發(fā)布時間：2018-05-18 16:07

  本文選題：彎曲菌 + 耐藥性�。� 參考：《中國農(nóng)業(yè)大學》2017年博士論文

【摘要】：以空腸彎曲菌(C.jejuni)和結腸彎曲菌(C.coli)為代表的彎曲菌屬細菌,是重要的食源性病原菌,可通過食物鏈傳播給人,引起人的腹瀉等多種疾病。隨著抗生素在畜牧養(yǎng)殖業(yè)和人醫(yī)臨床的大量使用導致了多藥耐藥和高水平耐藥彎曲菌的出現(xiàn)和播散,該類彎曲菌一旦傳播給人,將會給臨床治療彎曲菌病的用藥選擇帶來極大困難。目前在我國,畜禽源彎曲菌對多種抗生素的耐藥率明顯高于其他國家,而已知的耐藥機制不足以解釋我國彎曲菌耐藥率且耐藥水平普遍偏高的現(xiàn)象。本研究在調研我國部分地區(qū)畜禽源彎曲菌耐藥現(xiàn)狀的基礎上,深入研究了彎曲菌的新型耐藥機制。彎曲菌CmeABC外排泵是其固有的耐藥機制,并對其生理有著極其重要的作用。本研究在具有對環(huán)丙沙星極高水平耐藥表型的彎曲菌中發(fā)現(xiàn)CmeABC變異體,與C.jejuni參考菌株NCTC 11168相比,該CmeABC變異體中CmeB氨基酸同源性僅為81%,而CmeA和CmeC均98%。對其功能進行深入研究證實:CmeABC變異體可導致彎曲菌對多種抗菌藥物耐藥性增加,我們將該變異體命名為功能增強型外排泵(RE-CmeABC);RE-CmeABC可以拓寬彎曲菌對氟喹諾酮類藥物耐藥的突變選擇窗;在抗性壓力下大幅度地增加氟喹諾酮類耐藥突變出現(xiàn)的頻率;與C257T突變協(xié)同作用時導致彎曲菌對環(huán)丙沙星極高水平耐藥。細菌體內蓄積實驗顯示,RE-CmeABC對藥物的外排功能顯著強于野生型外排泵。通過蛋白結構預測和分子對接模擬,發(fā)現(xiàn)CmeB和RE-CmeB與藥物結合的氨基酸位點不同,RE-CmeB更傾向于用突變的氨基酸結合藥物,推測RE-CmeABC功能的增強與這些變化的氨基酸有關。同時,我們發(fā)現(xiàn)在2012-2014年分離的彎曲菌中,RE-cmeABC基因陽性率呈逐年增加趨勢,并且在C.jejuni中檢出率顯著高于C.coli。PFGE實驗證實RE-cmeABC基因陽性菌株能夠發(fā)生克隆傳播,自然轉化實驗確證RE-cmeABC能夠發(fā)生水平傳播,這在一定程度上解釋了 RE-cmeABC攜帶株增多的原因。該研究首次揭示了一種外排泵變異體可以增加細菌對多種藥物的耐藥性,有助于細菌在多種藥物選擇壓力下生存。近年來的研究顯示我國彎曲菌對慶大霉素的耐藥率有所上升,但是其流行的分子機制并未深入研究。2014年從我國山東、河南、廣東、寧夏和上海五個地區(qū)采集的607株畜禽源彎曲菌中,C.jejuni和C.coli對慶大霉素的耐藥率分別為15.6%(25/160)和79.9%(357/447)。對慶大霉素耐藥株進行耐藥基因PCR檢測分析,發(fā)現(xiàn)兩種慶大霉素耐藥基因aph(2")-If和aacA/aphD且aph(2")-If基因的檢出率顯著高于aacA/aphD:C.jejuni中,aph(2")-If基因的檢出率為10%(16/160),aacA/aphD基因為 2.5%(4/160),而C.coli中aph(2")-If和aacA/aphD基因的檢出率分別為57.7%(258/447)和15.2%(68/447)。自然轉化實驗證實aph(2")-If基因能夠轉移至敏感菌株NCTC 11168,對轉化子進行全基因組測序及分析發(fā)現(xiàn)在染色體基因組保守基因cj0299和panB間存在一個包括aph(2")-If基因在內的新型耐藥基因島,包含aph(2")-If,aac,aadA,aph2,aad9,aphA3,aphA7,cat等多個耐藥基因,能夠同時介導慶大霉素、卡那霉素、新霉素、阿米卡星、氯霉素等抗生素耐藥。功能性試驗證明aph(2")-If基因可導致彎曲菌同時對慶大霉素和卡那霉素高水平耐藥,本研究發(fā)現(xiàn)該基因可定位于彎曲菌染色體。此外,PFGE實驗結果顯示aph(2")-If基因既能發(fā)生克隆傳播也能發(fā)生水平傳播�！ち酌顾乜捎行е委煆澢鸬哪c炎,被視為治療由彎曲菌引起相關疾病的備選藥物,而本研究發(fā)現(xiàn)一株豬源C.coli具有磷霉素高水平耐表型(512μg/mL)。該菌株的磷霉素高水平耐藥表型能夠通過自然轉化方式轉移至敏感菌株C.jejuni NCTC 11168;轉化子全基因組測序分析發(fā)現(xiàn):在染色體cadF和CCO1582基因之間發(fā)現(xiàn)一段大約9 Kb包含11個ORF的外源DNA插入片段,其中一個ORF編碼136個氨基酸,與磷霉素耐藥蛋白FosA、FosB、FosX分別呈現(xiàn)26.9%、34.2%、63.9%的氨基酸同源性;隨后,對此ORF進行功能性驗證,發(fā)現(xiàn)該ORF可介導彎曲菌和大腸桿菌對磷霉素的高水平耐藥,將該新型磷霉素耐藥基因亞型命名為fosX~(CC);并證實在實驗條件下,fosX~(CC)基因可從C.coli轉移至C.jejuni。綜上所述,針對我國畜禽源彎曲菌多重耐藥情況嚴重的現(xiàn)象,對其耐藥機制進行深入研究,首次發(fā)現(xiàn)介導彎曲菌多重耐藥且參與彎曲菌對環(huán)丙沙星極高水平耐藥的外排泵變異體RE-CmeABC;首次在彎曲菌染色體上發(fā)現(xiàn)介導慶大霉素和卡那霉素耐藥的耐藥基因aph(2")-If及新型多重耐藥基因島;此外,在彎曲菌中發(fā)現(xiàn)磷霉素耐藥基因新亞型fosX~(CC)。上述研究結果在闡明彎曲菌耐藥新機制的同時也為彎曲菌耐藥性相關研究提供了新思路。
[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.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S852.61

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本文編號：1906499