一株新型牛源肺炎克雷伯菌噬菌体的分离鉴定

Isolation and identification of a novel bovin Klebsiella pneumoniae bacteriophage vB_Kpn_B01

摘要:

[背景] 肺炎克雷伯菌（Klebsiella pneumoniae）是一种广泛分布于环境中的重要致病菌，该菌较高的耐药性致其在养殖业中治疗较为困难。[目的] 分离一株裂解性肺炎克雷伯菌噬菌体，对分离株进行生物学特性鉴定和基因组学分析。[方法] 使用双层平板法从四川省某奶牛场中分离、纯化出一株裂解性噬菌体，测定其裂解谱、热稳定性、酸碱耐受度、最佳感染复数及一步生长曲线等生物学特性，并进行全基因组的测序及注释分析。[结果] 得到一株裂解性肺炎克雷伯菌噬菌体vB_Kpn_B01，该噬菌体拥有透明且无晕环的噬菌斑，热稳定性较高，在极酸或极碱环境下不进行裂解活动，特异性较强，属长尾噬菌体科（Siphoviridae）。vB_Kpn_B01全基因组大小为113 227 bp，GC含量为47.97%。注释结果显示噬菌体拥有149个编码序列和25个tRNAs，不含耐药基因及毒力基因。通过噬菌体的进化树分析发现，该噬菌体为Sugarlandvirus。[结论] vB_Kpn_B01拥有高效的生长特性和对不利环境的低耐受性，拥有裂解宿主菌的必备基因，并不含耐药基因及毒力基因，具有应用于畜牧业中防治多重耐药细菌的潜力。

Abstract:

[Background] Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can infect both humans and bovine. The wide spread of Klebsiella pneumoniae and its high drug resistance have caused great difficulties in the treatment of the disease. [Objective] To isolate a lytic Klebsiella pneumoniae bacteriophage. Its biological properties were studied, and the entire genome analysis of the bacteriophage was performed. [Methods] Klebsiella pneumoniae bacteriophage was isolated by the double-layer agar culture method from a dairy farm trough in Sichuan province. We used the same method to conduct host range, thermostability, pH, optimal multiplicity of infection (MOI), and one-step growth experiment. Phage morphology was observed by transmission electron microscopy (TEM). The whole genome of the bacteriophage was sequenced. [Results] A lytic Klebsiella pneumoniae bacteriophage vB_Kpn_B01 was isolated which had strong specificity. The morphology of plaque was round, transparent plaques with clear boundaries. Phage found in this study was double-stranded DNA bacterial viruses belonging to the Siphoviridae family, and the size of it was 113 227 bp. Comparative genome analysis revealed that vB_Kpn_B01 genome possesses the highest similarity to the bacteriophages in the genus of Sugarlandvirus. The genome of vB_Kpn_B01 comprises 149 coding sequences (CDS) and 25 tRNAs, but does not contain any known antibiotic resistant genes or virulent genes. [Conclusion] Phage vB_Kpn_B01 has a strong lysis ability and the necessary genes to lyse host bacteria. It has the potential to be used in the prevention and treatment of multi-drug resistant bacteria in animal husbandry.