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, 5 (8), e12078

Deep Sequencing of the Vaginal Microbiota of Women With HIV

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Deep Sequencing of the Vaginal Microbiota of Women With HIV

Ruben Hummelen et al. PLoS One.

Abstract

Background: Women living with HIV and co-infected with bacterial vaginosis (BV) are at higher risk for transmitting HIV to a partner or newborn. It is poorly understood which bacterial communities constitute BV or the normal vaginal microbiota among this population and how the microbiota associated with BV responds to antibiotic treatment.

Methods and findings: The vaginal microbiota of 132 HIV positive Tanzanian women, including 39 who received metronidazole treatment for BV, were profiled using Illumina to sequence the V6 region of the 16S rRNA gene. Of note, Gardnerella vaginalis and Lactobacillus iners were detected in each sample constituting core members of the vaginal microbiota. Eight major clusters were detected with relatively uniform microbiota compositions. Two clusters dominated by L. iners or L. crispatus were strongly associated with a normal microbiota. The L. crispatus dominated microbiota were associated with low pH, but when L. crispatus was not present, a large fraction of L. iners was required to predict a low pH. Four clusters were strongly associated with BV, and were dominated by Prevotella bivia, Lachnospiraceae, or a mixture of different species. Metronidazole treatment reduced the microbial diversity and perturbed the BV-associated microbiota, but rarely resulted in the establishment of a lactobacilli-dominated microbiota.

Conclusions: Illumina based microbial profiling enabled high though-put analyses of microbial samples at a high phylogenetic resolution. The vaginal microbiota among women living with HIV in Sub-Saharan Africa constitutes several profiles associated with a normal microbiota or BV. Recurrence of BV frequently constitutes a different BV-associated profile than before antibiotic treatment.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Relative abundance of taxa across samples.
The composition of the microbiota was visualized by bar-plots. Samples were clustered by the similarity of the relative organism abundance and their similarity was visualized using a neighbor-joining tree. The major clusters (>10 samples) are named after a taxa with a relative even abundance across samples in the cluster. The sample numbers are coloured according to Nugent categories with BV =  red, intermediate  =  green, normal  =  blue. Amsel criteria are shown for each sample with present  =  grey, absent  =  white and missing data  =  black. The complete legend is given in figure S2.
Figure 2
Figure 2. Association of vaginal pH and odor with members of the vaginal microbiota.
The flow chart explaining the relationship between microbiota and diagnostic criteria were generated using data mining techniques (Materials and Methods). To obtain the prediction about a clinical sample, follow the arrows like a flow chart from the organisms (rectangular boxes) to the prediction (ovals). Each decision in the tree is split on the proportion of the organism above; thus, if a clinical sample has proportions less than 13.6% L. crispatus and less than 48.8% of L. iners it is predicted that the sample is taken from a vaginal environment with a pH≥4.5. The accuracy of the decision tree is reported for the entire dataset and for 10-folds cross validation, which is an attempt to estimate the accuracy of the decision tree on new clinical samples.
Figure 3
Figure 3. Diversity of the vaginal microbiota and the impact of antibiotics.
The species diversity was measured by calculating Shannon's Diversity index . The data was partitioned either by Nugent category , or by treatment time-point. Metronidazole treatment (week 0) reduced the diversity at two weeks but a slightly higher diversity was observed at later follow-up time-points indicating a return to a more diverse microbiota.
Figure 4
Figure 4. Changes in microbiota profile after antibiotic intervention.
The network depicts the profile of the vaginal microbiota before (time  = 0) and after metronidazole intervention. A thin line represents one subject while a thick line represents two. Only women were included with a complete follow-up and when a microbiota was present similar to the major profiles at baseline (Figure 1). Gv indicates G. vaginalis, MS =  Multiple species, LV =  Lachnospiraceae & Veillonellaceae, Ls =  Lachnospiraceae, Pb =  P. bivia and U =  minor clusters.
Figure 5
Figure 5. Dynamics of the vaginal microbiota after antibiotic intervention.
The line graphs depict the changes in relative abundance for a select number of species after metronidazole intervention (week 0). After two weeks both patients are cleared of BV along with an increase in the relative abundance of L. iners at two weeks. However patient 43 experiences a recurrence and receives another course of metronidazole at 5 weeks. Of note, after an L. crispatus profile is established, BV does not recur in this subject.

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