56株食源性单增李斯特菌耐药性测定及耐药基因的检测
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摘要
为调查新疆部分食源性单增李斯特菌的药物敏感性和相关耐药基因的携带现况,采用微量肉汤稀释法(MIC)和K-B琼脂扩散法对56株单增李斯特菌进行18种抗菌药物敏感性试验。通过PCR方法进行12种相关耐药基因检测,并对耐药基因进行测序分析。结果表明,28.6%(16/56)的菌株微量肉汤稀释法检测耐药,而K-B琼脂扩散法检测19.6%(11/56)菌株耐药。56株单增李斯特菌均对青霉素、氨苄西林、亚胺培能、庆大霉素、万古霉素、左氧氟沙星、利福平敏感,对四环素、复方新诺明、链霉素、强力霉素、卡那霉素耐药。微量肉汤稀释法检测发现,菌株对四环素、复方新诺明、链霉素耐药率较高,分别为12.5%(7/56)、10.7%(6/56)、10.7%(6/56),多重耐药菌株占7.1%(4/56)。在18株表型耐药菌株中,61.1%(11/18)的菌株携带相应的耐药基因,四环素类耐药基因以tetM为主,红霉素耐药基因为ermB,氯霉素素耐药基因为cat。而在38株非耐药表型菌株中,氨基糖苷类耐药基因aac(6')-Ib-cr和红霉素耐药基因ermB的检出率分别高达57.9%和39.5%。扩增片段核苷酸序列与参考序列同源性为95%～100%。新疆食源性单增李斯特菌出现耐药,且耐药表型与耐药基因不完全符合,应引起关注。
The aim of the study was to understand the status of antibacterial drugs resistance and related resistance genes in food-borne Listeria monocytogenes in Xinjiang.Broth microdilution method and K-B agar diffusion method were used to determine 18 antibacterial drugs sensitivity against 56 Listeria monocytogenes.The distribution of 12 related resistance genes were detected by PCR.The resistance genes were sequenced and analyzed.In result,28.6%(16/56) resistant strains were detected by the broth microdilution method,and 19.6%(11/56) resistant strains were detected by the K-B agar diffusion method.56 strains were sensitive to penicillin,ampicillin,imipenem,gentamicin,vancomycin,levofloxacin and rifampin and resistant to tetracycline,trimethoprim-sufamethoxazole,streptomycin,doxycycline and kanamycin.By the broth microdilution method,the resistance rates to tetracycline,trimethoprim-sufamethoxazole,and streptomycin were 12.5%(7/56),10.7%(6/56),10.7%(6/56),respectively.The multi-drug resistance strains were 7.1%(4/56).Among the 18 resistant phenotype strains,61.1%(11/18) of the strains carried correspond resistance genes.The tetracycline resistance gene was mainly tet M,erythromycin resistance gene was erm B,and chloramphenicol resistance gene was cat.Among the 38 non-resistant phenotype strains,the detection rates of aminoglycoside resistance gene aac(6')-Ib-cr and erythromycin resistance gene erm B were as high as 57.9% and39.5%,respectively.The nucleotide sequence of the resistance genes had 95%—100% homology with the reference sequence.In conclusion,antibacterial drugs resistance of food-borne Listeria monocytogenes in Xinjiang and inconsistent between resistant phenotype and resistant genes should be concerned.
The aim of the study was to understand the status of antibacterial drugs resistance and related resistance genes in food-borne Listeria monocytogenes in Xinjiang.Broth microdilution method and K-B agar diffusion method were used to determine 18 antibacterial drugs sensitivity against 56 Listeria monocytogenes.The distribution of 12 related resistance genes were detected by PCR.The resistance genes were sequenced and analyzed.In result,28.6%(16/56) resistant strains were detected by the broth microdilution method,and 19.6%(11/56) resistant strains were detected by the K-B agar diffusion method.56 strains were sensitive to penicillin,ampicillin,imipenem,gentamicin,vancomycin,levofloxacin and rifampin and resistant to tetracycline,trimethoprim-sufamethoxazole,streptomycin,doxycycline and kanamycin.By the broth microdilution method,the resistance rates to tetracycline,trimethoprim-sufamethoxazole,and streptomycin were 12.5%(7/56),10.7%(6/56),10.7%(6/56),respectively.The multi-drug resistance strains were 7.1%(4/56).Among the 18 resistant phenotype strains,61.1%(11/18) of the strains carried correspond resistance genes.The tetracycline resistance gene was mainly tet M,erythromycin resistance gene was erm B,and chloramphenicol resistance gene was cat.Among the 38 non-resistant phenotype strains,the detection rates of aminoglycoside resistance gene aac(6')-Ib-cr and erythromycin resistance gene erm B were as high as 57.9% and39.5%,respectively.The nucleotide sequence of the resistance genes had 95%—100% homology with the reference sequence.In conclusion,antibacterial drugs resistance of food-borne Listeria monocytogenes in Xinjiang and inconsistent between resistant phenotype and resistant genes should be concerned.
