Shared Core and Host Specificities of Culturable Pathogenic Yeast Microbiome in Fresh and Dry Feces of Five Synanthropic Wild Birds (Rock Pigeon, European Starling, White Wagtail, Great Tit and House Sparrow)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Journal: Birds (ISSN 2673-6004)

Manuscript ID: birds-3565044

Type: Article

Title: Shared core and host specificities of culturable pathogenic yeast microbiome in fresh and dry feces of five synanthropic wild birds (Rock Pigeon, European Starling, White Wagtail, Great Tit and House Sparrow)

Authors: Anna Glushakova * , Aleksey Kachalkin

The study focused on comparing the diversity of ascomycetous, po-tentially pathogenic yeast species (Candida spp.) in the fresh and dry feces of five synanthropic birds (Rock Pigeon, European Starling, White Wagtail, Great Tit and House Sparrow). While this work is valuable, several concerns were raised throughout the text and need to be clarified by the authors before further consideration.

Please refer to the comments in the PDF file and revise accordingly before further consideration.

 

Comments for author File: Comments.pdf

Author Response

First of all, we would like to thank the Reviewer for the work, the valuable comments and the time.

Change to-->shoe soles carrying contaminants indoors

Corrected.

The abstract's opening paragraph is too generic. It lacks specific research aims and does not clearly state the hypotheses tested.

In our humble opinion, the aim of the study is concentrated in the Simple Summary. In the Abstract, we wanted to show that our small study is in the mainstream of research on many aspects related to public health.

Clarify whether Brilliance Candida Agar can reliably isolate all relevant Candida spp., or if it has known limitations in sensitivity.

We agree that the media we have chosen, like any other, has its limitations and disadvantages. These are stated by the manufacturer and determined by experienced researchers over many years of work. In all doubtful situations we have used controls and molecularly identified the strains. We have added information about some limitations of the media to the methods. Our previous study with pigeon droppings, using non-selective media, yielded similar Candida species (Glushakova, A. M., Rodionova, E. N., & Kachalkin, A. V. (2021). Yeasts in Feces of Pigeons (Columba livia) in the City of Moscow. Current Microbiology, 78(1), 238-243.). This gives us reason to believe that our results are quite relevant. In this work, we have intentionally limited the ability to isolate saprotrophic Candida species in order to focus on the potentially pathogenic ascomycetous species.

consider adding Oxford comma: “Great Tit, and House Sparrow”

Corrected.

Requires statistical backing in the abstract, not just later in the results

In our humble opinion, this information is logically arranged in the results. The significance criteria are also stated in the methods. The phrase “no significant differences” we used is familiar to readers and implies that we did not find any statistically significant differences in our work on this aspect.

better phrased as “pose a contamination risk”

Corrected.

You state that Uppsala samples contained Candida auris, yet this species is not reported in your own dataset—please clarify why it is referenced so heavily.

The information has been added. Thank you.

"Feces rapidly lose valuable substances" is vague—quantify or reference the claim about nitrogen loss impacting yeast survival.

The Introduction has been corrected and this vague sentence has been deleted. Thank you.

The study areas are broad and diverse—was any stratification done to avoid sampling bias from highly contaminated or urban-heavy sites?

The information has been added.

Please clarify whether the observed birds were definitively linked to the droppings, or if misidentification of species was possible (e.g., for small passerines).

The information has been added.

Brilliance Candida Agar cannot distinguish some yeast species—was this validated with all isolates, or only phenotypically atypical.

The information has been added.

Were storage conditions (humidity, contamination control) between fresh and dry samples rigorously standardized? If not, results on aging may be biased.

Sterility control of the air and surfaces in the laminar box was performed once a week according to the schedule in the laboratory. The stability of the environmental conditions was monitored and maintained throughout the experiment.

For BLAST and MycoID results—include identity % cut-offs used for species-level identification. Was 99.8% threshold sufficient?

Yes, the threshold value meets the currently accepted criteria for the differentiation of yeasts: Vu, D.; Groenewald, M.; Szöke, S.; Cardinali, G.; Eberhardt, U.; Stielow, B.; de Vries, M.; Verkleij, G.J.M.; Crous, P.W.; Boekhout, T.; Robert, V. DNA barcoding analysis of more than 9 000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation. Stud. Mycol. 2016, 85 (1), 91–105. https://pubmed.ncbi.nlm.nih.gov/28050055/

NMDS and LDA are appropriate, but there’s no mention of sample rarefaction or normalization. This is important for microbial abundance data.

The information has been added. Thank you.

The result that certain yeasts were "only found" in fresh feces could be due to lower detection limits in old feces. Clarify whether absence was due to total lack of growth or drop below detection.

