肠球菌多重PCR方法的建立及不同来源肠球菌种属分布和耐药特点初探

发布时间：2018-06-30 05:50

本文选题：肠球菌 + 多重PCR方法　；参考：《河北北方学院》2015年硕士论文

【摘要】：肠球菌为重要的人兽共患条件致病菌,可引起人和动物多部位的感染。肠球菌也是我国院内感染的重要病原菌之一,近年来,多重耐药肠球菌、万古霉素耐药肠球菌(vancomycin-resistant Enterococci,VRE)的出现,对临床肠球菌的感染控制提出了挑战。肠球菌属包括近40个种,人类及哺乳动物消化道均普遍携带。不同种属肠球菌的分布特征及药物敏感性有很大差异,如鹑鸡肠球菌、钻黄肠球菌因携带天然万古霉素耐药基因vanC表现出对万古霉素固有耐药。耐药基因水平转移是肠球菌耐药性获得的主要分子机制之一。不仅仅是人类机体,养殖动物、环境都可能作为肠球菌耐药基因的储存库。因此了解不同来源肠球菌的种属分布及其药物敏感性特征将有助于研究耐药肠球菌的流行规律和传播途径,帮助临床更有效的预防和控制耐药肠球菌的感染。本课题对来自社区人群及养殖场动物(鸡、猪)的粪便样本进行肠球菌的分离培养,通过建立的肠球菌种属鉴定的多重PCR方法对分离菌株进行种属鉴定;采用纸片扩散(disk diffusion method,K-B)法、琼脂稀释法对不同来源肠球菌进行抗菌药物的敏感性检测;应用建立的肠球菌属、常见肠球菌种及万古霉素耐药基因型检测的多重PCR方法对VRE疑似菌株进行耐药基因型检测,以期初步阐述我国不同来源肠球菌的种属分布及其药物敏感性特征。选择包括肠球菌属及6个肠球菌种(粪肠球菌、屎肠球菌、鹑鸡肠球菌、钻黄肠球菌、坚韧肠球菌、鸟肠球菌)在内的特异性扩增引物,以16S rDNA特异性扩增引物作为内参,建立鉴定肠球菌属及6个肠球菌种的8重PCR方法;选择肠球菌属、粪肠球菌、屎肠球菌、4个常见万古霉素耐药基因型(vanA、vanB、vanC1、vanC2/C3)特异性扩增引物,以1对16srdna引物作为内参建立用于肠球菌属、常见肠球菌种及万古霉素耐药基因型检测的8重pcr方法。经过扩增产物多对特异性引物反复试验,两套8重pcr体系最终各选用最佳的8对特异性引物,准确快速检测肠球菌种属及万古霉素耐药基因型。对220份社区人群及养殖场动物(鸡、猪)的粪便样本中肠球菌进行分离培养、种属鉴定及药物敏感性分析显示:肠球菌总分离率为70.91%(156/220),其中猪源样本肠球菌分离率最高(86.00%),人源样本肠球菌分离率最低(62.63%),且人源与猪源样本肠球菌分离率差异显著(p0.018);人源粪便样本中分离率最高的为屎肠球菌(31.36%),鸡源、猪源肠球菌中粪肠球菌分离率最高,分别为28.17%和32.00%;抗菌药物敏感性结果显示肠球菌对多种药物的耐药率在人源、鸡源、猪源3种来源间差异显著(p0.05),且3种来源肠球菌的多药耐药率差异有统计学意义(p0.05);人源肠球菌对红霉素(69.35%)、环丙沙星(37.10%)、氨苄西林(19.35%)等抗菌药物耐药率较其他来源的肠球菌要高;鸡源肠球菌对四环素(88.24%)、氟苯尼考(11.76%)、氯霉素(21.57%)等抗菌药物耐药率较其他来源的肠球菌要高;猪源肠球菌对抗菌药物耐药率总体较低,且其多药耐药率(7.84%)也低于人源(35.48%)及鸡源肠球菌(30.19%)。对41株动物源vre疑似菌株种属、耐药表型、耐药基因型研究显示:37株菌为vre,包括6株携带vanc1的鹑鸡肠球菌和31株携带vanc2/c3的钻黄肠球菌,全部对万古霉素中介耐药而对替考拉宁敏感,;4株菌为非vre,对万古霉素和替考拉宁均表现为敏感;未鉴定到种的肠球菌,未检测到万古霉素耐药基因。本课题成功建立了两套多重pcr检测方法,分别用于同时鉴定肠球菌种属和同时鉴定肠球菌属、常见肠球菌种及万古霉素耐药基因,为肠球菌种属鉴定及耐药基因的快速检测提供了实用方法;初步阐述了不同来源粪便样本中肠球菌种属分布特征;分析了不同来源肠球菌抗菌药物药物敏感性特征及vre的耐药机制,为描画我国不同地区、不同来源的肠球菌分布流行规律,有效控制耐药肠球菌的产生和传播提供了基础数据和信息支持。
[Abstract]:Enterococcus is an important pathogen of human zoonosis, which can cause infection in many parts of human and animal. Enterococcus is also one of the important pathogens in hospital infection in China. In recent years, the emergence of multiple resistant Enterococcus, vancomycin resistant Enterococcus (vancomycin-resistant Enterococci, VRE) and the control of clinical enterococcus infection The genus Enterococcus includes nearly 40 species, both human and mammalian digestive tract are generally carried. The distribution characteristics and drug sensitivity of different species of Enterococcus are very different, such as chicken Enterococcus, and the resistance to vancomycin is characterized by the resistance to vancomycin resistant gene vanC. One of the main molecular mechanisms of bacterial resistance is not only human organism, culture animal and environment, but also the distribution of species and drug sensitivity of enterococci from different sources and its drug sensitivity will help to study the prevalence and transmission of Enterococcus, and help the clinic to be more effective. To prevent and control the infection of Enterococcus resistant Enterococcus. We isolated and cultured the fecal samples from the community and the farm animals (chickens and pigs), and identified the isolates by the multiple PCR method established to identify the species of Enterococcus; disk diffusion method (K-B) method and agar dilution method were used. The sensitivity detection of antimicrobial agents for Enterococcus from different sources, the multiple PCR method of multiple Enterococcus, common Enterococcus and vancomycin resistant genotypes was used to detect the drug resistance genotypes of suspected VRE strains, in order to preliminarily explain the distribution of species and drug sensitivity characteristics of Enterococcus in China. Selective amplification primers including Enterococcus and 6 Enterococcus (Enterococcus faecalis, Enterococcus faecium, Enterococcus quail, Enterococcus dryococcus, Enterococcus yellowenterococcus, Enterococcus leathery, Enterococcus) were selected as the internal parameters of 16S rDNA specific amplification primers, and 8 PCR methods for identification of Enterococcus and 6 Enterococcus were established, and Enterococcus, Enterococcus faecalis, and feces were selected. Enterococcus, 4 common vancomycin resistant genotypes (vanA, vanB, vanC1, vanC2/C3) specific amplification primers. 