The Epidemic Characteristics Of Clostridium Perfringens And Analysis Of Its Bacteriophage's Genetic Background

Clostridium perfringens is a common bacterium in the intestines of livestock,poultry and humans,a variety of pathogenic toxins produced during its mass reproduction can lead to infection of livestock and seriously harm the development of animal husbandry.At present,the method of adding antibiotics in feed is usually used to prevent Clostridium perfringens of livestock and poultry,however,this method will undoubtedly lead to the formation of drug-resistant strains and disorder of intestinal flora,so it is urgent to find an effective and reliable antibiotic substitute.Phages can specifically infect and lyse Clostridium perfringens without affecting microorganisms other than the target bacteria,so that the natural microbial community is not disturbed,and can be used as an antibiotic substitute for the control of clostridium perfringens.However,Clostridium perfringens is an anaerobic bacterium,which has the problems of relatively difficult phage separation,narrow phage spectrum,rapid resistance development and lysogenization.Therefore,according to the epidemic characteristics of clostridium perfringens,specific phages were screened from the environment in this study,and protophages of Clostridium perfringens genome were analyzed.It provides theoretical basis and new research ideas for the research and development of phage products for the prevention and control of clostridium perfringens.This study is based on Shandong Province Veterinary Drug Resistance Monitoring Network(Varms),isolate the epidemic strains of Clostridium perfringens from different animal sources clinically through various identification methods such as bacterial isolation and culture,detection of alpha toxin genes and mass spectrometry,using(?,?,?,?)toxin gene PCR to identify toxin genotypes to grasp the current epidemic characteristics of Clostridium perfringens.Bacterial genome prophage sequences and phage characteristics were annotated using the PHASTER website,and virulence genes and drug resistance genes that may be present in the complete prophage region were screened through the online websites VFDB and CARD.The complete prophage sequence was homologously aligned with all Clostridium perfringens phage in the NCBI database.Based on popular strains,analysis of the prophage by whole genome sequencing.The isolated popular strains were used as host bacteria to isolate and identify their specific phages from environmental samples such as feces,sewage and rivers by co-cultivation.Through the lysis spectrum test,two bacteriophages with high lysis ability were screened,the biological characteristics were determined by the double-layer agar plate method,and the genetic background of the bacteriophage was further interpreted through phage function prediction and phylogenetic analysis.Results seventy-six strains of Clostridium perfringens were isolated and identified from viscera and feces samples from different animal sources in shandong,guangxi and hebei provinces: 63 strains from poultry(separation rate 17.3%),3 strains from sheep(separation rate 50%)and 10 strains from aquatic animals(separation rate 38.5%).C type 56.5%(43/76)was the most popular,followed by A type 43.4%(33/76).The positive gene of NetB toxin was detected only from A strain of chicken.One strain isolated from aquatic animal source carried cpe toxin gene.None of the isolates were detected with the 2 toxin gene.In the genome of Clostridium perfringens,a region integrated with a prophage and many coding genes related to the phage were found,and antibiotic resistance genes and toxin genes were not detected in the complete prophage region.With the isolated strain as the host,through co culture,ten high titer bacteriophages of Clostridium perfringens were isolated and purified from farm sewage,feces,slaughterhouse sewage and rivers.Among them,bacteriophage CPYS4 and CPYF3,CPJF7,CPH14 and CPYS8 showed typical lytic plaque,and CPYS48 and CPYF1,CPRJF10,CPRYF11 and CPYF4 had halo characteristics around the plaque.By cleavage spectrum test,Two bacteriophages CPYS4 and CPYS48 with high cleavage rate and cross cleavage of type A and C strains were preliminarily screened.The optimal temperature of the two phages was about 37? and the optimal pH was alkaline.The lysis period of CPYS4 phage was long,and the optimal number of infections was 0.1,the optimal multiplicity of phage CPYS48 infection was 1.The CPYS4 and CPYS48 linear double-stranded DNA genomes vary greatly in size.The bacteriophage CPYS4 genome is about 17491 bp,with an average G + C content of about28.23%,and contains 23 ORFs.It belongs to the order Virovirus and Breviviridae.The bacteriophage CPYS48 genome is about 51953 bp,with an average G + C content of about34.13%,containing 78 ORFs,belonging to the order Caunovirus,Myoviridae.No genes encoding toxins,antibiotic resistance genes and lysogenicity were found in the whole genomes of the two bacteriophages.Through phylogenetic relationships and genome-wide homology analysis,CPYS48 is a newly discovered bacteriophage.In summary,Clostridium perfringens isolated in this study was predominantly type C,followed by type A.The NetB toxin gene was only detected from C.perfringens type A in chickens,with a positive rate of22%.For the isolated strains,this study screened two strains with high lysis,good adaptability and safety.Among them,CPYS48 is a newly discovered bacteriophage,at the same time,the inducible prophage was found from the genome of C.perfringens.It provides basis and new ideas for the precise prevention and control of C.perfringens infection and bacteriophagetransformation by bacteriophage.