Applied Metagenomic Profiling of Domestic Cat Feces from Cali, Colombia: An Exploratory Approach

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This peer-reviewed article was devoted to the study of the gut microbiota of domestic cats and contributed to the development of the concept under the One Health paradigm. The authors present data from a molecular analysis of fecal biomaterial obtained from domestic cats in Cali, Valle del Cauca, Colombia. The results are of regional interest and contribute to the accumulation of data on the microbiota of the most actively used domestic animals-cats.

Despite the undoubted interest, this work requires significant revision.

1. Section “2.1. Study Site, Sample Collection, and Processing” should be revised. The authors need to present the overall design of the study, which would explain the information on the number of animals taken into the analysis, the quantitative composition of the groups - by outdoor activity, age, sex and diet; how often samples were collected - since the terminology "early" samples are encountered in the work.

2. L.101-103: Authors were asked to provide information on who performed the amplicon library and metagenomic analysis. If the authors performed these analyses themselves, the description of the experimental work should be detailed. In addition, it is not clear how many samples were used for the amplicon library or metagenomic analysis. If the samples for metagenomic analysis were pooled by 5 µl to a volume of 30 µl, were there 6 samples out of 10 taken in total? Then it is necessary to justify such a methodological approach, why and on what basis the samples were combined that differed so greatly in the composition of the microbiota, as the authors describe and prove in Fig. 3.

3. Throughout the text, the authors must provide all taxonomic names in accordance with the modern nomenclature: see https://www.bacterio.net/

4. Throughout the text, the authors must correct the spelling of all genera and species names used for bacteria in italics.

5. Figure 2. “The top 10 bacterial taxa by average relative abundance”. If the figures indicate average values, corrections must be made and the SD must be indicated.

6. Section “3.1.2. Variation in Relative Abundance of Dominant Bacterial Taxa According to Metadata Variables” needs to be completely rechecked and corrections made according to the figure. For example, the authors write: "It shows that Peptoclostridium … have a higher abundance in individuals without outdoor access", but according to Figure 3A, four out of the six taxa that were identified in representatives without Outdoor Access, the values for this taxon are low. 

The authors wrote "In contrast, Holdemanella, Clostridium perfringens, and Megasphaera elsdenii exhibit relatively higher abundance in individuals with outdoor access", but the taxon Peptoclostridium has the highest values, according to Figure 3A. 

There are even more inconsistencies in the descriptions of Figures 3B, 3C, and 3D.

L. 209-210: Not correct for Megamonas. This taxon is distinguished only among females. 

L. 215-217: Does not correspond to the figure at all: the first is more represented in young individuals, as in the two taxa described above, while the next two are detected only in young cats or adults.

L. 220: It is necessary to explain how these percentages were calculated. Does not correspond to the figure.

7. Figure 3. Why is it written that top-5, although information on 7 taxa is presented?

8. Gene names should be written in italics.

9. L. 323-325: What do “early samples” mean? Is this somehow related to the age of the animals? This information is not presented in the results of our own research.

10. L. 343: It is incorrect to use the old taxonomy of Cyanobacteria because at present, this phylum has been specifically described in the context of gut studies. 

11. L. 354, 363, and further throughout the text: All average values should be presented as mean and SD.

12. L. 395: The authors did not analyze host-associated factors in the article.

13. L. 405-406: not “elevated”, but “present only”. 

14. L. 416-417: Incorrect, since Megamonas only occurs in females, while Megasphaera, namely Megasphaera elsdenii according to the fig. 3B is higher in males.

General note: The authors must carefully review the entire text of the manuscript and align the description of the results and visualization.

After making corrections, the manuscript can be submitted for re-review.

Author Response

We sincerely thank the reviewer for their thoughtful and constructive comments, which greatly contributed to improving the quality and clarity of our manuscript. In response, we revised Section 2.1 to provide a clearer description of the study design, including the number of animals, sex distribution, age groups, diet types, and outdoor access. We clarified that each animal contributed a single fecal sample, and the term “early samples” was replaced with more precise language. We also added detailed information about the pooling strategy for metagenomic analysis and provided a clear rationale for this approach in both the Methods and Discussion sections.

Additionally, we updated all taxonomic names to align with current nomenclature (LPSN), ensured all genera and species names were italicized, and corrected graphical elements. Figure 2 was replaced with box plots to better reflect inter-individual variability and statistical patterns, and Figure 3 was improved for readability and analytical accuracy. We clarified inconsistencies in text-to-figure descriptions, removed ambiguous terminology (e.g., “host-associated factors”), and edited the results and discussion to reflect accurate metadata associations. These revisions, along with the updated conclusion and more robust interpretation of Clostridium perfringens gene profiles, enhance the scientific rigor and transparency of our study.

