畜禽源弯曲菌耐药分子机制研究

发布时间：2018-05-18 16:07

本文选题：弯曲菌 + 耐药性　；参考：《中国农业大学》2017年博士论文

【摘要】：以空肠弯曲菌(C.jejuni)和结肠弯曲菌(C.coli)为代表的弯曲菌属细菌,是重要的食源性病原菌,可通过食物链传播给人,引起人的腹泻等多种疾病。随着抗生素在畜牧养殖业和人医临床的大量使用导致了多药耐药和高水平耐药弯曲菌的出现和播散,该类弯曲菌一旦传播给人,将会给临床治疗弯曲菌病的用药选择带来极大困难。目前在我国,畜禽源弯曲菌对多种抗生素的耐药率明显高于其他国家,而已知的耐药机制不足以解释我国弯曲菌耐药率且耐药水平普遍偏高的现象。本研究在调研我国部分地区畜禽源弯曲菌耐药现状的基础上,深入研究了弯曲菌的新型耐药机制。弯曲菌CmeABC外排泵是其固有的耐药机制,并对其生理有着极其重要的作用。本研究在具有对环丙沙星极高水平耐药表型的弯曲菌中发现CmeABC变异体,与C.jejuni参考菌株NCTC 11168相比,该CmeABC变异体中CmeB氨基酸同源性仅为81%,而CmeA和CmeC均98%。对其功能进行深入研究证实:CmeABC变异体可导致弯曲菌对多种抗菌药物耐药性增加,我们将该变异体命名为功能增强型外排泵(RE-CmeABC);RE-CmeABC可以拓宽弯曲菌对氟喹诺酮类药物耐药的突变选择窗;在抗性压力下大幅度地增加氟喹诺酮类耐药突变出现的频率;与C257T突变协同作用时导致弯曲菌对环丙沙星极高水平耐药。细菌体内蓄积实验显示,RE-CmeABC对药物的外排功能显著强于野生型外排泵。通过蛋白结构预测和分子对接模拟,发现CmeB和RE-CmeB与药物结合的氨基酸位点不同,RE-CmeB更倾向于用突变的氨基酸结合药物,推测RE-CmeABC功能的增强与这些变化的氨基酸有关。同时,我们发现在2012-2014年分离的弯曲菌中,RE-cmeABC基因阳性率呈逐年增加趋势,并且在C.jejuni中检出率显著高于C.coli。PFGE实验证实RE-cmeABC基因阳性菌株能够发生克隆传播,自然转化实验确证RE-cmeABC能够发生水平传播,这在一定程度上解释了 RE-cmeABC携带株增多的原因。该研究首次揭示了一种外排泵变异体可以增加细菌对多种药物的耐药性,有助于细菌在多种药物选择压力下生存。近年来的研究显示我国弯曲菌对庆大霉素的耐药率有所上升,但是其流行的分子机制并未深入研究。2014年从我国山东、河南、广东、宁夏和上海五个地区采集的607株畜禽源弯曲菌中,C.jejuni和C.coli对庆大霉素的耐药率分别为15.6%(25/160)和79.9%(357/447)。对庆大霉素耐药株进行耐药基因PCR检测分析,发现两种庆大霉素耐药基因aph(2")-If和aacA/aphD且aph(2")-If基因的检出率显著高于aacA/aphD:C.jejuni中,aph(2")-If基因的检出率为10%(16/160),aacA/aphD基因为 2.5%(4/160),而C.coli中aph(2")-If和aacA/aphD基因的检出率分别为57.7%(258/447)和15.2%(68/447)。自然转化实验证实aph(2")-If基因能够转移至敏感菌株NCTC 11168,对转化子进行全基因组测序及分析发现在染色体基因组保守基因cj0299和panB间存在一个包括aph(2")-If基因在内的新型耐药基因岛,包含aph(2")-If,aac,aadA,aph2,aad9,aphA3,aphA7,cat等多个耐药基因,能够同时介导庆大霉素、卡那霉素、新霉素、阿米卡星、氯霉素等抗生素耐药。功能性试验证明aph(2")-If基因可导致弯曲菌同时对庆大霉素和卡那霉素高水平耐药,本研究发现该基因可定位于弯曲菌染色体。此外,PFGE实验结果显示aph(2")-If基因既能发生克隆传播也能发生水平传播。·磷霉素可有效治疗弯曲菌引起的肠炎,被视为治疗由弯曲菌引起相关疾病的备选药物,而本研究发现一株猪源C.coli具有磷霉素高水平耐表型(512μg/mL)。该菌株的磷霉素高水平耐药表型能够通过自然转化方式转移至敏感菌株C.jejuni NCTC 11168;转化子全基因组测序分析发现:在染色体cadF和CCO1582基因之间发现一段大约9 Kb包含11个ORF的外源DNA插入片段,其中一个ORF编码136个氨基酸,与磷霉素耐药蛋白FosA、FosB、FosX分别呈现26.9%、34.2%、63.9%的氨基酸同源性;随后,对此ORF进行功能性验证,发现该ORF可介导弯曲菌和大肠杆菌对磷霉素的高水平耐药,将该新型磷霉素耐药基因亚型命名为fosX~(CC);并证实在实验条件下,fosX~(CC)基因可从C.coli转移至C.jejuni。综上所述,针对我国畜禽源弯曲菌多重耐药情况严重的现象,对其耐药机制进行深入研究,首次发现介导弯曲菌多重耐药且参与弯曲菌对环丙沙星极高水平耐药的外排泵变异体RE-CmeABC;首次在弯曲菌染色体上发现介导庆大霉素和卡那霉素耐药的耐药基因aph(2")-If及新型多重耐药基因岛;此外,在弯曲菌中发现磷霉素耐药基因新亚型fosX~(CC)。上述研究结果在阐明弯曲菌耐药新机制的同时也为弯曲菌耐药性相关研究提供了新思路。
[Abstract]:Campylobacter of Campylobacter jejuni (C.jejuni) and Campylobacter colocamurus (C.coli), an important foodborne pathogen, can be transmitted to humans through the food chain, causing human diarrhoea and many other diseases. With the extensive use of antibiotics in animal husbandry and human medical clinics, multidrug resistance and high level resistant Campylobacter are produced. Now and disseminating, once the Campylobacter is transmitted to people, it will bring great difficulties to the treatment of clinical treatment of Campylobacter disease. At present, the resistance rate of Campylobacter to many kinds of antibiotics is obviously higher than that