The Detection Of CTX-M-Type Esbls Gene D Research Of Genetic Environ Ment In Escherichia Coli From Duck

Abstract:In recent years, due to the development and extensive use of the third generation cephalosporin and single ring beta-lactam antibiotics, many bacterias produce plasmid-mediated extended-spectrum beta-lactamase (ESBLs) which can hydrolyze the third generation cephalosporin, such as cefotaxime and single amine class antibiotic, such as aztreonam. Among those ESBLs, CTX-M type ESBLs present a rising trend, they spread all over the world widely and rapidly, even erupt in some areas. CTX-M-type ESBLs become prevalence in many countries and it is a great threat to human life safety and the prevention and control of animal disease. The purpose of this experiment is to explore beta-lactam of molecular mechanism of drug resistance and the spread diffusion mechanism of blaCTX-M.We analysed molecular genotype for beta-lactam of drug-resistant in E.coli from duck by ESBLs phenotypic confirmatory test, drug sestitive test and drug resistance gene detection. Genetic enviroment of blaCTX-M genes were analysed by PCR mapping and molecular cloning technology. Molecular epidemiology characteristics of CTX-M-type ESBLs were analysed by plasmid incompatibility group, Eric-PCR and multilocus sequence typing (MLST).1、ESBL-producing phenotypes were confirmed in34of53isolates by the phenotypic confirmatory test. The MIC of Cefotaxime, Cefotaxime/sulbactam, Ceftazidime, Ceftazidime/sulbactam, Cefepime, Kanamycin, Amikacin, Tetracycline, Doxycycline, Florfenicol, Enrofloxacin and Ciprofloxacin were determined by a broth microdilution method. The results showed that the majority of isolates (79.4%,27/34) were multidrug resistance. PCR was used to detected genotype. Thirty-three isolates harbored blaTEM-1genes and31isolates carried six blaCTX-M genes, including blaCTX-M-55, blaCTX-M-79, blaCTX-M-65, blaCTX-M-14, blaCTX-M-27and blaCTX-M-24.SHV-type gene was not detected.2、The blaCTX-M genes were often found to be flanked upstream by ISEcp1and IS26 insertion sequence, and downstream by an IS903element. PCR mapping was carried out on the strains.ISEcp1was identified upstream of blaCTX-M-55,blaCTX-M-79,-M-65, blaCTX-M-14, blaCTX-M-27and blaCTX-M-24.All ISEcpls provided-35(TTGAAA) and-10(TACAAT) promoter sequences. Between different blaCTX-M gene subtype and ISEcpl have different spacer (42-266bp). In addition, ISEcpl was truncated by IS26elements in five strains carrying blaCTX-M-65genes. IS903was identified downstream of blaCTX-M-65, blaCTX-M-79, blaCTX-M-14, blaCTX-M-27and blaCTX-M-24. Downstream of blaCTX-M-55genes adjoined ORF477. The genetic environment sequence of all strains were logged in GeneBank. The accession numbers for the nucleoide sequences are JX204385, JX290340, JX232215, JX232216and JX306032-JX306038. The strains of ESBLs gene were not detected by PCR. We got a new gene combination. The length of gene sequence is7853bp and the sequence number for KC493654. The genetic structure is hypothetical protein-inti1-qacE△1-sull-orf5-IS26-blaTEM-1DNA topoisomerase Ⅲ.3、The molecular epidemiology of31strains with blaCTX-M gene were investigated by the plasmid incompatibility group, Eric-PCR and MLST. Results of plasmid incompatibility group showed that most of strains contained two or more than two plasmid incompatibility group.87.1%of the strains contained the plasmid incompatibility group FII. A total of eight different clonal types (A, B, C, D, E, F, G and H) were observed among the31CTX-M-producing isolates. Two predominant clonal types were A (n=8) and E (n=8), respectively. Clonal type A produced CTX-M-65(n=8) and clonal type E produced CTX-M-79(n=5) and CTX-M-55(n=3). MLST analysis results revealed that31CTX-M-producing isolates belonged to seven different ST ST10、ST2711、ST163、ST162、ST156、ST224and ST3085. The main MLST was ST10(11/31). ST10was mainly associated with carrying CTX-M-65isolates. ST3085is a new type in this study and has aslo been deposited in MLST database.In summary, the CTX-M-type ESBLs have widely disseminated among duck E. coli strains in China. The reason is mainly due to two aspects. On the one hand, different genetic elements may be involved in the mobilization of blaCTX-M genes, such as ISEcpl and IS26. Especially ISEcpl inseryion sequence not only can make the downstream gene transfer, but also can enhance the expression of gene. On the other hand, the clonal and horizontal dissemination of blaCTX-M genes is a important factor, and the main clone types are A, E and ST10.