Isolation, identification and characterization of Clostridium perfringens from cooked meat-poultry samples and in silico biomodeling of its delta enterotoxin


Improper cooking of meat and poultry products may cause food borne gastroenteritis by Clostridium perfringens due to its enterotoxin. In this study we isolated Clostridium perfringens from cooked meat, pork and poultry samples from different areas of Coimbatore District, Tamilnadu, India. The presumptive, confirmed and completed characterization showed that isolated organism was Clostridium perfringens and it produces various toxins during sporulation. The toxin detected by simple immunodiffusion and various temperatures treatment showed its hyperthermophlicity. The antibiogram has indicated resistance of the organism to conventional antibiotics hence in silico study is significant. Comparative modeling of the δ-enterotoxin, most important toxin in gastroenteritis, was carried out with its protein sequence present in NCBI as no crystallographic structure of the same is available. PSI-BLAST analysis indicated the δ-toxin showed perfect homology (31% identity and 51 % similarity) with Staphylococcal β-hemolysin. This has confirmed by MSA and phylogentic analysis. The ORF has 1300 coding frames and predicted secondary structure consist of mainly randomcoil. The model refinement and validation is done by various empirical force fields and RMSD value, 1.43 A°, showed that the model is good. Most residues of the toxin are located in allotted regions of Ramchandran plot. The modeled structure could give novel ideas for molecular docking, development of new agonist and potential target for studies of drug- receptor interaction.

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Available from: Sinosh Skariyachan, May 09, 2014
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    • "Isolates were identified on the basis of their colony characteristics , Gram's staining, morphological features, spore staining and motility testing according to instructions given in Bergey's manual (Bergey 2009). Selected isolates were further characterized through their biochemical profile like double haemolysis on blood agar (García and Heredia 2011), reverse Christie Atkins Munch-Petersen (CAMP) test (Brady et al. 2010), lecithinase and proteolytic activity as well as gelatin liquefaction, nitrate reduction and lactose fermentation tests (Lindstrom et al. 2011; Skariyachan et al. 2010). "
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    ABSTRACT: A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates.
    Journal of Food Science and Technology -Mysore- 10/2014; 52(8). DOI:10.1007/s13197-014-1584-3 · 2.20 Impact Factor