emergence of bacterial resistance to many different antibiotics is considered as a great concern in human health . pseudomonas aeruginosa ( pa ) has been recognized as one of the significant pathogens of nosocomial infections ( 1 ) . key mechanism of antibiotic resistance in p. aeruginosa is the expulsion of antibiotics through multidrug resistance ( mdr ) efflux systems belonging to the resistance - nodulation - division ( rnd ) family ( 2 ) . mexab - oprm , mexcd - oprj , mexef - oprn , mexxy , mexjk and mexvw contribute the most significantly to antibiotic resistance ( 3 ) and play an important role in intrinsic and acquired multidrug resistance ( 2 ) . resistance of p. aeruginosa to multiple antibiotics is largely attributable to expression of the mexab - oprm efflux pump ( 4 ) . among all the rnd pumps of pa , mex - ab - oprm was the first efflux pump found to target multiple classes of antibiotics including -lactam ( carboxypenicillins , aztreonam , extended - spectrum cephalosporins , penems , the carbapenems such as meropenem and panipenem except imipenem and biapenem ) ; fluoroquinolones , tetracyclines , chloramphenicol , macrolides , novobiocin , trimethoprim and sulfonamides ( 5 , 6 ) . the mexab - oprm efflux pump belongs to the superfamily of ribonucleoproteins and consists of an inner membrane ( mexb ) , a periplasmic membrane fusion protein ( mexa ) and a channel - forming outer membrane protein , oprm ( 7 ) . in this study , we investigated the role of mex - ab - oprm efflux pump . transcription level of efflux pump genes mexa , mexb , oprm , mexr and ampc were analyzed using real - time pcr . p. aeruginosa strains were isolated from patients of children s hospital medical center during 6 months ( march and august 2012 ) . all bacterial isolates were identified at microbiology lab using standard biochemical identification methods ( 8) . a total of 45 samples were collected from various clinical specimens such as urine ( n=21 ) , exudates ( n=11 ) , eye ( n=2 ) , ear ( n=2 ) , csf ( n=2 ) , blood ( n=2 ) , trachea ( n=2 ) and lung secretions ( n=3 ) . p.aeruginosa pao1 strain which has entirely sequenced genome was used as the reference wild type strain throughout the study . antimicrobial susceptibility tests were performed using disk diffusion and minimum inhibitory concentration ( mic ) methods . antibiotics used in this study were chosen randomly from different antibiotic classes that were used mostly in this hospital . antibiotics used in the disk diffusion method were cephalothin ( 30 g ) , cefepime ( 30 g ) , ceftazidime ( 30 g ) , ciprofloxacin ( 5 g ) ( flouroquionolones ) , meropenem ( 10 g ) , imipenem ( 10 g ) ( carbapenems ) , piperacillin / tazobactam ( 10/100 g ) ( -lactams ) , gentamicin ( 10 g ) and amikacin ( 30 g ) ( aminoglycosides ) . antibiotics used in this study were cefuroxime , ceftazidim , cefazolin , ceftriaxone ( cephems ) , meropenem ( carbapenems ) , amikacin , tobramycin ( aminoglycosides ) , aztreonam ( monobactam ) , ampicillin , piperacillin ( penicillins ) , colistin ( lipopeptides ) , gatifloxacin , nalidixic acid ( flouroquionolones ) , piperacillin - tazobactam , tazobactam ( -lactams ) ( mast , uk ) . total rna was extracted using rna extraction kit ( fermentas , lithuania ) , and converted into cdna using the cdna synthesis kit ( fermentas , lithuania ) according to the manufacturer s instruction and the quality and purity of the rna obtained was evaluated using spectrophotometer . the final optimized pcr reaction consisted of 1 l of each primer ( 10pmol ) ( table 1 ) , 0.5 l dntp ( 10 mm ) , 0.5 l mgcl ( 100 mm ) , 0.2 l ( 1 unit ) taq dna polymerase ( metabion , germany ) , 2.5 l pcr buffer ( 10x ) , and 0.5 l of dna template ( 100g / ml ) in total volume of 25 l with double distilled water . the cycling program was adjusted as follows : initial denaturation at 94 c for 5 min followed by 30 cycles of 94 c for 45 sec , 5072 c ( 45 sec ) , 72 c ( 1 min ) and a final extension at 72 c for 10 min ( 9 ) . primers used in pcr and real - time pcr real - time quantification of cdna was carried out on an abi step one detection system ( applied biosystems , uk ) using the sybr green pcr master mix . real - time pcr was used to investigate the expression level of each gene in mexab - oprm efflux pump to expression each of these genes of separately measured by relative quantitation real - time pcr . the optimized reaction consisted of master mix ( 10x ) , 1 l of each primer ( 10 pmol ) , and 0.5 l of template dna ( 100 g / ml ) in a total volume of 20 l p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . ampc gene was considered as a housekeeping gene and all gene expressions were compared with ampc gene expression . the assay was performed three times for each sample and the mean of three obtained quantities was considered as quantity . cycle of threshold ( c 

