Abstract
The human gastrointestinal tract harbours a complex microbial community which exist in planktonic and sessile form. The degree to which composition and function of faecal and mucosal microbiota differ remains unclear. We describe the development and characterisation of an in vitro human gut model, which can be used to facilitate the formation and longitudinal analysis of mature mixed species biofilms. This enables the investigation of the role of biofilms in Clostridium difficile infection (CDI). A well established and validated human gut model of simulated CDI was adapted to incorporate glass rods that create a solid-gaseous-liquid interface for biofilm formation. The continuous chemostat model was inoculated with a pooled human faecal emulsion and controlled to mimic colonic conditions in vivo. Planktonic and sessile bacterial populations were enumerated for up to 46 days. Biofilm consistently formed macroscopic structures on all glass rods over extended periods of time, providing a framework to sample and analyse biofilm structures independently. Whilst variation in biofilm biomass is evident between rods, populations of sessile bacterial groups (log10 cfu/g of biofilm) remain relatively consistent between rods at each sampling point. All bacterial groups enumerated within the planktonic communities were also present within biofilm structures. The planktonic mode of growth of C. difficile and gut microbiota closely reflected observations within the original gut model. However, distinct differences were observed in the behaviour of sessile and planktonic C. difficile populations, with C. difficile spores preferentially persisting within biofilm structures. The redesigned biofilm chemostat model has been validated for reproducible and consistent formation of mixed species intestinal biofilms. This model can be utilised for the analysis of sessile mixed species communities longitudinally, potentially providing information of the role of biofilms in CDI.
Highlights
The human colon is a highly complex and diverse ecosystem lined by a mucous membrane
Bacterial populations within the gastrointestinal tract exist in two forms; planktonic free-floating communities within the lumen, and sessile bacteria within mucosal-associated biofilm communities
In vitro human gut model We have previously described the use of a triple-stage chemostat human gut model to study the interaction between antimicrobial agents, the indigenous gut microbiota and C. difficile [14,15,16]
Summary
The human colon is a highly complex and diverse ecosystem lined by a mucous membrane. Mucosal communities interact closely with host epithelial cells, and thereby may have a greater influence than planktonic populations on disease pathogenesis and immunomodulatory responses. Despite these potential important roles, many intestinal microbiota studies are derived from faecal samples and only provide information on planktonic populations. We describe the development and characterisation of an in vitro continuous chemostat human gut model that facilitates the formation of mature mixed species biofilms, which can be longitudinally harvested and analysed separately. This chemostat system was validated for the consistent formation of 18 independent rod-associated biofilm structures, in both single- and triplestage gut model systems
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