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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. difficil e 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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12866-016-0653-3) contains supplementary material, which is available to authorized users.

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