【作者】 叶青华；

【导师】 吴清平；

【作者基本信息】 华南理工大学 ， 微生物学， 2016， 博士

【摘要】 致病微生物尤其是肠杆菌科某些菌如沙门氏菌、小肠结肠炎耶尔森菌等引起的食源性感染是食源性疾病发生的突出因素,为有效遏制这些感染性疾病的发生,对这些致病菌的快速鉴定成为迫切需要解决的问题。并且随着抗生素在临床与农业生产的广泛使用,肠杆菌科细菌耐药株迅速出现,多重耐药严重,己成为21世纪最大的公共卫生安全问题之一。本论文针对目前我国在商品化的数值鉴定系统空白及食源性致病菌耐药性监测方面缺乏系统性研究的问题,以肠杆菌科细菌为研究对象研制出具有自主知识产权的肠杆菌科数值鉴定系统;在此基础上,以本系统鉴定的肠杆菌科细菌种为受试菌株,从细菌学分析、抗生素敏感性实验以及超广谱β-内酰胺酶(extended spectrumβ-lactamase,ESBL)、质粒介导头孢素酶(plasmid-mediated cephalosporinase,AmpC)的分子生物学分类等方面进行研究,探讨我国食品与广州珠江水样中肠杆菌科细菌的耐药现状、ESBL与AmpC的流行情况及基因型分布。具体研究内容和结果如下:1、建立了肠杆菌科数值鉴定系统理论模型,与现行商品化的数值鉴定系统相比增加了各菌属与小肠结肠炎耶尔森菌6种生物型的数学模块,通过优化后筛选出不同于目前市面上进口的任何相似产品的24种生化反应组合,研制出生化鉴定条;结合我国肠杆菌科细菌主要生化型特征制定了适合我国肠杆菌科数值鉴定系统结果评价标准;利用C++6编写了数值鉴定分析软件Numide v1.0,实现在线分析功能。2、对建立的数值鉴定系统进行置信度的验证与应用,API 20E(梅里埃,法国)和MID-GNA+MID-GNB(Microgen,英国)对某些16S rRNA基因测序结果为肠杆菌科细菌株鉴定为非肠杆菌科细菌,而本系统与测序结果一致,表明本系统针对性较强;本系统对小肠结肠炎耶尔森菌、沙门氏菌、阪崎肠杆菌、无丙二酸盐柠檬酸菌、克氏柠檬酸菌等的鉴定效果比进口API 20E、MID-GNA或/和MID-GNB产品更优,具有广泛应用价值。3、我国食品中小肠结肠炎耶尔森菌的生物型/血清型以1A/O:8为主;没有出现典型的致病菌株,但携带有除inv、ail、ystA、yadA、virF 5种典型毒力基因外的其他9种毒力基因,并分离到了1株1A型ail阳性菌株,具有潜在的致病性;全部菌株均为多重耐药株,耐3种抗生素以上分离株为92.2%;ERIC聚类结果比较分散,无明显优势ERIC型别,ERIC型别与生物型、血清型、毒力基因型、药敏型、菌株来源、采样地点均不具有相关性,具有遗传多样性特征。4、广州食品与珠江水样中分离的282株肠杆菌科细菌,55.3%的分离株为多重耐药株,耐3种抗生素以上分离株为42.2%;经双纸片确认和三维试验初筛分别有25、27株分离株产ESBL与AmpC酶,其中1株同时产ESBL和AmpC酶,产酶菌株的耐药性高于非产酶菌株;经PCR扩增及序列分析,产质粒编码ESBL和Amp C酶基因型分别以CTX-M型和DHA-1亚型为主;Ⅰ类整合子基因检出率为100%,Ⅱ类整合子基因无检出;接合实验中产酶分离株的接合率为25.5%,供体菌相应的β-内酰胺酶基因与β-内酰胺类抗生素的耐药性传递给了受体菌,且部分接合子出现了对非β-内酰胺类抗生素的耐药,表明质粒编码ESBL编码和AmpC酶基因可通过接合转移方式使ESBL和AmpC酶基因水平传播、β-内酰胺类抗生素耐药性横向传递,以及部分非β-内酰胺类抗生素的耐药性可以与β-内酰胺类抗生素的耐药性随质粒发生共转移,从而导致多重耐药性的蔓延。5、全国食品中分离的1024株肠杆菌科细菌,产ESBL酶菌株的检出率为9.4%,ESBL阳性样本率为16.8%;产ESBL酶分离株除对碳青霉稀类(5.2%)与头孢西丁(6.3%)的耐药率相对较低外,对其他抗生素的耐药率均在47.9%以上,94.8%的分离株耐7种及7种以上的抗生素;产ESBL酶菌株β-内酰胺酶以CTX-M和TEM基因型为主,87.1%的菌株携带2种及2种以上β-内酰胺酶基因;Ⅰ类整合子基因检出率为97.2%,Ⅱ类整合子基因无检出;接合率为30.6%,供体菌相应的β-内酰胺酶基因与β-内酰胺类抗生素的耐药性传递给了受体菌,且部分接合子出现了对非β-内酰胺类抗生素的耐药,进一步表明细菌的耐药性与耐药基因可通过质粒接合转移方式而蔓延。本论文研制的肠杆菌科数值鉴定系统不仅打破了西方发达国家的技术壁垒,而且为促进其它种类细菌检验的类似鉴定系统的开发和应用,建立系列的简便快速的细菌数值鉴定方法方面提供技术支持。同时本论文研究的质粒型产ESBL与AmpC酶的流行规律、耐药基因型分布及耐药的分子机制,为正确掌握我国食源性细菌耐药的发展趋势、合理使用抗生素及控制耐药基因的蔓延提供理论依据。更多还原

