【作者】 方媛；

【导师】 谢关林；

【作者基本信息】 浙江大学 ， 植物病理学， 2010， 博士

【摘要】 洋葱伯克氏菌群(Burkholderia cepacia complex,Bcc)的细菌主要存在于水体、医院和农业环境中,虽然部分Bcc被认为是有益菌,但不少是世界公认的植物和人体致病菌。了解水体、医院和一些植物上的Bcc菌的种类及其遗传多样性,对我国Bcc菌的利用与环境安全极其重要。然而,国内外尚无这类详细的研究报道。本研究以西湖水为主要研究对象,对水中的Bcc种类及其动态作了较深入研究；并从医院环境中获得的细菌中筛选出Bcc菌,调查了我国一些医院环境中主要存在的Bcc种；同时对由Bcc菌引起的杏果新病害——杏果腐病作了研究。对西湖水、医院及杏果中所获得的Bcc菌的种类、致病性及其致病基因进行了比较研究,同时针对Bcc菌抗药性强和防治难的特点,本研究还对防治Bcc菌的新物质进行了探索。取得了如下的主要研究结果：(1)采用培养和非培养的分子检测方法,在为期一年的时间内定点定时对西湖水中的Bcc菌种类及其遗传多态性作了研究；同时与来自医院和杏果Bcc菌进行了分析和比较。通过选择性培养基PCAT共获得776株疑似Bcc菌株,对这些菌疑似Bcc菌株作了recA基因的PCR特异性扩增,共确定309株Bcc菌,其中265株、42株、2株分别来源于西湖水、医院和杏果。采用限制性内切酶HaeⅢ对这309株Bcc菌的recA基因片段进行了限制性酶切(RFLP)分析。结果表明,在西湖水的265株Bcc菌中,发现7种不同的限制性酶切图谱(F,G,I, H, J, B, A);在医院来源的42株Bcc菌中,发现了3种不同的限制性酶切图谱(K,G,H)；杏果腐Bcc菌的限制性酶切图谱为H,这是国内外首次发现Bcc菌可引起杏果病害。这表明在这些环境中,Bcc菌群存在明显的遗传多态性。(2)采用recA-HaeⅢ限制性酶切分析、基因型特异性引物扩增、recA基因的序列分析和多位点序列分型(MLST)技术,对309株来源于西湖水、医院和杏的Bcc菌进行了种群分析。首次明确了在西湖水中存在B. multivorans, B. cenocepacia, B. stabilis, B. vietnamiensis和B. seminalis5个Bcc种,其中以B.cenocepacia的菌株数量最多,占分离自西湖水的Bcc总量的51%。在医院环境中存在B. cepacia, B. cenocepacia和Burkholderia contaminans3个种,其中也以B. cenocepacia的菌株数量最多,占分离自医院环境的Bcc总量的73.8%。而引起杏果腐的病原细菌鉴定为Bcc中的B. seminalis,这是B. seminalis首次作为植物病原菌被发现。MLST技术研究表明,该技术不仅可以区分Bcc的种,还可以区分种内菌株间的差异。(3)对分离自西湖水、医院和果腐杏的93株Bcc代表菌株进行了致病性及其毒力基因BCESM的检测和菌株蛋白酶活性研究。在测试的93个菌株中,只有17株对洋葱不致病,其余76株均能引起洋葱鳞茎不同程度的腐烂,说明这些菌株是洋葱的致病菌；在测试的7个Bcc种中,B. multivorans和B. vietnamiensis菌株没有产蛋白酶活性,其余种的大部分菌株均有产蛋白酶活性,说明西湖水中和果腐杏上的Bcc菌同样具有与对人体致病相关的毒力因子,与医院致病菌没有差异。在苜蓿模型上,来源于医院的B. cepacia,B. cenocepacia和B. contaminans菌株均能导致苜蓿幼苗发病,来源于西湖水的B. cenocepacia也能导致苜蓿幼苗发病,与医院临床致病菌表现出相似的症状,这表明来源于西湖水的这些Bcc菌株也是人体的潜在致病菌。在所有Bcc菌中,从A8和Y5上检测到毒力基因BCESM,确定了这两个菌株的高风险性。(4)分别检测了来自医院、西湖水和杏果的29株Bcc代表菌对4种抗生素(头孢西定、四环素、硫磺多年菌素、万古霉素)和壳聚糖的敏感性,结果发现Bcc菌对这4种抗生素都存在抗性,而壳聚糖被认为能有效抑制Bcc菌的生长,这一新发现为防治Bcc菌在医院的污染及其对CF病人的感染提供了新思路。更多还原

