Abstract

BackgroundClostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. In this study, antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. Three in vitro models were exposed to clindamycin to induce simulated CDI. All models were treated with vancomycin, and two received an FMT. Populations of total bacteria, Bacteroides spp., Lactobacillus spp., Enterococcus spp., Bifidobacterium spp., C. difficile, and Enterobacteriaceae were monitored using both methods. Total clostridia were monitored by selective culture. Using qPCR analysis, we additionally monitored populations of Prevotella spp., Clostridium coccoides group, and Clostridium leptum group.ResultsBoth methods showed an exacerbation of disruption of the colonic microbiota following vancomycin (and earlier clindamycin) exposure, and a quicker recovery (within 4 days) of the bacterial populations in the models that received the FMT. C. difficile proliferation, consistent with CDI, was also observed by both qPCR and culture. Pearson correlation coefficient showed an association between results varying from 98% for Bacteroides spp., to 62% for Enterobacteriaceae.ConclusionsGenerally, a good correlation was observed between qPCR and bacterial culture. Overall, the molecular assays offer results in real-time, important for treatment efficacy, and allow the monitoring of additional microbiota groups. However, individual quantification of some genera (e.g. clostridia) might not be possible without selective culture.

Highlights

  • Clostridioides difficile infection (CDI) has a high recurrent infection rate

  • Bacterial populations monitored by quantitative PCR (qPCR) and bacterial culture Gut microbiota variations were monitored in three chemostat models of the human colon, here described as A, B and C

  • All models were started with the same human faecal slurry and underwent clindamycin induced CDI, followed by treatment with vancomycin

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Summary

Introduction

Clostridioides difficile infection (CDI) has a high recurrent infection rate. Faecal microbiota transplantation (FMT) has been used successfully to treat recurrent CDI, but much remains unknown about the human gut microbiota response to replacement therapies. Antibiotic-mediated dysbiosis of gut microbiota and bacterial growth dynamics were investigated by two quantitative methods: real-time quantitative PCR (qPCR) and direct culture enumeration, in triple-stage chemostat models of the human colon. The chemostat model has been validated against the intestinal contents of sudden-death victims and consists on a reliable representation on the microbial content and bacterial activities of the human colon [8]. This model of the human colon has been used to investigate the propensity of different antibiotics to induce CDI and the efficacy of treatments [9,10,11,12,13,14,15]. Gut microbiota exposure to antibiotics can create ‘artefacts’, as the proportion of reads assigned to a bacterial population may increase as result of the depletion of abundant bacterial populations, and not due to bacterial expansion

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