shown in Fig.  4 , since relying solely on relative abundance can lead to inaccurate comparisons between samples (Fig. S 5 ) [ 14 ]. Whilst DFM was used here for absolute quantification of bacterial root colonisation, it can also be applied to other bacterial communities in any environment. Whilst in this study we limited the SynCom to six members to correspond to the available marker combinations, marked strains can of course be combined into larger communities. Furthermore, by varying the marked strains, large assemblies can be investigated. Techniques using DFM illustrated here provide the means for rapid assessment of microbial communities in diverse plant, animal, and environmental settings.

Availability of data and materials

All relevant data are within the manuscript and its supporting information files. All pTn7-SCOUT plasmids are available in Addgene (See Table S 3 ).

Abbreviations

Azorhizobium caulinodans ORS571 containing the landing pad

Azoarcus olearius DQS-4 labelled with sYFP2

Ampicillin resistance marker

Achromobacter xylosoxidans AT1 labelled with sYFP2 and mTagBFP

Combined population with exclusively Blue colour population

Differential fluorescent marking

Days post inoculation

Enterobacter cloacae AA4 labelled with mCherry and sYFP2

Events·g root −1

Events·mL −1

Fluorescence intensity

Fluorescently in situ hybridisation

Flippase recognition site

Gentamicin resistance marker

Kanamycin resistance marker

Mean generation time

Multicloning site

Ochrobactrum pituitosum AA2 labelled with mCherry

Pseudomonas fluorescens SBW25 labelled with mTagBFP

Plasmid Tn7 Suicidal low COpy for Universal Transfer

Combined population with exclusively Red colour population

Combined population with exclusively Red and Blue colour population

Ribosome biding site

Root growth inhibition

Rhizobium leguminosarum Bv. viciae 3841

Rlv3841 labelled with mTagBFP

Rlv3841 labelled with mCherry

Rlv3841 labelled with mCherry and mTagBFP

Rlv3841 labelled with mCherry and sYFP2

Rlv3841 not labelled

Rlv3841 labelled with sYFP2

Rlv3841 labelled with sYFP2 and mTagBFP

Restriction site

Combined population with exclusively Red and Yellow colour population

Standard error of the mean

Sinorhizobium meliloti CL150 containing the landing pad

Spectinomycin

Spectinomycin resistance marker

• Synthetic community

Combined population with exclusively Yellow colour population

Combined population with exclusively Yellow and Blue colour population

Tetracycline

Tetracycline resistance marker

Tryptone yeast

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Acknowledgements

We would like to thank Dr Euan James from The James Hutton Institute, UK for sharing Azoarcus olearius DQS-4. We would like to thank Prof. Roberto Kolter from Harvard University, USA for sharing Ochrobactrum pituitosum AA2, Enterobacter cloacae AA4, Pseudomonas putida AA7 and Herbaspirillum frisingense AA6. We would like to thank to Prof. Jose Manuel Palacios from Universidad Politécnica de Madrid, Spain, for sharing Rhizobium leguminosarum bv. viciae UPM791. We would like to thank Dr Silke Ruppel from Leibniz-Institute for Agricultural Landscape Research, Germany for sharing Kosakonia radicincitans DSM16656 T . We would like to thank Prof. Hebert P. Schweizer from Northern Arizona University, USA for sharing the pUC18T-mini-Tn7 plasmids and to the Enabling Nutrient in Symbioses in Agriculture (ENSA) project for sharing the Golden Gate destination vectors. We would like to thank Dr Alison East for her in deep critical revision of this manuscript. We would like to thank all past and present lab members of Prof. Philip Poole for their valuable discussion about this project during meetings and coffee breaks. We would like to thank to ChatGPT for its help in the definition of pTn7-SCOUT acronym.

This work was supported by the Biological Science Research Council [grant numbers BB/N013387/1, BB/T001801/1, BB/T006722/1, BB/W006219/1] granted to PSP. TLH is the recipient of an 1851 Royal Commission for the Exhibition of 1851 Research Fellowship (RF-2019–100238) and Wolfson College, University of Oxford Junior Research Fellowship. JD is the recipient of a Radford Scholarship, Department of Plant Sciences in conjunction with Worcester college, TJU is the recipient of the BBSRC grant BB/T008784/1.

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Beatriz Jorrin, Timothy L. Haskett, Hayley E. Knights, Anna Martyn, Thomas J Underwood, Jessic Dolliver, Raphael Ledermann & Philip S. Poole

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All authors listed have made a substantial direct and intellectual contribution to the work and approved it for publication. BJ and PSP conceived the study and designed the work. BJ, TLH, HEK, AM, TJU, JD and RL performed the experiments. BJ analysed the data and prepared the manuscript. BJ, PSP, TLH, HEK, RL, JD, AM and TJU critically reviewed the manuscript

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Supplementary Information

Additional file 1: fig s1..

