Study On Resistant Mechanism Of The Carbapenem-resistant Pseudomonas Aeruginosa

1.Drug sensitivity testing and homology analysis of Pseudomonas aeruginosa.Pseudomonas aeruginosa(PA) is one of nosocomial infection germs,and causes the clinical variety of infections.This study focused on 141 carbapenem-resistant Pseudomonas aeruginosa from January 2005 to December 2005 in Huashan Hospital,Fudan University.141 carbapenem-resistant Pseudomonas aeruginosa sensitivity testing showed that three models.Imipenem and meropenem which are resistant strains accouted for a maximum of 94(66.7%).The results of resistance rates of 141 Pseudomonas aeruginosa to antibiotics were:Polymyxin B＜cefepime＜piperacillin-tazobactam＜amikacin＜cefoperazone-sulbactam＜ceftazidime＜meropenem＜piperacillin＜aztreonam＜levofloxacin＜ciprofloxacin＜cefoperazone＜gentamicin＜imipenem.141 PA to various antibiotics in vitro activity of the comparison results showed that sensitivity of the imipenem-resistant strains to piperacillin-tazobactam and amikacin are still 46.1%and 45.4%,and not found polymycin B-resistant strains,suggesting that serious PA infection in patients with the treatment ofβ-lactams and aminoglycosides anti- Pseudomonas is a good medication combination.Polymyxin B can also be used as an alternative drug.Homology of 141 carbapenem-resistant strains were analysis by ERIC-PCR.the results showed that 141 carbapenem-resistant PA have 11 type which were A,B,C,E,D,F,G,H,I,J,K,and A,B,C type were mainly threetypes,respectively,64,31 and 29,suggesting that the hospital mainly to spread three kinds of cloning.Type A has existed in various departmens,but mainly distributed in brain surgery,the senior cadres ward and departments of the Center ICU;type B in the six departments,mainly in brain surgery,the senior cadres ward and neurology;type C in the nine departments,mainly distributed in brain surgery.In brain surgery,senior cadres ward,neurology,ICU and medicine there are A,B,C three major cloning.All of 141 carbapenem resistant PA are MDR,accounting for 100%;PDR for 19, accounting for 13.5%.19 PDR strains are mainly distributed in brain surgery,ICU,renal medicine and hand surgery.ERIC-PCR results showed five types,the main type A and B, respectively,six and seven.The five type are distributed in a number of departments,not showing a focus on the characteristics of a particular department.Therefore,the strengthening of these strains especially PDR and MDR strains monitoring,control drug-resistant PA in the spread of the ward and reduce the incidence of nosocomial infection is the urgent affairs.2.Study on Carbapenem-Resistant Pseudomonas aeruginosa Producing Metallo-β-Lactamase.In recent years,carbapenem antibiotics,such as imipenem,were the wider used in clinical practice,as a result,metallo-β-lactamases were produced,which can extensive hydrolyseβ-lactam antibiotics,bacterial producted MBLs were resistant to all ofβ-lactama antibiotics,and caused great difficulties to the clinical anti-infective chemotherapy.Since 1991,genotype and quantity of metallo-β-lactamases in PA were increased continuously. In this study,141 Carbapenem-Resistant Pseudomonas aeruginosa,metallo-β-lactamases were detected by EDTA synergy and Etest test.Genotype of bacterial producted MBLs were amplified by PCR and sequenced analysis,homology analysis using PFGE, resistance gene positioning by Southern Blotting and transconjection testing,integron of MBLs strains were amplified by PCR and sequenced analysis.The study results of EDTA synergy and Etest test showed that 4 strains is positive results in 141 carbapenem resistant PA.accounting for 2.8%.Another 137 resistance carbapenem PA was not detected in any of carbapenem,therefore,to product MBLs are not the main cause of resistance PA to carbapenem antibacterial in our hospital.IMP,VIM- type MBLs was amplified by PCR,and DNA sequencing was identified as PA produced VIM-2 type enzyme.Drug sensitivity test results show that imipenem and meropenem were resistant,suggested that metal enzyme can hydrolysis the two antimicrobial agents at the same time,but both sensitivity to Polymyxin B and aztreonam.4 strains of PA,are producing metal VIM-2 enzyme,transconjection testing showed that the donor bacteria can be transferred resistance,suggesting that the gene lied in the plasmid,and can transfer their resistance by plasmid.In addition,the study indicated that 4 MBLs strains are carried a classâ… integron,and plays an important role in multi-drug resistant bacterial.3.study on outer membrane protein mechanism of Carbapenem-Resistant Pseudomonas