Below is a detailed summary of the main changes made in response to the reviewer’s comments:

1. Section “2.1. Study Site, Sample Collection, and Processing” should be revised. The authors need to present the overall design of the study, which would explain the information on the number of animals taken into the analysis, the quantitative composition of the groups - by outdoor activity, age, sex and diet; how often samples were collected - since the terminology "early" samples are encountered in the work.

Answer: Thank you for this observation. The study included 10 individuals whose fecal samples met the required DNA concentration for sequencing. The cats were categorized based on outdoor access (5 with access, 5 without), sex (3 males, 7 females), age group (young, adult), and diet type (processed, raw). This information has been incorporated into a revised summary of the study design. We also clarified that only one fecal sample was collected per animal during the July–August 2023 period. Sample collection was conducted once per individual. The term "early samples" referred to samples G1–G4, based on their order of collection, and we have revised the terminology to avoid confusion.

2. L.101-103: Authors were asked to provide information on who performed the amplicon library and metagenomic analysis. If the authors performed these analyses themselves, the description of the experimental work should be detailed. Additionally, it is unclear how many samples were used for the amplicon library or metagenomic analysis. If the samples for metagenomic analysis were pooled by 5 µl to a volume of 30 µl, were there 6 samples out of 10 taken in total? Then it is necessary to justify such a methodological approach, why and on what basis the samples were combined that differed so greatly in the composition of the microbiota, as the authors describe and prove in Fig. 3.

Answer: The amplicon library preparation and shotgun metagenomic sequencing were performed by the technical team at Novogene (https://www.novogene.com/us-en/services/research-services/genome-sequencing/). For the metabarcoding analysis, all 10 DNA samples, each extracted individually from a different domestic cat, were processed independently. In the case of shotgun metagenomic analysis, the 10 samples were pooled by combining 5 µL from each, resulting in a total volume of 50 µL. This pooling strategy was adopted due to budgetary limitations and the exploratory nature of the study. Our primary objective was to obtain a broad overview of the functional genetic potential—particularly antimicrobial resistance and virulence genes—present in the gut microbiome of the domestic cat population studied. By pooling equal volumes of DNA from each individual, we aimed to generate a representative consensus metagenome. We acknowledge that this approach may obscure inter-individual variability and introduce bias, especially considering the observed differences in microbial community composition described. This limitation has been explicitly addressed in the revised Discussion section, and the rationale for this methodological decision has been incorporated into the Methods section to provide greater transparency.

3. Throughout the text, the authors must provide all taxonomic names in accordance with the modern nomenclature: see https://www.bacterio.net/

Answer: All bacterial taxonomic names in the manuscript have been revised to comply with the modern nomenclature standards as outlined by the List of Prokaryotic names with Standing in Nomenclature (LPSN, https://www.bacterio.net/). The phylum Proteobacteria has been updated to Pseudomonadota. Similarly, Cyanobacteria has been corrected to Cyanobacteriota, and Chloroflexi to Chloroflexota, in line with recent taxonomic revisions. The phylum Firmicutes has also been revised to Bacillota, reflecting the restructuring of this group in which major classes such as Bacilli and Clostridia are now classified under Bacillota. In addition, the species Clostridium difficile has been updated to Clostridioides difficile, following its reclassification based on phylogenomic evidence. All updates were made consistently throughout the manuscript in the text, tables, and figure captions. Where appropriate, we retained the former names in parentheses at first mention to aid reader understanding. These revisions ensure full alignment with current LPSN and GTDB guidelines.

4. Throughout the text, the authors must correct the spelling of all genera and species names used for bacteria in italics.

Answer: All genera and species names of bacteria are now correctly italicized throughout the text.

5. Figure 2. “The top 10 bacterial taxa by average relative abundance”. If the figures indicate average values, corrections must be made and the SD must be indicated.

Answer: the original bar plot displaying the average relative abundance of the top 10 bacterial taxa was removed and replaced with a box plot representing the log-transformed read counts per sample. While the bar plot provided a simplified overview of mean abundance, it did not adequately reflect the variability across individual samples or the presence of outliers. By contrast, the box plot offers a more informative visualization of the distribution of read counts for each taxon. It allows the reader to assess differences in spread, central tendency (median), and variability across samples. The use of a log₁₀ transformation helps normalize highly skewed distributions and improves interpretability, especially in the context of metagenomic data where certain taxa may dominate in only a few samples. This change enhances the statistical robustness and transparency of the data presentation and aligns with the reviewers’ emphasis on accurate and detailed result interpretation.

