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Thank you for your careful attention to the comments, and very nice handling of the data availability. I agree that the manuscript is ready to be published, thank you!
[# PeerJ Staff Note - this decision was reviewed and approved by Konstantinos Kormas, a PeerJ Section Editor covering this Section #]
Thank you for your attention to the previous comments. There are a few very minor remaining issues:
It is wonderful that you have included the github link, what a great resource. Are the sequence data and metadata also available? A data availability section was not apparent.
Some tables lack error / standard deviation (e.g. Tables 2 and 3)
Some results highlighted even in the abstract are only available in the supplement, the taxa differences listed in the abstract are in Supp Fig 1. Is that the intention, to have some of the highlighted results only available in the supplement?
Please consider rephrasing weather conditions > climate
thank you
Overall, there are still several noticeable spelling and grammatical errors, along with statements even in the abstract that are not consistent with the revised results. Please see below, and please return a carefully revised manuscript
1. The change in name from Propionibacterium to Cutibacterium means that the bacteria should be identified as Cutibacterium, rather than Propionibacterium/Cutibacterium. Perhaps in the first instance, you would like to describe Cutibacterium as formerly known as Propionibacterium.
2. Line 405: tree > three
3. Line 415: metropolitant > ? metropolitan maybe?
Perhaps other pervasive errors, beyond the scope of journal review to correct
4. Please label the x-axis in Figure 1C . If possible to denote the age group the sample comes from in Fig 1C, that might help with comparison of the data in other figures
5. Please label the x axis in Figure 4. Also, please define CST in the Fig 4 legend.
6. Abstract - the results section highlights the abundance of phyla without context for whether this is novel. This sounds like it is supported by what has been found in other studies. It might be worth highlighting taxa at species or genus level.
6a. Geography is listed as significant, without evidence to back this up. Perhaps highlight the PERMANOVA results (including the R2 value showing variance explained, not just the p-value), and consider highlighting a few taxa that are differentially abundant in each location.
6b. Age is listed as insignificant, but there are significant differences found in age as stated in your rebuttal and revised manuscript
7. Please add variance explained to PCA axes
8. Please consider the big picture results and make sure they are reflected in the abstract
Dear Authors,
We have received thoughtful and constructive review comments, and I would like to share these comments and ask that you please resubmit a tracked changes version of the manuscript so that we can follow your changes more easily in the next round. (This time we could not find a track changes version of the manuscript in the submission materials.) In summary, the data are worthwhile but will require substantial care in the revisions to address the reviewer comments and reach publication level.
[# PeerJ Staff Note: Please ensure that all review and editorial comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the response letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the response letter. Directions on how to prepare a response letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
Please rename Propionibacterium to Cutibacterium throughout the manuscript. Why rename it in the table and not text?
The weather of Ahmednagar is comparatively hot and dry than Pune --> Please rephrase.
317 The average humidity and rainfall received in Nashik are more (67%, ~99.3mm) than in Pune (60.3%, ~67.9 mm) and in Ahmednagar (59%, ~46.3 mm). --> please rephrase.
Please check English grammar!
Among all three cities, the population and urbanization are more in Pune (~ 31 lacks), followed by Nashik (~ 16 lacks) and Ahmednagar (~ 3 lacks). --> please rephrase to proper English.
What is "lacks"? 100 000? Please report in millions or report the full number.
Age: the alpha diversity for ppl aged 41-59 is half as much as those aged 16-40y. Are you sure there is no impact seen for age? The only reason for me to see no sign difference is the low sample size.
254 Staphylococcus and Corynebacterium were abundant on the skin of individuals from the Pune district (Figure 6I-J). --> These results resemble the most with the results found in Europe (Callewert et al 2013) and USA (Council et al 2016; Grice et al 2010). Pune is also the most urbanized location of the three; a city with many urbanization elements. I think the authors should discuss this better in the discussion section. For instance on this location:
325 individuals residing in Pune are inhabitants of a metropolis, adapted to the western lifestyle, developed industrial area and infrastructure.
Did the authors ask the participants for deodorant use? Did the people in Pune use deodorants more frequently as compared to the other 2 locations? I believe deodorant use is more common in Pune, which correlates to more Staph and Coryne presence in that city.
Figure 2: I appreciate the effort, but it still remains very difficult to interpret these results. I would really urge the authors to make a bargraph, as done in f.i. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3337431/ Fig 3. It would be great to see the individual bargraphs per person, and clustered per location. That is the only good way to represent the bacterial composition.
Also: Staphylococcus abundance in Fig 2 is around 1-1.5%, while in Table 3 this is 23.2%. Where does the difference come from?
Figure 6: please add statistics in the figures.
I would suggest to mention the y-axis as done in panel I. (no scientific number - just a comma number, for easy interpretation)
Can the authors also put the most abundant one in panel A, followed by the second abundant one in panel B, etc?
Figure 4: CST nr 1, 2, 3. Can the authors say what locations are 1, 2 and 3? Actually, I don't understand the CSTs, I'm afraid. I also did understand after looking it up in the manuscript. Can the authors explain more on this?
Figures: I would suggest to combine a couple of figures into multipanel figures.
288 Bangalore (India) detected dominance of four phyla viz., Actinobacteria, Firmicute, --> FirmicuteS
335 Callewaert et al., in 2013 studying interpersonal axillary diversity, did not observe a significant correlation with geographical location --> This study was done in Belgium, which is a small country and entirely urbanized. So it is difficult to say something about geographical location here.
Can I ask the authors to provide the track change word document next time? It was hard to find all the differences in the manuscript as compared to previous version.
