Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split University Hospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a bla(OXA-107) gene, encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolates formed three clusters belonging to the sequence group 2 (European clone 1) lineage

Bedenic, Branka

Goic-Barisic, Ivana

Kalenic, Smilja

Katic, Stjepan

Novak, Anita

Punda-Polic, Volga

Tonkic, Marija

Towner, Kevin J

PubMed

Edinburgh Research Explorer

     Edinburgh Research Explorer                                      Occurrence of OXA-107 and ISAba1 in carbapenem-resistantisolates of Acinetobacter baumannii from CroatiaCitation for published version:Goic-Barisic, I, Bedenic, B, Tonkic, M, Novak, A, Katic, S, Kalenic, S, Punda-Polic, V & Towner, KJ 2009,'Occurrence of OXA-107 and ISAba1 in carbapenem-resistant isolates of Acinetobacter baumannii fromCroatia' Journal of Clinical Microbiology, vol 47, no. 10, pp. 3348-9. DOI: 10.1128/JCM.02394-08Digital Object Identifier (DOI):10.1128/JCM.02394-08Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Journal of Clinical MicrobiologyGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 28. Apr. 2017JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2009, p. 3348–3349 Vol. 47, No. 100095-1137/09/$08.000 doi:10.1128/JCM.02394-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.Occurrence of OXA-107 and ISAba1 in Carbapenem-Resistant Isolatesof Acinetobacter baumannii from CroatiaIvana Goic-Barisic,1* Branka Bedenic,2 Marija Tonkic,1 Anita Novak,1 Stjepan Katic,2Smilja Kalenic,2 Volga Punda-Polic,1 and Kevin J. Towner3Department of Clinical Microbiology, Split University Hospital and School of Medicine, University of Split, Split,1 and Department ofClinical and Molecular Microbiology, Clinical Hospital Centre Zagreb and School of Medicine, University of Zagreb, Zagreb,2Croatia, and Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust,Queen’s Medical Centre, Nottingham, United Kingdom3Received 13 December 2008/Returned for modification 22 December 2008/Accepted 10 August 2009Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split UniversityHospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a blaOXA-107 gene,encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolatesformed three clusters belonging to the sequence group 2 (European clone 1) lineage.An increasing trend toward multidrug resistance, includingcarbapenem resistance, in Acinetobacter baumannii has beenobserved in the past two decades. Carbapenem resistancemechanisms in A. baumannii include the production of metallo--lactamases (MBLs) and carbapenem-hydrolyzing oxacilli-nases, efflux mechanisms, and the loss of outer membraneproteins, often in combination (6). These resistance mecha-nisms pose a serious therapeutic threat, since carbapenems arefrequently used to treat otherwise resistant A. baumannii in-fections.Since 2002, increasing numbers of carbapenem-resistant iso-lates of A. baumannii have been recovered at Split UniversityHospital in Split, Croatia. Meropenem is prescribed at thishospital more frequently than imipenem, while ertapenem hasonly recently been licensed in Croatia. There are few publisheddata concerning carbapenem resistance in multidrug-resistantisolates of A. baumannii from Croatia. The present study in-vestigated the epidemiology and carbapenem resistance mech-anisms of multidrug-resistant A. baumannii isolates endemic inthis regional teaching hospital.Between 2002 and 2007, 106 nonrepetitive isolates of A.baumannii with an unusual resistance profile were isolatedconsecutively from two adult surgical intensive care units(ICUs), a pediatric ICU, a neurosurgery ICU, and a generalsurgery ICU at different locations in Split University Hospital(a 1,651-bed university teaching hospital with facilities at threesites). The hospital serves a pediatric and adult population ofca.500,000 and is also a referral hospital for much of southernCroatia (with a total population of ca. 1 million inhabitants).Isolates were initially recovered on blood agar plates fromroutine blood cultures, urine samples, wound exudates, cathe-ter tip specimens, and bronchial secretions. Conventional bio-chemical tests and the API 20NE system (bioMe´rieux, Marcy-l’Etoile, France) were used to presumptively identify theisolates as members of the Acinetobacter calcoaceticus-A. bau-mannii complex. Isolates were confirmed to be