6. Section “3.1.2. Variation in Relative Abundance of Dominant Bacterial Taxa According to Metadata Variables” needs to be completely rechecked and corrections made according to the figure. For example, the authors write: "It shows that Peptoclostridium … have a higher abundance in individuals without outdoor access", but according to Figure 3A, four out of the six taxa that were identified in representatives without Outdoor Access, the values for this taxon are low. 

The authors wrote "In contrast, Holdemanella, Clostridium perfringens, and Megasphaera elsdenii exhibit relatively higher abundance in individuals with outdoor access", but the taxon Peptoclostridium has the highest values, according to Figure 3A. 

There are even more inconsistencies in the descriptions of Figures 3B, 3C, and 3D.

1. 209-210: Not correct for Megamonas. This taxon is distinguished only among females. 
2. 215-217: Does not correspond to the figure at all: the first is more represented in young individuals, as in the two taxa described above, while the next two are detected only in young cats or adults.
3. 220: It is necessary to explain how these percentages were calculated. Does not correspond to the figure.

Thank you for your observation regarding Figure 3. In response, we revised the graphical representation by replacing the bar plots with box plots using log₁₀-transformed read counts. This modification was made to enhance the clarity and statistical depth of the analysis. Bar plots, as originally presented, displayed only the mean relative abundance of each bacterial taxon within metadata categories (e.g., outdoor access, gender, age, and diet). While useful for a general overview, bar plots are limited in that they mask important information such as variability within groups, the presence of outliers, and zero-inflation, common characteristics in microbiome datasets. To address this limitation, we adopted box plots, which allow for a more comprehensive visualization of the data by showing: The median and interquartile range (IQR), offering a better sense of central tendency and spread; Outliers and distribution tails, revealing individual variation; and The full distribution of log-transformed read counts, thereby normalizing scale differences and making patterns more interpretable. This change enables a more accurate depiction of colonization patterns, highlights taxa that are conditionally rare or inconsistently detected, and supports a more reliable comparison across metadata categories. We believe this approach aligns better with current standards in microbiome data visualization and improves the scientific rigor of the manuscript. The figure legend and results section were updated accordingly to reflect this change. We hope this modification addresses your concerns and enhances the clarity of our findings.

7. Figure 3. Why is it written that top-5, although information on 7 taxa is presented?

Answer: The issue was resolved.

8. Gene names should be written in italics.

Answer: The issue was resolved.

9. L. 323-325: What do “early samples” mean? Is this somehow related to the age of the animals? This information is not presented in the results of our own research.

Answer: The issue was resolved. The term "early samples" referred to samples G1–G4, based on their order of collection, and we have revised the terminology to avoid confusion

10. L. 343: It is incorrect to use the old taxonomy of Cyanobacteria because at present, this phylum has been specifically described in the context of gut studies. 

Answer: The issue was resolved.

11. L. 354, 363, and further throughout the text: All average values should be presented as mean and SD.

Answer: The original bar plot displaying the average relative abundance of the top 10 bacterial taxa was removed and replaced with a box plot representing the log-transformed read counts per sample.

12. L. 395: The authors did not analyze host-associated factors in the article.

Answer: We have revised the text to clarify that although we analyzed metadata variables (age, sex, diet, outdoor access), we did not directly assess internal host physiology or immune parameters. The term "host-associated factors" has been removed or clarified where appropriate.

13. L. 405-406: not “elevated”, but “present only”. 

Answer: The issue was resolved.

14. L. 416-417: Incorrect, since Megamonas only occurs in females, while Megasphaera, namely Megasphaera elsdenii according to the fig. 3B is higher in males.

Answer: The issue was resolved.

General note: The authors must carefully review the entire text of the manuscript and align the description of the results and visualization.

