| Phages（bacteriophages） are viruses which can infect bacteria, mycetes, actinomycetes and spirochaetes.Phages can be divided into cleavage phage and temperate phages. About65%of the bacteria of which the genome have sequenced possess a significant amount of prophages or remnant prophage DNA,such as Shigatoxin-producing Escherichia coli（STEC）and salmonella. Shiga toxin-producing Escherichia coli （STEC） strains are food-borne pathogens whose ability to produce Shiga toxin （Stx） is due to integration of Stx-encoding lambdoid bacteriophages. These phages can change the phenotype of the host bacterium, enhance virulence or change the avirulent strains to pathogenic strains,advancing the development of population genetic diversity. Genes coded by phage are replicated and transcripted in the process of induction. The ability to type Stx phages induced from STEC isolates will enable definition of the level of heterogeneity among Stx phages and identification of specific phage genes that are disseminated across a bacterial population or even enable the discovery of phage-borne genes that are associated with enhanced pathogenicity fitness of their bacterial hosts.In this study,418strains of E.coli were act as experimental materials.Induced the endogenous prophages using norfloxacin,selected positive results using double layer agar plates.Meanwhile,selected λ-like phages using PCR by amplifying three genes of Q,stxlA,stx2A.The results showed that phage plaque of65strains of E.coli were clear. PCR positive result of Q gene,stxlA and stx2A was24,5and137. These results included three cases:only double layer agar plates results were positive but Q,and stx2A genes were negative results;only the Q and stx22A genes were positive results,but double layer agar plates results were negative; three kinds of test were positive situation.22strains of which both phage plaque and Q genes were positive.9strains of which both stx2A and Q genes were positive. There were44strains of which the plaque was positive but Q gene test was negative,there were15strains of which the plaque was positive but stx2A gene test was negative, there were2strains of which Q gene was positive but the plaque was negative; there are68strains of which stx2A were positive but the plaque screening was negative. Through statistics,71.43% of the Stx phages found in serotype O157strains and the rest distributed in serotypes0138and K88E. coli; Only45.45%of λ-like phage found in serotype0157strains and the rest was found in the unknown serotypes E. coli from duck, chicken and human. Stx phages that have similar gene structure,some can form plaque, and some can not form plaque. Two methods can complement each other, enhanced sensitivity and specificity of screening.38strains of Escherichia coli were selected as test materials. According to λ phage （GenBank accession number:NC002166, NC001416） and the Stx2phage （GenBank accession number:NC000924, NC010237） sequence,we chose int, N, O, P, Q, CI, stx2A, stx2B geneswhich were related to the core function. e and designed22pairs of primers.38strains of phage filtrate induced from Escherichia coli were amplified by PCR using these22pairs of primers,PCR products were purified, cloned into the vector pMD18-T, sequenced and BLAST analyzing homology. Results showed that detection rates of genes of int, N, O, P, Q, CI, stx2A, stx2B were as follows:16/38,6/38,16/38,18/38,10/38,14/38,4/38,5/38. The sequenced genes int, N, O, P, Q, CI, had98-99%,99-00%,98-99%,94-100%,96-99%,93-99%homology respectively compared with known sequence stx2phage （GenBank accessio number:NC000924, NC010237） and λ phage （GenBank acces-sion number:NC002166, NC001416） in NCBI. stx2A and stx2B had93%to100%homology compared with known sequence Min27and933w phage.The experiment results showed that the phage isolation had high homology compared with Stx2phage and933W phage, but their gene sequences were different. Further for each gene, using the Jotun Hein in DNAStar software made phylogenetic tree then used MegAlign to form a map to describe these phage genetic differences among populations. Q gene phylogenetic tree showed the standard strains0157and110E,66E were in a line, indicated these phages might be the same one, there were4strains of phage originated from the same branch with λ phage, the other strains existed a certain of distance, but the variability was little;while phylogenetic tree of stx gene showed the phages induced from the K88and0138strains were derived from the same branch with the known serotype strains0157. Thus, we can analyze relations between phages and their host bacteria’s virulence through the phylogenetic tree, with a view to lay the foundation for biological therapeutic agents of phage and prevention of their host bacteria. |
