pseudomonas aeruginosa is an important opportunistic pathogen with the ability to propagate on medical devices , hospital environments and even in disinfectants ( 1 ) . due to the intrinsic resistance to many antimicrobial agents , p. aeruginosa infections present serious therapeutic challenges both in the community and health centers . in addition , the organism can acquire resistance elements to multiple classes of antibacterial agents , even during the course of treatment ( 2 , 3 ) . p. aeruginosa isolates from burn wounds are often responsible for systemic sepsis , graft loss , prolonged hospital stay and mortality ( 4 ) . several mechanisms are responsible for resistance to -lactam antibiotics in p. aeruginosa including : genetic mutations that lead to stable over expression of chromosome - mediated ampc cephalosporinases , acquisition of transferable -lactamase genes , overproduction of efflux systems , and reduced permeability ( 5 ) . among the numerous -lactamases , ambler class a extended - spectrum -lactamases ( esbls ) and class b metallo -lactamases ( mbls ) are reported as rapidly growing enzymes in clinical isolates of p. aeruginosa . esbls are capable of hydrolyzing penicillins , cephalosporins , and aztreonam ( except for cephamycins or carbapenems ) . these enzymes are inhibited by -lactamase inhibitors such as clavulanic acid ( 6 , 7 ) . among more than 200 different known esbls in gram - negative bacteria , 32 are detected in p. aeruginosa belonging to class a ( tem , shv , ctx - m , per , veb , ges , bel ) , and class d ( oxa type ) -lactamases ( 8) . ampc -lactamases hydrolyze cephamycins ( e.g. cefoxitin and cefotetan ) , oxyimino - cephalosporins ( e.g. ceftazidime , cefotaxime , and ceftriaxone ) , and monobactams ( e.g. aztreonam ) ( 9 ) . in p. aeruginosa , decreased susceptibility to the extended - spectrum cephalosporins such as ceftazidime mostly results from over expression of the naturally occurring ampc -lactamases ( 10 ) . among the carbapenems , intermediate susceptibility to imipenem is widely associated with the modification of the outer membrane protein oprd , production of mbls and over expression of the cephalosporinases associated with decreased outer membrane permeability . recently , a novel resistance mechanism was shown in p. aeruginosa , where peculiar ampc -lactamases with expanded - spectrum activity toward imipenem may also contribute to decreased imipenem susceptibility ( 11 ) . the current study aimed to determine the prevalence of esbl and mbl production among ampc -lactamase producing p. aeruginosa isolated from burns and evaluate the presence of their related antibiotic resistance genes . fifty one non - duplicate p. aeruginosa isolates were randomly selected from a collection of burn isolates provided by shahid motahari hospital in 2011 . the antibiotic susceptibility patterns and mbl production were previously determined using disc diffusion and the double disc synergy tests , respectively ( 12 ) . the isolates were maintained in brain heart infusion broth ( oxoid , uk ) containing 10% dimethyl sulfoxide and stored at -20c until use . controls for polymerase chain reaction ( pcr ) tests were : p. aeruginosa pao1 containing blaampc ( provided by dr . abdi , al - zahra university , iran ) , dna from klebsiella pneumoniae 7881 harboring blatem , blashv and blactx - m , p. aeruginosa koas carrying blaper-1 and p. aeruginosa 10.2 harboring blaveb-1 were obtained from pasteur institute , tehran , iran . antibiotic susceptibility testing of the isolates was carried out by the disc diffusion method , according to the clinical and laboratory standards institute guidelines ( clsi ) ( 5 ) . the used antibiotic discs ( mast group ltd , merseyside , uk ) were : ceftazidime ( 30 g ) , aztreonam ( 30 g ) , carbenicillin ( 100 g ) , piperacillin ( 100 g ) , ticarcillin ( 75 g ) , cotrimoxazole ( 25 g ) , amikacin ( 30 g ) , cefepime ( 30 g ) , ciprofloxacin ( 5 g ) , tobramycin ( 10 g ) , meropenem ( 10 g ) , imipenem ( 10 g ) , cefoxitin ( 30 g ) , and piperacillin / tazobactam ( 100/10 g ) . minimum inhibitory concentrations ( mics ) for imipenem and ceftazidime were determined by the microdilution assay as recommended by the clsi ( 13 ) . the