本文選題：腸球菌 + 多重PCR方法��； 參考：《河北北方學(xué)院》2015年碩士論文

【摘要】：腸球菌為重要的人獸共患條件致病菌,可引起人和動物多部位的感染。腸球菌也是我國院內(nèi)感染的重要病原菌之一,近年來,多重耐藥腸球菌、萬古霉素耐藥腸球菌(vancomycin-resistant Enterococci,VRE)的出現(xiàn),對臨床腸球菌的感染控制提出了挑戰(zhàn)。腸球菌屬包括近40個種,人類及哺乳動物消化道均普遍攜帶。不同種屬腸球菌的分布特征及藥物敏感性有很大差異,如鶉雞腸球菌、鉆黃腸球菌因攜帶天然萬古霉素耐藥基因vanC表現(xiàn)出對萬古霉素固有耐藥。耐藥基因水平轉(zhuǎn)移是腸球菌耐藥性獲得的主要分子機制之一。不僅僅是人類機體,養(yǎng)殖動物、環(huán)境都可能作為腸球菌耐藥基因的儲存庫。因此了解不同來源腸球菌的種屬分布及其藥物敏感性特征將有助于研究耐藥腸球菌的流行規(guī)律和傳播途徑,幫助臨床更有效的預(yù)防和控制耐藥腸球菌的感染。本課題對來自社區(qū)人群及養(yǎng)殖場動物(雞、豬)的糞便樣本進行腸球菌的分離培養(yǎng),通過建立的腸球菌種屬鑒定的多重PCR方法對分離菌株進行種屬鑒定;采用紙片擴散(disk diffusion method,K-B)法、瓊脂稀釋法對不同來源腸球菌進行抗菌藥物的敏感性檢測;應(yīng)用建立的腸球菌屬、常見腸球菌種及萬古霉素耐藥基因型檢測的多重PCR方法對VRE疑似菌株進行耐藥基因型檢測,以期初步闡述我國不同來源腸球菌的種屬分布及其藥物敏感性特征。選擇包括腸球菌屬及6個腸球菌種(糞腸球菌、屎腸球菌、鶉雞腸球菌、鉆黃腸球菌、堅韌腸球菌、鳥腸球菌)在內(nèi)的特異性擴增引物,以16S rDNA特異性擴增引物作為內(nèi)參,建立鑒定腸球菌屬及6個腸球菌種的8重PCR方法;選擇腸球菌屬、糞腸球菌、屎腸球菌、4個常見萬古霉素耐藥基因型(vanA、vanB、vanC1、vanC2/C3)特異性擴增引物,以1對16srdna引物作為內(nèi)參建立用于腸球菌屬、常見腸球菌種及萬古霉素耐藥基因型檢測的8重pcr方法。經(jīng)過擴增產(chǎn)物多對特異性引物反復(fù)試驗,兩套8重pcr體系最終各選用最佳的8對特異性引物,準確快速檢測腸球菌種屬及萬古霉素耐藥基因型。對220份社區(qū)人群及養(yǎng)殖場動物(雞、豬)的糞便樣本中腸球菌進行分離培養(yǎng)、種屬鑒定及藥物敏感性分析顯示:腸球菌總分離率為70.91%(156/220),其中豬源樣本腸球菌分離率最高(86.00%),人源樣本腸球菌分離率最低(62.63%),且人源與豬源樣本腸球菌分離率差異顯著(p0.018);人源糞便樣本中分離率最高的為屎腸球菌(31.36%),雞源、豬源腸球菌中糞腸球菌分離率最高,分別為28.17%和32.00%;抗菌藥物敏感性結(jié)果顯示腸球菌對多種藥物的耐藥率在人源、雞源、豬源3種來源間差異顯著(p0.05),且3種來源腸球菌的多藥耐藥率差異有統(tǒng)計學(xué)意義(p0.05);人源腸球菌對紅霉素(69.35%)、環(huán)丙沙星(37.10%)、氨芐西林(19.35%)等抗菌藥物耐藥率較其他來源的腸球菌要高;雞源腸球菌對四環(huán)素(88.24%)、氟苯尼考(11.76%)、氯霉素(21.57%)等抗菌藥物耐藥率較其他來源的腸球菌要高;豬源腸球菌對抗菌藥物耐藥率總體較低,且其多藥耐藥率(7.84%)也低于人源(35.48%)及雞源腸球菌(30.19%)。對41株動物源vre疑似菌株種屬、耐藥表型、耐藥基因型研究顯示:37株菌為vre,包括6株攜帶vanc1的鶉雞腸球菌和31株攜帶vanc2/c3的鉆黃腸球菌,全部對萬古霉素中介耐藥而對替考拉寧敏感,;4株菌為非vre,對萬古霉素和替考拉寧均表現(xiàn)為敏感;未鑒定到種的腸球菌,未檢測到萬古霉素耐藥基因。本課題成功建立了兩套多重pcr檢測方法,分別用于同時鑒定腸球菌種屬和同時鑒定腸球菌屬、常見腸球菌種及萬古霉素耐藥基因,為腸球菌種屬鑒定及耐藥基因的快速檢測提供了實用方法;初步闡述了不同來源糞便樣本中腸球菌種屬分布特征;分析了不同來源腸球菌抗菌藥物藥物敏感性特征及vre的耐藥機制,為描畫我國不同地區(qū)、不同來源的腸球菌分布流行規(guī)律,有效控制耐藥腸球菌的產(chǎn)生和傳播提供了基礎(chǔ)數(shù)據(jù)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.
【學(xué)位授予單位】：河北北方學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

【參考文獻】

相關(guān)期刊論文 前4條

1 王亞賓;陳麗穎;胡慧;段志剛;崔保安;;豬源糞腸球菌和屎腸球菌多重PCR快速鑒定方法的建立[J];動物醫(yī)學(xué)進展;2010年S1期

2 朱成賓;竇露;夏永祥;;耐萬古霉素腸球菌的流行病學(xué)及臨床檢測方法研究進展[J];國際檢驗醫(yī)學(xué)雜志;2012年06期

3 李小權(quán);樊書娟;劉俐;肖謐;林曉潔;;新生兒鶉雞腸球菌腦膜炎1例[J];中國當代兒科雜志;2013年12期

4 王進;肖永紅;;Mohnarin 2006～2007年度報告:革蘭陽性菌耐藥監(jiān)測結(jié)果[J];中國抗生素雜志;2008年10期

，

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