Influenza A virus infection alters the resistance profile of gut microbiota to clinically relevant antibiotics

ABSTRACT Influenza A virus (IAV) infection triggers quantitative and qualitative modifications in lung and intestinal microbiota composition, which contain an important reservoir of antibiotic resistance genes. Analysis of genetic changes is a common practice in studies that analyze microbiota modifications. However, there is little evidence of functional changes linked to such microbiota modifications. This study evaluates some cecal microbiota’s functional changes, comparing sublethal IAV with mock-infected mice. Community-wide phenotypic metabolic profile (Biolog EcoPlatesTM) and relative antibiotic resistance changes to clinically relevant antibiotics (cenoantibiogram) have been performed in this context. Results reveal a temporal association between IAV infection and alterations in nutrient substrate profile usage as well as changes in antibiotic resistance of cecal microbiota. Alterations are transient and predominantly occur at early time points post-IAV infection. There is a functional rebalance in nutrient substrate usage and antibiotic resistance under the established culture conditions, accompanied by a decrease in microbial density of the cecal community on days 5 and 7 after the IAV infection. Our data underline that active IAV infections altering microbial populations are associated with changes in nutrient usage preferences and affect community behaviors toward specific antibiotics. These findings could have implications including activation of nutrient-related metabolic stress at the microbiota community level and additional antibiotic resistance selection mechanisms of clinically relevant infections. IMPORTANCE Influenza virus infection affects both lung and intestinal bacterial community composition. Most of the published analyses focus on the characterization of the microbiota composition changes. Here we assess functional alterations of gut microbiota such as nutrient and antibiotic resistance changes during an acute respiratory tract infection. Upon influenza A virus (IAV) infection, cecal microbiota drops accompanied by a decrease in the ability to metabolize some common nutrients under aerobic conditions. At the same time, the cecal community presents an increase in resistance against clinically relevant antibiotics, particularly cephalosporins. Functional characterization of complex communities presents an additional and necessary element of analysis that nowadays is mainly limited to taxonomic description. The consequences of these functional alterations could affect treatment strategies, especially in multimicrobial infections.

Major Comments 1.While the authors discuss potential bacteria taxa, such as Enterobacteriaceae, to be linked to antibiotic resistance genes, the presence of this bacteria within this sample set is unknown.The sample type analyzed by Yildez et al. was the small intestine, which has been previously demonstrated to have a different microbial composition from the cecum.It would be ideal to report the microbial composition of the samples analyzed within these analyses or further discuss the differences in sample type between the two studies and its effect on the interpretation of the data.
2. The analyses performed within this study were on aerobic bacteria.However, the majority of the gut microbiome is composed of obligate anaerobic bacteria.The limitation of only analyzing aerobic bacteria should be further discussed as these results do not represent numerous bacteria within the intestinal microbiota.
3. Within the discussion, it is stated that the relative number of bacteria in laminoculture and bacteria load based on OTU abundance was consistent in order to counter the limitation of only analyzing aerobic bacteria.Is the OTU abundance referring to the samples analyzed in Yildiz et al?If so, Yildiz et al. analyzes the small intestine, which has been shown to have a different number of OTUs, diversity, and composition compared to the cecum, which is the sample type analyzed within this study.
Therefore, the two sample types are not an ideal comparison.It would be ideal to report the number of OTUs from the cecum samples used in this study.
Minor Comments Within the introduction and the discussion, numerous paragraphs were 1-3 sentences in length.Suggest combining paragraphs so that the text is not disjointed.Line 61.Suggest inserting (IAV) after "Influenza".Line 290-292: Suggest rephrasing.The references stated discuss the prevalence of antibiotic-resistant genes in a specific bacterial species in children with and without antibiotic treatment and adults.However, it is not suggested within the reference that the recovery of the microbiome from respiratory illness is influenced by antibiotic resistance phenotypes.Thank you for submitting your manuscript to Microbiology Spectrum.Your work has been evaluated by two experts in the field.Both reviewers were interested in your work and found it has merit.Reviewers also pointed out important aspects that must be addressed before the manuscript is ready for publication.Please, see below my recommendations below, together with the reviewers's report.1) Two limitations are that the study does not provide any information about the taxonomy of the bacteria investigated and focuses only on aerobic microorganisms.These shortcomings raise the issue of whether the study offers a clear picture of the effect of IAV on the gut microbiome.As the requirement for publication in Microbiology Spectrum is technically sound work regardless of potential impact, your work could still be considered if you address the limitations of your study in the revised version.
2) Ensure the number of samples and the statistical analysis used is summarized in the figure legends and clearly explained in the figure legends.When overviewing reviewer 2 comments, I could not find the information requested.
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Silvia Cardona
Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.orgReviewer comments: Reviewer #1 (Comments for the Author): In the manuscript titled "Influenza A virus infection alters resistance profile of gut microbiota to clinically relevant antibiotics", the authors analyze cecum samples previously collected by Yildez et al. to analyze the metabolic and antibiotic resistance profile of culturable aerobic bacteria.The authors report a decreased bacterial load during peak viral titers within the lungs at 3, 5, and 7 days post-infection, and changes in the antibiotic resistance of cecum microbes, particularly cefoxitin, and ceftazidime.The study focuses on the importance of evaluating underlying changes to nutrient preferences and the prevalence of antibioticresistant genes during IAV infection.