 t 
 ) was considered as the average threshold cycle number from three independent experiments . the real - time pcr apparatus was programmed as follows : initial denaturation at 95 c ( 15 min ) followed by 40 cycles of 95 c ( 15 sec ) , 61 c ( 15 sec ) , 71 c ( 20 sec ) and melt curve at 61 c ( 1 min ) and 95 c for 15 sec . to obtain reproducibility of the reaction ampc gene was considered as a housekeeping gene and the results were compared with gene expression in a susceptible pa reference strain . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification had occurred , melting curves of each amplicon were assessed p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . real - time pcr efficiencies were acquired by amplification of a standardized dilution series of the template cdna and were determined for each gene as the slope of a linear regression model . pcr efficiency was determined by measuring the ct ct to a specific threshold for a serial dilution of cdna . the corresponding real - time pcr efficiencies were then calculated according to the equation : e = ( 101 )  100 ( 1012 ) . p. aeruginosa strains were isolated from patients of children s hospital medical center during 6 months ( march and august 2012 ) . all bacterial isolates were identified at microbiology lab using standard biochemical identification methods ( 8) . a total of 45 samples were collected from various clinical specimens such as urine ( n=21 ) , exudates ( n=11 ) , eye ( n=2 ) , ear ( n=2 ) , csf ( n=2 ) , blood ( n=2 ) , trachea ( n=2 ) and lung secretions ( n=3 ) . p.aeruginosa pao1 strain which has entirely sequenced genome was used as the reference wild type strain throughout the study . antimicrobial susceptibility tests were performed using disk diffusion and minimum inhibitory concentration ( mic ) methods . antibiotics used in this study were chosen randomly from different antibiotic classes that were used mostly in this hospital . antibiotics used in the disk diffusion method were cephalothin ( 30 g ) , cefepime ( 30 g ) , ceftazidime ( 30 g ) , ciprofloxacin ( 5 g ) ( flouroquionolones ) , meropenem ( 10 g ) , imipenem ( 10 g ) ( carbapenems ) , piperacillin / tazobactam ( 10/100 g ) ( -lactams ) , gentamicin ( 10 g ) and amikacin ( 30 g ) ( aminoglycosides ) . antibiotics used in this study were cefuroxime , ceftazidim , cefazolin , ceftriaxone ( cephems ) , meropenem ( carbapenems ) , amikacin , tobramycin ( aminoglycosides ) , aztreonam ( monobactam ) , ampicillin , piperacillin ( penicillins ) , colistin ( lipopeptides ) , gatifloxacin , nalidixic acid ( flouroquionolones ) , piperacillin - tazobactam , tazobactam ( -lactams ) ( mast , uk ) . total rna was extracted using rna extraction kit ( fermentas , lithuania ) , and converted into cdna using the cdna synthesis kit ( fermentas , lithuania ) according to the manufacturer s instruction and the quality and purity of the rna obtained was evaluated using spectrophotometer . the final optimized pcr reaction consisted of 1 l of each primer ( 10pmol ) ( table 1 ) , 0.5 l dntp ( 10 mm ) , 0.5 l mgcl ( 100 mm ) , 0.2 l ( 1 unit ) taq dna polymerase ( metabion , germany ) , 2.5 l pcr buffer ( 10x ) , and 0.5 l of dna template ( 100g / ml ) in total volume of 25 l with double distilled water . the cycling program was adjusted as follows : initial denaturation at 94 c for 5 min followed by 30 cycles of 94 c for 45 sec , 5072 c ( 45 sec ) , 72 c ( 1 min ) and a final extension at 72 c for 10 min ( 9 ) . real - time quantification of cdna was carried out on an abi step one detection system ( applied biosystems , uk ) using the sybr green pcr master mix . real - time pcr was used to investigate the expression level of each gene in mexab - oprm efflux pump to expression each of these genes of separately measured by relative quantitation real - time pcr . the optimized reaction consisted of master mix ( 10x ) , 1 l of each primer ( 10 pmol ) , and 0.5 l of template dna ( 100 g / ml ) in a total volume of 20 l p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . ampc gene was considered as a housekeeping gene and all gene expressions were compared with ampc gene expression . the assay was performed three times for each sample and the mean of three obtained quantities was considered as quantity . cycle of threshold ( c 

