弯曲菌宽谱烈性噬菌体生物学特性、全基因组测序分析及初步应用

Biological Characteristics,Whole-genome Sequencing Analysis and Preliminary Application of Campylobacter Phage with Broad Spectrum

为探索控制弯曲菌污染的新途径,以弯曲菌NCTC12662为宿主菌,从养殖场鸡粪便中分离纯化得到弯曲菌噬菌体,并对其进行电镜形态特征观察、生物学特性测定与基因组学分析,同时采用棋盘法,测试噬菌体与抗生素的联合应用效果。结果显示,从山东省胶东地区养鸡场粪便中分离到1株噬菌体P-61。透射电镜下显示该噬菌体颗粒具有1个直径约为102.6 nm的二十面体头部和1个长度约为112.9 nm的长尾部,属于有尾噬菌体目,长尾噬菌体科;裂解谱为48.48%,裂解效价为5.5×10^(11)pfu/mL,感染复数为0.01;一步生长曲线显示潜伏期为20 min,裂解期约为130 min,裂解量为240 pfu/cell;最适宜生长温度为42℃,pH耐受性较强,在pH 5~9范围内效价较稳定。噬菌体基因组全长为130425 bp,G+C含量为26.13%,共有176个开放阅读框、3个tRNA,软件预测基因组中不含毒力基因和抗生素耐药基因。利用棋盘法测得,当噬菌体为10^(6) pfu/mL时,环丙沙星最低浓度降至1/8 MIC,联合用药组具有明显的联合杀菌效果。综上可知,得到的噬菌体P-61具有效价高、裂解谱宽及对环境适应能力强等生物学特性,且基因组信息清晰,能与抗生素联合应用发挥协同作用,具有消毒、净化等临床应用可能性。本研究为噬菌体制剂的开发利用提供了生物资源。

In order to explore a new pathway to control Campylobacter(C.)contamination,C.phage was isolated and purified from chicken fecal samples taking C.NCTC12662 as the host bacteria,followed by electron microscopy observation and biological characterization and genomic analysis.Meanwhile,its effect produced jointly with antibiotic was tested using a checkerboard method.The results showed that a strain of C.phage P-61 was isolated.It was shown that,by a transmission electron microscope,the phage particle had an icosahedral head with a diameter of 102.6nm and a long tail with a length of 112.9 nm,falling into Caudovirales of Siphoviridae;the cleavage spectrum was 48.48%,with a titer of 5.5×10^(11)pfu/mL and multiplicity of infection(MOI)of 0.01;it was shown that,by one-step growth curve,the latent period was 20 min,the lysis period was about 130 min,and the lysis amount was 240 pfu/cell;the optimum temperature was 42℃ with strong pH tolerance,and the titer was stable within the range of pH 5 to9.The total length of phage genome was 130425 bp,and the G+C content was 26.13%,there were 176 open reading frames and 3 tRNAs,but no virulence gene and antibiotic resistance gene were contained in the genome as predicted by software.It was measured by the checkerboard method that the lowest concentration of Ciprofloxacin was reduced to 1/8 MIC when P-61 was 10^(6) pfu/mL,indicating an obviously combined bactericidal effect.In conclusion,the obtained phage P-61 had the biological characteristics of high titer,broad spectrum and strong adaptability to environment,and the genome information was clear,which could play a synergistic role in the combined application of antibiotics,indicating a possibility of clinical application such as disinfection and purification.Biological resources were provided by the study for future development and utilization of phage preparations.