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Thank you for your interesting study and manuscript. Both reviewers have provided constructive criticism to improve the visualization and interpretation of the data. With careful attention to these comments, improved figures and re-writing, the manuscript will be acceptable for publication. It is important not the overstate what can be learned from these data - for example, the V3-4 region of 16S is known to miss major skin taxa, please make sure these limitations and caveats are clearly stated in the manuscript.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the response letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the response letter. Directions on how to prepare a response letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
English language is good.
Some important literature is missing in the manuscript
The figures could have been better, fi. the bacterial composition in each region.
Number of samples is rather low.
more info - see below
see below
Authors described the underarm bacterial microbiome of Indian people in 3 distinct locations of India. Using amplicon sequencing of the V3-4 region of 16S rRNA. The author found clear associations with location. No impact of age or diet were found.
Comments
Minor comments
There have been other papers describing the underarm microbiome (eg. Callewaert et al., 2013). It would be meaningful to compare your results with previous results.
Would there be overlap between environmental bacteria and the skin microbiota? Is this a driving factor in the differences seen per location? Can authors hypothesize on this?
What is the Impact of gender? It is not reported in the manuscript, although in the discussion section, it was mentioned as not significantly associated?
Can authors include gender in Table 1 (or another table)?
Can the authors describe the characteristics of Pune, Ahmednagar, and Nashik? Are these big cities? How many inhabitants? I had to go look it up online: Pune (3000000), Ahmednagar (350000), and Nashik (1500000). How is rainfall, temperature, humidity etc different in each city? It is generally discussed in the discussion, but reader has no clue on the differences for each city.
Does skin diversity associate with city size (inhabitant number)?
Can the authors describe more about urbanization grade in each location? How are Pune, Ahmednagar, and Nashik different from each other? Urbanization paper: McCall et al, 2020
Age: no correlation found; but do the authors see pattern with sebum / apocrine sweat production (which is the highest at ~16-30y)? Are there more lipophilic bacteria around that age?
N=58 might be low. Did the authors perform a power calculation to verify if the number of samples is sufficient for each location?
Major comments
The V3-V4 region of the 16S rRNA region was amplified. It is known that this region gives an underrepresentation of the Propionibacterium/Cutibacterium genus (Meisel et al., 2016 JID); which are relatively important taxa on the human skin and in the underarms.
Cutibacterium is not mentioned once. Propionibacterium has been renamed to Cutibacterium recently (Scholz et al., 2016 Int. J. Syst. Evol. Microbiol).
Figure 1 should be a bargraph of the underarm microbiome of the 3 different locations. The current log scale makes it really difficult to interprete. Also, what are the other taxa?? This is not the best way to represent the bacterial composition.
Figure 2 is of poor quality. Can the author add/visualize statistics to the plots?
Figure 4 is again of poor quality.
How have the 10 taxa been chosen; is this the top 10 most abundant taxa, and then ranked according alphabet? I guess not, because Corynebacterium is missing.
Suggest to rank according abundance: so Staph first. Now it seems as if Tuberibacillus is the most important genus in the underarms of Indian people. Which I highly doubt, and to be honest, I cannot even know what are the most abundant taxa; because it is not reported ) (except the top 4 most abundant in Fig 1).
I would argue that Nashik, Ahmednagar and Pune are not so distinct from each other. They are actually geographically quite close to each other. In the same province / mid-West of India. What is the reasoning behind the choice for these 3 areas?
I suggest authors describe in much more detail why these 3 cities are so different from each other.
All in all an interesting paper, but it’s really hard to comprehend and interpret the results. No clear figure on bacterial composition; unclear why the 3 locations were chosen, which gender the volunteers were, etc.
Line 62: "The commensal....pathogens...immunities" requires citation
Lines 202-203 Indicate that analysis is available on github; however the provided link is not accessible.
Impact of diet on skin microbiome is less explored albeit an important area of exploration. The diets should be defined in more depth to make meaningful interpretation. Additionally, since the authors do review the literature in significant depth, it would be useful to discuss findings from other relevant studies as well.
In sample collection, authors’ state:
“To avoid the effect of geographical location and related climatic differences, the axillary sweat samples were collected within 1 to 2 days from all the volunteers.” It is unclear as a reader, what does “within 1-2 days of what reference point?” mean.
While the representation of figures in the microbiome field is always up to debate, it is always useful to have pictorial/graphical depictions of data wherever possible. It would be useful to have some of the tables, particularly Tables 3 and 4 as graphs. Further, some of the figures are of lower resolution, making them hard to read.
Please refer to general comments for detailed points.
The manuscript by Potbhare, et al (2021) is an important manuscript in the context of the Indian subcontinent as there are not many studies that investigate the composition of the skin microbiome in the defined population. The study samples 58 volunteers, of which 36 belong to Pune, making the data from Ahmednagar (n=11) and Nashik (n=11), slightly over-interpretative due to small sample sizes. Generally, the manuscript is written clearly, and the literature reviewed is appropriate in the context of skin microbiome. Since the first description of skin microbiome using culture-independent technique, several advances have been made in the field. The authors use 16s rRNA profiling to sequence the samples, which is fairly adequate in the context of this manuscript. However since then studies performing whole-metagenomic sequencing [Oh, et al., 2016] have described the microbiome at a deeper resolution. It is important to discuss these findings as well. The other issue, I have as a reviewer is that the authors’ only sample the axillary sweat region. The axillary sweat region consists of hair follicles, sebaceous glands and is rich in sweat glands as such making it a completely unique microenvironment that is not exactly representative of the skin microbiome. While the authors’ discuss the differences in body site, this important point is not discussed in great depth. As of current, the axillary microbiome is given its own unique position [Refer to: Li, et al.(2019), Taylor, et al (2003), Council, et al. (2016), Callewaert, et al. (2013)]. Therefore, this reviewer thinks that it would be more valuable and insightful to discuss the findings specifically in the context of the axillary microbiome.
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