Diversity of Culturable Yeasts in the Feces of Mew Gulls Breeding in Natural and Urban Habitats, with Insights into the Antifungal Susceptibility of the Observed Pathogens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The main objectives of the study are (1) to describe the microbial communities in feces during the breeding season (lines 87-89); (2) to compare the microbial communities between a natural and an anthropic environment (lines 90-93); (3) to describe the susceptibility of three yeasts to four common anti-microbials (lines 93-97). All three objectives were sufficiently accomplished. The main conclusion is that gulls breeding in areas with anthropogenic influences went to wintering areas with more pathogenic microorganisms than those breeding in natural environments with less anthropogenic influences.

The above conclusion postulates a logical problem that the authors do not address in the Discussion: How can it be explained that at the beginning of the breeding season (see Figs. 3 and 5, April) the feces of two gull species did not differ? If the gulls return with the same microbial communities, then somewhere during the wintering season they have been freed from pathogenic organisms and have recovered microorganisms typical of environments without anthropogenic influence. However, gulls have long since learnt to feed on landfill sites even in winter. It is therefore not clear how it is possible for the population in the anthropogenic environment to converge with the population in the non-anthropogenic environment during winter. This puzzling result needs to be explained in the discussion. If the same microbial communities are found in April, this might raise the question of the relevance of the topic of human impact on a longer time scale than the breeding season, for example the whole annual cycle.

Some reorganization of the text is required in the manuscript. For example, the introduction needs to provide a motivation for each of the three main objectives. However, the introduction to Objective 3 is too brief (lines 95-96). It needs to be expanded, for example by including several lines that are currently in the Discussion (lines 312-325, 330-338, 340-372). The Discussion will be reduced, but no matter: it must be improved by adding a comparative discussion of the results of the present study with other studies, whether conducted on gulls or other bird species.


Minor comments

Page 2, lines 63-67. These lines suggest that the finding of pathogenic yeasts in the feces of this gull species is an expected result. Add a few sentences to justify the reason for carrying out research that will confirm previous studies.

Page 2, lines 93-96. The third aim of the paper, namely to measure the susceptibility of three yeast species to four common antimicrobial agents, is not sufficiently justified in the introduction. My proposal is to include the lines that introduce the importance of C. parapsilosis and C. tropicalis (page 12, lines 312-325) and Rh. mucilaginosa (lines 330-342) to justify this objective in the introductory section. These lines seem more appropriate in the Introduction, right?

Page 4, lines 118-120. Table 1. Please comment on the values in this table in relation to the results. What was the purpose of measuring temperatures and wind speed to study microbes and their resistance against antimicrobial agents? Nothing was said about this in the Discussion I would say that they were measured to show that the differences in the results were not due to environmental differences between the colonies other than specifically natural vs. anthropogenic. However, it is not clear to me whether the differences in temperature and wind are analysed and discussed in the Results, Methods or Discussion. Please, comment about this Table in the Discussion.

Page 6, line 202. I'm not sure if it's three or four.

Page 9, line 234. Measuring both populations at the same time during the breeding season was a good idea. Kudos!

Page 11, lines 268, 270 and 276. Small percentages such as 10%, 6%, 8% and 4% may not be different from zero. Please add the 95% confidence interval (CI95%) to these values. Note that for the extreme percentages (<15%, >85%) the CI95% is not symmetrical. Symmetric intervals are usually only found for the middle percentages (e.g. 50%). When the sample size is very large, the skewness is reduced in small (<15%) or large (>85%) percentage intervals. In the case of this paper, it is very unlikely that the interval will be symmetrical. Also, the lower interval may not be significantly greater than zero, so it is worth checking. This is a minor aspect of the paper. However, it is important when comparing the results with other studies. Overall, to say that small percentages are different from zero requires a large sample size. Can you be sure that the 4 per cent that is reported in the manuscript is significantly greater than zero?

Page 12, lines 285-289. How do you know where the yeast comes from when growing in a natural environment? This study didn't measure it, right? Nor is the study that measured it quoted. It may be that this is a well-established fact and that as such it is in the textbooks. In any case, it is appropriate to add the source of the information contained in these lines.