引文
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[15]严纪文,王海燕,赖蔚苳,等.广东省几种重要食源性致病菌的耐药状况及耐药谱研究[J].中国卫生检验杂志,2007,17(11):2030-2032,2077.YAN Ji-wen,WANG Hai-yan,LAI Wei-dong,et al.Research of antimicrobial susceptibility and antimicrobial resistant types of several significant pathogens in food-born diseases in Gongdong Province[J].Chinese Journal of Health Laboratory Technology,2007,17(11):2030-2032,2077.(in Chinese)
[16]DERRA F A,KARLSMOSE S,MONGA D P,et al.Occurrence of Listeria spp.in retail meat and dairy products in the area of Addis Ababa,Ethiopia[J].Foodborne Pathogens and Disease,2013,10(6):577-579.
[17]张亚兰,冉陆,李迎惠,等.2003-2004年中国食品中单核细胞增生李斯特菌耐药监测[J].中国食品卫生杂志,2006,18(5):398-400.ZHANG Ya-lan,RAN Lu,LI Ying-hui,et al.Surveillance of drug resistance of Listeria monocytogenes from foods in China,2003-2004[J].Chinese Journal of Food Hygiene,2006,18(5):398-400.(in Chinese)
[18]杨洋,付萍,郭云昌,等.2005年中国食源性单核细胞增生李斯特菌耐药性趋势分析[J].卫生研究,2008,37(2):183-186.YANG Yang,FU Ping,GUO Yun-chang,et al.Analysis of antibiotic susceptibility of foodborne Listeria monocytogenes in China[J].Journal of Hygiene Research,2008,37(2):183-186.(in Chinese)
[19]何建华.一株多重耐药单核细胞增生李斯特菌的耐药基因分析[D].广州:华南理工大学,2013.HE Jian-hua.Study on the resistance genes of a multidrug-resistant Listeria monocytogenes[D].Guangzhou:South China University of Technology,2013.(in Chinese)
[20]闫鹤,陈妙瑞,石磊.食源性单核细胞增生李斯特菌四环素、红霉素耐药基因研究[J].现代食品科技,2010,26(8):772-775.YAN He,CHEN Miao-rui,SHI Lei.Tetracycline and erythromycin resistant genes in foodborne Listeria monocytogeies[J].Modern Food Science and Technology,2010,26(8):772-775.(in Chinese)
[21]BISWAS T,HOUGHTON J L,GARNEAU-TSODIKOVA S,et al.The structural basis for substrate versatility of chloramphenicol acetyltransferase CATI[J].Protein Science,2012,21(4):520-530.
[22]刘艳红.沙门氏菌和大肠杆菌耐药性及耐药基因的研究[D].沈阳:沈阳农业大学,2016.LIU Yan-hong.Research of drug resistance and resistance genes of Salmonella and Escherichia coli[D].Shenyang:Shenyang Agricultural University,2016.(in Chinese)
[2]邵美丽,刘思国,尹录.单增李斯特菌四环素耐药基因tetM膜接合转移的研究[J].中国兽医科学,2010,40(6):589-592.SHAO Mei-li,LIU Si-guo,YIN Lu.Study on filtermating transfer of tetracycline resistant gene tetMin Listeria monocytogenes[J].Chinese Veterinary Science,2010,40(6):589-592.(in Chinese)
[3]YUCEL N,CITAK S,ONDER M.Prevalence and antibiotic resistance of Listeria species in meat products in Ankara,Turkey[J].Food Microbiology,2005,22(2-3):241-245.
[4]王茂起,刘秀梅,王竹天.中国食品污染监测体系的研究[J].中国食品卫生杂志,2006,18(6):491-497.WANG Mao-qi,LIU Xiu-mei,WANG Zhu-tian.Studies on national surveillance system for food contaminations and foodborne diseases in China[J].Chinese Journal of Food Hygiene,2006,18(6):491-497.(in Chinese)
[5]姚琳,江艳华,李风铃,等.鲜活贝类中单核细胞增生李斯特菌的分离鉴定及毒力基因与耐药性分析[J].中国食品卫生杂志,2017,29(1):5-8.YAO Lin,JIANG Yan-hua,LI Feng-ling,et al.Identification,virulence gene determination and antibiotic resistance of Listeria monocytogenes in fresh shellfish from shellfish beds[J].Chinese Journal of Food Hygiene,2017,29(1):5-8.(in Chinese)
[6]KINGA W,KATARZYNA D,JACEK O.Prevalence,characterization,and antimicrobial resistance of Listeria monocytogenes isolates from bovine hides and carcasses[J].Applied and Environmental Microbiology,2012,78(6):2043-2045.
[7]CHARPENTIER E,COURVALIN P.Antibiotic resistance in Listeria spp.[J].Antimicrobial Agents and Chemotherapy,1999,43(9):2103-2108.
