Clostridium difficile infection (CDI) has reached an epidemic state in many developed countries with high incidence and severe disease. The binary toxin positive strain PCR ribotype (RT)027 has caused many outbreaks and remains highly prevalent in North America. This strain has never established in Australia, however, recently there have been reports of increased CDI incidence due to other strains that produce binary toxin. One such strain that has increased in prevalence Australia-wide is RT251. Herein, we genotypically and phenotypically characterised RT251 strains isolated in Australia and North America to ascertain virulence factors and the evolutionary history of this lineage.
C. difficile RT251 strains were isolated by toxigenic culture (n=124). Genetic characterisation was performed using toxin gene profiling, whole genome sequencing (WGS), in silico multilocus sequence typing (MLST) and core-genome single nucleotide variant (SNV) analyses. Antimicrobial resistance was determined using agar incorporation methods, in vitro toxin production was confirmed by Vero cell and HT-29 cytotoxicity assays. Motility assays were performed and pathogenicity was observed in a murine model on selected isolates.
WGS and MLST clustered RT251 in the same evolutionary clade (clade 2) as RT027. Core-genome analyses revealed RT251 strains from Australia were highly clonal, separated by <10 SNVs. All Australian strains were motile and shared a common ancestor with a strain from Virginia, USA, isolated in 2012 (2-7 SNVs), indicating this lineage was of North American origin. All isolates were susceptible to metronidazole and vancomycin; one showed clindamycin and erythromycin resistance. Despite comparatively lower levels of in vitro toxin production, RT251 infection resembled infection with epidemic RT027 in vivo. Mice showed marked weight loss, severe disease within 48h post-infection and required euthanising.
Our findings indicate Australian C. difficile RT251 strains share a common ancestry with RT027 and are likely of North American origin. How RT251 entered Australia requires further investigation, however, these data emphasise the importance of ongoing surveillance for new strains of C. difficile.