| It is a serious problem for anti - infective therapy to theβ - lac-tam resistance of Gram - negative bacillus. At present β - lactamase is thought to be the primary mechanism of β - lactam resistance in the Gram - negative bacillus. Most of the resistances are led by AmpCβ -lactamase. AmpCβ - lactamase belong to the group of Bush - J - M,, and it is a serine cephalosporinase that can t be inhibited by clavu-lanic acid, and it is synthesized by the induction of β - lactam. AmpCβ -lactamase is often detected in the Enterobacter,Citrobacter, Serration marcescans, Morganella morganii, Pseudomonas aeruginosa, Escherichia coli,Shigella and klebsiella.The expression of inducible AmpCβ - lactamase coding by chro-matosome is controlled by multiplex amp operon, and it is made of five unlinkage genes , such as ampC, ampR, ampD, ampE and ampG. ampC is the structural gene of AmpCβ - lactamase, which code the protein of β - lactamase; The genes of ampR,ampD,ampE and ampG are regulatory , which are concerned with the control of synthesization for AmpCβ - lactamase. The ranks of ampD correlate tightly with the phenotype of AmpCβ - lactamase. There are four phenotypes in the Enterobacter expressing AmpCβ - lactamase ; wild type ( normal in-ducible AmpC β -lactamase) yper - inducible type (the basic express level of AmpC β - lactamase increase and the high level of AmpC β - lactamase express in the lower dosage of inducer) , temperature sensitive type (the inducible ability will lose in the impropriety temperature) and stably derepressed type (the high level of AmpC β -lactamase express continuously). All of them impaired the clinic therapy most is stably derepressed type , in which the mutation of ampD manifest the amino acid substitute or the amino acid mutation for the end of hydroxy.Most of the opportunistic pathogen in the Enterobacters were neglected for known as non - pathogenicity . As the development of medical techniques, such as some operations別xamining instruments ,cath-etering, transfusing and usage of antibioltic and immunosuppressive a-gent frequently, and it provides the conditions for the opportunistic pathogen infection. Serration marcescans and Cloaca enterobacters often cause the lower respiratory tract infection, especially in the patient of trachea incision or mechanical ventilation. Therefore, it is difficult for solving the drug resistance from higher expressing AmpC β - lactamase thoroughly . On the one hand , we should actively search and devise the new type of antibiotic and β - lactamase inhibitor; On the other hand, an internist should use the antibiotic rationally in order to avoiding the synthesis of higher expressing AmpCβ - lactamase. In this experiment, we select the ceftazidime as the inducor and compare of the AmpCβ - lactamase induction to Serratia marcescans and Cloaca enterobacter , and we hope to provid the science foundation for the rational antibiotic usage.Materials and instruments1. strainsWe collect the 23 strains of Serratia marcescans and 40 strains of Cloaca enterobacter from June,2001 -June,2002 in the respiratory ward.2. reagent ceftazidime,cefoxitm,cefazolin,benzylpenicillin,M -H medium NAPI20e - strains identification strip, drug - sensitive slip, E - test strip,clavulanic acid N Coomassie brilliant blue G250, ampholine ( pH = 3.5 -10) ,Iodin, Potassium iodide, starch,TaqDN A polymerase,acryl-amide.3. instrumentshomoiothermy incubator,homoiothermy water bath shake apparatus ,hypothermia high speed centrifuge, profound hypothermua refrigerator ( -83℃) ,752 ultraviolet - visible spectrophotometer ,LKB elec-trophresis apparatus ,electrophoresis chamber and chiller ,PCR apparatus.Methods1. strains collection and identification2. antibiotic sensibility experiment and cefoxitin induction3. ceftazidime screening experiment4. extract β - lactamase5. isoelectric focusing electrophoresis and clavulanic acid inhibit experiment6. detect AmpCβ - lactamase activity7. three dime... |
