五株克雷伯氏菌噬菌体的生物学特性及比较基因组学研究

Biological characteristics and comparative genomics of five Klebsiella phage isolates

【目的】耐药性克雷伯氏菌属(Klebsiella)的细菌作为人类感染的重要病原,是临床治疗重要的挑战。本研究对多株克雷伯氏菌裂解性噬菌体的生物学特性和基因组特征进行比较分析,为其应用提供更多科学数据。【方法】使用双层平板法从人类和动物新鲜粪便、污水中分离纯化裂解性克雷伯氏菌噬菌体;通过磷钨酸染色和透射电镜观察其形态;采用双层平板噬菌斑法确定其宿主范围,测定温度和pH稳定性、一步生长曲线和体外抑菌效果等生物学特性;基于全基因组测序对分离株进行比较基因组学分析;通过体内抑菌试验评估噬菌体对多重耐药变栖克雷伯氏菌(Klebsiella variicola)BS375-3感染的大蜡螟(Galleria mellonella)幼虫的保护作用。【结果】5株噬菌体分别属于Schitoviridae(pKP-BM327-1.2)、Autographiviridae(pKP-M186-2.1、pKP-M186-2.2和pKV-BS375-3.1)、Drexlerviridae(pKP-BS317-1.1)家族;噬菌体pKV-BS375-3.1可裂解受试菌中的8株,pKP-BM327-1.2可裂解受试菌中的3株,pKP-M186-2.1、pKP-M186-2.2和pKP-BS317-1.1则分别裂解受试菌中的1株;5株噬菌体感染10–20 min后即进入指数增长期,在–20–37℃、pH 6–10环境下均能够保持稳定活性;感染变栖克雷伯氏菌BS375-3后经噬菌体pKV-BS375-3.1处理[感染复数(multiplicity of infection,MOI)=100]的大蜡螟幼虫96 h内存活率达到80%(8/10);5株噬菌体基因组长度在42–77 kb之间,未携带抗生素抗性基因和毒力基因,基于内溶素(endolysin)的溯源分析显示该蛋白在克雷伯氏菌噬菌体中呈现多样性,属内呈保守性。【结论】5株克雷伯氏菌噬菌体均具有较好的体外抑菌活性,生物学特性稳定,endolysin在噬菌体属内呈现保守性。宿主谱宽、潜伏期短的噬菌体pKV-BS375-3.1在治疗Klebsiella pneumoniae和K.variicola临床感染方面具有潜在应用前景。

[Objective]As a major pathogen of human infection,multidrug-resistant Klebsiella poses a great challenge in clinical treatment.To provide more scientific data for the application of phages,we analyzed and compared the biological and genomic characteristics of several strains of Klebsiella phages.[Methods]Klebsiella phages were isolated from human and animal fresh feces and sewage by the double plate method.The phage morphology was observed by phosphotungstic acid staining and transmission electron microscopy.The host spectrum,thermal stability,and pH stability of the phages were measured,and the one-step growth curve and in vitro bacteriostatic curve were established.The comparative genomics analysis was carried out for the whole genomes of the phage isolates.We further evaluated the protective effect of the phage isolates by the survival rate of Galleria mellonella larvae infected with the multidrug-resistant mutant Klebsiella variicola BS375-3 in vivo.[Results]The five phage isolates belonged to Schitoviridae(pKP-BM327-1.2),Autographiviridae(pKP-M186-2.1,pKP-M186-2.2,and pKV-BS375-3.1),and Drexlerviridae(pKP-BS317-1.1).The phage isolates pKV-BS375-3.1 and pKP-BM327-1.2 could lyse eight and three bacterial hosts,respectively,while pKP-M186-2.1,pKP-M186-2.2,and pKP-BS317-1.1 could only lyse one bacterial host.The five phage isolates entered the exponential growth stage 10–20 min after inoculation and maintained stable activity at–20℃–37℃and pH 6–10.The survival rate of G.mellonella larvae infected with the K.variicola BS375-3 treated with pKV-BS375-3.1(MOI=100)reached 80%(8/10)after 96 h.The genomes of the five phage isolates had the length of 42–77 kb and did not carry resistance genes or virulence genes.The traceability analysis based on endolysin showed that the protein demonstrated diversity in Klebsiella phages and was conserved within the genus.[Conclusion]All the five Klebsiella phage isolates had good antibacterial activity in vitro and stable biological characteristics.The endolysins of the phage isolates were conserved within the genus.The phage pKV-BS375-3.1 with a wide host range and short latency has a potential application prospect in the clinical treatment of Klebsiella pneumoniae and K.variicola infections.