the increasing rate of occurrence is mainly due to the ability of a. baumannii to survive in different environments , including nutritionally poor ones , and ability to acquire different antimicrobial resistance genes . for the last 2 decades , increasing numbers of carbapenem - resistant a. baumannii strains have been reported worldwide . resistance to carbapenems may result from metallo - beta - lactamases ( mbls ) synthesis , hydrolyzing almost all beta - lactam antibiotics . mbls are not inhibited by classic beta - lactams inhibitors ( clavulanic acid , tazobactam , sulbactam ) . however , mbls use zn ions as cofactors and can therefore be inhibited in vitro by edta . mbls - positive a. baumannii strains are a serious therapeutic problem due to co - resistance to numerous antibiotic groups and difficulties in treating the infections caused by them . the aim of this study was to evaluate the antimicrobial susceptibility patterns of a. baumannii isolates obtained from poland and to estimate their genetic relatedness and ability to synthesize mbls . the study included 78 a. baumannii clinical isolates resistant to at least 1 carbapenem , collected between 2008 and 2009 from patients hospitalized at dr . identification of the isolates to the species level was done using the i d 32 e and api 20 ne ( biomrieux ) tests , according to the manufacturer s recommendations . antimicrobial susceptibility testing for meropenem , imipenem , gentamicin , amikacin , tobramycin , ciprofloxacin , levofloxacin , and trimethoprim / sulfamethoxazole was performed by the agar disc diffusion method according to the recommendations of the national reference centre for antimicrobial susceptibility testing . resistance to imipenem , meropenem and doripenem was also determined using etest ( biomrieux ) according to the manufacturer s instructions . results were interpreted using clinical breakpoints as defined by the clinical and laboratory standards institute . dna for pcr - rapd typing purpose was isolated using genematrix bacterial & yeast genomic dna purification kit ( eurx ) . pcr - rapd reaction was conducted using the method previously described by carr et al . with gcttgtgaac primer . final volume of the reaction ( 25 l ) consisted of 0.4 l of taq dna polymerase ( 5 u/l , solis biodyne ) , 2.5 l of buffer b ( 0.8 m tris - hcl , 0.2 m ( nh4)2so4 , 0.2% w / v tween-20 , solis biodyne ) , 3.5 l of mgcl2 ( 25 mm , solis biodyne ) , 0.25 l of dntp mixture ( 20 mm , solis biodyne ) , 1.8 l of 10  primer ( 100 pmol/l , genomed ) , 15.55 l of water ( molecular biology grade water , eppendorf ) and 1 l of bacterial dna . amplification was done in a geneamp pcr system 2700 thermocycler ( applied biosystems ) according to the following temperature profile : first denaturation step at 92c for 30 seconds , 34 cycles , each consisting of 3 steps at 92c denaturation for 30 seconds , annealing at 40c for 1 minute and primer extension at 72c for 3 minutes . the final step included 30 seconds at 92c , 1 minute at 40c , and the final extension of the primer for 10 minutes at 72c . pcr - rapd reaction products were separated by electrophoresis in 2% agarose gel ( 1xtbe ) at 13.5 v / cm for 2.5 hours in the sub - cell gt ( biorad ) apparatus . after the electrophoresis step , gel was stained with ethidium bromide solution for 20 minutes , washed for 20 minutes in deionized water and observed in uv light . molecular sizes of the obtained pcr - rapd reaction products were compared to molecular size marker 1003000 bp ( solis biodyne ) . visualizations of the stained gel were collected on gel doc 2000 system and quantity one program ( biorad ) . mbls activity was phenotypically investigated using : ( i ) different double disk synergy tests for ceftazidime , imipenem , and meropenem , along with edta or 2-mercaptopropionic acid ( 2-mpa ) discs ( ddst ) ; ( ii ) imipenem / edta combination disc test ( cdt ) ; and ( iii ) etest mbl ( biomrieux ) . bacterial suspension of 0.5 macfarland was placed on mueller - hinton agar in all mbls detection methods . in double disc synergy tests with edta and 2-mpa , ceftazidime ( 30 g ) , imipenem ( 10 g ) and meropenem ( 10 g ) discs were placed 2 cm apart from the sterile disc with edta and 2-mpa addition , respectively . bacteria inhibition zone deformation or enhancement for any used antibiotic disc from the side of mbls inhibitor was interpreted separately as the a. baumannii strain s ability to mbls production in disc diffusion methods . at least 5 mm bacteria inhibition zone size enhancement for imipenem impregnated with edta was also interpreted as mbls - positive strain in the method of comparison inhibition zones diameters . using etest mbl imipenem and imipenem / edta mic values ratio higher or equal to 8 , or deformation of the inhibition zone , was interpreted as a positive result . aeruginosa strains producing imp- or vim - like mbls ( carrying blaimp or blavim genes ) and atcc 27853 served as positive and negative controls of the reaction , respectively . a duplex pcr assay was performed to detect the occurrence of imp- and vim - like mbls genes as previously described . pcr reaction products were separated by electrophoresis on 1% agarose gel in 1xtbe at 9 v / cm for 1 hour in mini sub dna cell ( biorad ) apparatus . visualizations were recorded and documented in gel doc 2000 system using quantity one ( biorad ) program . the study included 78 a. baumannii