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

Abstract

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

CLC Number:

S852.61

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.

References

[1] ROBERTS B N,CHAKRAVARTY D,GARDNER J C,et al.Listeria monocytogenes response to anaerobic environments[J].Pathogens,2020,9(3):210.
[2] LECUIT M.Listeria monocytogenes,a model in infection biology[J].Cellular Microbiology,2020,22(4),e13186.
[3] 汪国俊,黄金梅,季强,等.单增李斯特菌组氨酸激酶AgrC对毒力的影响[J].中国兽医科学,2021,51(10):1270-1278. WANG G J,HUANG J M,JI Q,et al.Role of histidine kinase AgrC in virulence of Listeria monocytogenes[J].Chinese Veterinary Science,2021,51(10):1270-1278.(in Chinese)
[4] MUCHAAMBA F,STEPHAN R,TASARA T. Listeria monocytogenes cold shock proteins:Small proteins with a huge impact[J].Microorganisms,2021,9(5):1061.
[5] 杭奕.单增李斯特菌谷氧还蛋白介导的抗氧化应激及感染机制研究[D].杭州:浙江农林大学,2020. HANG Y.Mechanism of Listeria monocytogenes glutaredoxin-mediated bacterial oxidative tolerance and infection[D].Hangzhou:Zhejiang A&F University,2020.(in Chinese)
[6] VAZQUEZ-BOLAND J A,KUHN M,BERCHE P,et al.Listeria pathogenesis and molecular virulence determinants[J].Clinical Microbiology Reviews,2001,14(3):584-640.
[7] LI W,BAI L,FU P,et al.The epidemiology of Listeria monocytogenes in China[J].Foodborne Pathogens and Disease,2018,15(8):459-466.
[8] 张敬蕊,刘力,王怡倩,等.1例产后哺乳期腹泻患者分离单增李斯特菌的生物学特征分析[J].中国人兽共患病学报,2020,36(2):169-172. ZHANG J R,LIU L,WANG Y Q,et al.Biological characteristics of Listeria monocytogenes ST145 strain isolated from of a lactation diarrhea case[J].Chinese Journal of Zoonoses,2020,36(2):169-172.(in Chinese)
[9] LUTH S,HALBEDEL S,ROSNER B,et al.Backtracking and forward checking of human listeriosis clusters identified a multiclonal outbreak linked to Listeria monocytogenes in meat products of a single producer[J].Emerging Microbes & Infections,2020,9(1):1600-1608.
[10] 中华人民共和国国家卫生和计划生育委员会国家食品药品监督管理总局.GB 4789.30-2016.食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验[S].2016. NATIONAL HEALTH AND FAMILY PLANNING COMMISSION OF THE PEOPLE'S REPUBLIC OF CHINA AND STATE FOOD AND DRUG ADMINISTRATION.GB 4789.30-2016,National standards for food safety,microbiology detection,Listeria monocytogenes test[S].2016.(in Chinese)
[11] DOUMITH M,BUCHRIESER C,GLASER P,et al.Differentiation of the major Listeria monocytogenes serovars by multiplex PCR[J].Journal of Clinical Microbiology,2004,42(8):3819-3822.
[12] 闫军.北京市售食品中单增李斯特菌致病力及比较基因组学研究[D].大庆:黑龙江八一农垦大学,2018. YAN J.Research of virulence and comparative genomics of Listeria monocytogene from market-sold food in Beijing city[D].Daqing:Heilongjiang Bayi Agricultural University,2018.(in Chinese)
[13] 凌志婷.单核细胞增生李斯特菌组氨酸激酶基因yycH响应胆盐胁迫的分子机制[D].扬州:扬州大学,2022. LING Z T.Molecular mechanism of Listeria monocytogenes histidine kinase gene yycH in response to bile salt stress[D].Yangzhou:Yangzhou University,2022.(in Chinese)
