本文關鍵詞： 鼠傷寒沙門菌 最小抑菌濃度 耐藥基因 PMQR mcr-1　出處：《揚州大學》2017年碩士論文　論文類型：學位論文

【摘要】：鼠傷寒沙門菌是一種重要的人獸共患病原菌,特別在歐美等發(fā)達國家?guī)缀趺磕甓加猩⒃诒┌l(fā)的報道。鼠傷寒沙門菌來源廣泛,可在人和動物之間通過食物鏈發(fā)生交叉感染�？咕幬锏氖褂檬欠乐紊抽T菌病的重要措施,但由于其不合理使用導致耐藥菌株不斷出現(xiàn),耐藥性不斷增強,并出現(xiàn)多重耐藥的現(xiàn)象。此外,耐藥基因的傳播也是耐藥菌株不斷出現(xiàn)的原因之一。耐藥基因通常位于可移動元件上,攜帶耐藥決定因子的質粒是傳播耐藥性的重要因素。由此帶來的耐藥問題、食品安全問題也越來越嚴重。而目前國內關于鼠傷寒沙門菌耐藥分布情況及其質粒介導耐藥基因的研究相對較少。本研究以330株鼠傷寒沙門菌為研究對象,通過測定這些菌株對18種抗菌藥物的最小抑菌濃度,分析不同來源的鼠傷寒沙門菌耐藥特性和多重耐藥狀況;調查質粒介導的喹諾酮類藥物耐藥基因(PMQR)在鼠傷寒沙門菌中的流行和分布情況;通過質粒接合試驗獲得新型多粘菌素耐藥基因mcr-1陽性接合子,并對供受體菌及接合子進行氨基糖苷類、喹諾酮類、苯丙醇類、β-內酰胺類、磺胺類、硝基呋喃類、喹VA啉類、四環(huán)素類藥物的敏感性試驗,測定其對各種抗菌藥物的影響。本研究將有助于進一步了解鼠傷寒沙門菌分離株耐藥流行狀況、耐藥特點及其質粒介導耐藥基因的分布。1不同來源鼠傷寒沙門菌分離株耐藥性測定與分析本研究對330株不同來源的鼠傷寒沙門菌最小抑菌濃度(MIC)進行測定,結果顯示其對阿莫西林/克拉維酸的耐藥率最高,達到75.8%,其次為四環(huán)素(73.9%)、氨芐西林(73.6%);對鏈霉素、氯霉素、頭孢唑啉、萘啶酸、復方新諾明和多粘菌素E的耐藥率均在50%以上;對氨曲南、卡那霉素、慶大霉素、恩諾沙星和喹乙醇的耐藥率分布在20.1%-38.8%之間;環(huán)丙沙星和呋喃妥因的體外抑菌效果較好,耐藥率均未達到10%;而美羅培南和阿米卡星的體外抑菌效果最好,幾乎沒有耐藥菌株出現(xiàn)。鼠傷寒沙門菌分離株主要來源于養(yǎng)殖環(huán)節(jié)(豬場)、屠宰環(huán)節(jié)(屠宰場)、市場環(huán)節(jié)(農貿市場)和人,因此對各個環(huán)節(jié)鼠傷寒沙門菌耐藥性進行研究。結果顯示不同環(huán)節(jié)的鼠傷寒沙門菌對阿莫西林/克拉維酸、四環(huán)素、氨芐西林、氯霉素、卡那霉素、環(huán)丙沙星、阿米卡星、美羅培南和呋喃妥因的耐藥性相似。而對余下的8種抗菌藥物,其耐藥性表現(xiàn)差異較大。對于同一個環(huán)節(jié)而言,鼠傷寒沙門菌對各抗菌藥物的耐藥性與本研究中菌株的總體耐藥情況較為相似,其中養(yǎng)殖環(huán)節(jié)中鼠傷寒沙門菌的耐藥率較其他三個環(huán)節(jié)都高,其次是市場環(huán)節(jié),屠宰環(huán)節(jié)菌株的耐藥率與人源菌株較為相似。鼠傷寒沙門菌多重耐藥現(xiàn)象較為普遍。其中有260株分離株對3類及以上抗菌藥物表現(xiàn)出耐藥性,多重耐藥率達83.9%(260/310)。多重耐藥的優(yōu)勢表型為氨芐西林-阿莫西林/克拉維酸-鏈霉素-四環(huán)素-復方新諾明-氯霉素-多粘菌素E。市場銷售環(huán)節(jié)中鼠傷寒沙門菌多重耐藥現(xiàn)象最為嚴重(100%),養(yǎng)殖和人環(huán)節(jié)次之,多重耐藥率分別為99.0%和76.1%,屠宰環(huán)節(jié)多重耐藥率最低為72.3%。對2014-2016年間在定點豬場收集的鼠傷寒沙門菌進行耐藥性監(jiān)測發(fā)現(xiàn),定點豬場鼠傷寒沙門菌對各抗菌藥物的MICmin、MIC50值要普遍高于本研究中鼠傷寒沙門菌總體統(tǒng)計狀況,說明該定點豬場中鼠傷寒沙門菌在這段時間耐藥現(xiàn)象較為嚴重。2鼠傷寒沙門菌分離株中PMQR基因的流行和分布情況本研究對330株不同源鼠傷寒沙門菌中PMQR基因進行檢測,PMQR基因的陽性率高達51.2%。在所有的PMQR基因中,oqxAB陽性率最高,為29.7%;aac(6-Ib-cr和qnrS的陽性率次之(19.4%和10.9%),未檢測到qnrM、qnrC和qnrD基因。oqxAB的陽性率顯著高于其它PMQR基因,尤其是在豬源分離株中。通過對qnr基因陽性菌株進行測序,確定了其基因亞型。在本研究中,共測出qnrB基因亞型2種,分別為qnrB4和qnrB26;qnrS基因亞型也為2種,分別為qnrS1和qnrS2。雙陽性的菌株中qnrS+aac(6')-Ib-cr和oqxAB+aac(6')-Ib-cr共同存在的現(xiàn)象最為常見,分別占9.6%和9.9%。對PMQR陽性菌株的基因型和表型相關性分析顯示:對喹諾酮類藥物耐藥的鼠傷寒沙門菌中,攜帶有PMQR基因的菌株占73.6%;幾乎所有攜帶PMQR基因的表型敏感性菌株對環(huán)丙沙星敏感性下降(DCS,decreased ciprofloxacin susceptibility),所對應的MIC值的范圍在0.12-1 μg/mL之間。oqxAB陽性菌株對喹乙醇的MIC值結果顯示,有90.0%的oqxAB陽性菌株對喹乙醇表現(xiàn)耐藥或者中介,88.6%的oqxAB陰性菌株對喹乙醇表現(xiàn)敏感或者中介,說明330株鼠傷寒沙門菌的基因型與喹乙醇的耐藥表型基本對應。3鼠傷寒沙門菌質粒介導的新型多粘菌素耐藥基因mcr-1的初步研究應用PCR方法對新型質粒介導的多粘菌素耐藥基因mcr-1進行檢測,發(fā)現(xiàn)330株鼠傷寒沙門菌中,僅一株菌含有此基因。通過對該mcr-1陽性菌株的全基因組測序結果進行分析,發(fā)現(xiàn)該鼠傷寒沙門菌全長5,232,290 bp,GC含量為51.4%,多位點序列型為ST34。進一步研究發(fā)現(xiàn),該菌基因組中含有IncHI2、IncX1、IncI2、IncHI2A和IncQ1共5個復制子,提示其可能攜帶不止一個質粒,且mcr-1基因位于其中一個質粒上;通過質粒接合試驗,獲得1株mcr-1陽性接合子。藥物敏感性試驗結果顯示,該接合子對多粘菌素E和鏈霉素表現(xiàn)耐藥,所對應的MIC值分別為8 μg/mL和64 μg/mL,與受體菌相比,分別升高了 16倍和64倍。接合子對環(huán)丙沙星、恩諾沙星和萘啶酸的敏感性出現(xiàn)不同程度降低,與受體菌相比,接合子MIC值分別升高16、8和8倍。這表明質粒在進行接合轉移時,除了介導產生多粘菌素E的耐藥性外,質粒上還包含了鏈霉素的耐藥基因,與mcr-1基因一同發(fā)生了轉移,因此接合子表現(xiàn)出對這兩種藥物的耐藥性。
