| Escherichia coli is a conditioned pathogen that can cause septicemia,urinary tract infections and other diseases in animals and humans.The abuse and overuse of antibiotics in clinic aggravated the emergence of drug-resistant bacteria.Beta lactam and fluoroquinolones have been used as important therapeutic drugs.The selective pressure produced by the abuse of these drugs produces multidrug-resistant bacteria.One of its mechanisms is the production of beta lactamases mediated by plasmids that can hydrolyze the beta lactam ring.ESBLs can hydrolyze beta lactam ring and spread among bacteria in the form of plasmid mediated,and the bacteria carrying the enzyme can hydrolyze the corresponding antibiotics and lead to the failure of the treatment.This is one of the main reasons for the resistance of Gram-negative bacteria.The genes encoding ESBLs exist on the plasmids,and there are many genotypes.CTX-M,SHV,TEM and OXA are found,and CTX-M is the main type of beta lactamase at present.In the past few decades,the presence of ESBLs-producing Escherichia coli has been reported in different environments,including food,food-like animals,different water environments,especially sewage.The drug-resistant strains of human and animal sources will eventually enter the water environment through various emission channels,and the resistance genes they carry are likely to be further spread among the bacteria in the aquatic environment.Municipal wastewater treatment plant is an important reservoir for human and animal derived microbes,which can be reentered into the surrounding environment through the outlet of the sewage plant,such as water and food,and may in turn reinfect humans and animals.As a result,once the ESBL Enterobacteriaceae enters the human and animal intestines through contaminated food or drinking water,it is likely to cause the resistance genes to spread in humans and animals,and even cause serious infection.There are a large number of bacteria and antibiotics in sewage,which are very conducive to bacterial conjugation and promote the horizontal transfer of mobile elements carrying drug resistant genes.The transmission of drug resistance through mobile elements is the main mechanism of drug resistance diffusion,and mobile originals,including conjugation plasmids and transposons,have the ability to obtain and transmit foreign genes.In this study,ESBLs Escherichia coli isolated from a sewage treatment plant in Tai'an was studied,and the resistance phenotype and the resistance genes of lactam and quinolones were detected,and the multiple drug resistance and its influence on the surrounding environment were analyzed.In order to provide scientific basis for the prevention and treatment of colibacillosis and the risk assessment of clinical medication,and provide reference for the rational and effective use of antibiotics in aquaculture industry.The conjugation test was carried out to compare the changes of resistance phenotype and resistance gene of Escherichia coli to explore the role of mobile plasmids in the process of drug resistance transmission.1.Epidemic characteristics of ESBL Escherichia coli produced from sewageTo understand the prevalence and characteristics of Enterobacter ESBL producing in Tai'an sewage treatment plant.A total of 160 samples were collected from the sewage treatment plant.Through the isolation and culture of selective medium,140 positive samples were obtained by 16 S rRNA PCR detection,sequencing and gene bank comparison,and the separation rate was up to 87.5%.By ESBLs confirmatory test(double disc synergy test),100 ESBL Escherichia coli isolates were isolated and the isolation rate was 62.5%.After ERIC analysis,a strain of more than 90% similarity was removed,and the remaining 50 strains of ESBL producing E.coli were used as follow-up experimental samples.The results show that the sewage of Tai'an sewage treatment plant has been seriously polluted by Enterobacter ESBL,and becomes the storage of resistance gene,and the treatment of sewage can not eliminate all ESBL Enterobacteriaceae.2.Resistance of ESBL Escherichia coli from sewageChoose 19 antibiotic drug sensitive tablets commonly used by locals and animals in accordance with CLSI(M100-S222012)standard operating standard.According to the clinical laboratory standard committee of the United States,the results were divided into sensitive(S),drug resistance(R)and mediator(I),and the drug resistance spectrum and multidrug resistance of each test strain were also analyzed.The results showed that the drug resistance of Escherichia coli isolated from ESBL was more serious and showed multiple multi-drug resistance(MDR resistant).This experiment provides a reliable basis for rational use of antibiotics in clinic.3.Epidemiological analysis of ESBL Escherichia coli produced in a sewage plantBLA gene was detected in 50 strains of Enterobacter ESBL producing bacteria isolated from sewage treatment plants.Among them,the detection rate of blaTEM and blaCTX-Mgene was 100%(50/50),and only two strains did not detect blaSHV gene.Most of the 50 BLA genes detected were blaTEM-1,blaSHV-11 and blaCTX-M-15.No qrA gene was detected.The qrB gene(A5,A22,B2,B4,C5)in five strains and qrS genes in 32 strains were detected.The resistance genes carried on the plasmid were detected by PCR reaction.The detection results were in accordance with the genome carrying situation,indicating that all the resistant genes were located on the plasmid and carried by the plasmid.The results show that the selective pressure caused by the abuse of beta lactam and fluoroquinolones leads to the production of bacteria carrying a variety of resistant genes and virulence genes.We should further monitor the transmission of blaCTX-M,blaSHV and blaTEM genotypes,so as to provide reliable evidence for rational use of antibiotics in clinic.4.The production of ESBL Escherichia coli from sewage to transport drug resistance through conjugation level.Conjugation is the most common way to transmit resistance at the gene level,and plays a very important role in the spread of drug-resistant genes.It is proved that the resistance genes can be transmitted through the level.Filter mating method(mating method)was used to engage in conjugation tests.By ERIC-PCR and drug selective plates,50 strains of ESBLs and cefotaxime were successfully joined together successfully,and the success rate was up to 70%.35 strains of Escherichia coli with successful conjugation were tested for the drug resistant phenotype of 15 antimicrobial agents.The results showed that the zygotes showed a certain resistance phenotype.The resistance gene carried by the butt zygote was detected by PCR,and all the resistance genes were carried out.It was found that all the TEM and CTX-M genes were successfully joined,only two of the SHV strains were not joined successfully.Both the donor and its zygote did not carry OXA and qnrA genes,and only one of the 24 strains carrying the qnrS gene were transferred and 2 strains were transferred.No gene transfer was observed in the donor strains carrying qnrB gene.The results showed that ESBL producing Escherichia coli transmitted resistance through conjugation level.5.Full sequence analysis of ESBLs resistant plasmidsThe encoded ESBLs gene is usually located on the plasmid and transferred and propagated through conjugation and transposition.We selected the zygote WA8 as the analysis object.The shotgun method was used to construct the library method for DNA sequencing and bioinformatics analysis was used to analyze the coding gene and structure.Plasmid wwA8(GenBank MG773378)is a closed loop DNA molecule,83157 BP,GC content is 52.74%,which contains 45 prediction genes.WwA8 carried three known drug resistant genes,blaCTX-M-15,blaTEM-1,QnrS1.The resistance phenotype and resistancegene test results of extracorporeal conjugative bacteria were in agreement with the plasmid sequencing results,which proved that the E.coli resistance gene of water source had the ability to transfer in vitro. |
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