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题名: 我国鱼类病原菌耐药性、耐药质粒及几种药物抗菌作用的研究
作者: 李爱华
答辩日期: 1998
导师: 沈韫芬 ; 王伟俊
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 鱼类病原菌 ; 耐药性 ; 耐药质粒 ; 嗜水气单胞菌 ; 河弧菌 ; 环丙沙星 ; 氯霉素 ; 最低抑菌浓度 ; 最低杀菌浓度 ; 抗生素后效应 ; 耐药突变 ; 抗性发展 ; 药代动力学 ; 质粒特征图谱
摘要: 本论文包括以下几方面的研究内容:我国鱼类病原细菌的耐药性调查和分析:病原细菌对常用药物的抗性发展研究;药物的血液浓度与耐药性的关系;耐药质粒的检测;耐药质粒的生物学特性;耐药质粒的消除;质粒特征与耐药性的关系;海水、半咸水对抗菌药物抗菌作用的影响;环丙沙星和氯霉素的杀菌作用及抗生素后效应研究。具体结果如下:(1) 从不同地区分离收集到的35株鱼类病原菌(包括嗜水气单胞菌、迟缓爱德华氏菌、鲁氏耶尔森菌、弧菌、假单胞菌和未定种病原菌)中,对青霉素和半合成青霉素、头孢菌素及万古霉素耐药的菌株比例最高,为85.7-94.3%。其中26株嗜水气单胞菌(A.hydrophila)对青霉素类和头孢菌素类药物全部具抗性,对万古霉素也多数不敏感。21种药物中最敏感的是氟哌酸、氯霉素、呋喃唑酮,总体耐药率分别只有5.7%、11.4%、11.4%;而A.hydrophila中这一数值分别为7.7%、7.7%和7.7%。氨基糖甙类药物和四环素类药物的耐药频率介于其间。对磺胺药的耐药菌比例也很高,为57.1%,其中有73.1%的A.hydroghila对磺胺及复方磺胺具有抗性,但耐磺胺比耐复方磺胺的现象更为普遍。同时对3种常用药物具有抗性的菌株比例最高。嗜水气单胞菌对环丙沙星和氯霉素非常敏感,MIC_(50)分别为:0.025μg/ml和0.25 ug/ml;MIC_(90)分别为0.12μg/ml和4.0μg/ml。四环素、土霉素、呋喃唑酮、链霉素的MIC_(50)虽然不高,分别只有1.0μg/ml、3.2μg/ml、0.8μg/ml、12.8 μg/ml,但MIC_(90)却很高,分别为8.0μg/ml、12.8μg/ml、6.4μg/ml以及50μg/ml以上。磺胺药的敏感性最低,虽然最低的MIC为4.Oμg/ml,但MIC_(50)高达62.5μg/ml,MIC_(90)更是在250μg/ml以上,对大多数病原菌已不敏感。(2)嗜水气单胞菌在分别含8 * MIC环丙沙星、链霉素的培养基中的自发突变率较低,分别为8.5 * 10~(-9)、4.2 * lO~(-8);河弧菌(V. fluvialis)相应的数值为3.4 * lO~(-8)、6.4 * 10~(-7)。突变株的MIC增加了8-16倍.在本此试验的检测灵敏度范围内,未能检测到氯霉素的耐药突变子,其自发突变率分别小于1.1 * 10~(-9)和小于6.6 * lO~(-9)。环丙沙星选择出的突变株对其它药物的敏感性无明显变化,但对四环素类药物的敏感性有所下降,说明存在一定的交叉耐药性。经环丙沙星、链霉素、氯霉素选择了11代得到的A.hydrophila耐药突变株其MIC分别增加了128倍,32倍和32倍。而选择出的V. fluvialis其MIC均增加了64倍。经在无药培养基中连续传代8次,这些突变株的抗性均维持稳定。(3)药代动力学研究表明,氯霉素通过腹腔注射吸收良好,在草鱼和异育银鲫体内的半衰期分别为11.9h和11.1h。根据文献中耐药菌的判断标准以及本次试验中所得到的MIC_(90)计算了不同给药剂量所需的给药频率,即给药方案。(4)海水由于富含钙离子或镁离子可影响环丙沙星的抑菌效果,然而钙离子或镁离子与环丙沙星的结合是可逆的,一旦用蒸馏水稀释后,环丙沙星将从结合状态逐渐转变为游离状态,又恢复杀菌活力。生理盐水对环丙沙星的抑菌作用也有不良影响。此外,生理盐水、半咸水和海水对氯霉素、四环素、土霉素和磺胺的抗菌作用也有不同程度的影响,这种影响与细菌的种类也有关系。呋喃唑酮的抗菌作用不受影响。(5)环丙沙星对A.hydrophila的MBC=2 * MIC,为0.0244μg/ml:对V. fluvialis的MBC=4 * MIC,为O.0244μg/ml。2倍MIC以上的氯霉素对A.hydrophila和V. fluvialis也具有杀菌作用,对A.hydrophila的MBC为O.78μg/ml;对V. fluvialis的MBC为1.56μg/ml,但即使16* MIC的氯霉素28 ℃处理28小时也不能将细菌完全杀灭,这些是对氯霉素抗菌作用新的认识。环丙沙星对静止期的A.hydrophila和V. fluvialis也有杀灭作用。lO倍MIC浓度的环丙沙星对A.hydrophila作用2小时,即可将细菌数量减少到10%以下,随着作用时间的延长,细菌数量进一步减少。说明环丙沙星对敏感的A.hydrophila具有强大而快速的杀灭作用。(6)环丙沙星和氯霉素都具有抗生素后效应,而且随着药物处理时间的延长,两种药物的抗生素后效应越明显。10 * MIC环丙沙星处理0.5h、1.0h、2.0h后,PAE在0.22-1.1小时之间;4 * MIC氯霉素处理O.5h、1.Oh、2.Oh后,PAE在0.95-2.3小时之间,两种药物均表现为处理时间越长,PAE越大。1/4 * MIC、4 * MIC、10 * MIC的环丙沙星处理2小时后,PAE在O.64-0.90小时之间,组间差异不明显。(7)采用细菌接合试验从只从l株A.hydrophila CJ26中检测到了可自身转移的耐药质粒。该耐药质粒被命名为pWH9601,它具有四环素和磺胺两种抗性标记。这是国内关于鱼类病原菌耐药质粒的首次报道。该质粒最快可在15分钟内完成传递;传递频率与温度有关,但在12 ℃时也可发生转移,温度在20 ℃以上时,转移频率明显升高。质粒pWH96101对四环素的抗性可被1 μg/ml浓度的四环素类药物所诱导。(8)采用两种方法试验了氟哌酸和利福平对耐药质粒的消除效果,获得了A.hydrophiia CJ26的R质粒消除株,但两种药物对该质粒的消除效果均不够理想。不过,本研究发展了一种用于该项研究更简便、更有效的试验方法,可为今后的研究提供便利。质粒消除株在与原质粒携带株的混合培养过程中,前者又可逐渐获得耐药质粒。说明质粒消除剂的作用只是暂时的,需要寻找消除作用更强的药物。(9)对12株A.hydrophila的质粒特征分析发现,这些菌株均只含有1-2种质粒或不含质粒。根据质粒的有无、大小及组成,质粒图谱分为4种类型。质粒特征与耐药谱之间存在一定的相关性,但其耐药性可能与质粒无关。质粒特征与菌株来源有关,而且发现多数A.hydrophila具有一共同的小质粒,这在流行病学上可能具有一定意义。
