本文選題：海水浴場大腸桿菌 + 耐藥基因��； 參考：《大連海洋大學(xué)》2017年碩士論文

【摘要】：海水環(huán)境已逐步成為抗性細(xì)菌和抗性基因的重要“儲存庫”,細(xì)菌耐藥性可通過水平基因轉(zhuǎn)移方式在海洋環(huán)境中進(jìn)行傳播擴(kuò)散。海水浴場是重要的娛樂場所,也是病原微生物傳播和聚集的地方,通過娛樂用水而傳播的微生物疾病已成為公眾關(guān)注的首要問題之一。海洋環(huán)境糞便污染指示物--大腸桿菌(Escherichia coli)可通過海水吞咽引發(fā)游泳人群致命性腸道疾病。為防止及減緩細(xì)菌耐藥性的播散,就必須全面了解細(xì)菌耐藥性的產(chǎn)生和播散機(jī)制。因此,本項(xiàng)目以大連付家莊海水浴場為研究區(qū)域,研究大腸桿菌在海水浴場中的分布特征及耐藥性狀況,分析整合子-基因盒系統(tǒng)介導(dǎo)的大腸桿菌多重耐藥性;利用質(zhì)粒接合轉(zhuǎn)移實(shí)驗(yàn)揭示質(zhì)粒介導(dǎo)大腸桿菌耐藥基因的水平傳遞。研究結(jié)果如下:1.采用濾膜法對大連付家莊海水浴場海水和沙灘樣品中的疑似大腸桿菌進(jìn)行初篩得到373株菌,經(jīng)生理生化鑒定在海水樣品中共獲得69株大腸桿菌,在沙灘樣品中獲得13株大腸桿菌,鑒定率分別為27.8%和10.4%。采用K-B紙片擴(kuò)散法對海水浴場82株分離大腸桿菌進(jìn)行10種抗生素的耐藥性檢測,藥敏結(jié)果顯示,海水中耐藥性大腸桿菌的檢出率為37.7%。對除氨曲南外的9種抗生素都存在耐藥性,對四環(huán)素(24.6%),甲氧芐啶(24.6%)和復(fù)方新諾明(23.2%)的耐藥率較高,對慶大霉素耐藥率為10.1%,對鏈霉素、環(huán)丙沙星、頭孢噻吩、氯霉素及左氧氟沙星的耐藥率較低(10%)。海水中大腸桿菌多重耐藥率達(dá)57.7%,大腸桿菌多重耐藥譜顯示其最多對8種抗生素耐藥。沙灘中濕砂分離菌只檢出對環(huán)丙沙星耐藥,耐藥率為37.5%,干砂分離菌只檢出對甲氧芐啶耐藥,耐藥率為40%,無多重耐藥現(xiàn)象。上述結(jié)果表明海水浴場海水中多重耐藥情況明顯。2.基于上述大腸桿菌耐藥表型結(jié)果,選取四環(huán)素類、β-內(nèi)酰胺類、磺胺類和喹諾酮類抗性基因,采用PCR方法對海水和沙灘中耐藥性大腸桿菌基因組和質(zhì)粒DNA進(jìn)行抗性基因檢測。結(jié)果顯示海水大腸桿菌基因組DNA中四環(huán)素類抗性基因的檢出率為88.2%,其中tetA基因檢出率為64.7%,tetB基因檢出率為41.2%;質(zhì)粒中四環(huán)素類抗性基因的檢出率為64.7%,tetA基因(29.4%)檢出率低于tetB基因(41.2%)。β-內(nèi)酰胺類抗性基因在基因組DNA和質(zhì)粒DNA中檢出率分別為80%和20%�；前奉惪剐曰蛟诨蚪M和質(zhì)粒DNA中檢出率分別為50%和66.7%。僅在質(zhì)粒DNA上檢測到喹諾酮類的qnrS型抗性基因,其余基因均未檢出。而沙灘大腸桿菌僅在質(zhì)粒DNA上檢測到磺胺類抗性基因sul1,其余抗性基因均未檢出。3.為研究整合子介導(dǎo)的海水大腸桿菌多重耐藥性,對15株多重耐藥菌進(jìn)行整合酶基因檢測,9株基因組DNAⅠ類整合酶陽性,在整合酶陽性分離株中有3株擴(kuò)增出Ⅰ類整合子可變區(qū);13株質(zhì)粒DNAⅠ類整合酶陽性,在整合酶陽性分離株中有3株擴(kuò)增出Ⅰ類整合子,多重耐藥菌株中I類整合子檢測率較高。對整合子可變區(qū)測序結(jié)果顯示得到8種不同的基因盒,分別是編碼磺胺類耐藥基因二氫葉酸還原酶基因(dfr16/A12/A17,dhfr12/17),編碼氨基糖苷類耐藥基因核苷轉(zhuǎn)移酶基因(aadA2/5),以及編碼林可酰胺核苷酸轉(zhuǎn)移酶基因(linF)。上述結(jié)果表明整合子可能介導(dǎo)了大腸桿菌的多重耐藥。4.從大腸桿菌的分離菌中選取質(zhì)粒上含有磺胺類耐藥基因的菌株,將其與受體菌E.coli C600進(jìn)行接合實(shí)驗(yàn),同時(shí)測定接合時(shí)間。結(jié)果表明含耐磺胺類耐藥基因質(zhì)粒的12株大腸桿菌中有6株接合成功,接合率為50%,發(fā)生接合轉(zhuǎn)移的時(shí)間為2~7h,接合子中檢測到磺胺類抗性基因,但耐藥表型未呈現(xiàn)。上述結(jié)果表明環(huán)境菌株耐藥性基因可通過質(zhì)粒接合在同種屬菌株中進(jìn)行傳遞。
[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.

【學(xué)位授予單位】：大連海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R126.4

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