ampc is responsible for resistance to cephalosporins and esbls confer resistance to all -lactams except for the carbapenem family .
carbapenemases , particularly metallo -lactamases ( mbl ) , hydrolyze all -lactam antibiotics with the exception of monobactams .
coexistence of multiple -lactamases in clinical isolates of p. aeruginosa is common , causing resistance to almost all -lactam antibiotics ( 7 ) .
another important factor contributing to the pathogenesis of p. aeruginosa in causing fatal infections is its potential to form biofilms on biotic and abiotic surfaces ( 8) .
the bacterial populations in biofilms are usually more resistant to antibiotics and host - mediated clearance strategies compared to their planktonic counterparts , giving rise to chronic infections that are notoriously difficult to eradicate ( 9 , 10 ) . bacteria growing in biofilms produce one or more extracellular polymeric matrices which hold the cells of the biofilm community together .
polysaccharides are important components of the biofilm matrix , as they contribute to the overall biofilm architecture and to the resistance of biofilm - grown bacteria to certain antibacterial agents ( 11 ) .
at least three exopolysaccharides have been shown to be involved in biofilm formation by p. aeruginosa , including alginate , psl , and pel ( 12 ) . among these , psl is a mannose - rich polymer with an essential role in the initial steps of biofilm formation by non - mucoid p. aeruginosa as well as in its maintenance .
psl forms a helical structure around p. aeruginosa cells which increases the cell - to - surface and cell - to - cell interactions necessary for biofilm formation ( 13 , 14 ) .
synthesis of psl is mediated by the psl gene cluster ( psla - pslo ) and psla has been reported to be the first and most important gene necessary for psl synthesis ( 15 , 16 ) .
due to the suggested role of psla in the initial steps of biofilm formation by p. aeruginosa , we studied the association between biofilm formation and the psla gene carriage in burn isolates of p. aeruginosa .
we also evaluated the potential to form biofilm in relation to antibiotic resistance and the production of ampc , mbl and esbl -lactamases among the isolates .
sixty - two isolates of p. aeruginosa were collected from hospitalized burned patients in shahid motahari hospital , iran , from august to october 2011 ( 17 ) .
the isolates were maintained in brain - heart infusion broth ( becton dickinson , franklin lakes , nj ) containing 10% dimethyl sulfoxide ( dmso ) at -20c until use .
the antibiotic susceptibility of the isolates had been previously determined for 13 antibiotics by the disc diffusion method ( 17 ) .
staphylococcus epidermidis strains rp62a and rp62na were used as positive and negative controls for biofilm production , respectively ( 18 ) .
biofilm formation was determined by the microtiter plate assay , as previously reported ( 18 ) .
briefly , 200 l of a 1:100 dilution of each overnight grown bacterial culture in trypticase soy broth ( tsb ) ( merck , germany ) was inoculated into four wells of a 96-well flat bottomed polystyrene plate ( greiner bio - one inc .
following incubation at 37c for 22 - 24 hours , the cultures were removed and the wells were washed twice with 200 l of phosphate buffered saline ( pbs , ph = 7.4 ) and dried at room temperature .
biofilms were stained with 0.1% safranin ( merck , germany ) solution in water for 15 minutes and the plates were washed in distilled water and dried at room temperature . the optical density ( od ) of the biofilms
was measured at 492 nm using an elisa reader ( stat fax 2100 , awareness tech inc .
biofilm formation was considered negative at ods below 0.12 , weakly positive at ods 0.12 - 0.24 and strong positive at ods > 0.24 ( 18 ) .
each test was repeated on three different days and the results were reported as the mean of the obtained values .
pcr amplification of the psla gene was carried out using the following primers : psla - f , 5-cactggacgtctactccgacgatat-3 ; psla - r , 5-gtttcttgatcttgtgcagggtgtc-3 ( bioneer , korea ) , generating an amplification product of 1119 bp ( 20 ) .
the reaction mixture ( 25 l ) contained 1 l of the extracted dna , 1.5 mm mgcl2 , 0.4 mm of each dntp , 10 pm of each primer and one unit of taq dna polymerase ( cinnagen , iran ) .
amplifications were performed in a thermal cycler ( peqlab , germany ) using the following program : an initial incubation at 94c for 10 minutes , followed by 30 cycles of one minute denaturation at 94c , 30 seconds annealing at 55c and one minute extension at 72c followed by 10 minutes at 72c .
the amplification products were separated on 1% agarose gels , stained with red safe ( intronbio , korea ) , and visualized using an image analysis system ( uvitec , st john s innovation centre , uk ) .
briefly , the turbidity of overnight grown bacteria in mueller - hinton ( mh ) broth ( merck , germany ) was adjusted to mcfarland standard 0.5 before inoculating mh agar plates . an imipenem disc ( 10 g )
was placed on the bacterial lawn 10 mm apart from a blank disc ( edge to edge ) , to which , 10 l of an edta solution ( 0.5 m , ph : 8) was added before incubation at 37c for 24 hours .
presence of an extended growth inhibition zone between the two discs was interpreted as positive for mbl production .