[14] BÉCAVIN C,BOUCHIER C,LECHAT P,et al.Comparison of widely used Listeria monocytogenes strains EGD,10403S,and EGD-e highlights genomic variations underlying differences in pathogenicity[J].mBio,2014,5(2):e00969-14.
[15] 辛永萍,单颖,夏叶,等.一株单核细胞增生李斯特菌分离株的分子分型鉴定及其生物学特性[J].畜牧兽医学报,2020,51(5):1101-1109. XIN Y P,SHAN Y,XIA Y,et al.Genotyping analysis and biological characteristics of a clinical Listeria monocytogenes isolate[J].Chinese Journal of Animal and Veterinary Sciences,2020,51(5):1101-1109.(in Chinese)
[16] ROUSSEL S,FELIX B,GRANT K,et al.Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping[J].BMC Microbiology,2013,13:14.
[17] WERBROUCK H,BOTTELDOORN N,CEELEN L,et al.Characterization of virulence properties of Listeria monocytogenes serotype 4b strains of different origins[J].Zoonoses Public Health,2008,55(5):242-248.
[18] WIKTORCZYK-KAPISCHKE N,SKOWRON K,GRUDLEWSKA-BUDA K,et al.Adaptive response of Listeria monocytogenes to the stress factors in the food processing environment[J].Frontiers in Microbiology,2021,12:710085.
[19] GLASER P,FRANGEUL L,BUCHRIESER C.Comparative genomics of Listeria species[J].Science.2001,294(5543):849-852.
[20] VILCHIS-RANGEEL R E,ESPIONOZA-MELLADO M D R,SALINAS-JARAMILLO I J,et al.Association of Listeria monocytogenes LIPI-1 and LIPI-3 marker llsX with invasiveness[J].Current Mcrobiology,2019,76(5):637-643.
[21] CHEN G Y,PENSINGER D A,SAUER J D.Listeria monocytogenes cytosolic metabolism promotes replication,survival,and evasion of innate immunity[J].Cellular Mcrobiology, 2017,19(10):e12762.
[22] FERREIRA A,O'BYRNE C P,BOOR K J,et al.Role of sigma(B) in heat,ethanol,acid,and oxidative stress resistance and during carbon starvation in Listeria monocytogenes[J].Applied and Environmental Microbiology,2001,67(10):4454-4457.
[23] OLLINGER J,BOWEN B,WIEDMANN M,et al.Listeria monocytogenes sigmaB modulates PrfA-mediated virulence factor expression[J].Infection and Immunity,2009,77(5):2113-2124.
[24] 李秀娟,赵冬,潘琢,等.29种毒力基因在91株食源性单核细胞增生李斯特氏菌中的分布[J].中国人兽共患病学报,2017,33(11):972-978. LI X J,ZHAO D,PAN Z,et al.Distribution characterization of 29 virulence genes in 91 foodborne Listeria monocytogenes strains[J].Chinese Journal of Zoonoses,2017,33(11):972-978.(in Chinese)
[25] 郝民,阮明捷,王恒伟,等.北京市朝阳区单核细胞增生李斯特菌关键毒力基因缺失与致病性关联性研究[J].中国食品卫生杂志,2022,34(1):75-81. HAO M,RUAN M J,WANG H W,et al.Study of association between deletion of key virulence genes and pathogenicity of Listeria monocytogenes in Chaoyang district of Beijing[J].Chinese Journal of Food Hygiene,2022,34(1):75-81.(in Chinese)
[26] LEE S.Bacteriocins of Listeria monocytogenes and their potential as a virulence factor[J].Toxin,2020,12(2):103.
[27] PHELPS C C,VADIA S,ARNETT E,et al.Relative roles of listeriolysin O,InlA,and InlB in Listeria monocytogenes uptake by host cells[J].Infection and Immunity,2018;86(10):e00555-18.

