Mechanism Of MexAB-OprM Efflux Pump In Carbapenem-resistant Pseudomonas Aeruginosa

Part?Overexpression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosaObjective Efflux pump systems are one of the most important mechanisms conferring multidrug resistance in P.aeruginosa.MexAB-OprM efflux pump is one of the largest multi-drug resistant efflux pumps with high level expression,which is controlled by regulatory genes mex R,nal C,and nal D.In order to understand the expression of MexAB-OprM efflux pump in carbapenem-resistant Pseudomonas aeruginosa(CAPA),and the influence of regulatory genes mex R,nal C,nal D to the resistant mechanism.Methods This study investigated the role of efflux pump MexAB-OprM in 75 strains of carbapenem-resistant P.aeruginosa and evaluated the influence of point mutation of the regulatory genes.The minimum inhibitory concentrations of imipenem and meropenem,with or without MC207110,an efflux pump inhibitor,were determined by broth microdilution method to select the positive strains for an over-expressed active efflux pump.Carba NP test and EDTA-disk synergy test were used for detection of carbapenemase and metallo-?-lactamases,respectively.The gene mex A,responsible for the fusion protein structure,and the reference gene rpo D of the MexAB-OprM pump were amplified by real-time PCR.The quantity of relative m RNA expression wasdetermined simultaneously.By PCR method,the efflux regulatory genes mex R,nal C,and nal D and outer membrane protein Opr D2 were amplified for the strains showing over expression of MexAB-OprM,and subsequently analyzed by BLAST.Results Among the 75 P.aeruginosa strains,the prevalence of efflux pump positive phenotype was 17.3%(13/75).Carba NP test and EDTA-disk synergy test were all negative in the 13 strains.PCR assay results showed that 10 strains overexpressed the MexAB-OprM efflux pump,and were all positive for the regulatory genes mex R,nal C,and nal D.Sequence analysis indicated that of the 10 isolates,9 had a mutation(Gly?Glu)at 71 st amino acid position in Nal C,and 8 also had a mutation(Ser?Arg)at 209 th position in Nal C.Only one strain had a mutation(Thr?Ile)at the 158 th amino acid position in Nal D,whereas 8 isolates had mutations in Mex R.Conclusion overexpression of efflux pump MexAB-OprM plays an important role in carbapenem-resistant P.aeruginosa.The mutations of regulatory genes may be a main factor contributing to overexpression of MexAB-OprM.Part? MexAB-OprM efflux pump system in imipenem resistant pseudomonas aeruginosaObjective Pseudomonas aeruginosa is one of the main opportunistic pathogen in clinical,more attention paid to the study of its resistance mechanism because of the highresistant rate to the carbapenems.MexAB-OprM efflux pump is one of the important multi-drug resistant efflux pumps to carbapenems,and its related to the meropenem but no to the imipenem.In our study,we research one strain of pseudomonas aeruginosa which imipenem inhibited by efflux pump inhibitor but meropenem not inhibited.We used homologous recombination technology to knock out the fusion protein gene mex A of MexAB-OprM efflux pump,to study the function of the MexAB-OprM to imipenem resistance in pseudomonas aeruginosa.Methods We carried the homologous recombination method according to the RecBCD system of the bacterial to realize allelic replacement of target gene.Firstly,upstream and downstream homologous arm(A,B fragment)of mex A gene were amplified by PCR,and the fusion fragment of AB fragment were amplified by overlap PCR.The suicide vector p LP12 T was ligated with the AB fragment,ligation product were transformed into competent cell of E.coli DH5? ?pir.And the product screed by PCR which using primer p LP-UF/ p LP-UR,and extracted plasmid p LP12-mex A transformed into E.coli ?2163 by electroporation.Then the recombination plasmids were transferred into pseudomonas aeruginosa.Mating mixtures were then deposited on L-B plate which wit DAP and D-glucose,donor strain E.coli ?2163 cannot grow on LB plates without(DAP).Under pressure from antibiotic selection(Cm=200?g/ml),only the inserted mutate strain which integrated plasmids into specific chromosomal loci could survived.Then the inserted mutant