 t 
 ) was considered as the average threshold cycle number from three independent experiments . the real - time pcr apparatus was programmed as follows : initial denaturation at 95 c ( 15 min ) followed by 40 cycles of 95 c ( 15 sec ) , 61 c ( 15 sec ) , 71 c ( 20 sec ) and melt curve at 61 c ( 1 min ) and 95 c for 15 sec . to obtain reproducibility of the reaction ampc gene was considered as a housekeeping gene and the results were compared with gene expression in a susceptible pa reference strain . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification had occurred , melting curves of each amplicon were assessed p. aeruginosa strain pao1 was used as a standard strain for normalization of relative mrna levels . real - time pcr efficiencies were acquired by amplification of a standardized dilution series of the template cdna and were determined for each gene as the slope of a linear regression model . pcr efficiency was determined by measuring the ct ct to a specific threshold for a serial dilution of cdna . the corresponding real - time pcr efficiencies were then calculated according to the equation : e = ( 101 )  100 ( 1012 ) . totally , 45 strains of p. aeruginosa were isolated from patients aged 2 months to 12 years who were referred to children s medical center hospital , tehran , iran . the majority of isolates were collected from patients hospitalized in picu ( n=18 , 39% ) , nicu ( n=5 , 11% ) followed by emergency ward ( n=3 , 7% ) , nephrology ( n=3 , 7% ) , surgery ( n=5 , 11% ) , neurology ( n=3 , 7% ) , urology ( n=5 , 11% ) and infectious ward ( n=3 , 7% ) . the most antibiotic resistance based on clsi reference guidelines for disk diffusion method was detected for cephalothin ( n=41 , 92% ) whereas mics should the following frequencies for antibiotic resistance : cefuroxime ( n=41 , 91% ) , ceftazidime ( n=42 , 93% ) , amikacin ( n=38 , 84.5% ) , cefazolin ( n=40 , 89% ) , aztreonam ( n=42 , 93% ) , piperacillin ( n=39 , 86.5% ) , tazobactam ( n=41 , 91% ) and piperacillin - tazobactam ( n=42 , 93% ) . antibiotics such as colistin ( 15% ) , ceftriaxone ( 33% ) and tobramycin ( 22% ) showed the highest susceptibility rates against the isolates ( table 2 ) . pattern of determined antibiotic resistance among 

 p. aeruginosa 

 strains using disk diffusion and mic methods the best annealing temperature was obtained at 61 c . the results of real - time pcr for the genes of mexab - oprm efflux pump have been shown in table 3 . according to the results of antibiotic resistance via disk diffusion method , overexpression of mexab - oprm genes was associated to the resistance towards cephalosporin while via mic method overexpression of mexab - oprm was seen in those showing resistance towards special antibiotics especially fluoroquinolones , cephalosporin and beta lactams . the expression of mexab - oprm efflux pump genes in 

 p. aeruginosa 

 strains isolated from children the reproducibility of the expression levels of each gene was measured three times . p. aeruginosa clinical isolates demonstrated increased level of mexa (  2 folds ) , mexb ( from 2.2 to 12.0 folds ) , oprm (  2 folds ) and ampc (  10 folds ) at transcriptional mrna level , respectively . confirmation of specific amplifications during real - time pcr on mexab - oprm genes of efflux pump among 45 resistant isolates , overexpression of mexa gene was observed in 25 isolates ( 55.5% ) , mexb in 24 isolates ( 53.3% ) and oprm in 16 isolates ( 35.5% ) . in 28 isolates ( 62% ) , overexpression was observed in one of the three genes of mexab - oprm efflux pump . the ratio values obtained for each of the four genes in samples showed that the expression levels of mexa gene ( n=25 , 89% ) was between 1.8 and 11.7 ( mean= 6.7 ) , mexb ( n=24 , 85% ) between 1.3 and 14 ( mean= 7.6 ) and oprm gene ( n=16 , 57% ) between 1 and 9 ( mean= 5 ) . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification ( fig . totally , 45 strains of p. aeruginosa were isolated from patients aged 2 months to 12 years who were referred to children s medical center hospital , tehran , iran . the majority of isolates were collected from patients hospitalized in picu ( n=18 , 39% ) , nicu ( n=5 , 11% ) followed by emergency ward ( n=3 , 7% ) , nephrology ( n=3 , 7% ) , surgery ( n=5 , 11% ) , neurology ( n=3 , 7% ) , urology ( n=5 , 11% ) and infectious ward ( n=3 , 7% ) . the most antibiotic resistance based on clsi reference guidelines for disk diffusion method was detected for cephalothin ( n=41 , 92% ) whereas mics should the following frequencies for antibiotic resistance : cefuroxime ( n=41 , 91% ) , ceftazidime ( n=42 , 93% ) , amikacin ( n=38 , 84.5% ) , cefazolin ( n=40 , 89% ) , aztreonam ( n=42 , 93% ) , piperacillin ( n=39 , 86.5% ) , tazobactam ( n=41 , 91% ) and piperacillin - tazobactam ( n=42 , 93% ) . antibiotics such as colistin ( 15% ) , ceftriaxone ( 33% ) and tobramycin ( 22% ) showed the highest susceptibility rates against the isolates ( table 2 ) . pattern of determined antibiotic resistance among 

