牛源金黄色葡萄球菌凝固酶分型与致病因子检测
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摘要
为了解青海和甘肃地区奶牛养殖场的金黄色葡萄球菌(S.aureus)基因型分布状况,本研究对来自该两个地区的210份奶样进行了S.aureus的分离与鉴定,利用凝固酶基因(Coa)扩增和限制性内切酶AluⅠ酶切方法对所有分离株进行了凝固酶多态性分型。并对不同凝固酶基因型的S.aureus进行了白细胞毒素F (LukF)、白细胞毒素S (LukS)、α溶血素(α-hemolysin)、β溶血素(β-hemolysin)等主要致病因子检测。结果显示,共分离鉴定出35株S.aureus (青海13株,甘肃22株),所有分离株均能够扩增出Coa基因片段,并有5种PCR分型结果(本文简称PCR1～PCR5型),其中PCR 1、4和5型只有1种亚型,而PCR 2型有3种亚型,PCR 3型有2种亚型。PCR3型是青海(6/13)和甘肃地区(8/22)主要流行的基因型。致病因子检测结果显示,所有分离株均携带致病因子LukS和α溶血素;致病因子LukF和β溶血素检出率也很高,分别是30株(85.7%)和31株(88.6%)。β溶血素基因在甘肃地区检出率为95.5%(21/22),青海地区检出率为76.9%(10/13),致病因子LukF在甘肃地区检出率为91.0%(20/22),青海地区检出率为76.9%(10/13),且在各基因型中均有分布,甘肃地区S.aureus致病因子LukF和β溶血素检出率高于青海地区。本研究首次对青海和甘肃地区致奶牛乳房炎S.aureus进行了鉴定分析,为这两个地区奶牛乳房炎的防治提供了参考依据。
In order to understand the epidemic characteristics of Staphylococcus aureus in dairy farms in Qinghai and Gansu provinces, S.aureus isolates were isolated and identified from 210 dairy cow mastitis milk samples collected from Qinghai and Gansu provences, the coagulase polymorphism of all the isolates were typed by the coagulase gene amplification and restriction endonuclease Alu Ⅰ digestion method, and the main pathogenic factors of all the isolates were detected, including LukF, LukS,α-hemolysin, and β-hemolysin. The results showed that 35 isolates of S.aureus(13 isolates from Qinghai and 22 isolates from Gansu) were identified. The coagulase gene were able to be amplified in all isolates. There were 5 coagulase genotypes, of which only 1 subtype in type 1, 4 and 5, 3 subtypes in type 2, and 2 subtypes in type 3. Type 3 was the major genotype in Qinghai(6/13) and Gansu(8/22) provinces. The detection results of pathogenic factors showed that all the isolates carried LukS andα-hemolysin. The detection rates of LukF and β-hemolysin were 30(85.7%) and 31(88.6%), respectively. Of which β-hemolysin in Gansu and Qinghai provinces were 95.5%(21/22) and 76.9%(10/13), respectively, while the LukF in Gansu and Qinhai provinces were 91%(20/22) and 76.9%(10/13), respectively. The detection rates of virulence factors LukF and β hemolysin in Gansu were higher than those in Qinghai.
In order to understand the epidemic characteristics of Staphylococcus aureus in dairy farms in Qinghai and Gansu provinces, S.aureus isolates were isolated and identified from 210 dairy cow mastitis milk samples collected from Qinghai and Gansu provences, the coagulase polymorphism of all the isolates were typed by the coagulase gene amplification and restriction endonuclease Alu Ⅰ digestion method, and the main pathogenic factors of all the isolates were detected, including LukF, LukS,α-hemolysin, and β-hemolysin. The results showed that 35 isolates of S.aureus(13 isolates from Qinghai and 22 isolates from Gansu) were identified. The coagulase gene were able to be amplified in all isolates. There were 5 coagulase genotypes, of which only 1 subtype in type 1, 4 and 5, 3 subtypes in type 2, and 2 subtypes in type 3. Type 3 was the major genotype in Qinghai(6/13) and Gansu(8/22) provinces. The detection results of pathogenic factors showed that all the isolates carried LukS andα-hemolysin. The detection rates of LukF and β-hemolysin were 30(85.7%) and 31(88.6%), respectively. Of which β-hemolysin in Gansu and Qinghai provinces were 95.5%(21/22) and 76.9%(10/13), respectively, while the LukF in Gansu and Qinhai provinces were 91%(20/22) and 76.9%(10/13), respectively. The detection rates of virulence factors LukF and β hemolysin in Gansu were higher than those in Qinghai.