Page 12, lines 327-330. Contrast these results with those obtained in other bird studies. Lines 330-338 are not for this purpose.

Page 13. Lines 340-342. These sentences justify the third main objective of this study: to describe the susceptibility of three yeast species to four routinely used anti-microbials (page 2, lines 93-97). I recommend that these sentences be relocated to the introduction.

Page 13, lines 343-354. The whole paragraph is a statement of results. Avoid repeating them. Especially if they are not commented on, discussed or compared with the results of other papers. For example, rather than repeating the results, try to put them in the context of other similar studies. It is desirable that the comparison is quantitative. Was the yeast resistance lower or higher (by how much?) than that found in other studies of antimicrobial resistance in bird feces collected from natural and anthropogenic sites? Lines 63-66 cite a number of papers with which you can compare your results in the Discussion, perhaps in the form of a table or a graph. Try to take the Discussion to a new upper level.

Page 13, lines 361-363. The change in yeast diversity over time is a very important point. Strangely, it is not mentioned in the abstract of the paper. Why not? Well, it is exactly where it should be, as it is one of the most important results. On the other hand, the other lines (lines 361 - 374) are not part of the conclusions, because they are the experimental results. They are summarised in the abstract. Rejected or accepted hypotheses are what the conclusions are about. Try to transfer the results to the abstract and remove them from section 5 "Conclusions". It may be that the abstract has reached its maximum word count. If so, as a compromise, try to reduce the first 14 lines of the conclusions to one or two sentences. For example: "During the breeding season, the yeast composition of excreta increases or decreases depending on whether the breeding colony is in a natural or anthropogenic environment. Yeasts in the anthropogenic environment are more resistant to several antimicrobials than yeasts in the natural environment."

Page 13, lines 375-377. These lines indicate a clear pattern, but in a hesitant way: "seem to be", "potentially". Perhaps the study does not allow you to say conclusively what these lines say. But if it does not, then it is not a conclusion. Improve the style.

Page 13, lines 377-379. Same comment as above: check this sentence to make sure that it is a conclusion that has been drawn from the results. The content of these lines 377 - 379 and the content of lines 357 - 359 are quite similar. Check these sentences. It is possible that the conclusion is already contained in reference [74] (line 359). If this is the case, this sentence would not be a conclusion of your manuscript.

Page 18, line 575. Typo: delete "    134" in the doi code

Page 18, line 580. Typo: delete "    140" in the doi code

Author Response

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

The main objectives of the study are (1) to describe the microbial communities in feces during the breeding season (lines 87-89); (2) to compare the microbial communities between a natural and an anthropic environment (lines 90-93); (3) to describe the susceptibility of three yeasts to four common anti-microbials (lines 93-97). All three objectives were sufficiently accomplished. The main conclusion is that gulls breeding in areas with anthropogenic influences went to wintering areas with more pathogenic microorganisms than those breeding in natural environments with less anthropogenic influences.

The above conclusion postulates a logical problem that the authors do not address in the Discussion: How can it be explained that at the beginning of the breeding season (see Figs. 3 and 5, April) the feces of two gull species did not differ? If the gulls return with the same microbial communities, then somewhere during the wintering season they have been freed from pathogenic organisms and have recovered microorganisms typical of environments without anthropogenic influence. However, gulls have long since learnt to feed on landfill sites even in winter. It is therefore not clear how it is possible for the population in the anthropogenic environment to converge with the population in the non-anthropogenic environment during winter. This puzzling result needs to be explained in the discussion. If the same microbial communities are found in April, this might raise the question of the relevance of the topic of human impact on a longer time scale than the breeding season, for example the whole annual cycle.

Some reorganization of the text is required in the manuscript. For example, the introduction needs to provide a motivation for each of the three main objectives. However, the introduction to Objective 3 is too brief (lines 95-96). It needs to be expanded, for example by including several lines that are currently in the Discussion (lines 312-325, 330-338, 340-372). The Discussion will be reduced, but no matter: it must be improved by adding a comparative discussion of the results of the present study with other studies, whether conducted on gulls or other bird species.

Response: We tried to do our best to reorganize and improve the manuscript accoding to commrnts and suggestions.

