Prevalence And Transmission Of CTX-M Type ESBLs In Escherchia Coli From Meat Of Livestock And Poultry

Antibiotics are widely used in animal production,and result in serious bacterial resistance of animal source recently.β-lactam antibiotics can inhibit the activity of the bacterial adhesion peptide transpeptidase,thereby blocking the synthesis of bacterial cell wall and presenting broad-spectrum and efficient antibacterial activities.β-lactam antibiotics are widely used in animal husbandry industry to treat diseases such as bovine mastitis,gastrointestinal and respiratory infections caused by bacteria in livestock and poultry.However,the overuse of β-lactam antibiotics in animals not only leads to bacterial resistance,but also increases the possibility of endogenous infection of livestock,and long-term usage will cause the decline in immunity and antibiotics residues in animal products as well as the environment.Antibiotics residues in the animal products and environment as well as the resistant bacteria it carries may influence human health through the food chain,and increase the difficulty of treatment for bacterial infection diseases.Meat of livestock and poultry is an important part of human diet and principal source of human high quality protein,and is also an important way through which resistant bacteria spread to human.Escherichia coli is inherent bacteria in animal intestinal tract,is also one of the main pathogen causing human diarrhea;therefore,.monitoring the resistance level and studying the resistance origin as well as transmission mechanisms of E.coli in meat of livestock and poultry is of great importance to guide the rational use of antibiotics in animal husbandry and safeguard the human health.Extended-spectrum β-lactamase,mediated by plasmids,is the significant cause of gram-negative bacteria such as E.coli resistant to β-lactam antibiotics.The thesis took livestock and poultry meat from Shenzhen major markets as the research object,studied on the isolation and identification of E.coli and horizontal transfer of resistant plasmids,as well as the genetic environment of ESBLs resistant genes,so as to clarify the resistance origin and dissemination mechanism of E.coli from livestock and poultry meat.The thesis includes the following sections:In the first section,the experiment was conducted to understand the resistance of E.coli from livestock and poultry meat in Shenzhen,monitoring the detection rate,prevalent characteristic,resistant phenotype and genptype,in order to provide theoretical basis for rational use of β-lactam antibiotics in animal production,reducing resistance and controlling the spread of bacterial resistance.First,80 E.coli were isolated from 120 samples of meat of livestock and poultry,which antibiotic resistance to 13 antimicrobial agents was detected using agar dilution method according to the recommendation of the Clinical Laboratory Standards Institute(CLSI).The results showed that 80 E.coli were resistant to 12 antibiotics at different levels,but sensitive to meropenem.55 strains which were resistant to cefotaxime showed different degree resistance to other classes of antibiotics,such as tetracycline(78%),sulfamethoxazole/trimethoprim(65%),nalidixic acid(71%),chloramphenicol(58%),ciprofloxacin(56%),kanamycin(47%)and fosfomycin(22%),and most isolates showed multi-drug resistance.Second,ESBLs genes and fosfomycin resistance genes were detected in 55 cefotaxime-resistant E.coli by PCR.Results showed that blaCTX-M gene was detected in 37 strains,blaCMY-2 gene was detected in 16 strains,blaACT-2 was detected in 4 strains,and fosfomycin resistance gene(fosA3)was detected in 12 strains among 55 cefotaxime-resistant E.coli.In addition,fosfomycin resistance gene,fosA3,was detected only in strains which were positive for blaCTX-M gene,not found in blaCMY-2r blaACT-2 positive strains.In conclusion,the antibiotics resistance of E.coli from meat of livestock and poultry in Shenzhen was serious,and most showed multi-drug resistance to common antibiotics.Type CTX-M and TEM were the main genotype of ESBLs-producing E.coli,and CTX-M type ESBLs was an important factor causing E.coli of animal origin resistant to cephalosporin antibiotics.In the second section,the experiment aimed to study horizontal transfer mechanism of resistant plasmids among the blaCTX-M gene positive E.coli,so as to illustrate the resistance origin and transmission mechanism of E.coli from meat of livestock and poultry,and provide scientific basis to antibiotics rational use in clinic as well as controlling the drug-resistant genes to transfer to human from the livestock and poultry.Firstly,in order to demonstrate the blaCTX-M gene can transfer among bacteria,test took blaCTX-M positive E.coli as donor and E.coli J53 as recipient to do