enterococci are gram - positive , facultative anaerobic bacteria that are members of the normal flora in the human gastrointestinal tract but have been recognized as important pathogens worldwide [ 1 , 2 ] . enterococcus faecalis and enterococcus faecium are the most prevalent species isolated from the urinary tract and wound infections , which affect mainly patients admitted to the medical care centers [ 3 - 6 ] . the disruption of the normal skin barrier , the immunocompromised state , prolonged hospitalization and antibiotic therapy put burns patients at a high risk of acquiring nosocomial infections including enterococcal infections . enterococcal infections can be difficult to treat because they have a remarkable ability of adaptation when are exposed to antibiotics ; they have intrinsic resistance to several antimicrobial agents and have a tremendous capacity to acquire high levels of resistance to antibiotics [ 8 , 9 ] . vancomycin - resistant enterococci ( vre ) and high - level gentamicin - resistant ( hlgr ) isolates have emerged as important pathogen in iran as well as in other countries , which create serious challenges for treating the infected patients [ 9 - 11 ] . there are several reports on the endemicity of vre and hlrg in iran but there is a lack of information on enterococcal stains isolated form burn centers [ 3 , 12 ] . this study was designed to determine the molecular characteristics and antimicrobial resistance of enterococcal strains isolated from patients in a burn center in iran . twenty - seven enterococcal isolates were collected from wound specimens of patients with burn injury , during april to september 2012 , in a burn hospital in tehran . only one isolate per patient identification of enterococci was performed based on a series of conventional microbiological tests including gram reaction , catalase reaction , presence of pyrrolidonyl arylamidase ( pyr ) , growth on bile - aesculin agar and 6.5% nacl media , motility test , arginine decarboxylation in moeller decarboxylase media , pyruvate utilization , and fermentation of arabinose , raffinose , mannitol and ribose . to confirm the identity of the isolates as e. faecalis or e. faecium the ddle gene was amplified by a polymerase chain reaction ( pcr ) method as described by dutka - malen et al . . the antibiotics ( mast group ltd . , merseyside , uk ) tested were ciprofloxacin ( 5 g ) , erythromycin ( 15 g ) , tetracycline ( 30 g ) , and quinupristin- dalfopristin ( 15 g ) . high - level resistance to gentamicin was also determined by disk diffusion method on mueller hinton agar ( conda s.a . , the minimal inhibitory concentration ( mic ) of vancomycin was determined by the standard agar dilution test on brain heart infusion agar ( conda s.a . , all antibiotic susceptibilities were performed and interpreted according to the criteria of the clinical and laboratory standards institute ( clsi ) guidelines . dna extraction was performed as described previously and this dna was used as a template for pcr analysis . all strains were screened for genes encoding resistance to aminoglycoside [ aac(6')-ie - aph(2'')-ia , aph ( 3 ' ) , ant ( 4 ' ) ] , vancomycin ( vana , vanb ) , tetracycline ( tetk , tetl , tetm , teto ) , and resistance to erythromycin ( erma , ermb , ermc ) by pcr and multiplex pcr - based methods , using specific primers [ 3 , 11,12 , 15 , 16 ] . the rapd - pcr assay was carried out in 25 l reaction volumes containing 0.5 m of each primer ( 5-agcgggccaa-3 and 5- acggccgacc-3 ) , 12.5 l of pcr master ( sinaclone inc , iran ) and 5 l of dna template . cycling conditions were as follow : initial denaturation at 95c for 5 min , followed by 30 cycles of 95c for 1 min , 38.5c for 1 min , and 72c for 2 min , and final extension at 72c for 10 min . pcr products were then separated by electrophoresis in 1.4% agarose gel with 0.5x tbe buffer . dna bands were observed by staining with kbc power load dye ( kawsar biotech co. iran ) and photographed under uv illumination . rapd patterns were analyzed by visual inspection . only the obvious , prominent and reproducible bands from repeated experiments ( at least twice ) were considered and any pattern differing by one or more bands was classified as a distinct rapd type . twenty - seven enterococcal isolates were collected from wound specimens of patients with burn injury , during april to september 2012 , in a burn hospital in tehran . only one isolate per patient identification of enterococci was performed based on a series of conventional microbiological tests including gram reaction , catalase reaction , presence of pyrrolidonyl arylamidase ( pyr ) , growth on bile - aesculin agar and 6.5% nacl media , motility test , arginine decarboxylation in moeller decarboxylase media , pyruvate utilization , and fermentation of arabinose , raffinose , mannitol and ribose . to confirm the identity of the isolates as e. faecalis or e. faecium the ddle gene was amplified by a polymerase chain reaction ( pcr ) method as described by dutka - malen et al . . the antibiotics ( mast group ltd . , merseyside , uk ) tested were ciprofloxacin ( 5 g ) , erythromycin ( 15 g ) , tetracycline ( 30 g ) , and quinupristin- dalfopristin ( 15 g ) . high - level resistance to gentamicin was also determined by disk diffusion method on mueller hinton agar ( conda s.a . , the minimal inhibitory concentration ( mic ) of vancomycin was determined by the standard agar dilution test on brain heart infusion agar ( conda s.a . , all antibiotic susceptibilities were performed and interpreted according to the criteria of the clinical and laboratory standards institute ( clsi ) guidelines . dna extraction was performed as described previously and this dna was used as a template for pcr analysis . all strains were screened for genes encoding resistance to aminoglycoside [ aac(6')-ie - aph(2'')-ia , aph ( 3 ' ) , ant ( 4 ' ) ] , vancomycin ( vana , vanb ) , tetracycline ( tetk , tetl , tetm , teto ) , and resistance to erythromycin ( erma , ermb , ermc ) by pcr and multiplex pcr - based methods , using specific primers [ 3 , 11,12 , 15 , 16 ] . the rapd - pcr assay was carried out in 25 l reaction volumes containing 0.5 m of each primer ( 5-agcgggccaa-3 and 5- acggccgacc-3 ) , 12.5 l of pcr master ( sinaclone inc , iran ) and 5 l of dna template . cycling conditions were as follow : initial denaturation at 95c for 5 min , followed by 30 cycles of 95c for 1 min , 38.5c for 1 min , and 72c for 2 min , and final extension at 72c for 10 min . pcr products were then separated by electrophoresis in 1.4% agarose gel with 0.5x tbe buffer . dna bands were observed by staining with kbc power load dye ( kawsar biotech co. iran ) and photographed under uv illumination . rapd patterns were analyzed by visual inspection . only the obvious , prominent and reproducible bands from repeated experiments ( at least twice ) were considered and any pattern differing by one or more bands was classified as a distinct rapd type . distribution of species according to conventional microbiological tests and pcr method included 19 ( 70.3% ) e. faecalis and 8 ( 29.7% ) e. faecium . the prevalence of antimicrobial resistance pattern and rapd types have been summarized in table i. in the present study , no vre was recovered and all enterococcal isolates showed complete sensitivity to vancomycin with mic  0.5 g / ml . the distribution of antimicrobial resistance genes , virulence genes and biofilm production among enterococcal species . e , erythromycin ; g , gentamicin ; syn , quinupristin - dalfopristin ; tet , tetracycline ; cip , ciprofloxacin all isolates at least exhibited resistance to one antibiotic and 6 different profiles were observed on the basis of their antibiotic resistance patterns . high - level resistance to gentamicin and multiple drug resistance were observed in 96.2% of isolates . the most frequent profile was resistance to erythromycin , gentamicin , tetracycline , quinupristin - dalfopristin and ciprofloxacin that was observed in 74% of isolates . the most prevalent aminoglycoside resistance gene was aac(6')-ie - aph(2'')-ia , found in 96.2% ( 26/27 ) of the isolates . these genes were detected in all hlgr isolates . the ant ( 4 ' ) gene was detected in 62.9% ( 17/27 ) of isolates . the most prevalent tetracycline resistance gene was tetm , found in 85.1% ( 23/27 ) of the isolates followed by tetl and teto found in 7.4% ( 2/27 ) of the isolates . the erma and ermb genes were detected in 33.3% ( 9/27 ) and 44.4% ( 12/27 ) of the isolates respectively . the aph ( 3 ' ) , tetk , ermc , vana , and vanb genes were not detected in any of the enterococcal isolates in this study . to determine the degree of clonality among enterococcal isolates 1 ) . one clusters of isolates shared the same rapd patterns , while 16 isolates had unique rapd patterns . a dominant rapd type designated as a , that consisted 11 isolates of e. faecalis and were hlgr . lanes 1and 2 ; dna size marker ( 100bp ) , lanes a - q ; enterococcal isolates with different rapd patterns . among enterococci isolates , e. faecalis and e. faecium are the most common species caused enterococcal nosocomial infections . in our study , the most prevalent species was e. faecalis ( 70.3% ) followed by e. faecium ( 