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Abstract

Clostridium difficile is a major cause of healthcare-associated infection and inflicts a considerable financial burden on
healthcare systems worldwide. Disease symptoms range from self-limiting diarrhoea to fatal Pseudomembranous colitis.
Whilst C. difficile has two major virulence factors, toxin A and B, it is generally accepted that other virulence components of the
bacterium contribute to disease. C. difficile colonises the gut of humans and animals and hence the processes of adherence and
colonisation are essential for disease onset. Bacteria within biofilms are protected from multiple stresses, including immune
responses and antimicrobial agents. Increased antibiotic resistance and chronic recurrent infections have been attributed to
the ability of bacterial pathogens to form biofilms. While biofilms have been well studied for several gut pathogens, little is
known about biofilm formation by anaerobic gut species. We have limited understanding of how the causative bacterium C.
difficile colonizes the host or how it can resist antibiotics and persist within the gut. While persistent infections have been
previously linked to biofilm-formation by pathogens, biofilm development by C. difficile has not been characterized. Our
work demonstrates the ability of this anaerobic pathogen to form complex biofilms, the involvement of important clostridial
pathways in biofilm development and perhaps a connection between formation of spores which are believed to mediate
persistence, and biofilm formation. Importantly, we show that bacterial sensitivity to antibiotics is reduced in clostridial
biofilms. Biofilm formation may be a mechanism employed by C. difficile to survive in hostile environments such as the human
gut. Here we tested this hypothesis by comparing flagellated parental strains to strains in which flagella genes were inactivated
using ClosTron technology. Our focus was on a UK-outbreak, PCR-ribotype 027 (B1/NAP1) strain, R20291. We compared the
flagellated wild-type to a mutant with a paralyzed flagellum and also to mutants (fliC, fliD and flgE) that no longer produce
flagella in vitro and in vivo. Our results with R20291 provide the first strong evidence that by disabling the motor of the
flagellum, the structural components of the flagellum rather than active motility, is needed for adherence and colonisation of
the intestinal epithelium during infection. Comparison to published data on 630Δerm and our own data on that strain revealed
major differences between the strains: the R20291 flagellar mutants adhered less than the parental strain in vitro, whereas we
saw the opposite in 630Δerm. We also showed that flagella and motility are not needed for successful colonization in vivo
using strain 630Δerm. Finally we demonstrated that in strain R20291, flagella do play a role in colonisation and adherence
and that there are striking differences between C. difficile strains. The latter emphasises the overriding need to characterize
more than just one strain before drawing general conclusions concerning specific mechanisms of pathogenesis. In addition,
we also demonstrate that clinical C. difficile strains, 630 and the hypervirulent strain R20291, form structured biofilms in vitro,
with R20291 accumulating substantially more biofilm. Microscopic analyses show multiple layers of bacteria encased in a
proteinaceous biofilm matrix. Employing isogenic mutants, we show that virulence-associated protein, cwp84, and a putative
quorum sensing regulator, luxS are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in spo0A,
a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between
sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to
high concentrations of vancomycin, a drug commonly used for treatment of CDI. Biofilm formation by C. difficile is a complex
multifactorial process and may be a crucial mechanism for clostridial persistence in the host.

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