Answer: We carefully revised all figure legends, captions, and corresponding text descriptions for full consistency and accuracy.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study highlights the applied metagenomic analysis of domestic cat feces from Cali, Colombia. There are several points to consider:

1. In the abstract section, please add more information about the cats studied (number, age range, study location).
2. In the abstract section, please also include a final conclusion of the study, whether the composition of the gut microbiota was affected by age, gender, or diet in the study, and what the final recommendation of the study is.
3. In the introduction, it is important to point out the actual value of studying the gut microbiota in cats, especially since they are among the animals that most closely accompany humans and affect their health. It is essential to diagnose the conditions they are exposed to that may negatively affect their owners.
4. In the Materials and Methods section and under the figures, the number of samples studied or the number of cats from which the samples were taken should be stated.
5. How many males and females were? What was the diets of the cats studied? How was outdoor access determined? Where is the data on these factors studied?
6. Line 146, "10 fecal" What are these samples? What are groups 1 to 13 mentioned in the results and Figure 1? This should be explained in the Materials and Methods section.
7. The abstract section stated that it would evaluate how diet, age, gender, and access to fresh air influence gut microbiome composition. These factors were not well defined in the study, and therefore, it was not sufficiently demonstrated how gut microbiota composition varied according to these factors. What are processed foods and natural foods? Further clarification and explanation are needed in the discussion section on how age, gender, diet, and access to outdoor air affect the composition of the gut microbiota.
8. How can the identification of antibiotic resistance and virulence genes in Clostridium perfringensbe practically applied? Please explain in the discussion section.
9. Please re-edit the conclusion section, focusing on the results and their evidence.
10. line 513, "Ultimately, this study not only contributes to the expanding field of microbiome science but also highlights the importance of including underrepresented geographic regions in global microbial research efforts", Did the geographic region affect the study results? Are comparisons made between different geographic regions?

 

Comments for author File: Comments.pdf

Author Response

1. In the abstract section, please add more information about the cats studied (number, age range, study location).

Response 2: Thank you for your suggestion. The requested changes have been implemented and are now reflected in lines 15 to 17 of the revised manuscript.

1. In the abstract section, please also include a final conclusion of the study, whether the composition of the gut microbiota was affected by age, gender, or diet in the study, and what the final recommendation of the study is.

Response 2: Thank you for your suggestion. The requested changes have been implemented and are now reflected in lines 25 to 29 of the revised manuscript.

1. In the introduction, it is important to point out the actual value of studying the gut microbiota in cats, especially since they are among the animals that most closely accompany humans and affect their health. It is essential to diagnose the conditions they are exposed to that may negatively affect their owners.

Response 3: Thank you for your suggestion. The requested changes have been implemented and are now reflected in lines 38 to 42 of the revised manuscript.

1. In the Materials and Methods section and under the figures, the number of samples studied or the number of cats from which the samples were taken should be stated.

Response 4; Thank you for your suggestion. This information has already been included in the revised manuscript, both in the Materials and Methods section and in the figure captions.

1. How many males and females were? What was the diets of the cats studied? How was outdoor access determined? Where is the data on these factors studied?

Response 5: Thank you for raising these points. Additional details regarding sex, diet, and outdoor access have been incorporated into the revised manuscript. Specifically, the sample included 7 female and 3 male cats. Diet was categorized as either processed (commercial dry or wet food) or raw (meat-based, home-prepared meals), based on owner-reported information. Outdoor access was determined through a structured survey, classifying cats as either having regular access to outdoor environments (n = 5) or being kept exclusively indoors (n = 5). This metadata is now described in the “Metadata Collection” subsection of the Materials and Methods section and also referenced in the Results and Discussion where relevant. Line 99 - 104

1. Line 146, "10 fecal" What are these samples? What are groups 1 to 13 mentioned in the results and Figure 1? This should be explained in the Materials and Methods section.

Response 6: Thank you for this observation. We have clarified in the Materials and Methods section that 14 samples were initially collected, but only 10 fecal samples met the minimum DNA concentration required for sequencing. These were labeled G1, G4, G5, G8, G9, G10, G11, G12, G13, and G14. The remaining samples (G2, G3, G6, G7) were excluded from further analysis. This explanation has been added to the Sample Collection subsection to improve clarity. Line 99 - 107

1. The abstract section stated that it would evaluate how diet, age, gender, and access to fresh air influence gut microbiome composition. These factors were not well defined in the study, and therefore, it was not sufficiently demonstrated how gut microbiota composition varied according to these factors. What are processed foods and natural foods? Further clarification and explanation are needed in the discussion section on how age, gender, diet, and access to outdoor air affect the composition of the gut microbiota.

