| Campylobacter jejuni is a gram-negative microaerophilic bacterium and one of the most common pathogens causing human intestinal diseases.In recent years,due to the abuse of antibiotics,the resistance of Campylobacter jejuni to antibiotics has continued to increase.The WHO has listed Campylobacter jejuni as an important antibiotic-resistant bacterium,and an urgent need for new antimicrobial agents.Phage are bacterial virus,which can specifically recognize and infect the host bacteria,replicates in large numbers in its body,eventually lysis and killing the host bacteria.Compared with antibiotics,phage products have many advantages,such as simplicity,specificity,rapid proliferation,and strong antibacterial ability.In this study,the phage of Campylobacter jejuni was isolated and screened from chicken feces and sewage samples,and the basic biological characteristics and genomic characteristics of phages were identified.The optimal application conditions of Campylobacter jejuni were studied,and the effect of phage on Campylobacter jejuni was evaluated.1、Isolation and selection of Campylobacter jejuni phageUsed Campylobacter jejuni NCTC11168 as the host strain,and with the method of double-layer plate,244 positive samples were detected from 1480 chicken feces and sewage samples.The positive rate of feces samples was 19.69%(189/960),and the positive rate of sewage samples was 10.58%(55/520).After preliminary selecting and purification,45 broad-spectrum Campylobacter jejuni phages were obtained.The positive rate and lysis spectrum of phage in different samples were significantly different.The phage positive rate of fecal samples(19.69%)was higher than that of sewage samples(10.58%).However,the phages isolated from sewage samples have stronger lysis ability than that from fecal samples.The highest phage positive rate(43.46%)was found in fecal samples collected from the the live poultry market.The phages in feces samples collected from the small broiler farm had the strongest lysis ability.The positive rate(36.74%)and the phage lysis ability of phage which isolated from broiler rearing on floor were both higher than those from rearing in battery(2.00%)and free range(7.00%).The lysis spectrum analysis showed that 45 Campylobacter jejuni phages displayed strong lytic ability,the lysis rates of Campylobacter jejuni isolates from chicken,human and cattle were 60.71%(34/56),76.19%(16/21)and 75.00%(6/8).After further analysis of the lysis spectrum data,9 broad-spectrum phages were selected(CP7,CP13,CP15,CP23,CP32,CP33,CP35,CP39,CP47).Among them,the highest lysis rate was CP39(53.33%),the lowest lysis rate was CP47(32.22%),and the lysis spectrum of CP7 was the most different from the other 8 phages.After the combination of 9 phages,the lysis rate increased from 32.22%to 70.00%.2、Characterization of Campylobacter jejuni phage CP7 and CP39In order to study the basic biological characteristics of Campylobacter jejuni phage,and determine the genetic background and genomic characteristics of phage,the phage CP7 and CP39 were selected.Identification of biological characteristics,such as spectrum determination,plaque,morphological identification,optimal multiplicity of infection(MOI),and one-step growth curve,thermal stability,pH tolerance and genomic enzyme cutting analysis.Meanwhile,the whole genome sequencing and functional annotation of phage CP39 were carried out to compare the evolutionary relationship between CP39 and other Campylobacter phages.Transmission electron microscopy showed that both phage CP7 and CP39 belong to family of Myoviridae with a typical terminal bleb and empty head.The genome size of phage CP7 and CP39 is similar,about 140kb,and both of thembelong to group Ⅲ Campylobacter phage.The genome can be digested by the restriction endonucleases Hind Ⅲ,Hha Ⅰ,Dra Ⅰ,EcoR Ⅴ and TaqⅠ,and can not be digested by other restriction endonucleases.The phage CP7 and CP39 had strong tolerance to pH,and still have a relatively high titer in pH 3 and pH 12.The optimum temperature of phage CP7 and CP39 was below 50℃,but CP7 was more tolerant of high temperature than CP3 9.The latent period of phage CP7 and CP3 9 was 45 min,the burst period of CP7 was 120 min,the average burst size was 354/cell,the burst period of CP39 was 60 min,and the average burst size was 177/cell.Genome sequencing showed that the whole genome size of phage CP39 was 130.715 kb,and the content of G+C was 26%.The whole genome of CP39 contains a total of 168 ORFs and 3 tRNAs.54 ORFs were known for function,and 114 ORFs were hypothetical protein expression sequences.The phylogenetic tree was constructed by the major capsid protein compared with the existing 11 Campylobacter phages.It was found that the phage CP39 and Campylobacter phage of group III were in the same cluster,and in the same branch with phage vB_CjeM_Losl.CP39 has a distant relationship with Campylobacter phage of group II.Whole genome analysis of phage CP39 did not find antibiotic resistance genes and virulence genes.3、Preliminary application of Campylobacter jejuni phage CP7 and CP39After identification of the basic biological characteristics of phage CP7 and CP39,a series of single-factor experiments were studied to explore the optimal application conditions of phage.The optimal time for phage incubation was 4 h and the optimal titer was 1×1010 pfu/ml,the optimal temperature was 25℃.Based on the researches above,the efficacy of phage on the reduction of Campylobacter jejuni in chicken feces samples was determined.The results of reduction experiment showed that the reduction of Campylobacter jejuni by phage was between 1-5 log.Among the 200 samples,37.5%(75)samples were reduced about 5 log,the average content of Campylobacter jejuni was reduced to 0.15 lg cfu/g,and 57.0%(114)samples were reduced about 2 log,the average content of Campylobacter jejuni was reduced to 3.46 lg cfu/g,and 5.5%(11)samples were reduced about 1 log,the average content of Campylobacter jejuni was reduced to 4.71 lg cfu/g. |
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