Comparative Induction Of Imipenem, Cefepime, Ceftriaxone And Aztreonam To Enterobacter Cloacae AmpC β-lactamase

PrefaceIt is a serious problem for drug - resistance of the gram - negative bacilli with β -lactam antibiotics using widely. AmpC β - lactamase and ESBLs are the major cause of the gram - negative bacilli resist-ance to the third cephalosporins. The inducible formation of AmpC β - lactamase is of special importance in clinical medicine and in the development of β - lactam antibiotics and has received relatively high attention.AmpC β - lactamase belong to the group of Bush - J - M₁ ,and it is a serine cephalosporinase that can' t be inhibited by clavulanic acid, and it is synthesized by the induction of β - lactam antibiotics. AmpC β - lactamase is common in the Enterobacter cloacae, Citrobacter, Serratia marcescens, pseudomonas aeruginosa, et al.Enterobacter Cloacae should be the opportunistic pathogen. In recent years, Enterobacter Cloacae is most common nosocomial patho-gen isolated from patients due to use widely of cephalosporins and to be selected. In this experiment, the comparative in vitro induction ac-tivity of Imipenem, Cefepime, Ceftriaxone and Aztreonam was evalua-ted with clinical isolates of Enterobacter Cloacae. At the same time, the mutation of ampD was detected. We hope the study could provide some reference for clinical medicine.Materials1. Bacterial strainsThe original strains were isolated from the patients hospitalized in the respiratory ward from June,2000 June 2002.2. Reagent;Cefoxitin, Imipenem, Cefepime, Ceftriaxone and Aztreonam , ce-fazolin, benzylpenicillin, M -H medium, API 20E - strains identifi-cation strip, drug - sensitive slip, E - test strip, clavulanic acid, Coomassie brilliant blue G₂₅₀ ampholine(pH =3. 5 - 10) ,Iodin, Po-tassium Iodide, starch, Taq DNA polymerase, acrylamide.3. Instruments:homoiothermy incubator, hypothermia high speed centrifuge, 752 ultraviolet - visible spectrophotometer , LKB electrophoresis appara-tus , electrophoresis chamber and chiller, PCR apparatus.Methods1. collection and identification of strains2. antibiotic sensibility experiment and cefoxitin induction experi-ment3. antibiotic (IPM, FEP, CRO, ATM ) screening experiment4. extraction of 3 - lactamase5. isoelectric focusing electrophoresis and clavulanic acid inhibit experiment6. detection of AmpCβ - lactamase activity7. three dimensional test detecting phenotype mutation8. extraction of the strains DNA9. Polymerase chain reaction (PCR)10. Purification and DNA sequence of products in PCR11. Statistics analysis:Results1. The result of cefoxitin induction experimentIn the 25 strains of Enterobacter Cloacae, there are 12 stains of wild type, 4 strains of hyper - inducible type and 8 strains of dere-pressed type Enterobacter Cloacae.2. Drug sensitivity.There isn't obvious alteration in MIC with IPM, FEP, CRO and ATM for wild type strains whether it is induced or not. But, for the hyper - inducible type, there is apparent increasing in MIC after anti-biotics inducing, especially CRO and ATM , up to 10.7 -128 times.3. The detection AmpCβ- lactamase induction activity of IPM, FEP,CRO and ATM to isolated strains.AmpCβ ?lactamase activity is nearly none in the wild type of En-terobacter Cloacae . For the hyper - inducible type Enterobacter Cloa-cae, the average of AmpCβ - lactamase activity is high, especially IPM, the average of that is 1. 842 ?. 689mmol/mg ?min after in-duced by the 2MIC . Statistics analysis show that it is marked differ-ence when comparing AmpCβ - lactamase activity in the hyper - in-ducible type by the 2MIC ATM and CRO for that of MIC and 1/2 MIC in the hyper - inducible type Enterobacter Cloacae( p < 0. 05 ). How-ever, it is no difference in the group of MIC and 1/2MIC.4. The mutation rate of hyper - inducible type Enterobacter Cloa-caeStatistics analysis show that it is marked difference when compa-ring the mutation rate in the group of 2MIC ATM and CRO for MIC.and 1/2 MIC( p <0.05). There is no statistics difference in the group of MIC and 1/2M...