Major Comments
While the authors provide interesting insight into the changes in nutrient preferences and antibiotic-resistant genes during IAV infection, there is a limited association with influenza virus infection.Since the major difference between the two groups being compared is influenza A virus infection, it would be informative to correlate the major findings reported in this paper with the virus shedding and pathogenicity observed post influenza A virus infection reported in Yildez et al.The majority of the significant findings within this paper occur between 3 and 7 dpi, which correlates with high viral loads in the lungs and particularly, weight loss and a large number of infiltrating immune cells within the lungs at 7 dpi.
Minor Comments Line 74.Suggest removing "The" before influenza viruses Line 84-88: Suggest combining this paragraph with the previous paragraph about influenza viruses in lines 74-78 Line 129-131: In addition to correlating with reduced genome counts in the small intestine, days 3 and 7 had high viral titers within the lungs of infected mice in Yildez et al.
Line 151-153: Similar to the comment above, since the major difference between the two groups is IAV infection, days 3 and 7 had the highest viral titers during infection which may be important to mention since the largest changes are observed at those days.
Line 157: Suggest adding "the" before substrates Line 215 -225: Suggest combining this paragraph with the previous paragraph Reviewer #2 (Comments for the Author): The manuscript by Robas et al focuses on the differences in the cecum's metabolic and antibiotic resistance profile from mice cecal samples that were exposed to mock or IAV infection.The authors have reported changes in viable bacterial load at different days post-infection.Additionally, they report changes in carbon and nitrogen sources as well as alterations in antibiotic resistance of cecal bacterial communities to specific antibiotics like cephalosporins.This is a relevant and interesting study where the authors have used microbiological assays to understand the effect of IAV infection on changes to nutrient preferences and the prevalence of antibiotic-resistant genes in gut microbiome.Overall, the manuscript is well-written and easy to follow.However, I have a few general comments for the authors: Major Comments 1.The authors have neither analyzed nor have reported the microbial composition of the samples used in this study which makes it difficult to interpret the results.Especially since there is a lot of speculation made in the discussion section, it will be immensely helpful to pinpoint which bacterial species is in fact responsible for differences observed for the antibiotic resistance profile in mock versus IAV infected samples.
2. Figure 1: No error bars have been shown.Also, it will be useful to mention the number of samples that have been used to plot the bar graph.
3. Since the authors have only focused on aerobic bacteria in their study, which further raises the question if what they are observing is in fact giving a clear picture of the effect of IAV on the gut microbiome since a vast number of gut microbiome is composed of obligate anaerobic bacteria.
4. Figure 3B: Can the authors please justify the reason for plotting data for samples with a non-significant p-value Figure 3C: please re-plot this data.
Overall, it will be helpful to see data points for bar graphs throughput the paper, as has been shown for Figure 3A.

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Madrid, 28 of August, 2023
Dear Dr Cardona, Below you can find our point-by-point response to your comments as well as the response to the reviewer´s critiques.We have done our best to address all the issues you have raised.