1 pairs of 16SrDNA primers were used as the internal parameters to establish 8 PCR methods for Enterococcus, common Enterococcus and vancomycin resistance genotypes. Repeated experiments were carried out by increasing the yield and more specific primers, and two sets of 8 PCR systems. The best 8 pairs of specific primers were selected for the accurate and rapid detection of Enterococcus and vancomycin resistant genotypes. 220 community population and the fecal samples from the animal (chicken, pig) were isolated and cultured. The identification and drug sensitivity analysis showed that the total isolation rate of Enterococcus was 70.91% (156/220), in which the pig samples were found. The isolation rate of Enterococcus was the highest (86%), the isolation rate of Enterococcus was the lowest (62.63%), and the isolation rate of Enterococcus was significantly different between human and pig sources (p0.018). The highest separation rate in human fecal samples was Enterococcus faecium (31.36%), chicken source and Enterococcus faecalis were the highest, 28.17% and 32%, respectively. The sensitivity results showed that the resistance rate of Enterococcus to a variety of drugs was significant (P0.05) in human source, chicken source and pig source (P0.05), and the multidrug resistance rate of 3 sources of Enterococcus was statistically significant (P0.05); the drug resistance rate of human Enterococcus to erythromycin (69.35%), ciprofloxacin (37.10%) and ampicillin (19.35%) was compared with the others. The source of Enterococcus was higher; the resistance rate of chicken Enterococcus to tetracycline (88.24%), florfenicol (11.76%), chloramphenicol (21.57%) was higher than that of other Enterococcus, and the rate of antibiotic resistance of swine Enterococcus was lower, and the rate of multidrug resistance (7.84%) was lower than that of human (35.48%) and chicken Enterococcus (30.19%). 41 animals were found. The drug-resistant phenotype and drug resistance genotype of source VRE showed that 37 strains were VRE, including 6 strains of vanc1, and 31 strains of Enterococcus aureus carrying vanc2/c3, all of which were resistant to vancomycin and sensitive to teicoplanin; 4 strains were non VRE, and all were sensitive to vancomycin and teicorannin. Two multiple PCR methods were successfully established for the identification of Enterococcus and simultaneous identification of Enterococcus, common Enterococcus and vancomycin resistance genes, which provided a practical method for the identification of enterococci and the rapid detection of resistance genes. The distribution characteristics of Enterococcus species in different sources of feces were described, and the antimicrobial susceptibility characteristics of Enterococcus and the resistance mechanism of VRE were analyzed. The basic data and information were provided to describe the distribution and epidemic of Enterococcus in different regions and different sources, and to effectively control the production and transmission of antibiotic resistant Enterococcus. Support.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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