clinical isolates resistant to at least 1 carbapenem , collected between 2008 and 2009 from patients hospitalized at dr . identification of the isolates to the species level was done using the i d 32 e and api 20 ne ( biomrieux ) tests , according to the manufacturer s recommendations . antimicrobial susceptibility testing for meropenem , imipenem , gentamicin , amikacin , tobramycin , ciprofloxacin , levofloxacin , and trimethoprim / sulfamethoxazole was performed by the agar disc diffusion method according to the recommendations of the national reference centre for antimicrobial susceptibility testing . resistance to imipenem , meropenem and doripenem was also determined using etest ( biomrieux ) according to the manufacturer s instructions . results were interpreted using clinical breakpoints as defined by the clinical and laboratory standards institute . dna for pcr - rapd typing purpose was isolated using genematrix bacterial & yeast genomic dna purification kit ( eurx ) . pcr - rapd reaction was conducted using the method previously described by carr et al . with gcttgtgaac primer . final volume of the reaction ( 25 l ) consisted of 0.4 l of taq dna polymerase ( 5 u/l , solis biodyne ) , 2.5 l of buffer b ( 0.8 m tris - hcl , 0.2 m ( nh4)2so4 , 0.2% w / v tween-20 , solis biodyne ) , 3.5 l of mgcl2 ( 25 mm , solis biodyne ) , 0.25 l of dntp mixture ( 20 mm , solis biodyne ) , 1.8 l of 10  primer ( 100 pmol/l , genomed ) , 15.55 l of water ( molecular biology grade water , eppendorf ) and 1 l of bacterial dna . amplification was done in a geneamp pcr system 2700 thermocycler ( applied biosystems ) according to the following temperature profile : first denaturation step at 92c for 30 seconds , 34 cycles , each consisting of 3 steps at 92c denaturation for 30 seconds , annealing at 40c for 1 minute and primer extension at 72c for 3 minutes . the final step included 30 seconds at 92c , 1 minute at 40c , and the final extension of the primer for 10 minutes at 72c . pcr - rapd reaction products were separated by electrophoresis in 2% agarose gel ( 1xtbe ) at 13.5 v / cm for 2.5 hours in the sub - cell gt ( biorad ) apparatus . after the electrophoresis step , gel was stained with ethidium bromide solution for 20 minutes , washed for 20 minutes in deionized water and observed in uv light . molecular sizes of the obtained pcr - rapd reaction products were compared to molecular size marker 1003000 bp ( solis biodyne ) . visualizations of the stained gel were collected on gel doc 2000 system and quantity one program ( biorad ) . mbls activity was phenotypically investigated using : ( i ) different double disk synergy tests for ceftazidime , imipenem , and meropenem , along with edta or 2-mercaptopropionic acid ( 2-mpa ) discs ( ddst ) ; ( ii ) imipenem / edta combination disc test ( cdt ) ; and ( iii ) etest mbl ( biomrieux ) . bacterial suspension of 0.5 macfarland was placed on mueller - hinton agar in all mbls detection methods . in double disc synergy tests with edta and 2-mpa , ceftazidime ( 30 g ) , imipenem ( 10 g ) and meropenem ( 10 g ) discs were placed 2 cm apart from the sterile disc with edta and 2-mpa addition , respectively . bacteria inhibition zone deformation or enhancement for any used antibiotic disc from the side of mbls inhibitor was interpreted separately as the a. baumannii strain s ability to mbls production in disc diffusion methods . at least 5 mm bacteria inhibition zone size enhancement for imipenem impregnated with edta was also interpreted as mbls - positive strain in the method of comparison inhibition zones diameters . using etest mbl imipenem and imipenem / edta mic values ratio higher or equal to 8 , or deformation of the inhibition zone , was interpreted as a positive result . p. aeruginosa strains producing imp- or vim - like mbls ( carrying blaimp or blavim genes ) and atcc 27853 served as positive and negative controls of the reaction , respectively . a duplex pcr assay was performed to detect the occurrence of imp- and vim - like mbls genes as previously described . pcr reaction products were separated by electrophoresis on 1% agarose gel in 1xtbe at 9 v / cm for 1 hour in mini sub dna cell ( biorad ) apparatus . visualizations were recorded and documented in gel doc 2000 system using quantity one ( biorad ) program . about 85% of the isolates were obtained from patients from the anesthesiology and intensive care clinic , pediatric intensive care ward , and pediatric surgery clinic . most of the isolates were derived from throat and wound swabs and bronchoalveolar lavage ( figures 1 , 2 ) . seventy - seven ( ~99% ) of the isolates were multi - drug resistant ( mdr ) , showing reduced susceptibility to antimicrobial agents from 3 classes regularly used for treatment . resistance to at least 1 carbapenem was confirmed in all the isolates . among the examined 78 carbapenem - resistant a. baumannii strains , all of the examined strains were resistant to amikacin and ciprofloxacin , 98.7% to trimethoprim / sulfamethoxazole , 61.5% to levofloxacin , 53.4% to gentamicin , and 15.4% to netilmicin . only 1 isolate ( ~1% ) was resistant to tobramycin and colistin . five of the pcr - rapd patterns were represented by 1 isolate / pattern , while 13 included 2 isolates / pattern . each double disc diffusion method with ceftazidime , imipenem , meropenem and edta revealed 66.7% of mbls - positive strains . the enhancement of bacteria growth inhibition