江苏地区奶牛源链球菌分子流行病学调查及四环素耐药基因分析

发布时间：2018-03-06 16:19

本文选题：奶牛乳房炎　切入点：调查　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：奶牛乳房炎是影响奶牛养殖业最重要的疾病之一,同时也是奶牛常发病之一,给乳品生产和奶牛养殖造成巨大经济损失。不同种类的病原菌感染是引起奶牛乳房炎的最重要原因。为了解江苏地区奶牛乳房炎流行病学以及病原微生物感染现状,本研究通过对江苏省4个地区的5个规模化奶牛养殖场进行采样并进行病原菌的分离,同时对分离的病原菌的药物敏感性进行检测并针对四环素严重的耐药现状进行四环素相关耐药基因的检测与分析。研究内容一、江苏地区奶牛乳房炎流行病学调查通过BMT检测试剂对江苏省4个地区的奶牛养殖场的奶牛进行奶牛乳房炎检测,根据检测结果采集隐形及临床奶牛乳房炎乳样,并对这些乳样病原菌的分离、纯化并利用MALID-TOF-MS方法对江苏地区乳房炎源链球菌进行种属鉴定。从2014年10月30日至2016年8月17日,共在江苏地区四个城市的牛场(A、B、C、D)中采集临床和隐形奶牛乳房炎奶样,一共采集奶样536份,共分离葡萄球菌382株(47.75%),链球菌207株(25.88%),杆菌211株(26.38%)。在受鉴定的207株链球菌中共鉴定奶牛乳房炎相关链球菌无乳链球菌165株(79.71%),停乳链球菌3株(1.45%),乳房链球菌3株(1.45%)。研究结果表明引起江苏地区奶牛乳房炎的链球菌主要为无乳链球菌。研究内容二、乳房炎源性链球菌耐药性检测根据CLSI规定的K-B纸片法对256株乳房炎源性链球菌进行8类20种药物进行药物敏感性检测,结果显示链球菌对四环素、苯唑西林、大观霉素和多粘菌素B均表现出非常严重的耐药现象,对青霉素、诺氟沙星、环丙沙星等药物的中介率较高。多重耐药方面主要的多重耐药表现为三重、四重、五重和六重耐药,其中四重耐药菌株数量最高(19.92%),十重以上耐药现象占总受检数的10.33%。万古霉素、头孢菌素和红霉素、克拉霉素和克林霉素以及呋喃妥因敏感性较高。研究结果表明江苏地区奶牛乳房炎源链球菌耐药现状严重,大环内酯类药物和万古霉素以及呋喃妥因仍可作为临床用药。研究内容三、乳房炎源性链球菌四环素耐药基因检测本研究对四环素耐药基因(tetM、tetO、tetKA、tetL)等基因进行PCR扩增。参考文献设计相关引物,从链球菌基因组中扩增出tetM、tetO、tetK tetL等耐药基因,经BLAST分析表明扩增的片段与GenBank中参考序列同源性均在99%以上。对256株链球菌进行四环素耐药基因的分子流行病学检测,根据检测结果显示tetM基因的检出率最高,为39.06%,tet0的检出率为15.63%,tetK的检出率为0.78%,tetL未在本次检测中发现。研究结果表明江苏地区乳房炎源性链球菌对四环素的严重耐药现状主要由相关耐药基因tetM和tetO引起。同时四环素耐药率高于耐药基因的检出率,分析原因或与其他同机制耐药基因编码或其他耐药机制导致。本研究表明四环素耐药现状严重、耐药机制十分复杂,临床管理和治疗十分困难。
[Abstract]:Cow mastitis is one of the most important diseases affecting dairy cattle breeding, and it is also one of the common diseases in dairy cattle. In order to understand the epidemiology of dairy cow mastitis and the present situation of pathogenic microorganism infection in Jiangsu province, the most important cause of dairy cow mastitis was caused by different kinds of pathogenic bacteria. In this study, five large-scale dairy cattle farms in 4 regions of Jiangsu Province were sampled and the pathogens were isolated. At the same time, the drug sensitivity of isolated pathogenic bacteria was detected and the tetracycline related drug resistance gene was detected and analyzed according to the current situation of tetracycline serious drug resistance. Epidemiological investigation of dairy cow mastitis in Jiangsu province dairy cow mastitis was detected in dairy cattle farms in 4 areas of Jiangsu Province by using BMT reagent. According to the test results, invisible and clinical milk samples of dairy cow mastitis were collected. The strains of Streptococcus mastitis were isolated, purified and identified by MALID-TOF-MS from October 30th 2014 to August 17th 2016. A total of 536 milk samples were collected from clinical and invisible dairy cow mastitis in four cities of Jiangsu province. A total of 382 strains of Staphylococcus, 207 strains of streptococcus and 211 strains of bacilli were isolated. 165 strains of streptococcus associated with mastitis were identified, 3 strains of Streptococcus brevis and 1.45% of Streptococcus mastococcus were identified. The results show that the main streptococcus causing mastitis of dairy cattle in Jiangsu area is Streptococcus acuminata. Resistance of Streptococcus mastitis to tetracycline and oxacillin were detected by K-B disk method according to CLSI. The drug resistance to penicillin, norfloxacin, ciprofloxacin and other drugs is high. The main multidrug resistance in multidrug resistance is triple and quadruple. Among the five and six resistant strains, the four resistant strains were the highest in number (19.922), and the ten or more resistant strains accounted for 10.33% of the total tested drug resistance. Vancomycin, cephalosporin and erythromycin, Clarithromycin, clindamycin and furantoin were highly sensitive. Macrolides, vancomycin and furantoin can still be used as clinical drugs. PCR amplification of tetracycline resistance gene of Streptococcus mastitis. TetMmitt OttetetK tetL was amplified from the genome of Streptococcus mastitis by PCR. The results of BLAST analysis showed that the homology between the amplified fragments and the reference sequence in GenBank was above 99%. The results of molecular epidemiological analysis of tetracycline resistance gene in Streptococcus spp showed that the detection rate of tetM gene was the highest. The detectable rate of tetet0 was 15.63 tetK and 0.78 tetL was not found in this test. The results showed that the severe resistance of Streptococcus mastitis to tetracycline was mainly caused by tetM and tetO. The rate of tetracycline resistance was higher than that of resistance gene. This study shows that tetracycline resistance is serious, drug resistance mechanism is very complex, clinical management and treatment is very difficult.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23

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