Investigation of the antimicrobial resistance, molecular epidemiology, toxin genes and phages in clinical isolates of clostridium difficile

Clostridioides (Clostridium) difficile, the leading cause of healthcare-associated infections and globally recognised as a significant threat to public health, it is an anaerobic, spore-bearing, gram-positive bacillus that can cause life-threatening infections. The aim of this study was to isolate C. difficile and investigate toxin genes, antimicrobial resistance, clonal relationship and bacteriophages of the isolates. Anaerobic culture of 825 faecal samples investigated for toxins between December 2017 and October 2020 was performed and isolates were identified by MALDI-TOF MS (Bruker, Germany) in addition to conventional methods. Forty-three C. difficile isolates were investigated for antimicrobial resistance by gradient test and agar dilution method, toxin genes and bacteriophages by polymerase chain reaction (PCR) and clonal relationship by variable-field gel electrophoresis (PFGE). The sensitivities of the isolates for vancomycin, metronidazole, moxifloxacin and clindamycin were 100%, 100%, 90% and 60%, respectively. The minimum inhibitory concentration (MIC) values of erythromycin, tigecycline and fidaxomicin were determined between 0.25-≥256 µg/mL, ≤0.016-0.047 µg/mL and 0.008-0.25 µg/mL, respectively. Fourteen (32.6%) of the isolates were positive for tcdA, tcdB, cdtA, cdtB genes, 11 (25.6%) were positive for tcdA, tcdB, cdtB genes and 18 (41.9%) were positive for tcdA, tcdB genes. By PCR, it was determined that 31 of the isolates carried Myovirus, 17 carried Syphovirus and, 10 carried both holin genes together. The 38 isolates positive by PCR were induced with Mitomycin C and phage lysates were shown to infect at least one host. The similarity of 43 isolates by PFGE was 39.3%, the presence of multiple clonal groups and only six isolates in total were found to be 100% similar regardless of source (i.e. clinic and outpatient). According to the literature, this is the first study in Turkey in which clonal relationships, susceptibility to fidaxomycin and bacteriophages of C. difficile isolates were determined by PFGE. We believe that the findings obtained and comprehensive studies with more clinical isolates may contribute to a better understanding of the epidemiology, prevention and treatment of C. difficile infections and reduce the burden of CDE on the global health system.