Development of acute Pseudomonas aeruginosa and Acinetobacter baumannii lung mono-challenge models in mice using oropharyngeal aspiration
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Antimicrobial-resistant pathogens such as Pseudomonas aeruginosa and Acinetobacter baumannii can cause potentially fatal infections in susceptible individuals, with respiratory tract infections among the most common clinical presentations. The development of novel treatments or prophylactic interventions to combat these infections is urgently needed and requires robust, reliable animal models for their preclinical evaluation. In particular, the bacterial burden needs to be accurately determined before and after administration of the potential therapy under evaluation to quantify the effectiveness of the treatment. We provide two reliable, non-invasive murine acute lung challenge models with either P. aeruginosa or A. baumannii using an oropharyngeal aspiration technique, which has been widely overlooked in studies testing vaccines or treatments for these pathogens. Here, we show that this non-surgical technique to deliver suspensions into mouse lungs does not significantly impact animal welfare (based on welfare monitoring and weight) and allows uniform bilateral distribution of the bacterial dose, resulting in even bioburden in both lungs. The optimal timepoint for humane killing and organ harvest was 24 h after challenge for both pathogens, and at least 4×10 6 and 10 7 c.f.u. per mouse were needed to obtain a reproducible P. aeruginosa or A. baumannii bioburden, respectively. These mouse challenge models offer a valuable tool to assess therapeutic interventions against P. aeruginosa or A. baumannii infections.
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The work presented is clear and the arguments well formed. This study would be a valuable contribution to the existing literature. This is a study that would be of interest to the field and community.
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Dear Dr McClean, Thank you for submitting your manuscript, titled (Development of acute Pseudomonas aeruginosa and Acinetobacter baumannii lung infection models in mice using oropharyngeal aspiration) to Access Microbiology Journal. After careful consideration of the feedback provided by the reviewers, we have come to a decision. Both reviewers are of the opinion that the manuscript is not yet ready for publication and requires substantial revision and additional experimental work before it can be considered further. Reviewer Feedback Summary 1. Both reviewers have raised concerns about the work in the manuscript. Significantly they have noted that there is insufficient optimization of the model for the two pathogens used in the study. To address this, you will need to provide a clearer justification of how the work described …
Dear Dr McClean, Thank you for submitting your manuscript, titled (Development of acute Pseudomonas aeruginosa and Acinetobacter baumannii lung infection models in mice using oropharyngeal aspiration) to Access Microbiology Journal. After careful consideration of the feedback provided by the reviewers, we have come to a decision. Both reviewers are of the opinion that the manuscript is not yet ready for publication and requires substantial revision and additional experimental work before it can be considered further. Reviewer Feedback Summary 1. Both reviewers have raised concerns about the work in the manuscript. Significantly they have noted that there is insufficient optimization of the model for the two pathogens used in the study. To address this, you will need to provide a clearer justification of how the work described advances the current state of knowledge of this already established model and ensure that optimization steps for the pathogens are clearly detailed and validated. 2. A significant criticism highlighted by the reviewers is the lack of evidence that infection has been successfully established using your model. It is essential to provide rigorous assessments of infection in your experiments, along with appropriate controls, to substantiate your claims. 3. The manuscript does not include a direct comparison between your model and other established infection or colonization models. The reviewers have emphasized the importance of including such comparisons to contextualize your findings and demonstrate the relevance and advantages of your model. 4. Reviewer 2 has specifically pointed out that the number of mice used in each experiment is insufficient for robust statistical analysis. You will need to increase the number of animals used in each group and perform a more comprehensive statistical evaluation to ensure that your conclusions are statistically sound. 5. Both the introduction and discussion sections require substantial rewriting. The introduction should be reframed to clearly state the novelty and significance of your work, while the discussion should address the reviewers' concerns, including comparisons with other models and a thorough interpretation of your results in the context of existing literature. Decision and Next Steps Given these significant concerns, the manuscript is not acceptable for publication in its current form. However, we are willing to offer you the opportunity to revise your manuscript under the following conditions: 1. Major Revisions: You must address all the points mentioned above. This includes conducting additional experiments to provide robust evidence of infection, increasing the sample size, optimizing the model for the pathogens used and making direct comparisons with other models. The introduction and discussion sections must also be completely rewritten to address the reviewers' concerns comprehensively. 