Flow cytometry gating strategy. Employing the CellStream® Analysis 1.3.384 software, the gating strategy was implemented to delineate the Colour and Combined populations. The initial step involved defining the Bacteria population by selecting the concentrated events area when plotting size (FSC – 456/51) against granularity (SSC – 773/56). Subsequently, the Bacteria population was gated based on FSC (threshold > 0) and the aspect-ratio of SSC (threshold > 0.4) establishing the Singlets population. Then Singlets population was further refined based on their fluorescence emission to depict the different Colour populations: Red, Yellow and Blue, corresponding to the fluorescent emission of mCherry, sYFP2 and mTagBFP, respectively. For mCherry, fluorescent emission was detected at 611/31, with a threshold above 550 FI units to define Red population. For sYFP2, emission was detected at 528/46, and the events above 500 FI units were designated as Yellow population. Emission for TagBFP was acquired at 457/51, and events exhibiting fluorescence above 450 FI units were categorised as the Blue population. Combining in one or two of the different Colour populations led to the definition of six distinct Combined populations: R (Red), Y (Yellow), B (Blue), RY (Red and Yellow), RB (Red and Blue) and YB (Yellow and Blue).

Additional file 2: Fig S2.

Fluorescent proteins spectra. The excitation (EX) (dot) and emission (EM) (dash) spectra are shown for three fluorescent proteins: mTagBFP (blue), sYFP2 (yellow) and mCherry (red) (data sourced from fpbase.org). Vertical lines indicate the laser wavelength (nm), whilst the light bars represent the filters used in the Amnis® Cellstream® flow cytometer to detect mTagBFP (blue, 405 nm – 457/51), sYFP2 (yellow, 488 – 528/46) and mCherry (red, 561 – 528/46).

Additional file 3: Fig S3.

Confocal microscopy images of Rhizobium leguminosarum 3841 unlabelled and labelled with different DFM combinations. A) bright channel. B) bright, red, yellow and blue channel. C) red, yellow and blue channel. D) red channel. E) yellow channel. F) blue channel. WT: R. leguminosarum 3841 (Rlv3841) not labelled. R: Rlv3841 labelled with mCherry. Y: Rlv3841 labelled with sYFP2. B: Rlv3841 labelled with mTag. RY: Rlv3841 labelled with mCherry and sYFP2. RB: Rlv3841 labelled with mCherry and mTag. YB: Rlv3841 labelled with sYFP2 and mTag.

Additional file 4: Fig S4.

Stereomicroscope images of Rhizobium leguminosarum bv. viciae 3841 RB within nodules on pea roots inoculated with OxCom6 at 13 and 14 dpi. In the first column, bright images are shown. In the second column the 560/40—630/74 channel was utilised to observe mCherry expression. The third column utilises the 405/20—460/40 channel to visualise TagBFP expression.

Additional file 5: Fig S5.

Absolute and relative values of community assembly of Enterobacter cloacae AA4. This figure represents the absolute (blue) and relative values (orange) of E. cloacae AA4 labelled with mCherry and sYFP2 (EcAA4 RY ) colonising pea roots (A), barley roots (B) and growing on rich media (C). egr (events•g root −1 ). emL (event•mL −1 ). Data shows that for EcAA4 RY that the absolute and relative values showed a different tendency on pea roots and on rich media where in both of them looks like there is a decrease when checking relative values whereas absolute values shows that the strains maintain steady.

Additional file 6: Table S1.

Primers used in this study

Additional file 7: Table S2.

Plasmids use in this study

Additional file 8: Table S3.

pTn7-SCOUT plasmids developed in this study

Additional file 9: Table S4.

Strains used in this study

Additional file 10: Table S5.

Flow repository codes for flow cytometry data used in this study

Additional file 11: Table S6.

Events per gram of root of non-inoculated pea and barley roots for each Combined population.

Additional file 12: Table S7.

Mean Generation time of each OxCom6 strain unlabelled and labelled with its DFM pattern

Additional file 13: Table S8.

Colonisation of pea roots (egr) by each OxCom6 strains when inoculated alone (single colonisation) or in competition with unlabelled strain (competitive colonisation)

Additional file 14: Table S9.

Comparison between colony formation units and flow cytometry data for each OxCom6 strain.

Additional file 15: Supplementary Methods.

Description of the assembly of Golden Gate plasmids used in this study.

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Jorrin, B., Haskett, T.L., Knights, H.E. et al. Stable, fluorescent markers for tracking synthetic communities and assembly dynamics. Microbiome 12 , 81 (2024). https://doi.org/10.1186/s40168-024-01792-2

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DOI : https://doi.org/10.1186/s40168-024-01792-2

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