大连付家庄海水浴场中大肠杆菌耐药性及质粒介导的耐药性传递研究

发布时间：2018-05-02 10:33

本文选题：海水浴场大肠杆菌 + 耐药基因　；参考：《大连海洋大学》2017年硕士论文

【摘要】：海水环境已逐步成为抗性细菌和抗性基因的重要“储存库”,细菌耐药性可通过水平基因转移方式在海洋环境中进行传播扩散。海水浴场是重要的娱乐场所,也是病原微生物传播和聚集的地方,通过娱乐用水而传播的微生物疾病已成为公众关注的首要问题之一。海洋环境粪便污染指示物--大肠杆菌(Escherichia coli)可通过海水吞咽引发游泳人群致命性肠道疾病。为防止及减缓细菌耐药性的播散,就必须全面了解细菌耐药性的产生和播散机制。因此,本项目以大连付家庄海水浴场为研究区域,研究大肠杆菌在海水浴场中的分布特征及耐药性状况,分析整合子-基因盒系统介导的大肠杆菌多重耐药性;利用质粒接合转移实验揭示质粒介导大肠杆菌耐药基因的水平传递。研究结果如下:1.采用滤膜法对大连付家庄海水浴场海水和沙滩样品中的疑似大肠杆菌进行初筛得到373株菌,经生理生化鉴定在海水样品中共获得69株大肠杆菌,在沙滩样品中获得13株大肠杆菌,鉴定率分别为27.8%和10.4%。采用K-B纸片扩散法对海水浴场82株分离大肠杆菌进行10种抗生素的耐药性检测,药敏结果显示,海水中耐药性大肠杆菌的检出率为37.7%。对除氨曲南外的9种抗生素都存在耐药性,对四环素(24.6%),甲氧苄啶(24.6%)和复方新诺明(23.2%)的耐药率较高,对庆大霉素耐药率为10.1%,对链霉素、环丙沙星、头孢噻吩、氯霉素及左氧氟沙星的耐药率较低(10%)。海水中大肠杆菌多重耐药率达57.7%,大肠杆菌多重耐药谱显示其最多对8种抗生素耐药。沙滩中湿砂分离菌只检出对环丙沙星耐药,耐药率为37.5%,干砂分离菌只检出对甲氧苄啶耐药,耐药率为40%,无多重耐药现象。上述结果表明海水浴场海水中多重耐药情况明显。2.基于上述大肠杆菌耐药表型结果,选取四环素类、β-内酰胺类、磺胺类和喹诺酮类抗性基因,采用PCR方法对海水和沙滩中耐药性大肠杆菌基因组和质粒DNA进行抗性基因检测。结果显示海水大肠杆菌基因组DNA中四环素类抗性基因的检出率为88.2%,其中tetA基因检出率为64.7%,tetB基因检出率为41.2%;质粒中四环素类抗性基因的检出率为64.7%,tetA基因(29.4%)检出率低于tetB基因(41.2%)。β-内酰胺类抗性基因在基因组DNA和质粒DNA中检出率分别为80%和20%。磺胺类抗性基因在基因组和质粒DNA中检出率分别为50%和66.7%。仅在质粒DNA上检测到喹诺酮类的qnrS型抗性基因,其余基因均未检出。而沙滩大肠杆菌仅在质粒DNA上检测到磺胺类抗性基因sul1,其余抗性基因均未检出。3.为研究整合子介导的海水大肠杆菌多重耐药性,对15株多重耐药菌进行整合酶基因检测,9株基因组DNAⅠ类整合酶阳性,在整合酶阳性分离株中有3株扩增出Ⅰ类整合子可变区;13株质粒DNAⅠ类整合酶阳性,在整合酶阳性分离株中有3株扩增出Ⅰ类整合子,多重耐药菌株中I类整合子检测率较高。对整合子可变区测序结果显示得到8种不同的基因盒,分别是编码磺胺类耐药基因二氢叶酸还原酶基因(dfr16/A12/A17,dhfr12/17),编码氨基糖苷类耐药基因核苷转移酶基因(aadA2/5),以及编码林可酰胺核苷酸转移酶基因(linF)。上述结果表明整合子可能介导了大肠杆菌的多重耐药。4.从大肠杆菌的分离菌中选取质粒上含有磺胺类耐药基因的菌株,将其与受体菌E.coli C600进行接合实验,同时测定接合时间。结果表明含耐磺胺类耐药基因质粒的12株大肠杆菌中有6株接合成功,接合率为50%,发生接合转移的时间为2~7h,接合子中检测到磺胺类抗性基因,但耐药表型未呈现。上述结果表明环境菌株耐药性基因可通过质粒接合在同种属菌株中进行传递。
[Abstract]:The marine environment has gradually become an important "storage pool" of resistant bacteria and resistance genes. Bacterial resistance can spread through the horizontal gene transfer mode in the marine environment. The sea baths are important places of entertainment, and also the spread and aggregation of pathogenic microorganisms. One of the first issues of public concern, the marine environmental pollution indicator, E. coli (Escherichia coli), can cause fatal intestinal disease in swimmers through swallowing. In order to prevent and slow down the spread of bacterial resistance, a comprehensive understanding of the production and dissemination mechanism of bacterial resistance must be fully understood. Therefore, the project is paid in Dalian. The distribution of Escherichia coli in the bathing field and the drug resistance of E. coli in the Zhuang Haishui bathing field were studied. The multiple resistance of Escherichia coli mediated by the integron gene box system was analyzed, and plasmid conjugation transfer experiment was used to reveal the horizontal transfer of plasmid mediated Escherichia coli resistance gene. The results were as follows: 1. the filter membrane method was used in the study. 373 strains of Escherichia coli were screened in the seawater and beach samples of the bathing beach, and 69 Escherichia coli were obtained by physiological and biochemical identification in sea water samples. 