Isolation,Identification and Biological Characteristics Analysis of a Strain of Listeria monocytogenes from Environment

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	LI Rui, ZHANG Yiyang, LUO Xuehui, ZHOU Manlin, WU Shaomi, YANG Kun, QIAO Zilin. Effect of Hypoxia on Proliferation and Oxidative Stress Injury of Pulmonary Artery Smooth Muscle Cells in Yaks [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2245-2254.
[2]	LU Yukui, LIU Jiaqi, ZHONG Jiacheng, CHEN Jidang, ZHU Wanjun, ZHANG Yishan, ZHANG Jipei. Isolation,Identification and Pathogenicity Analysis of Avibacterium paragallinarum from Geese [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2562-2572.
[3]	WU Zhijuan, CHAI Zhixin, WANG Jikun, WANG Jiabo, ZHONG Jincheng, XIN Jinwei. Expression Analysis of Specific Matrix Metalloproteinases and Functionally Related Genes of Skeletal Muscles in Yak [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1774-1784.
[4]	ZENG Dongdong, LIU Caixia, KOU Lijun, QIN He, JIN Ruijie, WANG Jing, REN Jingjing, MA Xun, JIANG Jianjun. Construction and Biological Characteristics of Lm4b_02324 Gene Deletion Strains of Listeria monocytogenes [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1981-1990.
[5]	LIU Caixia, SHI Weidi, KOU Lijun, MA Xun, WANG Jing, GAO Shengjie, LYU Shuangfei, CHEN Xuke, ZENG Dongdong, KONG Cuilian, REN Huijie. Regulation of Transcription of Key Virulence Factors of Listeria monocytogenes by LIPI-4 EⅡC [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1340-1351.
[6]	LI Zhijie, ZHAO Ying, MA Xin, WANG Meng, YU Sijiu, WANG Libin, PAN Yangyang. Research Progress on Isolation Methods and Main Reproductive Functions of Exosomes from Animal Follicular Fluid [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1480-1488.
[7]	WANG Leping, PAN Yan, TAN Yizhou, LI Lei, HAN Kaiou, CAO Yajie, LI Jun, ZHOU Yuqing, LIAO Yuying, MA Dongxin, GONG Yu, PENG Hao, WANG Xiaoye. Isolation,Identification and Genome Analysis of Escherichia coli Phage vB_EcoP_GN07 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1522-1531.
[8]	CAO Yingying, LIANG Liang, ZENG Yirong, ZHONG Qinghua, YUAN Xiaofang, TANG Haibo, LENG Jing. Isolation,Identification and Biological Characteristics of Salmonella from Tupaia belangeri chinensis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1718-1728.
[9]	WANG Yunlu, MENG Zhaoyi, YAO Yilong, GUO Min, NIU Jiaqaing, SUOLANG Sizhu, XU Yefen. Screening and Targeting Verification of lncRNA of "Sponge Adsorption" bta-miR-146a in Yak [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 859-869.
[10]	CHENG Longfei, LIAO Weilian, CHEN Hongmei, JIANG Nansong, FU Qiuling, WAN Chunhe, LIU Rongchang, SHI Shaohua, HUANG Yu, FU Guanghua. Screening and Biological Characteristics of Broad-spectrum Riemerella anatipestifer Phage [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1169-1176.
[11]	GAN Lu, ZHENG Huizhen, NUO Mingdalai, LIU Yan, JIN Min, HE Wenwen, WEN Licui, LI Yongchang, GAILIKE·Bayinchahan. Propagation of Trypanosoma evansi Yili Strain and Cloning, Expression and Bioinformatics Analysis of Its PFR Gene [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 469-478.
[12]	LIU Bin, LI Meng, XI Qianyun, SUN Jiajie, LUO Junyi, ZHANG Yongliang, CHEN Ting. Research Progress on Biological Characteristics of Hermetia illucens L. and Its Application in Poultry Production [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 586-597.
[13]	YIN Yangyan, PAN Yan, LI Xiaoning, LI Jun, LIAO Yuying, LI Changting, WEI Tianchao, PENG Hao. Study on Biological Characteristics and Antibacterial Activity in vitro of a Lactobacillus plantarum Strain [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 666-673.
[14]	WANG Bingyi, MA Hongcai, ZOU Minghao, FAN Shijie, YUAN Zhenjie, Labaciren, DONG Hailong, ZENG Jiangyong. Isolation,Identification and Drug Resistance Analysis of Streptococcus from Yak [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 745-753.
[15]	ZHONG Huachen, WANG Lifang, GUO Chenyang, LIU Jialin, SONG Jie. Isolation,Identification and Drug Resistance Analysis of Pathogenic Bacteria from Milk Samples and Environment of Mastitis in Inner Mongolia [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 817-826.