Current Resistance Status And Effect Of AmpD Gene Mutation In AmpC β-lactamases Producing Enterobacteriaceae Strains

Objective 1) To investigate the drug resistance of AmpC β-lactamases producing Enterobacteriaceae strains to instruct clinical application of antibiotics. 2) To value phenotype screening method for the detection of strains which produce AmpC β-lactamases. 3) To study the relationg between the mutatiaon of ampD and expression of ampC gene in Enterobacter cloacae.Methods 1) Shandard disk diffusion susceptibility tests were used as initial screen for the isolates from 2001 to 2003. Adopting modified three-dimensional extract test was adopted to detect AmpC β -lactamase high producing strains. All of used isolates were identified by VITEK 32 system. The minimal inhibitory concentrations (MIC) of them to 11 antibiotics, including Imipenem, Piperacillin, Cefotaxime, Ceftazidime, Cefepime, Aztreonam, Amikacin, cefoxitin, Ceftriaxone, amoxilin/clavulanic acid and Piperacillin/Tazobactam were detected by agar dilution method. 2) Phenotype screening was performed to detect AmpC β -lactamase high producing strains. The result was compared to adopting modified three-dimensional extract test. 3) AmpD gene was amplified by PCR and verificated by DNA sequencing.Results 1) 252 Enterobacteriaceae strains with decreased susceptibilities to cefotaxime, ceftazidime, ceftriaxone or aztreonam were detected. Among 252 Enterobacteriaceae strains, 36 of them were identified as AmpC P-lactamases high producing strains by adopting modified three-dimensional extract test, including 22 strains of Enterbacter cloacae, 10 strains of Citrobacter freundii, 3 strains of Serratia and 1 strains of Klebsiella pneumonia. AmpC P-lactamases high producing strains were mainly isolated from ICU ward, accounting for 44.4%. The resistance to Cefoxitin, Piperacillin and amoxilin/clavulanic acid were the highest, which were 100% all.The resistance to Cefotaxime, Ceftazidime, Ceftriaxone were rather high, accounting for 97.2%, 88.9%, 86.1% respectively. And resistance to Imipenem, Cefepime and Amikacin was 0.0%, 8.3% and 19.4% respectively. 2) Sensitivity and specificity were 86.8% and 95.5% respectively for the phenotype screening. The coincidence rate of Phenotype screening with three-dimensional test were 92.4% (P<0.05). 3) 4 strains were found to be positive by PCR for ampD. Compared with ampD14 gene from Kopp, the ampD genes of 3 stably derepressed strains had point mutations and amino acid alteration such as Thr-55→Asn, Ile-56→Leu, Ala-71→Arg, Leu-72→Val, Glu-106→Asp,Asp-148—Gly.Conclusion 1) The rate of AmpC p-lactamases high producing Enterobacteriaceae strains is rather high. ICU and Senilis wards are the most serious place where AmpC P-lactamases high producing Enterobacteriaceae strains prevails. 2) Drug resistance of AmpC P-lactamases high producing Enterobacteriaceae strains to P-lactamantibiotics is serious. It is important to strengthen resistance monitoring and apply antibiotics reasonably with drug sensitivity test. 3) Phenotype screening is suitable for screening strains producing AmpC p-lactamases in routine in all hospital. 4) The mutations had been found in 3 strains possibly related with stably derepressed AmpC enzymes.