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 30-g cefoxitin disc ( mast , uk ) on the surface of an mh agar plate , previously inoculated with a lawn of escherichia coli atcc 25922 and incubated overnight at 37c . a flattening or an indentation of cefoxitin inhibition zone adjacent to the disc containing the test strain indicated ampc -lactamase production .
esbl production was detected by the double - disc synergy test ( ddst ) , as described before ( 17 ) .
discs containing ceftazidime ( 30 g ) and cefepime ( 30 g ) were placed 15 mm apart from an amoxicillin / clavulanic acid disc ( 20 + 10 g ) on bacterial lawns before overnight incubation at 37c .
esbl production was detected when synergy was observed between the inhibition zones of cephalosporins and amoxicillin / clavulanic acid discs .
phenotypic confirmatory test for esbl production was performed by placing a ceftazidime disc ( 30 g ) alone and ceftazidime with clavulanic acid ( 10 g ) on bacterial lawns before incubation at 37c overnight .
an increase of 5 mm in the inhibition zone around the combination disc was considered as esbl production .
comparison of -lactamase production between biofilm - negative and biofilm - forming isolates was carried out by the mann - whitney test , using spss v19 .
sixty - two isolates of p. aeruginosa were collected from hospitalized burned patients in shahid motahari hospital , iran , from august to october 2011 ( 17 ) .
the isolates were maintained in brain - heart infusion broth ( becton dickinson , franklin lakes , nj ) containing 10% dimethyl sulfoxide ( dmso ) at -20c until use .
the antibiotic susceptibility of the isolates had been previously determined for 13 antibiotics by the disc diffusion method ( 17 ) .
staphylococcus epidermidis strains rp62a and rp62na were used as positive and negative controls for biofilm production , respectively ( 18 ) .
biofilm formation was determined by the microtiter plate assay , as previously reported ( 18 ) .
briefly , 200 l of a 1:100 dilution of each overnight grown bacterial culture in trypticase soy broth ( tsb ) ( merck , germany ) was inoculated into four wells of a 96-well flat bottomed polystyrene plate ( greiner bio - one inc .
, the cultures were removed and the wells were washed twice with 200 l of phosphate buffered saline ( pbs , ph = 7.4 ) and dried at room temperature .
biofilms were stained with 0.1% safranin ( merck , germany ) solution in water for 15 minutes and the plates were washed in distilled water and dried at room temperature . the optical density ( od ) of the biofilms
was measured at 492 nm using an elisa reader ( stat fax 2100 , awareness tech inc .
biofilm formation was considered negative at ods below 0.12 , weakly positive at ods 0.12 - 0.24 and strong positive at ods > 0.24 ( 18 ) .
each test was repeated on three different days and the results were reported as the mean of the obtained values .
pcr amplification of the psla gene was carried out using the following primers : psla - f , 5-cactggacgtctactccgacgatat-3 ; psla - r , 5-gtttcttgatcttgtgcagggtgtc-3 ( bioneer , korea ) , generating an amplification product of 1119 bp ( 20 ) .
the reaction mixture ( 25 l ) contained 1 l of the extracted dna , 1.5 mm mgcl2 , 0.4 mm of each dntp , 10 pm of each primer and one unit of taq dna polymerase ( cinnagen , iran ) .
amplifications were performed in a thermal cycler ( peqlab , germany ) using the following program : an initial incubation at 94c for 10 minutes , followed by 30 cycles of one minute denaturation at 94c , 30 seconds annealing at 55c and one minute extension at 72c followed by 10 minutes at 72c .
the amplification products were separated on 1% agarose gels , stained with red safe ( intronbio , korea ) , and visualized using an image analysis system ( uvitec , st john s innovation centre , uk ) .
briefly , the turbidity of overnight grown bacteria in mueller - hinton ( mh ) broth ( merck , germany ) was adjusted to mcfarland standard 0.5 before inoculating mh agar plates . an imipenem disc ( 10 g )
was placed on the bacterial lawn 10 mm apart from a blank disc ( edge to edge ) , to which , 10 l of an edta solution ( 0.5 m , ph : 8) was added before incubation at 37c for 24 hours .
presence of an extended growth inhibition zone between the two discs was interpreted as positive for mbl production .
