本文關鍵詞： 水產養(yǎng)殖 氣單胞菌 多重耐藥性 耐藥基因 可移動元件 水平傳播　出處：《集美大學》2017年碩士論文　論文類型：學位論文

【摘要】：隨著抗菌藥物在水產養(yǎng)殖中的廣泛使用,水產養(yǎng)殖源細菌的耐藥性逐漸變強,耐藥多樣性也漸漸增加,進而促使動物細菌性疾病的控制效果變差。氣單胞菌是主要的水產動物致病菌之一,容易引起敗血癥、癤瘡和潰瘍等出血性疾病�？咕幬锏氖褂�,是誘導氣單胞菌耐藥性的主要因素。本文以水產養(yǎng)殖源氣單胞菌為研究對象,對其耐藥性、耐藥基因的分布特征及傳播機制進行研究分析。本研究中,49株氣單胞菌對8類17種抗菌藥物的藥敏結果顯示,對AMX的耐藥率高達100%,對SMZ、TMP、SXT和RIF的耐藥率介于60%~90%之間,而對CTX、NEO、DOX以及喹諾酮類的ENR、NOR和OFL的耐藥率均低于15%;在所有菌株中,41株菌株對3類及以上的抗菌藥物具有抗性,24株菌株對9種及以上抗菌藥物具有抗性。最小抑菌濃度(MIC)檢測結果顯示,TMP和SMZ的MIC50值最高(2048μg/mL),其余按MIC50值高低依次為STR(64μg/m L)、CHL(32μg/mL)、NEO(16μg/m L)、TET(16μg/mL)、FFL(16μg/mL)、DOX(4μg/mL)、ENR(1μg/mL)。最小殺菌濃度(MBC)檢測結果顯示,TET、TMP、SMZ ENR和NEO的MBC50與MIC50相等,CHL、STR和DOX的MBC50(64、256、和8μg/m L)則高于MIC50。抑菌圈直徑與MIC值的相關性研究發(fā)現(xiàn),兩者之間均呈對數(shù)或指數(shù)負相關。25種耐藥基因的PCR檢測結果顯示,基因組中,blaTEM的檢出率最高(83.7%);其次是sul1和aadA,檢出率分別是69.4%和61.2%;再者是arr2/3、aphA1、tetC、sul2、dfrA5和floR,檢出率均高于40%。質粒中,sul1的檢出率最高,為51%;其次是aphA1與aadA,分別為40.8%和38.8%;blaTEM、tetB、qnrB、qnrS、dfrA7、cat1和cmlA均未檢出,其余耐藥基因檢出率均在35%以下。5類可移動元件的PCR檢測結果顯示,在基因組中,intI1檢出率最高(65.3%);其次是IS26、ISCR2和tnpU,分別是63.3%、57.1%和51%;其余均低于50%。質粒中,intI1檢出率依舊最高,為57.1%;其次是IS26、ISCR2和merA,檢出率介于20%~35%之間;其余均低于15%。在1型整合子的可變區(qū)中,共發(fā)現(xiàn)13種基因盒排列,dfrA12-orfF-aadA2比例最高。ISCR2的可變區(qū)主要為兩種:ISCR2+glmM+sul2(10株)和ISCR2-like+strB+strA+sul2(2株)。本研究中共獲得16株大腸桿菌轉化子、21株氣單胞菌轉化子和4株大腸桿菌接合子。轉化實驗結果顯示,耐藥基因aadA、aphA1、sul2、tetA和floR較易通過轉化的方式傳遞給大腸桿菌,其中tetA基因陽性轉化子均表達四環(huán)素抗性;維氏氣單胞菌轉化子新增表型以RIF抗性為多,嗜水氣單胞菌轉化子多新增STR、KAN和CHL抗性,結果表明這4種藥物抗性較易通過轉化在種內進行傳播。接合實驗結果顯示,只有嗜水氣單胞菌成功與大腸桿菌接合,基因strA-B和floR較易通過接合在種間進行傳播,且基因型陽性接合子均表達出對應抗性。此外,所有的轉化子與接合子中,ISCR2的檢出率均是最高的。
[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.
【學位授予單位】：集美大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S941.4

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