Persistence and In Vivo Evolution of Vaginal Bacterial Strains over a Multiyear Time Period

ABSTRACT It is not clear whether the bacterial strains that comprise our microbiota are mostly long-term colonizers or transient residents. Studies have demonstrated decades-long persistence of bacterial strains within the gut, but persistence at other body sites has yet to be determined. The vaginal microbiota (VMB) is often dominated by Lactobacillus, although it is also commonly comprised of a more diverse set of other facultative and obligate anaerobes. Longitudinal studies have demonstrated that these communities can be stable over several menstrual cycles or can fluctuate temporally in species composition. We sought to determine whether the bacterial strains that comprise the VMB were capable of persisting over longer time periods. We performed shotgun metagenomics on paired samples from 10 participants collected 1 and 2 years apart. The resulting sequences were de novo assembled and binned into high-quality metagenome assembled genomes. Persistent strains were identified based on the sequence similarity between the genomes present at the two time points and were found in the VMB of six of the participants, three of which had multiple persistent strains. The VMB of the remaining four participants was similar in species composition at the two time points but was comprised of different strains. For the persistent strains, we were able to identify the mutations that were fixed in the populations over the observed time period, giving insight into the evolution of these bacteria. These results indicate that bacterial strains can persist in the vagina for extended periods of time, providing an opportunity for them to evolve in the host microenvironment. IMPORTANCE The stability of strains within the vaginal microbiota is largely uncharacterized. Should these strains be capable of persisting for extended periods of time, they could evolve within their host in response to selective pressures exerted by the host or by other members of the community. Here, we present preliminary findings demonstrating that bacterial strains can persist in the vagina for at least 1 year. We further characterized in vivo evolution of the persistent strains. Several participants were also found to not have persistent strains, despite having a vaginal microbiota (VMB) with similar species composition at the two time points. Our observations motivate future studies that collect samples from more participants, at more time points, and over even longer periods of time. Understanding which strains persist, what factors drive their persistence, and what selective pressures they face will inform the development and delivery of rationally designed live biotherapeutics for the vagina.

2. Please address comments from reviewer 2. "The authors might choose to qualify that the absence of detected shared strains might in some case be limited by data quality and the limits of inStrain, and that persistence might be even more common than suggested by these results." Thank you for the privilege of reviewing your work. Below you will find instructions from the mSystems editorial office and comments generated during the review.

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Response to Reviewer comment:
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Reviewer #1 (Comments for the Author):
Please adhere to a 99.999% ANI threshold as suggested by Reviewer 1.
We have removed the dotted lines from Figure 1 indicating likely persistence and have similarly revised the text which now reports these strains and their ANI but does not conclude that they have persisted.
P4 L 102-107: "An additional six strains (four Gardnerella, one L. iners, and one Megasphaera) were estimated to have an ANI between 99.95 and 99.99% ANI. These could represent persistent strains whose sequence divergence has been inflated either due to recombination or as a result of the low and variable sequence coverage of these species. Conversely, these observations could represent instances where a closely related strain of the same species displaced the original." Reviewer #2 (Comments for the Author): The authors might choose to qualify that the absence of detected shared strains might in some cases be limited by data quality and the limits of inStrain and that persistence might be even more common than suggested by these results.
We have revised the text as suggested to qualify the limitations of our analysis.
P5 L 148-150: "We may have also failed to detect persistent strains due to a lack of sequence depth, and persistence may be more common than suggested by our results." Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems production staff to make sure that all elements meet the technical requirements for publication. They will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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