引文
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[8]倪春霞,蒲万霞,胡永浩,等.奶牛乳房炎金黄色葡萄球菌凝固酶基因型研究[J].中国农业科学,2011,44(2):417-422.
[9]Harmsen D,Claus H,Witte W,et al.Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and data-base management[J].J Clin Microbiol,2003,41(12):5442-5448.
[10]任宪刚,朱战波,崔莉,等.不同凝固酶基因型牛源金黄色葡萄球菌主要致病因子的检测及分析[J].中国预防兽医学报,2008,30(8):608-611.
[11]朱战波,任宪刚,崔玉东,等.牛源金黄色葡萄球菌凝固酶基因的分型[J].中国预防兽医学报,2007,27(5):728-730.
[12]Rodrigues da Silva E,da Silva N.Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil[J].Can J Vet Res,2005,69(4):260-264.
[13]Nocadello S,Minasov G,Shuvalova L,et al.Crystal structures of the components of the Staphylococcus aureus leukotoxin ED[J].Acta Crys A,2016,72(1):113-117.
[2]Tesfaye G Y,Regassa F G,Kelay B.Milk yield and associated economic losses in quarters with subclinical mastitis due to Staphylococcus aureus in Ethiopian crossbred dairy cows[J].Trop Anim Health Prod,2010,42(5):925-931.
[3]Larsen H D,Sloth K H,Elsberg C,et al.The dynamics of Staphylococcus aureus intramammary infection in nine Danish dairy herds[J].Vet Microbiol,2000,71(1):89-101.
[4]Subramanian A,Chitalia V K,Bangera K,et al.Evaluation of HiaureusTM coagulase confirmation kit in identification of Staphylococcus aureus[J].Jcdr,2017,11(2):8-15.
[5]Hussain A,Ahmed M D,Mushtaq M H,et al.Molecular characterization of coagulase genes of Staphylococcus aureus isolated from mastitic river buffaloes[J].J Anim Plant Sci,2016,26(2):408-414.
[6]Goh S H,Byrne S K,Zhang J L,et al.Molecular typing of Staphylococcus aureus on the basis of coagulase gene polymorphisms[J].J Clin Microbiol,1992,30(7):1642-1645.
[7]Kim S M,Lee D C,Park S D,et al.Genotype,coagulase type and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolated from dermatology patients and healthy individuals in Korea[J].J Bacteriol,2009,39(4):307-316.
[8]倪春霞,蒲万霞,胡永浩,等.奶牛乳房炎金黄色葡萄球菌凝固酶基因型研究[J].中国农业科学,2011,44(2):417-422.
[9]Harmsen D,Claus H,Witte W,et al.Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and data-base management[J].J Clin Microbiol,2003,41(12):5442-5448.
[10]任宪刚,朱战波,崔莉,等.不同凝固酶基因型牛源金黄色葡萄球菌主要致病因子的检测及分析[J].中国预防兽医学报,2008,30(8):608-611.
[11]朱战波,任宪刚,崔玉东,等.牛源金黄色葡萄球菌凝固酶基因的分型[J].中国预防兽医学报,2007,27(5):728-730.
[12]Rodrigues da Silva E,da Silva N.Coagulase gene typing of Staphylococcus aureus isolated from cows with mastitis in southeastern Brazil[J].Can J Vet Res,2005,69(4):260-264.
[13]Nocadello S,Minasov G,Shuvalova L,et al.Crystal structures of the components of the Staphylococcus aureus leukotoxin ED[J].Acta Crys A,2016,72(1):113-117.