英文摘要: Following topics were included in this paper: Investigations and analysis of drug resistance of fish bacterial pathogens from China; resistance development of fish pathogens against common antimicrobials; relationship of serum drug concentration and drug resistance; detection and characterization of R plasmid; curing of R plasmid, relationship of plasmid profiles and pattern of antibiotic resistance; effect of seawater and brackish water on antimicrobial activity of aquacultural antibacterials; bactericidal activity and post-antibiotic effect of ciprofloxacin and chloramphenicol. The main results were given as follows: (1) 35 strains of fish bacterial pathogens were collected from region of China including A. hydrophila, Edwardsiella ictaluri, Yersinia ruckeri, Vibrio, Pseudomonas and others). Strains resistant to penicillin, cephalosporins and vancomycin had the highest proportion (85.7%-94.3%). All A. hydrophila tested were resistant to penicillin and cephalosporins, most of them were also resistant to vancomycin. Among 21 antimicrobials studied, norfloxacin, chloramphenicol and furazolidone showed the strongest antibacterial activity against all organism tested, only 5.7%, 11.4%, 11.4% strains were resistant to them, respectively. Two out of 26 strains of A. hydrophila were resistant to drugs mentioned above. The tetracycline and aminoglycoside antibiotics were also effective against fish pathogens, but the level of activity was lower than that of the previous three. There were 57.1% of all the fish pathogens and 73.1% of A. hydrophila resistant to sulfonamides (SA) and SA/TMP, but more strains showed resistance to SMZ than to SMZ/TMP. A major part of the strains carried more than one resistance marker, especially 3 con, non resistance markers. A. hydrophila were highly sensitive to ciprofloxacin and chloramphenicol with MIC_(50) of O.025μg/ml, O.25μg/ml, respectively, MIC_(90) of O.12μg/ml, 4.0μg/ml, respectively. MIC_(50) of tetracycline, oxytetracycline, furazolidone, streptomycin were low, 1.0μg/ml, 34.2 μg/ml, O.8μg/ml, 12.8g/ml, respectively, but their MIC_(90) were high, 8.0μg/ml, 12.8μg/ml, 6.4μg/ml, 50μg/ml, respectively. Sulfonamide were not sensitive to most tested strains, concentrations of 62.5μg/ml and 250μg/ml were needed to inhibit 50% and 90% of bacterial growth, respectively. (2) The frequency of spontaneous mutations of A. hydrophila to 8 * MIC ciprofloxacin, streptomycin was low (8.5 * 10~(-9), 4.2 * l0~(-8), respectively); The frequency of resistance of V. fluvialis to ciprofloxacin and streptomycin, at eight times the MIC, was also not high (3.4 * 10~(-8), 