农田中洋葱伯克氏菌基因型及其与医院同类致病菌的比较研究

发布时间：2018-06-24 12:10

  本文选题：洋葱伯克氏菌群 + 鉴定　； 参考：《浙江大学》2006年博士论文

【摘要】：洋葱伯克氏菌(Burkholderia cepacia)是一种广泛存在于农业环境和医院的革兰氏阴性细菌,现被国际上划分为9个基因型,合称洋葱伯克氏菌群(B.cepacia complex,简称Bcc)。Bcc虽然引起洋葱酸皮病,但国内主要作为生物农药在应用。目前国内外报道该菌引起洋葱伯克氏综合症而致多人死亡。现有研究显示Bcc菌中生物农药菌、植物致病菌和人体条件致病菌难以区分。我国无人涉足Bcc菌基因型及其与医院致病菌的比较研究。我国农田中存在哪些Bcc基因型、它们是否与医院同类致病菌一样危险等问题急待解决。 本研究从广西、浙江、海南、河北4省共采集农田灌溉水和土壤样本43份,用选择培养基Bcc富集液分离Bcc菌株,经表型特征鉴定后确定所分离的细菌菌株中有26个为Bcc菌株。从辽宁、浙江2省的医院获取并用表型特征鉴定Bcc菌株41个。研究表明Biolog和脂肪酸分析(FAMEs)均只能将Bcc鉴定到种的水平,而无法鉴定到基因型。 由于各基因型Bcc菌株间同源性极高,常用的细菌分子鉴定手段16S rRNA基因序列比较无法区分各基因型。本研究采用recA基因区分Bcc各基因型菌株,同时比较了recA特异引物PCR扩增和recA全序列分析的方法与表型鉴定方法的差异,经表型鉴定方法定为Bcc的67个菌株中只有59个被确证为Bcc菌株,其中农田菌株25个,医院菌株34个,Biolog和FAMEs对Bcc菌的误鉴定率为11.94%。农田菌株中检测到基因型Ⅰ、Ⅱ、Ⅳ和Ⅴ,其中基因型Ⅰ为优势类型,其次是基因型Ⅴ;医院菌株中检测到基因型Ⅰ、Ⅱ、ⅢA、ⅢD和Ⅴ,其中基因型ⅢA为优势类型,它是引起洋葱伯克氏综合症的主要基因型,但同时也发现医院存在较高比率的基因型Ⅰ菌。recA特异引物PCR扩增法是针对医院Bcc菌株的基因型鉴定设计的,该法操作简便、准确率较高可以作为初步鉴定Bcc基因型的主要手段,但是由于一些基因型recA特异引物的缺少(如基因型Ⅸ)和对农田菌株灵敏度不够高(如基因型Ⅱ),因此recA全序列分析是鉴定和确证Bcc基因型不可或缺的手段。研究还表明一些菌株的基因型与其在TSA平板上的菌落特征有一定的对应关系,通常基因型Ⅰ菌株菌落均为黄绿色、基因型Ⅳ菌落接近无色、基因型Ⅲ的菌落多为乳白色。
[Abstract]:Burkholderia cepacia is a Gram-negative bacterium widely found in agricultural environment and hospital. It is divided into 9 genotypes in the world. It is called B.cepacia complex (BCC). BCC causes onion skin disease. However, it is mainly used as biological pesticide in China. At present, it is reported at home and abroad that the bacteria causes onion Burke's syndrome and causes many deaths. Current studies show that biological pesticide bacteria, plant pathogens and human conditioned pathogens in BCC bacteria are difficult to distinguish. A comparative study of BCC genotype and hospital pathogenic bacteria in China. The problems of which BCC genotypes exist in farmland in China and whether they are as dangerous as the similar pathogenic bacteria in hospitals need to be solved urgently. In this study, 43 farmland irrigation water and soil samples were collected from Guangxi, Zhejiang, Hainan and Hebei provinces. BCC strains were isolated by BCC enrichment medium. 26 of them were identified as BCC strains. 41 strains of BCC were obtained from hospitals in Liaoning and Zhejiang provinces and identified with phenotypic characteristics. Biolog analysis and fatty acid analysis (FAMEs) showed that BCC could only be identified at the species level, but not genotypes. Because of the high homology among the BCC strains, the 16s rRNA gene sequence of the commonly used bacterial molecular identification method can not distinguish the genotypes. In this study, recA gene was used to distinguish BCC genotypes, and the differences between recA specific primer PCR amplification and recA full sequence analysis were compared with phenotypic identification methods. Only 59 of the 67 strains identified as BCC by phenotypic identification method were identified as BCC strains, including 25 strains from farmland and 34 strains from hospitals. The false identification rate of BCC bacteria was 11.94% by Biolog and FAmes. Genotypes 鈪，

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