Toxinotyping and Protein-Protein Interaction (PPI) Networks and Functional Annotation of the Detected Toxin Genes of Clostridium perfringens Isolated from food samples in Duhok Province, Iraq

Abstract

lostridium perfringens is a zoonotic pathogen causing health problems like gas gangrene, food poisoning, and other enteric infections, especially in livestock, causing global economic losses. In the present article, C. perfringens isolates were obtained from 94 food samples, including chicken shawerma, meat shawerma, chicken kabab, meat kabab; and canned fish and chickens collected from 6 markets and stores, and 34 restaurants within Duhok province, Iraq during the period from June 1 st , to August 31, 2022. The strains were isolated on CHROMagar TM C. perfringens agar and blood agar, and confirmed by PCR depending on 16 rRNA, and toxin typing based on genes encoding alpha (CPA), beta (CPB), epsilon (ETX), iota (IAP), enterotoxin (CPE), and necrotic betalike (NetB) toxins. From the 62 samples collected from the restaurants, 44 were grown on CHROMagar TM C. perfringens as orange colonies, representing 71%, and confirmed with PCR. All the samples collected from canned foods were negative for C. perfringens. Out of 28 isolates from meat kabab, 26 (92.9%) carried alpha toxin gene, while only 2 (7.15%) samples carried both alpha and epsilon toxin genes, which represent type D strains. From the 8 isolates isolated from chicken shawerma, only alpha (CPA) gene was detected in 6 (75%) samples, while alpha toxin and enterotoxin genes were detected in 2 (25%) isolates which refer to type F type. All the other samples were diagnosed as type A as they carried only the alpha toxin gene from the six toxins investigated. Protein-Protein Interaction (PPI) Networks and Functional Annotation of the Detected Toxin Genes revealed that the top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were glycerophospholipid metabolism; activation of this pathway led to induction of the toxin production in C. perfringens which in turn causes gangrene related diseases in humans and animals. To conclude that the prevalence of C. perfringens was very high in food samples from restaurants, especially in meat kabab and chicken kabab, followed by a relatively lower rate in meat shawerma and chicken shawerma. However, the vast majority of the isolates from food samples were C. perfringens type A, but D and F types were also detected, as besides CPA toxin, they also produce ETX and CPE toxins. None of the investigated imported canned foods (tuna, sardine, and processed chicken) carried C. perfringens.