zones was strongest for imipenem and ceftazidime . using the same method but with 2-mpa , 88.9% of the strains were classified as mbls - positive and the deformation of the bacteria growth inhibition zones were observed more frequently for imipenem and meropenem . in the comparison method of bacterial growth inhibition zones diameters between imipenem / edta and imipenem discs , using etest mbl , positive results were obtained for 88.9% of strains ( table 1 ) . the presence of imp - like mbls coding genes was identified in 8 isolates , while none of the isolates contained the blavim-4 gene . the blaimp - like - positive isolates were mainly derived from patients of the anesthesiology and intensive care clinic . the most common are : bacteremias , pneumonias , meningitis , urinary tract and wound infections . carbapenem - resistant a. baumannii has increasingly become a serious therapeutic problem worldwide [ 1113 ] . in this regard , limited data has been published on carbapenem - resistant a. baumannii in poland . the existence of isolates with undistinguished molecular patterns indicated the occurrence of bacterial spread between patients . the spread between different hospital wards and clinics might be due to transfer of the patients or mediated by health - care personnel and/or medical devices . this idea is supported by the isolation , from a bath - tub siphon trap , of an a. baumannii strain in which pcr - rapd pattern was observed , as well as strains derived from patients cured in 4 different wards . among the strains examined with phenotypic methods , the highest percentage of mbls producers was obtained using the comparison method of bacterial growth inhibition zones between imipenem / edta and imipenem discs . the obtained results indicate high significance of mbls in occurrence of a. baumannii resistance to carbapenems . the existence of 18 mbls - positive a. baumannii isolates was observed using phenotypic methods ; 8 ( 44.4% ) of them were confirmed as imp - like enzymes producers . a study from korea demonstrated that 48.4% of the a. baumannii isolates resistant to carbapenems produced blaimp-1 , while blavim was produced by only 3.2% of the isolates . another study reported the presence of blaimp and blavim in 61% and 29% of a. baumannii isolates , respectively . blavim-4 in the isolates showing positive results in phenotypic methods probably indicated the existence of other genes , like blasim . the carbapenem - resistant a. baumannii strains with no phenotypic or genotypic sign of mbls production may possess other enzymes mediating carbapenem resistance , such as oxa - type lactamases . naturally occurring oxa carbapenemases are oxa-51-like enzymes . to date , up to 45 variants of oxa-51 have been identified in a. baumannii isolates from medical centers worldwide . acquired oxa carbapenemases can be divided into 3 clusters , based upon the variant sequence homology : oxa-23 , oxa-40 , and oxa-58 . phenotypic methods have been previously reported to be sensitive for the detection of mbls in a. baumannii . the technique is very easy and economical and can be incorporated into the routine testing of any busy microbiology laboratory . the genotypic methods are based on analysis of genetic material that is unique and invariable for every organism , in contrast to phenotypic features . mbls detection using the e - test and other phenotypic methods is not reliable because there can be false - positive results . a. baumannii isolates resistant to carbapenems are mainly obtained from throat and wound swab samples derived from patients of the anesthesiology and intensive care clinic and pediatric surgery clinic . pcr - rapd demonstrates a high level of genetic diversity among the isolates , although intra - hospital spread of some strains is noted . mbl - coding genes represent an important mechanism of carbapenem resistance among a. baumannii isolates from poland .
backgroundacinetobacter baumannii is an opportunistic microorganism with an increasing role in nosocomial outbreaks . for the last 2 decades , 
 a growing number of carbapenem - resistant a. baumannii strains have been identified , including the metallo - beta - lactamases ( mbls ) producers . 
 the study aimed to investigate the genetic relatedness of , and mbls production among , a collection of a. baumannii isolates from poland.material/methodsthis study involved 78 clinical isolates of carbapenem - resistant a. baumannii . 
 strain typing of the isolates was performed using pcr - rapd . 
 the presence of mbls was phenotypically determined using different double disc synergy tests ( ddst ) , the imipenem / edta combination disk test ( cdt ) and etest mbl . blaimp and blavim genes were detected using a duplex pcr assay.resultsthe isolates were divided into 18 pcr - rapd patterns . among 18 examined isolates , 94.4% were mbl - positive by the phenotypic method relying on comparing the bacteria growth inhibition zones diameters between imipenem / edta and imipenem discs , 88.9% using etest mbl , 66.7% using the double disc synergy test with ceftazidime , imipenem , meropenem and edta , and 88.9% using a corresponding method with 2-mpa . 
 the existence of blaimp was identified in 8 ( 10.3% ) strains.conclusionsmbls production was an important mechanism of carbapenem resistance among a. baumannii isolates in poland . 
 laboratories should routinely screen for mbls among a. baumannii isolates .