13 strains of Escherichia coli were obtained in the beach samples. The identification rate was 27.8% and 10.4%. was used to isolate 82 strains of Escherichia coli in the sea baths by K-B paper diffusion method. The drug sensitivity test of 10 antibiotics showed that the detection rate of resistant Escherichia coli in seawater was 37.7%. against 9 kinds of antibiotics, and the resistance rate to tetracycline (24.6%), trimethoprim (24.6%) and compound novamoxin (23.2%) was higher, and the resistance rate to gentamicin was 10.1%, and streptomycin and cyclopropane were used for streptomycin. The resistance rate of ciprofloxacin, cefthiophene, chloramphenicol and levofloxacin was lower (10%). The multidrug resistance rate of Escherichia coli in seawater was 57.7%. The multiple resistance spectrum of Escherichia coli showed that the most resistant to 8 antibiotics was found in the sandy beach. The resistance rate of sands isolated in the sand was only 37.5%, and the dry sand isolates were only resistant to trimethoprim. The resistance rate was 40%, and there was no multidrug resistance. The results showed that the multidrug resistance in sea water was obviously.2. based on the results of the Escherichia coli resistance phenotype. The resistance genes of tetracycline, beta lactam, sulfonamides and quinolones were selected, and the PCR method was used for the drug resistant Escherichia coli genome and plasmid DNA in sea water and sand. Resistance gene detection. The results showed that the detection rate of tetracycline resistant genes in the genomic DNA of marine Escherichia coli was 88.2%, of which the detection rate of tetA gene was 64.7%, the detection rate of tetB gene was 41.2%, the detection rate of the tetracycline resistant gene in the plasmid was 64.7%, and the detection rate of the tetA gene (29.4%) was lower than that of the tetB gene (41.2%). The detection rates of sex genes in genomic DNA and plasmid DNA were 80% and 20%. sulfonamides resistance genes in genome and plasmid DNA respectively, 50% and 66.7%. were detected only on plasmid DNA, and the other genes were not detected. The other genes were not detected. And the Escherichia coli in sandy beach only detected the sulfonamides resistant base on plasmid DNA. For sul1,.3. was not detected by the other resistance genes as the integron mediated multiple resistance of marine Escherichia coli. 15 strains of multiple resistant strains were detected by integrase gene, and 9 strains of DNA I integrase positive. 3 strains of integron variable region were amplified in the integrase positive isolates, and 13 plasmids DNA I integrase was positive. Among the integrase positive isolates, 3 strains of integron were amplified, and the detection rate of class I integrons in multiple resistant strains was higher. 8 different gene boxes were found for the integron variable region sequencing results, which encode the sulfonamide resistant gene dihydrofolate reductase gene (dfr16 /A12/A17, dhfr12/17), and encode aminoglycoside resistant groups. The results show that the integron may mediate the multidrug resistant.4. of Escherichia coli from the isolates of Escherichia coli to select the strains containing the sulfonamides resistance gene from the Escherichia coli isolates, and to join the receptor bacteria E.coli C600 for the conjugation experiment, and the results show that the integron may mediate the multidrug resistant.4. of Escherichia coli. The results showed that 6 of the 12 strains of Escherichia coli containing the resistant gene plasmid containing sulfonamides were engaged successfully, the conjugation rate was 50%, the time of joint transfer was 2~7h, the sulfonamide resistance gene was detected in the zygote, but the resistance phenotype was not presented. Transmission is carried out in the genus strains.

【学位授予单位】：大连海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R126.4

【参考文献】