鼠伤寒沙门菌分离株耐药性和其质粒介导耐药基因研究

发布时间：2018-01-20 20:49

本文关键词： 鼠伤寒沙门菌最小抑菌浓度耐药基因 PMQR mcr-1　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：鼠伤寒沙门菌是一种重要的人兽共患病原菌,特别在欧美等发达国家几乎每年都有散在暴发的报道。鼠伤寒沙门菌来源广泛,可在人和动物之间通过食物链发生交叉感染。抗菌药物的使用是防治沙门菌病的重要措施,但由于其不合理使用导致耐药菌株不断出现,耐药性不断增强,并出现多重耐药的现象。此外,耐药基因的传播也是耐药菌株不断出现的原因之一。耐药基因通常位于可移动元件上,携带耐药决定因子的质粒是传播耐药性的重要因素。由此带来的耐药问题、食品安全问题也越来越严重。而目前国内关于鼠伤寒沙门菌耐药分布情况及其质粒介导耐药基因的研究相对较少。本研究以330株鼠伤寒沙门菌为研究对象,通过测定这些菌株对18种抗菌药物的最小抑菌浓度,分析不同来源的鼠伤寒沙门菌耐药特性和多重耐药状况;调查质粒介导的喹诺酮类药物耐药基因(PMQR)在鼠伤寒沙门菌中的流行和分布情况;通过质粒接合试验获得新型多粘菌素耐药基因mcr-1阳性接合子,并对供受体菌及接合子进行氨基糖苷类、喹诺酮类、苯丙醇类、β-内酰胺类、磺胺类、硝基呋喃类、喹VA啉类、四环素类药物的敏感性试验,测定其对各种抗菌药物的影响。本研究将有助于进一步了解鼠伤寒沙门菌分离株耐药流行状况、耐药特点及其质粒介导耐药基因的分布。1不同来源鼠伤寒沙门菌分离株耐药性测定与分析本研究对330株不同来源的鼠伤寒沙门菌最小抑菌浓度(MIC)进行测定,结果显示其对阿莫西林/克拉维酸的耐药率最高,达到75.8%,其次为四环素(73.9%)、氨苄西林(73.6%);对链霉素、氯霉素、头孢唑啉、萘啶酸、复方新诺明和多粘菌素E的耐药率均在50%以上;对氨曲南、卡那霉素、庆大霉素、恩诺沙星和喹乙醇的耐药率分布在20.1%-38.8%之间;环丙沙星和呋喃妥因的体外抑菌效果较好,耐药率均未达到10%;而美罗培南和阿米卡星的体外抑菌效果最好,几乎没有耐药菌株出现。鼠伤寒沙门菌分离株主要来源于养殖环节(猪场)、屠宰环节(屠宰场)、市场环节(农贸市场)和人,因此对各个环节鼠伤寒沙门菌耐药性进行研究。结果显示不同环节的鼠伤寒沙门菌对阿莫西林/克拉维酸、四环素、氨苄西林、氯霉素、卡那霉素、环丙沙星、阿米卡星、美罗培南和呋喃妥因的耐药性相似。而对余下的8种抗菌药物,其耐药性表现差异较大。对于同一个环节而言,鼠伤寒沙门菌对各抗菌药物的耐药性与本研究中菌株的总体耐药情况较为相似,其中养殖环节中鼠伤寒沙门菌的耐药率较其他三个环节都高,其次是市场环节,屠宰环节菌株的耐药率与人源菌株较为相似。鼠伤寒沙门菌多重耐药现象较为普遍。其中有260株分离株对3类及以上抗菌药物表现出耐药性,多重耐药率达83.9%(260/310)。多重耐药的优势表型为氨苄西林-阿莫西林/克拉维酸-链霉素-四环素-复方新诺明-氯霉素-多粘菌素E。市场销售环节中鼠伤寒沙门菌多重耐药现象最为严重(100%),养殖和人环节次之,多重耐药率分别为99.0%和76.1%,屠宰环节多重耐药率最低为72.3%。对2014-2016年间在定点猪场收集的鼠伤寒沙门菌进行耐药性监测发现,定点猪场鼠伤寒沙门菌对各抗菌药物的MICmin、MIC50值要普遍高于本研究中鼠伤寒沙门菌总体统计状况,说明该定点猪场中鼠伤寒沙门菌在这段时间耐药现象较为严重。2鼠伤寒沙门菌分离株中PMQR基因的流行和分布情况本研究对330株不同源鼠伤寒沙门菌中PMQR基因进行检测,PMQR基因的阳性率高达51.2%。在所有的PMQR基因中,oqxAB阳性率最高,为29.7%;aac(6-Ib-cr和qnrS的阳性率次之(19.4%和10.9%),未检测到qnrM、qnrC和qnrD基因。oqxAB的阳性率显著高于其它PMQR基因,尤其是在猪源分离株中。通过对qnr基因阳性菌株进行测序,确定了其基因亚型。在本研究中,共测出qnrB基因亚型2种,分别为qnrB4和qnrB26;qnrS基因亚型也为2种,分别为qnrS1和qnrS2。双阳性的菌株中qnrS+aac(6')-Ib-cr和oqxAB+aac(6')-Ib-cr共同存在的现象最为常见,分别占9.6%和9.9%。对PMQR阳性菌株的基因型和表型相关性分析显示:对喹诺酮类药物耐药的鼠伤寒沙门菌中,携带有PMQR基因的菌株占73.6%;几乎所有携带PMQR基因的表型敏感性菌株对环丙沙星敏感性下降(DCS,decreased ciprofloxacin susceptibility),所对应的MIC值的范围在0.12-1 μg/mL之间。oqxAB阳性菌株对喹乙醇的MIC值结果显示,有90.0%的oqxAB阳性菌株对喹乙醇表现耐药或者中介,88.6%的oqxAB阴性菌株对喹乙醇表现敏感或者中介,说明330株鼠伤寒沙门菌的基因型与喹乙醇的耐药表型基本对应。3鼠伤寒沙门菌质粒介导的新型多粘菌素耐药基因mcr-1的初步研究应用PCR方法对新型质粒介导的多粘菌素耐药基因mcr-1进行检测,发现330株鼠伤寒沙门菌中,仅一株菌含有此基因。通过对该mcr-1阳性菌株的全基因组测序结果进行分析,发现该鼠伤寒沙门菌全长5,232,290 bp,GC含量为51.4%,多位点序列型为ST34。进一步研究发现,该菌基因组中含有IncHI2、IncX1、IncI2、IncHI2A和IncQ1共5个复制子,提示其可能携带不止一个质粒,且mcr-1基因位于其中一个质粒上;通过质粒接合试验,获得1株mcr-1阳性接合子。药物敏感性试验结果显示,该接合子对多粘菌素E和链霉素表现耐药,所对应的MIC值分别为8 μg/mL和64 μg/mL,与受体菌相比,分别升高了 16倍和64倍。接合子对环丙沙星、恩诺沙星和萘啶酸的敏感性出现不同程度降低,与受体菌相比,接合子MIC值分别升高16、8和8倍。这表明质粒在进行接合转移时,除了介导产生多粘菌素E的耐药性外,质粒上还包含了链霉素的耐药基因,与mcr-1基因一同发生了转移,因此接合子表现出对这两种药物的耐药性。
[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.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

【参考文献】