Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes▿

rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification—providing an assessment that is independent of a reverse primer—and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity

A Multi-Omic Systems-Based Approach Reveals Metabolic Markers of Bacterial Vaginosis and Insight into the Disease

<div><h3>Background</h3><p>Bacterial vaginosis (BV) is the most common vaginal disorder of reproductive-age women. Yet the cause of BV has not been established. To uncover key determinants of BV, we employed a multi-omic, systems-biology approach, including both deep 16S rRNA gene-based sequencing and metabolomics of lavage samples from 36 women. These women varied demographically, behaviorally, and in terms of health status and symptoms.</p> <h3>Principal Findings</h3><p>16S rRNA gene-based community composition profiles reflected Nugent scores, but not Amsel criteria. In contrast, metabolomic profiles were markedly more concordant with Amsel criteria. Metabolomic profiles revealed two distinct symptomatic BV types (SBVI and SBVII) with similar characteristics that indicated disruption of epithelial integrity, but each type was correlated to the presence of different microbial taxa and metabolites, as well as to different host behaviors. The characteristic odor associated with BV was linked to increases in putrescine and cadaverine, which were both linked to <em>Dialister</em> spp. Additional correlations were seen with the presence of discharge, 2-methyl-2-hydroxybutanoic acid, and <em>Mobiluncus</em> spp., and with pain, diethylene glycol and <em>Gardnerella</em> spp.</p> <h3>Conclusions</h3><p>The results not only provide useful diagnostic biomarkers, but also may ultimately provide much needed insight into the determinants of BV.</p> </div

Heterogeneity of Vaginal Microbial Communities within Individuals▿ #

Recent culture-independent studies have revealed that a healthy vaginal ecosystem harbors a surprisingly complex assemblage of microorganisms. However, the spatial distribution and composition of vaginal microbial populations have not been investigated using molecular methods. Here, we evaluated site-specific microbial composition within the vaginal ecosystem and examined the influence of sampling technique in detection of the vaginal microbiota. 16S rRNA gene clone libraries were prepared from samples obtained from different locations (cervix, fornix, outer vaginal canal) and by different methods (swabbing, scraping, lavaging) from the vaginal tracts of eight clinically healthy, asymptomatic women. The data reveal that the vaginal microbiota is not homogenous throughout the vaginal tract but differs significantly within an individual with regard to anatomical site and sampling method used. Thus, this study illuminates the complex structure of the vaginal ecosystem and calls for the consideration of microenvironments when sampling vaginal microbiota as a clinical predictor of vaginal health

Richness and Diversity of Each Sample.

<p>Rarefaction curves showing the richness of microbiomes for all samples colored by Nugent score (A; green = 0–3, orange = 4–6, red = 7–10) or Amsel classification (B; red = positive, green = negative) are presented along with Shannon diversity indexes (C), with samples grouped by Nugent score or Amsel criteria and colored as in rarefaction curves.</p

Network path lengths to BV.

<p>Measured as ¹Days since ²Times weekly ³Times monthly ⁴In past 6 months.</p

Quantitative PCR Enumeration of Lactobacillus inners by Sample.

1<p>Only samples found to contain <i>L. iners</i> are shown</p