aeruginosa.Carbapenem antibiotic is an important antibiotics to treatment of PA infection,with the antimicrobial agents in a broad clinical application,resistance of PA to carbapenem are the rapidly rising.Carbapenem is a kind of small molecular weight hydrophilic ofβ-lactam antibiotics,and can be passed on the outer membrane of the bacteria which are OprC,OprD₂,OprE.In this study,of 141 carbapenem resistant(140 meropenem resistancee) PA,outer membrane OprD₂ encoding gene was amplified and studied by PCR,useing SDS-PAGE analysis OprD₂ deletion,Results showed that the gene encoding of OprD₂ happened significant gene mutation in 141 imipenem resistant strains,variable sites show diversity.OprD₂ gene encoding of 34 strains has a large fragment missing,OprD₂ encoding gene of 6 strains has insert fragment,OprD₂ the gene encoding sequencing of 96 resistant strains were found small fragments missing or different location of multi-point mutation. the gene encoding of 4 MBLs strains and 1 imipenem sensitive(meropenem resistant) strains is normal,suggesting that OprD₂ gene mutation is the main mechanism for PA to imipenem resistance.The results revealed that OprD₂ of 15 imipenem resistant strains were reduced by SDS-PAGE.the sequencing of the encoding gene of OprD₂ were found three fragments in a big loss,and the remaining 12 are small fragments missing or different location of multi-point mutation,suggesting that OprD₂ gene mutation is the molecular basis of OprD₂ loss.4.Study on multi-drug effiux pump mechanism of Carbapenem-resistant Pseudomonas aeruginosaFor understanding the relationship between efflux pump system and carbapenem resistance,multidrug efflux pump of 141 carbapenem resistance PA(of which 95 imipenem resistance) have been studied.MIC of 141 multi-drug resistant PA to meropenem is determined by Agar dilution method with and without efflux pump inhibitor MC207110(20μg/ml).The results indicated that effiux pump inhibitor MC207110 can make MIC of 94 meropenem resistant strains to meropenem lower four times or more than single drug,accounting for 69.1%.Randomly 20 meropenem resistant strains were selected,which is meropenem MIC decreased four times or more by effiux pump inhibitor test.using real-time PCR,four kinds of effiux pump mRNA expression levels are increased in 20 meropenem resistant strains.MexEF-OprN have ten,MexCD-oprJ have five,MexAB-OprM have four,MexXY-OprM at least,only three.Among them,two MexA and MexC gene mRNA expression levels increased at the same time.Results indicated resistance of clinical resistant bacteria to Meropenem may caused by the over-expression of these efflux pump.efflux pump over-expression often contribute to the upper control gene mutation.,of 20 meropenem resistant PA,the four types of effiux pump control gene sequencing results showed,three are MexR mutation,one is nalC mutation,not found nalD mutation in over-expression of MexAB-OprM.5 MexCD-OprJ over-expression strains have occurred nfxB mutation,10 MexEF-OprN high expression strains have mexT mutation,3 MexXY-OprM high expression strains have mexZ gene mutation,suggesting that these efflux pump system over expression was mainly due to the corresponding regulation gene mutation.5.Study on resistant mechanism of Pandrug-resistant Pseudomonas aeruginosaResistance rate of Pseudomonas aeruginosa to the carbapenem increased significantly in recent years,even appeared the pandrug resistant PA(PDR-PA)to all of the anti-bacteria drug resistance.To understand the pandrug resistant mechanism,we screened 19 PDR-PA ESBLs,and results showed that 17 VEB-3 positive,1 OXA-10 positive at the same time. Plasmid AmpC enzyme were negative in 19 pandrug resistant strains,carbapenem enzyme tests were negative.OprD₂ encoding gene sequencing analysis revealed that mutation were in all of strains.Pump inhibitor(MC207110) and meropenem collaborative detected 19 pandrug resistant strains effiux pump showed that 16 meropenem MIC values more than four-fold lower,suggesting that these strains have effiux pump mechanism.19 have gyrA mutation occurred,14 of them have parC mutation simultaneously,and found no gyrB and parE mutation,Qnr gene tests were negative.16 kinds of aminoside modifying enzyme gene test results show that 19 strains pandrug PA have ant(3")â… ,aac(3)â…¡,ant(4')â…¡,ant(2") and ant(6)â… five kinds were positive,respectively,19,18,1,1 and 1.â… type integron of 15 PDR-PA were positive.These results suggested that the resistance of pan-resistant Pseudomonas aeruginosa was caused by comprehensive the multi-drug resistance mechanism.