6.4 * 10~(-7), respectively). No mutants to chloramphenicol with A. hydrophila and V. fluvialis were detected in this experiment, the frequency was less than 1.1 * 10~(-9), 6.6 * 10~(-9) respectively. Mutants selected with ciprofloxacin and streptomycin were 8-16 times less susceptible than the corresponding parent was. During 11 serial transfer, the activities of the antibacterials against the test organisms typically decreased slowly in a stepwise fashion, and many MICs were 32-128 times higher at the end of the experiment than they were at the beginning, but mutants selected with ciprofloxacin, streptomycin and chloramphenicol showed no significant change in susceptibility to any other antibacterial except tetracycline. Cross-resistance was present between ciprofloxacin and tetracycline. The MICs of all antibacterials were similar for resistant mutants before and after 10 transfer in drug-free broth. (3) Studies on pharmacokinetics of chloramphenicol showed that chloramphenicol can be absorbed well with a half-life time of 11.9h and 11.lh f6r grass carp and crucian carp. Based on MIC of this drug against resistant strains reported in documents and on MIC_(90) obtained in this study, dosage regimens (including dosage and dose interval) were designed. (4) Antibacterial activity of ciprofloxacin can be weakened by seawater resulting from rich Ca~(2+) and Mg~(2+) in it, but this effect was not permanent, when it was diluted with distilled water, ciprofloxacin can be freed from integrated state and resume bactericidal activity. Antibacterial activities of tetracycline, oxytetracycline, sulfonamide, chloramphenicol, not furazolidone, were also weakened by seawater and/or brackish water. O. 85% NaCl can also exert negative effect on the bactericidal ability of ciprofloxacin. (5) Minimal bactericidal concentration (MBC) of ciprofloxacin against A. hydrophila was O. 02441μg/ml, two times of MIC (Minimal inhibitory concentration) and O. 02444μg/ml for V. fluvialis, 4 XMIC. Chloramphenicol at more than 2 times MIC and at 2 times MIC displayed bactericidal ability during a treatment of 18 hours at 28 ℃, too. MBC of chloramphenicol to A. hydrophila and V. fluvialis was O. 781μg/ml and 1.561μg/ml, respectively, but it cannot kill all of the bacteria even at the concentration of 16 * MIC for 28 hours at 28 ℃. This was new recognition for chloramphenicol. In addition, ciprofloxacin was founded to be active against non-dividing A. hydrophila and V. fluvialis. After 2 hour' s exposure to ciprofloxacin at concentration of 10 times MIC, A. hydrophila was killed by 90% and more bacteria were killed with increased time of exposure. These results suggested that ciprofloxacin possesses strong and rapid bactericidal activity. (6) It was showed by two different