[8]FOTHERGILL A W.Antifungal susceptibility testing:Clinical Laboratory and Standards Institute(CLSI)methods[J].Clinical Microbiology Newsletter,2015,18(21):161-167.
[9]王天姝,王艳,叶正兴,等.中国部分食品分离单增李斯特菌的抗菌药物敏感性及耐药基因检测[J].疾病监测,2013,28(3):224-229.WANG Tian-shu,WANG Yan,YE Zheng-xing,et al.Detection of drug susceptibility and resistant genes in selected food borne Listeria monocytogens in China[J].Disease Surveillance,2013,28(3):224-229.(in Chinese)
[10]赵悦,付萍,裴晓燕,等.中国食源性单核细胞增生李斯特菌耐药特征分析[J].中国食品卫生杂志,2012,24(1):5-8.ZHAO Yue,FU Ping,PEI Xiao-yan,et al.Characteristic analysis of antimicrobial resistance of Listeria monocytogenes in foods[J].Chinese Journal of Food Hygiene,2012,24(1):5-8.(in Chinese)
[11]李迎慧,冉陆.李斯特菌的耐药性及耐药基因[J].国外医学卫生学分册,2004,31(2):120-124.LI Ying-hui,RAN Lu.Resistance and resistance genes of Listeria monocytogenes[J].Foreign Medical Hygiene Volumes,2004,31(2):120-124.(in Chinese)
[12]WIEDMANN M,BRUCE J L,KEATING C,et al.Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential[J].Infection and Immunity,1997,65(7):2707-2716.
[13]AYAZ N D,EROL I.Relation between serotype distribution and antibiotic resistance profiles of Listeria monocytogenes isolated from ground Turkey[J].Journal of Food Protection,2010,73(5):967-972.
[14]SAFDAR A,ARMSTRONG D.Antimicrobial activities against 84 Listeria monocytogenes isolates from patients with systemic listeriosis at a Comprehensive Cancer Center(1955-1997)[J].Journal of Clinical Microbiology,2003,41(1):483-485.
[15]严纪文,王海燕,赖蔚苳,等.广东省几种重要食源性致病菌的耐药状况及耐药谱研究[J].中国卫生检验杂志,2007,17(11):2030-2032,2077.YAN Ji-wen,WANG Hai-yan,LAI Wei-dong,et al.Research of antimicrobial susceptibility and antimicrobial resistant types of several significant pathogens in food-born diseases in Gongdong Province[J].Chinese Journal of Health Laboratory Technology,2007,17(11):2030-2032,2077.(in Chinese)
[16]DERRA F A,KARLSMOSE S,MONGA D P,et al.Occurrence of Listeria spp.in retail meat and dairy products in the area of Addis Ababa,Ethiopia[J].Foodborne Pathogens and Disease,2013,10(6):577-579.
[17]张亚兰,冉陆,李迎惠,等.2003-2004年中国食品中单核细胞增生李斯特菌耐药监测[J].中国食品卫生杂志,2006,18(5):398-400.ZHANG Ya-lan,RAN Lu,LI Ying-hui,et al.Surveillance of drug resistance of Listeria monocytogenes from foods in China,2003-2004[J].Chinese Journal of Food Hygiene,2006,18(5):398-400.(in Chinese)
[18]杨洋,付萍,郭云昌,等.2005年中国食源性单核细胞增生李斯特菌耐药性趋势分析[J].卫生研究,2008,37(2):183-186.YANG Yang,FU Ping,GUO Yun-chang,et al.Analysis of antibiotic susceptibility of foodborne Listeria monocytogenes in China[J].Journal of Hygiene Research,2008,37(2):183-186.(in Chinese)
[19]何建华.一株多重耐药单核细胞增生李斯特菌的耐药基因分析[D].广州:华南理工大学,2013.HE Jian-hua.Study on the resistance genes of a multidrug-resistant Listeria monocytogenes[D].Guangzhou:South China University of Technology,2013.(in Chinese)
[20]闫鹤,陈妙瑞,石磊.食源性单核细胞增生李斯特菌四环素、红霉素耐药基因研究[J].现代食品科技,2010,26(8):772-775.YAN He,CHEN Miao-rui,SHI Lei.Tetracycline and erythromycin resistant genes in foodborne Listeria monocytogeies[J].Modern Food Science and Technology,2010,26(8):772-775.(in Chinese)
[21]BISWAS T,HOUGHTON J L,GARNEAU-TSODIKOVA S,et al.The structural basis for substrate versatility of chloramphenicol acetyltransferase CATI[J].Protein Science,2012,21(4):520-530.
[22]刘艳红.沙门氏菌和大肠杆菌耐药性及耐药基因的研究[D].沈阳:沈阳农业大学,2016.LIU Yan-hong.Research of drug resistance and resistance genes of Salmonella and Escherichia coli[D].Shenyang:Shenyang Agricultural University,2016.(in Chinese)