牛奶源金黄色葡萄球菌血清型、毒力基因及PFGE分型
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摘要
目的:研究生鲜牛奶中金黄色葡萄球菌分离、荚膜多糖血清型分布、毒力基因携带及脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分型情况。方法:从河南省4个地区奶牛养殖场采集生鲜牛奶样品按照国标法进行分离,扩增耐热核酸酶基因nuc鉴定金黄色葡萄球菌,利用聚合酶链式反应方法测定荚膜多糖血清型和毒力基因携带情况,采用PFGE分析菌株间的相关性和遗传关系。结果:从350份生鲜牛奶样品中分离鉴定到80株金黄色葡萄球菌,分离率为22.86%。荚膜多糖血清型测定发现,cap5(60%)是流行血清型。从这些阳性菌株中,发现有62株(77.5%)携带有毒力基因,毒力基因set、hlb、hld、lukED、ebp、clfA和clfB,检出率分别为40.00%、51.25%、57.50%、60.00%、58.75%、57.50%和58.75%。此外,47株(58.75%)菌携带不少于6个毒力基因,流行的毒力基因谱型为set-hla-hlb-hld-lukED-cna-ebp-clfA-clfB。PFGE结果显示,获得72株菌的PFGE图谱,按90%的相似性可分为12个簇和46种PFGE型。D簇(3种PFGE型)、G簇(3种PFGE型)和J簇(5种PFGE型)菌株中均检出一定基因类型的毒力基因,表明河南地区生鲜牛奶中金黄色葡萄球菌毒力基因广泛存在于多种PFGE型别中。结论:生鲜牛奶均有一定程度的金黄色葡萄球菌污染,多数菌株携带毒力基因,且毒力基因的类型较为复杂,这对消费这些牛奶的人群构成潜在的健康威胁。PFGE分型菌株主要以克隆形式进行传播,且克隆型具有多样性和差异性,故临床应加强生奶及乳品血清型、毒力基因检测及分子分型研究。
Objective: To investigate the serotyping, virulence genes and pulsed field gel electrophoresis (PFGE) typing of Staphylococcus aureus in fresh milk for a better understanding of the epidemiological characteristics of S. aureus. Methods:S. aureus strains were isolated from fresh milk samples collected from dairy farms in four regions of Henan province and they were identified by PCR amplification of the thermostable nuclease gene (nuc). The capsular polysaccharide (Cap) serotypes and virulence genes were determined by PCR. The correlation and genetic relatedness among these S. aureu isolates were analyzed by PFGE. Results: A total of 80 S. aureus strains were isolated from 350 fresh milk samples, with a separation rate of 22.86%. Cap5 (60%) was found to be a popular serotype. Of the positive isolates, 62 (77.5%) carried virulence genes, and the prevalence of the virulence genes set, hlb, hld, lukED, ebp, clfA and clfB were 40.00%, 51.25%, 57.50%, 60.00%, 58.75%, 57.50% and 58.75%, respectively. In addition, 47 (58.75%) strains carried at least 6 virulence genes, set-hla-hlb-hld-lukED-cna-ebp-clfA-clfB were found to be the prevalent virulence genotypes. PFGE profiles of 72 strains were obtained. Based on a 90% similarity, they were divided into 12 clusters and 46 PFGE types. The strains in clusters D, G and J were detected to carry some virulence genotypes, suggesting that virulence genes widely existed in a variety of PFGE types of S. aureus isolated from fresh milk in Henan. Conclusions: Fresh milk samples were contaminated by S. aureu to a certain degree. Most of the isolates carried virulence genes, and the virulence genotypes were complicated, posing a potential threat to the health of consumers. The PFGE subtypes were mainly transmitted in the form of clones, and the clone types were diversified and differentiated. Therefore, further clinical studies are needed for serotyping, virulence gene detection and molecular typing of S. aureus in raw milk and dairy products.
