藏猪链球菌2型血清学调查及肠球菌耐药性研究

发布时间：2018-04-17 21:52

  本文选题：藏猪 + 链球菌　； 参考：《华中农业大学》2017年硕士论文

【摘要】：猪链球菌病(Streptococcus suis S.Suis)是因为猪链球菌感染而导致的分布极其广泛的传染病,对养殖业和人类影响极大。该病含有35个血清型,其中2型(S.Suis 2)发病率最高,主要以淋巴结炎、关节炎、脑膜炎及败血症为主要临床特征,已被国家规定为二类动物疫病。然而在近期的临床实践中发现部分西藏林芝地区的藏猪出现类似于S.Suis的临床症状,故此拟通过血清学调查进一步掌握该病在林芝地区藏猪的流行情况,以期为后续的防疫工作和综合性措施提供依据。肠球菌(Enterococci)存在广泛,为条件性致病菌。然而近年不科学的抗生素应用使肠球菌耐药越发严重,不仅对高水平氨基糖苷类药物耐药,对大环内酯类、β-内酰胺类、四环素类及糖肽类抗生素等的耐药率也较高,肠球菌引起的各种传染病也随之增多。然而,要想研究其耐药性,分离肠球菌是必不可少的一步,而肠球菌又与其他球菌表型相似,故很难区分,加之其分布随地区的不同而不同,西藏林芝地区对此研究甚少,故本研究就肠球菌的分离鉴定及其耐药问题展开研究。本研究分别对2014年12月份到2015年1月份间在西藏林芝地区所分离的438份血清进行ELISA试验,完成对猪链球菌2型在该地区阳性率的初步调查,这对增强当地居民对猪链球菌病的防控意识,具有重要的公共卫生学意义。同时,本研究对2015年7月到9月份及2016年6月到8月期间所采取的藏猪内脏及粪便进行肠球菌分离,并对分离菌株进行耐药及MIC试验。以此为基础,筛选出耐药性肠球菌菌株并进行基因检测。试验结果与结论如下:1.猪链球菌的检测结果本试验采用双抗原夹心酶联免疫法(ELISA)对林芝县及米林县共438份血清进行检测。检测结果显示:所有血清的抗体阳性率为60.05%(263/438)。其中,雌性、雄性分别为61.50%和58.96%,林芝县与米林县的阳性率分别为61.79%和57.81%。应用SPSS 13.0软件统计分析得出,在本研究中,性别及两地区间无显著性差异(P≥0.05)。本研究说明林芝地区藏猪S.Suis 2隐性感染率较高,当地居民应加强该病的防控意识。2.藏猪源肠球菌的分离情况本研究采用胰酶大豆琼脂(Tryptic Soy Ager,TSA)固体培养基(加入2%兔血清,50μg/ml氨曲南及10μg/ml多粘菌素B)用于选择性分离培养肠球菌,进而通过触酶试验并结合生化特征初步鉴定为肠球菌,最后进一步结合16SrDNA以及肠球菌特保守的tuf基因通过pcr进一步验证得出如下结果:2015年从105个样品中共分离出51株、4种藏猪源肠球菌(48.6%):E.faecalis 15株(29.4%),E.faecium 4株(7.8%),E.hirae 27株(52.9%),E.durans 2株(3.9%)及其他肠球菌3株(5.9%),E.durans为首次在藏源猪获得;2016年从120个样品中共分离出45株、2种藏猪源肠球菌(37.5%):E.faecalis 12株(26.7%),E.faecium 6株(13.3%)及其他肠球菌27株(60%)。3.肠球菌耐药检测情况本研究通过采用K-B药敏片法及MIC法对2015-2016年两年分离菌株进行6类10种抗菌药物的敏感性测定。结果显示:所有肠球菌对卡那霉素、庆大霉素及链霉素高水平耐药率高达60%到80%以上,而对万古霉素的敏感率均为100%,红霉素及四环素的耐药率均接近50%。2016年与2015年相比,氨苄西林的耐药率明显增加。头孢呋辛、青霉素及左氧氟沙星的耐药率均为10%左右,敏感率较高,且两年增减幅度不大。此外,两年份的菌株均表现为严重的多重耐药性,尤其为粪肠球菌,高达5耐,未发现6耐菌株。说明当地居民以后应多加注意并科学用药。4.肠球菌耐药基因的检测与分析结合K-B药敏片法及MIC的结果,本研究分别对两年份5类药物10种耐药基因(aac(6’)-Ie-aph(2’)-Ia,aph(3’)-IIIa,aph(2’)-Ic,ant(4’)-Ia,ant(6’)-Ia,ant(3’)-(9),TEM,ermB,mef A,tetM)进行检测,结果发现:菌株的耐药表型及耐药基因之间并不呈正相关。比如2015年耐高水平链霉素菌株为31株,但并无携带ant(6’)-Ia和ant(3’)-(9)的肠球菌。2015年部分菌株携带aac(6’)-Ie-aph(2’)-Ia,ant(4’)-Ia,ermB及tetM的阳性率高达100%,没有发现aph(3’)-IIIa,aph(2’)-Ic,ant(6’)-Ia,ant(3’)-(9)及mef A耐药菌株,但2016年分离的肠球菌中发现有6株携带aph(3’)-IIIa,3株携带ant(6’)-Ia,但没有检测到ant(4’)-Ia,其余分离率与2015年相比有微增长现象。虽然分离率不高,但E.faecalis与E.faecium均比其他肠球菌的分离率高。该结果说明E.faecalis与E.faecium的耐药性比较严重,且2016年与2015年相比并没有下降趋势。
[Abstract]:Streptococcus suis (Streptococcus suis S.Suis) is extremely widespread due to Streptococcus suis infection of infectious diseases, greatly on aquaculture and human impacts. The disease contains 35 serotypes, including type 2 (S.Suis 2) the highest incidence, mainly in lymphadenitis, arthritis, meningitis and sepsis were the main clinical features, has been the provisions of the state for the two categories of animal diseases in clinical practice. However, recently found in the Linzhi area part of Tibet Tibet pig had clinical symptoms similar to S.Suis, therefore intends to further understand the serological investigation of the epidemic situation in Linzhi area of Tibet pig, in order to provide the basis for further prevention work and comprehensive measures of Enterococcus. (Enterococci) exists widely, for opportunistic bacteria. However antibiotics not scientific that enterococci become more and more serious, not only for the high level aminoglycoside drugs Resistance to macrolides, beta lactams, tetracyclines resistant and glycopeptide antibiotics such as to have a higher rate of various infectious diseases caused by enterococci are increasing. However, in order to study the drug resistance of enterococci is a necessary step, but also with other similar phenotypes aureus Enterococcus, so it is difficult to distinguish, and its distribution varied by region, Linzhi