毒力因子参与不同源肠球菌的机会感染机制及整合子介导的耐药机制的初探

Enterococcus is established as an important opportunistic pathogen which included a large number of virulence factors and unique resistance mechanisms. With the emergence of vancomycin-resistant Enterococcus faecium and the NDM-1 gene Enterococcus faecium, the study on multi-drug resistant and transmission mechanism of drug-resistant genes between the different isolates in enterococcus is to very important. The integron of bacterials is DNA system which carry gene cassette encoding antibiotic resistance, integrase has an important molecular foundation of bacteria to capture the resistance gene, the upstream promoter helps integron express the resistance gene, which causes the transmission of bacteria multidrug resistant. In order to study the species of virulence factors and resistance mechanisms by integron, the samples were collected from the different ways, including animals faeces, environments and hospitals air. And the resistance phenotype and genotype of isolates were also studied. The virulence factors were detected by PCR and the pathogenic including different virulence factors isolates were studied by animals experiments. The distribution of integrons in the isolates was detected by real-time PCR. Sequence analysis method was selected to identify the resistance gene-cassette in isolates. The correlation between resistance and virulence of different sources of isolates were studied by this experiment and statistical analysis. It has practical importance to prevent drug resistance gene and virulence factors spreading between different isolates and different animals, animals and human beings through integron gene-cassette system and genetic factors.

肠球菌是一种重要的机会致病菌，其机会感染的原因在于拥有众多的毒力因子和独特的耐药机制。随着耐万古霉素肠球菌的产生以及屎肠球菌中"NDM-1"基因的发现，关于肠球菌多重耐药性及耐药基因在不同菌株间传播机制的研究变得尤为重要，而整合子可以通过质粒、转座子使耐药基因在细菌间/中进行水平转移，从而使细菌的耐药性广泛传播。为查明毒力因子参与的机会感染机制和整合子-基因盒系统介导的多重耐药机制，本研究以动物源（包括致病株）、相关环境源和医源性肠球菌为研究对象，筛选多重耐药菌株；采用PCR筛选其毒力因子，观察不同毒力因子基因型菌株的致病性；采用Real-time PCR检测耐药整合子的分布，分析其耐药基因盒种类及特征；对比分析不同源分离株的耐药性与毒力的相关性。以期从动物性食品源头入手，阻断肠球菌毒力基因通过各种遗传因子，耐药基因通过整合子-基因盒在不同菌株之间、不同动物之间、动物性食品和人之间的散播。

抗生素的滥用现象在我国畜牧业生产中尤为严重，有专家预言，我国有可能率先进入“后抗生素时代”。肠球菌作为卫生部全国细菌耐药性监测的指示菌之一，多年的监测结果表明，其医院内感染及多重耐药性在我国呈逐年上升趋势，但畜牧业生产中尚未见相关的耐药监测数据。而肠球菌作为一种重要的机会致病菌，其之所以能引起感染主要在于拥有众多的毒力因子和独特的耐药机制。因此，本项目以不同动物源肠球菌为研究对象，采用常规微生物学手段结合PCR检测及序列分析，建立了一套肠球菌属细菌快速分离鉴定方法，分离到不同源肠球菌1910株，对其生物学特性进行了系统研究，并分离到1株犊牛源致病性耐万古霉素粪肠球菌；筛选出多重耐药肠球菌菌株285株，其对8种抗生素的耐药率达1.40%～92.28%，存在1重到8重等不同程度的耐药现象，5重耐药现象最多达31.39%；耐药基因型和表型结果基本一致，其中tetM、tem、ermB、aac(6’)-aph(2’’)和ant(6)-Ⅰ基因的存在分别是分离株对四环素、青霉素、红霉素和氨基糖苷类耐药的主要机制；265株多重耐药粪肠球菌中I类整合酶基因、qacE△1-sul 1基因的阳性率分别为36.60%和71.13%，该基因阳性菌株对多种抗菌药物的耐药率均高于阴性菌株，I类整合子携带有aadA2编码氨基糖苷类耐药基因盒，其与aph(3')-III、 aac(6')/aph2''和ant(6')-I耐药基因相关，从5株分离株中检测到II类整合酶基因；分离株中携带ace，asa1，asa373，efaA，clyA，gelE，sprE，esp和hyl等不同的毒力因子及组合的比例高达64%；通过分析不同分离株的ERIC-PCR分子指纹聚类结果，同时结合携带不同耐药基因和毒力因子组合菌株的动物感染试验、细胞毒性试验结果发现，相同动物来源耐药组毒力强于非耐药组，相同耐药组不同动物来源进行比较，猪源毒力强于蜣源。研究结果为从动物性食品源头入手，阻断肠球菌毒力基因通过各种遗传因子以及耐药基因通过整合子-基因盒在不同菌株之间、不同动物之间、动物性食品和人之间的散播奠定了理论基础。