techniques that ciprofloxacin at concentration of 10 * MIC and chloramphenicol at concentration of 4 * MIC can cause post-antibiotic effect (PAE). There was a progressive increase in the PAE from O.22h to 1.1h for ciprofloxacin, O.95-2.3h for chloramphenicol as the duration of drugs exposure was increased from O.5 hours to 2 hours. In addition, ciprofloxacin can produce PAEs of O. 64h-0.90h against A. hydrophila at concentrations of 1/4 *MIC, 4 * MIC, 10 * MIC after 2 hours of exposure, but there was no significant difference among concentrations. (7) A drug resistance plasmid(R plasmid), pWH9601, was detected from one strain of A. hydrophila which carried a resistance marker of tetracycline and sulfonamide. This was the first report on R plasmid from fish bacterial pathogens in China. The R plasmid can transfer from A. hydrophila to Escherichia coli during conjugation of 15 minutes. Transfer of pWH9601 can occur at 12 ℃, and frequency of transfer increased with rising of temperature. Resistance levels of pWH9601 can be induced by 1μg/mi of tetracycline. (8) A. hydrophila CJ26 carrying R plasmid of pWh9601 was cured of their resistance markers by treatment with subinhibitory levels of norfloxacin and rifampicin by two techniques. A. hydrophila with the elimination of pWH9601 was obtained. But the rate of elimination of R plasmid with the two agents tested is not satisfied. But the technique developed in this study was simple and more efficient than classic methods, which will be helpful for the similar study in the future. When A. hydrophila CJ26 and A. hydrophila whose R plasmid was cured were cultured together at a ratio of 1:1 at 28 ℃ for 18 hours, organism resistant to TC and SA increased significantly, which indicated the efficacy of plasmid-curing agents by be transient if it can not be 100% efficient. So there is a need for more potent plasmid-curing agents. (9) Plasmid profile analysis showed that 12 representative strains of A. hydrophila carried no or 1-2 plasmids with different size. Based on the size and content of plasmids, four plasmid groups were identified. There was an association between plasmid profiles and pattern of resistance to antimicrobials, but it seemed to be not possible to relate the presence of a plasmid with antibiotic resistance in these strains. Most of A. hydrophila tested in this study, including a strain isolated in 1958, harboured a common small plasmid with about size of 2. 5kb. There was a strong association between plasmid profiles and sources of the strains of bacteria.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12520
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
我国鱼类病原菌耐药性、耐药质粒及几种药物抗菌作用的研究.李爱华[d].中国科学院水生生物研究所,1998.20-25
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