Response 7: We have revised both the Materials and Methods and Discussion sections to better define and discuss the evaluated factors. Age was categorized into young (8–24 months) and adult (>24 months). Sex, outdoor access (yes/no), and diet type (processed vs. raw) were recorded through owner surveys. Processed food refers to commercial dry or wet cat food, while raw food includes home-prepared, meat-based diets. The influence of these variables on gut microbiota composition is now more thoroughly addressed in the Discussion section, where we report that diet and outdoor access showed stronger associations with microbial composition than age and gender.

1. How can the identification of antibiotic resistance and virulence genes in Clostridium perfringensbe practically applied? Please explain in the discussion section.

Response 8: A new paragraph was added to the Discussion section explaining the significance of detecting resistance and virulence genes. These findings have potential applications in veterinary care, particularly in monitoring antimicrobial resistance in domestic animals and assessing zoonotic risk. This information contributes to understanding how companion animals may serve as reservoirs for resistance determinants within household environments. Line 551-559

1. Please re-edit the conclusion section, focusing on the results and their evidence.

Response 9: The conclusion has been revised to directly reflect the key findings of the study, emphasizing the evidence-based impact of diet and outdoor access on gut microbiota, the detection of potential zoonotic genes, and the importance of broadening microbiome studies to include data from underrepresented regions such as Latin America.Line 627 - 632

1. line 513, "Ultimately, this study not only contributes to the expanding field of microbiome science but also highlights the importance of including underrepresented geographic regions in global microbial research efforts", Did the geographic region affect the study results? Are comparisons made between different geographic regions?

Response 10: We acknowledge the reviewer’s point. The sentence has been revised for accuracy. While direct comparisons between geographic regions were not conducted in this study, we highlight that this research provides valuable baseline data from an underrepresented region, which is essential for building a more inclusive and diverse understanding of the global microbiome landscape.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript addresses a highly relevant and timely topic concerning the gut microbiome of domestic cats, in the context of environmental, dietary, and epidemiological factors. The authors employ two complementary methodologies—16S rRNA gene sequencing and shotgun metagenomics—to comprehensively profile the microbial communities and assess their functional potential. The study was conducted in Colombia, a region underrepresented in global microbiome databases, which adds significant value to the scientific community. The approach aligns well with the One Health framework, integrating animal, environmental, and public health perspectives.

The manuscript follows a classical structure with a clearly defined aim, and the conclusions are well supported by the presented results. Overall, I find the work to be scientifically sound and particularly valuable due to its regional novelty, as this is the first such metagenomic study performed on feline fecal samples in Cali, Colombia. It offers new data relevant to global microbiome research and zoonotic risk assessment.

I have no major concerns regarding the figures; however, Figure 3 is nearly unreadable in its current form. The labels are too small and only legible after significantly enlarging the file. I strongly recommend improving the resolution and adjusting the font size for better clarity and accessibility.

The authors successfully detected antimicrobial resistance genes (ErmG, ErmQ) and virulence factors (pfoA, plc, colA, among others). However, the interpretation remains somewhat superficial. The manuscript does not indicate whether these genes were associated with specific environmental or dietary factors, nor does it specify in how many samples they were detected. I recommend adding this information—ideally in a summary table—since it would greatly enhance the value and interpretability of the findings.

As the authors themselves correctly note, future studies should include a larger number of animals to increase the statistical power and generalizability of the findings. Moreover, I would like to highlight the labor-intensive and logistically challenging nature of collecting samples during home visits. I recognize the likely long waiting times and unpredictability associated with feline defecation, which adds to the methodological complexity and demonstrates the authors’ strong commitment to quality data collection.

Author Response

We sincerely thank the reviewer for their thoughtful and constructive feedback, which helped us significantly improve the clarity, depth, and impact of our manuscript. In response to the comments, we enhanced the resolution and readability of Figure 3 by increasing its font size and overall image quality. We also addressed the interpretation of antimicrobial resistance and virulence genes by adding a detailed explanation in the Discussion section. While direct associations with metadata variables could not be established due to the pooled nature of the metagenomic sequencing, we elaborated on indirect relationships supported by 16S rRNA data—particularly the higher abundance of Clostridium perfringens in raw-fed and outdoor-access cats.

Furthermore, we included a new discussion paragraph that outlines three testable hypotheses regarding the ecological dynamics of C. perfringens, virulence traits, and AMR genes about lifestyle and environmental factors. These additions aim to strengthen the scientific value of the study and clarify its relevance within a One Health context. We also acknowledge the logistical challenges of sample collection and appreciate the reviewer’s recognition of the efforts involved.

