Journal of Bacteriology & Parasitology

The role of flagella in Clostridium difficile pathogenesis and biofilm formation: Comparison between a non-epidemic and an epidemic strain

3^rd International Congress on Bacteriology and Infectious Diseases

August 04-06, 2015 Valencia, Spain

SozaBaban

Posters-Accepted Abstracts: J Bacteriol Parasitol

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. difficilecolonises 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. We have limited understanding of how the causative bacterium C.
difficilecolonizes the host or how it can resist antibiotics and persist within the gut. 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. 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. Employing isogenic mutants, we show
those virulence-associated proteins, 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.