A. baumanniiby the identification of an OXA-51-type enzyme (10) (seebelow) and, for selected isolates, by tRNA spacer fingerprint-ing (2).Routine susceptibility testing used a disk diffusion method,while MICs were determined by broth microdilution withMueller-Hinton broth in 96-well microtiter plates (1). Resis-tance to imipenem and/or meropenem was confirmed usingEtests (AB Biodisk, Solna, Sweden). The isolates were alsotested for possible MBL production by using MBL Etests.Multidrug resistance was defined as resistance to three or moreantimicrobial classes.Crude bacterial DNA templates from bacterial cells wereprepared by boiling. Specific primers to detect the presence ofblaOXA23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like genes (10) and ISAba1 (9) were used in PCR mixtures(50-l final volumes) containing 25 l of PCR master mix(Roche Diagnostics, Burgess Hill, United Kingdom), 20 l ofultrapure water, 1 l of each primer (50 M), and 2 l of theDNA template. Cycling conditions comprised 95°C for 5 min,followed by 30 cycles of 95°C for 1 min, 50°C for 1 min, and72°C for 2 min, with final elongation at 72°C for 10 min, exceptthat an annealing temperature of 58°C was used to investigatethe possible location of ISAba1 upstream of blaOXA-51-likegenes. Amplicons from selected isolates were purified usingthe QIAquick PCR purification kit (Qiagen, Hilden, Germany)and sequenced using an ABI Prism 377 genetic analyzer (Ap-plied Biosystems, Warrington, United Kingdom). A. bauman-nii sequence groups were determined as described by Turton etal. (8). Pulsed-field gel electrophoresis was performed follow-ing macrorestriction with ApaI (5), with subsequent clusteranalysis as described previously (5).All 106 isolates were multidrug resistant (Table 1), but sul-bactam and colistin nonsusceptibility was not detected. Theimipenem MICs for the isolates were 2 to 32 g/ml, and themeropenem MICs were 8 to 128 g/ml. Cumulative percent-ages of A. baumannii isolates inhibited by imipenem and mero-penem are shown in Table 2. All 106 isolates were negative forMBL production according to the results of MBL Etests, but* Corresponding author. Mailing address: Department of ClinicalMicrobiology, Split University Hospital, Spinciceva 1, 21 000 Split,Croatia. Phone: 385 21 556 201. Fax: 385 21 547 901. E-mail:ivanagoicbar@net.hr. Published ahead of print on 19 August 2009.3348PCR revealed that each isolate possessed a blaOXA-51-likegene. No other class D oxacillinase genes were detected byPCR. All isolates with high-level carbapenem resistance (thosefor which the imipenem or meropenem MIC was 8 g/ml[94% of isolates]) had ISAba1 inserted upstream of theblaOXA-51-like gene. Pulsed-field gel electrophoresis genotyp-ing revealed that the 106 isolates formed three related clusters(with 85% similarity) belonging to the sequence group 2(European clone 1) lineage (i.e., that including the Europeanclone 1 reference strain RUH 2037). Sequencing of theblaOXA-51-like genes (both strands) from six representative iso-lates for which meropenem MICs were elevated revealed thepresence of the unusual OXA-107 enzyme.The carbapenem-hydrolyzing class D -lactamases of A.baumannii form four phylogenetic subgroups: OXA-23-like,OXA-24 like, OXA-51-like, and OXA-58-like enzymes (6).Since the discovery of OXA-51 in 2004, at least 39 relatedenzymes in this group have been described (3). The first de-scription of carbapenem-resistant clinical isolates of A. bau-mannii in Croatia reported the presence of an OXA-69-likeoxacillinase (4). The unusual OXA-107 enzyme was first de-tected in A. baumannii isolates from Poland and Slovenia (3)and is closely related to OXA-69, with an amino acid change atposition 167 that replaces leucine with valine (3). OXA-107 isnot distinguished from OXA-69 by the multiplex PCR methodof Turton et al. (8). In the present study of isolates withblaOXA-107 as the sole detectable carbapenemase gene, imi-penem and/or meropenem resistance was associated withISAba1 in 94% of cases, supporting the suggestion of Turton etal. (9) that the presence of ISAba1 upstream of a blaOXA-51-like gene is associated with high-level meropenem resistance.Sequence group 2 (European clone 1) is one of two majorlineages of multidrug-resistant A. baumannii that are wide-spread in Europe (7), including Bulgaria, Germany, Greece,The Netherlands, Norway, Poland, and Slovenia (7), but OXA-107 appears to be