Minor comments

Page 2, lines 63-67. These lines suggest that the finding of pathogenic yeasts in the feces of this gull species is an expected result. Add a few sentences to justify the reason for carrying out research that will confirm previous studies.

Response: The information has been added.

Page 2, lines 93-96. The third aim of the paper, namely to measure the susceptibility of three yeast species to four common antimicrobial agents, is not sufficiently justified in the introduction. My proposal is to include the lines that introduce the importance of C. parapsilosis and C. tropicalis (page 12, lines 312-325) and Rh. mucilaginosa (lines 330-342) to justify this objective in the introductory section. These lines seem more appropriate in the Introduction, right? Thank you very much for very valuable comment.

Response: The Introduction has been reconstructed.

Page 4, lines 118-120. Table 1. Please comment on the values in this table in relation to the results. What was the purpose of measuring temperatures and wind speed to study microbes and their resistance against antimicrobial agents? Nothing was said about this in the Discussion I would say that they were measured to show that the differences in the results were not due to environmental differences between the colonies other than specifically natural vs. anthropogenic. However, it is not clear to me whether the differences in temperature and wind are analysed and discussed in the Results, Methods or Discussion. Please, comment about this Table in the Discussion.

Response: Comment has been added to the Discussion.

Page 6, line 202. I'm not sure if it's three or four.

Response: We are sorry. Of course, four. Thank you very much.

Page 9, line 234. Measuring both populations at the same time during the breeding season was a good idea. Kudos!

Response: We are very grateful for your evaluation. It encourages us to continue working.

Page 11, lines 268, 270 and 276. Small percentages such as 10%, 6%, 8% and 4% may not be different from zero. Please add the 95% confidence interval (CI95%) to these values. Note that for the extreme percentages (<15%, >85%) the CI95% is not symmetrical. Symmetric intervals are usually only found for the middle percentages (e.g. 50%). When the sample size is very large, the skewness is reduced in small (<15%) or large (>85%) percentage intervals. In the case of this paper, it is very unlikely that the interval will be symmetrical. Also, the lower interval may not be significantly greater than zero, so it is worth checking. This is a minor aspect of the paper. However, it is important when comparing the results with other studies. Overall, to say that small percentages are different from zero requires a large sample size. Can you be sure that the 4 per cent that is reported in the manuscript is significantly greater than zero?

Response: The confidence interval is not applicable to this analysis as there was no variability in resistance in the samples (strains) tested - the strain was either resistant to one or other antibiotic or not. Unfortunately, the proportion of resistant yeast and bacterial strains among the natural isolates has increased. This is due to incorrect dosing of prescribed drugs and non-compliance with doctors' orders when patients stop taking antibiotics before the prescribed period (as soon as they feel an improvement), as well as the sale of antibiotics without a prescription in some countries. Inadequate decontamination of clinical waste is also a common cause of the spread of resistant clinical strains in nature (e.g. antibiotic resistance genes from bacteria are often found in soil and water near hospitals). Resistant yeast strains are now also being found in the Antarctic. It is suspected that they have "emerged" there with the arrival of large numbers of researchers and tourists and are being transmitted by birds.

Page 12, lines 285-289. How do you know where the yeast comes from when growing in a natural environment? This study didn't measure it, right? Nor is the study that measured it quoted. It may be that this is a well-established fact and that as such it is in the textbooks. In any case, it is appropriate to add the source of the information contained in these lines.

Response: Yes, you are right. These are very common species which distribution is very well known. The references have been added. Thank you.

Page 12, lines 327-330. Contrast these results with those obtained in other bird studies. Lines 330-338 are not for this purpose.

Response: Corrected.

Page 13. Lines 340-342. These sentences justify the third main objective of this study: to describe the susceptibility of three yeast species to four routinely used anti-microbials (page 2, lines 93-97). I recommend that these sentences be relocated to the introduction.

Response: The sentences have been relocated.

