Clostridium difficile (C. difficile) is a gram-positive, toxin-producing bacillus which is an intestinal pathogen in both humans and animals and causes a range of digestive disorders including inflammation of the bowel, abdominal pain, fever and diarrhea. C. difficile toxins include enterotoxin (Toxin A), cytotoxin (Toxin B) and a binary toxin. Two large protein toxins A and B are encoded by separate genes, tcdA and tcdB. Clostridium difficile infection (CDI) mainly caused by the activity of the genes tcdA and tcdB. The binary toxin is encoded by the genes cdtA and cdtB. The binary toxin caused increased adherence of bacteria to intestinal epithelium. The aim of the present study was isolation of C. difficile from feces of calves, and study of the frequency of C. difficile virulence genes.
150 samples of fresh feces from calves were collected and C. difficile was isolated from feces of calves using bacterial culture methods. DNA was extracted by a genomic DNA purification kit. Then PCR method was used for definitive diagnosis of C. difficile. Multiplex PCR method performed for identification of tcdA, tcdB, cdtA and cdtB genes. In the final stage antimicrobial resistance determining was carried out by standard Bauer-Kirby disk diffusion method.
C. difficile was isolated from 90 samples (60%). The tcdA was observed in 8 isolates (8.8%), tcdB in 16 isolates (17.7%), cdtA in 8 isolates (8.8%) and cdtB in 14 isolates (15.5%). Only 1 isolated (1.1%) was containing all four genes tcdA, tcdB, cdtA and cdtB, 2 isolates (2.2%) only had both tcdA and tcdB genes, and there was no sample positive only for both cdtA and cdtB. The highest rate of drug resistance was against clindamycin (100%) and the highest rate of drug sensitivity was against ciprofloxacin (50%).
The results showed high incidence of C. difficile and also high antibiotic resistance of this bacterium, but frequency of strains containing virulence genes (tcdA, tcdB, cdtA and cdtB) was low.