食品中克罗诺杆菌分离菌株生物被膜形成、耐药性及毒力基因检测
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摘要
研究食品中克罗诺杆菌分离菌株的生物被膜形成、耐药性以及携带毒力基因情况。在成都市周边农贸市场和路边小摊采集食品样品129份,采用DFI阪崎肠杆菌显色培养基分离克罗诺杆菌;通过16S rRNA序列比对分析鉴定分离菌株;采用试管法和微孔板法分析菌株生物被膜形成能力,同时研究温度对细菌成膜能力影响;采用纸片法检测分离菌株对18种抗生素的耐药性;采用PCR方法检测分离菌株携带cpa、hly、sip和omp X毒力基因情况。结果发现从129份食品样本中共检出克罗诺杆菌43株,检出率为33.3%。43株克罗诺杆菌食品分离菌株的成膜率为90.7%,并且温度对细菌成膜影响明显。四种毒力基因中,omp X检出率为100%; cpa检出率为13.9%; hly检出率为11.6%; sip基因未检出。耐药表型检测发现43株克罗诺杆菌食品分离菌株对青霉素、克林霉素、万古霉素、苯唑西林和杆菌肽B的耐药率为100%,对利福平的耐药率达97.7%;对红霉素的耐药率为7%;对环丙沙星、庆大霉素、四环素、氯霉素、亚胺培南、磺胺甲恶挫、呋喃妥因、头孢西丁、链霉素、阿米卡星、氧氟沙星等100%敏感。本研究表明克罗诺杆菌食品分离菌株具有较好的形成生物被膜能力,对常见的抗生素耐药率较高,并且分离菌株携带一定的毒力基因,对食品安全造成潜在威胁。
In this study,Cronobacter spp. food isolates were identified,and the biofilm formation ability,antimicrobial susceptibility and virulence genes were investigated.All 129 food samples were collected from food markets and food stands from Chengdu city in 2016.Cronobacter spp. were isolated and identified by DFI selective medium and 16 S rRNA sequencing analysis.The 96-well microplate and tube test were used to detect biofilm formation ability.The influence of temperature on the biofilm formation was also explored.The antimicrobial susceptibility of Cronobacter spp.isolates to 18 antibiotics was detected by disk diffusion method.The virulence genes( cpa,hly,sip,and omp X) were detected by PCR.The results showed 43 Cronobacter spp.strains were identified from 129 food samples.The Cronobacter spp.isolation rate was 33.3%.The biofilm formation rate was90.7% and the temperature had a significant effect on bacterial biofilm formation. The detection rate of omp X gene in 43 Cronobacter spp.strains was 100%; cpa was 13.9%,hly was 11.6%,and the sip gene was not detected in all isolates.The drug resistance rates to penicillin,clindamycin,vancomycin,oxacillin and bacitracin B for all 43 Cronobacter were 100%.The drug resistance rate to rifampicin was 97.7%. For erythromycin,the resistance rate was 7%. All the Cronobacter food isolates were sensitive to ciprofloxacin,gentamicin,tetracycline,chloramphenicol,imipenem,sulfamethoxazole,nitrofurantoin,cefoxitin,streptomycin,amikacin,and ofloxacin,completely.The study indicated Cronobacter spp.food isolates had better biofilm formation ability and had some resistance to most antibiotics. The food safety were threatened by Cronobacter spp. isoaltes with virulence genes.
In this study,Cronobacter spp. food isolates were identified,and the biofilm formation ability,antimicrobial susceptibility and virulence genes were investigated.All 129 food samples were collected from food markets and food stands from Chengdu city in 2016.Cronobacter spp. were isolated and identified by DFI selective medium and 16 S rRNA sequencing analysis.The 96-well microplate and tube test were used to detect biofilm formation ability.The influence of temperature on the biofilm formation was also explored.The antimicrobial susceptibility of Cronobacter spp.isolates to 18 antibiotics was detected by disk diffusion method.The virulence genes( cpa,hly,sip,and omp X) were detected by PCR.The results showed 43 Cronobacter spp.strains were identified from 129 food samples.The Cronobacter spp.isolation rate was 33.3%.The biofilm formation rate was90.7% and the temperature had a significant effect on bacterial biofilm formation. The detection rate of omp X gene in 43 Cronobacter spp.strains was 100%; cpa was 13.9%,hly was 11.6%,and the sip gene was not detected in all isolates.The drug resistance rates to penicillin,clindamycin,vancomycin,oxacillin and bacitracin B for all 43 Cronobacter were 100%.The drug resistance rate to rifampicin was 97.7%. For erythromycin,the resistance rate was 7%. All the Cronobacter food isolates were sensitive to ciprofloxacin,gentamicin,tetracycline,chloramphenicol,imipenem,sulfamethoxazole,nitrofurantoin,cefoxitin,streptomycin,amikacin,and ofloxacin,completely.The study indicated Cronobacter spp.food isolates had better biofilm formation ability and had some resistance to most antibiotics. The food safety were threatened by Cronobacter spp. isoaltes with virulence genes.
