【作者】 罗远婵；

【导师】 谢关林；

【作者基本信息】 浙江大学 ， 植物病理学， 2006， 博士

【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。更多还原

【Abstract】 The Burkholderia cepacia, is a gram-negative bacterium widely distributed in agricultural and clinical environment. It has been divided into 9 genomovars called Burkholderia cepacia complex (Bcc). The strains of Bcc had been mainly used as biological control agent in China, though some of them were the onion pathogens or opportunistic pathogens of humans causing "cepacia sydrome" resulted in many deaths of the patients with cystic fibrosis in several countries. It has been known that the strains of plant pathogens, human opportunistic pathogens and biological control agent are undistinguishable. Little is known about the genomovars of Bcc from farmland and hospital in China up-to-now. It is extremely necessary to know the genomovars of Bcc existed in Chinese farmland and determine whether they had the same risk as the dangerous clinical strains.Forty-three samples of soil and irrigation water were collected from the farmland of Guangxi, Zhejiang, Hainan, and Hebei province. Twenty-six isolates from the farmland were identified as Bcc strains after growing on the Bcc selective medium and the phenotypic tests.And 41 clinical isolates from hospital of Liaoning and Zhejiang province were identified as Bcc strains. The phenotypic tests including Biolog and FAMEs only can identify the strains at Bcc level but not the genomovarsThe sequencing analysis of 16S rRNA gene commonly used for idetification of bacteria can not distinguish the genomovars of Bcc due to their high homoeology. Comparison of the identification by PCR with Bcc-specific and. genomovar-specific . primers and sequencing analysis based on recA gene and by Biolog and FAMEs were conducted at the present stduy. The results showed that 59 strains(25 from the farmland and 34 from hospital) out of the 67 strains identified by Biolog and FAMEs were confirmed as Bcc by the molecule biological methods. The misidentification percentage of Biolog and FAMEs were 11.94 %. It has been found that the Bcc strains from the farmland were genomovar I, II, IV and V, and the genomovar I was the most predominant one followed by the genomovar V The Bcc strains from thehospital were genomovar I, II, IIIA, HID, and V, and the genomovarlllA was the most predominant while the genomovar I also had higher isolation frequency in the hospital. PCR with Bcc-specific and genomovar-specific primers based on recA gene were the major methods for primarily identification of Bcc genomovars due to their simple procedure and high reliability. However, the sequencing analysis based on recA still was a important supplementary me"ans for low sensitivity of PCR with some recA gene genomovar-specific primers for Bcc strains from the farmland (eg. genomovar II) and lack of genomovar DC-specific primers. The results also showed that some genomovars had their unique colony characteristics, the colonies of genomovar I were olivaceous, colorless of genomovar IV colonies, and opal of the most genomovarlllcolonies..The results for distinguishing the genomovar I strains of the predominant Bcc from the farmland and the hospital showed that all the methods of RFLP and sequencing analysis of recA gene, and hypersensitive reaction in tobacco could not distinguish them, however, the toxicity to mammal tested by alfalfa plant model, the replacement of mammal model, could distinguish the virulent and avirulent ones, which laid the firm foundation for us to safely use the Bcc strains in biocontrol and bioremediation. It was the first report of distinguishing the genomovar I strains of Bcc from the two different sources by using alfalfa plant model to test the toxicity to mammal and the further study on its mechanism was necessary. The toxicity to mammal of the different Bcc genomavars from the farmland was firstly studied in China by alfalfa model, the replacement of mammal model. It showed that the toxicity of the Bcc strains from the farmland was distinctly weaker than the clinical strains. The disease infection percentage of the all Bcc strains from the farmland tested including the genomovar I, H and genomovar IVon the alfalfa seedlings did not show significant difference compared with the check at P=l% level. The percentage of the all Bcc clinical strains of genomovar I and genomovar III on the alfalfa seedlings showed significant difference compared with the check at P=l% level. The infection perentage of the Bcc strains of genomovar V from the farmland on the alfalfa seedlings was variable, 4 of them showed significant difference compared with thecheck at P=l% level and 3 of them were not. However, all the Bcc strains of genomovar V from the farmland showed significant difference in the disesaese infection compared with the Bcc strains of same genomovar from the clinical at P=l% level. Fifty-nine Bcc strains were tested for their antagonism against the 3 phytobacterial and 1 fungal pathogens in vitro. It revealed that the antagonism of the Bcc strains against the phytobacteria was low and higher against the fungal pathogen. The strain 91 from the farmland showed safety to mammal by test on the plant model and high effectivity in suppressing the Botrytis cinerea, causing vegetable gray mold had high potential to develop a biological control agent.更多还原