Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease

Background: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. Results: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. Conclusions: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc

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

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. Previously it has been suggested that flagella might be implicated in colonisation. 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 colonisation 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