The Cloning And Homology Analysis Of Antimicrobial Agents Resistance Determinant Genes In Escherichia Coli Strains Isolated From Animals

The antibiotics in preventing and treating Escherichia coli diseases is indispensable.However, the person and the animal infectious diseases caused by resistant strains increase yearby year because of the abuse of antibiotics. The problem of Escherichia coli resistance andmultiple resistance to antimicrobial agents is extremely serious.We isolated and identified 62 isolates of Eseherichia coli from 96 supposed Escherichia colifrom pigs, chickens, dogs and cats in JiLin province to investigate the level of antibiotic-resistanceEscherichia coli and provide positive samples to oligonucleotide array detecting antibioticsresistance gene in Escherichia coli, so that solve the problem of resistance to antimicrobial agentsultimately. 20 isolates were selected randomly to evaluated the level of Escherichia coli resistanceto Norfloxacin, Gentamicin, Trimethoprim-sulfamethoxazole, Deoxycycline, and Florfenicol.20 Eseherichia coli isolates were Quinolone resistant, of which 6 strains were highlyresistant; 19 isolates were Aminoglycoside resistant, of which 12 isolates were highly resistant; Allisolates were high-level resistance to Sulfonamides; 14 isolates were Tetracycline resistant, ofwhich 11 strains were highly resistant; 1 strain Chloramphenicol resistant. Among the total 20isolates,5 strains displayed coresistance to 3 families of antimicrobial agents; 13 strains showedcoresistance to 4 families of antimicrobial agents;1 strains showed coresistance to 5 families ofantimicrobial agents. Escherichia coli isolates showed a high, multiple-antimicrobialagents-resistant phenotype.The specific primers were designed to PCR antibiotics resistance gene fragments includingGyrA, GyrB, ParC, AacC4, Sul2 and TetA, which were cloned and sequenced subsequently.Thesequencing results were analyzed with the softwares Clustalx1.83 and MEGA3.1.The resultssuggested the mutations of resistance genes GyrA(83Ser→Leu, 87Asp→Asn) andParC(80Ser→Ile, Arg) are correlated with Quinolone-resistantce Escherichia coli;the presence ofAacC4, Sul2 and TetA resistance genes are correlated with Escherichia coli resistance toAminoglycoside, Sulfonamides, Tetracycline, respectively. The sequence of the number 5 strainwith the mutations of resistance genes GyrA (83Ser→Leu, 87Asp→Asn) andParC(80Ser→Ile, Arg) was aligned to standard sequence, the homology reach to 98ï¼…, which implyGyrA carried by the number 5 strain can provide necessary reference for the preparation ofpositive samples to oligonucleotide array detecting Quinolone resistance gene in Escherichiacoli. AacC4 carried in number O78^R strain, Sul2 carried in the number 9,14, JX¹¹ strains and TetAcarried in number E_IR strain can provide positive samples to oligonucleotide array detecting resistance genes of Aminoglycoside, Sulfonamides and Tetracycline in Escherichia coli,respectively.Sequence Analysis also showed that the number O78^R strain have the mutations of83Ser→Leu, 104Asp→Gly and 181Asn→Asp in GyrA but no 87Asp→Asn, and that the number 8strain which is low-level resistance to Quinolone only has one mutation in GyrA but also has themutation of ParC(80Ser→Ile), which are never reported in previous studies. The results ofSequence analysis showed GyrA and ParC may have new mutations which is relative toEscherichia coli resistant to Quinolone, so we should consider this possibility when preparingoligonucleotide array detecting Quinolone resistance gene GyrA in Escherichia col.