小肠结肠炎耶尔森氏菌烈性噬菌体Yen-yong1的生物学特性及其对生猪肉片中病菌的清除作用

Biological characteristics of virulent phage Yen-yong1 and its sterilization effect against Yersinia enterocolitica in raw pork slices

摘要:

目的 小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)是耶尔森氏菌病(yersiniosis)的主要致病因子，是全球最重要的食源性致病菌之一。本研究的目的在于分离、鉴定其烈性噬菌体，并探讨噬菌体的应用潜力。方法 以Yersinia enterocolitica CICC 10869为指示宿主，采用双层琼脂平板法从浙江省宁波市北仑区路林市场下水道中分离得到一株烈性噬菌体Yersinia phage Yen-yong1 (简称Yen-yong1)，对其进行形态学观察、宿主范围测定、全基因组测序、基因功能注释、系统进化地位分析、理化耐受性测定以及生物被膜去除能力测定，并检测其对生猪肉片中小肠结肠炎耶尔森氏菌的清除作用。结果 Yen-yong1可在1.5 h内使菌液裂解澄清。透射电镜下，Yen-yong1呈长尾病毒样(Siphovirus-like)，椭圆形头部长(83.32±0.15) nm、宽(59.57±2.28) nm；尾长(168.04±3.21) nm。Yen-yong1的dsDNA基因组全长51 321 bp，G+C含量为47.80%，包含93个开放阅读框(open reading frames, ORFs)，且不含影响应用的溶原、耐药或毒力因子基因。Yen-yong1与公共数据库中的其他噬菌体基因组间的最高核苷酸序列相似度低至20.64%，远低于属界限值(70.00%)；与其他噬菌体间共享的核心基因(core gene)占比最高值仅为20.00%，小于亚科阈值(27.00%-79.00%)。Yen-yong1在有尾纲(Caudoviricetes)中代表一个新的属和亚科。Yen-yong1具有种特异性，仅裂解小肠结肠炎耶尔森氏菌，在4株供试小肠结肠炎耶尔森氏菌菌株中可裂解3株。Yen-yong1在-80-60 ℃、pH 2.0-13.0、0-3%氯化钠的胁迫下均保持高水平的溶菌活性。此外，Yen-yong1能够破坏细菌生物被膜。在25 ℃下，当噬菌体与宿主的感染复数(multiplicity of infection, MOI)为100时，Yen-yong1对生猪肉片中小肠结肠炎耶尔森氏菌CICC 10869、CICC 21669和CMCC 52202株的清除率分别为93.48%、52.77%和43.24%；在4 ℃下，3株供试菌株均可增殖，而在MOI为100时，Yen-yong1对生猪肉片中CICC 10869、CICC 21669和CMCC 52202株的清除率分别为98.28%、78.35%和96.33%。结论 Yen-yong1代表一个以往未知的进化谱系，其具有优良的生物学特性以及在常温和低温下良好的保鲜潜力。本研究拓宽了对噬菌体的认知，丰富了噬菌体库和基因库，为食源性耶尔森氏菌病的防控奠定了基础。

Abstract:

Objective Yersinia enterocolitica is the primary pathogenic agent of yersiniosis, and it is one of the major foodborne pathogens worldwide. The purpose of this study is to isolate and identify a virulent phage against Y. enterocolitica and explore its application potential.Methods A novel virulent phage, named Yersinia phage Yen-yong1 (abbreviated as Yen-yong1), was isolated from the sewers of Lulin Market in Beilun District, Ningbo City, Zhejiang Province, China, by the double-layer agar method with Y. enterocolitica CICC 10869 as the indicator host. The morphology, host range, genome sequence, gene functions, phylogenetic position, physical and chemical tolerance, and biofilm removal ability of Yen-yong1 were studied. Additionally, the sterilization effect of Yen-yong1 on Y. enterocolitica in raw pork slices was evaluated.Results Yen-yong1 clarified the turbid CICC 10869 culture within 1.5 h. Yen-yong1 was Siphovirus-like, consisting of an elliptical head with a length of (83.32±0.15) nm and a width of (59.57±2.28) nm and a long tail with a length of (168.04±3.21) nm. The double-stranded DNA genome of Yen-yong1 was 51 321 bp, with the G+C content of 47.80% and 93 open reading frames (ORFs). No known lysogeny-associated genes, resistance genes, or virulence genes detrimental to application were found in Yen-yong1. The highest nucleotide sequence similarity between Yen-yong1 and the other phages in public databases was 20.64%, which was <70.00% (the threshold to define a genus). Yen-yong1 shared 20.00% of core genes with the closest relative, which was lower than the ICTV criteria for subfamily classification (27.00%?79.00%). The results suggested that Yen-yong1 represented a novel genus and a novel subfamily belonging to Caudoviricetes. Yen-Yong1 was species-specific and capable of lysing 3 out of 4 tested Y. enterocolitica strains. Yen-yong1 maintained high bacteriolytic activity at -80?60 ℃ and pH 2.0?13.0, as well as in the presence of 0?3% NaCl. Yen-yong1 can disrupt the biofilms of Y. enterocolitica. At 25 ℃ and MOI=100, the sterilization rates of Yen-yong1 against Y. enterocolitica CICC 10869, CICC 21669, and CMCC 52202 in raw pork slices were 93.48%, 52.77%, and 43.24%, respectively. All the 3 tested Y. enterocolitica strains proliferated at 4 ℃. At 4 ℃ and MOI=100, the sterilization rates of Yen-yong1 against Y. enterocolitica CICC 10869, CICC 21669, and CMCC 52202 in raw pork slices were 98.28%, 78.35%, and 96.33%, respectively.Conclusion Yen-Yong1 reveals a previously unknown evolutionary lineage. Yen-Yong1 has excellent biological characteristics and potential applications. This study enriches the knowledge of Y. enterocolitica phages, phage database, and gene database, laying a foundation for the application of phages in the prevention and control of food-borne yersiniosis.