Isolation And Identification Of Clostridium Perfringens Phage And Its Antibacterial Application In Raw Poultry Meat

Clostridium perfringens（Cp）is an important zoonotic Gram-positive anaerobic bacterium capable of producing spores.It is widely present in soil,sewage,sediments,and the digestive tract of humans and animals and spread through the environment.Cp can secrete a variety of toxins,more than 20 kinds of which have been reported.According to the main pathogenic toxins（α,β,ε,ι）secreted by Cp,Cp can be divided into five types:A,B,C,D,and E,which cause human food poisoning,animal necrotizing enteritis,dysentery,sudden death syndrome and so on.Food poisoning incidents caused by Cp outbreak all over the world emerge one after another.Poor temperature control during food cooking and improper storage of meat products are the main reasons for the outbreak of gastroenteritis,which seriously threatens human health.Cp contamination in raw poultry meat mainly comes from intestinal Cp during slaughtering and processing.However,current chemical disinfectant have poor control of Cp and are prone to residues.Therefore,effective Cp biological control methods are urgently needed to reduce Cp contamination,protect human health.（1）Tissue and fecal samples from different farms were collected,and 52 Cp strains were identified,including 29 chicken-derived Cp strains,15 cattle-derived Cp strains,and 8sheep-derived Cp strains.Antimicrobial resistance of the 52 Cp isolates were identified.The results showed that all 52 isolates were belonged to type A Cp,and the antimicrobial resistance analysis showed that Cp from different sources showed different degrees of resistance to 10 antibiotics.Chicken-derived Cp showed the highest resistance to tetracycline（12/29,41.38%）,while sheep-derived and bovine-derived Cp showed high resistance to clindamycin,62.50%（5/8）and 73.33%（8/11）,respectively.Among the isolates from different sources,the multidrug resistance of bovine origin was the highest,followed by chicken origin and sheep origin.The isolates mainly showed multidrug resistance to 2 and 3antibiotics.The drug resistance gene analysis showed that tetracycline tet A was the main drug resistance gene,with a detection rate of 94.23%,followed by tet B and tet M,which were65.38%and 17.31%,respectively.This study defined the virulence types of Cp from different sources（chicken,sheep,and cow）,which are highly resistant and generally carry tetracycline-resistant genes,thereby increasing the risk of drug resistance transmission,and providing information for the prevention and control of food-borne diseases caused by Cp.（2）Bacteriophages have been highly concerned by the food and pharmaceutical industries because of their unique antibacterial properties.Obtaining specific phages for drug-resistant Cp can provide bacteriostatic resources for the effective prevention and control of Cp.According to the epidemic Cp strain and the reference strain ATCC 13124,specific phage was isolated from chicken fecal samples.The host range,lysis efficiency and biological characteristics of the phage were analyzed.The results showed that the isolated lytic Cp phage v B＿Cpe P＿HN02 was a short-tailed phage（HN02 for short）.Phage HN02 has good p H（5～11）and temperature tolerance（<70°C）and show a host range of 23.08%.The phage could adsorb the host strain rapidly and display a long outbreak period.The average outbreaks reached 27.67 PFU/cell.The genome of phage HN02 is a linear double-stranded DNA with a full length of 17,754 bp（Gen Bank MW815121）and a GC content of 28.2%.The genome contains 22 open reading frames（ORFs）,of which only 10 ORFs have some homology to known sequences.A phylogenetic tree was constructed based on the major capsid proteins of the phage,and the results showed that phage HN02 is highly homologous to the lytic Cp phages phi24R and CPD2.In conclusion,phage HN02 is a member of Podoviridae family and has good lytic activity against Cp,laying a foundation for the application of phage in Cp prevention and control.（3）Phage HN02 can effectively inhibit the growth of Cp,and can provide technical resources for the prevention and control of Cp in the slaughtering and processing of raw chickens.Firstly,the safety of Cp phage HN02 in vivo was analyzed by chick model.High titer phage HN02 was prepared by fermentation and used in chicken to analyze its potential bacteriostatic efficacy.The results showed that the phage HN02 had no significant side effect on the daily behavior of the chicks.Anatomical analysis showed that there were no abnormalities in the organs and tissues.The anaerobic fermentation technology of Cp phage was established and optimized,and high titer of Cp phage（>10⁹PFU/m L）could be obtained by adding 20 mmol/m L Ca²⁺for 12 h at 37°C.Phage HN02 can effectively inhibit the growth of Cp on the surface of fresh chicken,and high concentration of HN02(10¹⁰ PFU/m L)can completely inhibit Cp in 48 h.It can be seen that phage HN02 is safe,and also can be used for bacteriostasis and preservation in slaughtering and processing,providing a new strategy for the prevention and control of Cp contamination in food.