水产养殖源气单胞菌耐药基因的分布特征和传播机制

发布时间：2018-01-21 04:37

本文关键词： 水产养殖气单胞菌多重耐药性耐药基因可移动元件水平传播　出处：《集美大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着抗菌药物在水产养殖中的广泛使用,水产养殖源细菌的耐药性逐渐变强,耐药多样性也渐渐增加,进而促使动物细菌性疾病的控制效果变差。气单胞菌是主要的水产动物致病菌之一,容易引起败血症、疖疮和溃疡等出血性疾病。抗菌药物的使用,是诱导气单胞菌耐药性的主要因素。本文以水产养殖源气单胞菌为研究对象,对其耐药性、耐药基因的分布特征及传播机制进行研究分析。本研究中,49株气单胞菌对8类17种抗菌药物的药敏结果显示,对AMX的耐药率高达100%,对SMZ、TMP、SXT和RIF的耐药率介于60%~90%之间,而对CTX、NEO、DOX以及喹诺酮类的ENR、NOR和OFL的耐药率均低于15%;在所有菌株中,41株菌株对3类及以上的抗菌药物具有抗性,24株菌株对9种及以上抗菌药物具有抗性。最小抑菌浓度(MIC)检测结果显示,TMP和SMZ的MIC50值最高(2048μg/mL),其余按MIC50值高低依次为STR(64μg/m L)、CHL(32μg/mL)、NEO(16μg/m L)、TET(16μg/mL)、FFL(16μg/mL)、DOX(4μg/mL)、ENR(1μg/mL)。最小杀菌浓度(MBC)检测结果显示,TET、TMP、SMZ ENR和NEO的MBC50与MIC50相等,CHL、STR和DOX的MBC50(64、256、和8μg/m L)则高于MIC50。抑菌圈直径与MIC值的相关性研究发现,两者之间均呈对数或指数负相关。25种耐药基因的PCR检测结果显示,基因组中,blaTEM的检出率最高(83.7%);其次是sul1和aadA,检出率分别是69.4%和61.2%;再者是arr2/3、aphA1、tetC、sul2、dfrA5和floR,检出率均高于40%。质粒中,sul1的检出率最高,为51%;其次是aphA1与aadA,分别为40.8%和38.8%;blaTEM、tetB、qnrB、qnrS、dfrA7、cat1和cmlA均未检出,其余耐药基因检出率均在35%以下。5类可移动元件的PCR检测结果显示,在基因组中,intI1检出率最高(65.3%);其次是IS26、ISCR2和tnpU,分别是63.3%、57.1%和51%;其余均低于50%。质粒中,intI1检出率依旧最高,为57.1%;其次是IS26、ISCR2和merA,检出率介于20%~35%之间;其余均低于15%。在1型整合子的可变区中,共发现13种基因盒排列,dfrA12-orfF-aadA2比例最高。ISCR2的可变区主要为两种:ISCR2+glmM+sul2(10株)和ISCR2-like+strB+strA+sul2(2株)。本研究中共获得16株大肠杆菌转化子、21株气单胞菌转化子和4株大肠杆菌接合子。转化实验结果显示,耐药基因aadA、aphA1、sul2、tetA和floR较易通过转化的方式传递给大肠杆菌,其中tetA基因阳性转化子均表达四环素抗性;维氏气单胞菌转化子新增表型以RIF抗性为多,嗜水气单胞菌转化子多新增STR、KAN和CHL抗性,结果表明这4种药物抗性较易通过转化在种内进行传播。接合实验结果显示,只有嗜水气单胞菌成功与大肠杆菌接合,基因strA-B和floR较易通过接合在种间进行传播,且基因型阳性接合子均表达出对应抗性。此外,所有的转化子与接合子中,ISCR2的检出率均是最高的。
[Abstract]:With the widespread use of antimicrobial agents in aquaculture, the antimicrobial resistance of aquaculture bacteria has gradually become stronger, and the diversity of drug resistance has gradually increased. Aeromonas is one of the main pathogenic bacteria in aquatic animals, which can easily cause septicemia, furuncle and ulcers, and the use of antimicrobial agents. Aeromonas aeruginosa is the main factor to induce antimicrobial resistance. In this study, the drug resistance of Aeromonas spp., the distribution characteristics of drug resistance genes and the transmission mechanism were analyzed. The susceptibility of 49 strains of Aeromonas to 8 classes of 17 antimicrobial agents showed that the drug resistance rate to AMX was as high as 100, and to SMZTMP. The drug resistance rates of SXT and RIF were between 60% and 90%, but the resistance rates to CTX neo DOX and quinolones ENRNOR and OFL were lower than 15%. Among all the strains, 41 strains were resistant to 3 or more kinds of antimicrobial agents. 24 strains were resistant to 9 or more antimicrobial agents. The MIC50 value of TMP and SMZ was the highest (2048 渭 g / mL), and the rest was STR(64 渭 g / mL (CHL(32 渭 g / mL). NEO(16 渭 g / mL, TET(16 渭 g / mL, FFL16 渭 g / mL DOX4 渭 g / mL. The results of ENR(1 渭 g / mL 路minimum bactericidal concentration (MBC) showed that the MBC50 of TMP ENR and NEO were equal to MIC50. The results showed that the MBC50 / mL of STR and DOX was higher than that of MIC50. The correlation between the diameter of inhibition circle and MIC value was found. There was a logarithmic or exponential negative correlation between the two genes. The results of PCR analysis showed that the highest detectable rate of blaTEM was 83.7% in the genome. The detection rate of sul1 and aada was 69.4% and 61.2 respectively. Furthermore, the detection rates of arr2 / 3aphA1 tetCnsul2dfrA5 and flor were higher than those of 40.The detection rate of sul1 was the highest (51%) in plasmids. AphA1 and aada were 40.8% and 38.8 respectively. Neither the cmlA nor the cmlA were detected. The detection rate of other drug-resistant genes was below 35%. The results of PCR analysis showed that the highest detectable rate of I1 was 65.3% in the genome. The second was IS26, ISCR2 and TnpU, which were 63.3% and 57.1%, and 51.1%, respectively. The others were lower than 50. The detection rate of intI1 in plasmid was still the highest, 57.1; The second was IS26, ISCR2 and merA, the detection rate was between 20% and 35%. In the variable region of type 1 integron, 13 gene-boxes were found. The variable region of the highest proportion of dfrA12-orfF-aadA2. ISCR2 is mainly two species of glmM sul2(10 strain: ISCR2. And ISCR2-like strB strA sul2(2. In this study, 16 Escherichia coli transformants were obtained. 21 strains of Aeromonas mutans and 4 strains of Escherichia coli conjugate. TetA and floR were easily transferred to Escherichia coli by transformation, in which tetA gene positive transformants expressed tetracycline resistance. The new phenotypes of the transformants of Aeromonas Vickers were more resistant to RIF, and the transformants of Aeromonas hydrophila increased the resistance to STRN KAN and CHL. The results showed that the four drug resistance were easily transmitted by transformation. The results of conjugation test showed that only Aeromonas hydrophila successfully conjugated with Escherichia coli. The gene strA-B and floR were more easily transmitted between species by conjugation, and the genotypic positive conjugates expressed the corresponding resistance. In addition, all the transformants and conjugates expressed the corresponding resistance. The detection rate of ISCR2 was the highest.
【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S941.4

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