Below is a detailed summary of the main changes made in response to the reviewer’s comments:

 

This manuscript addresses a highly relevant and timely topic concerning the gut microbiome of domestic cats, in the context of environmental, dietary, and epidemiological factors. The authors employ two complementary methodologies—16S rRNA gene sequencing and shotgun metagenomics—to comprehensively profile the microbial communities and assess their functional potential. The study was conducted in Colombia, a region underrepresented in global microbiome databases, which adds significant value to the scientific community. The approach aligns well with the One Health framework, integrating animal, environmental, and public health perspectives.

The manuscript follows a classical structure with a clearly defined aim, and the conclusions are well supported by the presented results. Overall, I find the work to be scientifically sound and particularly valuable due to its regional novelty, as this is the first such metagenomic study performed on feline fecal samples in Cali, Colombia. It offers new data relevant to global microbiome research and zoonotic risk assessment.

I have no major concerns regarding the figures; however, Figure 3 is nearly unreadable in its current form. The labels are too small and only legible after significantly enlarging the file. I strongly recommend improving the resolution and adjusting the font size for better clarity and accessibility.

Answer: Thank you for the observation. The figures have been carefully reviewed, and both the resolution and font size have been improved to ensure better clarity and readability.

 

The authors successfully detected antimicrobial resistance genes (ErmG, ErmQ) and virulence factors (pfoA, plc, colA, among others). However, the interpretation remains somewhat superficial. The manuscript does not indicate whether these genes were associated with specific environmental or dietary factors, nor does it specify in how many samples they were detected. I recommend adding this information—ideally in a summary table—since it would greatly enhance the value and interpretability of the findings.

Answer: It is important to clarify that, due to the pooled nature of the shotgun metagenomic sequencing, it is not possible to establish direct associations between specific antimicrobial resistance or virulence genes and individual metadata variables such as diet, outdoor access, age, or sex. Since the DNA from all ten fecal samples was combined into a single metagenomic library, individual-level resolution was lost, preventing precise attribution of genetic elements to specific cats or metadata categories. However, indirect hypotheses can still be proposed based on the taxonomic analysis conducted through 16S rRNA gene sequencing, which was performed on individual samples. In particular, Clostridium perfringens—the species in which multiple resistance and virulence genes were detected in the pooled metagenomic sample—was more abundant in cats that were raw-fed and had outdoor access. This pattern suggests that these lifestyle and environmental factors may play a role in promoting colonization by potentially pathogenic and antimicrobial-resistant strains. Although this cannot be confirmed with the current pooled data, the correlation between C. perfringens abundance and specific metadata categories provides a rationale for the observed functional gene content and supports the development of testable hypotheses in future studies with individual-level metagenomic sequencing.

The following information was included in the discussion: “The findings of this exploratory study generate several testable hypotheses regarding the ecological and epidemiological dynamics of Clostridium perfringens and its associated genetic determinants within the feline gut microbiome. First, given the observed association between higher C. perfringens abundance and raw feeding, one hypothesis is that raw diets in domestic cats increase the risk of colonization by toxigenic and antimicrobial-resistant strains of C. perfringens. This could be due to direct bacterial contamination of raw animal products or the absence of processing steps that would normally reduce microbial loads. Second, the presence of antimicrobial resistance genes (ErmG, ErmQ) and virulence factors (pfoA, plc, colA, nanJ, nagI) in cats with outdoor access suggests the hypothesis that environmental exposure may act as a significant vector for the acquisition of resistance and virulence traits. This may occur through contact with contaminated soil, water, or other animals carrying resistant or pathogenic microorganisms. A third hypothesis is that cats living under combined conditions of raw feeding and outdoor access represent a higher-risk reservoir for zoonotic transmission of clinically relevant bacteria and genes, which could have implications for both veterinary and public health. Additionally, the absence of clear patterns related to age and sex may suggest that diet and environment are more influential factors than host biology in shaping the gut resistome and virulome, though larger-scale studies would be needed to confirm this. These hypotheses warrant further investigation through longitudinal studies with larger sample sizes and individual level metagenomic sequencing to assess causality and transmission dynamics”.

 

As the authors themselves correctly note, future studies should include a larger number of animals to increase the statistical power and generalizability of the findings. Moreover, I would like to highlight the labor-intensive and logistically challenging nature of collecting samples during home visits. I recognize the likely long waiting times and unpredictability associated with feline defecation, which adds to the methodological complexity and demonstrates the authors’ strong commitment to quality data collection.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised version of the manuscript under review generally complies with the earlier recommendations. However, the authors should correct and clarify the following comments that were not taken into account in the revised version.