results showed a zone diameter of < 18 mm for cefoxitin ( 14 ) . briefly , a blank disc moistened with sterile saline , was inoculated with a few colonies of the test strain . the disc was then placed next to a cefoxitin disc ( 30 g ) on the surface of a muller hinton agar plate ( leofilchem , italy ) previously inoculated with a lawn of escherichia coli atcc 25922 . an indentation of the cefoxitin inhibition zone adjacent to the disc containing the test strain was an indication of ampc -lactamase production . ampc production was also determined using a modified three - dimensional extract test ( tdet ) ( 15 ) . briefly , bacterial colonies from overnight cultures on mueller hinton agar were transferred to a pre - weighed sterile micro centrifuge tube . the tube was weighed again to obtain 10 to 15 mg of bacterial wet weight per sample . the bacteria were then suspended in peptone water ( merck , germany ) and centrifuged at 3000 rpm ( sigma 1 - 13 microcentrifuge , germany ) for 15 minutes . crude cell extracts were prepared by 10 cycles of alternate freeze - thawing at -78c ( dry ice / ethanol bath ) and 37c . linear slits ( 3 cm ) were cut into plates inoculated with each test organism using a sterile scalpel blade , 3 mm away from a cefoxitin disc in an outward radial direction . approximately 40 to 50 l of the prepared extract was loaded into each slit and the plates were kept upright for 5 to 10 minutes followed by incubation at 37c overnight . ampc production was recorded when clear distortion of cefoxitin inhibition zones were observed ( figure 1 ) . esbl production was detected by the conventional double disc synergy test ( ddst ) using ceftazidime ( 30g ) and cefotaxime discs ( 30 g ) with or without clavulanic acid ( 10 g ) as recommended by the clsi . an increase of  5 mm in the inhibition zones of either cephalosporin in combination with clavulanic acid compared to the cephalosporin alone was interpreted as esbl production ( 13 ) . briefly , a loopful of an overnight grown culture of each test isolate as well as the control strains were suspended in 500 l of sterile double distilled water , boiled at 100c for 10 minutes and were centrifuged at 12000  g for 10 minutes . pcr amplifications were performed using specific primers for ambler class a ( blatem , blashv , blactx - m , blaveb-1 , blaper-1 ) and class c ( blaampc ) -lactamase genes ( table 1 ) . ampc amplifications were initially performed using a pair of external primers ( preampc - pa1 and postampc - pa2 ) yielding a 1243-base pair product encompassing the entire ampc gene of p. aeruginosa , but excluding its promoter sequences . another pair of primers ( ampc - pa - a and ampc - pa - b ) was also used as internal primers for ampc gene yielding a 1063 bp product ( table 1 ) . pcr experiments were carried out in 25 l volume reaction mixtures containing 10 pm of each primer , 200 m dntp , 1.4 mm mgcl2 , 1 l of crude dna template and 1u of taq dna polymerase in the reaction buffer provided by the manufacturer ( cinnagen , tehran , iran ) . amplifications were carried out in thermocycler ( techne , uk ) using the following program : five minutes denaturation at 95c followed by 30 cycles of 95c for one minute , one minute at the annealing temperature ( 43 to 63c for esbls , 62c and/or 65c for ampc ) , and one minute at 72c with a final extension period of 10 minutes at 72c . comparisons of -lactamase phenotypes and presence of the related genes were performed using the chi - square test . correlation between antibiotic resistance and -lactamase gene carriage was analyzed by the nonparametric mann - whitney u test . all statistical analyses were carried out using spss software version 19 . a p value of < 0.05 was considered statistically significant . a ; clear distortion of inhibition zone indicates ampc -lactamase production , b , c and d ; absence of distortion indicates negative test . fifty one non - duplicate p. aeruginosa isolates were randomly selected from a collection of burn isolates provided by shahid motahari hospital in 2011 . the antibiotic susceptibility patterns and mbl production were previously determined using disc diffusion and the double disc synergy tests , respectively ( 12 ) . the isolates