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Virulence of  Clostridium difficile  is primarily attributed to the large clostridial toxins A and B while the role of binary toxin (CDT) remains unclear. The prevalence of human strains of  C .  difficile  possessing only CDT genes (A − B − CDT + ) is generally low (< 5%), however, this genotype is commonly found in neonatal livestock both in Australia and elsewhere. Zoonotic transmission of  C .  difficile  has been suggested previously. Most human diagnostic tests will not detect A − B − CDT +  strains of  C .  difficile  because they focus on detection of toxin A and/or B. We performed a prospective investigation into the prevalence and genetic characteristics of A − B − CDT +   C .  difficile  in symptomatic humans. All glutamate dehydrogenase or toxin B gene positive faecal specimens from symptomatic inpatients over 30 days ( n  = 43) were cultured by enrichment, and  C .  difficile  PCR ribotypes (RTs) and toxin gene profiles determined. From 39 culture-positive specimens, 43  C .  difficile  isolates were recovered, including two A − B − CDT +  isolates. This corresponded to an A − B − CDT +  prevalence of 2/35 (5.7%) isolates possessing at least one toxin, 2/10 (20%) A − B −  isolates, 2/3 CDT +  isolates and 1/28 (3.6%) presumed true CDI cases. No link to Australian livestock-associated  C .  difficile  was found. Neither A − B − CDT +  isolate was the predominant A − B − CDT +  strain found in Australia, RT 033, nor did they belong to toxinotype XI. Previous reports infrequently describe A − B − CDT +   C .  difficile  in patients and strain collections but the prevalence of human A − B − CDT +   C .  difficile  is rarely investigated. This study highlights the occurrence of A − B − CDT +  strains of  C .  difficile  in symptomatic patients, warranting further investigations of its role in human infection.

Prevalence of binary toxin positive Clostridium difficile in diarrhoeal humans in the absence of epidemic ribotype 027