Objective: To investigate the serotyping, virulence genes and pulsed field gel electrophoresis (PFGE) typing of Staphylococcus aureus in fresh milk for a better understanding of the epidemiological characteristics of S. aureus. Methods:S. aureus strains were isolated from fresh milk samples collected from dairy farms in four regions of Henan province and they were identified by PCR amplification of the thermostable nuclease gene (nuc). The capsular polysaccharide (Cap) serotypes and virulence genes were determined by PCR. The correlation and genetic relatedness among these S. aureu isolates were analyzed by PFGE. Results: A total of 80 S. aureus strains were isolated from 350 fresh milk samples, with a separation rate of 22.86%. Cap5 (60%) was found to be a popular serotype. Of the positive isolates, 62 (77.5%) carried virulence genes, and the prevalence of the virulence genes set, hlb, hld, lukED, ebp, clfA and clfB were 40.00%, 51.25%, 57.50%, 60.00%, 58.75%, 57.50% and 58.75%, respectively. In addition, 47 (58.75%) strains carried at least 6 virulence genes, set-hla-hlb-hld-lukED-cna-ebp-clfA-clfB were found to be the prevalent virulence genotypes. PFGE profiles of 72 strains were obtained. Based on a 90% similarity, they were divided into 12 clusters and 46 PFGE types. The strains in clusters D, G and J were detected to carry some virulence genotypes, suggesting that virulence genes widely existed in a variety of PFGE types of S. aureus isolated from fresh milk in Henan. Conclusions: Fresh milk samples were contaminated by S. aureu to a certain degree. Most of the isolates carried virulence genes, and the virulence genotypes were complicated, posing a potential threat to the health of consumers. The PFGE subtypes were mainly transmitted in the form of clones, and the clone types were diversified and differentiated. Therefore, further clinical studies are needed for serotyping, virulence gene detection and molecular typing of S. aureus in raw milk and dairy products.
引文
[1]WANG X,MENG J H,ZHOU T,et al.Antimicrobial susceptibility testing and genotypic characterization of Staphylococcus aureus from food and food animals[J].Foodborne Pathogens and Disease,2012,9(2):95-101.DOI:10.1089/fpd.2011.0987.
[2]王琼,唐俊妮.金黄色葡萄球菌肠毒素及移动基因元件研究进展[J].食品科学,2016,37(3):241-246.DOI:10.7506/spkx1002-6630-201603042.
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[6]XING X N,ZHANG Y,WU Q,et al.Prevalence and characterization of Staphylococcus aureus isolated from goat milk powder processing plants[J].Food Control,2016,59:644-650.DOI:10.1016/j.foodcont.2015.06.042.
[7]SUDAGIDAN M,AYDIN A.Virulence properties of methicillinsusceptible Staphylococcus aureus food isolates encoding Panton-Valentine Leukocidin gene[J].International Journal of Food Microbiology,2010,138(3):287-291.DOI:10.1016/j.ijfoodmicro.2010.01.023.
[8]BASANISI M G,BELLA G L,NOBILI G,et al.Genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and dairy products in South Italy[J].Food Microbiology,2017,62:141-146.DOI:10.1016/j.fm.2016.10.020.
[9]MENDES R E,DESHPANDE L M,COSTELLO A J,et al.Genotypic characterization of methicillin-resistant Staphylococcus aureus recovered at baseline from phase 3 Pneumonia clinical trials for ceftobiprole[J].Microbial Drug Resistance,2016,22(1):53-58.DOI:10.1089/mdr.2014.0307.
[10]ALIBAYOV B,BABA-MOUSSA L,SINA H,et al.Staphylococcus aureus mobile genetic elements[J].Molecular Biology Reports,2014,41(8):5005-5018.DOI:10.1007/s11033-014-3367-3.
[11]SCHMIDT T,KOCK M M,EHLERS M M.Molecular characterization of Staphylococcus aureus isolated from bovine mastitis and close human contacts in South African dairy herds:genetic diversity and inter-species host transmission[J].Frontiers in Microbiology,2017,8:1-15.DOI:10.3389/fmicb.2017.00511.
[12]SONG Z,GU F F,GUO X K,et al.Antimicrobial resistance and molecular characterization of Staphylococcus aureus causing childhood pneumonia in Shanghai[J].Frontiers in Microbiology,2017,8:1-7.DOI:10.3389/fmicb.2017.00455.
[13]王娟,黄秀梅,曲志娜,等.生鲜牛奶中金黄色葡萄球菌的分离及耐药性分析[J].中国人兽共患病学报,2014,30(12):1214-1217.
[14]赵俊利,李培锋,李志芳,等.内蒙古地区临床型奶牛子宫内膜炎葡萄球菌毒力基因检测及耐药性研究[J].畜牧兽医学报,2014,45(2):295-304.