1. The article uses the terms "later samples" (L. 195) and "earlier samples" (L. 387); however, the authors do not indicate the indicators by which the samples were divided into these two categories in the description of the methods. It is necessary to clarify and make additions to the text.

2. L. 191: Clarify the name of sample G6; there is no such sample in Table 1, so clarify and make corrections.

3. L. 197, 393, 399: Replace "G1 to G4" and "G1–G4" with "G1 and G4" because G2 and G3 samples are not included in the study according to Table 1.

4. L. 232, 269, 277, 294, 297, 375, 443, 462, 468, 470, 509, 558, 597: Replace Clostridium perfringens with C. perfringens. According to the rules of writing bacterial nomenclature, when a bacterial species is mentioned repeatedly, the genus name is abbreviated to the first letter. Exceptions may be made for names used in figures, tables, and titles of sections and subsections. 

5. L. 240, 293, 394, 516: Bacteroides, Clostridium perfringens, Bifidobacterium, Erm – write italic.

The authors write the names of bacterial phyla in italics in the abstract and in regular font in the main text of the manuscript. It should be written uniformly. There is no strict rule for writing the name of bacterial taxa: you can write in regular font and in italics, and you only need to choose a single option for the entire text of the manuscript.

6. L. 386: the authors indicate an obsolete name for the taxon Bacteroidota (formerly Bacteroidetes). It is advisable to introduce uniformity into the text and give obsolete names for all taxa as they are mentioned or to remove this description from the text.

After correcting the comments, the manuscript can be recommended for publication.

Author Response

1. The article uses the terms "later samples" (L. 195) and "earlier samples" (L. 387); however, the authors do not indicate the indicators by which the samples were divided into these two categories in the description of the methods. It is necessary to clarify and make additions to the text.

Response 1: We appreciate the reviewer’s observation. The term “later samples” has been removed from the manuscript to avoid confusion, as it did not correspond to any biological or experimental grouping but rather to the sample indexing order established at the beginning of the study. Initially, 14 fecal samples were collected; however, only 10 of them met the minimum DNA concentration required for high-throughput sequencing. The retained samples included: G1, G4, G5, G8, G9, G10, G11, G12, G13, and G14. Samples G2, G3, G6, and G7 were excluded from the analysis due to insufficient DNA yield. Since the distinction between “earlier” and “later” samples was only related to the original indexing and not to any biological or temporal factor, we believe this classification does not influence the results or conclusions of the study. We have revised the text accordingly for greater clarity.

2. 191: Clarify the name of sample G6; there is no such sample in Table 1, so clarify and make corrections.

Response 2: The suggested corrections have been implemented. The text was revised and rewritten from lines 188 to 199 to provide a clearer description of the samples included in the study-

3. 197, 393, 399: Replace "G1 to G4" and "G1–G4" with "G1 and G4" because G2 and G3 samples are not included in the study according to Table 1.

Response 3: The suggested corrections have been implemented. The text was revised and rewritten from lines 386 to 399 to provide a clearer description of the samples included in the study

4. 232, 269, 277, 294, 297, 375, 443, 462, 468, 470, 509, 558, 597: Replace Clostridium perfringens with C. perfringens. According to the rules of writing bacterial nomenclature, when a bacterial species is mentioned repeatedly, the genus name is abbreviated to the first letter. Exceptions may be made for names used in figures, tables, and titles of sections and subsections.

Response 4: The suggested corrections have been implemented 

5. L. 240, 293, 394, 516: Bacteroides, Clostridium perfringens, Bifidobacterium, Erm – write italic.

The authors write the names of bacterial phyla in italics in the abstract and in regular font in the main text of the manuscript. It should be written uniformly. There is no strict rule for writing the name of bacterial taxa: you can write in regular font and in italics, and you only need to choose a single option for the entire text of the manuscript.

Response 5: The suggested corrections have been implemented 

5. 386: the authors indicate an obsolete name for the taxon Bacteroidota (formerly Bacteroidetes). It is advisable to introduce uniformity into the text and give obsolete names for all taxa as they are mentioned or to remove this description from the text.

Response 6:The suggested corrections have been incorporated into Figure 1.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The proposed amendments have been made and the inquiries have been answered. 

Author Response

1. In the abstract section, please add more information about the cats studied (number, age range, study location).

Response 2: Thank you for your suggestion. The requested changes have been implemented and are now reflected in lines 15 to 17 of the revised manuscript.

