【作者】 任晶；

【导师】 安利国； 宋林生； 党宏月；

【作者基本信息】 山东师范大学 ， 细胞生物学， 2006， 硕士

【摘要】 我们利用DAPI染色荧光显微镜技术和微生物培养的方法分析2004年9月和10月两个月份胶州湾海水样品中的总微生物和可培养细菌的数量和分布特征。DAPI染色荧光的计数结果显示总微生物数量为10~6-10~7 cells/ml,与世界上其它位于温带地区的半封闭性海湾微生物数量的研究结果相似。其中微生物密度最高(10~7/ml)的区域分布于胶州湾东北部娄山河和李村河河口及红岛渔业养殖区附近。这些区域的可培养细菌密度在整个胶州湾中也是最高,最高的站位(A5站,李村河口附近)达到5.16×10~5/ml。因此,污染类型和程度以及地理和水文学特征是决定胶州湾微生物数量和分布的重要因素。通过T-RFLP分子生物学方法和聚类分析方法研究胶州湾水体的微生物群落结构特征,可将胶州湾内外的细菌群落分为3组,胶州湾湾口及湾外的D1, D3, D5, D6, D7站位被划分为一组,其细菌群落更接近于受到湾外海水环境影响的细菌群落特征;位于胶州湾最内侧的站位,如A3, A5, B2,Y1被划为一组,代表胶州湾的“土著”细菌群落;C1, C3, C4站位则代表着介于二者之间的过渡性细菌群落。而同时进行的胶州湾光合细菌群落结构的T-RFLP分析未见其与地理分布和环境有明显相关关系。抗性细菌的分布和数量主要受到水产养殖业中抗生素的使用,以及生活和工业污水排放状况的影响,是自然水体环境的重要水质标准之一。对自然水体中的抗性细菌及其抗性基因的研究不仅对渔业养殖具有重要的意义而且还直接关系到人类的公共卫生健康。胶州湾土霉素和氯霉素抗性细菌密度最高的区域均位于A5站,李村河口附近,说明环境是影响和决定抗性细菌分布和数量的重要因素。通过对抗性细菌16S rDNA序列分析和系统进化树的构建,胶州湾内的大部分抗生素抗性细菌属于γ-蛋白菌亚纲(γ-Proteobacteria),其中氯霉素抗性细菌则表现出非常明显的假单胞菌属(Pseudomonas sp.)细菌优势。多重PCR方法分析胶州湾抗性细菌中土霉素抗性基因-tet基因和氯霉素抗性基因-cat基因的分布特征。在对84株土霉素抗性基因的分析中,所有六种tet基因型-tetA, tetB, tetC, tetD, tetE和tetG均被检测到,其中含量最高的是tetA(30.6%), tetB(24.5%),和tetG(36.7%)。在对60株氯霉素抗性基因的分析中,仅有12株氯霉素抗性细菌被检测到携带有cat基因,且只包括catⅠ和catⅢ型。但更多还原

【Abstract】 DAPI staining epifluorescence microscopy and bacterial cultivating method wereused to analyze the total microbial abundance and culturable microbial abundance in theseawater of Jiaozhou Bay in September and October of 2004. The abundance of totalmicrobes is in the range of 10~6-10~7 cells/ml, similar to those of most of the worldsemi-enclosed bays in the temperate areas. The highest microbial densities (10~7/ml)occurin the northeastern part of the Jiaozhou Bay, around the mouth of rivers Loushan andLicun and the Hongdao aquacultural farming areas where the culturable microbialabundance reached 5.16×10~5/ml (A5 station, around the mouth of Licun river). Thus, thedegree and characteristics of pollutants, along with geographical and hydrological effects,may be important determinants affecting the abundance and distribution of bacteria in theJiaozhou Bay. Bacterial communities inside and outside of Jiaozhou Bay can be groupedinto 3 classes based on T-RFLP and cluster analyses. Stations at the water channel of thebay mouth and outside, such as D1, D3, D5, D6 and D7, can be grouped together to standfor the bacterial community more related to the environment outside of the Jiaozhou Bay.Stations of the innermost side of Jiaozhou Bay, such as A3, A5, B2 and Y1, can begrouped together to stand for the Jiaozhou Bay residential bacteria community. StationsC1, C3 and C4 can be grouped together and may stand for the transitional bacterialassembly between the residential community and the outside community. However, thereis no obvious relationship between cyanobacterial diversity and geographic settings inJiaozhou Bay.The distribution and abundance of antibiotics resistant bacteria, which is mainlyinfluenced by the usage of antibiotics in mariculture and the condition of sewagedischarge, is one of the important standards in natural aquatic environments. The study onresistant bacteria and their resistance genes in natural aquatic environments is not onlysignificant to mariculture but also influential to the public hygiene of human beings. Thehighest oxytetracycline and chloramphenicol resistant bacterial density occurs in stationA5, around the mouth of Licun River, suggesting that the environment is the mostimportant factor influencing and determining the abundance and distribution of antibioticresistance bacteria. Based on the 16S rDNA analysis it is discovered that most of theresistant bacteria in Jiaozhou Bay belong to the γ-Proteobacteria subdivision. Obviously,Pseudomonas sp. is the predominant species in all Chloramphenicol resistant isolates.Multiplex-PCR method is used to characterize the oxytetracycline resistance genes (tetgenes) and Chloramphenicol resistance genes (cat genes) in Jaiozhou Bay. In the analysisof 84 oxytetracycline resistant isolates, tetA, tetB, tetC, tetD, tetE and tetG are alldetected. The predominant tet genes in these oxytetracycline resistance isolates are tetA(30.6%), tetB (24.5%), and tetG (36.7%). In the characterization of cat genes in 60Chloramphenicol resistant bacteria, only 12 isolates had cat genes, and only cat I and catIII were detected. But there is not obvious relationship between the types of cat/tet genesand the species of the resistant bacteria. Since there are also many resistant bacteriawhich were not detected harboring any cat/tet genes, other or even novel resistancemechanisms might also exist. And the Multi-drug resistance mechanism might alsocontribute to the antibiotic resistance in Jiaozhou Bay.更多还原