area, Tibet has little study about it, so the research on isolation and identification of resistance of Enterococcus is studied. In this study were ELISA trial in 438 serum samples in 2014 December to 2015 January, isolated in Linzhi area of Tibet, the completion of the preliminary investigation of Streptococcus suis 2 type the positive rate in the region, which is to enhance the awareness of prevention and control of local residents of Streptococcus suis, has important significance in public health. At the same time, the study on July 2015 to September and 201 6 years from June to August during the Tibetan pig offal and feces were adopted in enterococci separation, and drug resistance and MIC test of isolates. On this basis, screening of drug resistance of Enterococcus strains and gene detection. The experimental results and conclusions are as follows: 1. the results of the test of Streptococcus suis by double antigen sandwich ELISA (ELISA) in Linzhi county and Milin County, a total of 438 serum samples were detected. The test results show that: the positive rate of antibody in all serum was 60.05% (263/438). Among them, female and male were 61.50% and 58.96%, the positive rate of Linzhi county and Milin county were 61.79% and 57.81%. SPSS 13 software was used in statistical analysis. In this study, there was no significant difference between gender and interval (P = 0.05). This study shows that the Linzhi area of Tibet pig S.Suis 2 recessive infection rate is high, the local residents should strengthen the awareness of disease prevention and control of Tibet pig intestinal.2. The separation aureus used in this study (Tryptic Soy Ager tryptic soy agar, TSA solid medium (2%) with rabbit serum, 50 g/ml aztreonam and 10 g/ml polymyxin B) was used to isolate enterococci selectivity, then through catalase test and combined with the identification of biochemical characteristics of enterococci, furthermore tuf gene 16SrDNA and Enterococcus special conservative through the PCR verify the results obtained are as follows: 105 in 2015 from a sample of the isolated 51 strains of Enterococcus, 4 Tibet pig (48.6%): E.faecalis 15 (29.4%) strains, 4 strains of E.faecium (7.8%), 27 strains of E.hirae (52.9%), 2 strains of E.durans (3.9%) he and 3 strains of Enterococcus (5.9%), E.durans for the first time in 2016 from the source of Tibetan Pig; 120 samples isolated from 45 strains of Enterococcus, 2 Tibet pig (37.5%): E.faecalis 12 (26.7%) strains, 6 strains of E.faecium (13.3%) and 27 strains of Enterococcus Enterococcus (60%).3. The study of drug resistance detection by using K-B method and MIC method of drug sensitivity of 2015-2016 strains were 6 years of 10 kinds of antibacterial drug sensitivity test. The results showed that all the isolates to kanamycin, gentamicin and high-level streptomycin resistance rate is as high as 60% to more than 80%, while the sensitive rate to vancomycin were 100%, resistance to erythromycin and tetracycline were close to 50%.2016 compared with 2015, ampicillin resistant rate increased significantly. Cefuroxime, penicillin and levofloxacin resistance rate was about 10%, higher sensitivity, and two changes little. In addition, two copies of the strains showed multiple drug resistance is serious, especially for Enterococcus faecalis, as high as 5 resistant, not found in the 6 resistant strains. After the detection and analysis of local residents should pay more attention and scientific use of.4. Enterococcus resistance gene with K-B susceptibility method and MIC 鐨勭粨鏋，

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