河南睢县儿童腹泻患者EAEC的分离及特征分析

发布时间：2018-05-25 22:04

本文选题：致泻性大肠杆菌 + 肠集聚性大肠杆菌　；参考：《中国疾病预防控制中心》2013年硕士论文

【摘要】：致泻性大肠杆菌(Diarrheagenic Escherichia coli)引起的感染性腹泻是发展中国家面临的一个重要公共卫生问题。肠集聚性大肠杆菌(enteroaggregative Escherichia coli, EAEC)是一类新发现的致泻性大肠杆菌,其特征是对HEp-2细胞的“叠砖样”集聚性粘附表型(aggregative 'stacked-brick' pattern)。EAEC于1987年首次报道,并从一名患有顽固性腹泻的智利儿童体内分离到。EAEC致病机制相对复杂,且不同菌株间的异质性(多态性)非常大。EAEC目前已在世界各发达和发展中国家引起散发或暴发流行。本研究对河南省睢县腹泻患者粪便标本进行了EAEC分离,并对分离自河南、北京、山西的腹泻患者粪便的EAEC菌株进行了血清分型、HEp-2细胞粘附分析、毒力及粘附相关基因检测分析以及PFGE及MLST分子分型分析。血清型检测发现,本研究中的53株EAEC菌株分属于11种O血清群,16种H型,共可确定24株菌株的12种O：H血清型,其中03：H2(6株,占11.32%)是主要的血清型,表明EAEC的血清型众多,仅仅依靠血清型检测已不再是EAEC的有效检测手段。HEp-2细胞粘附试验中,53株aggR阳性的菌株中,36株表现为集聚性粘附(aggregative adherence,AA)粘附,其余菌株为不粘附；79株aggR阴性、astA阳性的菌株中,9株表现为AA粘附,6株表现为局灶性粘附(localized adherence,LA)粘附,其余64株均表现为不粘附。AA粘附表型与aggR基因型并不存在绝对的对应关系,但aggR基因仍然是典型EAEC有效的检测标识。除毒力基因cdt、ipaH及set2外,53株aggR阳性的菌株中存在28个检测毒力基因中的25个,菌株中最多的同时存在14个毒力基因,其中aap及pCVD432存在于所有的菌株中,79株aggR阴性、astA阳性的菌株中,除aggA、aafA、agg3A、 hdaA、aggB、aggR、cdt、cnf、ipaH、set2及sfaD-sfaE外,共可检测到17个毒力基因,菌株中最多的同时存在7个毒力基因。由此可见,EAEC是一类高度异质性的大肠杆菌,不同菌株所携带的毒力基因种类差异较大,在所检测的毒力基因中,没有一种毒力基因为具有AA粘附表型的EAEC菌株所特有。 PFGE可将我国分离的51株EAEC菌株及EAEC国际参考菌株042及17-2分成48个不同的PFGE带型,表明EAEC菌株呈现高度多态性。MLST分析表明,53株EAEC菌株分属于20个不同的ST型别,其中包括两个新的等位基因型,分别为adk127和fumC133和三个新的ST型,分别为ST3748、ST3749及ST3750。ST10为优势ST型别,占18.87%,加强对该序列群EAEC菌株的研究,具有重要意义。本研究中分离的EAEC菌株,与国际暴发菌株相比,亲缘关系较远,但不排除我国出现EAEC暴发疫情的可能。
[Abstract]:Infectious diarrhea caused by diarrhoeal Escherichia coli (Escherichia) is an important public health problem in developing countries. Enteroaggregative Escherichia coli, EAEC) is a kind of newly discovered cathartic Escherichia coli, characterized by the "stacked-brick 'pattern).EAEC" agglomerative adhesion phenotype of HEp-2 cells, which was first reported in 1987. The pathogenetic mechanism of .EAEC isolated from a Chilean child suffering from refractory diarrhea is relatively complex, and the heterogeneity (polymorphism) among different strains is very large. EAEC has caused sporadic or epidemic outbreaks in developed and developing countries all over the world. In this study, EAEC was isolated from fecal specimens of diarrhea patients in Suixian County, Henan Province, and EAEC strains isolated from diarrhea patients' feces from Henan, Beijing and Shanxi were analyzed for serotyping of HEp-2 cells. Virulence and adhesion related gene analysis and PFGE and MLST molecular typing analysis. The serotype analysis showed that 53 EAEC strains in this study belong to 11 O serogroup 16 H serotypes, and 12 O: h serotypes of 24 strains can be identified, among which 03:H2(6 strain (11.32g) is the main serotype, indicating that EAEC serotypes are numerous. Serotyping alone is no longer an effective method for the detection of EAEC. 36 of 53 aggR positive strains in HEp-2 cell adhesion test showed aggregate adhesion. Among the 79 strains with negative aggR A positive, 9 strains showed AA adhesion, 6 strains showed focal adhesion, and the other 64 strains showed non-adhesion. AA adhesion phenotype did not correlate with aggR genotypes. However, aggR gene is still an effective detection marker for typical EAEC. There were 25 out of 28 virulence genes detected in 53 strains of aggR positive except for the virulence gene cdttteripaH and set2, and 14 virulence genes were found at the same time in most of the strains. Aap and pCVD432 were found in 79 strains with negative aggR A positive in all strains. 17 virulence genes were detected except for aggAaafAagg3A, hdaAaggBaggBaggRaggRdtcdtcnfnfnfnfimipaHset2 and sfaD-sfaE, and 7 virulence genes were found at the same time among the most virulent genes in the strain. It can be seen that EEAEC is a class of highly heterogeneous Escherichia coli, and the types of virulence genes carried by different strains vary greatly. None of the virulence genes detected is unique to EAEC strains with AA adhesion phenotype. PFGE could divide 51 EAEC strains isolated from China and EAEC international reference strains 042 and 17-2 into 48 different PFGE bands, indicating that EAEC strains showed high polymorphism. MLST analysis showed that 53 EAEC strains belonged to 20 different St types. There were two new allelic genotypes, adk127 and fumC133, and three new St genotypes, ST3748, ST3749 and ST3750.ST10, respectively, which accounted for 18.870.It is of great significance to strengthen the research on the EAEC strains of this sequence group. The EAEC strains isolated in this study were more closely related to those of the international outbreak strains, but the possibility of EAEC outbreak in China was not ruled out.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R725.7

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