Association between cigarette smoking and the vaginal microbiota: a pilot study

Smoking has been identified in observational studies as a risk factor for bacterial vaginosis (BV), a condition defined in part by decimation of Lactobacillus spp. The anti-estrogenic effect of smoking and trace amounts of benzo[a]pyrene diol epoxide (BPDE) may predispose women to BV. BPDE increases bacteriophage induction in Lactobacillus spp. and is found in the vaginal secretions of smokers. We compared the vaginal microbiota between smokers and non-smokers and followed microbiota changes in a smoking cessation pilot study. In 2010–2011, 20 smokers and 20 non-smokers were recruited to a cross-sectional study (Phase A) and 9 smokers were enrolled and followed for a 12-week smoking cessation program (Phase B). Phase B included weekly behavioral counseling and nicotine patches to encourage smoking cessation. In both phases, participants self-collected mid-vaginal swabs (daily, Phase B) and completed behavioral surveys. Vaginal bacterial composition was characterized by pyrosequencing of barcoded 16S rRNA genes (V1-V3 regions). Vaginal smears were assigned Nugent Gram stain scores. Smoking status was evaluated (weekly, Phase B) using the semi-quantitative NicAlert® saliva cotinine test and carbon monoxide (CO) exhalation. In phase A, there was a significant trend for increasing saliva cotinine and CO exhalation with elevated Nugent scores (P value <0.005). Vaginal microbiota clustered into three community state types (CSTs); two dominated by Lactobacillus (L. iners, L. crispatus), and one lacking significant numbers of Lactobacillus spp. and characterized by anaerobes (termed CST-IV). Women who were observed in the low-Lactobacillus CST-IV state were 25-fold more likely to be smokers than those dominated by L. crispatus (aOR: 25.61, 95 % CI: 1.03-636.61). Four women completed Phase B. One of three who entered smoking cessation with high Nugent scores demonstrated a switch from CST-IV to a L.iners-dominated profile with a concomitant drop in Nugent scores which coincided with completion of nicotine patches. The other two women fluctuated between CST-IV and L. iners-dominated CSTs. The fourth woman had low Nugent scores with L. crispatus-dominated CSTs throughout. Smokers had a lower proportion of vaginal Lactobacillus spp. compared to non-smokers. Smoking cessation should be investigated as an adjunct to reducing recurrent BV. Larger studies are needed to confirm these findings.https://doi.org/10.1186/1471-2334-14-47

Alteration in the gut microbiome is associated with changes in bone metabolism after laparoscopic sleeve gastrectomy

Laparoscopic sleeve gastrectomy (LSG), the most common bariatric surgical procedure, leads to durable weight loss and improves obesity-related comorbidities. However, it induces abnormalities in bone metabolism. One unexplored potential contributor is the gut microbiome, which influences bone metabolism and is altered after surgery. We characterized the relationship between the gut microbiome and skeletal health in severe obesity and after LSG. In a prospective cohort study, 23 adults with severe obesity underwent skeletal health assessment and stool collection preoperatively and 6&nbsp;mo after LSG. Gut microbial diversity and composition were characterized using 16S rRNA gene sequencing, and fecal concentrations of short-chain fatty acids (SCFA) were measured with LC-MS/MS. Spearman's correlations and PERMANOVA analyses were applied to assess relationships between the gut microbiome and bone health measures including serum bone turnover markers (C-terminal telopeptide of type 1 collagen [CTx] and procollagen type 1&nbsp;N-terminal propeptide [P1NP]), areal BMD, intestinal calcium absorption, and calciotropic hormones. Six months after LSG, CTx and P1NP increased (by median 188% and 61%, P&nbsp;&lt; .01) and femoral neck BMD decreased (mean -3.3%, P&nbsp;&lt; .01). Concurrently, there was a decrease in relative abundance of the phylum Firmicutes. Although there were no change in overall microbial diversity or fecal SCFA concentrations after LSG, those with greater within-subject change in gut community microbial composition (β-diversity) postoperatively had greater increases in P1NP level (ρ&nbsp;= 0.48, P&nbsp;= .02) and greater bone loss at the femoral neck (ρ&nbsp;= -0.43, P&nbsp;= .04). In addition, within-participant shifts in microbial richness/evenness (α-diversity) were associated with changes in IGF-1 levels (ρ&nbsp;= 0.56, P&nbsp;&lt; .01). The lower the postoperative fecal butyrate concentration, the lower the IGF-1 level (ρ&nbsp;= 0.43, P&nbsp;= .04). Meanwhile, the larger the decrease in butyrate concentration, the higher the postoperative CTx (ρ&nbsp;= -0.43, P&nbsp;= .04). These findings suggest that LSG-induced gut microbiome alteration may influence skeletal outcomes postoperatively, and microbial influences on butyrate formation and IGF-1 are possible mechanisms

Histogram of the LDA scores computed for microbial genera differentially abundant between nested and standard PCRs in stool samples.

<p>The taxa shown in red are the ones with significantly higher abundance by nested PCRs while the taxa shown in green are the ones with significantly higher abundance by standard PCR. The g or f before the taxon name means genus or family.</p

Box plots comparing nested to standard PCRs in number of OTUs and Shannon’s index.

<p>Note, *P<0.05, **P<0.01, ***P<0.001, the P values were calculated by the Wilcoxon Matched-Pairs Signed Ranks test.</p

Clusters of vagina samples based on bacterial genus relative abundance.

<p>Heatmaps were based on the hierarchical clustering solution (Bray-Curtis) distance metric and average clustering method. Row represents different sample ID (The number before the period is the subject ID; The text after the period is the PCR method used.). Columns represent the predominant bacterial genera with mean relative abundance of 0.01 or greater. The colors in the heatmaps represent the relative abundance of each genus, as indicated in the upper left corner of each panel.</p

Nested PCR Biases in Interpreting Microbial Community Structure in 16S rRNA Gene Sequence Datasets

<div><p>Background</p><p>Sequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention.</p><p>Results</p><p>In this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon’s index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool).</p><p>Conclusions</p><p>Nested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.</p></div

Principal coordinates analysis (PCoA) of weighted UniFrac distance.

<p>Proportion of variance explained by each axis is denoted in the corresponding axis labels. Each symbol (designated by the combination of color and shape) represents each subject with the open symbols for the nested PCRs and the closed symbols for the standard PCRs. For example, the blue circles represent subject 1 with open blue ones for two nested PCR results and closed blue ones for three standard PCR results.</p

The details for nested and standard PCR design and number of successful PCR reactions in vagina and stool samples.

<p>Note, Primer pair 1 is 515F and 1492R; primer pair 2 is the tagged 515F and 806R.</p><p>The details for nested and standard PCR design and number of successful PCR reactions in vagina and stool samples.</p