[Abstract]:Salmonella typhimurium is an important zoonotic pathogens, particularly in Europe and other developed countries almost every year there are scattered reports. An outbreak of Salmonella typhimurium wide source, can the food chain cross infection between human and animal. Through the use of antibiotics is an important measure for prevention of Salmonella disease, but because of the unreasonable use of lead resistant strains emerged, resistance increased, and the emergence of the phenomenon of multiple drug resistance. In addition, one of the reasons for the transfer of resistant gene is the emergence of drug resistant strains. Drug resistance genes are usually located in the movable element, plasmid carrying resistant determinant factor is an important factor in the spread of drug resistance. The problems of drug resistance the food safety problem is becoming more and more serious. At present, the domestic research on the distribution and drug resistance of Salmonella typhimurium plasmid mediated resistance gene of the relatively small. Study on 330 strains of Salmonella typhimurium as the research object, through the determination of the minimum inhibitory concentration of these strains to 18 antimicrobial agents, analysis of drug resistance of Salmonella typhimurium from different sources and multiple drug resistance; quinolone resistance gene in plasmid mediated (PMQR) prevalence and distribution in Salmonella typhimurium in the plasmid; joint test of drug resistance gene mcr-1 in new polymyxin positive zygote, and donor strains and transconjugants were aminoglycosides, quinolones, phenyipropanol, beta lactam, sulfonamides, nitrofuran, quinoline VA phenanthroline, tetracycline sensitivity test, determination of the antibacterial effect of all. This study will help to further understand the prevalence of drug-resistant strains isolated from Salmonella typhimurium, drug resistance and plasmid mediated resistance gene distribution of.1 from different sources of Salmonella typhimurium Study on the separation and determination of the minimum inhibitory concentration of 330 Salmonella typhimurium strains from different sources and analysis of drug resistance (MIC) were measured. The results show that the amoxicillin / clavulanic acid resistance was the highest, reached 75.8%, followed by tetracycline (73.9%), ampicillin (73.6%); streptomycin, chloramphenicol, cefazolin. Nalidixic acid, cotrimoxazole and polymyxin