副溶血弧菌噬菌体Vpas_PP24的分离鉴定及生物学特性

Isolation, identification, and characterization of Vibrio parahaemolyticus phage Vpas_PP24

摘要:

【背景】副溶血弧菌是南美白对虾养殖中常见的致病菌，传统的抗生素防治办法不仅低效，而且越来越难以满足食品安全和绿色环保及可持续发展的要求。副溶血弧菌的生物防治是南美白对虾养殖业可持续发展的必由之路。噬菌体是天然安全的活体抗菌剂，因其对特定细菌的专一性感染和高效性裂解而备受关注。【目的】分离一株能高效裂解副溶血弧菌的烈性噬菌体，为探索副溶血弧菌的噬菌体防治方法提供基础研究。【方法】以28株病虾来源的副溶血弧菌为宿主菌，用双层琼脂平板法从海鲜市场污水中分离副溶血弧菌噬菌体；点斑法测定噬菌体的裂解谱，并对筛选到的宽裂解谱噬菌体进行透射电镜(transmission electron microscopy，TEM)观察、生物学特性测定和全基因组序列分析。【结果】分离筛选到一株副溶血弧菌烈性噬菌体，命名为Vpas_PP24。透射电镜观察显示该噬菌体头部为二十面体，有一长尾，头部长约92 nm，宽约46 nm，尾部长约147 nm，属于有尾噬菌体目长尾噬菌体科。其基因组全长83 482 bp，预测有118个开放阅读框(open reading frames，ORFs)，具有已知功能的有13个，不含非编码RNA、毒力基因及抗生素抗性基因。基因组一致性对比显示噬菌体Vpas_PP24可能为弧菌噬菌体的一个新种。Vpas_PP24对28株副溶血弧菌的裂解率为54%，对其他种属的116株弧菌的总裂解率为16%；最佳感染复数(multiplicity of infection，MOI)为0.000 1，效价可达3.0×10¹⁰ PFU/mL。一步生长曲线显示Vpas_PP24的潜伏期为10 min，暴发期为150 min，暴发量为30 PFU/cell。该噬菌体在温度<50 ℃、pH 4.0-11.0范围内活性稳定，对糜蛋白酶、木瓜蛋白酶和对虾肝胰腺酶提取液的水解作用不敏感，但蛋白酶K可快速使其失活，紫外辐照也能使Vpas_PP24失活。宿主菌对该噬菌体的不敏感突变频率为2×10^-5。【结论】分离筛选到一株裂解谱较宽、基因型较新、生物学性质较稳定的副溶血弧菌噬菌体，该噬菌体具有进一步开发成为新型副溶血弧菌抗菌剂的潜力。

Abstract:

[Background] Vibrio parahaemolyticus is a major pathogen of Penaeus vannamei. Posing a threat to food safety, environment, and sustainable development, conventional antibiotic therapy is no longer effective. Biocontrol of this bacteria seems to inevitable for the sustainable development of P. vannamei aquaculture. Phage, a natural safe antibacterial, is highly specific and typically only infects or kills an individual species of bacteria with high efficiency. [Objective] To identify virulent phage that can efficiently lyse V. parahaemolyticus so that the set of methodology was established to explore the phage agents for the control of V. parahaemolyticus. [Methods] Double-layer agar technique was used to enrich phages from sewage samples of seafood market with 28 strains of V. parahaemolyticus derived from diseased shrimps. The lytic spectra of phages were determined through spot test. The one with broad spectrum was characterized by transmission electron microscopy, biological characterization, and whole-genome sequencing. [Results] Vpas_PP24, a virulent phage against V. parahaemolyticus was screened out. It had an icosahedral head with about 92 nm in length and around 46 nm in width and a long tail of about 147 nm in length, which was thus identified as a member of Siphoviridae in Caudovirales. The full-length genome of Vpas_PP24 was 83 482 bp, and it harbored 118 open reading frames (ORFs), with 13 known protein-coding genes, and no non-coding RNA, virulence genes, or resistance genes. Genome alignment suggested that Vpas_PP24 may be a new phage species against Vibrio. The phage could lyse 15 (54%) of the 28 V. parahaemolyticus strains and 18 (16%) of 116 other Vibrio species. The optimal multiplicity of infection (MOI) of Vpas_PP24 was 0.000 1 and titer was 3.0×10¹⁰ PFU/mL. One-step growth curve indicated that it had a latent period of 10 min, rise period of 150 min, and burst size of 30 PFU/cell. The optimal temperature and pH for the phage growth were<50 ℃ and pH 4.0–11.0, respectively. The phage was tolerant to chymotrypsin, papain, and shrimp hepatopancreas extract but sensitive to proteinase K. Vpas_PP24 was also sensitive to ultraviolet radiation. The emergence frequency of Vpas_PP24-insensitive mutants was 2×10^-5. [Conclusion] A virulent phage Vpas_PP24 with new genotype was screened out. It has the potential to be further developed into novel anti- V. parahaemolyticus agent, thanks to its broad lytic spectrum, biological properties, as well as its promising stability.