Detection And Expression Of Resistant Genes Of Escherichia Coli From Chicken And Correlation Analysis Of Resistance Genes And Resistance Phenotype

Chicken pathogenic Escherichia coli (CPEC) is a main pathogen causing infections in poultry, which results in serious threat to poultry industry. At present, because of severe condition of antimicrobial resistance in E.coli, it is important significance to strengthen the study about bacterial resistance. The objective of this study was to detect the resistance phenotype, resistance genes of APEC isolated from diseased chicken in Xuzhou and analyse the correlation between drug-resistant phenotype and resistance genes based on construction resistance gene recombinant bacteria, which lay the foundation for providing theoretical support for clinical treatment, developing new antimicrobial agents based on potential drug targets.Firstly, CPEC was isolated and indentified, which was served as experimental strain for following study. Bacteria were isolated from the liver samples of sick chicken, then identified by the Gram staining test, biochemical characteristics and 16S rDNA genes sequence analysis. The results showed that two strains named CE and SE were isolated and were pathogenic to the experimental chicken. Both isolates were G- brevibacterium with scattered arrangement. They formed typical greenish-black colonies with metallic luster by cultured on EMB medium and their biochemical characteristics conformed to the Escherichia sp. according to Berger’s manual bacteria identification. The products of 16S rDNA of CE and SE strain were 1502 bp and 1503 bp, respectively, which showed 98.7%-99.8% homology with the Escherichia coli type strain ATCC 11775T and ATCC 8739T. Phylogenetic trees analysis showed that CE strain was clustered closely with E.coli type strain ATCC 11775T, while SE strain was clustered closely with type strain ATCC 8739T. The results showed that CE and SE strains both belong to CPEC.Secondly, drug-resistant phenotype were performed by K-B disc agar diffusion method and drug resistance-related genes were detected by PCR. The result showed that two isolates were multiple drug-resistance to 6 classes 16 kinds of antimicrobial agents (Beta lactam, aminoglycoside, large ring lactone class, tetracycline, quinolone and sulfonamides), and had corresponding resistance genes (Bla-TEM、Aac(3)、TetA) and drug multiresistant gene AcrA. Bio、informatics analysis indicated that resistance genes were highly conserved, with 99.0-100.0% homology between both isolates and referent strains. Bla-TEM gene encoded beta-lactamase with a predicted molecular weight of 31.5kDa, Aac(3) gene encoded aminoglycoside-3-N-acetyl transferase (Antibiotic_NAT) with a predicted molecular weight of 30.7 kDa, Aac(3) gene encoded multiple drug-resistant proteins (mdtA) with a predicted molecular weight of 42.4 kDa, and TetA gene encoded tetracycline efflux pump proteins (mdtl) with a predicted molecular weight of 45.2 kDa.Next, three resistance genes recombinant plasmid named PET-28a-Bla-TEM, PET-28a-Aac(3) and PET-28a-TetA were constructed by gene recombination technology and transferred into E.coli BL21 (DE3), then identified by restriction enzyme digestion and PCR amplification. After induced recombinant bacterium by IPTG (1mmol/L) for 6h, three expression proteins named Bla-TEM, Aac(3) and TetA were obtained, which molecular weights were 31.5kDa,30.7kDa and 45.2kDa, respectively.Finally, in order to understand the correlation between resistance genes and resistance phenotype, E.coli BL21 (DE3),which was sensitive to β-lactams, aminoglycosides and tetracyclines antimicrobials, was served as original strain to construct three resistance genes recombinant strains. Drug resistance phenotypes of three recombinant strains and original strain were compared, and the effect of antibiotics on those bacterial growth was detected. The result showed that Bla-TEM recombinant strain resistance to beta-lactam antibiotic improved, piperacillin from sensitive to drug resistance, ampicillin, amoxicillin, cefoperazone, cefotaxime and cefixime from sensitive to medium sensitive; Aac(3) recombinant strain resistance to aminoglycoside antibiotics improved, amikacin, gentamicin and netilmicin from sensitive to drug-resistance; TetA recombinant strain resistance didn’t change for tetracycline class antibiotics. Ampicillin had no obvious effect on Bla-TEM recombinant bacterial growth, cultured in without added and added ampicillin medium for 8h, OD600nm were 1.875 and 1.865, respectively. Kanamycin had low level inhibition for Aac(3) recombinant bacteria growth, cultured in without added and added kanamycin medium for 8h, OD600nm were 1.879 and 1.475. Tetracycline significantly inhibit the growth of TetA recombinant bacteria, cultured in without added and added tetracycline medium for 8h, OD600nm were 1.845 and 0.542. Results indicated that there existed certain relevance between resistance gene and the resistant phenotype.In conclusion, the present study clearly confirmed that CPEC isolates were multidrug resistant, and there was certain correlation between resistance genes and resistance phenotype.