产气荚膜梭菌噬菌体鉴定及其环境消减效果评价

doi:10.11843/j.issn.0366-6964.2022.10.027

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (10): 3550-3560.doi: 10.11843/j.issn.0366-6964.2022.10.027

产气荚膜梭菌噬菌体鉴定及其环境消减效果评价

吴立婷¹, 刘半红^1,2, 田源^1,3, 王娟⁴, 包红朵¹, 周艳¹, 庞茂达¹, 王冉¹, 张辉^1*

1. 江苏省农业科学院农产品质量安全与营养研究所, 江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地, 南京 210014;
2. 陕西科技大学食品与生物工程学院, 西安 710021;
3. 江苏大学食品与生物工程学院, 镇江 212013;
4. 西北农林科技大学, 杨凌 712100

收稿日期:2022-02-22 出版日期:2022-10-23 发布日期:2022-10-26
通讯作者: 张辉,主要从事致病微生物监测及生物防控技术开发,E-mail:Huiz@jaas.ac.cn
作者简介:吴立婷(1990-),女,江苏南京人,硕士生,主要从事食源性病原菌的耐药性研究,E-mail:1206186910@qq.com;刘半红(1998-),女,河南周口人,硕士生,主要从事食源性致病菌致病及防控研究,E-mail:2585294839@qq.com。
基金资助:
国家重点研发计划项目（2018YFE0101900）；江苏省农业科技自主创新资金项目（cx（21）1004）；江苏省重点研发计划项目（BE2022361）

Identification and Environmental Inhibition Effect of Clostridium perfringens Phage

WU Liting¹, LIU Banhong^1,2, TIAN Yuan^1,3, WANG Juan⁴, BAO Hongduo¹, ZHOU Yan¹, PANG Maoda¹, WANG Ran¹, ZHANG Hui^1*

1. Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology, Institute of Food Safe and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi'an 710021, China;
3. Jiangsu University-School of Food and Biological Engineering, Zhenjiang 212013, China;
4. Northwest A&F University, Yangling 712100, China

Received:2022-02-22 Online:2022-10-23 Published:2022-10-26

摘要/Abstract

摘要： 分离获得1株禽源产气荚膜梭菌（Clostridium perfringens，Cp）噬菌体，拟鉴定其生物学特性及裂解力，并在环境中应用评价其对Cp的消减效果。通过双层及单层平板法从肉鸡肠道组织样品中分离并纯化1株Cp噬菌体。运用透射电镜观察该噬菌体形态，分析其MOI、一步生长曲线、pH及温度耐受特性、宿主谱及体外抗菌活性。通过基因组测序分析其基因组信息，最终在模拟环境下评价噬菌体对Cp污染量消减效果。扫描电镜结果显示，该噬菌体为长尾噬菌体，潜伏期<20 min，爆发量为28.5 PFU·cell^-1，最佳MOI为0.01，能够特异性裂解禽源Cp菌株，对待检的43株Cp裂解率高达83.72%。基因组分析表明，该噬菌体为双链DNA噬菌体，全长41 590 bp，拥有59个开放阅读框，其中33个ORF与已知功能蛋白具有一定同源性，将该噬菌体命名为vB_CpeS_SD72。小亚基末端酶构建进化树分析表明，噬菌体与Clostridium phage vB CpeS-CP51同源性较高。在环境消减测试中观察到噬菌体作用0.5 h，杀菌效率可达98.36%；2 h能够抑制99.60%的Cp。研究证实鉴定出1株强裂解性的新型Cp噬菌体能够在环境中有效抑制Cp的污染，为食品动物养殖及畜禽产品中Cp污染和控制提供了可靠的候选者。

关键词: 产气荚膜梭菌噬菌体, 生物学特性, 基因组, 环境消减

Abstract: This study attempted to isolate a novel C. perfringens (Cp) phage from poultry. Biological characteristics and genomic information have been analyzed, which can provide new strategy for Cp infection and control. A Cp phage was isolated and purified from broiler intestinal tissue samples by double-layer and single-layer plate methods. The morphology of the phage was observed by transmission electron microscope, and the MOI, one-step growth curve, pH and temperature tolerance characteristics, host range and antibacterial activity in vitro were analyzed. The genomic information was analyzed by genome sequencing, and finally the effect of phage on reducing the pollution of Cp was evaluated in a space simulated environment. The results showed that the electron microscope showed that the phage was a long tailed phage, the latent period was less than 20 min, the burst size was 28.5 PFU·cell^-1, and the best MOI was 0.01. It could specifically inhibit avian Cp strains, and the host range was as high as 83.72%. Genome analysis showed that the phage was a double stranded DNA phage with a total length of 41 590 bp and 59 open reading frames (ORF), of which 33 ORFs had certain homology with known functional proteins. The phage was named as vB_CpeS_SD72. The phylogenetic tree analysis of terminase small subunit showed that the phage had high homology with Clostridium phage vB CpeS-CP51. In the environmental control test, it was observed that the phage could inhibit 98.36% Cp at 30 min and nearly completely inhibited within 2 h. A novel Cp phage with strong lytic activity can effectively inhibit Cp contamination in the environment, which provide a powerful strategy candidate source for Cp pollution and control in food, animal breeding and livestock and poultry products.

Key words: C. perfringens phage, biological characteristics, genome, environmental inhibition

中图分类号:

S851
Q939.48

吴立婷, 刘半红, 田源, 王娟, 包红朵, 周艳, 庞茂达, 王冉, 张辉. 产气荚膜梭菌噬菌体鉴定及其环境消减效果评价[J]. 畜牧兽医学报, 2022, 53(10): 3550-3560.

WU Liting, LIU Banhong, TIAN Yuan, WANG Juan, BAO Hongduo, ZHOU Yan, PANG Maoda, WANG Ran, ZHANG Hui. Identification and Environmental Inhibition Effect of Clostridium perfringens Phage[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3550-3560.

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产气荚膜梭菌噬菌体鉴定及其环境消减效果评价

Identification and Environmental Inhibition Effect of Clostridium perfringens Phage

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