【作者】 郭鹏；

【导师】 刘永杰；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 嗜水气单胞菌（Aeromonas hydrophila,Ah）普遍存在于淡水、土壤、人畜粪便等各种环境中,可引起多种水产动物的败血症以及畜禽和人类腹泻。嗜水气单胞菌是重要的水生病原菌,给水产养殖业造成极大的经济损失,由于抗生素在渔业方面的滥用,导致嗜水气单胞菌产生耐药性,对水产养殖业的进一步发展产生影响.外膜蛋白作为革兰氏阴性菌外膜的重要结构对细菌耐药性的形成有一定的影响.对43株嗜水气单胞菌的外膜蛋白（OMP）进行SDS-PAGE分析,根据不同菌株蛋白质电泳条带之间的差异性可以将其分成多个型：OMP I-OMPⅥ.使用统计软件SPSS13.0进行聚类分析,以期在流行病学调查方面提供进一步了解。采用微量双倍稀释法测定43株嗜水气单胞菌最小抑菌浓度。药敏试验结果参照美国NCCLS标准判定各菌株是否耐药。在所选的7种抗生素中,环丙沙星、恩诺沙星、氟苯尼考对临床分离的43株嗜水气单胞菌的作用较好,抑菌率分别达97.67%（42／43）、97.67%（42／43）和95.35%（41／43）,而其它几种抗生素的抑菌效果较差,红霉素、泰乐菌素,抑菌率只有9.30%（4／43）、6.98%（3／43）,所有43株菌株对氨苄西林和磺胺间甲基嘧啶则完全耐药。多数菌株具有多重耐药性,两个与多重耐药输出泵相关的基因AHA₀858和aheABC在嗜水气单胞菌各菌株中广泛分布,检出率分别为77%（33／43）和81%（35／43）。实验中发现嗜水气单胞菌的外膜蛋白电泳图谱与细菌的耐药性有一定的关系,外膜蛋白型相同的菌株在耐药性也基本一致.同其他菌株相比,菌株NB-1,A.Pun和MR-1于30kD～45kD处部分外膜蛋白条带缺失,并且其对氟苯尼考（florfenicol）耐药性也大幅增强,提示部分外膜蛋白的缺失可能与嗜水气单胞菌的耐氟苯尼考有一定的关系.同时发现部分具有完全一致OMP型的菌株在某些抗生素的耐药性上也存在差异,表明嗜水气单胞菌的耐药性有其他因素影响.为了对嗜水气单胞菌的耐药机理进行更进一步的研究,本实验通过对嗜水气单胞菌J-1株（诺氟沙星敏感株）利用半数最小抑菌浓度的方法诱变形成稳定的诺氟沙星耐药株,出发菌株J-1对诺氟沙星的最小抑菌浓度为0.78μg／mL,诱变后形成的诺氟沙星耐药菌株NJ-1最小抑菌浓度为90μg／mL.利用比较蛋白质组学的方法对嗜水气单胞菌J-1和诺氟沙星耐药诱变株NJ-1进行二维电泳的研究,软件分析比较两者的蛋白质电泳图谱的差异蛋白点。通过双向电泳、质谱测定肽质量指纹图谱,鉴定出嗜水气单胞菌外膜蛋白中4个与嗜水气单胞菌诺氟沙星耐药性形成相关的蛋白,包括寡聚ABC转运器蛋白、玻璃酸酶蛋白、OmpAⅡ和二氢吡啶二羧酸合酶。细菌对诺氟沙星耐药性的形成可能与细菌外膜通透性改变从而减少抗生素的摄入以及产生灭活酶或钝化酶修饰抗生素有关.本研究为进一步揭示嗜水气单胞菌外膜蛋白与耐药性相关性提供了理论依据.更多还原

【Abstract】 Aeromonas hydrophila is a ubiquitous organism of many environments such as fresh water, sewage and soil which can cause the septicemia of aquatic animals as well as the diarrhea of human. A. hydrophila is the causative agent of fatal hemorrhagic septicemia in fish. A. hydrophila gained the resistance to antibiotics since its abuse in the fishery which would have the impact to further development. As the important structure of outer membrane of gram-negative bacteria, outer membrane protein has certain correlation with the resistance of bacteria.The SDS-PAGE profiles of outer membrane proteins （OMP） of 43 A. hydrophila strains showed diverse patterns and they could at least be divided into several types:OMP I-OMPVI. Cluster analysis of the OMP profiles was carried out by SPSS 13.0 to get further information about the epidemiologic survey of A. hydrophila. The minimum inhibitory concentrations （MIC） of 43 strains were determined by double microdilution method. The results were determined by referring to NCCLS. Ciprofloxacin, enrofloxacin and florfenicol had the better antibacterial effect to the 43 clinical isolates of A. hydrophila than the other 4 antibiotics of all 7 antibiotics selected. And the inhibitory rate of them was 97.67 %（42/43）,97.67%（42/43） and 95.35%（41/43）. The inhibitory rate of erythromycin and tylosin was 9.30%（4/43）,6.98%（3/43）. All the strains of 43 A. hydrophila are uniformly resistant to ampicillin and sulfamonomethoxine. A large number of isolates were resistant to more than one antibiotic. Positive rate of AHA₀858 and aheABC, two genes related to multiple drug-efflux pump, were 77% （33/43） and 81%（35/43） which means these two genes were widely distributed in A. hydrophila. The OMP profiles had a correlation with the resistance of A. hydrophila to a certain extent, and the strains with the similar SDS-PAGE profiles had similar resistances. Compared with other strains, the strains NB-1, A.Pun and MR-1 have lost part of their outer membrane proteins with the molecular weights between 30kD and 45kD and their resistance to florfenicol substantially increased, which suggest that the loss or reduction of OMP might have relationship with resistance to florfenicol. Some strains which showed completely identical OMP types had some differences in their resistance to antibiotics, which indicate that there might be other factors that affect the drug-resistance of A. hydrophila.To get further knowledge about the mechanism of the resistance of A. hydrophila, the strain J-1 has been mutated by using the method of half minimum inhibit concentration with norfloxacin. The MIC of J-1 to norfloxacin was 0.78μg/mL while the resistant strain NJ-1 was 90μg/mL. A comparative proteomics study has been carried out to find out the mechanism of A. hydrophila’s resistance to norfloxacin. Four proteins which have a certain correlation with the resistance to norfloxacin have been identified by the method of 2-DE and peptides mass fingerprint （PMF）, including oligopeptide ABC transporter, ABC transporter permease protein,OmpAⅡand dihydrodipicolinate synthase. The change of outer membrane permeability and the generation of inactive enzyme or modifying enzyme might be the reason that bacteria have the resistance to norfloxacin. The research was aim to know more about the correlation between A. hydrophila outer membrane protein and the resistance.更多还原