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 30-g cefoxitin disc ( mast , uk ) on the surface of an mh agar plate , previously inoculated with a lawn of escherichia coli atcc 25922 and incubated overnight at 37c . a flattening or an indentation of cefoxitin inhibition zone adjacent to the disc containing the test strain indicated ampc -lactamase production .
esbl production was detected by the double - disc synergy test ( ddst ) , as described before ( 17 ) .
discs containing ceftazidime ( 30 g ) and cefepime ( 30 g ) were placed 15 mm apart from an amoxicillin / clavulanic acid disc ( 20 + 10 g ) on bacterial lawns before overnight incubation at 37c .
esbl production was detected when synergy was observed between the inhibition zones of cephalosporins and amoxicillin / clavulanic acid discs .
phenotypic confirmatory test for esbl production was performed by placing a ceftazidime disc ( 30 g ) alone and ceftazidime with clavulanic acid ( 10 g ) on bacterial lawns before incubation at 37c overnight .
an increase of 5 mm in the inhibition zone around the combination disc was considered as esbl production .
comparison of -lactamase production between biofilm - negative and biofilm - forming isolates was carried out by the mann - whitney test , using spss v19 .
of the 62 test isolates , 27 ( 43.5% ) formed biofilm , of which 18 ( 66.7% ) were strong and 9 ( 33.3% ) were weak producers with an average od492 of 0.582  0.015 and 0.194  0.001 , respectively .
presence of the psla gene was observed in all biofilm producers , showing a strong association between psla gene carriage and biofilm formation .
the amplification product of psla gene for a number of p. aeruginosa burn isolates ( 1119 bps ) is shown in figure 1 .
no significant difference was observed between the antibiotic susceptibility profiles of biofilm - positive and biofilm - negative isolates .
production of esbl , mbl and ampc -lactamases by non - biofilm and biofilm producers is presented in figure 2 . among the biofilm producing isolates , 19 ( 70.3% ) were mbl producers , 17 ( 62.9% ) produced ampc , and 9 ( 33.3% ) were positive for esbl production .
production of ampc , mbl and esbl in biofilm - negative isolates occurred in 12 ( 34.2% ) , 11 ( 31.4% ) and 7 ( 20% ) isolates , respectively .
as observed , mbl and ampc productions were significantly higher in biofilm - positive strains ( p = 0.003 and p = 0.02 , respectively ) .
coproduction of ampc and mbl was significantly higher in biofilm - positive isolates ( n = 12 , 44.4% ) compared to biofilm negative ones ( n = 7 , 20% ) ( p = 0.00 ) .
finally , three biofilm - positive ( 11.1% ) and one biofilm - negative isolates ( 2.8% ) produced all three -lactamases .
the degree of biofilm formation in relation to -lactamase(s ) production is shown in figure 3 .
as observed , strong biofilms were formed by the isolates that coproduced mbl and ampc as well as the strains that had all three -lactamases ( od492 : 0.46  0.020 and 0.36  0.060 , respectively ) .
the isolates that produced one -lactamase type formed weak biofilms ( mbl producers od492 : 0.17  0.015 , esbl producers od492 : 0.15  0.005 and ampc producers od492 : 0.13  0.012 ) .
non--lactamase - producing strains did not form biofilms ( od492 : 0.043  0.001 ) .
c+ , control ; l , 1 kb dna ladder ; lanes 2 to 20 , some test isolates .
p. aeruginosa is capable of causing chronic infections mostly due to its potential to form biofilms ( 16 ) .
the hallmarks of a mature biofilm include production of an extracellular matrix and increased resistance to antibiotics ( 11 ) . in a recent study , 96% of p. aeruginosa
burn wound isolates were shown to form moderate to strong biofilms in vitro ( 21 ) .
showed the high potential of biofilm formation by clinical isolates of p. aeruginosa regardless of the specimen source ( 22 ) .
the association between the potential to form strong biofilms by p. aeruginosa and antibiotic resistance has also been shown ( 23 ) .
we found no significant difference between the antibiotic susceptibility profiles in biofilm - positive and biofilm - negative burn isolates of p. aeruginosa . in this research , 43.5% of our p. aeruginosa
burn isolates formed biofilms , the majority of which ( 66.7% ) were strong biofilms .
presence of the psla gene was shown as a good predictor of biofilm formation in non - mucoid isolates of p. aeruginosa in a number of studies ( 13 , 15 , 20 ) .