Of course, we cannot exclude the presence of errors and biases in our study. However, the large number of replicates and the similar trends found for the dry droppings of different bird species give us reason to believe that our results are relevant and reproducible.

Figure 2 is central to showing inter-species variation but lacks effect size estimates. Include them in the legend or main text.

Figure 2 has been corrected.

“except for the Rock Pigeon feces (p = 0.001)” → incomplete comparison; consider clarifying

The capture to the Figure has been corrected.

“seperatibility” → “separability”

Corrected.

color of points indicates the 'age' of feces” → improve clarity: “colors represent feces age”

Corrected.

The exception of Rock Pigeon 90-day feces retaining high P. kudriavzevii levels is interesting but speculative. Was this statistically tested?

High P. kudriavzevii level was statistically significant. The sentence was corrected.

Lack of citation, add.

The text has been corrected.

could be better as “investigated”

Corrected

You claim "shared core" pathogenic yeast microbiome—was this formally tested using core microbiome analysis?

We refer to a complex of potentially pathogenic ascomycetous yeasts found in the feces of all bird species examined.

The dietary observations are interesting, but anecdotal. If used as an explanatory factor, they should be backed with quantitative dietary data or citations.

The text has been corrected.

awkward phrasing; consider “in human-modified environments”

The term antropophyzed was suggested to us once by the Reviewer (for another study dedicated to anthropogenic influence). We find it rather accurate.

The suggestion that Arx. bovina is thermophilic and gut-adapted is speculative—clearly label this as a hypothesis or conduct follow-up studies.

The thermophilic properties of this species have been studied, we write about them above with references. And for the phylogenetically closest species, the possibilities for stimulating digestion in pigs have been demonstrated: Hu, J., Chen, J., Hou, Q., Xu, X., Ren, J., Ma, L., & Yan, X. (2023). Core-predominant gut fungus Kazachstania slooffiae promotes intestinal epithelial glycolysis via lysine desuccinylation in pigs. Microbiome, 11(1), 31.

PS Arxiozyma slooffiae = former Kazachstania slooffiae

The isolated strains from pigeon feces grew stably at +43, the type strain can grow at +45. Of course, we cautiously assume that this species is both thermophilic and belongs to the core mycobiome of the pigeon gut. However, we consider it wrong not to mention the identified fact. The information has been added to the text.

inconsistent abbreviation; clarify earlier

Corrected.

may be a mistranslation or incorrect; possibly meant “nematodes”

Corrected.

The link between Debaryomyces spp. and invertebrate diet is intriguing but tenuous without gut content confirmation.

The text has been corrected.

The sentence "This study could be a starting point..." reads more like grant-writing language. Refocus it on current study implications.

In our humble opinion, this sentence is important for the discussion and comparison of the data.

plural: “yeasts”

Corrected

The statement that “pigeons are most dangerous” needs to be more cautious—this is correlative, not causal, and could be sensationalized if misinterpreted.

The statement was corrected a bit. Thank you.

clearer as “particulate matter from dry feces”

Corrected.

“should become the absolute norm…” → overly informal; consider “should be encouraged as a standard practice…”

Corrected. Thank you.

The conclusion should clearly highlight the public health significance of pathogenic yeasts in bird feces, especially in urban settings. Moreover, include a statement on how these findings can inform urban hygiene policy or risk mitigation for vulnerable populations.

The conclusion has been corrected.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Overall, this is a very good manuscript addressing a topic of high relevance to public health and the One Health framework. The study focuses on an emerging and important issue, is based on a solid and replicable methodology, and provides results that merit publication. However, there are several points that should be addressed to improve clarity and scientific rigor before acceptance.

The abstract would benefit from more quantitative detail regarding the study population, including the number of birds sampled by species and location, as well as the total number of isolates recovered. This would help readers appreciate the scope and robustness of the study from the outset.

The introduction is comprehensive, well-supported by updated literature, and successfully frames the issue from a One Health perspective. Nonetheless, while the public health problem is convincingly established, the main objective of the study becomes somewhat diluted among multiple complementary hypotheses. I recommend closing the introduction with a concise paragraph clearly stating the main aim of the study (with optional secondary aims), independent of the subsequent discussion about feces aging.

The methods section is meticulous and a clear strength of the work. However, one specific detail could be improved for clarity. The statement regarding the temperature measurement of fresh feces with a pyrometer would fit more logically in the "Sampling" subsection, where defecation was directly observed. This would help to clarify that the samples were freshly excreted and reliably attributed to the correct bird species. On that note, more detail is needed on how species assignment of droppings was ensured in cases where birds from different species might have been present simultaneously. It is important to describe how these potential confounding factors were minimized.

A noteworthy methodological strength is the screening of the operators’ hands to confirm the absence of opportunistic yeasts before sampling. This is an excellent and rarely implemented contamination control measure that should be highlighted more prominently.