引文
[1]覃小玲,任国谱.乳制品中阪崎肠杆菌的研究进展[J].中国乳品工业,2011(11):45-46,49.
[2]黄启红,蔡大川,张志军,等.食品中克罗诺杆菌的分离和鉴定[J].食品研究与开发,2016,37(9):192-194.
[3]韩冉.阪崎克罗诺杆菌间毒力比较与致病因子研究[D].天津:天津科技大学,2014.
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[8]李燕杰,杜冰,董吉林,等.食品中细菌生物被膜及其形成机制的研究进展[J].现代食品科技,2009,25(4):435-438.
[9]Gupta T B,Mowat E,Brightwell G,et al.Biofilm formation and genetic characterization of New Zealand Cronobacter isolates[J].Journal of Food Safety,2017(396):e12430.
[10]郑金华,张新峰,陆娟娟,等.2011年-2014年泰安市婴幼儿食品中阪崎肠杆菌的检测及耐药性和毒力基因研究[J].中国卫生检验杂志,2016,26(5):670-672.
[11]甘辛,白莉,闫韶飞,等.2012~2014年我国婴幼儿食品中克罗诺氏菌耐药特征分析[J].食品安全质量检测学报,2015,6(9):3491-3496.
[12]周显凤,高建新,龙慧,等.婴幼儿配方奶粉中肺炎克雷伯菌与阪崎肠杆菌分离株的药敏分析[J].现代预防医学,2012,39(6):1511-1513.
[13]陈卓,任立松,马龙,等.阪崎肠杆菌新疆分离株的药敏分析[J].现代预防医学,2011,38(17):3539-3541.
[14]熊明华,朱继荣,王光利,等.一种快速经济提取革兰氏阴性和革兰氏阳性细菌基因组DNA的方法[J].基因组学与应用生物学,2016,35(2):385-390.
[15]Lehner A,Tasara T,Stephan R.16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification[J].BMCMicrobiology,2004,4(1):1-7.
[16]唐俊妮,康名松,陈焕春,等.葡萄球菌核酸酶对金黄色葡萄球菌和其他细菌生物被膜形成的抑制作用[J].中国科学:生命科学,2011,41(7):586-592.
[17]Rodrigues L B,Dos Santos L R,Tagliari V Z,et al.Quantification of biofilm production on polystyrene by Listeria,Escherichia coli and Staphylococcus aureus isolated from a poultry slaughterhouse[J].Br J Microbiol,2010,41(4):1082-1085.
[18]杜玄,贺苏皖,田万帆,等.培养条件对阪崎克罗诺杆菌食品分离菌株生物被膜形成的影响[J].食品安全质量检测学报,2018,9(4):699-705.
[19]李远宏,姜华,焦阳,等.食品香辛料和调味品中克罗诺杆菌的分离与鉴定[J].食品工业科技,2017,38(19):125-130.
[20]陈万义,任婧,吴正钧,等.生鲜蔬菜中阪崎克罗诺杆菌的分离与鉴定[J].食品科技,2014,39(1):304-308.
[21]潘琢,郭玉梅,徐保红,等.营养面条生产过程中克罗诺杆菌污染状况与溯源研究[J].中国食品卫生杂志,2018,30(1):54-58.
[22]王琼,唐俊妮,陈娟,等.金黄色葡萄球菌生物被膜检测及控制方法研究进展[J].食品工业科技,2014,35(22):371-375.
[23]王倩宁.羊奶粉生产环节克罗诺杆菌污染情况及分离菌株特性研究[D].杨陵:西北农林科技大学,2014.
[24]黄玉兰,雷高鹏,张林,等.2010-2014年及2016年四川省婴幼儿食品及临床分离克罗诺杆菌耐药分析[J].中国食品卫生杂志,2017,29(3):299-301.
[25]张翼,陈雅蘅,周帼萍,等.克罗诺杆菌的生物膜检测和药敏性分析[J].食品科学,2015,36(21):129-134.
[26]崔晶花,杨小蓉,杜小莉,等.60株克罗诺杆菌的药敏分析[J].疾病监测,2012,27(5):409-411.
[2]黄启红,蔡大川,张志军,等.食品中克罗诺杆菌的分离和鉴定[J].食品研究与开发,2016,37(9):192-194.