were maintained in brain heart infusion broth ( oxoid , uk ) containing 10% dimethyl sulfoxide and stored at -20c until use . controls for polymerase chain reaction ( pcr ) tests were : p. aeruginosa pao1 containing blaampc ( provided by dr . abdi , al - zahra university , iran ) , dna from klebsiella pneumoniae 7881 harboring blatem , blashv and blactx - m , p. aeruginosa koas carrying blaper-1 and p. aeruginosa 10.2 harboring blaveb-1 were obtained from pasteur institute , tehran , iran . antibiotic susceptibility testing of the isolates was carried out by the disc diffusion method , according to the clinical and laboratory standards institute guidelines ( clsi ) ( 5 ) . the used antibiotic discs ( mast group ltd , merseyside , uk ) were : ceftazidime ( 30 g ) , aztreonam ( 30 g ) , carbenicillin ( 100 g ) , piperacillin ( 100 g ) , ticarcillin ( 75 g ) , cotrimoxazole ( 25 g ) , amikacin ( 30 g ) , cefepime ( 30 g ) , ciprofloxacin ( 5 g ) , tobramycin ( 10 g ) , meropenem ( 10 g ) , imipenem ( 10 g ) , cefoxitin ( 30 g ) , and piperacillin / tazobactam ( 100/10 g ) . minimum inhibitory concentrations ( mics ) for imipenem and ceftazidime were determined by the microdilution assay as recommended by the clsi ( 13 ) . the results showed a zone diameter of < 18 mm for cefoxitin ( 14 ) . briefly , a blank disc moistened with sterile saline , was inoculated with a few colonies of the test strain . the disc was then placed next to a cefoxitin disc ( 30 g ) on the surface of a muller hinton agar plate ( leofilchem , italy ) previously inoculated with a lawn of escherichia coli atcc 25922 . an indentation of the cefoxitin inhibition zone adjacent to the disc containing the test strain was an indication of ampc -lactamase production . ampc production was also determined using a modified three - dimensional extract test ( tdet ) ( 15 ) . briefly , bacterial colonies from overnight cultures on mueller hinton agar were transferred to a pre - weighed sterile micro centrifuge tube . the tube was weighed again to obtain 10 to 15 mg of bacterial wet weight per sample . the bacteria were then suspended in peptone water ( merck , germany ) and centrifuged at 3000 rpm ( sigma 1 - 13 microcentrifuge , germany ) for 15 minutes . crude cell extracts were prepared by 10 cycles of alternate freeze - thawing at -78c ( dry ice / ethanol bath ) and 37c . linear slits ( 3 cm ) were cut into plates inoculated with each test organism using a sterile scalpel blade , 3 mm away from a cefoxitin disc in an outward radial direction . approximately 40 to 50 l of the prepared extract was loaded into each slit and the plates were kept upright for 5 to 10 minutes followed by incubation at 37c overnight . ampc production was recorded when clear distortion of cefoxitin inhibition zones were observed ( figure 1 ) . esbl production was detected by the conventional double disc synergy test ( ddst ) using ceftazidime ( 30g ) and cefotaxime discs ( 30 g ) with or without clavulanic acid ( 10 g ) as recommended by the clsi . an increase of  5 mm in the inhibition zones of either cephalosporin in combination with clavulanic acid compared to the cephalosporin alone was interpreted as esbl production ( 13 ) . briefly , a loopful of an overnight grown culture of each test isolate as well as the control strains were suspended in 500 l of sterile double distilled water , boiled at 100c for 10 minutes and were centrifuged at 12000  g for 10 minutes . pcr amplifications were performed using specific primers for ambler class a ( blatem , blashv , blactx - m , blaveb-1 , blaper-1 ) and class c ( blaampc ) -lactamase genes ( table 1 ) . ampc amplifications were initially performed using a pair of external primers ( preampc - pa1 and postampc - pa2 ) yielding a 1243-base pair product encompassing the entire ampc gene of p. aeruginosa , but excluding its promoter sequences . another pair of primers ( ampc - pa - a and ampc - pa - b ) was also used as internal primers for ampc gene yielding a 1063 bp product ( table 1 ) . pcr experiments were carried out in 25 l volume reaction mixtures containing 10 pm of each primer , 200 m dntp , 1.4 mm mgcl2 , 1 l of crude dna template and 1u of taq dna polymerase in the reaction buffer provided by the