an uncommon enzyme and may represent amore recent evolutionary adaptation to antibiotic challengewith carbapenems.To our knowledge, this is the first extensive characterizationof clinical A. baumannii isolates producing a class D carbap-enemase in Croatia. Colistin and sulbactam are often the onlyremaining therapeutic options for the patients involved. Thefact that clonally related isolates were obtained over a numberof years suggests that these strains persisted unnoticed in thehospital’s ICUs, which then served as reservoirs for patientcolonization, followed by patient-to-patient transmission orcommon-source acquisition (e.g., through contaminated me-chanical ventilation equipment). Enhanced infection controlmeasures should be employed to limit the spread of A. bau-mannii strains within the hospital, and the consumption ofmeropenem should be restricted in order to reduce the selec-tion pressure in the hospital environment. Further studies inother hospitals in Croatia are needed to evaluate possibleinterhospital spread of multidrug-resistant A. baumanniistrains.Nucleotide sequence accession number. The blaOXA-51-likegene sequence determined in this study has been deposited inGenBank under accession number EF650033.We gratefully acknowledge helpful discussions with B. Evans (Uni-versity of Edinburgh).This work was supported, in part, by the UK Medical ResearchCouncil (grant no. RA0119) and the Croatian Ministry of Science,Education and Sport (grant no. 108-1080-0015). We otherwise declarethat we have no conflicting or dual interests in relation to this work.REFERENCES1. Clinical and Laboratory Standards Institute. 2007. Performance standardsfor antimicrobial susceptibility testing, 17th informational supplement,M100-S17. CLSI, Wayne, PA.2. Ehrenstein, B., A. T. Bernards, L. Dijkshoorn, P. Gerner-Smidt, K. J.Towner, P. J. Bouvet, F. D. Daschner, and H. Grundmann. 1996. Acineto-bacter species identification by using tRNA spacer fingerprinting. J. Clin.Microbiol. 34:2414–2420.3. Evans, B. A., A. Hamouda, K. J. Towner, and S. G. B. Amyes. 2008. OXA-51-like -lactamases and their association with particular epidemic lineagesof Acinetobacter baumannii. Clin. Microbiol. Infect. 14:268–275.4. Goic-Barisic, I., B. Bedenic, M. Tonkic, S. Katic, S. Kalenic, and V. Punda-Polic. 2007. First report of molecular characterization of carbapenem-resis-tant Acinetobacter baumannii in different intensive care units in UniversityHospital Split, Croatia. J. Chemother. 19:416–418.5. Gouby, A., M. J. Carles-Nurit, N. Bouziges, G. Bourg, R. Mesnard, and P. J.Bouvet. 1992. Use of pulsed-field gel electrophoresis for investigation ofhospital outbreak of Acinetobacter baumannii. J. Clin. Microbiol. 30:1588–1591.6. Poirel, L., and P. Nordmann. 2006. Carbapenem resistance in Acinetobacterbaumannii: mechanisms and epidemiology. Clin. Microbiol. Infect. 12:826–836.7. Towner, K. J., K. Levi, and M. Vlassialdi. 2008. Genetic diversity of carbap-enem-resistant isolates of Acinetobacter baumannii in Europe. Clin. Micro-biol. Infect. 14:161–167.8. Turton, J. F., S. N. Gabriel, C. Valderrey, M. E. Kaufmann, and T. L. Pitt.2007. Use of sequence-based typing and multiplex PCR to identify clonallineages of outbreak strains of Acinetobacter baumannii. Clin. Microbiol.Infect. 13:807–815.9. Turton, J. F., M. E. Ward, N. Woodford, M. E. Kaufmann, R. Pike, D. M.Livermore, and T. L. Pitt. 2006. The role of ISAba1 in expression of OXAcarbapenemase genes in Acinetobacter baumannii. FEMS Microbiol. Lett.258:72–77.10. Woodford, N., M. J. Ellington, J. M. Coelho, J. F. Turton, M. E. Ward, S.Brown, S. G. B. Amyes, and D. M. Livermoore. 2006. Multiplex PCR forgenes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J.Antimicrob. Agents 27:351–353.TABLE 1. MICs for 106 A. baumannii isolates from SplitUniversity HospitalAntibioticMIC (g/ml)Range 50% 90%Imipenem 2–32 4 8Meropenem 8–128 8 32Ceftazidime 128–128 128 128Ceftriaxone 64–128 128 128Cefepime 32–128 64 128Ciprofloxacin 4–64 16 32Amikacin 32–128 64 128Gentamicin 8–128 128 128Piperacillin-tazobactam 64–128 128 128Ampicillin-sulbactam 1–4 1 2Colistin 0.19–0.50 0.19 0.38TABLE 2. Cumulative percentages of 106 A. baumannii isolatesfrom Split University Hospital that were inhibitedby imipenem and meropenemCarbapenem% Inhibited at antibiotic concn (g/ml) of:0.5 1 2 4 8 16 32 64 128Imipenem 31 71 85 93 100Meropenem 64 80 87 98 100VOL. 47, 2009 NOTES 3349