[15]凡琴,刘书亮,吴聪明,等.牛乳源金黄色葡萄球菌耐药性变迁及β-内酰胺类药物耐药基因分析[J].食品科学,2015,36(3):147-151.DOI:10.7506/spkx1002-6630-201503028.
[16]DE B E,ZWARTKRUISNAHUIS J T M,WIT B,et al.Prevalence of methicillin-resistant Staphylococcus aureus in meat[J].International Journal of Food Microbiology,2009,134(1):52-56.DOI:10.1016/j.ijfoodmicro.2008.12.007.
[17]OTHMAN H E,MERZA N S,JUBRAEL J M S.Nucleotide sequence analysis of methicillin resistance Staphylococcus aureus in Kurdistan region-Iraq[C]//The International Scientific Conference.Zakho,Journal of University of Zakho,2014:65-73.
[18]GOERKE C,ESSER S,KUMMEL M,et al.Staphylococcus aureus strain designation by agr and cap polymorphism typing and delineation of agr,diversification by sequence analysis[J].International Journal of Medical Microbiology,2005,295(2):67-75.DOI:10.1016/j.ijmm.2005.01.004.
[19]ARGUDIN M A,MENDOZA M C,GONZALEZHVIA M A,et al.Genotypes,exotoxin gene content,and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers[J].Applied and Environmental Microbiology,2012,78(78):2930-2935.DOI:10.1128/AEM.07487-11.
[20]JARRAUD S,MOUGEL C,THIOULOUSE J,et al.Relationships between Staphylococcus aureus genetic background,virulence factors,agr groups (alleles),and human disease[J].Infection and Immunity,2002,70(2):631-641.DOI:10.1128/IAI.70.2.631-641.2002.
[21]BABA-MOUSS A,SINA H,SCHEFTEL J M,et al.Staphylococcal Panton-Valentine leucocidin as a major virulence factor associated to furuncles[J].PLoS ONE,2011,6(10):e25716.DOI:10.1371/journal.pone.0025716.
[22]LINA G,PIEMONT Y,GODAILGAMOT F,et al.Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[J].Clinical Infectious Diseases,1999,29(5):1128-1132.DOI:10.1086/313461.
[23]OTE I,TAMINIAU B,DUPREZ J N,et al.Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis[J].Veterinary Microbiology,2011,153(3/4):285-292.DOI:10.1016/j.vetmic.2011.05.042.
[24]MCDOUGAL L K,STEWARD C D,KILLGORE G E,et al.Pulsedfield gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States:establishing a national database[J].Journal of Clinical Microbiology,2003,41(11):5113-5120.DOI:10.1128/JCM.41.4.1574-1585.2003.
[25]刘冬香,刘书亮,王印,等.四川省动物性食品中金黄色葡萄球菌的分离鉴定及污染分析[J].食品科学,2009,30(8):251-254.DOI:10.3321/j.issn:1002-6630.2009.08.057.
[26]张倩,屈勇刚,于会举,等.石河子地区奶牛乳房炎源金黄色葡萄球菌的分离鉴定及耐药性分析[J].黑龙江畜牧兽医,2017(2):102-105.
[27]刘秀丽,郝永清,郭宇.奶牛乳房炎金黄色葡萄球菌荚膜多糖研究概况[J].动物医学进展,2011,32(3):117-120.
[28]张静,于三科,王新,等.原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析[J].中国兽医学报,2012,32(5):759-764.
[29]章乐怡,李毅,马雪莲.食物中毒及食品中金黄色葡萄球菌的肠毒素及基因的研究[J].中国卫生检验杂志,2009,19(12):2851-2853.
[30]谢秀兰,刘溪源,王建东,等.奶牛乳房炎金黄色葡萄球菌8种毒力基因的PCR检测[J].中国畜牧兽医,2014,41(10):47-50.
[31]杨峰,王旭荣,李新圃,等.牛源金黄色葡萄球菌耐药性与相关耐药基因和菌株毒力基因的相关性研究[J].中国兽医科学,2016,46(2):247-252.DOI:10.16656/j.issn.1673-4696.2016.02.020.
[32]王桂琴,余婷,孙栎,等.宁夏地区奶牛子宫内膜炎金黄色葡萄球菌耐药性分析及脉冲场凝胶电泳分型研究[J].畜牧与兽医,2013,45(5):21-25.
