奶牛乳腺炎性金黄色葡萄球菌耐药基因检测、分子分型和耐甲氧西林菌株全基因组测序

发布时间：2018-06-12 06:35

  本文选题：奶牛乳腺炎 + 耐甲氧西林金黄色葡萄球菌　； 参考：《中国农业大学》2016年博士论文

【摘要】：乳腺炎是奶牛养殖业中造成经济损失最大的疾病,金黄色葡萄球菌是其主要致病菌之一,在泌乳牛头混合奶样中的分离率高达20-50%。目前,抗生素仍是预防和治疗金黄色葡萄球菌的唯一有效药物,在适应性变异和抗生素的选择压力下,各地分离菌株已对多种抗生素产生耐药,给临床治疗带来很大困难。为调查我国奶牛隐性乳腺炎中金黄色葡萄球菌的耐药现状和耐药基因分布,明确其分子流行病学特点,分析牛源MRSA菌株的基因组特征。采集2010-2013年间北京、山西、山东、内蒙古、浙江和新疆六省区的2103份奶样,从确诊的1069份隐性乳腺炎样本中分离鉴定出219株金黄色葡萄球菌,采用纸片扩散法测试分离菌株对兽医临床常用10种药物的敏感性,并使用多重PCR方法对其中81株菌进行三类六种耐药基因检测,结果显示,六省区样本中70%以上的分离株对红霉素、克林霉素、青霉素、复方新诺明、多西环素和四环素耐药,并且60%的菌株对5种以上抗生素多重耐药,但各省区分离株对不同药物的敏感性存在差异；耐药基因调查显示,被检测的90%以上的分离株携带耐药基因,ermC, aac(6')/aph(2")、aph(3')-Ⅲ、tetK和tetM耐药基因是主要流行基因；此外,从中鉴定出mecA阴性耐甲氧西林菌株。进一步,使用MLST分型方法对已完成耐药基因检测的菌株进行分子分型,并对鉴定的MRSA菌株进行SCCmec分型,结果表明该区域分离株的流行序列型为ST97和ST398,属于CC5和CC398克隆复合群;6株MRSA菌株属于CC5,其中3株为ST97-SCCmec Ⅳ,其它为ST965-SCCmec Ⅳ、 ST6-SCCmec Ⅳ和ST9-SCCmec NT;同时,使用eBURST v3软件分析MLST数据库中医源和牛源MRSA相关性,分析表明医源和牛源MRSA形成是相对独立的,已报道的牛源MRSA为MSSA菌株获得SCCmec而产生的,未大范围扩散。此外,基于Hiseq 2000和PacBio SMRT技术的测序数据,使用de novo拼接方法获得多重耐药MRSA菌株Z35和Z43的全基因组序列并进行SCCmec特征分析,结果显示Z35为ST9-SCCmec ⅩⅡ,携带重组酶基因复合体ccrC2; Z43为ST97-SCCmec Ⅳ,该基因组序列与S. aureus Mu50同源性较高,SCCmec Ⅳ结构中存在约15 Kb的J2区,含有多种耐药基因。本研究表明我国六省区奶牛隐性乳腺炎性金黄色葡萄球菌的流行序列型为ST97和ST398,属于CC5和CC398克隆复合群；ermC、aac(6')/aph(2")、aph(3')-Ⅲ和tetM耐药基因是该区域金黄色葡萄球菌耐药的主要基因;牛源MRSA是由不同序列型的MSSA获得SCCmec而产生的,未造成大范围扩散；牛源ST9-MRSA-SCOmec ⅩⅡ和ST97-MRSA-SCCmecⅣ的全基因组测序显示其分别携带重组酶基因复合体ccrC2和含有多种耐药基因的J2区。
[Abstract]:Mastitis is the most economic loss disease in dairy cattle breeding, Staphylococcus aureus is one of the main pathogenic bacteria, the isolation rate is as high as 20-50 in mixed milk samples of lactating cattle. At present, antibiotics are still the only effective drugs for the prevention and treatment of Staphylococcus aureus. Under the adaptive variation and the pressure of antibiotic selection, the strains isolated from various places have become resistant to many kinds of antibiotics, which brings great difficulties to clinical treatment. In order to investigate the present situation of drug resistance and gene distribution of Staphylococcus aureus in cow recessive mastitis in China, the molecular epidemiological characteristics of Staphylococcus aureus and the genomic characteristics of bovine MRSA strain were analyzed. From 2010 to 2013, 2103 milk samples were collected from Beijing, Shanxi, Shandong, Inner Mongolia, Zhejiang and Xinjiang provinces, and 219 strains of Staphylococcus aureus were isolated from 1069 confirmed occult mastitis samples. The susceptibility of the isolated strains to 10 kinds of drugs commonly used in veterinary clinic was tested by disk diffusion method, and the multiplex PCR method was used to detect the three types and six drug resistance genes of 81 strains. The results showed that, More than 70% of the isolates in the six provinces were resistant to erythromycin, clindamycin, penicillin, compound sulfamine, doxycycline and tetracycline, and 60% of the isolates were multidrug resistant to more than 5 antibiotics. However, the susceptibility of the isolates to different drugs was different in different provinces, and the drug-resistance gene survey showed that more than 90% of the isolates were carrying the drug resistance gene, aac(6')/aph(2 "Hapaphora 3tetK and tetM resistance genes" were the main prevalent genes, in addition, more than 90% of the isolates carried the drug resistance gene, and the drug resistance genes were found to be the main prevalent genes. MecA negative methicillin-resistant strains were identified. Furthermore, MLST typing method was used to type the strains that had completed the detection of drug resistance genes, and SCCmec genotyping was used to identify MRSA strains. The results showed that the epidemic sequence types of the isolates were ST97 and ST398.The MRSA strains belonging to CC5 and CC398 clone complex belonged to CC5, three of them were ST97-SCCmec 鈪，

本文编号：2008734