Editor´s comments:
Thank you for submitting your manuscript to Microbiology Spectrum.Your work has been evaluated by two experts in the field.Both reviewers were interested in your work and found it has merit.Reviewers also pointed out important aspects that must be addressed before the manuscript is ready for publication.Please, see below my recommendations below, together with the reviewers's report.
1) Two limitations are that the study does not provide any information about the taxonomy of the bacteria investigated and focuses only on aerobic microorganisms.These shortcomings raise the issue of whether the study offers a clear picture of the effect of IAV on the gut microbiome.As the requirement for publication in Microbiology Spectrum is technically sound work regardless of potential impact, your work could still be considered if you address the limitations of your study in the revised version.
Thank you for the suggestions.In this current version, we present the metagenomic/taxonomic analysis of the small intestine material on days 3, 5, 7, 14, and 28 as well as the fecal material on days 7 and 14.We have included this analysis as Supplementary Figure 1 material.Although the bacteria found in this analysis are not from the cecum, we can infer that changes in bacteria composition occur across the digestive microbiota and that bacteria composition changes at the community level are connected to the functional changes described in the manuscript.Unfortunately, we could not analyze the bacteria composition and taxonomic analysis of the cecum samples used for the functional assays presented due to the sample availability.
When comparing the composition of the microbiota between Mock and HALO IAV infected mice, we can appreciate differences at day 7 in the small intestine (SI) that specifically affect Bacteroidia, Bacilli, Gammaproteobacteria, and Verrucomicrobiae classes (Panels A) and C) in Supplementary Figure 1).A more specific analysis (panel E) shows the prevalence of the Muribaculaceae family within the Bacteroidales order and Bacteroidia class.This family of bacteria is reduced at 7 dpi (days post-infection).
Lactobacillaceae and Spreptococcaceae within the Lactobacillares order and Bacilli class are increased at 7 dpi.At the same time, the Gammaproteobacteria class shows an increase at 7 dpi.In particular Cardiobacteriales order and specifically the Cardiobacteriaceae family; Chromatiales order and specifically the Halothiobacillaceae family; the highest increase, was observed in the Enterobacteriales order and Enterobacteriaceae family; Pasteurellales order and specifically Pasteurellaceae family; high increase of Pseudomonadales order and specifically Moraxellaceae family; Vibrionales order and specifically Vibrionaceae family increases also.In the case of the Verrucomicrobiae, Verrucomicrobiales order, and Verrucomicrobiaceae family experience an increase also at 7 dpi.In panels C) and D), we can see data corresponding to individual mice (dots) that present considerable variability between animals.
Although upstream from the cecum, these differences in the small intestine between mice could explain the high variability of our functional assays.
Based on SI observations, we also analyzed the fecal microbiota, downstream from the cecum, on days 7 and 14 trying to see some correlation between fecal and the upstream microbiota at the SI.No clear differences could be observed in Bacteroidia, but a reduction in Lactobacillus at 7 dpi could be observed.A small but significant increase in the Gammaproteobacteria and Verrucomicrobiae classes can be observed at 7 dpi (Panel F Supp Fig 1).This correlation between SI and fecal microbiota could indicate a similar phenotype at the cecum, used for the functional assays.As mentioned in the manuscript Discussion section, Enterobacteriaceae belonging to Gammaproteobacteria (lines 313, 380, and 397) have been linked to overgrowth and increased antibiotic resistance.However, 16S gene analysis cannot determine whether this is the source of the resistance.Metagenomic analyses, cannot guarantee the identification of the members in the communities responsible for the functional changes analyzed in the manuscript either.The presence or absence of specific taxa can be related to particular functions, but the experimental determination of such functions in native conditions within the community complexity is a challenge to be addressed in the future.
We have included in different places across the manuscript the limitation of extracting, manipulating, and culturing the microorganisms in the samples under aerobic conditions.We understand the limitations of this approach however, all the samples were manipulated the same way and the changes observed are indicative of metabolic and antibiotic resistance changes that could be further described in the future by considering also anaerobiosis.This limitation does not subtract from the relevance of functional changes at the community level and could be measured and considered to interpret phenotypes.Reproduction of the native environment of the microorganisms present in the cecal samples is not trivial and will require culture conditions to be defined, particularly the culture media and the atmospheric conditions.
2) Ensure the number of samples and the statistical analysis used are summarized in the figure legends and clearly explained in the figure legends.When overviewing reviewer 2 comments, I could not find the information requested.
We appreciate this observation.We have made a thorough rewriting of figure legends trying to clarify the analysis conditions, including the number of samples in each experiment presented in the figures.
We'd also like to highlight that, in response to a suggestion from Reviewer 2, we have incorporated individual values into the analysis.During this process, we identified an error in the duplication of nutrient uptake data from day 3 in samples obtained from IAV-infected animals.After rectifying this duplication, we proceeded to update the panels in Figure 2 and adjusted the accompanying result descriptions accordingly.