The study population should be described more clearly at the start of the methods section. For instance, stating upfront that 48 Rock Pigeon, 47 European Starling, 38 White Wagtail, 32 Great Tit, and 30 House Sparrow droppings were collected over nine days, and that 195 subsamples for each feces “age” category (fresh, 45 days, and 90 days old) were analyzed in triplicate, would help readers to quickly understand the study design.

There is some ambiguity regarding how the three swabs per fecal sample were treated in the statistical analysis. It is not clear whether these were considered technical or biological replicates. This point should be clarified to ensure there is no pseudoreplication and to explain how dependence between repeated measures was handled.

Although beta diversity is thoroughly analyzed using NMDS and Bray–Curtis dissimilarity, and species-specific differences are evaluated with Kruskal–Wallis and Dunn’s tests, the frequent use of the term "diversity" throughout the manuscript suggests that alpha diversity should also be explicitly addressed. Including indices such as Chao-1 for richness and Shannon for evenness would significantly strengthen the interpretation of community structure.

In the statistical methods section, “liner discriminant analysis” should be corrected to “linear discriminant analysis (LDA).” While minor, such terminology errors should be avoided in a scientific manuscript.

There are some inconsistencies in taxonomic nomenclature. After the first full mention of a genus name, it should be abbreviated to a single letter (e.g., N. glabratus instead of Nak. glabratus), in line with standard conventions. This inconsistency appears in the results section and should be revised throughout.

Several figures appear pixelated upon enlargement. To ensure readability and professional presentation, figures should be replaced with higher-resolution versions (≥300 dpi) or vector images where possible.

The discussion is well-referenced and based on an extensive review of the literature. Nonetheless, its phrasing could be improved for clarity and tone. Informal expressions such as “It turned out that…” or starting sentences with “And…” (e.g., “And they were generally characterized…”) should be avoided. Phrases like “We find interesting…” are too vague and should be rephrased to state the actual observation or interpretation.

Regarding bird species names, these are repeatedly mentioned in full (and capitalized) throughout the manuscript. Using abbreviations, such as “European Starling (ES)”, could streamline the text and improve readability, especially in figure captions or repeated references.

Several hypotheses proposed in the discussion are intriguing, such as the link between diet and microbiota composition (e.g., pigeons consuming more anthropogenic “junk” food). However, the manuscript does not present direct evidence for this claim, such as systematic behavioral observations or diet analysis. These ideas should be framed clearly as speculative or future research directions rather than as established findings. For example, it could be rephrased as “Based on field observations, we hypothesize that...”

Additionally, the presence of Candida parapsilosis in large numbers among isolates suggests an opportunity to mention the emerging concern of antifungal resistance, particularly to fluconazole. Incorporating a brief comment on environmental acquisition of resistance would enhance the One Health implications of the study.

Finally, in discussing public health recommendations, the suggestion to leave shoes at the door is well noted. However, it would also be helpful to mention hand hygiene when handling footwear, especially in households with immunocompromised individuals.

The conclusions summarize the results effectively. However, they could be expanded slightly to include the main future research directions mentioned in the discussion and to re-emphasize the value of a One Health approach in studying fungal pathogens, particularly given the influence of climate change and global warming on fungal ecology and host-pathogen interactions.

Comments on the Quality of English Language

Some rephrasing is needed for clarity, especially through the Discussion section.

Author Response

First of all, we would like to thank the Reviewer for the work, the valuable comments and the time.

Overall, this is a very good manuscript addressing a topic of high relevance to public health and the One Health framework. The study focuses on an emerging and important issue, is based on a solid and replicable methodology, and provides results that merit publication. However, there are several points that should be addressed to improve clarity and scientific rigor before acceptance.

We would like to thank the Reviewer for the time, work, important comments and evaluation of our study.

The abstract would benefit from more quantitative detail regarding the study population, including the number of birds sampled by species and location, as well as the total number of isolates recovered. This would help readers appreciate the scope and robustness of the study from the outset.

The information has been added.

The introduction is comprehensive, well-supported by updated literature, and successfully frames the issue from a One Health perspective. Nonetheless, while the public health problem is convincingly established, the main objective of the study becomes somewhat diluted among multiple complementary hypotheses. I recommend closing the introduction with a concise paragraph clearly stating the main aim of the study (with optional secondary aims), independent of the subsequent discussion about feces aging.

Corrected.

The methods section is meticulous and a clear strength of the work. However, one specific detail could be improved for clarity. The statement regarding the temperature measurement of fresh feces with a pyrometer would fit more logically in the "Sampling" subsection, where defecation was directly observed. This would help to clarify that the samples were freshly excreted and reliably attributed to the correct bird species. On that note, more detail is needed on how species assignment of droppings was ensured in cases where birds from different species might have been present simultaneously. It is important to describe how these potential confounding factors were minimized.

