Mechanisms Of Resistance To Amoxicillin-clavulanate In Escherichia Coli Isolates

Objectives: To study the resistant mechanisms of amoxicillin/clavulanate -resistant Escherichia coli isolated from West China Hospital of Sichuan University. To investigate the distribution and transmissibility of bla_TEM, bla_SHV, bla_OXA and plasmid-mediated AmpCβ-lactamase (pAmpC) in these isolates. To investigate the distribution of integrons in the isolates and to identify the cassettes in integrons. So as to provide evidence for clinical therapy in infections caused by amoxicillin/clavulanate-resistant Escherichia coli, and decrease the incidence of resistance and control the spread of resistance genes.Methods: Among 276 isolates showing resistant to ampicillin/sulbactam, 52 strains were resistant to amoxicillin/clavulanate by disk diffusion method (zone diameter≤13mm). The susceptibility of amoxicillin/clavulanate -resistant E.coli isolates revealed by disk diffusion method to antibacterial agents were assayed by agar dilution testing and the genotypes were detected by PCR analysis. The genes of TEM-type enzymes in isolates of which phenotype was consistant with inhibitor-resistant TEMβ-lactamase (IRT) was cloned and expressed. We used multiplex PCR to discriminate bla_TEM, bla_SHV, bla_OXA genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR can differentiate the six plasmid-mediated ampC-specific families in E.coli isolates with six sets of ampC-specific primers resulting in easily distinguished amplicons ranging from 190bp to 520bp. Integrons integrase PCR, restriction fragment-length polymorphism (RFLP), conserved-segment PCR, and DNA sequencing were determined the prevalence and contents of integrons.Results: Among 52 amoxicillin/clavulanate-resistant E.coli isolates confirmed by disk diffusion method, the resistant rate to amoxicillin/clavulanate was 76.9% (40/52) and the susceptible rate was 21.2%(11/52) by agar dilution testing. The susceptible rate to piperacillin/tazobactam was 80.8%(42/52). Forty-nine out of 52 isolates were resistant to cefazolin. The resistant rate to cefoxitin and ceftazidime were: 73.1% and 26.9%, respectively. Multiplex PCR detected bla_TEM and bla_OXA genes in 46 and 6 strains, respectively, and only one isolate had a bla_SHV gene. The cocurrent presence of bla_TEM and bla_OXA genes were detected in 5 strains, and only one strain harboured bla_TEM and bla_SHV genes simultaneously. Among the isolates with TEM-type enzymes, TEM-1 was seen frequently and inhibitor-resistant TEMβ-lactamase (IRT) was not found. The incidence rate of plasmid-mediated AmpCβ-lactamases was 30.8%(16/52) and CMY-2 was the unique genotype. Thirteen strains had bla_TEM and pAmpC genes simultaneously; three isolates had pAmpC exclusively. Class 1 integrons were detected in 36 of 52 (69.2%) isolates by PCR using primers targeted to conserved regions of class 1, 2 and 3 integrase genes. DNA sequencing analysis revealed amplicons contained aadA4, aadA5, aadB, cmlA, cmlA1, and dfr17 gene cassettes that confer resistance to the aminoglycoside, chloramphenicol and trimethoprim respectively.Conclusions: In West China Hospital, hyperproduction of TEM-1β-lactamases are the main mechanism in amoxicillin/clavulanate-resistant Escherichia coli isolates, while producing pAmpC and OXA-1β-lactamases are important mechanisms of resistance. Class 1 integrons were found in amoxicillin/clavulanate-resistant Escherichia coli isolates frequently. dfr17 and aadA5 were found in this study, which confers resistance to trimethoprim and spectinomycin respectively, and mainland of China had never been reported them.