Selection of validated hypervariable regions is crucial in 16S-based microbiota studies of the female genital tract

Next-generation sequencing-based methods are extensively applied in studies of the human microbiota using partial 16 S rRNA gene amplicons. However, they carry drawbacks that are critical to consider when interpreting results, including differences in outcome based on the hypervariable region(s) used. Here, we show that primers spanning the V3/V4 region identify a greater number of taxa in the vaginal microbiota than those spanning the V1/V2 region. In particular, taxa such as Gardnerella vaginalis, Bifidobacterium bifidum and Chlamydia trachomatis, all species that influence vaginal health and disease, are not represented in V1/V2-based community profiles. Accordingly, missing or underestimating the frequency of these species overestimates the abundance of other taxa and fails to correctly assess the bacterial diversity in the urogenital tract. We elaborate that covering these taxa using the V3/V4 region leads to profound changes in the assignment of community state types. Altogether, we show that the choice of primers used for studying the vaginal microbiota has deep implications on the biological evaluation of the results

S100A8 and S100A9 are important for postnatal development of gut microbiota and immune system in mice and infants

BACKGROUND & AIMS: After birth, the immune system matures via interactions with microbes in the gut. The S100 calcium binding proteins S100A8 and S100A9, and their extracellular complex form, S100A8-A9, are found in high amounts in human breast milk. We studied levels of S100A8-A9 in fecal samples (also called fecal calprotectin) from newborns and during infancy, and their effects on development of the intestinal microbiota and mucosal immune system.background METHODS: We collected stool samples (n=517) from full-term (n=72) and preterm infants (n=49) at different timepoints over the first year of life (days 1, 3, 10, 30, 90, 180, and 360). We measured levels of S100A8-A9 by ELISA and analyzed fecal microbiomes by 16S sRNA gene sequencing. We also obtained small and large intestine biopsies from 8 adults and 10 newborn infants without inflammatory bowel diseases (controls) and 8 infants with necrotizing enterocolitis and measured levels of S100A8 by immunofluorescence microscopy. Children were followed for 2.5 years and anthropometric data and medical information on infections were collected. We performed studies with newborn C57BL/6J wild-type and S100a9(-/-) mice (which also lack S100A8). Some mice were fed or given intraperitoneal injections of S100A8 or subcutaneous injections of Staphylococcus aureus. Blood and intestine, mesenterial and celiac lymph nodes were collected; cells and cytokines were measured by flow cytometry and studied in cell culture assays. Colon contents from mice were analyzed by culture-based microbiology assays. RESULTS: Loss of S100A8 and S100A9 in mice altered the phenotypes of colonic lamina propria macrophages, compared with wildtype mice. Intestinal tissues from neonatal S100-knockout mice had reduced levels of CX3CR1 protein, and Il10 and Tgfb1 mRNAs, compared with wildtype mice, and fewer T-regulatory cells. S100-knockout mice weighed 21% more than wildtype mice at age 8 weeks and a higher proportion developed fatal sepsis during the neonatal period. S100-knockout mice had alterations in their fecal microbiomes, with higher abundance of Enterobacteriaceae. Feeding mice S100 at birth prevented the expansion of Enterobacteriaceae, increased numbers of T-regulatory cells and levels of CX3CR1 protein and Il10 mRNA in intestine tissues, and reduced body weight and death from neonatal sepsis. Fecal samples from term infants, but not preterm infants, had significantly higher levels of S100A8-A9 during the first 3 months of life than fecal samples from adults; levels decreased to adult levels after weaning. Fecal samples from infants born by cesarean section had lower levels of S100A8-A9 than from infants born by vaginal delivery. S100 proteins were expressed by lamina propria macrophages in intestinal tissues from infants, at higher levels than in intestinal tissues from adults. High fecal levels of S100 proteins, from 30 days to 1 year of age, were associated with higher abundance of Actinobacteria and Bifidobacteriaceae, and lower abundance of Gammaproteobacteria-particularly opportunistic Enterobacteriaceae. A low level of S100 proteins in infants' fecal samples associated with development of sepsis and obesity by age 2 years.results CONCLUSION: S100A8 and S100A9 regulate development of the intestinal microbiota and immune system in neonates. Nutritional supplementation with these proteins might aide in development of preterm infants and prevent microbiota-associated disorders in later years

Gut dysbiosis with Bacilli dominance and accumulation of fermentation products precedes late-onset sepsis in preterm infants

Background: Gut dysbiosis has been suggested as a major risk factor for the development of late-onset sepsis (LOS), a main cause of mortality and morbidity in preterm infants. We aimed to assess specific signatures of the gut microbiome including metabolic profiles in preterm infants <34 weeks of gestation preceding LOS. Methods: In a single center cohort fecal samples of preterm infants were prospectively collected during the period of highest vulnerability for LOS (day 7, 14, 21 of life). Following 16S rRNA gene profiling, we assessed microbial community function using microbial metabolic network modeling. Data were adjusted for gestational age and use of probiotics. Results: We studied stool samples of 71 preterm infants with LOS and 164 unaffected controls (no LOS/necrotizing enterocolitis). The bacteria isolated in diagnostic blood culture in most cases corresponded to the genera in the gut microbiome. LOS cases had a decelerated development of microbial diversity. Before onset of disease, LOS cases had specific gut microbiome signatures with higher abundance of Bacilli (specifically coagulase-negative Staphylococci, CoNS) and a lack of anaerobic bacteria. In-silico modeling of bacterial community metabolism suggested accumulation of the fermentation products ethanol and formic acid in LOS cases before the onset of disease. Conclusions: Intestinal dysbiosis preceding LOS is characterized by an accumulation of Bacilli and their fermentation products and a paucity of anaerobic bacteria. Early microbiome and metabolic patterns may become a valuable biomarker to guide individualized prevention strategies of LOS in highly vulnerable populations

Thermoneutral housing exacerbates nonalcoholic fatty liver disease in mice and allows for sex-independent disease modeling.

Nonalcoholic fatty liver disease (NAFLD), a common prelude to cirrhosis and hepatocellular carcinoma, is the most common chronic liver disease worldwide. Defining the molecular mechanisms underlying the pathogenesis of NAFLD has been hampered by a lack of animal models that closely recapitulate the severe end of the disease spectrum in humans, including bridging hepatic fibrosis. Here we demonstrate that a novel experimental model employing thermoneutral housing, as opposed to standard housing, resulted in lower stress-driven production of corticosterone, augmented mouse proinflammatory immune responses and markedly exacerbated high-fat diet (HFD)-induced NAFLD pathogenesis. Disease exacerbation at thermoneutrality was conserved across multiple mouse strains and was associated with augmented intestinal permeability, an altered microbiome and activation of inflammatory pathways that are associated with the disease in humans. Depletion of Gram-negative microbiota, hematopoietic cell deletion of Toll-like receptor 4 (TLR4) and inactivation of the IL-17 axis resulted in altered immune responsiveness and protection from thermoneutral-housing-driven NAFLD amplification. Finally, female mice, typically resistant to HFD-induced obesity and NAFLD, develop full disease characteristics at thermoneutrality. Thus, thermoneutral housing provides a sex-independent model of exacerbated NAFLD in mice and represents a novel approach for interrogation of the cellular and molecular mechanisms underlying disease pathogenesis