【作者】 黎满香；

【导师】 薛立群；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2010， 博士

【摘要】 粪肠球菌(Enterococcus faecalis)是医院交叉感染的主要病原并具多重耐药性,在人医方面早已引起广泛的关注,然而在动物感染方面研究并不多。本实验对感染猪的粪肠球菌的部分特征及诊断方法做了相关的研究。1.本研究从2003年到2008年送检湖南农业大学的湖南各地病料中共分离到细菌42株,菌落形态及染色镜检均与粪肠球菌参考株ATCC 29212相似,Lancefield分群显示分离菌以D群为主(41/42),符合粪肠球菌特征,分离菌所检测的生化特性和《常见细菌系统鉴定手册》所记录的粪肠球菌的生化特性也一致。为了进一步确定分离菌属何种菌,采用16S rDNA测序鉴定的方法对分离菌进行鉴定,结果显示42株分离菌与同步测序的ATCC 29212同源性在99.2%到100%之间,与所选择粪肠球菌参考序列的同源性在98.7%到100%之间,NCBI在线BLAST分析发现42株均与Genebank收录的粪肠球菌序列同源性最高,而与其他肠球菌则有多处变异,结合染色形态和生化特征最终将这42株菌株鉴定为粪肠球菌,并将测序结果上传到Genebank。系统进化分析发现湖南分离株16S rDNA序列与E.faecalis参考序列进化关系与地域分布无关,来源于不同宿主的菌株的16S rDNA变异并不大,而与对比的其他四种肠球菌则有多处变异,这些变异区域在这四种肠球菌中却是保守的。2.16S rDNA测序的方法作为日常鉴定是成本高且耗时长的,为此我们建立了基于粪肠球菌16S rDNA的PCR检测方法,通过比较肠球菌属其他菌的16S rDNA差异设计特异性引物,经PCR检测与16SrDNA测序结果比较、用其他种属细菌阴性对照、扩增片段测序后表明该PCR方法具有很好特异性,最小可检出134 CFU,而且用培养基上的细菌作为模板时不需要特殊处理,操作简易,并可不经细菌培养直接对病料进行检测,适用于日常实验室的检测。3.42株分离株的8种毒力基因esp、cylA、asa1、ace、efaA、EF0591、EF3314、gelE中EF3314是普遍存在的(检出率100%),efaA也有相当高的检出率(92.86%),这与Creti报道的意大利医院分离株很相似,但ace基因差异很大,在意大利分离株检出率是100%,湖南分离株仅61.90%。湖南省猪源分离株的esp检出率(35.71%)和Creti及Shankar(丹麦)报导的人源分离株相似,Shankar的猪源分离株仅8%(6/75)检出携带esp基因。同样一批菌株在绵羊血琼脂平板上以α-溶血为主导,而在家兔血琼脂平板中以β-溶血为主,并且兔血检出溶血的比例比绵羊血的高,还表现出溶血基因型与溶血表型不一致,‘即cylA+菌株却表现为不溶血,cylA-菌株却表现为溶血。大多菌株(34/42)的明胶分解试验获得与理论预计的一致,即gelE+对应明胶分解(+),gelE-对应明胶不分解(-),然而有4株gelE+菌不能分解明胶的现象,另有4株gelE-菌能分解明胶,出现基因型与表型不一致的情况。此外我们还对引物进行兼并,构建二重PCR,在优化条件后获得良好的扩增效果,提高了检测的效率。4.对其中的HN45号菌株的生长曲线、半数致死量、在感染动物体内的动态分布及感染动物的组织病理学变化等方面研究时发现,该菌株在TSB培养基的对数生长期在3.5到5.5h左右,之后进入一个缓慢增殖阶段。该菌株对小鼠毒力较弱,半数致死量为5.76×109,这比其它细菌高出一个数量级。然而在腹腔攻毒后1h即可在动物体内心脏、肝脏、脾脏、肺脏、肾脏中检出该细菌,2h即可在脑中检出细菌,3h即可在尾静脉血液中检出,有较强的组织穿透能力。致死的小鼠心脏、肾脏以及肝脏都有明显眼观病变,如出血、充血现象,微观病变可见肝细胞变性坏死,肺泡膈增生,浆液性渗出,少量炎性细胞浸润等。更多还原