【Abstract】 Pathogenic microorganism especially for some Enterobacteriaceae such as Salmonella,Yersinia enterocolitica are very important factors for foodborne diseases.The realization of rapid methods to detect pathogenic bacteria is central to preventing of diseases caused by foodborne pathogens.With the widely use of antibiotics in clinical and agricultural production,increasing prevalence of resistance to antimicrobial agents in Enterobacteriaceae and multiresistant has become one of the biggest public health safety issues in the 21 st century.To date,there was no commercial numerical identification system made by Chinese and systematic surveillance data of antimicrobial resistance for food-borne pathogens in China.Thus,this thesis was developed a independent intellectual property rights of numerical identification system for identification of Enterobacteriaceae in China.In addition,this thesis also analysed antimicrobial resistance of these isolates,and investigated the prevalence and genotypes of extended spectrum β-lactamase(ESBL)and plasmid-mediated AmpC β-lactamases(AmpC).The specific contents are as follows: 1.The theoretical model for numerical methods were established,which was increased six kinds of mathematical model for biotype of Yersinia enterocolitica compared with commercial numerical identification systems.A set of biochemical tests containing of twenty-four individual tests which are inoculated with a bacterial suspension was different from any kind of market product.Considering the main biochemical type of Enterobacteriaceae in China,valuation criteria was formulated.Using programming language of C++6,computer service of Numide v1.0 was establish to realize on-line analysis.2.The method based numerical identification was evaluated and applicated.Some isolates were identificated to Enterobacteriaceae by this method and sequencing based 16 S rRNA gene,but the results for these isolates were not Enterobacteriaceae identificated by API20E(BioMérieux,France)and MID-GNA+MID-GNB(Microgen,England).The results indicated that the method has strongly pertinence.Some isolates such as Yersinia enterocolitica,Salmonella spp,Enterobacter sakazakii,Citrobacter amalonaticus and Citrobacter koseri were more accurate using this method than API and MID.Thus,this method has extensive application.3.The primary biotype/serotype was 1A/O:8 among Yersinia enterocolitica isolates from retail food in China.There was no classic pathogenic,but some isolates were found to carry the other nine kinds of virulence genes except inv,ail,ystA,yadA and virF.In addition,one isolate harbored ail virulence gene which was belonged to 1A biotype.These isolates harbored virulence gene may be associated with the specific environmental niche.All strains were multidrug-resistant and 92.2% of the strains were resistant up to 3 antibiotics.The results of enterobacterial repetitive intergenic consensus-polymerase chain reaction(ERIC-PCR)was showed that ERIC types exhibited high genetic heterogeneity,and there was no direct relationship among the ERIC types in terms of their origins,isolation sites,serovars,virulence profiles,or even antibiotic profiles.4.25.5% of isolates from retail food and Zhujiang water in Guangzhou were sensitive to 16 antibiotics,whlie 55% of isolates were multidrug-resistant and 42.2% of the strains were resistant up to 3 antibiotics.The results of double disk confirmatory test and three-dimensional test showed that 25 and 27 of isolates were ESBL-and AmpC-β-lactamase-producing strains respectively,and 1 isolate was produced two enzymes at the same time.A higher resistant rate was observed inβ-lactamase-producing postive strains than that in negative strains.The results based PCR and PCR-sequencing showed that CTX-M and DHA-1 were the predominate genotype in ESBL-and AmpC-producing strains respectively.Production of plasmid-mediated ESBL and AmpC-lactamases is the primary mechanism of β-lactam resistance in these isolates.Class 1 integrons were present in all isolates,while Class 2 integrons was absent.In the conjugation experiments,25.5% of isolates were detcected transconjugants,and transconjugants received the gene ofβ-lactamase,resistant ofβ-lactam antibiotics,and some non-β-lactam antibiotics.These results indicated that plasmids could not only carried genes of ESBL or/and AmpCβ-lactamase,but aslo could spreaded horizontally among Enterobacteriaceae,which was contributed to resistance to antimicrobials for β-lactam and some non-β-lactam,thus multidrug resistance was prevalence.5.16.8% of retail food samples collected in China were positive for ESBL,and 9.6% of the isolates were ESBL-producing strains.Except for carbapenems(5.2%)and cefoxitin(6.3%),resistant rate to other antimicrobials were up to 47.9%.42.2% of the strains were resistant up to 7 antibiotics.CTX-M and TEM were the predominate genotypes in ESBL-producing strains and 87.1% of islates were procued more than two genes of β-lactamase.97.2% of isolates were deceted Class 1 integrons,while Class 2 integrons was absent.In the conjugation experiments,30.6% of isolates were detcected transconjugants,and transconjugants received the gene ofβ-lactamase,resistant ofβ-lactam antibiotics,and some non-β-lactam antibiotics.These results indicated that resistance to antimicrobial agents and genes encoded ESBL can be spread by plasmid transfer.Numerical identification system developed in this paper,it was not only to break the technical barriers of the western developed countries,but also to provide technical support for promoting a series of other kinds of similar identification system development and application.At the same time,studies for epidemic regularity,resistant genotype distribution and the molecular mechanism of antimicrobial resistance of the plasmid ESBL-and AmpCproduction,it was not only to correctly understand the development trend of antimicrobial resistance for foodborne pathogens,but also to provide the theory basis for reasonable using antibiotics and controlling the spread of antimicrobial resistant genes.更多还原