E resistance rate was more than 50%; to aztreonam, kanamycin, gentamicin, resistant to enrofloxacin and olaquindox rate distribution between 20.1%-38.8%; good antibacterial effect of ciprofloxacin and nitrofurantoin, drug resistance rate did not reach 10%; and in vitro antibacterial effect of meropenem and Amikacin best, almost no drug resistant strains of Salmonella typhimurium isolates. Mainly from farming areas (farm), slaughter part (slaughterhouse), market segments (farmers market) and so on, the A link of drug resistance in Salmonella typhimurium were studied. The results showed that different aspects of Salmonella typhimurium to amoxicillin / clavulanic acid, tetracycline, ampicillin, chloramphenicol, kanamycin, ciprofloxacin, meropenem and Amikacin, resistance to nitrofurantoin. Similar to 8 kinds of antibiotics remaining, the resistance for performance differences. The same link, Salmonella typhimurium on the overall resistance of the antimicrobial drug resistance and strain in this study is similar to the resistance breeding process in Salmonella typhimurium rate than the other three sectors are high, the second is the market segment, resistance rate and human strains strains are similar. The slaughter part of rats multi drug resistant Salmonella typhi is a common phenomenon. There are 260 strains of 3 or more antibiotics showed multidrug resistance, multidrug resistance rate was 83.9% (260/310). The advantage of multiple drug resistance phenotype to ampicillin, amoxicillin / clavulanic acid, streptomycin tetracycline and cotrimoxazole and chloramphenicol - polymyxin E. market sales in Salmonella typhimurium multidrug resistance phenomenon is most serious (100%), and the second part of breeding, multiple drug resistance rate were 99% and 76.1%, part of the slaughter the lowest rate of multiple drug resistance for 72.3%. drug resistance monitoring for 2014-2016 years in the designated farm collection of Salmonella typhimurium, designated swine Salmonella typhimurium on the antibacterial drugs MICmin, MIC50 values are generally higher than the overall statistics of Salmonella typhimurium in this study illustrate the prevalence and distribution of the sentinel pig in Salmonella typhimurium in the more serious this time the drug resistance of Salmonella typhimurium isolates of.2 PMQR gene in this study of 330 strains of Salmonella typhimurium PMQR homologous gene in PMQ were detected. The positive rate of R gene reached 51.2%. in all of the PMQR genes, oqxAB positive rate was 29.7%; the highest AAC (6-Ib-cr and qnrS positive rate times (19.4% and 10.9%), qnrM was not detected, the positive rate of qnrC and qnrD gene of.OqxAB was