Abstract
Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically–relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile–derived metabolites in culture supernatants, including hexanoyl– and pentanoyl–amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300–fold increases in the expression of sporulation–associated genes were observed in FW media–grown cells, along with reductions in motility and toxin genes’ expression. Moreover, the expression of classical stress–response genes did not change, showing that C. difficile is well–adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility.
Highlights
The Gram–positive spore–forming anaerobe Clostridium difficile is recognised as one of the major causes of health-care associated infections1,2 and exerts a negative and well–publicised impact on hospital morbidity and mortality rates1
Using our novel approach we demonstrate for the first time that expression of genes essential for pathogenesis are significantly differentially expressed in the human faecal water milieu
We characterised the faecal water (FW) using LC–MSn and demonstrated that it contained components identified in previous investigations32,34–36
Summary
The Gram–positive spore–forming anaerobe Clostridium difficile is recognised as one of the major causes of health-care associated infections and exerts a negative and well–publicised impact on hospital morbidity and mortality rates. While it could be argued that many of the experimental systems that exist to study C. difficile pathogenesis have rather limited relevance to the human gut, recent work with in vitro continuous flow bioreactors has elegantly demonstrated the increased competitive fitness of ribotype 027 C. difficile strains in a mixed microbiota model and shown that microbial communities representative of key features of the gut can be cultivated and manipulated successfully. While it could be argued that many of the experimental systems that exist to study C. difficile pathogenesis have rather limited relevance to the human gut, recent work with in vitro continuous flow bioreactors has elegantly demonstrated the increased competitive fitness of ribotype 027 C. difficile strains in a mixed microbiota model and shown that microbial communities representative of key features of the gut can be cultivated and manipulated successfully18 Such in vitro models have been used to investigate and model antibiotic exposure, intestinal biofilm development and genomic stability of C. difficile during simulated infection experiments. Using our novel approach we demonstrate for the first time that expression of genes essential for pathogenesis are significantly differentially expressed in the human faecal water milieu
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