2. Re-evaluation: After you have made these substantial revisions and conducted the necessary additional experiments, you may resubmit your manuscript for further consideration. Please note that the revised manuscript will be sent back to the original reviewers for evaluation. Alternatively, if you believe that the extensive experimental work required to address these issues is not feasible at this time, you may choose to withdraw the manuscript. I understand that the revisions may require considerable effort and I want to be transparent that the manuscript will only be considered for publication if the reviewers' concerns are fully addressed. Please let us know how you wish to proceed. If you choose to undertake the revisions, we encourage you to carefully consider the reviewers' comments and make the necessary improvements to your manuscript. Should you decide to resubmit, please include a detailed point-by-point response to the reviewers' comments, outlining how each concern has been addressed. We appreciate your interest in publishing with Access Microbiology and we look forward to your response. Best regards, Dr Zina Alfahl
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Comments to Author
This paper has been written well with good English and put together in the correct paper format. Unfortunately, there are some key flaws in the paper. The main issue is that I am not convinced this is a better method to use than non-surgical intratracheal or intranasal. Overall, the paper needs to be rewritten to improve the flow as sections about P. aeruginosa and A. baumannii are very different and seem to be written by different people. The use of the method is not convincing as other methods were not conducted in parallel for comparison. I have not been convinced it's better than intranasal administration or non-surgical intratracheal administration. I am also not convinced a true infection has been established. This seems to be an acute challenge model rather than infection model. No output of …
Comments to Author
This paper has been written well with good English and put together in the correct paper format. Unfortunately, there are some key flaws in the paper. The main issue is that I am not convinced this is a better method to use than non-surgical intratracheal or intranasal. Overall, the paper needs to be rewritten to improve the flow as sections about P. aeruginosa and A. baumannii are very different and seem to be written by different people. The use of the method is not convincing as other methods were not conducted in parallel for comparison. I have not been convinced it's better than intranasal administration or non-surgical intratracheal administration. I am also not convinced a true infection has been established. This seems to be an acute challenge model rather than infection model. No output of infection has been confirmed. Additionally, the use of the word colonisation is not accurate throughout. Please see more detailed feedback below. Finally, I think you need to discuss the drawbacks and limitations of your method. Title Upon first reading I assumed the infection was a co-infection model. Could this be adjusted to more clearly state this is a mono-infection model. Introduction A high proportion of the references are from over 5 years ago and almost half are from 10+ years ago. I think more up-to-date references for statistics within the introduction would be more informative. Overall, the introduction would be written with better structure as its quite jumbled. It seems rushed and doesn't have a good flow. The sections introducing P. aeruginosa (paragraph 1) compared to A. baumannii (paragraph 2) are completely different they introduce the pathogens in completely different ways. It seems like they were written by different people. Makes the introduction disjointed and difficult to follow. Try to follow a similar structure to in both pathogens and look for similarities between the two. I would introduce them together as it will probably make it more clear why you have chosen to develop a model for these two pathogens. The statistics that have been quoted seem a little bit random. Unclear why it has been missed out that P. aeruginosa is among the 5 most common causes of hospital acquired infections. Also, its main association is with nosocomial infections and people with CF and this doesn't come across as a key factor. It would be good if there was a little bit more about the pathogens themselves in the introduction. Even one sentence for each just introducing them in terms of gram-negative/positive, where are they commonly found? Are the commensals? This will make it more accessible for a range of readers. There is a lot in the introduction about A. baumannii and MDR and VAP - I think P. aeruginosa has similar issues and arises in similar patients groups. Also, the MDR of P. aeruginosa could be discussed. Methods are in general good. Seems like it would be very difficult to do this technique quickly based on only 1min of isoflurane administration, the mice will wake up very quickly. Also as mentioned below the technique seems to require 2 people to hold the mouse in position making this quite a difficult technique to do in a short time. Unclear why different mice were used for each bacteria. Maybe both pathogens could be conducted in both mouse strains. Unclear why 2 different methods of homogenisation of tissues were used. Also unclear why some CFU are /mg and some /ml. Line 122 - the growth curves take a long time to do and I don't think they have been utilised to their best advantage. If I were to replicate this I wouldn't know which dose to give for the high and low dose. Results Some key flaws in the results. 