Study Of Gram Negative Bacilli β-lactamases Resistant Mechanism

Because of the good curative effect and low toxicity,β-lactam antibiotics are the important drug for infectious diseases.Penicillins,cephalosporins and carbopenes are all belong toβ-lactam antibiotics.But bacteria become more resistant quickly.for the past few years,the major problem faced by the medical faculty is to control the nosocomial infection due to multidrug resistant gram negative bacteria.The production of extended-spectrumβ-lactamases(ESBLs)is the most common mechanism of resistance to the third generation cephalosporins in Enterobacteriaceae bacteria.The prevalence of ESBL-producing strains differs from country to country and from institute to institute.Pseudomonas aeruginosa is one of the most frequent gram negative bacilli species for hospital onset of infection.It is usually resistant to many kinds of antimicrobial agents for its intrinsic and acquired drug resistance.The death rate is very high for its infection.To search for the resistant mechanism and develope new antibiotics are the persistent topics during the fighting between human and for infectious diseases.In this researchment,the sensitivity to 10 kinds of drugs were determined by the agar dilution method for 181 Enterobacteriaceae bacteria stains and 75 P.aeruginosa isolates,which were ESBLs positive screened by routine methods.Ten kinds of ESBLs genes were identified by PCR-sequencing.Resuts showed that resistance of Enterobacteriaceae bacteria to cephalosporins and quinolones were higher,but sensitivity rates to drugs consist ofβ-lactamases inhibitor are higher,moreover,they are all sensitive carbopenems.But resistant rates of P.aeruginosa to 10 drgs were all high.The most effective drug was meropenem,but its inhibitory rate was only 69.7%. 181 Enterobacteriaceae bacteria stains were all detected ESBLs genes which are major tem-1 and ctx-m-3 conformed by sequencing.There were 82 strains were both tem and ctx-m positive.Four tem positive.strains detected oxa-Ⅰgene.The other 7 genotype were not detected.40 Pseudomonas aeruginosa strains detected ESBLs genes,tem-1(20 strains)was the most frequently detected ESBL and oxa-10(13 strains)was the second,shv,ctx-m and ges genes were not detected in Pseudomonas aeruginosa.For ESBLs are not the most importmentβ-lactamases of Pseudomonas aeruginosa,AmpC and metallo-β-lactamases(MBLs)were screened in 75 Pseudomonas aeruginosa strains.Cefoxitin susceptibility test,triple-disks test and modified three dimensional test were used for AmpC screening.The inhibitor-potentiated disk diffusion(IPD)test and double disk synergy test(DDS) were used for MBLs screening.Results showing that 40 strains yielded AmpC and 38 yielded MBL.Combinate with ESBLs,11 strains were positive of ESBLs,AmpC and MBLs simultaneously and 30 strains were two types positive.Membrane permeability depressing and multidrug efflux pumps increasing are all important for carbopenem resistance in Pseudomonas aeruginosa.The expression level of oprD and 4 kinds of efflux pumps were measured by real-time RT-PCR in 30 carbopenems resistant strains.There Were 14 stains over expressed efflux pumps and 12 strains had diminished expressioh of oprD in carbopenems resistant strains. Integrate theβ-lactamase analysis in part 1,there may be abnomal expression of efflux pumps and oprD and one or moreβ-lactamase in 1 strain.That is to say,the resistant mechanism is very complicated.The interaction among the different mechanism results in different resistant patterns.Compared to laboratory isolates,the resistant mechanism of the clinical isolates is more anfractuous.Some resistant patterns related to special mechanism showed in laboratory isolates was not sure in clinical isolates.for example,overexpression of mexCD resulted in hypersensitive to impenem in laboratory strains,but 3 mexCD overexpression strains weren't show susceptible to impenem in this study.Cefepime was usually recommended to treat AmpC positive bacteria infection,but 4 of 8 AmpC positive strains were resistant to cefepime.In conclusion,in spite of strong resistant to the third and fourth generation cephalosporins,Enterobacteriaceae bacteria still maintain higer sensitive rates to drugs consist ofβ-lactamases inhibitor and all ssensitive carbopenems.So we still have drugs for its infection.But resistant status of P.aeruginosa is very serious.The most effective drug was meropenem,but its inhibitory rate was only 69.7%.β-Lactamase production is the most common mechanism ofβ-lactam drug resistance in gram-negative bacteria.Partial carbopenems resistant strains had abnomal expression of efflux pumps and oprD.There may be multiple mechanisims in one strain. Generally,the strains not only producing one or moreβ-lactamase but also abnomal expressing effiux pumps and oprD were more resistant.Developing new antimicrobial which can against all resistant mechanism is an imminent problem.The whole society should pay more attention to rational utility of antimicrobials and prevent the production and dissemination of multidrug resistant bacteria.