Clostridium (Clostridiodes) difficile clade 3 ribotype (RT) 023 strains that fail to produce black colonies on bioMérieux ChromID agar have been reported, as well as variant strains of C. difficile that produce only toxin A. We have recently isolated strains of C. difficile from the environment in Western Australia (WA) with similar characteristics. The objective of this study was to characterize these strains. It was hypothesized that a putative β-glucosidase gene was lacking in these strains of C. difficile, including RT 023, leading to white colonies.

) by PCR and were a novel RT (QX 597). All isolates were susceptible to all antimicrobials tested except one with low-level resistance to clindamycin (MIC = 8 mg/L). The four tcdA-positive strains were motile. All isolates contained neither bgl locus but only bgl K or a putative β-glucosidase gene by PCR. Whole-genome sequencing showed the 17 strains belonged to novel multi-locus sequence types 632, 848, 849, 850, 851, 852 and 853, part of the evolutionarily divergent clade C-III. Four isolates carried a full-length tcdA but not tcdB nor binary toxin genes.

) strains of C. difficile from environmental samples.

White colonies of C. difficile from environmental samples could be overlooked when using ChromID C. difficile agar, leading to false-negative results, however, whether these strains are truly pathogenic remains to be proven.