Environmental reservoirs of hypervirulent Clostridioides difficile: RT078 strains in wastewater and first detection of RT027 in shellfish in Taiwan

Clostridioides difficile infection is a global health concern, with increasing community-associated cases. Environmental sources may contribute to transmission. This study assessed hypervirulent C. difficile in wastewater and shellfish in southern Taiwan. From April to September 2020, water samples were collected from two domestic wastewater treatment plants (WWTPs), one hospital WWTP, and shellfish from an aquafarm in Tainan City. C. difficile isolates underwent molecular identification, toxin gene detection, ribotyping, antimicrobial susceptibility, and multi-locus variable-number tandem repeat analysis (MLVA). Ninety-seven C. difficile isolates representing 24 ribotypes were classified into 29 distinct strains—4, 13, and 12 from domestic WWTPs, hospital WWTP, and shellfish, respectively. Among 17 WWTP strains, 64.7% were toxigenic. Hypervirulent RT078 family strains (RT598, RT126, and RT127) with Δ39 base-pair deletion in toxin production control protein C gene were detected in WWTPs. One RT027 strain was discovered in clams—the first environmental report of RT027 in Taiwan. MLVA revealed this strain’s association with the American Type Culture Collection reference strain but distinct from other clinical strains. Five hypervirulent isolates harbored Thr82→Ile mutation in DNA gyrase subunit A, conferring high-level fluoroquinolone resistance (minimum inhibitory concentration >32 mg/L) while remaining susceptible to metronidazole and vancomycin. This first detection of hypervirulent RT078 in wastewater and RT027 in shellfish in Taiwan indicates that environmental reservoirs may contribute to community-associated infections, highlighting the need for expanded surveillance.