of other countries in our country, and the known resistance mechanism is not enough to explain the resistance rate of Campylobacter in our country and the high level of drug resistance is generally high. Elephant. On the basis of investigating the current situation of resistance to Campylobacter of livestock and poultry in some parts of China, this study has studied the new mechanism of resistance to Campylobacter. The CmeABC outer row pump of Campylobacter is its inherent mechanism of resistance and plays an important role in its physiology. This study has been found in the Campylobacter with a high level resistant phenotype to ciprofloxacin. The present CmeABC variant, compared with the C.jejuni reference strain NCTC 11168, is only 81% of the CmeB amino acid homology in the CmeABC variant, while CmeA and CmeC 98%. have further confirmed that the CmeABC variant can lead to the increasing resistance of Campylobacter to a variety of antimicrobial agents. We named the variant as a function enhanced outer row pump (RE-C). MeABC); RE-CmeABC can widen the mutation selection window for fluoroquinolone drug resistance of Campylobacter; increase the frequency of fluoroquinolone resistant mutations at the resistance pressure; lead to the high level of Campylobacter against ciprofloxacin when synergistic with C257T mutations. The accumulation of microbacteria shows that RE-CmeABC is out of the drug. By protein structure prediction and molecular docking simulation, it is found that the amino acid loci of CmeB and RE-CmeB are different from those of drugs. RE-CmeB is more inclined to use mutated amino acid binding drugs. It is suggested that the enhancement of RE-CmeABC function is related to these changes of amino acids. At the same time, we found that in 2012-2014 years In the isolated Campylobacter, the positive rate of RE-cmeABC gene increased year by year, and the detection rate in C.jejuni was significantly higher than that of the C.coli.PFGE test. The positive strain of RE-cmeABC gene could be cloned, and the natural transformation test confirmed that RE-cmeABC could be transmitted horizontally, which explained the increase of RE-cmeABC carrying strain to a certain extent. The study has revealed for the first time that a variant of an outer row pump can increase the resistance of bacteria to a variety of drugs and help the bacteria survive under the pressure of a variety of drugs. Recent studies have shown that the resistance rate of Campylobacter to gentamicin has increased in recent years, but its popular molecular mechanism has not been studied in.2014 from me. In Shandong, Henan, Guangdong, Ningxia and Shanghai, the resistance rates of C.jejuni and C.coli to gentamicin were 15.6% (25/160) and 79.9% (357/447) respectively. The resistance genes of gentamicin resistant strains were detected and analyzed, and two kinds of gentamycin resistance genes APH (2 ") -If and aacA/aphD and APH (2") were found. The detection rate of If gene was significantly higher than that in aacA/aphD:C.jejuni. The detection rate of APH (2 ") -If gene was 10% (16/160), aacA/aphD gene was 2.5% (4/160), while the detection rates of APH (2) -If and aacA/aphD genes in C.coli were 57.7% (258/447) and 15.2%, respectively. The whole genome sequencing and analysis of the transformants showed that there was a new drug resistant gene island including APH (2 ") -If gene between cj0299 and panB, including APH (2") -If, AAC, aadA, aph2, aad9, aphA3, aphA7, and other multidrug resistant genes, which could simultaneously mediate gentamicin, kanamycin, neomycin, Amie APH (2 ") -If gene could lead to high level resistance to gentamicin and kanamycin. This study found that the gene could be located in the Campylobacter chromosome. In addition, the PFGE experiment showed that the APH (2") -If gene could both be cloned and spread horizontally. Hormone can effectively treat enteritis caused by Campylobacter, which is considered as a candidate for the treatment of related diseases caused by Campylobacter, and this study found that a pig source C.coli has a high phenotypic resistance of phosphomycin (512 mu g/mL). The high level resistance phenotype of the strain can be transferred to the sensitive strain C.jejuni NCTC 11168 by natural transformation. The whole genome sequencing analysis of the transformants found that a segment of exogenous DNA inserted between the chromosome cadF and the CCO1582 gene was found to be about 9 Kb containing 11 ORF, one of which encodes 136 amino acids, and the amino acid homology of 26.9%, 34.2%, 63.9% of the phosphamycin resistant protein FosA, FosB, and FosX, respectively. Subsequently, the ORF was functional test. It was found that the ORF could mediate the high level resistance of Campylobacter and Escherichia coli to fosfomycin, and named the new phosphamycin resistant gene subtype fosX~ (CC). Under the actual experimental conditions, the fosX~ (CC) gene could be transferred from C.coli to C.jejuni., and it was resistant to the multidrug resistance of Campylobacter in China. The drug mechanism was studied for the first time. For the first time, the multidrug resistance of Campylobacter was found to be involved in the mutant RE-CmeABC with high level resistance to ciprofloxacin, and the drug resistant gene APH (2 ") -If and a new multidrug resistant gene Island, which were resistant to gentamicin and kanamycin, were found on the chromosomes of Campylobacter for the first time. The new subtype fosX~ (CC) of the phosphamycin resistance gene was found. The results mentioned above provide a new idea for the new mechanism of resistance to Campylobacter and the study of resistance related to Campylobacter.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.61

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