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 .
 background and objectives : pseudomonas aeruginosa ( pa ) is one of the most important causes of nosocomial infections and has an intrinsic resistance to many antibiotics . among all the resistance - nodulation - division ( rnd ) 
 pumps of p. aeruginosa , mexab - oprm is the first efflux pump found to target multiple classes of antibiotics . 
 this study was aimed to evaluate the expression level of genes expressing mexab - oprm in clinical isolates of p. aeruginosa.materials and methods : in this study , 45 p. aeruginosa strains were isolated from patients admitted to children s medical center hospital , an iranian referral hospital . disk diffusion and minimum inhibitory concentration ( mic ) methods 
 were used for determination of the patterns of resistance to antibiotics . 
 real - time pcr was used to investigate the expression level of genes of mexab - oprm efflux pump.results:among 45 resistant pa isolates , the frequency of genes overexpression was as follows : mexa ( n=25 , 55.5% ) , mexb ( n=24 , 53.3% ) and oprm ( n=16 , 35.5% ) . 
 in addition , in 28 strains ( 62% ) overexpression was observed in one of the studied three genes of mexab - oprm efflux pump.conclusion:in our study 28 isolates ( 62% ) had increased expression level of efflux pumps genes , mexab - oprm . although the efflux pumps play important roles in increasing the resistance towards different antibiotics but the role of other agents and mechanisms in evolution of resistance should not be ignored . since the concomitant overproduction of other mex efflux systems might have additive effects on antibiotic resistance , the co - expressing of a multicomponent efflux pump is recommended . on the other hand , 
 the concomitant overproduction of two mex pumps might have additive effects on resistance to antibiotic . therefore co - expressing of mex efflux systems is recommended . 
INTRODUCTION
MATERIALS AND METHODS
Bacterial strains.
Antimicrobial susceptibility tests.
Isolation of total RNA and cDNA synthesis for RT-PCR.
PCR reaction.
Real-time PCR reaction.
RESULTS
Demographic data.
Antibacterial susceptibility testing.
PCR and Real-time PCR.
DISCUSSION
CONCLUSION