This paper talks about phenotypic and genotypic differences between Lactobacillus crispatus strains isolated from Lactobacillus dominated vaginal microbial community (LVM) and dysbiotic vaginal microbial community (DVM). The study findings show that DVM strains have a significantly higher number of glycosyl transferease gene compared to LVM isolates suggesting a 'fitness advantage' in their environment. Furthermore, the authors have claimed first-ever evidence of glycogen utilization by Lactobacill. This paper reports important findings L. crispatus characteristics associated with their environment however, some observations needs further clarification and needs improvement.

Here are my comments:

1) The first part of the title involves the methodological approach and findings from this study and immediately draws the attention of the researchers in this study area. The abstract is well written however lacks the research gaps. Most of the relevant references are included.

2) The introduction provides a very good background in the context of the present study in a logical and coherent way. It discusses the beneficial role of Lactobacillus crispatus in the vaginal environment and presents the research gaps in the literature which is why some L. crispatus strains are dominant than others in the vaginal environment and proposes that these strains could be different phenotypically as well as genotypically. This study clearly describes the objective which is to compare the phenotypic and genotypic differences between Lactobacillus strains isolated from the healthy and dysbiotic microbial community and identify the factors that could lead to the successful establishment in the vaginal environment.

3) The second paragraph started with the idea about the potential therapeutic application of L. crispatus and also uses this idea to lead to the present literature gaps, however, this idea is missed in the rest of the manuscript. If one aspect of this research is to discuss the fact that some observed phenotypic and genotypic differences can make some strains a better probiotic candidate, in my opinion, it would be a good idea to address this point in the discussion section or else this idea can be omitted from the introduction.

4) The third paragraph highlights some factors that might be helpful in the successful dominance of L. crispatus in the vaginal environment. In proposing these points, authors have mentioned that "there is a general consensus that vaginal lactobacilli (including L. crispatus) ferment glycogen thus producing lactic acid". However, there are contrasting reports about the glycogen degradation capacity of Lactobacilli and this is still debatable due to lack of concrete evidence showing glycogen degradation ability. In the absence of a concrete report, the scientific community is still reluctant to accept that Lactobacilli are able to degrade glycogen (ref: 17). However, it has been shown that human vaginal secretions have amylase enzymes that can degrade glycogen (ref: 37). My suggestions to the authors would be either provide a solid evidence to support the claim or to remove this from the introduction.

4) All the methods are provided with appropriate details and are sufficient to reproduce the findings. API CH50 method although sensitive but can be subjected to the reader's biases. How this bias was addressed? The number of technical and biological replicates performed should be included.

5) Proper positive and negative controls are included in growth curve experiments. Horse serum in NYC media has amylase activity and can digest supplemented glycogen. It could be possible that observed growth on glycogen supplemented media could be due to the glucose released by the action of serum amylase to the glycogen. Therefore additional tests are required to confirm that glucose released from this activity is not responsible for the growth. TLC analysis of the NYC + glycogen at 0 hrs and 24 hrs can tell us if serum amylase is digesting the supplemented glycogen.

6) Authors in describing the growth in glycogen supplemented condition discussed the growth pattern until 36 hours of incubation while the growth curve shows only 24 hours results. If authors wish to describe results up to 36 hours, please include the same time point in the curves as well.

7) Figure 4: The title is "Correlation between growth on glycogen and N-terminal mutation in a putative type I pullulanase protein" however the figure shows the growth pattern of 28 different L crispatus isolates out of which some have N-terminal mutation in a putative type I pullulanase gene. As this figure is a compilation of 28 strains and both categories are included altogether, this figure does not stand out to show the correlation between two groups. Two separate figures or even a single figure with two separate categories would be more useful.

8) Authors have discussed that a significantly higher number of glycosyltransferase (GT) genes in DVM isolates compared to LVM isolates provides a fitness advantage to DVM isolates by modulating surface glycome through phase variation. However, a phenotypic trait associated with this gene, biofilm-forming ability, is not different between the two groups suggesting potentially other roles related to this gene. Does phase variation affects the cell wall glycoconjugates which in turn help them to adapt better in the vaginal environment could be a future area of research.

9) It is reported that transposases genes are more dominant in DVM isolates in the results section but was completely missed in the discussion. Is this an interesting finding? What could be the possible roles associated with this gene and how they are important in context of vaginal environment?

10) The authors discussed that growth in glycogen supplemented media is correlated with the presence of a pullulanase enzyme. However, the catalytic domain to break alpha 1,4 bond ​of glycogen is absent in this enzyme without which a complete digest of glycogen is not possible. Some other enzyme activity must be present to break alpha 1, 4 bonds as well which is not discussed. In order to show that the pullulanase enzyme was responsible to break the glycogen, incubating cell-free supernatant with glycogen should give the different breakdown products which can be visualized by the TLC or any other techniques.

11) As authors have claimed that this is the first evidence to show Lactobacilli showing growth on glycogen, I suggest them to report this finding in order to make the results more convincing. In addition, how their methodology helped them to get this success? What was the missing in the previous researches, was methodology flawed or media was not proper? What made them a success? Please refer to some previous unsuccessful results and discuss how your approach helped to show these different findings.

Overall, the study is well designed and started with a defined set of questions and properly addressed these questions using appropriate methods. The conclusions are well supported by the findings and address the research questions. This study contributes to filling in the literature gaps in whether the Lactobacilli strains isolated from DVM or LVM community are different and possible factors that might contribute to their establishment in DVM state. However, I suggest them to perform some additional tests to make their results more credible.