牛源大肠杆菌Ⅰ型整合子的检测及对氟苯尼考耐药机制的研究

发布时间：2018-05-03 05:42

本文选题：大肠杆菌 + 耐药性　；参考：《宁夏大学》2016年硕士论文

【摘要】：近年来,大肠杆菌耐药现象日益严重,且各种耐药机制通常会共同存在、彼此协同,导致其耐药的复杂性,其复杂的耐药性也加大了临床治疗的困难。因此研究宁夏地区牛源大肠杆菌整合子及外排泵机制对细菌耐药性产生的影响,不仅对临床合理用药具有指导意义,而且为控制细菌耐药性的形成和传播提供理论依据。本研究以临床分离鉴定的245株奶牛乳房炎大肠杆菌为试验菌,采用K-B法测定245株大肠杆菌对16种抗菌药的耐药性;采用PCR技术对菌株的Ⅰ型整合子进行检测。药敏试验结果显示,245株大肠杆菌对氨苄西林耐药率最高,达到51.48%,其次对链霉素、四环素、多西环素和氯霉素的耐药率也较高,分别为37.96%、35.51%、36.33%、33.06%,诺氟沙星耐药率最低,仅为7.16%。整合子检测结果显示,有35株大肠杆菌携带Ⅰ型整合子,检出率为14.29%;35株Ⅰ型整合子阳性菌株中32株菌扩增出7种类型基因盒,通过对基因盒进行测序分析,发现其以介导甲氧苄啶和氨基糖苷类耐药的dfrA和aadA为主,其中,基因盒排列dfrA17-aadA5和dfrA7较为流行。以35株整合子阳性菌株为试验菌,对其进行ESBLs基因的检测。结果显示,35株整合子阳性菌株中全部检测出ESBLs基因,其中blaTEM、blaCTX-M、blaOXA三种基因的检出率分别为88.57%(31/35)、91.43%(32/35)、14.29%(5/35),而未检出blaSHV基因。35株菌中有24株同时携带blaCTX-M和blaTEM基因,1株同时携带blaCTX-M和blaOXA基因,4株同时携带blaCTx-M、blaTEM及blaOXA三种ESBLs基因。采用PCR方法对245株大肠杆菌进行floR基因的检测；以floR基因阳性且耐氟苯尼考的菌株及大肠杆菌标准株(ATCC25922)为试验菌,运用HPLC技术测定氟苯尼考在细菌内的积聚量。结果floR基因检出率为7.35%；菌内氟苯尼考积聚量的测定结果显示：使用CCCP前后,大肠杆菌标准株(ATCC2592)对氟苯尼考的积聚量并无太大变化,而耐药菌株对氟苯尼考的积聚量受CCCP的影响较大。综上所述,本研究通过分析研究宁夏地区牛源大肠杆菌耐药性、Ⅰ型整合子及外排泵机制对细菌耐药性产生的影响,初步揭示了大肠杆菌在宁夏地区奶牛场的耐药情况和Ⅰ型整合子的流行情况以及氟苯尼考的外排泵耐药机制,为监控宁夏地区牛源大肠杆菌耐药性的流行趋势、控制耐药性的形成和传播以及耐药机制的研究提供理论依据。
[Abstract]:In recent years, the drug resistance of Escherichia coli is becoming more and more serious, and all kinds of drug resistance mechanisms usually exist together, together with each other, resulting in the complexity of drug resistance, and its complex drug resistance has also increased the difficulty of clinical treatment. Therefore, the study of the influence of the integron and the efflux pump mechanism of bovine Escherichia coli in Ningxia on the bacterial resistance is not only Clinical rational use of drugs is of guiding significance, and provides a theoretical basis for controlling the formation and transmission of bacterial resistance. In this study, 245 colitis colibacilli were identified by clinical isolation as experimental bacteria, and the resistance of 245 strains of Escherichia coli to 16 antibiotics was determined by K-B; and PCR technique was used to carry out the type I integron of the strain. The drug sensitivity test showed that the resistance rate of 245 strains of Escherichia coli to ampicillin was the highest, reaching 51.48%. Secondly, the resistance rate of streptomycin, tetracycline, doxycycline and chloramphenicol was also high, 37.96%, 35.51%, 36.33%, 33.06%, and norfloxacin was the lowest, only the 7.16%. integron test results showed that 35 Escherichia coli were carried. The detection rate of type I integron was 14.29%. 7 types of gene boxes were amplified by 32 strains of type I integron positive strains. By sequencing and analysis of the gene boxes, it was found that dfrA and aadA, which mediate the resistance of trimethoprim and aminoglycoside, were mainly dfrA and aadA, and the gene box row dfrA17-aadA5 and dfrA7 were more popular. 35 strains of integron Yang were integrated. The ESBLs gene was detected by the strain of the strain. The results showed that the ESBLs gene was detected in all 35 zygote positive strains, of which the detection rates of three genes of blaTEM, blaCTX-M and blaOXA were 88.57% (31/35), 91.43% (32/35), 14.29% (5/35), but 24 strains of.35 strains were not detected at the same time carrying blaCTX-M and blaTEM. Gene, 1 strains carried blaCTX-M and blaOXA gene simultaneously, 4 strains carried blaCTx-M, blaTEM and blaOXA three ESBLs genes simultaneously. The PCR method was used to detect the floR gene of 245 strains of Escherichia coli; the floR gene positive and the florfenicol resistant strain and the Escherichia coli standard strain (ATCC25922) were tested as the test bacteria, and the florfenicol was determined by HPLC technique. The accumulation of the floR gene was 7.35%. The results of the accumulation of fluorfenicol in the bacteria showed that the accumulation of florfenicol was not very significant before and after the use of CCCP, and the accumulation of florfenicol was greatly influenced by the drug resistant strain on florfenicol. To sum up, this study passed the study. The drug resistance of Escherichia coli in Ningxia, the effect of type I integron and the efflux pump mechanism on the bacterial resistance were analyzed. The drug resistance of Escherichia coli in Ningxia dairy farm and the epidemic situation of type I integron and the resistance mechanism of florfenicol out of the efflux pump were preliminarily revealed in order to monitor the bovine coliform rod in Ningxia area. It provides a theoretical basis for studying the epidemic trend of bacterial resistance, controlling the formation and spread of drug resistance and the mechanism of drug resistance.

【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61

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