Toxin gene profiling of Clostridium difficile in food and food products of animal origin
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Date
2017-07
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Assam Agricultural University, Khanapara,Guwahati
Abstract
The present work was undertaken with a view to investigate the presence of Clostridium difficile in food and food products of animal origin and profiling of toxin genes. A total of 235 samples were collected from different sources including raw meat (beef, pork, chicken and chevon), meat products (dry beef, dry pork, cooked chicken, chicken sausage and chicken salami), raw cow milk, milk product (Indian paneer) and fish products (dry fish and canned fish). Out of the total 235 samples, initially 56 (23.83%) samples revealed suspected colonies of C. difficile. All the suspected colonies exhibited the typical colony morphology with horse manure odour and typical Gram-positive rod shaped cell with sub-terminal spores. Among 56 tentative isolates, 17 (7.23%) could be confirmed as C. difficile, based on the detection of species specific gluD (GDH) and tpi (triose phosphate isomerase) genes. The 17 confirmed C. difficile isolates comprised of 15 (8.24%) from raw meat and meat product samples and remaining two (11.11%) from fish product samples. None of the samples from cooked chicken, raw chevon, chicken sausage, chicken salami, canned fish, cow milk and paneer yielded any C. difficile isolates. All the 17 C. difficile isolates were screened for detection of glutamate dehydrogenase (GDH) protein and toxin A and /or toxin B by enzyme immuno assay (EIA), out of which all isolates were found positive for GDH protein and six were found to be phenotypically positive for toxin production. The C. difficile isolates were characterized by PCR for detection of toxin genes (tcdA, tcd B and binary toxin) and PaLoc region. Antimicrobial resistance pattern was also tested against nine different antimicrobial agents by E-test. Altogether, six C. difficile isolates were found to be toxigenic. Out of which two were from chicken samples and four from pork samples. All the toxigenic isolates from chicken (2) samples possessed both tcdA and tcdB (A+ B+), while all the pork isolates (4) carried variant toxin genes (A-B+). All the (A-B+) isolates from pork were found to harbour the binary toxin genes (cdtA and cdtB). Based on the detection of PaLoc region comprising regulatory genes tcdC, tcdR and tcdE revealed that the two toxigenic chicken isolates (A+ B+) possessed tcdC and tcdE ,while the remaining four toxigenic pork isolates (A-B+) carry tcdR and tcdE, respectively. All the 17 C. difficile isolates showed higher resistance pattern to ciprofloxacin (70.59%), followed by cefotaxime (58.82%), clindamycin (29.41%) and tetracycline (17.64%) but 100 per cent sensitive to chloramphenicol, moxifloxacin, tigecycline, metronidazole and vancomycin. All the beef isolates were clindamycin- resistant with an MIC of 96 - 128 mg/ml. While, all C. difficile isolates from pork and dry fish were sensitive to the antimicrobials tested except ciprofloxacin and cefotaxime, the four chicken isolates were sensitive to the antimicrobials except ciprofloxacin.
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