Limitations of 16S rRNA Gene Sequencing to Characterize Lactobacillus Species in the Upper Genital Tract

Abstract

The endometrial cavity is an upper genital tract site previously thought as sterile, however, advances in culture-independent, next-generation sequencing technology have revealed that this low-biomass site harbors a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most abundant non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy women and women with urogenital conditions. The aim of this study was to evaluate our ability to characterize members of the genital tract microbial community to species-level taxonomy using variable regions of the 16S rRNA gene. Samples were interrogated for the presence of microbial DNA using next-generation sequencing technology that targets the V5–V8 regions of the 16S rRNA gene and compared to speciation using qPCR. We also performed re-analysis of published data using alternate variable regions of the 16S rRNA gene. In this analysis, we explore next-generation sequencing of clinical genital tract isolates as a method for high throughput identification to species-level of key Lactobacillus sp. Data revealed that characterization of genital tract taxa is hindered by a lack of a consensus protocol and 16S rRNA gene region target allowing comparison between studies.

The vaginal microbiota of pregnant women varies with gestational age, maternal age, and parity

“…Network analyses of the 25 most relatively abundant ASVs revealed that Lactobacillus ASVs were consistently network hubs, defined as ASVs closest to the center of the network, across term gestation (Figure 8A-D) . It is worth mentioning that there was limited resolution to differentiate Lactobacillus spp ., given that the V4 hypervariable region of the 16S rRNA gene was targeted for sequencing (130). Nevertheless, Lactobacillus ASVs were clearly split between a primary group (ASVs 2, 7, 12, 13, 15, and 20) that included a mixture of L. crispatus, L. jensenii, and L. gasseri , and a secondary group (ASVs 1 and 6) comprised exclusively of L. iners (Figure 8A-D) .…”

Sex Differences in Association Between Gut Microbiome and Essential Hypertension Based on Ambulatory Blood Pressure Monitoring

“…49 Subsequently, most studies investigating the GM association with human hypertension over the last decade or so have either recruited individuals of either sex 16,50 or did not perform sex-stratified analysis. [8][9][10][11][12][13]17 Moreover, 16S rRNA gene sequencing may lack the taxonomic resolution 18,51 to identify sex-specific associations between GM and hypertension. We found that hypertension had a strong positive correlation with inflammationinducing opportunistic pathogen, R gnavus.…”

“…Improving the resolution for identifying microbial species is a crucial step toward deducing the therapeutic potential of GM. 18 Additional limitations of previous clinical studies are (1) inclusion of participants taking antihypertensive medications that are known to influence the GM 19 ; (2) lack of stratification based on ethnicity and sex 7,20 ; and (3) lack of adjustment for covariates that can potentially confound hypertension [21][22][23][24] or GM analysis, 25 such as sodium intake, 21 smoking, 22 menopause, 23 and fatty liver status. 24 More importantly, it remains to be investigated whether there are sex differences in the relationships between GM and hypertension, 7,20 since differences in intrinsic and lifestyle factors, such as hormones, age, inflammation, and diet, are well-known to impact the incidence and pathology of hypertension among men and women.…”

Compositional Changes in the Vaginal Bacterial Microbiome of Healthy Pregnant Women across the Three Gestational Trimesters in Ismailia, Egypt

“…However, this technique has limited taxonomic power, particularly on the species level and is not precise enough when considering taxa with a minor abundance [ 71 , 72 , 73 ]. Some authors may use species-specific quantitative real-time PCR assays to confirm the identity and relative abundance [ 74 ]. However, this approach is hampered by the limited multiplex capability of real-time PCR [ 75 , 76 ].…”