Differential clustering of fecal and mucosa-associated microbiota in 'healthy' individuals.

OBJECTIVE: Fecal samples are often used to characterize gut microbiota. However, whether or not fecal microbiota differs from mucosa-associated microbiota remains largely unknown. This may be specifically relevant in conditions that are characterized by complex mucosal microbe-host interactions, such as Crohn's disease. We aimed to determine the degree of agreement between fecal and mucosal microbiota profiles in 'healthy' individuals, using two commonly used collection procedures. METHODS: The gut microbiota composition of fecal samples (sent at ambient temperature before storage at -70°C) and of colonic biopsies (obtained at endoscopy and immediately stored at -70°C) was determined by sequencing the 16S rRNA gene. Altogether 31 randomly selected 'healthy' individuals from the population-based colonoscopy (Popcol) study were included. RESULTS: The fecal samples were characterized by a reduced degree of richness (P < 0.0001) and diversity (P = 0.016), and also differences in several phyla, including a lower relative abundance of Proteobacteria (P < 0.0001) and Verrucomicrobia (P = 0.008) than in biopsies. Only three of 30 individuals had a similar fecal and mucosal microbiota profile, based on weighted UniFrac analysis. A difference in Crohn's disease dysbiosis-associated bacteria was observed, including a lower relative abundance of Faecalibacterium (P = 0.004) and a higher relative abundance of Ruminococcus (P = 0.001) in feces than in biopsies. CONCLUSIONS: The observed differences between fecal samples, transported at ambient temperature, and the colonic mucosa-associated microbiota have implications for the interpretation of the previous literature, and may be specifically relevant to studies on Crohn's disease.

Composition of the vaginal microbiota in women of reproductive age--sensitive and specific molecular diagnosis of bacterial vaginosis is possible?

BACKGROUND AND OBJECTIVE: Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis. MATERIALS AND METHODS: Vaginal samples from 163 women (79 control, 73 BV and 11 intermediate (Lactobacillary grade II flora) cases) were analyzed using 454 pyrosequencing of the hypervariable regions V3-V4 of the 16S rRNA gene and 16 quantitative bacterial species/genus-specific real-time PCR assays. Sensitivities and specificities of potential BV markers were computed using the Amsel criteria as reference standard for BV. The use of quantitative thresholds for prediction of BV, determined for both relative abundance measured with 454 pyrosequencing and bacterial load measured with qPCR, was evaluated. RESULTS: Relative to the healthy women, the BV patients had in their vaginal microbiota significantly higher prevalence, loads and relative abundances of the majority of BV associated bacteria. However, only Gardnerella vaginalis, Atopobium vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 detected at or above optimal thresholds were highly predictable for BV, with the best diagnostic accuracy shown for A. vaginae. The depletion of Lactobacillus species combined with the presence of either G. vaginalis or A. vaginae at diagnostic levels was a highly accurate BV predictor. CONCLUSIONS: Quantitative determination of the presence of G. vaginalis, A. vaginae, Eggerthella, Prevotella, BVAB2 and Megasphaera type 1 as well as the depletion of Lactobacillus was highly accurate for BV diagnosis. Measurements of abundance of normal and BV microbiota relative to total bacteria in vaginal fluid may provide more accurate BV diagnosis, and be used for test-of-cure, rather than qualitative detection or absolute counts of BV related microorganisms.