In order to understand the appropriate use of potentially probiotic Gram-positive microbes through their introduction in the gut microbiome, it is necessary to understand the influence of individual bacteria on the host-response system at a cellular level. In the present study, we have shown that lipopolysaccharides, flagellated Gram-negative bacteria, potentially probiotic Gram-positive bacteria and yeast interact differently with human intestinal epithelial cells with a custom-designed expression microarray evaluating 17 specific host-response pathways. Only lipopolysaccharides and flagellated Gram-negative bacteria induced inflammatory response, while a subset of Gram-positive microbes had anti-inflammatory potential. The main outcome from the study was the differential regulation of the central mitogen-activated protein kinase signalling pathway by these Gram-positive microbes versus commensal/pathogenic Gram-negative bacteria. The microarray was efficient to highlight the impact of individual bacteria on the response of intestinal epithelial cells, but quantitative real-time polymerase chain reaction validation demonstrated some underestimation for down-regulated genes by the microarray. This immune array will allow us to better understand the mechanisms underlying microbe-induced host immune responses.
The microbiota of the mouth disperses into the lungs, and both compartments share similar phyla. Considering the importance of the microbiota in the maturation of the immunity and physiology during the first days of life, we hypothesized that primo-colonizing bacteria of the oral cavity may induce immune responses in bronchial epithelial cells. Herein, we have isolated and characterized 57 strains of the buccal cavity of two human newborns. These strains belong to Streptococcus, Staphylococcus, Enterococcus, Rothia and Pantoea genera, with Streptococcus being the most represented. The strains were co-incubated with a bronchial epithelial cell line (BEAS-2B), and we established their impact on a panel of cytokines/chemokines and global changes in gene expression. The Staphylococcus strains, which appeared soon after birth, induced a high production of IL-8, suggesting they can trigger inflammation, whereas the Streptococcus strains were less associated with inflammation pathways. The genera Streptococcus, Enterococcus and Pantoea induced differential profiles of cytokine/chemokine/growth factor and set of genes associated with maturation of morphology. Altogether, our results demonstrate that the microorganisms, primo-colonizing the oral cavity, impact immunity and morphology of the lung epithelial cells, with specific effects depending on the phylogeny of the strains.
INTRODUCTIONA randomized, double‐blind, placebo‐controlled, crossover clinical study (NCT01879098) on healthy, middle‐aged adults investigated the effects of three probiotic strains, Bifidobacterium animalis subsp. lactis B94, Lactobacillus plantarum HA119, and Bacillus subtilis R0179 on bile acid metabolism, satiety, and inflammation. In order to substantiate positive outcomes, probiotic strains must persist during transit through the gastrointestinal tract of participants. The objective of this sub‐study was to detect and quantify HA119 and R0179 at the strain level in fecal samples using previously designed primers for real‐time PCR (qPCR).METHODOLOGYParticipants of the clinical study were randomized to receive one capsule per day of a probiotic strain or placebo for six weeks. Following a four week washout period, participants were crossed to the other treatment (probiotic to placebo or placebo to probiotic). Fecal samples were collected at the baseline and final time points of each intervention for a total of four stool samples per participant. DNA isolated from fecal samples of participants in the L. plantarum HA119 (n = 136) and B. subtilis R0179 (n = 124) groups were analyzed by a SYBR‐green based qPCR method for HA119 and R0179, respectively. Strain‐specific primer targets included a gene encoding a phage protein for HA119, and a hypothetical protein for R0179. All fecal samples from participants in the B. lactis B94 group were not analyzed for B94 due to the lack of strain‐specific primers.RESULTSThe HA119 strain was detected in 97% of the participants in the HA119 arm. The mean HA119 level in the feces from the final time point of probiotic intervention was 7.34 ± 0.86 log bacteria/g of feces. R0179 was detected in 97% of the participants in the R0179 group at mean 6.52 ± 0.38 log bacteria/g of feces in the final time point of probiotic intervention. The absence of the probiotic strains in the feces following the four week washout period suggests that the duration of the washout period was adequate between the intervention periods.CONCLUSIONSOverall, these results demonstrate the use of strain‐specific primers for the detection and quantification of probiotic strains HA119 and R0179 in complex fecal matrices containing numerous gut microbes. The ability to show persistence of specific probiotic strains in feces could help monitor participant compliance in clinical trials. This qPCR study also suggests that a four week washout period between intervention periods is sufficient for crossover clinical studies with probiotic intervention.Support or Funding InformationLallemand Health Solutions
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