Reviewer comments: Reviewer #1 (Comments for the Author):
In the manuscript titled "Influenza A virus infection alters resistance profile of gut microbiota to clinically relevant antibiotics", the authors analyze cecum samples previously collected by Yildez et al. to analyze the metabolic and antibiotic resistance profile of culturable aerobic bacteria.The authors report a decreased bacterial load during peak viral titers within the lungs at 3, 5, and 7 days post-infection, and changes in the antibiotic resistance of cecum microbes, particularly cefoxitin, and ceftazidime.The study focuses on the importance of evaluating underlying changes to nutrient preferences and the prevalence of antibiotic-resistant genes during IAV infection.

Major Comments
• While the authors provide interesting insight into the changes in nutrient preferences and antibiotic-resistant genes during IAV infection, there is a limited association with Influenza virus infection.Since the major difference between the two groups being compared is Influenza A virus infection, it would be informative to correlate the major findings reported in this paper with the virus shedding and pathogenicity observed post-influenza A virus infection reported in Yildez et al.Most of the significant findings within this paper occur between 3 and 7 dpi, which correlates with high viral loads in the lungs and particularly, weight loss and a large number of infiltrating immune cells within the lungs at 7 dpi.
We appreciate the reviewer´s comment.We have included this point addressing some of the minor comments below as well as the discussion, where both viral titers, as well as pathogenicity described by Yildiz et al., are correlated with the observations made in our manuscript.

Minor Comments
• Line 74.Suggest removing "The" before influenza viruses.Thank you for the suggestion.The text has been fixed.
• Line 84-88: Suggest combining this paragraph with the previous paragraph about influenza viruses in lines 74-78 Thanks for suggesting.Paragraphs have been merged.
• Line 129-131: In addition to correlating with reduced genome counts in the small intestine, days 3 and 7 had high viral titers within the lungs of infected mice in Yildez et al.
Thank you for the suggestion.We have addressed this suggestion in the manuscript.
• Line 151-153: Similar to the comment above, since the major difference between the two groups is IAV infection, days 3 and 7 had the highest viral titers during infection which may be important to mention since the largest changes are observed at those days.
Following the reviewer's suggestion, a comment about the high viral titers on days 3 and 7 has been included in the indicated location of the manuscript.
Thank you for the suggestion, the text has been changed.
• Line 215 -225: Suggest combining this paragraph with the previous paragraph.
Thanks for noticing.The idea of splitting paragraphs was to emphasize changes in different antibiotic families, however, following the reviewer´s suggestions, the paragraphs between lines 215 and 225 have been merged with the previous paragraph.

Reviewer #2 (Comments for the Author):
The manuscript by Robas et al focuses on the differences in the cecum's metabolic and antibiotic resistance profile from mice cecal samples that were exposed to mock or IAV infection.The authors have reported changes in viable bacterial load at different days postinfection.Additionally, they report changes in carbon and nitrogen sources as well as alterations in antibiotic resistance of cecal bacterial communities to specific antibiotics like cephalosporins.This is a relevant and interesting study where the authors have used microbiological assays to understand the effect of IAV infection on changes to nutrient preferences and the prevalence of antibiotic-resistant genes in gut microbiomes.Overall, the manuscript is well-written and easy to follow.However, I have a few general comments for the authors: We appreciate the reviewer´s comment about the value of the work and the effort to present the analysis in a way easy to follow.