【Abstract】 The bacteria of Burkholderia cepacia complex (Bcc) were commonly found in water bodies, hospitals and agricultural environments. They were known as phytopathogens and opportunistic human pathogens although some Bcc strains were considered as beneficial agents. It was very important to understand the Bcc species and their genetic diversity in water bodies, hospitals and some plants for utilization of the Bcc strains and environmental safety of Bcc in China. However, little information is available for Bcc populations in water bodies, hospitals and plants in China. A survey of distribution of Bcc species and seasonal variation during one year period were conducted in water of West Lake at present study. The Bcc strains from some hospitals in China were isolated and identified. The rotted fruit of apricot——a new plant disease caused by Bcc isolates was studied simultaneously. All Bcc isolates from water of West Lake, hospitals and apricot were compared for their genomovars, pathogenicity and virulence genes. As the intrinsic resistance of Bcc to antibiotics and the difficulty to control when infected by some Bcc strains, a new chemical for control of Bcc was explored in this study. The main results were as follows:(1) The cultured and uncultured-molecular methods were performed for examination of Bcc species and genetic variety in West Lake during one year by fixed locations and time for sample collection. The Bcc isolates from the lake were analyzed and compared with those recovered from hospitals and apricot. Total 776 putative Bcc isolates was recovered on PC AT selected medium and 309 of them were identified as Bcc by recA gene-specific PCR amplification, of which 265,42,2 were from water of West Lake, hospitals, apricot, respectively. The restriction fragment length polymorphism (RFLP) assay with enzyme HaeⅢwas performed to detect genetic variability among 309 Bcc isolates. The results showed that 7 different RFLP patterns (F, G, I, H, J, B, A) were found among 265 Bcc isolates from water of West Lake, and three different patterns (K, G, H) were also detected among 42 Bcc isolates from hospitals, the 2 phytobacterial strains of apricot belong to RFLP pattern H. It was the first report about Bcc recovered as apricot fruit pathogen. Our results revealed considerable variability among the Bcc isolates.(2) The identification of Bcc species was performed among 309 Bcc isolates from the water of West Lake, hospitals and apricot fruits by a combination of recA-HaeⅢRFLP assays, species-specific PCR tests, recA gene sequence analysis and multilocus sequence typing (MLST) scheme. Five species including B. multivorans, B. cenocepacia, B. stabilis, B. vietnamiensis and B. seminalis were recovered from the water of West Lake for the first time and B. cenocepacia was the dominant species from water of West Lake with 51% isolation frequecncy. Three species including B. cepacia, B. cenocepacia and Burkholderia contaminans were recovered from hospital environments and B. cenocepacia was also predominant with 73.8% of the total isolates tested. The phytobacterium of apricot fruit was identified as B. seminalis. It is a first report about B. seminalis recovered as phytobacterium. Our result showed that MLST scheme could not only differentiate the Bcc species but also distinguished the difference strains within a species.(3) The pathogenicity of 93 Bcc representatives recovered from the water of West Lake, hospitals and apricot fruit were evaluated. Seventeen of them were non-pathogenic while 76 isolates induced rot symptoms on onion bulbs in varying severity index, indicating these isolates were onion phytopathogens. B. multivorans and B. vietnamiensis didn’t show protease activity in all 7 species tested, while most isolates of other 5 species showed protease activity. It was noted that the isolates from water of West Lake and apricot friut shared the same virulence factors with the isolates from hospital, indicating no significant difference in pathogenicity among them. B. cepacia, B. cenocepacia and B. contaminans isolates from hospital induced symptoms on alfalfa seedlings and B. cenocepacia isolates from water of West Lake did produce symptoms similar as formers, indicating that these isolates from water of West Lake may be potential human opportunistic pathogens. Virulence gene BCESM was detected from two isolates A8 and Y5 which had a high risk of the Bcc strains to human beings.(4) The susceptibility of 29 Bcc representatives recovered from the water of West Lake, hospital and apricot to 4 antibiotics (Cefoxitin, Tetracycline, Polymycin B sulfate, Vancomycin) and chitosan solution was evaluated. The Bcc isolates tested were shown resistant to these 4 antibiotics, but susceptible to chitosan solution. Chitosan was considered the best effective one for Bcc inhibition in this study and could be served as a good candidate for novel antimicrobial agents against Bcc infections in CF patients and plant diseases.更多还原