[3]韩冉.阪崎克罗诺杆菌间毒力比较与致病因子研究[D].天津:天津科技大学,2014.
[4]Kim K,Kim K P,Choi J,et al.Outer membrane proteins A(OmpA)and X(OmpX)are essential for basolateral invasion of Cronobacter sakazakii[J].Applied&Environmental Microbiology,2010,76(15):5188.
[5]De K G,Bolton A,Martin G,et al.Invasion of rabbit ileal tissue by Enterobacter cloacae varies with the concentration of OmpX in the outer membrane[J].Infection&Immunity,1994,62(11):4722-4726.
[6]Cruz A,Xicohtencatl-Cortes J,González-Pedrajo B,et al.Virulence traits in Cronobacter species isolated from different sources[J].Canadian Journal of Microbiology,2011,57(9):735.
[7]Franco A A,Kothary M H,Gopinath G,et al.Cpa,the outer membrane protease of Cronobacter sakazakii,activates plasminogen and mediates resistance to serum bactericidal activity[J].Infection&Immunity,2011,79(4):1578.
[8]李燕杰,杜冰,董吉林,等.食品中细菌生物被膜及其形成机制的研究进展[J].现代食品科技,2009,25(4):435-438.
[9]Gupta T B,Mowat E,Brightwell G,et al.Biofilm formation and genetic characterization of New Zealand Cronobacter isolates[J].Journal of Food Safety,2017(396):e12430.
[10]郑金华,张新峰,陆娟娟,等.2011年-2014年泰安市婴幼儿食品中阪崎肠杆菌的检测及耐药性和毒力基因研究[J].中国卫生检验杂志,2016,26(5):670-672.
[11]甘辛,白莉,闫韶飞,等.2012~2014年我国婴幼儿食品中克罗诺氏菌耐药特征分析[J].食品安全质量检测学报,2015,6(9):3491-3496.
[12]周显凤,高建新,龙慧,等.婴幼儿配方奶粉中肺炎克雷伯菌与阪崎肠杆菌分离株的药敏分析[J].现代预防医学,2012,39(6):1511-1513.
[13]陈卓,任立松,马龙,等.阪崎肠杆菌新疆分离株的药敏分析[J].现代预防医学,2011,38(17):3539-3541.
[14]熊明华,朱继荣,王光利,等.一种快速经济提取革兰氏阴性和革兰氏阳性细菌基因组DNA的方法[J].基因组学与应用生物学,2016,35(2):385-390.
[15]Lehner A,Tasara T,Stephan R.16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification[J].BMCMicrobiology,2004,4(1):1-7.
[16]唐俊妮,康名松,陈焕春,等.葡萄球菌核酸酶对金黄色葡萄球菌和其他细菌生物被膜形成的抑制作用[J].中国科学:生命科学,2011,41(7):586-592.
[17]Rodrigues L B,Dos Santos L R,Tagliari V Z,et al.Quantification of biofilm production on polystyrene by Listeria,Escherichia coli and Staphylococcus aureus isolated from a poultry slaughterhouse[J].Br J Microbiol,2010,41(4):1082-1085.
[18]杜玄,贺苏皖,田万帆,等.培养条件对阪崎克罗诺杆菌食品分离菌株生物被膜形成的影响[J].食品安全质量检测学报,2018,9(4):699-705.
[19]李远宏,姜华,焦阳,等.食品香辛料和调味品中克罗诺杆菌的分离与鉴定[J].食品工业科技,2017,38(19):125-130.
[20]陈万义,任婧,吴正钧,等.生鲜蔬菜中阪崎克罗诺杆菌的分离与鉴定[J].食品科技,2014,39(1):304-308.
[21]潘琢,郭玉梅,徐保红,等.营养面条生产过程中克罗诺杆菌污染状况与溯源研究[J].中国食品卫生杂志,2018,30(1):54-58.
[22]王琼,唐俊妮,陈娟,等.金黄色葡萄球菌生物被膜检测及控制方法研究进展[J].食品工业科技,2014,35(22):371-375.
[23]王倩宁.羊奶粉生产环节克罗诺杆菌污染情况及分离菌株特性研究[D].杨陵:西北农林科技大学,2014.
[24]黄玉兰,雷高鹏,张林,等.2010-2014年及2016年四川省婴幼儿食品及临床分离克罗诺杆菌耐药分析[J].中国食品卫生杂志,2017,29(3):299-301.
[25]张翼,陈雅蘅,周帼萍,等.克罗诺杆菌的生物膜检测和药敏性分析[J].食品科学,2015,36(21):129-134.
[26]崔晶花,杨小蓉,杜小莉,等.60株克罗诺杆菌的药敏分析[J].疾病监测,2012,27(5):409-411.