significantly higher than that of other PMQR genes, especially in pig source separation strain. By sequencing the qnr gene positive strains to determine the genotype. In this study, the qnrB gene were detected in 2 subtypes, respectively qnrB4 and qnrB26; qnrS gene subtype 2, respectively qnrS+aac qnrS1 and qnrS2. double positive strains (6'and -Ib-cr) oqxAB+aac (6') -Ib-cr common phenomenon is most common, accounted for 9.6% and analysis of 9.9%. genotype on PMQR positive strains and phenotypic correlations showed that the quinolone resistance in Salmonella typhimurium, carrying PMQR gene of strains accounted for 73.6%; almost all carrying PMQR gene sensitive phenotype Strain decreased susceptibility to ciprofloxacin (DCS decreased, ciprofloxacin susceptibility), a range corresponding to the MIC value of the.OqxAB between the 0.12-1 g/mL positive strains of olaquindox MIC values showed that the positive strain oqxAB 90% of olaquindox resistance or dielectric, oqxAB negative strains 88.6% sensitive or intermediary of olaquindox, that preliminary study on the application of PCR method in new drug resistance gene mcr-1 in polymyxin genotype 330 strains of Salmonella typhimurium and olaquindox resistance phenotype corresponding.3 Salmonella typhimurium plasmid mediated to polymyxin novel plasmid mediated resistance gene in mcr-1 were detected, 330 strains of Salmonella typhimurium, this gene contains only one strains. Through the analysis of the results of whole genome sequencing of the mcr-1 positive strains of Salmonella typhimurium, found that the total length of 5232290 BP, the content of GC is 51 .4%, multilocus sequence typing for ST34. further study found that the bacteria genome contains IncHI2, IncX1, IncI2, IncHI2A and IncQ1 5 replicon, suggesting that it may carry more than one plasmid and the mcr-1 gene in one plasmid by plasmid conjugation experiment;, obtained 1 strains of mcr-1 zygote. Drug sensitivity test showed that the zygote of polymyxin E and streptomycin resistance, the MIC values were 8 g/mL and 64 g/mL, compared with the host strain were increased by 16 times and 64 times. Zygote sensitivity to ciprofloxacin, enrofloxacin ciprofloxacin and nalidixic acid appeared decreased in different degree compared with the host strain, zygote MIC increased 16,8 and 8 times respectively. This indicated that the plasmid in conjugation, mediated drug resistance in addition to polymyxin E, plasmid also contains a streptomycin resistant gene, together with mcr-1 gene The transfer was born, so the zygote showed resistance to the two drugs.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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