MexAB-OprM主动外排系统在全耐药铜绿假单胞菌耐药中的作用及机制

MexAB-OprM*邓芳　徐元宏　沈继龙MexAB-OprM()L--L---(MC-207,110)(CIP),26MIC;RT-PCRMexAB-OprMMexBmexB16SrDNA,mexB16SrDNA,mexBmRNA,MexAB-OprM;PCRMexAB-OprMmexR,BlastGenBankMC-207,11020(76.9%)CIPMIC4;2622MexAB-OprM,15mexR,13,,2,;336G-A603G-A660G-A584C-G653T-A,103Ala-Gly126Val-Glu,470474DOI:10.3969/j.issn.1671-2587.2010.01.003*(No.09020103002):230001,()();();():(1974-),,,,,,(Tel)13966535780:,,,(E-mail)[e-mail]/L,(P0.05),(P0.05)1mol/L,,,,[6,7],,(25.325.62)mol/L,(8.320.97)mol/L,,,,1,,,.[J].,2008,16(6):1442-1446.2OhtakeS,ScheborC,PalecekS,etal.Phasebehavioroffreeze-driedphospholipids-cholesterolmixturesstabi-lizedwithtrehalose[J].BiochimBiophysActa,2005,1713(1):57-64.3,,,.[J].,2008,21(1):28-31.4,,,.[J].,2008,10(2):106-109.5QuanG,ZhangL,GuoY,etal.Intracellularsugarsim-provesurvivalofhumanredbloodcellscryopreservedat-80inthepresenceofpolyvinylpyprolidoneandhumanserumalbum[J].CryoLetters,2007,28(1):95-108.6,,,.[J].,2006,14(3):592-596.7,,,.[J].,2006,14(3):605-609.(:2009-09-07)(:)6JClinTransfusLabMed,Jan.2010,Vol12,No.1GGC,6668AlaMexAB-OprM,mexRMexAB-OprMRT-PCRR378.99R392.11A1671-2587(2010)01-0006-05TheContributionandMechanismofMexAB-OprMActiveEffluxSysteminPan-resistantPseudomonasAeruginosaDENGFang,XUYuan-hong,SHENJi-long.DepartmentofMicrobiologyandParasitologyofAnhuiMedicalUniversity,Hefei230001AbstractObjectiveToinvestigatetheeffectofMexAB-OprMactiveeffluxsysteminpan-resistantPseudomonasaeruginosa(PRPA)andmechanismsofoverexpressionofMexAB-OprM.MethodsToobservetheinhibitionofeffluxpumpinhibitors(EPIs)MC-207,110.ThelevelsofmexBmRNAofwildstrain(PA01)and26strainsofPRPAwerede-terminedbyRT-PCR,the16SrRNAgenewasalsoamplifiedascontrol,thelevelsofrelativemexBmRNAwereanalyzedaccordingtotheratioofelectrophoresisscanresultsoftheirPCRproductsandthe16SrDNAs.mexRwasamplifiedwithPCRandPCRproductswereanalyzedwithDNAsequencing.ResultsTheexcretionofCIPcanbeinhibitedbyMC-207,110in20strains(76.9%)ofPRPA.ThelevelsofmexBmRNAin22strainsofPRPAweresignificantlyhigherthanthatofPA01.Among22strainsoverexpressingMexAB-OprM,15carriedmutationsinmexR.Thepointmutationswere336G-A,603G-A,660G-A,584C-G,653T-A,thetwolattersresultedinthesubstitutionofaminoacids,whichwere103Ala-Gly,126Val-Glurespectively.Theinsertionmutation(insertionofGGCbetween470and474)resultedininsertionofGlybe-tween66and68.ConclusionsTheoverexpressionofMexAB-OprMactiveeffluxsystemareassociatedwithresistanceofpseudomonasaeruginosa.ThemutationsofmexRgenemaybeoneofthereasonsforhighlevelexpressionofMexAB-O-prM.KeywordsPseudomonasaeruginosaActiveeffluxMexAB-OprMRT-PCR(Pseudomonasaeruginosa,PA),,(multidrugresistantPseudomonasaeruginosa),(pan-re-sistantPseudomonasaeruginosa,PRPA)PA,[1]PRPA,MexAB-OprMPA,MexAB-OprMmexR,mexRMexAB-OprM1TrizolReagentInvitrogen,ReverseTranscriptionSystemPromega,mexBmexR16SrDNADNAMarkerrTaqDNAPoly-merasedNTPMixture10PCRBuffer220061200712,26,MicroScanWalkAWay-40()ATCC27853,PA013(MIC)(CLSI)MIC,ATCC27853,(PIP)/(P/T)(CAZ)(FEP)(ATM)(IMP)(MEM)(GM)(AMK)(CIP)(LVX)(GAT)4MC207,110MIC(CIP)MC207,110(20g/ml),MIC5RT-PCRMexAB-OprM5.1:1,7201011211mexB16SrDNAmexR()(bp)mexB5-GTGTTCGGCTCGCAGTACTC-35-AACCGTCGGGATTGACCTTG-35424416SrDNA5-GGAGGAAGGTGGGGATGACG-35-ATGGTGTGACGGGCGGTGTG-361241mexR5-ACCAATGAACTACCCCGTGA-35-AATGTTCTTAAATATCCTCAA-3544535.2RNA:PA5mlLB,37200r/min,2ml10000r/min1min;1mlTrizol,,10min,5min,0.2ml(1/5),15s,3min,12000r/min,415min;EP,0.2ml,,10min,12000r/min,410min;,1ml75%RNA,,7500r/min,45min;,RNA,20lRNADEPCRNA,RNA,A260/A2802.0(0.1%DEPC)5.3cDNAPCR:RNA5l,7010min;20l,ddH2O4.9l,MgCl24l,Buffer2l,dNTPs2l,Rand-primer1l,Rnsin0.5l,AMV0.6l;10min,4260min,955min,42l,25l,dH2O17.375l,10Buffer2.5l,dNTPs2l,1l(10mol/L),2l,TaqDNA0.125lPCR:9510min;9530s,30s,7230s,30;725minABI7000DNAPCR1%(0.1l/ml),BIO-RADGelDocEQ,PCR6PCRmexR25l,dH2O17.375l,10Buffer2.5l,dNTPs2l,1l(10mol/L),2l,TaqDNA0.125lPCR:9510min;9530s,5430s,7230s,30;725minABI7000DNAPCR1%(0.1l/ml)PCR,GenBank1MC207,110PAMC207,11026PACIPMIC2,20(76.9%)MIC42mexBmRNAmexB16SrDNA244241bp,PA01mexBmRNA1.19(2,1),mexBmRNA1.19,1.19,mexB,22MexAB-O-prM3mexR22MexB-OprMmexR(2),PCR,15mexR,13,2(3)2GenBankGQ301036GQ301037PA,PA,PA,12,7[2-4],RND,MexAB-OprMMexCD-O-prJMexEF-OprNMexXY-OprMMexJK-Opr8JClinTransfusLabMed,Jan.2010,Vol12,No.1226MICMIC(mg/Liter)CIPCIP+MC-207,110MexBPA0120.51.191440.53.531516160.48181643.08191642.97211681.74251623.49301682.823216160.77351623.263716160.09381643.28401642411640.92531643.9561642.67571642.15581643.02591641.43621624.75651642.08661681.5811642.85901641.8951623.231001641.351091643.08:MexBmexBmRNAM:DL2000,12:mexB16SrDNA,34:PA14mexB16SrDNA1MexBmRNAM:DL2000,1-10:mexRPCR2mexBPCRMMexGHI-OpmDMexVW-OprMPA:,OprMOprJOprN,,;,MexBMexDMexFMexY,,;,Mex-AMexCMexEMexX,,,MexAB-OprM13mexRPA14584C-G,653T-A103AlaGly126ValGluPA18,PA35470474GGC6668AlaPA19,PA21,PA25,PA30,PA38,PA53,PA56,PA57,PA58,PA62,PA95,PA109336G-A,603G-A,653T-A,660G-A126Val-GluPA40,PA59,PA65,PA66,PA81,PA90,PA100:mexR(U23763)RND,MexAB-OprM[2-4]MexAB-OprMPA,,,L--L---(MC-207,110),(MexAB-OprM,MexCD-OprJ,MexEF-OprNMexXY-OprM)920101121[5],,,,MC-207,110MexAB-Opr