[33]VANDERHAEGHEN W,CERPENTIER T,ADRIAENSEN C,et al.Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows[J].Veterinary Microbiology,2010,144(1):166-171.DOI:10.1016/j.vetmic.2009.12.044.
[2]王琼,唐俊妮.金黄色葡萄球菌肠毒素及移动基因元件研究进展[J].食品科学,2016,37(3):241-246.DOI:10.7506/spkx1002-6630-201603042.
[3]LIM S K,NAM H M,JANG G C,et al.Transmission and persistence of methicillin-resistant Staphylococcus aureus in milk,environment,and workers in dairy cattle farms[J].Foodborne Pathogens Disease,2013,10(8):731-736.DOI:10.1089/fpd.2012.1436.
[4]CAN H Y,CELIK T H.Detection of enterotoxigenic and antimicrobial resistant S.aureus in Turkish cheeses[J].Food Control,2012,24(1/2):100-103.DOI:10.1016/j.foodcont.2011.09.009.
[5]KUMMEL J,STESSL B,GONANO M,et al.Staphylococcus aureus entrance into the dairy chain:tracking S.aureus from dairy cow to cheese[J].Frontiers in Microbiology,2016,7:1603.DOI:10.3389/fmicb.2016.01603.
[6]XING X N,ZHANG Y,WU Q,et al.Prevalence and characterization of Staphylococcus aureus isolated from goat milk powder processing plants[J].Food Control,2016,59:644-650.DOI:10.1016/j.foodcont.2015.06.042.
[7]SUDAGIDAN M,AYDIN A.Virulence properties of methicillinsusceptible Staphylococcus aureus food isolates encoding Panton-Valentine Leukocidin gene[J].International Journal of Food Microbiology,2010,138(3):287-291.DOI:10.1016/j.ijfoodmicro.2010.01.023.
[8]BASANISI M G,BELLA G L,NOBILI G,et al.Genotyping of methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and dairy products in South Italy[J].Food Microbiology,2017,62:141-146.DOI:10.1016/j.fm.2016.10.020.
[9]MENDES R E,DESHPANDE L M,COSTELLO A J,et al.Genotypic characterization of methicillin-resistant Staphylococcus aureus recovered at baseline from phase 3 Pneumonia clinical trials for ceftobiprole[J].Microbial Drug Resistance,2016,22(1):53-58.DOI:10.1089/mdr.2014.0307.
[10]ALIBAYOV B,BABA-MOUSSA L,SINA H,et al.Staphylococcus aureus mobile genetic elements[J].Molecular Biology Reports,2014,41(8):5005-5018.DOI:10.1007/s11033-014-3367-3.
[11]SCHMIDT T,KOCK M M,EHLERS M M.Molecular characterization of Staphylococcus aureus isolated from bovine mastitis and close human contacts in South African dairy herds:genetic diversity and inter-species host transmission[J].Frontiers in Microbiology,2017,8:1-15.DOI:10.3389/fmicb.2017.00511.
[12]SONG Z,GU F F,GUO X K,et al.Antimicrobial resistance and molecular characterization of Staphylococcus aureus causing childhood pneumonia in Shanghai[J].Frontiers in Microbiology,2017,8:1-7.DOI:10.3389/fmicb.2017.00455.
[13]王娟,黄秀梅,曲志娜,等.生鲜牛奶中金黄色葡萄球菌的分离及耐药性分析[J].中国人兽共患病学报,2014,30(12):1214-1217.
[14]赵俊利,李培锋,李志芳,等.内蒙古地区临床型奶牛子宫内膜炎葡萄球菌毒力基因检测及耐药性研究[J].畜牧兽医学报,2014,45(2):295-304.
[15]凡琴,刘书亮,吴聪明,等.牛乳源金黄色葡萄球菌耐药性变迁及β-内酰胺类药物耐药基因分析[J].食品科学,2015,36(3):147-151.DOI:10.7506/spkx1002-6630-201503028.
[16]DE B E,ZWARTKRUISNAHUIS J T M,WIT B,et al.Prevalence of methicillin-resistant Staphylococcus aureus in meat[J].International Journal of Food Microbiology,2009,134(1):52-56.DOI:10.1016/j.ijfoodmicro.2008.12.007.