1) There is no direct comparison with other methods. Why were the intranasal and non-invasive intratracheal technique not conducted at the same time to show this method is truly optimal. Without this information I don't think any claims can be made that this method is better. 2) isolation of CFU in the stomach is a big drawback of this method. One of the main goals of a model is to minimise contamination of non-target tissues. 3) Very inconsistent in the description of results between the development of the P. aeruginosa model and the A. baumannii model. For instance, strain type (one a CF isolate, one a reference strain), day of CFUs (day 7 for A. baumannii is the first thing that's done, but this was never done for P. aeruginosa), growth curves not discussed for both, discussion of weight loss (for P. aeruginosa final weight is given as a percentage i.e 93.03%, for A. baumannii its described as 10% weight loss), p numbers given in A. baumannii section but not in P. aeruginosa. There are many examples of inconsistency between the way these results sections are written, as with the introduction it seems like these have been written by different people and therefore these sections are disjointed and difficult to follow. Seems unusual to discuss the far timepoints before the short timepoints. This is development of an acute infection model I would expect the early timepoints to be discussed first and be the focus of the results. I don't think its relevant to discuss day 7 results for an acute model. Line 30 - 'less stressful than other techniques'. This is a big statement - no data shows this is actually less stressful than other techniques. No biological read out has been investigated to show this. What about intranasal administration? I would think this was the least stressful of them all. Line 42 - This is quite an old stat '13% of global deaths in children under 5' is there no more recent statistics? Also seems random for the first sentence. Line 45 - 'Worryingly, more than 40% of P. aeruginosa-associated deaths are due to infections in the lower respiratory tract [11, 47 12], which are a predictor of poor prognosis for hospitalised patients' - P. aeruginosa-associated deaths are a predictor of poor prognosis? Yes because the patient has died. This doesn't make sense. Line 50 - remove 'and' Line 55 - 'leading causes of hospital-acquired pneumonia' - this is also true for P. aeruginosa. Line 65 - ' are included in four classification groups' - this is confusing as a reader I have no idea what you are referring to here. Line 68 - 'ESKAPE pathogens, SPICE/SPACEK microorganisms' - These need to be described in more detail - I am quite lost reading this sentence. Line 73/74 - 'acute pneumonia infection murine models are needed' - this implies there are not currently any infection models available. Intranasal dosing and intratracheal dosing are common using for this at the moment. Line 75 - For acute infections as shown in this paper, intranasal is always conducted as the most common method of administration (as far as I can see in publications). The reference 29 not relevant. Some studies have shown now that intranasal is just as good as intratracheal (PMID: 33833346) Line 88 'we have developed' - this technique is similar to other methods under a different name - intratracheal (i.t.) instillation (PMID: 27390765). Also, the intratracheal instillation method has been extensively studied by others and has been optimised not to have any stomach contamination. ' This study shows that the non-invasive intratracheal instillation method has good accuracy and reliability, which can reduce the use of mice, do less harm to the mice, and then improve 3R animal welfare.' (PMID: 36323359). Line 88 - 'technically easy' in figure 1 it seems 3 hands are required for this procedure so 2 people are required to conduct this procedure making it not technically easy when other procedures can be conducted by 1 person. Line 160 - A non-lethal dose is often required for experiments, yes, but im not sure you've convinced me that you are inducing an infection model? Simply giving the mice a pathogen doesn't mean they are infected. There are no measurements of immune response induced etc. This could be a challenge rather than infection. Line 161 - 'The optimal bacterial dose was determined using growth curves' This needs to be rewritten. The CFU were determined by growth curves, not the optimal dose. Line 169 - severity of what? Line 195 and Line 207 - be very careful in your use of the word colonisation, not just here but throughout the paper. I would say in every instance it has been used incorrectly. This method