Page 13, lines 343-354. The whole paragraph is a statement of results. Avoid repeating them. Especially if they are not commented on, discussed or compared with the results of other papers. For example, rather than repeating the results, try to put them in the context of other similar studies. It is desirable that the comparison is quantitative. Was the yeast resistance lower or higher (by how much?) than that found in other studies of antimicrobial resistance in bird feces collected from natural and anthropogenic sites? Lines 63-66 cite a number of papers with which you can compare your results in the Discussion, perhaps in the form of a table or a graph. Try to take the Discussion to a new upper level.

Response: We tried to improve the discussion.

Page 13, lines 361-363. The change in yeast diversity over time is a very important point. Strangely, it is not mentioned in the abstract of the paper. Why not? Well, it is exactly where it should be, as it is one of the most important results. On the other hand, the other lines (lines 361 - 374) are not part of the conclusions, because they are the experimental results. They are summarised in the abstract. Rejected or accepted hypotheses are what the conclusions are about. Try to transfer the results to the abstract and remove them from section 5 "Conclusions". It may be that the abstract has reached its maximum word count. If so, as a compromise, try to reduce the first 14 lines of the conclusions to one or two sentences. For example: "During the breeding season, the yeast composition of excreta increases or decreases depending on whether the breeding colony is in a natural or anthropogenic environment. Yeasts in the anthropogenic environment are more resistant to several antimicrobials than yeasts in the natural environment."

Response: The changes have been made to the text.

Page 13, lines 375-377. These lines indicate a clear pattern, but in a hesitant way: "seem to be", "potentially". Perhaps the study does not allow you to say conclusively what these lines say. But if it does not, then it is not a conclusion. Improve the style.

Response: The conclusion has been corrected. Thank you for help.

Page 13, lines 377-379. Same comment as above: check this sentence to make sure that it is a conclusion that has been drawn from the results. The content of these lines 377 - 379 and the content of lines 357 - 359 are quite similar. Check these sentences. It is possible that the conclusion is already contained in reference [74] (line 359). If this is the case, this sentence would not be a conclusion of your manuscript.

Response: The conclusion has been corrected.

Page 18, line 575. Typo: delete "    134" in the doi code

Response: Corrected

Page 18, line 580. Typo: delete "    140" in the doi code

Response: Corrected

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present an interesting study on the diversity of yeast found in the feces of common gulls (Larus canus L.) from both natural and urban habitats. I greatly enjoyed reading and learning from this paper about the differences in yeast communities between the two environments, revealing that while natural habitats host a greater variety of yeasts, urban environments show a higher prevalence of pathogenic species. Notably, many of these pathogens resist conventional antibiotics, raising concerns about their potential role in spreading antifungal resistance. The paper is important for public health awareness and wildlife management – with a stress on urban animals. I was especially impressed by the linking of yeast diversity to environmental factors. The findings can serve as a basis for further studies on the impact of urbanization on microbial communities and the mechanisms of antifungal resistance, thereby advancing the field of public health.

I found the study to be well planned, implemented, analyzed, presented with appropriate visualizations, and the appropriate conclusions drawn from the results. However, the authors might consider integrating their findings with broader ecological and evolutionary theories to enhance their discussion. Also, although I did not have a problem with the text as is, the authors should consider providing more detailed descriptions of the methodologies used for yeast isolation and identification and the statistical analyses performed.

A few minor glitches to be corrected:

Lines 264, 272, 277 – please italicize the Latin names

 

Line 325 – Please give the Common name and then Latin in parentheses of the birds

Author Response

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

We tried to do our best to improve the manuscript according to comment and suggestions.

The glitches have been corrected.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The new version of the manuscript has improved the shortcomings of the astringent introduction and the overgeneralized discussion, which is now more grounded. The manuscript also acknowledges some serious limitations of the study, so the research is in the best possible condition. Listing the shortcomings in the main text is very valuable: the basis for future studies starts with this list.

 

All but one minor problem about the statistical variability of a binomial variable (0,1) are met. Never mind, because the statistical summary of a binomial variable is only a minor problem, although it permeates most published papers. Misguided arguments against calculating the asymmetric variability of a binomial variable (0,1) range from "it is no longer a binomial variable because we have calculated the arithmetic mean of several percentages" to "there was no variability in variables that were either zero or one". Hopefully someone will take the trouble to write a short technical note on the calculation of asymmetric intervals of binomial variables.