1. In the abstract section, please also include a final conclusion of the study, whether the composition of the gut microbiota was affected by age, gender, or diet in the study, and what the final recommendation of the study is.

Response 2: Thank you for your suggestion. The requested changes have been implemented and are now reflected in lines 25 to 29 of the revised manuscript.

1. In the introduction, it is important to point out the actual value of studying the gut microbiota in cats, especially since they are among the animals that most closely accompany humans and affect their health. It is essential to diagnose the conditions they are exposed to that may negatively affect their owners.

Response 3: Thank you for your suggestion. The requested changes have been implemented and are now reflected in lines 38 to 42 of the revised manuscript.

1. In the Materials and Methods section and under the figures, the number of samples studied or the number of cats from which the samples were taken should be stated.

Response 4; Thank you for your suggestion. This information has already been included in the revised manuscript, both in the Materials and Methods section and in the figure captions.

1. How many males and females were? What was the diets of the cats studied? How was outdoor access determined? Where is the data on these factors studied?

Response 5: Thank you for raising these points. Additional details regarding sex, diet, and outdoor access have been incorporated into the revised manuscript. Specifically, the sample included 7 female and 3 male cats. Diet was categorized as either processed (commercial dry or wet food) or raw (meat-based, home-prepared meals), based on owner-reported information. Outdoor access was determined through a structured survey, classifying cats as either having regular access to outdoor environments (n = 5) or being kept exclusively indoors (n = 5). This metadata is now described in the “Metadata Collection” subsection of the Materials and Methods section and also referenced in the Results and Discussion where relevant. Line 99 - 104

1. Line 146, "10 fecal" What are these samples? What are groups 1 to 13 mentioned in the results and Figure 1? This should be explained in the Materials and Methods section.

Response 6: Thank you for this observation. We have clarified in the Materials and Methods section that 14 samples were initially collected, but only 10 fecal samples met the minimum DNA concentration required for sequencing. These were labeled G1, G4, G5, G8, G9, G10, G11, G12, G13, and G14. The remaining samples (G2, G3, G6, G7) were excluded from further analysis. This explanation has been added to the Sample Collection subsection to improve clarity. Line 99 - 107

1. The abstract section stated that it would evaluate how diet, age, gender, and access to fresh air influence gut microbiome composition. These factors were not well defined in the study, and therefore, it was not sufficiently demonstrated how gut microbiota composition varied according to these factors. What are processed foods and natural foods? Further clarification and explanation are needed in the discussion section on how age, gender, diet, and access to outdoor air affect the composition of the gut microbiota.

Response 7: We have revised both the Materials and Methods and Discussion sections to better define and discuss the evaluated factors. Age was categorized into young (8–24 months) and adult (>24 months). Sex, outdoor access (yes/no), and diet type (processed vs. raw) were recorded through owner surveys. Processed food refers to commercial dry or wet cat food, while raw food includes home-prepared, meat-based diets. The influence of these variables on gut microbiota composition is now more thoroughly addressed in the Discussion section, where we report that diet and outdoor access showed stronger associations with microbial composition than age and gender.

1. How can the identification of antibiotic resistance and virulence genes in Clostridium perfringensbe practically applied? Please explain in the discussion section.

Response 8: A new paragraph was added to the Discussion section explaining the significance of detecting resistance and virulence genes. These findings have potential applications in veterinary care, particularly in monitoring antimicrobial resistance in domestic animals and assessing zoonotic risk. This information contributes to understanding how companion animals may serve as reservoirs for resistance determinants within household environments. Line 551-559

1. Please re-edit the conclusion section, focusing on the results and their evidence.

Response 9: The conclusion has been revised to directly reflect the key findings of the study, emphasizing the evidence-based impact of diet and outdoor access on gut microbiota, the detection of potential zoonotic genes, and the importance of broadening microbiome studies to include data from underrepresented regions such as Latin America.Line 627 - 632

1. line 513, "Ultimately, this study not only contributes to the expanding field of microbiome science but also highlights the importance of including underrepresented geographic regions in global microbial research efforts", Did the geographic region affect the study results? Are comparisons made between different geographic regions?

Response 10: We acknowledge the reviewer’s point. The sentence has been revised for accuracy. While direct comparisons between geographic regions were not conducted in this study, we highlight that this research provides valuable baseline data from an underrepresented region, which is essential for building a more inclusive and diverse understanding of the global microbiome landscape.

Author Response File: Author Response.pdf