| BackgroundBacterial resistance is a serious public health problem in world,which can lead to prolonged hospitalization time,increased the burden of medical care,even increased mortality and other serious consequences.Extended-spectrum beta-lactamases is a class of enzymes that can hydrolyze β-lactam drugs.The coding gene of ESBL can cause drug resistance from plasmid transport between the same or different strains,which is the main reason lead to Enterobacteriaceae bacteria antibiotic resistance.The most common bacteria about ESBL producing strains are Escherichia coli.The positive rate of ESBL-producing Escherichia coli in hospital patients was 31.5%to 55.8%.In the rural population of Shandong Province,the positive rate was 42%.The enzymes TEM,SHV and CTX-M were previously recognized as the main ESBL.Especially,CTX-M has recently become more prominent,and considered the most prevalent(3-lactamases found in clinical isolates of E.coli globally.Shandong province is a big pig province in China.In addition to large-scale farms,there are a large number of decentralized households.Due to close contact with the animals,those farmers are the focus crowd about animal-derived bacteria spreading.Investigation this population can help to understand the epidemic status,drug resistance and molecular typing characteristics about ESBL-producing Escherichia coli among rural residents household with pigs in Shandong Province.Furthermore,finding the channel of drug-resistant transmission can provide reference to control the antibiotic resistance.Objectives1.To evaluate the prevalence of ESBL-producing Escherichia coli among rural residents household with pigs in Shandong Province.2.To master the antibiotic resistance and drug resistance gene characteristics of ESBL-producing Escherichia coli among rural residents household with pigs in Shandong Province.3.Describe the molecular typing characteristics of ESBL-producing Escherichia coli among rural residents household with pigs in Shandong Province.4.Through analyzing the characteristics about enzymes TEM,SHV,CTX-M and the characteristics about molecular typing of ESBL-producing Escherichia coli,to explore the transmission route of drug-resistant bacteria,then provide reference to control the antibiotic resistance.Methods1.Sample collection:This study is based on the "IMPACT" project(Sino-Swedish international cooperation project).The project selected a pig breeding county in Shandong Province,a breeding town of 12 villages as a research site.We randomly selected 4 villages from the 12.Forty adults were randomly sampled in each village.Their fecal samples were collected by sampling tube.Meanwhile,record the residents’ age,sex and pig status.2.Isolation and identification of ESBL producing Escherichia coli:Isolated of ESBL-producing E.coli through ESBL plates,after incubation 18-24 hours at 35℃,selected different colonies based on color and morphology.Species were identified by MALDI-TOF Mass Spectrometry.3.ESBL confirmation and antimicrobial susceptibility test:ESBL confirmation testing and antimicrobial susceptibility testing were performed according to CLSI guidelines.ESBL activity was confirmed by double-disc diffusion and the antimicrobial susceptibility test was carried out by agar dilution.4.Genetic confirmation:The enzymes TEM,SHV and CTX-M were determined by Polymerase chain reaction(PCR)and DNA sequencing.5.Molecular typing:The MLST molecular typing and PFGE molecular typing were used to analyze the genetic evolution of the strains.Main results1.Carrying rate about ESBL-producing Escherichia coliOf the 160 rural residents,130 carrying ESBL-producing Escherichia coli,with a carrying rate of 81.3%,130 positive residents were carrying 184 ESBL-producing Escherichia coli.ESBL carrying rates in four villages were 75.0%,80.0%,77.5%and 92.5%,respectively.ESBL carrying rates in female and male were 84.5%and 78.7%.ESBL carrying rates in household with pigs and household without pigs were 83.3%and 80.0%.ESBL carrying rates in different age groups(<50,50-59,≥60)were 86.8%,87.0%and 63.2%,respectively.2.Antimicrobial susceptibility about ESBL-producing Escherichia coliTo study the antimicrobial sensitivity of the strains to 14 kinds of antimicrobial agents,remove the animal drug Florfenicol,the remaining 13 species belonging to 11 categories.Analysis of the resistance of ESBL-producing Escherichia coli to these 11 antimicrobial agents showed a multidrug resistance rate of 91.8%.The results of antimicrobial susceptibility showed that the proportion of resistance to 3-5 drugs was 56.0%(103/184),the proportion of resistance to 6-8 drugs was 35.9%(66/184).Pig-breeding is a risk factor for residents that can lead they heavily resistant to florfenicol(MIC=64),(OR=2.121,95%CI:1.034～4.353),meanwhile,pig-breeding is a risk factor for residents who in>5 drug resistance status(OR=1.995,95%CI:1.080～3.686).The antimicrobial susceptibility test results showed that the resistance rates of ESBLs-producing Escherichia coli to Cefotaxime,Tetracycline,Co-trimoxazole,Ciprofloxacin,Florfenicol,Gentamicin,Piperacillin-tazobactam,Fosfomycin,Amoxicillin-clavulanic acid,Nitrofurantoin,Amikacin,Cefoxitin,Meropenem,Tigecycline were 100.0%,87.0%,82.1%,64.7%,56.5%,53.8%,31.0%,29.9%,20.1%,13,0%,7.1%,4.9%,0.0%,0.0%,respectively.To analyze the expression of drug resistance gene CTX-M，SHV and TEM in Escherichia coli producing ESBL,the results showed that the detection rate of CTX-M was 100.0%,including 10 kinds of CTX-M subtypes,the most abundant were CTX-M-14(68/184 isolates,37.0%),followed by CTX-M-55,CTX-M-65 and CTX-M-15,accounting for 17.4%(32/184),16.3%(30/184)and 14.1%(26/184),respectively.The enzymes of TEM and SHV were not detected.3.Molecular typingFifty-four strains of Escherichia coli were randomly selected for PFGE typing analysis.The 54 strains of Escherichia belong to 35 PFGE genotypes.There is homologous between ESBL-producing Escherichia coli isolated from different village residents,even between household with pigs and household without pigs.A total of 172 strains of ESBL-producing Escherichia coli were matched with the ST sequence in the MLST gene pool and belonged to 81 ST sequences.The other 12 strains were not found in the MLST gene pool,belonging to the newly discovered ST type,the next step will be submitted to the MLST website for new naming.ST10 was the most abundant type(14 isolates,8.1%)，followed by ST38(10 isolates,5.8%),ST48(10 isolates,5.8%),ST6338(6 isolates,3.5%),ST46(5 isolates,2.9%),and ST648(5 isolates,2.9%).The proportion of each of the remaining 75 species ST types was less than 2.9%.Conclusion1.ESBL-producing Escherichia coli were widely popular in Shandong pig breeding county rural residents.Antibiotics resistance situation is severe in Shandong pig breeding county rural residents.2.The molecular typing of ESBL-producing Escherichia coli in this study is diversity.3.The animal origin drug-resistant genes CTX-M-55 and CTX-M-65 are popular among Shandong pig breeding county rural residents,and ST 10 was more common,which is widely used in livestock and poultry.This can initially prove that drug resistance has a tendency to spread from livestock to humans. |
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