manufacturer ( cinnagen , tehran , iran ) . amplifications were carried out in thermocycler ( techne , uk ) using the following program : five minutes denaturation at 95c followed by 30 cycles of 95c for one minute , one minute at the annealing temperature ( 43 to 63c for esbls , 62c and/or 65c for ampc ) , and one minute at 72c with a final extension period of 10 minutes at 72c . comparisons of -lactamase phenotypes and presence of the related genes were performed using the chi - square test . correlation between antibiotic resistance and -lactamase gene carriage was analyzed by the nonparametric mann - whitney u test . all statistical analyses were carried out using spss software version 19 . a p value of < 0.05 was considered statistically significant . a ; clear distortion of inhibition zone indicates ampc -lactamase production , b , c and d ; absence of distortion indicates negative test . disc diffusion results showed that all test isolates were resistant to three or more antibiotic classes ; therefore , they were identified as multidrug resistant ( mdr ) . antibiotic resistance rates shown in figure 2 were : piperacilin , carbenicillin , tobramycin and cefoxitin ( 100% ) , imipenem , ticarcillin , aztreonam and cotrimoxazole ( 98% ) , amikacin , cefepime and ciprofloxacin ( 96% ) , meropenem ( 94% ) , ceftazidime ( 92% ) and piperacillin / tazobactam ( 88% ) . the mic results showed 88.2% resistance to ceftazidime and 98% to imipenem , confirming the results obtained by the disc diffusion method . ampc production was observed in 35 isolates ( 68.6% ) using the ampc disc test and only 18 ( 51.4% ) by the tdet test suggesting the superiority of the ampc disc test to tdet . esbl production was observed in 20 isolates ( 39.2% ) and 19 isolates ( 37.3% ) were mbl producers . of the 35 ampc producers , two ( 5.7% ) had the esbl phenotype , 13 ( 37.1% ) produced mbl and six ( 17.1% ) were positive for all three enzymes . blaampc was present in 31 isolates ( 60.8% ) , two of which were susceptible to ceftazidime ( table 2 ) . there was a significant correlation between the ampc disc test phenotype and ampc gene carriage ( p < 0.05 ) , while no relationship was observed between tdet and presence of the ampc gene . among the class a -lactamases , 20 isolates ( 39.2% ) harbored blatem and 11 ( 21.6% ) carried blaper-1 genes . shv , ctx - m and veb-1 genes were not detected in any of the isolates . overall , there was correlation between -lactamase productions with the presence of their related genes in the test isolates ( p < 0.05 ) . susceptible ) ( mic , minimum inhibitory concentration )   extended - spectrum -lactamase caz , ceftazidime ; ipm , imipenem ; mem , meropenem ; prl , piperacilin ; ptz , piperacillin - tazobactam ; tc , ticarcillin ; py , carbenicillin ; ak , amikacin ; tn , tobramycin ; atm , azteonam ; cpm , cefepime ; cip , ciprofloxacin ; ts , cotrimoxazole ; fox , cefoxitin . multiple -lactamase producing p. aeruginosa cause major therapeutic failure and pose significant clinical challenges if remain undetected ( 17 ) . among more than 800 -lactamases identified in gram - negative bacteria , at least 120 were detected in p. aeruginosa , among which , ampc , esbls , and mbls are clinically significant ( 8) . the current clsi guidelines do not describe a method for detection of ampc lactamases . ampc disc test was originally introduced to detect plasmid - mediated ampc -lactamases ( 18 ) . however , black et al . reported the detection of chromosomally mediated inducible ampc -lactamases in a number of bacteria including p. aeruginosa , by the ampc disc test ( 19 ) . in the present study , using the ampc disc test , ampc production ( 68.6% ) was higher among the p. aeruginosa isolates from burns compared to those of other reports ( 17.3 - 59.4% ) ( 17 , 20 - 23 ) . on the other hand , only half of the isolates which were positive in ampc disc test showed ampc production by tdet . basak et al . also showed that the ampc disc test was superior to tdet in screening for ampc production ( 20 ) . the current study results showed a significant association between the ampc phenotype determined by the ampc disc test and ampc gene carriage ( p < 0.05 ) . the rate of ampc gene