strains were spread on L-B plates supplemented with L-arabinose for counterselecting the deletion mutant(double-crossover recombination).All of the deletion mutants were amplified by PCR and subsequent sequencing to confirmed the succeed of mex A gene knocked out.And the strain which knock-out mex A gene were tested related expression of mex A and the sensitivity to the imipenem.Results Thefragment A(618bp)and B(416bp)were upstream and downstreamhomologous arm of mex A,and the overlap PCR resulted the AB fusion fragment(993bp).The AB fragment was successful ligated with suicide vector p LP12 T,the PCR results show that the ligation production longer than un-ligation production.The ligation production p LP12-mex A were successful conjugated into the pseudomonas aeruginosa by using highly efficient conjugation system provided by E.coli ?2163.Under pressure from antibiotic selection(Cm=200?g/ml),only the inserted mutate strain which integrated plasmids into specific chromosomal loci could survived.The PCR results show that the inserted mutant with two band,but the wild strain only with one band.Then the inserted mutant strains were spread on L-B plates supplemented with L-arabinose for counterselecting the deletion mutant.Only the deletion mutant and the wild strain can survived.The according the PCR results screening the deletion mutant,and the deletion mutant with 1403 bp fragment and the wild strain with 2159 bp fragment.The PCR product subsequent sequencing to confirmed the succeed of mex A gene knocked out.The Real-time PCR of the mex A related expression were nearly to zero,this result to further prove the mex A gene was knocked out.And the imipenem MIC were dramatic decline after knocked out the mex A gene.Conclusion We successfully deleted mex A gene from pseudomonas aeruginosa using the linearization suicide vector p LP12 T according to the principle of the homologous recombination.The imipenem MIC were dramatic decline after knocked out the mex A gene,that demonstrated the MexAB-OprM efflux pump were influence the pseudomonas aeruginosa resistant to imipenem.Part?Study on drug resistance mechanism of Pseudomonas aeruginosa to imipenem using RNA-seqObjective There exist different between the pseudomonas aeruginosa resistance mechanism to impenem and meropenem.This study was to comprehensive understand the mechanism of pseudomonas aeruginosa to impenem.Methods we conducted RNA-seq technology to compare the differential gene between A and B strains,to explore the pseudomonas aeruginosa resistant mechanism to impenem.high-throughput sequencing A,B strains,Results A,B strains were made three repeated samples,extracted the total RNA of bacteria,the RNA quality test results were qualified.The results of RNA-seq sequencing data were tested and evaluated,which indicated that the sequencing quality was good and the data could be compared.In the screening results of the differential gene,there were 630 differentially expressed genes in IPMS(sample A)and IPMR(sample B),of which 297 differential genes were up-expression and 333 differential genes were down-expression.Among them,29 genes were associated with drug resistance genes,15 of them were up-regulated and 14 genes were down-regulated.(P <0.05).The difference gene was analyzed by GO enrichment,there have 162 GO functional annotations,and 7 functional annotations significantly enrichment(P<0.05).The difference gene was analyzed by KEGG enrichment,and the functional annotations of 83 signal pathways were obtained.Four of them were obtained by the method of KEGG enrichment.KEGG was significantly enriched,including nine genes in drugresistance-related pathways.Conclusion The main mechanism of Pseudomonas aeruginosa resistance to imipenem was as follows: the expression of outer membrane protein opr D gene was down-regulated;the mex R gene negative regulated efflux pump MexAB-OprM,decreased expression of mex R gene led to the high expression of MexAB-OprM efflux pump;mex T gene positive regulation of efflux pump Mex EF-Opr N,Mex T high expression can indirectly induce efflux pump Mex EF-Opr N high expression;ampG gene coding Of the AmpG protein,induced Pseudomonas aeruginosa beta lactamase production.