| 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 ?-lactamase(ESBL)and plasmid-mediated AmpC ?-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-?-lactamase-producing strains respectively,and 1 isolate was produced two enzymes at the same time.A higher resistant rate was observed in?-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 ?-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 of?-lactamase,resistant of?-lactam antibiotics,and some non-?-lactam antibiotics.These results indicated that plasmids could not only carried genes of ESBL or/and AmpC?-lactamase,but aslo could spreaded horizontally among Enterobacteriaceae,which was contributed to resistance to antimicrobials for ?-lactam and some non-?-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 ?-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 of?-lactamase,resistant of?-lactam antibiotics,and some non-?-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. |
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