carriage ( 60.8% ) in the present study was similar to a report from taiwan , where presentation of ampc gene in p. aeruginosa was 59.6% ( 16 ) . however , in a study conducted in iran , fazeli et al . reported 100% ampc gene carriage among 72 pediatric p. aeruginosa isolates from various clinical specimens , but did not show the ampc phenotype ( 24 ) therefore , the current study believes that it is important to associate the enzyme phenotype with the presence of resistance genes . high level expression of ampc can prevent recognition of esbls leading to false negative results ( 25 , 26 ) . esbl production in our isolates ( 39.2% ) was higher compared to the other reports from iran ( 2.2 - 23.3% ) and other countries ( 1.81 - 17% ) ( 16 , 27 - 31 ) . coproduction of ampc and esbl ( 3.9% ) in the isolates of this study was similar to the findings of upadhyay et al . ( 3.3% ) , but much lower than several other reports ( 24.5 - 26% ) ( 17 , 22 , 23 ) . finally , similar to a report from brazil , the isolates of the present study did not harbor shv and ctx - m genes ( 32 ) . in the past 30 years , extensive use of carbapenems to treat ampc and esbl producing p. aeruginosa , has led to increased levels of bacterial resistance mostly due to production of mbls ( 9 , 33 , 34 ) . coproduction of ampc and mbl was detected in 25.5% of the isolates which was low compared to the other studies worldwide ( 45.5 - 46.6% ) ( 22 , 23 ) . alarmingly , 11.8% of the isolates of the current study produced ampc along with esbl and mbl . detection of multiple -lactamases in highly resistant bacteria could be useful for the selection of suitable antibiotic therapy and avoiding treatment failure as well as reducing mortality rates in hospitalized patients . in conclusion , the present study showed a high prevalence of ampc production among p. aeruginosa isolates from burn wounds ( 68.6% ) . more importantly , multiple -lactamase production was observed in 60% of the ampc producers , the majority of which also produced mbl ( 25.5% ) . co - production of ampc with esbl occurred in 3.9% of the isolates and 11.9% produced the three -lactamases .
background : pseudomonas aeruginosa is one of the most common causes of nosocomial infections . resistance of p. aeruginosa to -lactam antibiotics may be the result of acquired resistance through mutation and over production of various antibiotic inactivating enzymes . 
 this research aimed to determine the prevalence of extended - spectrum -lactamases ( esbl ) and metallo -lactamase ( mbl ) production as well as the presence of their related genes among ampc -lactamase producing p. 
 aeruginosa isolated from burns.objectives:the current study aimed to determine the prevalence of class a esbl and mbl production in relation to the presence of their related genes among ampc -lactamase producing p. aeruginosa isolated from burns.materials and methods : the antimicrobial susceptibility of 51 p. aeruginosa isolates from patients with burns was examined against 13 antibiotics by the disc diffusion method . 
 minimum inhibitory concentrations ( mic ) for imipenem and ceftazidime were measured by the microdilution method . 
 ampc production was detected by ampc disc and the modified three - dimensional extract tests . 
 esbl phenotype was confirmed by the double disc synergy test ( ddst ) . 
 presence of -lactamase genes was detected by specific primers and polymerase chain reaction ( pcr).results : all isolates were multidrug resistant . 
 ampc , esbl and mbl production were observed in 35 ( 68.6% ) , 20 ( 39.2% ) and 19 ( 37.3% ) isolates , respectively . 
 overall , 43 isolates ( 84.3% ) carried -lactamase genes , out of which 31 ( 60.8% ) harbored blaampc , 20 ( 39.2% ) had blatem and 11 ( 21.6% ) carried blaper-1 genes . among the ampc producers , 
 two isolates ( 6.5% ) carried blaampc + blaesbl , 13 ( 41.9% ) had blaampc + blambl and six ( 19.4% ) produced the three enzymes.conclusions:a high prevalence of multiple -lactamase production was observed among the ampc producers ( 60% ) , of which the majority co - produced ampc and mbl . 
 the current study results showed correlation between -lactamase production and the presence of antibiotic resistance genes in the isolates .