1株环境源单增李斯特菌的分离鉴定及生物特性分析

doi:10.16431/j.cnki.1671-7236.2023.04.026

摘要/Abstract

摘要： 【目的】探究牦牛屠宰场中存在的单增李斯特菌对牦牛肉造成污染的风险。【方法】通过细菌分离培养、形态观察、生化试验及分子学方法对牦牛屠宰场50份污水样品中的单增李斯特菌进行分离鉴定;通过PCR方法对分离株血清型和毒力基因进行鉴定和检测,并测定分离株在不同生长条件下D_{600 nm}值;通过细胞的黏附和侵袭试验及小鼠半数致死量(LD₅₀)确定分离株的毒力。【结果】从50份污水样品中分离出1株革兰氏阳性短杆状细菌。分离株在李斯特菌显色培养基上为蓝绿色菌落,镜检为革兰氏阳性两端钝圆的短杆状菌,疑似为单增李斯特菌。生化试验结果显示,分离株可水解七叶苷,发酵葡萄糖、麦芽糖和鼠李糖,MR、VP、动力、过氧化氢酶试验为阳性,甘露醇和木糖发酵试验为阴性。系统进化树显示,分离株与单增李斯特菌EDG-e具有高度同源性,序列相似性为99%,血清型鉴定其血清型为1/2a。毒力基因检测结果显示,分离株携带inlB、inlC、inlJ、actA、plcA、prfA、mpl、hly、inlA、SigmaB毒力基因。抗应激能力试验显示,分离株在低温、强酸、强碱、高盐、乙醇胁迫和氧化等极端环境中的生长速度极显著高于标准株ATCC 19111(P＜0.01)。细胞试验显示,分离株对RAW246.7细胞的黏附率为8.3%,侵袭率为2.5%,其黏附和侵袭能力均极显著高于标准株ATCC 19111(P＜0.01)。对Caco-2细胞的黏附率为6.2%,侵袭率为1.5%,黏附和侵袭能力均极显著高于标准株ATCC 19111(P＜0.01)。动物试验结果显示,分离株对小鼠的LD₅₀为10^4.7 CFU。【结论】本研究获得的单增李斯特菌分离株血清型为1/2a,为引起人类李氏杆菌病的主要血清型,其抗应激能力较强,属于强毒株。本研究为牧区单增李斯特菌的监测提供了分子生物学基础,同时为保障牦牛肉食品安全提供理论依据。

关键词: 单增李斯特菌; 牦牛; 分离鉴定; 生物特性

Abstract: 【Objective】 The purpose of this experiment was to explore the risk of contamination of yak meat caused by Listeria monocytogenes in yak slaughterhouses.【Method】 Listeria monocytogenes was isolated and identified in 50 sewage samples of yak slaughterhouse by means of isolation culture,morphological observation,biochemical test and molecular method.The serotype and virulence genes of the isolate were identified and detected by PCR,and D_{600 nm}value of the isolate was determined under different growth conditions.The virulence of the isolate was determined by cell adhesion and invasion tests and mouse median lethal dose (LD₅₀).【Result】 One Gram-positive short-rod-shaped bacterium was isolated from 50 sewage samples.The isolates showed bluish-green colonies on Listeria chromogenic medium,and were Gram-positive short rod-shaped bacteria with blunt round ends,which was suspected to be Listeria monocytogenes.The results of biochemical tests showed that the isolates could hydrolyze aestin and ferment glucose,maltose and rhamnose.MR,VP,kinetic and catalase tests were positive,while mannitol and xylose fermentation tests were negative.Phylogenetic tree showed that the isolates had a high homology with Listeria monocytogenes EDG-e,the sequence similarity was 98%,and the serotype was identified as 1/2a.Virulence gene test results showed that the isolates carried inlB,inlC,inlJ,actA,plcA,prfA,mpl,hly,inlA and SigmaB virulence genes.The stress resistance test showed that the growth rate of the isolated strain was extremely significantly higher than that of the standard strain ATCC 19111 in extreme environment such as low temperature,strong acid,strong alkali,high salt,ethanol stress and oxidation (P＜0.01).The cell test showed that the adhesion rate and invasion rate of RAW246.7 cells were 8.3% and 2.5%,and the adhesion and invasion ability of RAW246.7 cells were extremely significantly higher than that of standard strain ATCC 19111 (P＜0.01).The adhesion rate and invasion rate of Caco-2 cells were 6.2% and 1.5%,respectively.The adhesion and invasion ability of Caco-2 cells were extremely significantly higher than those of standard strains ATCC 19111 (P＜0.01).Animal tests showed that the LD₅₀ of the isolated strain in mice was 10^4.7 CFU.【Conclusion】 The serotype of Listeria monocytogenes isolated in this study was 1/2a,which was the main serotype causing human listeriosis,it had strong anti-stress ability and belonged to strong strain.This study provided molecular biological basis for monitoring Listeria monocytogenes in pastoral areas and theoretical basis for ensuring food safety of yak meat.

Key words: Listeria monocytogenes; yak; isolation and identification; biological characteristics

中图分类号:

S852.61

田常青, 张坤中, 齐玉梅, 史文静, 董志杰, 张浩浩, 何曾文, 芝吉, 赵学慧, 崇倩, 薛慧文, 苟惠天. 1株环境源单增李斯特菌的分离鉴定及生物特性分析[J]. 中国畜牧兽医, 2023, 50(4): 1543-1555.

TIAN Changqing, ZHANG Kunzhong, QI Yumei, SHI Wenjing, DONG Zhijie, ZHANG Haohao, HE Zengwen, ZHI Ji, ZHAO Xuehui, CHONG Qian, XUE Huiwen, GOU Huitian. Isolation,Identification and Biological Characteristics Analysis of a Strain of Listeria monocytogenes from Environment[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1543-1555.

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