Corrected. The information has been added.

A noteworthy methodological strength is the screening of the operators’ hands to confirm the absence of opportunistic yeasts before sampling. This is an excellent and rarely implemented contamination control measure that should be highlighted more prominently.

Corrected. The information has been added.

The study population should be described more clearly at the start of the methods section. For instance, stating upfront that 48 Rock Pigeon, 47 European Starling, 38 White Wagtail, 32 Great Tit, and 30 House Sparrow droppings were collected over nine days, and that 195 subsamples for each feces “age” category (fresh, 45 days, and 90 days old) were analyzed in triplicate, would help readers to quickly understand the study design.

Corrected. Thank you very much.

There is some ambiguity regarding how the three swabs per fecal sample were treated in the statistical analysis. It is not clear whether these were considered technical or biological replicates. This point should be clarified to ensure there is no pseudoreplication and to explain how dependence between repeated measures was handled.

Corrected.

Although beta diversity is thoroughly analyzed using NMDS and Bray–Curtis dissimilarity, and species-specific differences are evaluated with Kruskal–Wallis and Dunn’s tests, the frequent use of the term "diversity" throughout the manuscript suggests that alpha diversity should also be explicitly addressed. Including indices such as Chao-1 for richness and Shannon for evenness would significantly strengthen the interpretation of community structure.

Some information on alpha diversity has been added.

In the statistical methods section, “liner discriminant analysis” should be corrected to “linear discriminant analysis (LDA).” While minor, such terminology errors should be avoided in a scientific manuscript.

Corrected. I profusely apologize.

There are some inconsistencies in taxonomic nomenclature. After the first full mention of a genus name, it should be abbreviated to a single letter (e.g., N. glabratus instead of Nak. glabratus), in line with standard conventions. This inconsistency appears in the results section and should be revised throughout.

Corrected. I profusely apologize.

Several figures appear pixelated upon enlargement. To ensure readability and professional presentation, figures should be replaced with higher-resolution versions (≥300 dpi) or vector images where possible.

Corrected.

The discussion is well-referenced and based on an extensive review of the literature. Nonetheless, its phrasing could be improved for clarity and tone. Informal expressions such as “It turned out that…” or starting sentences with “And…” (e.g., “And they were generally characterized…”) should be avoided. Phrases like “We find interesting…” are too vague and should be rephrased to state the actual observation or interpretation.

Corrected.

Regarding bird species names, these are repeatedly mentioned in full (and capitalized) throughout the manuscript. Using abbreviations, such as “European Starling (ES)”, could streamline the text and improve readability, especially in figure captions or repeated references.

I think that's a reasonable solution, thank you. But since I'm not an ornithologist, I'm afraid of getting it wrong and would rather leave the full names.

Several hypotheses proposed in the discussion are intriguing, such as the link between diet and microbiota composition (e.g., pigeons consuming more anthropogenic “junk” food). However, the manuscript does not present direct evidence for this claim, such as systematic behavioral observations or diet analysis. These ideas should be framed clearly as speculative or future research directions rather than as established findings. For example, it could be rephrased as “Based on field observations, we hypothesize that...”

Corrected. Thank you very much.

Additionally, the presence of Candida parapsilosis in large numbers among isolates suggests an opportunity to mention the emerging concern of antifungal resistance, particularly to fluconazole. Incorporating a brief comment on environmental acquisition of resistance would enhance the One Health implications of the study.

The information has been added.

Finally, in discussing public health recommendations, the suggestion to leave shoes at the door is well noted. However, it would also be helpful to mention hand hygiene when handling footwear, especially in households with immunocompromised individuals.

The information has been added. Thank you very much.

The conclusions summarize the results effectively. However, they could be expanded slightly to include the main future research directions mentioned in the discussion and to re-emphasize the value of a One Health approach in studying fungal pathogens, particularly given the influence of climate change and global warming on fungal ecology and host-pathogen interactions.

We tried to improve the conclusion. Thank you very much for help.

 

Comments on the Quality of English Language

Some rephrasing is needed for clarity, especially through the Discussion section.

Corrected.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

My concerns were clearly addressed; no further comment.

Author Response

First of all, we would like to thank the Reviewer for the work, the valuable comments and the time.

My concerns were clearly addressed; no further comment.

Thank you very much for help.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have answered satisfactorily all my concerns, and the article is now suitable for publication after some minor English and style corrections throughout the manuscript. 

Author Response

First of all, we would like to thank the Reviewer for the work, the valuable comments and the time.

The authors have answered satisfactorily all my concerns, and the article is now suitable for publication after some minor English and style corrections throughout the manuscript.

Thank you very much for help. We tried to do our best to improve English and style.

Author Response File: Author Response.docx