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, 77 (21), 7749-56

Correlation Between Protection Against Sepsis by Probiotic Therapy and Stimulation of a Novel Bacterial Phylotype

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Correlation Between Protection Against Sepsis by Probiotic Therapy and Stimulation of a Novel Bacterial Phylotype

Jacoline Gerritsen et al. Appl Environ Microbiol.

Abstract

Prophylactic probiotic therapy has shown beneficial effects in an experimental rat model for acute pancreatitis on the health status of the animals. Mechanisms by which probiotic therapy interferes with severity of acute pancreatitis and associated sepsis, however, are poorly understood. The aims of this study were to identify the probiotic-induced changes in the gut microbiota and to correlate these changes to disease outcome. Duodenum and ileum samples were obtained from healthy and diseased rats subjected to pancreatitis for 7 days and prophylactically treated with either a multispecies probiotic mixture or a placebo. Intestinal microbiota was characterized by terminal-restriction fragment length polymorphism (T-RFLP) analyses of PCR-amplified 16S rRNA gene fragments. These analyses showed that during acute pancreatitis the host-specific ileal microbiota was replaced by an "acute pancreatitis-associated microbiota." This replacement was not reversed by administration of the probiotic mixture. An increase, however, was observed in the relative abundance of a novel bacterial phylotype most closely related to Clostridium lituseburense and referred to as commensal rat ileum bacterium (CRIB). Specific primers targeting the CRIB 16S rRNA gene sequence were developed to detect this phylotype by quantitative PCR. An ileal abundance of CRIB 16S rRNA genes of more than 7.5% of the total bacterial 16S rRNA gene pool was correlated with reduced duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, improved pancreas pathology, and reduced proinflammatory cytokine levels in plasma. Our current findings and future studies involving this uncharacterized bacterial phylotype will contribute to unraveling one of the potential mechanisms of probiotic therapy.

Figures

Fig. 1.
Fig. 1.
Correlations between the T-RFLP profiles obtained from the ileal samples mapped onto the first and third principal components of PCA. Healthy rats are indicated with circles. Diseased rats are indicated with asterisks (placebo-treated animals) or squares (probiotic-treated animals).
Fig. 2.
Fig. 2.
Effect of pancreatitis and treatment with either placebo or the probiotic mixture on the relative ileal abundance of T-RF 457, corresponding to the as yet uncharacterized bacterial phylotype referred to as CRIB. Indicated are healthy control (white bar) and diseased animals, treated either with placebo (light gray bar) or the probiotic mixture (dark gray bar), respectively. Box-and-whisker diagrams represent median with interquartile range (boxes) and minimum and maximum nonoutlier values (whiskers). An asterisk indicates a significant difference (P < 0.05).
Fig. 3.
Fig. 3.
Neighbor-joining tree based on the 16S rRNA gene sequence of CRIB and other related clostridia. E. coli was included as an outgroup. Alignment and phylogenetic analysis were performed with the ARB software (16). The tree was calculated for E. coli positions 55 to 926. The reference bar indicates 10% sequence divergence. GenBank accession numbers are given in parentheses.
Fig. 4.
Fig. 4.
Associations between different measures of disease outcome and the relative ileal abundance of CRIB in diseased rats after 7 days of acute pancreatitis. Animals were treated with either placebo (gray dots) or probiotics (squares). Indicated are total bacterial counts in the duodenum (a), bacterial translocation to the MLNs (b), and total pathology of the pancreas (c). The relative abundance of CRIB was determined by qPCR analysis. Bacterial counts are expressed in log10 CFU per gram of sample. Pearson's correlation coefficients are provided, with corresponding P values. The dotted lines indicate the divisions between the samples with a low (<7.5%) or high (>7.5%) relative ileal abundance of CRIB (>7.5%).
Fig. 5.
Fig. 5.
Effect of low (<7.5%; white bars) or high (>7.5%; gray bars) relative ileal abundance of CRIB in diseased rats on plasma cytokine levels of IL-1β (a), IL-18 (b), TNF-α (c), IL-10 (d), CXCL1 (e), and IL-6 (f). The relative abundance of CRIB was determined by qPCR analysis. Box-and-whisker diagrams represent median with interquartile range (boxes) and minimum and maximum nonoutlier values (whiskers). Significant differences: *, P < 0.05; †, P < 0.10.

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