is trying to induce an acute infection model not a colonisation or chronic infection model. These pathogens are rapidly cleared they are not colonising the organs. Line 250 - The other techniques are also largely non-fatal, particularly intranasal. Also, other techniques are non-invasive. Line 253 - with comparable results to models established using other bacterial delivery - I don't think you can make this claim because you didn't do it, in your hands. Line 255 - 268 - Not sure why this has been discussed. Seems random and unnecessary. Line 270 - ' organs was optimal at 24 h' im not sure what is an optimal burden… do you mean reached a peak at 24h? Line 278 - other P. aeruginosa acute lung infection models - how are these pathogens administered? Did they look at reproducibility? Can you truly make the claim that this method is the best for an acute infection model? Line 282 - If the mice aren't sick, how do you know they are truly infected? Usually this can be investigated by output such as neutrophilia etc. Line 284 - other non-surgical techniques have been used and better delivery of inoculum have been reported. 'Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63 ± 8% of dose to lungs, and a nonsurgical intratracheal dosing approach further increased lung delivery to 92 ± 6% of dose.' (PMID: 37401383). Line 288 - Not really surprising as intranasal and non-surgical intratracheal administration have similar benefits. Line 306 - at 3 what? Line 307 - prolonged colonisation - I don't think CFUs to day 3 would support prolonged colonisation. Line 316 - reference cited as others in text please Line 320 - 'without surgical trauma' what method did Tansho-Nagakawa et al. use? Was this with surgical administration? Line 321 - the goal here, as far as I can ell is not to establish a long-term infection? So its unclear why this is being discussed. Line 331 - change hours to h for consistency. Figures Some groups have very low numbers for instance Fig S3A looks to only have 2 mice per group. Please specify how many times the experiment was conducted and how many mice were per group. (n=3, 4 mice per group). Overall the numbers of mice used in these experiments were very low. Table 1 Its confusing that the low and high dose are just described as , what actual doses were used and why were 2 doses used for a medium dose. This is quite confusing. If I were to repeat this I wouldn't know which dose to give for high and low doses. Figure 1 Legend could be expanded further to explain the technique- this looks like it needs 3 hands and requires 2 people to conduct? Figure 2 Not many mice used per group, is this enough for statical significance? How many experiments were conducted and how many mice in each experiment? For instance (n=2, 3 mice per group)? its confusing that there are 3 mice for left lung, 3 mice for right lung and 6 mice for stomach? Surely there should be 6 mice in each group? Figure 3 This is not colonisation. Colonisation is when you establish growth of a microbe in a commensal fashion and is not causing infection. This is bacterial burden. Figure 4 and 5 - how many experiments do these graphs represent?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
The study showed the aspiration model which has been used in the lab. The study focussed on optimization but did not show clearly the factors and criteria of optimization. 1. Methodological rigour, reproducibility and availability of underlying/supporting data. You may use the results of SciScore to support your assessment; the results can be found under 'Manuscript Analysis Services' in Editorial Manager. Methods were not clear. * Which kind of optimization has been made? * How was severity score calculated? * How was humane endpoint defined? * Any histogram has been done? * How were data analyzed? Used software? * 2. Presentation of results * The supporting data for the optimization was not presented. * The data of severity was not shown 3. How the style and organization of the paper communicates …
Comments to Author
The study showed the aspiration model which has been used in the lab. The study focussed on optimization but did not show clearly the factors and criteria of optimization. 1. Methodological rigour, reproducibility and availability of underlying/supporting data. You may use the results of SciScore to support your assessment; the results can be found under 'Manuscript Analysis Services' in Editorial Manager. Methods were not clear. * Which kind of optimization has been made? * How was severity score calculated? * How was humane endpoint defined? * Any histogram has been done? * How were data analyzed? Used software? * 2. Presentation of results * The supporting data for the optimization was not presented. * The data of severity was not shown 3. How the style and organization of the paper communicates and represents key findings * Key finding should be emphasized on abstract * The abstract should be written clearly on methods used, optimization used, significant findings 4. Literature analysis or discussion * What is new from this study? * Other studies using similar models should be discussed. 5. Any other relevant comments In the abstract, you should clearly show the objectives, methods/optimization, and respective results.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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