Spore Production of Toxigenic and Non-Toxigenic Clostridium difficile Isolates in Sub-MIC of Vancomycin, Clindamycin, and Ceftazidime

The non-toxigenic variant of Clostridium difficile is prevalent in clinical samples. The reason for the high prevalence of these strains in the clinical diarrhea specimens has not yet been studied. Evaluation of spore production in non-toxigenic C. difficile isolates (A-/B-/CDT-) compared to toxigenic isolates (A+/B+/CDT-) in the absence and presence of antibiotics. Minimum inhibitory concentration (MIC) for bacteria was performed by the microdilution technique. About ~106 bacteria from 18-hour culture were inoculated to pre-reduced media containing ½ × MIC of vancomycin (VAN), clindamycin (CLI) and ceftazidime (CAZ). After 24, 48, and 96 hours, one milliliter of broth culture was added and heated for killing vegetative forms. One hundred microliters of appropriate dilution were cultured on Columbia blood agar in triplicate. After 72 hours, the number of viable spores was counted based on the colony forming unit. The result showed the spore production of non-toxigenic C. difficile isolates in free antibiotic media and ½ × MIC of antibiotics was similar to toxigenic isolates. The VAN, CLI, and CAZ inhibited spore production in non-toxigenic isolates as much as toxigenic isolates of C. difficile. It seems the non-toxigenic C. difficile isolates are able to produce spores in the absence and presence of antibiotic in a similar manner to toxigenic isolates. Generally, their ability to produce toxin is lost, but they are able to remain, sporulate, and survive in hospitalized patients who receive antibiotics.