Detecting And Genotyping Of AmpC Beta Lactamases In E. Coli And Klebsiella And Antibiotic Resistance Analysis

OBJECTIVE:To detect the phenotype and genotype of plasmids-inmediated AmpCβ-lactamases and ESBLs in 123 cefoxitin-resistant stains of E. Coli and Klebsiella and analyze drugs resistance for main mechanism of drug resistance interpretation and medicate reasonably in clinic.METHODS:Three-dimensional test and Cloxacillin-potentiated disk diffusion test were use to detect the phenotype of AmpCβ-lactamases and two disk confirming test was used to detect the phenotype of ESBLs, and their antibacterial activities in vitro of 15 antibiotics were analyzed by disk diffused method. PCR was used to determine genotype of AmpC and ESBLs. Software EpiInfor2000 was used to analyze data and WHONE5.4 was used to analyze drug resistance.RESULTS:Among 123 isolates strains, 57(46.3%)were detected to produce AmpCβ-lactamases with three-dimensional test. 69(56.1%)were detected to produce AmpCβ-lactamases with Cloxacillin-potentiated disk diffusion test. There was no significant difference between the two groups(P<0.05). 87(70.7%)of them were detected to produce ESBLs with two disk confirming test. PCR results shows that the amount of strains carrying DHA, ACT, CMY and FOX type of AmpC gene are 51, 63, 10 and 2 respectively, percentage is 41.5%, 51.2%, 8.1% and 1.6% respectively. The amount of strains with TEM, SHV, CTX and OXA type of ESBLs gene are 75, 48, 12 and 12 respectively, percentage is 61%, 39%, 9.8% and 9.8% respectively. Among 69 strains of E. Coli, carrying DHA, ACT, CMY, and FOX type AmpC gene are 12, 39, 6 and 1 respectively, percentage is 17.4%, 56.5%, 8.7% and 1.4% respectively. Among 69 strains of E. Coli, carrying TEM, SHV, CTX and OXA type ESBLs gene are 45, 6, 12 and 6,percentage is 65.2%, 8.7%, 17.4% and 8.7% respectively. Among 54 strains of Klebsiella, carrying DHA, ACT, CMY and FOX type AmpC gene are 39, 24, 4 and 1 respectively, percentage is 72.2%, 44.4%, 7.4% and 1.9% respectively. Among 54 strains of Klebsiella, carrying TEM, SHV, CTX and OXA type ESBLs gene are 30, 42, 0 and 6 respectively, percentage is 55.6%, 77.8%, 0%, and 11.1% respectively. Drug susceptibility test results show that resistance rates of single-AmpC-positve strains to aztreonam, ceftriaxone, Cefotaxime and piperacillin were higher than that of enzyme-negative strains. Resistance rates of single-AmpC-positve strains and enzyme-negative strains to ampicillin, ampicillin/sulbactam, ciprofloxacin and cefazolin were all comparatively higher, resistance rates to amikacin, cefepime, imipenem, cefoperazone/sulbactam and piperacillin/tazobactam were all comparatively lower. Resistance rates of AmpC-positve strains to piperacillin, cefoperazone/sulbactam, and piperacillin/tazobactam were from 43.9% to 70.2%, but the resistant rates of enzyme-negative strains were all lower (25.8%～51.5%).There was significant difference between two groups (P<0.05). Resistance rates of AmpC-positve to ampicillin, ampicillin/sulbactam, ciprofloxacin, cefazolin, cefepime, ceftriaxone, aztreonam and cefotaxime were all higher(72.2%～100%), and resistant rates to amikacin and imipenem were all lowe(r0%～49%). Resistance rates of AmpC-negative strains were(63.6%～97.0%)and (0～36.4%). There was no significant difference between two groups (P>0.05). The rates of four multi-drug resistance(70.2%) and eight multi-drug resistance(61.4%) in AmpC-positve strains were obviously higher than those (45.3% and 30.3%) in AmpC-negative strains. There was significant difference between two groups (P<0.05).CONCLUSIONS: There was no significant difference between the result of three-dimensional test and that of Cloxacillin-potentiated disk diffusion test. Because three-dimensional test was very complicated and not easy to handle, we suggest clinic laboratory should accept Cloxacillin-potentiated disk diffusion test to detect the phenotype of AmpC. With PCR results:Positive rate of AmpC-producing strains(68.3%) and ESBLs strains(76.4%)were high in 123 strains isolation, which show that drug-resistance-enzyme was available widely in clinic isolation. ACT type(41.5%)and DHA type(51.2%)were the main genotyping of AmpC gene in all strains. TEM type(61%) and SHV(39%)type were the main genotyping of ESBLs gene in all isolation. ACT type Ampc gene(56.5%) and TEM type ESBLs gene(65.2%) were the main molecular types in E. Coli . DHA type Ampc gene(72.2%) and SHV type ESBLs gene (77.8%) were the main molecular types in Klebsiella. Drug susceptibility test results show that drug-resistance-enzyme such as AmpC and ESBLs have been one of the most important reason of drug resistance. We suggest to chose imipenem for infections treatment caused by AmpC-positive and ESBLs-positive strains, or chose cefepime for single-AmpC-positve strains, and we could also chose cefoperazone/ sulbactam, piperacillin/tazobactam and amikacin for infection treatment according to drug susceptibility test. The AmpC-positive strains is more likely to obtain mulit-resistance than the AmpC-negative strains, so we should pay more attention to identify bacteria, analyze drug susceptibility, and detect phenotype and genotype of drug-resistance-enzyme. We should chose antibiotics reasonable which will help us to postpone the resistance of bacteria and improve the effect of clinic anti-infectious treatment. This will be beneficial to control and inspect happen and transformation of hospital infections.