conjugation.Results showed that 15 out of 37 blaCTX-M positive strains can transfer resistance into the recipient.Compared with the CTX-M-9 type gene,CTX-M-1 type is easier to transfer the drug-resistant plasmids by the conjugation.Secondly,replicon typing of plasmids was performed in 15 conjugants by PCR,so as to analyze the major replicon typing of these conjugative plasmids.Results showed that 10 isolates could be typed,including three main types,namely that 6 isolates belonged to Inc Iltype,3 isolates belonged to Inc FII type and one belonged to Inc FIV type.And the last five were fail to be typed.Thirdly,the genetic environment of blaCTX-M and fosA3 gene was analyzed by the insertion sequence of IS26 and ISEcp1 etc.Results showed that IS26 was detected in the upstream of CTX-M-55 gene in 5 conjugants,and ORF477 was detected in the downstream at the same time.In addition,IS26 was detected in the upstream of fosA3 gene in other 2 conjugants.However,ISEcpl,ISCR1 and IS903 were not detected in the 15 conjugamts.Fourthly,test analyzed the transmission mechanism of conjugative plasmids in E.coli by S1-PFGE and Southern-blot hybridization.Results showed that plasmids of many different sizes existed in most isolates,while the blaCTX-M gene was mainly located in four different sizes of conjugative plasmids,namely 78kb,104kb,130kb and 138kb.In conclusion,the spread of blaCTX-M gene was mainly based on plasmids horizontal transfer,and few on clone spread.Bacteria such as E.coli can transfer conjugative plasmids to the recipient through conjugation,so as to spread the resistant genes among different bacteria.In the third section,the experiment aimed to analyze the homology among the blaCTX-M positive E.coli strains,and study the transmission route of conjugative plasmids in E.coli from meat of livestock and poultry,so as to provide theoretical basis for guiding rational use of antibiotics in animal husbandry and controlling the widespread of bacterial resistance.Firstly,in order to clarify the molecular epidemiology,15 transferable E.coli strains were subjected to phylogenetic grouping.Results showed that 9 isolates belonged to phylogenetic group B1,followed by group A and group D,and none belonged to group B2.Our data indicated that the transferable isolates tested in this study mostly belonged to commensal strains,not virulent strains.Secondly,MLST typing was performed in transferable E.coli,and results showed that 11 ST types were detected in the 15 conjugative E.coli strains,including ST156(n=3),ST155(n=2),ST88(n=2)and no ST131 was detected in these isolates.Thirdly,PFGE was performed among blaCTX-M positive E.coli and transferable isolates,so as to study transmission route of blaCTX-M gene among E.coli from meat of livestock and poultry.PFGE results showed that 29 different PFGE types were detected among the 34 blaCTX-M positive E.coli with most isolates present unique PFGE type.Furthermore,14 different PFGE types were detected in 15 conjugative E.coli,illustrating that E.coli harboring blaCTX-M gene showed low homology,while clone spread was also existing on rare occasion.In conclusion,the spread of blaCTX-M gene in E.coli from meat of livestock and poultry was mainly based on plasmids horizontal transfer and few on clone spread meanwhile.The horizontal transfer of resistant genes made the spread of resistance wider and more complex,aggravating on the slide of bacterial resistance.Conclusions:Escherichia coli from meat of livestock and poultry present a high degree of multi-drug resistance to antibiotics,and ESBLs genes were widespread among these strains,the blaCTX-M gene was the main reason for E.coli high-level resistant to cephalosporin antibiotics from meat of livestock and poultry in Shenzhen.It was easy for E.coli to transfer the resistance of cephalosporin antibiotics to each other by conjugation.The spread of blaCTX-M gene was mainly based on plasmids horizontal transfer,however,clone spread was also existed.The blaCTX-M gene was usually located in Inc I1 and Inc FII type plasmids,which was mainly in Inc I1 type.Insertion sequence IS26 and ISEcpl were closely related to the spread of blaCTX-M gene,the diversity of genetic environment accelerated the quickly transmission of resistance among bacteria.It would be a serious public health problem that E.coli from meat of livestock and poultry carrying blaCTX-M gene,reminding us that we should strengthen surveillance on antibiotics useage in animal husbandry,control the generation of resistant bacteria from the source.Meanwhile,we should enhance on resistance monitoring and research of resistance mechanism in E.coli from meat of livestock and poultry,so as to provide scientific basis for guiding rational use of antibiotics in clinic and preventing resistant genes from transferring to human.