【Abstract】 Enterococcus faecalis is one of the main pathogen causing Nosocomial infections and resistant to multiple antibiotics. Although it has caused worldwide concern in human infection, there is a little literature about Enterococcal infection in swine, Exceptionally,We made a study on characteristics and diagnosis method of Enterococcus faecalis isolates.Enterococcus faecalis strains (n=42) was isolated from clinical diseased pig in Hunan province from 2003 to 2008, The colonial morphology and the shape of Cram strain bear a close analogy to ATCC 29212 strain, Lancefield test showed that 97.6% (41/42) strains belongs to group D. Biochemistric characteristics of those strains are concordant with Enterococcus faecalis characteristics recorded in Bergey’s manual of systematic bacteriology. The sequence analysis of 16S rDNA revealed that all Hunan strains share a high identity with reference strain ATCC 29212 (≥99.2%) and the homology ranged from 98.7% to 100% when comparing with selected reference sequences. The 16S rDNA sequence of Hunan isolates had highest identity with E. faecalis published in genebank by BLAST in NCBI. Finally,42 isolates were identified as Enterococcus faecalis and the sequencing result was submited to genebank. The phyletic evolution of all compared E. faecalis strains have no connection with the geographic distribution and host, more variation was observed in the 16S rDNA between E. faecalis and others four Enterococcus, including E.faecium、E.canis、E.avium、E.hirae, but variation region are conservative in those four species.PCR assay based on 16S rDNA was developed because of 16S rDNA sequencing is time-consuming and expensive for daily detection. Specific primer was designed by comparing the difference between E. faecalis and others’enterococcus. The PCR assay presented good specificity and sensitivity after compared detection result with 16S rDNA sequencing, used others genus as negative controls, amplification fragment sequencing and sensitivity testing. In addition, the method needs no special treatment of PCR templates when bacterial culture was detected and can detect sample directly。For 42 strains, the existence of esp、cylA、asa1、ace、efaA、EF0591、EF3314、gelE genes were detected by PCR scanning. EF3314 (100%) and efaA (92.86%) are commonly found in those E. faecalis, The incidence is similar to Italianate strains reported by Creti’s, but obvious difference was discovered in ace gene,100%(Creti’s report) and 59.52%(Hunan) respectively. The esp gene positive rate of Hunan strain (n=42) is 38.10%, the proportion is more like human strains reported by Creti and Shankar but differ greatly to swine strains (8%, Shankar). To the same strains, a-hemolysis more likely occurred in sheep blood agar plates, but in rabbits blood mainly presentedβ-hemolysis. Genotype is nonconsistent with phenotype in hemolysis test, which cylA+strains failed to decompose erythrocyte, cylA—strains decomposed erythrocyte. For most strains (34/42), genotype is consistent with phenotype in gelatin degradation test, which gelE+strains decompose gelatin but gelE-not. However, there are still 4 gelE+strains failed to decompose gelatin,4 gelE—strains decomposed gelatin. In addition, the multiplex PCR for virulence-related genes detection was developed to simplify detection procedure in this study.The growth curve determination of strain HN45 show that the bacterial comes to log phase growth after 3.5 hour cultured and continuing to 5.5 hour, and then reached plateau phase. HN45 had lower toxicity (LD50=5.76×109) to mice, but It is amazing that the bacterial was detected in heart, liver, spleen, lungs, kidney by Gram staining and PCR assay after intraperitoneal challenge 1 hour,2 hour later detected in brain and 3h in tail vein blood. Obvious pathological change was observed in heart, liver, lungs of dead mice, as such hemorrhage, hyperemia, the degeneration and necrosis of kidney epithelial cells and hepatic cells, hyperplasia of interalveolar septum and serous effusion.更多还原