鲍曼不动杆菌噬菌体SWH-Ab-3的生物学特性及其全基因组初步研究

Biological characteristics and preliminary genome analysis of Acinetobacter baumannii bacteriophage SWH-Ab-3

目的从医院污水中分离鲍曼不动杆菌裂解性噬菌体,探讨其生物学特性及基因组基本信息。方法以临床分离的鲍曼不动杆菌为指示菌,采用双层琼脂平板法分离、纯化噬菌体,观察噬菌斑特征;利用透射电子显微镜观察噬菌体的形态特征;采用点板试验测定噬菌体的裂菌谱,并观察其最佳感染复数(MOI)、一步生长曲线以及对热、酸碱等理化因素的耐受性。提取噬菌体全基因组DNA,采用Illumina Hiseq2000高通量测序,并对其基因组序列进行注释、比对分析。结果从污水中成功分离出1株鲍曼不动杆菌的裂解性噬菌体,命名为SWH-Ab-3;噬菌体头部直径约为70 nm,尾长约80 nm,初步判断其属肌尾噬菌体科;该噬菌体裂菌谱较窄(6株/42株),最佳感染复数为0.1,潜伏期为40 min,爆发期为50 min,裂解量为31.4 PFU/cell,对热和酸碱环境的耐受性较高。SWH-Ab-3基因组长度为41 730 bp,G+C%含量为39.38%,共编码53个ORF(33个已知功能基因,20个假想蛋白),其序列与Abp1和vB_AbaP_B3的同源性最高(≥94.0%),但基因组长度、G+C%含量和形态特征差异显著。结论 SWH-Ab-3为1株新分离的鲍曼不动杆菌裂解性噬菌体,属肌尾噬菌体科,对热、酸碱等理化因素较耐受,可能通过穿孔素和裂解酶组成的'二元裂解系统'发挥裂菌作用,裂解能力较强,但裂菌谱较窄。

Objective To investigate the biological characteristics and basic genomic information of Acinetobacter baumannii(A.baumannii)lytic bacteriophage isolated from hospital sewage.Methods Bacteriophage was isolated and purified with indicator of A.baumannii by double-layer agar plate method,and plaque characteristics of the phage were observed.The structure of purified phage was observed with transmission electron microscopy.The host spectrum was determined by point plate test,and optimal multiplicity of infection(MOI),one-step growth curve,temperature and pH stability of the phage were investigated.The whole genome DNA of phage was extracted and sequenced by Illumina Hiseq2000 high-throughput sequencing technology,and then the genome sequence was annotated and analyzed by alignment.Results A strain of lytic bacteriophage against the A.baumannii,named SWH-Ab-3,was isolated from sewage.The phage had a head of about 70 nm in diameter and a tail of about 80 nm in length.SWH-Ab-3 was identified to belong to Myoviridae family.The bacteriophage had a narrower lytic spectrum of A.baumannii(6/42),optimal MOI of 0.1,latent period of 40 min and burst period of 50 min,and the burst size about 31.4 PFU/cell,and could stand the temperature up to 60℃and keep stable titer under acid or alkali environment.Its genome size was 41 730 bp with G+C content of 39.38%.There were 53 ORF encoded,of which 33 were known functional genes and 20 were hypothetical proteins.The BLASTn alignment found that SWH-Ab-3 had the highest homology with Abp1 and vB_AbaP_B3(>94%),but the length of the genome,the G+C%content and the morphological characteristics of the phage were significant.Conclusion SWH-Ab-3 is a new lytic phage of A.baumannii,belonging to the Myoviridae family.It is tolerant to heat,acid and alkali.It may lyse the host bacteria through'two-element lysis system',composed of holin and lyase.It has stronger lytic ability,but narrower lytic spectrum.