Resistance Mediated By AmpC β-Lactamase And ESBLs In Enterobacter Cloacae

[OBJECTIVE] To investigate the rates of occurrence of high-level AmpC P -lactamase producers and ESBL-producers among clinical isolates of Enterobacter cloacae. To establish a convenient method for detecting resistance mediated by high-level AmpC P -lactamase in Enterobacter cloacae. To study the impact of the expression of AmpC P -lactamase and ESBL on antibiotics susceptibility of Enterobacter cloacae. To compare the potential of six P -lactam antibiotics ( piperacillin/tazobactam, cefoperazone/ sulbactam, cefotaxime, ceftazidime, cefepime and imipenem) to select mutants with derepressed AmpC P -lactamases synthesis from Enterobacter cloacae. To explore the effect of amino acid substitution at position 70 of Enterobacter cloacae AmpC P -lactamase on its substrate specificity.[METHODS] Standard disk diffusion susceptibility tests were used as initial screen tests. P -lactamases of the isolates with decreased susceptibilities to ceftazidime, cefotaxime. ceftriaxone or aztreonam were investigated by three-dimensional extract test, isoelectric focusing overlay technique, conjugation experiment. PCR and DNA sequencing were used to determine the genotypes of ESBL. ERIC-PCR typing was used to investigate the epidemiological characteristics of the resistant isolates. A new cloxacillin potentiated disc diffusion test was introduced to detect high-level AmpC P -lactamase producing isolates of Enterobacter cloacae and it results were compared with the results of three-dimensional extract tests and isoelectric focusing overlay technique. In order to make clear the impact of the expression of AmpC P -lactamase and ESBL on antibiotics susceptibility of Enterobacter cloacae, the MICs of 16 antimicrobial agents against total 106 isolates were determined by standard agar dilution susceptibility tests. The potential of piperacillin/tazobactam,cefoperazone/sulbactam, cefotaxime, ceftazidime, cefepime and imipenem to select mutants with derepressed AmpC P -lactamase synthesis from Enterobacter cloacae was determined in 10 strains with inducible AmpC P -lactamase synthesis and 5 ESBL-producing strains by subculturing the strains daily with the tested antibiotics at doubling concentrations starting at 0.25 X MIC. Three-dimensional extract test was used to assess the expression of AmpC P -lactamase and standard agar dilution susceptibility tests were used to analyze the resistant phenotypes of mutants selected by antibiotics. ampC gene of a clinical isolate of Enterobacter cloacae was cloned by cloning PCR method, PCR site-directed mutagenesis was used to construct three mutant AmpC P -lactamases by replacing Thr70 with He, Asn or Ser respectively. Agar dilution susceptibility tests were used to analyze the phenotypic characterization of wild and mutant AmpC P -lactamases. The antibiotics hydrolysis by wild or mutant P -lactamases was measured by ultraviolet spectrophotometric method. [ RESULTS ] Among total 106 clinical isolates of Enterobacter cloacae, 17(16.0%)only produced high-level AmpC P -lactamase, 11(10.4%) only produced ESBLs, 14(13.2%)produced both high-level AmpC P -lactamase and ESBLs. Among all 25 ESBL-producers, 18 (72.0%) produced CTX-M-3 and 7(28.0%) produced SHV-12. This is the first report of the prevalence of CTX-M-3 and SHV-12 among clinical isolates of Enterobacter cloacae in China. ERIC-PCR typing revealed the multiclonal constitution of the strains with decreased susceptibilities to ceftazidime , cefotaxime , ceftriaxone or aztreonam. Among all 106 isolates tested, cefoxitin and ampicillin/sulbactam showed the highest resistance rates (91.51% and 93.39%, respectively), no isolate were found resistant to carbapenems. The susceptible rates of 17 isolates only producing high-level AmpC P -lactamase to cefepime, amikacin, gentamicin and ciprofloxacin were 100% , 94.12%, 70.59% and 76.47%, respectively. The susceptible rates of 11 isolates only producing ESBLs to amikacin, piperacillin/tazobactam and cefoperazone/sulbactam were higher than 60%. The susceptible rates of 14 isolates producing both high-leve...