29.7% ) isolated from burn wound infections . similar results were reported for clinical isolates by other studies [ 3 , 11 , 16 , 17 ] . in the treatment of enterococcal infections , the combination of gentamicin with a beta lactam antibiotic or a glycopeptide is used to obtain a synergistic bactericidal effect . however , strains that are highly resistant to gentamicin are no longer susceptible to the combination therapy . several studies have demonstrated that vre have caused both outbreaks and endemic infections in burn units [ 5 , 6 ] , however , in this study , there were no vre isolates and more than 96% of isolates were hlgr and 74% of them were simultaneously resistant to erythromycin , tetracycline , quinupristin - dalfopristin and ciprofloxacin . our study is the first investigation on enterococcal resistance among burn patients in iran and the findings indicated that vancomycin keeps its therapeutic effects against enterococcal infections . genes encoding aminoglycoside - modifying enzymes ( ames ) are often carried on transposable elements . one of the most prevalent ames genes among enterococci is aac(6')-ie - aph(2'')-ia encoding a bifunctional enzyme aac(6')-ie - aph(2'')-ia which confers resistance to virtually all of the clinically available aminoglycosides including gentamicin [ 11 , 19 ] . in the current study , as in many other reports , the aac(6')- ie - aph(2'')-ia gene was the most prevalent ame gene , encountered in 96.2% of isolates followed by the ant ( 4 ' ) gene and was detected in 62.9% of isolates [ 16 , 20 ] . acquired resistance to tetracyclines and macrolides in enterococci is often by mobile genetic elements . in the present study , as in many previous reports , the most common tetracycline and erythromycin resistance mechanism was mediated by the tetm and ermb genes , respectively [ 21 - 25 ] . to take preventive measures and apply infection control , identification of the source of infection and nature of outbreaks are required . rapd - pcr has become a reliable tool for the differentiation and identification of enterococci from clinical origin [ 26 - 28 ] . in this study , rapd - pcr profiles showed that eleven e. faecalis isolates shared identical banding patterns . in addition , the results of rapd typing corresponded with antibiogram findings and the resistance genes patterns for eight of these eleven isolates . this could be explained by the predominance or an outbreak of a particular clonal group of e. faecalis with high - level gentamicin resistance in the assessed burn unit . in conclusion , we found that over the examination time period one rapd genotype was the common type and was disseminated among patients in the burn unit . interestingly , most of these strains had an identical or very similar antibiotic and gene resistance pattern .
summarythis study was designed to determine the molecular characteristics and antimicrobial resistance of enterococcal strains isolated from patients admitted to an iranian hospital . 
 enterococcal strains were isolated from the burn patients . 
 all strains were screened for genes encoding resistance to aminoglycoside [ aac(6')-ie - aph(2'')-ia , aph ( 3 ' ) , ant ( 4 ' ) ] , resistance to vancomycin ( vana , vanb ) , resistance to tetracycline ( tetk , tetl , tetm , teto ) , and resistance to erythromycin ( erma , ermb , ermc ) by pcr and multiplex pcr - based methods . 
 genetic diversity was evaluated via random amplified polymorphic dna ( rapd)-pcr . all enterococcal isolates showed complete sensitivity to vancomycin with mic  0.5g / ml . resistance to gentamicin , tetracycline , erythromycin , ciprofloxacin or quinopristin - dalfopristin was detected , whilst more than 96.2% of isolates were high - level gentamicinresistant ( hlgr ) and multiple drug resistant . 
 the most prevalent aminoglycoside resistance gene was aac(6')-ie - aph(2'')-ia , that was found in 96.2% ( 26/27 ) of the isolates . 
 the most prevalent tetracycline resistance genes were tetm , found in 85.1% ( 23/27 ) followed by tetl and teto found in 7.4% ( 2/27 ) of the isolates . 
 the erma and ermb genes were detected in 33.3% ( 9/27 ) and 44.4% ( 12/27 ) of the isolates respectively . 
 rapd - pcr analysis yielded 17 distinct profiles among 27 investigated isolates . 
 one cluster of isolates shared the same rapd pattern , while 16 isolates had unique rapd pattern . 
 our study showed that during the examination time period one rapd genotype was the common type and was disseminated among patients in the burn unit . 
 interestingly , most of these strains had an identical or very similar antibiotic and gene resistance pattern .