aeruginosa burn isolates , suggesting a strong correlation between biofilm formation and psla gene carriage .
we also observed that 14.2% of the biofilm - negative isolates harbored the psla gene .
hou et al . found that psla gene was present in 31% of biofilm - negative ophthalmic p. aeruginosa isolates ( 20 ) .
this may suggests that gene presence does not necessarily result in its expression and biofilm formation is regulated by a complicated network of factors in addition to the psla gene .
the same observation has been made for biofilm formation in s. epidermidis ( 18 ) .
presence of different types of -lactamases including ampc , esbl and mbl and the association of some of these enzymes with biofilm formation in p. aeruginosa has been shown in a number of studies ( 5 , 24 , 25 ) . in our study ,
mbl , ampc and esbl production occurred in 48.3% ( 30/62 ) , 46.7% ( 29/62 ) and 25.8% ( 16/62 ) of the isolates , respectively .
however , mbl and ampc production were significantly higher in biofilm - positive strains than biofilm - negative ones ( 70.3% vs. 31.4% , 62.9% vs. 34.2% , respectively ) .
coproduction of different -lactamases has been observed by other investigators ( 5 , 7 , 26 ) .
we detected mbl and ampc coproduction in 30.6% of our isolates ( 19/62 ) , the majority of which were biofilm - positive ( 63.1% ) .
of the four isolates ( 6.4% ) that produced all three enzymes , 3 ( 75% ) were biofilm - positive .
figure 3 shows the degree of biofilm formation in relation with -lactamase production among our isolates .
showed that p. aeruginosa isolates harboring mbl gene produced strong to moderate biofilms in vitro ( 24 ) . in another study
, a highly significant association was found between the degree of biofilm formation and mbl production in p. aeruginosa ( 25 ) .
similar results have been shown for proteus mirabilis , where the potential to form biofilm was significantly higher in -lactamase ( ampc and esbl)-producing strains ( 27 ) .
however , another study showed that esbl ( but not mbl or ampc ) inhibited biofilm formation by impairing the twitching motility which plays an important role in micro - colony formation in p. aeruginosa ( 28 ) . in conclusion , biofilm formation correlated with psla gene carriage as well as mbl and ampc -lactamase production in burn isolates of p. aeruginosa .
more importantly , the isolates with multiple -lactamase phenotypes produced strong biofilms in comparison to the strains with one type of -lactamase that formed weak biofilms or -lactamase - negative isolates that did not form biofilm at all .
 background : pseudomonas aeruginosa is an important nosocomial pathogen characterized by its innate resistance to multiple antimicrobial agents . 
 plasmid - mediated drug resistance also occurs by the production of extended - spectrum -lactamases ( esbl ) , metallo -lactamases ( mbl ) , and ampc -lactamases . 
 another important factor for establishment of chronic infections by p. aeruginosa is biofilm formation mediated by the psl gene cluster.objectives:the aim of this study was to evaluate biofilm formation and presence of the psla gene in burn isolates of p. aeruginosa as well as the association of antibiotic resistance , mbl , esbl and ampc -lactamase production with biofilm formation among the isolates.materials and methods : sixty - two burn isolates of p. aeruginosa were obtained from shahid motahari hospital in tehran from august to october 2011 . 
 antibiotic susceptibility was determined by the disc diffusion assay . 
 mbl , ampc and esbl production were screened using the double disc synergy test , ampc disc test and combined disc diffusion assay , respectively . the potential to form biofilm 
 was measured using the microtiter plate assay and psla gene was detected using specific primers and pcr.results:biofilm formation was observed in 43.5% of the isolates , of which 66.7% produced strong and 33.3% formed weak biofilms . 
 all biofilm - positive and 14.2% of biofilm - negative isolates harbored the psla gene . 
 mbl , ampc and esbl production were significantly higher in the biofilm - positive isolates ( 70.3% , 62.9% and 33.3% , respectively ) compared to the biofilm - negative strains ( 31.4% , 34.2% and 20% , respectively ) . 
 overall , 19 isolates ( 30.6% ) co - produced mbl and ampc , among which the majority were biofilm - positive ( 63.1% ) . 
 finally , four isolates ( 6.4% ) had all three enzymes , of which 3 ( 75% ) produced biofilm.conclusions:biofilm formation ( both strong and weak ) strongly correlated with psla gene carriage . 
 biofilm formation also correlated with mbl and ampc -lactamase production . 
 more importantly , multiple--lactamase phenotype was associated with formation of strong biofilms . 