Major Comments
1.The authors have neither analyzed nor reported the microbial composition of the samples used in this study which makes it difficult to interpret the results.Especially since there is a lot of speculation made in the discussion section, it will be immensely helpful to pinpoint which bacterial species is, in fact, responsible for differences observed for the antibiotic resistance profile in mock versus IAV-infected samples.
Thank you for the comment.As pointed out above in response to the editor´s comments, the bacteria composition and taxonomic analysis of the samples used in the functional assays could not be performed due to sample availability.Instead, we are presenting the metagenomic analysis of small intestine (upstream of cecum) composition at days 3, 5, 7, 15, and 28 of mock and IAV-infected mice.Although they may present differences compared to the bacteria composition at the cecum, we can observe changes in composition that correlate with the pathology observed in the animal.This data could be an indicator and a reference to establish the changes occurring during infection.
In addition, since the largest changes are occurring at day 7 dpi in the classes already mentioned, we could compare the composition of the fecal microbiota, just after the cecum at days 7 and 14 post-infection.The increased amount of Enterobacteriaceae at 7 dpi in IAV-in the small intestine and fecal samples, upstream and downstream of the place where the samples were obtained for the functional assays of our manuscript.As stated before, an Enterobacteria increase could be associated with an increase in betalactam resistance increase.However, this is just speculation, and more effort in developing functional assays is needed.
Despite the composition containing the bacteria responsible for the functional changes described in the manuscript, this type of analysis cannot identify the bacteria or group of bacteria responsible for the changes in nutrient uptake and cenoantibiogram resistance.
This association remains a challenge in future descriptions of microbial community functional changes, and we hope to address specific associations in the future.
2. Figure 1: No error bars have been shown.Also, it will be useful to mention the number of samples that have been used to plot the bar graph.
Thank you for noticing.When error bars are not presented, it means that the detection limit of the assay has been reached.No sample was available to repeat the analysis, however, the analysis allows us to detect differences in the amount of the bacteria able to grow in the given experimental conditions between IAV and mock-infected mice.
The number of samples for each condition was 3. It has been included in the text.
3. Since the authors have only focused on aerobic bacteria in their study, it further raises the question if what they are observing is giving a clear picture of the effect of IAV on the gut microbiome since a vast number of gut microbiome is composed of obligate anaerobic bacteria.
We appreciate this comment.As indicated above in the editor´s general comments, we have included in several places across the manuscript the caveat of manipulating samples in an aerobic atmosphere.This limitation was applied to all the samples that would suffer the same aerobic restrictions.Still, we can see specific nutrient uptake and antibiotic resistance changes between IAV and mock-infected animals, which is one main argument in this manuscript.We cannot be sure that these functional changes will be the ones occurring inside the animals, but functional changes are occurring at the community level when comparing IAV and mock-infected animals at certain days postinfection.Some of them may be biologically relevant.Not only anaerobic bacteria may suffer by working in aerobic conditions, but also culture media, the presence of other microorganisms in the community, or host factors from the mice can influence functional changes.Addressing these issues will require technical improvements in the future.The main message of our work focuses on the fact that IAV infection causes functional alterations in the cecum (and probably other intestinal parts) microbiota at the community level with potential biological relevance.
4. Figure 3B: Can the authors please justify the reason for plotting data for samples with a non-significant p-value.
Thank you for the comment.The reason to include these values is to have a reference for those that indeed present differences.The statistical analysis was systematically performed for each IAV vs. mock-infected group.All the comparisons for the conditions under study were analyzed and presented as shown in the panel.

Figure 3C:
Please re-plot this data.
Overall, it will be helpful to see data points for bar graphs throughout the paper, as has been shown in Figure 3A.Thank you for your suggestions.Individual values have been added to the figures.We want also to note that this suggestion allowed us to identify a duplication of one mouse sample in the IAV-infected group.As mentioned before, we have corrected this mistake and present Figure 2 panels B) and C) with these updated changes also in the text.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred

Table 1
Legend: Suggest stating the meaning of acronyms used within the table, such as BLEE.