Distribution of Clostridium Difficile Ribotypes in Macedonian Patients and their Antimicrobial Susceptibility

Clostridium difficile is a major nosocomial pathogen. In Europe, this bacterium is mostly characterised by PCR ribotyping. Most of the Clostridium difficile infections (CDI) are treated with vancomycin or metronidazole, although prolonged antibiotic use is considered as one of the main risk factors for CDI.

This study aimed to detect the presence of various C. difficile ribotypes in hospitalised patients and to investigate their toxigenicity and antibiotic susceptibility.

All stool samples obtained from each patient were inoculated on Columbia blood agar and cycloserine cefoxitine fructose agar (CCFA) for isolation of C. difficile. Glutamate dehydrogenase and toxins A and B were investigated by immunochromatographic tests. Final confirmation of the isolates was performed by Vitek 2 and MALDI-TOF. A total of 21 isolates were collected for further investigation. PCR ribotyping was performed as described by Janezic and Rupnik. PCR ribotype profiles were analysed using software (Bionumerics, Applied Maths). Antibiotic susceptibility was determined by E-tests for metronidazole, vancomycin, tetracycline, clindamycin, erythromycin, imipenem, ciprofloxacin and moxifloxacin.

About 48% of C. difficile isolates belonged to ribotype 001/072. So, this ribotype was the most common ribotype in this study. The remaining 52% of C. difficile isolates consisted of 10 different ribotypes: 017, SLO 160, SLO 187, SLO 120, 255/258, 014/020, 046, 002, 070 and 027. Furthermore, 20 (95.2 %) out of 21 isolates of C. difficile were toxigenic. Toxins A and B were detected simultaneously in 90.5 % of C. difficile isolates. Two isolates from the ribotype 017 were toxin B positive only. Treatments with any of the following antimicrobials: clindamycin, erythromycin, ciprofloxacin and moxifloxacin (as well as many other antibiotics), could be a risk factor for CDI due to the high resistance of the strains in this study. About 90% of the strains from the most common ribotype 001/072 have MICs for clindamycin and erythromycin > 256 µg/ml.

All strains isolated are highly resistant to ciprofloxacin. All strains were susceptible to vancomycin (median MIC was 0.63 µg/ml) and metronidazole (median MIC was 0.084 µg/ml), so these two antimicrobials remain optimal treatment option for CDI.