 p. aeruginosa 

 strains using disk diffusion and mic methods the results of real - time pcr for the genes of mexab - oprm efflux pump have been shown in table 3 . according to the results of antibiotic resistance via disk diffusion method , overexpression of mexab - oprm genes was associated to the resistance towards cephalosporin while via mic method overexpression of mexab - oprm was seen in those showing resistance towards special antibiotics especially fluoroquinolones , cephalosporin and beta lactams . the expression of mexab - oprm efflux pump genes in 

 p. aeruginosa 

 strains isolated from children the reproducibility of the expression levels of each gene was measured three times . p. aeruginosa clinical isolates demonstrated increased level of mexa (  2 folds ) , mexb ( from 2.2 to 12.0 folds ) , oprm (  2 folds ) and ampc (  10 folds ) at transcriptional mrna level , respectively . confirmation of specific amplifications during real - time pcr on mexab - oprm genes of efflux pump among 45 resistant isolates , overexpression of mexa gene was observed in 25 isolates ( 55.5% ) , mexb in 24 isolates ( 53.3% ) and oprm in 16 isolates ( 35.5% ) . in 28 isolates ( 62% ) , overexpression was observed in one of the three genes of mexab - oprm efflux pump . the ratio values obtained for each of the four genes in samples showed that the expression levels of mexa gene ( n=25 , 89% ) was between 1.8 and 11.7 ( mean= 6.7 ) , mexb ( n=24 , 85% ) between 1.3 and 14 ( mean= 7.6 ) and oprm gene ( n=16 , 57% ) between 1 and 9 ( mean= 5 ) . primer dimers and other artifacts were evaluated by melting curve analysis . to confirm that specific amplification ( fig . p. aeruginosa is one of the most important causes of nosocomial infections due to the presence of various resistant elements ( 11 ) . during last decades , the emergency of multidrug resistant p. aeruginosa has been observed worldwide . in this study , 2835% of studied p. aeruginosa isolates were resistant to carbapenems which was in accordance with previous studies ( 13 , 14 ) . high frequency of cephalosporin resistance was also observed among p. aeruginosa isolates similar to the other reports ( 14 , 15 ) . among all isolates , resistance to piperacillin / tazobactam was low ( 2627% ) which was in agreement with our previous report ( 13 ) and was higher than ghazi et al . report ( 16 ) . in this study colistin showed 15% resistance which can be suggested as an effective antibiotic for treatment of pa infections . this finding is similar to the results of study performed by alekshun et al . in us ( 17 ) . at present , the efflux pump has been recognized as one of the significant complexes involved in resistance to most of the classes of antibiotics ( 11 , 18 , 19 ) . it has been reported that the prevalence of efflux pump overexpression in clinical p. aeruginosa strains of ranged from 1475% ( 20 , 21 ) . there are rare reports on prevalence of efflux pump overexpression in our country ( 22 , 23 ) and there is no investigation on pa strains isolated from children . in the present study the increased expression level of mexab - oprm genes of efflux pump simultaneously was 25% which was relatively more than dumas et al . ( 24 ) . in this study , 28 of 45 patients ( 62% ) showed an increased expression level of efflux pumps mex - ab - oprm genes that was similar to the studies reported more than 50% overexpression level of these genes ( 2427 ) . in arabestani study all the isolates ( n=31 ; 100% ) showed overexpression of efflux pump mexab - oprm genes ( 23 ) . according to aghazadeh et al . report , overexpression of mexa was 74% among the isolates ( 28 ) . since the concomitant overproduction of two mex pumps might have additive effects on being resistant to antibiotics ( 29 ) evaluating the co - expression of multi - component efflux pumps other than mexab - oprm is recommended . development of novel antibiotics that can bypass the effects of efflux pumps is still a challenging task . further studies on involved mechanisms and structure - function association of bacterial efflux systems as well as the interactions between the pumps and other resistance mechanisms are highly recommended .