[17]OTHMAN H E,MERZA N S,JUBRAEL J M S.Nucleotide sequence analysis of methicillin resistance Staphylococcus aureus in Kurdistan region-Iraq[C]//The International Scientific Conference.Zakho,Journal of University of Zakho,2014:65-73.
[18]GOERKE C,ESSER S,KUMMEL M,et al.Staphylococcus aureus strain designation by agr and cap polymorphism typing and delineation of agr,diversification by sequence analysis[J].International Journal of Medical Microbiology,2005,295(2):67-75.DOI:10.1016/j.ijmm.2005.01.004.
[19]ARGUDIN M A,MENDOZA M C,GONZALEZHVIA M A,et al.Genotypes,exotoxin gene content,and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers[J].Applied and Environmental Microbiology,2012,78(78):2930-2935.DOI:10.1128/AEM.07487-11.
[20]JARRAUD S,MOUGEL C,THIOULOUSE J,et al.Relationships between Staphylococcus aureus genetic background,virulence factors,agr groups (alleles),and human disease[J].Infection and Immunity,2002,70(2):631-641.DOI:10.1128/IAI.70.2.631-641.2002.
[21]BABA-MOUSS A,SINA H,SCHEFTEL J M,et al.Staphylococcal Panton-Valentine leucocidin as a major virulence factor associated to furuncles[J].PLoS ONE,2011,6(10):e25716.DOI:10.1371/journal.pone.0025716.
[22]LINA G,PIEMONT Y,GODAILGAMOT F,et al.Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia[J].Clinical Infectious Diseases,1999,29(5):1128-1132.DOI:10.1086/313461.
[23]OTE I,TAMINIAU B,DUPREZ J N,et al.Genotypic characterization by polymerase chain reaction of Staphylococcus aureus isolates associated with bovine mastitis[J].Veterinary Microbiology,2011,153(3/4):285-292.DOI:10.1016/j.vetmic.2011.05.042.
[24]MCDOUGAL L K,STEWARD C D,KILLGORE G E,et al.Pulsedfield gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States:establishing a national database[J].Journal of Clinical Microbiology,2003,41(11):5113-5120.DOI:10.1128/JCM.41.4.1574-1585.2003.
[25]刘冬香,刘书亮,王印,等.四川省动物性食品中金黄色葡萄球菌的分离鉴定及污染分析[J].食品科学,2009,30(8):251-254.DOI:10.3321/j.issn:1002-6630.2009.08.057.
[26]张倩,屈勇刚,于会举,等.石河子地区奶牛乳房炎源金黄色葡萄球菌的分离鉴定及耐药性分析[J].黑龙江畜牧兽医,2017(2):102-105.
[27]刘秀丽,郝永清,郭宇.奶牛乳房炎金黄色葡萄球菌荚膜多糖研究概况[J].动物医学进展,2011,32(3):117-120.
[28]张静,于三科,王新,等.原料乳和临床乳房炎金黄色葡萄球菌毒力基因检测及药敏分析[J].中国兽医学报,2012,32(5):759-764.
[29]章乐怡,李毅,马雪莲.食物中毒及食品中金黄色葡萄球菌的肠毒素及基因的研究[J].中国卫生检验杂志,2009,19(12):2851-2853.
[30]谢秀兰,刘溪源,王建东,等.奶牛乳房炎金黄色葡萄球菌8种毒力基因的PCR检测[J].中国畜牧兽医,2014,41(10):47-50.
[31]杨峰,王旭荣,李新圃,等.牛源金黄色葡萄球菌耐药性与相关耐药基因和菌株毒力基因的相关性研究[J].中国兽医科学,2016,46(2):247-252.DOI:10.16656/j.issn.1673-4696.2016.02.020.
[32]王桂琴,余婷,孙栎,等.宁夏地区奶牛子宫内膜炎金黄色葡萄球菌耐药性分析及脉冲场凝胶电泳分型研究[J].畜牧与兽医,2013,45(5):21-25.
[33]VANDERHAEGHEN W,CERPENTIER T,ADRIAENSEN C,et al.Methicillin-resistant Staphylococcus aureus (MRSA) ST398 associated with clinical and subclinical mastitis in Belgian cows[J].Veterinary Microbiology,2010,144(1):166-171.DOI:10.1016/j.vetmic.2009.12.044.
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