Study On The Control And Genotyping Of β-lactamases In Enterobacter Cloacae

The primary resistant mechanism is that bacteria can produce β -lactamases. Now the noticeable β -lactamases were extended-spectrum 2 -lactamases (ESBLs) and AmpC enzymes leading the bacteria resistant to the third cephalosporins and other β -lactam antibiotics. These problems also exist in Enterobacter cloacae so that the clinical treatment is more difficult.ESBLs belong to Bush classification 2be β -lactamases. They mainly hydrolyze the third and the fourth generation cephalosporins and can be inhibited by β -lactamase inhibitors. AmpC enzymes belong to Bush classification 1 β -lactamases. They can hydrolyze the third generation cephalosporins and can not be inhibited by β -lactamase inhibitors. AmpC enzymes are encoded by ampC gene and can be induced by β -lactam antibiotics. The characteristics of these two kinds of enzymes are different and it needs different antibiotics to treat the infections due to them. So it is very important to clear out the rates of AmpC enzymes, ESBLs and both produced in Enterobacter cloacae. There is no method commended by National Committee for Clinical Laboratory Standards (NCCLS) to detect AmpC enzymes and ESBLs in Enterobacter cloacae. To set up a convenient method to detect AmpC enzymes and ESBLs has important significance in application of clinical antimicrobial agents.The most important question in the infections caused by enterobacteriaceae is theycan produce AmpC enzymes and ESBLs. Enterobacter cloacae, Serratia, Morganella, Citrobacter freundii, and Klebsiella pneumoniae all have this question and Enterobacter cloacae are the typical one. The infections caused by Enterobacter cloacae are severe,and the resistance is higher in China than in foroign.So we studiedon the functions ofAmpC enzymes and ESBLs, expression and control of ampC gene and genotyping of ESBLs in Enterobacter cloacae.l.Set up a new phenotype screening method for detecting AmpC enzyme produced by Enterobacter cloacaeWe set up a new convenient phenotype screening method to detect AmpC enzyme. Phenotype screening and three-dimensional tests were performed to detect 144 strains of Enterobacter cloacae. Isoelectric focus and cloxacillin inhibiting test was detected in part of strains. The results of phenotype screening showed 35 strains (24.31%) produced stably derepressed AmpC enzymes. Phenotype screening compared with three-dimensional test, the coincidence rate was 89.58% (129/144) and 5 strains had opposite results. Isoelectric point straps of 4 strains were not inhibited by cloxacillin. The other strain had 5 isoelectric point straps which 3 straps were inhibited by cloxacillin. Phenotype screening is reliable to detect AmpC enzyme and easy to use in clinical laboratories.2.The status of resistance and ampC gene expression in Enterobacter cloacaeDisk diffusion tests were performed to detect the susceptibility of antimicrobial agents against Enterobacter cloacae. AmpC gene was amplified by PCR and verificated by DNA sequencing. AmpC gene expression was analyzed according to antimicrobial agent sensitive phenotype. The sensitive rates of 144 strains to imipenem, cefepime and cefoperazone/sulbactam were 98.61%, 65.97% and 63.89%, respectively. The sensitive rates to other antimicrobial agents were lower. 120 out of 144 strains were found to be positive by PCR for ampC. Stably derepressed strains, hyperinducible strains and unexpressing or lower level expressing strains account for 27.50% (33/120), 37.50%(45/120) and 35.00% (42/120), respectively. The hyperinducible strains were highly sensitive to all the antimicrobial agents except amoxicillin/clavulanic acid and cefuroxime, while the stably derepressed strains were only sensitive to imipenem and cefepime. But the sensitivity to cefepime descended if the strains also produced ESBLs. The resistant status of Enterobacter cloacae is severe. To clear out the expression status of ampC gene is helpful to select the antimicrobial agents in the treatment of clinical infection.3. Study on the relation between the muta...