Occurrence of OXA-107 and ISAba1 in carbapenem-resistant isolates of Acinetobacter baumannii from Croatia

     Edinburgh Research Explorer                                      Occurrence of OXA-107 and ISAba1 in carbapenem-resistantisolates of Acinetobacter baumannii from CroatiaCitation for published version:Goic-Barisic, I, Bedenic, B, Tonkic, M, Novak, A, Katic, S, Kalenic, S, Punda-Polic, V & Towner, KJ 2009,'Occurrence of OXA-107 and ISAba1 in carbapenem-resistant isolates of Acinetobacter baumannii fromCroatia', Journal of Clinical Microbiology, vol. 47, no. 10, pp. 3348-9. https://doi.org/10.1128/JCM.02394-08Digital Object Identifier (DOI):10.1128/JCM.02394-08Link:Link to publication record in Edinburgh Research ExplorerDocument Version:Publisher's PDF, also known as Version of recordPublished In:Journal of Clinical MicrobiologyGeneral rightsCopyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)and / or other copyright owners and it is a condition of accessing these publications that users recognise andabide by the legal requirements associated with these rights.Take down policyThe University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorercontent complies with UK legislation. If you believe that the public display of this file breaches copyright pleasecontact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately andinvestigate your claim.Download date: 28. Nov. 2025JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2009, p. 3348–3349 Vol. 47, No. 100095-1137/09/$08.000 doi:10.1128/JCM.02394-08Copyright © 2009, American Society for Microbiology. All Rights Reserved.Occurrence of OXA-107 and ISAba1 in Carbapenem-Resistant Isolatesof Acinetobacter baumannii from CroatiaIvana Goic-Barisic,1* Branka Bedenic,2 Marija Tonkic,1 Anita Novak,1 Stjepan Katic,2Smilja Kalenic,2 Volga Punda-Polic,1 and Kevin J. Towner3Department of Clinical Microbiology, Split University Hospital and School of Medicine, University of Split, Split,1 and Department ofClinical and Molecular Microbiology, Clinical Hospital Centre Zagreb and School of Medicine, University of Zagreb, Zagreb,2Croatia, and Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust,Queen’s Medical Centre, Nottingham, United Kingdom3Received 13 December 2008/Returned for modification 22 December 2008/Accepted 10 August 2009Carbapenem-resistant isolates of Acinetobacter baumannii from intensive care units at Split UniversityHospital, Split, Croatia, were studied. Most (100 of 106) had ISAba1 inserted upstream of a blaOXA-107 gene,encoding an unusual OXA-51-type oxacillinase. Pulsed-field gel electrophoresis revealed that the isolatesformed three clusters belonging to the sequence group 2 (European clone 1) lineage.An increasing trend toward multidrug resistance, includingcarbapenem resistance, in Acinetobacter baumannii has beenobserved in the past two decades. Carbapenem resistancemechanisms in A. baumannii include the production of metallo--lactamases (MBLs) and carbapenem-hydrolyzing oxacilli-nases, efflux mechanisms, and the loss of outer membraneproteins, often in combination (6). These resistance mecha-nisms pose a serious therapeutic threat, since carbapenems arefrequently used to treat otherwise resistant A. baumannii in-fections.Since 2002, increasing numbers of carbapenem-resistant iso-lates of A. baumannii have been recovered at Split UniversityHospital in Split, Croatia. Meropenem is prescribed at thishospital more frequently than imipenem, while ertapenem hasonly recently been licensed in Croatia. There are few publisheddata concerning carbapenem resistance in multidrug-resistantisolates of A. baumannii from Croatia. The present study in-vestigated the epidemiology and carbapenem resistance mech-anisms of multidrug-resistant A. baumannii isolates endemic inthis regional teaching hospital.Between 2002 and 2007, 106 nonrepetitive isolates of A.baumannii with an unusual resistance profile were isolatedconsecutively from two adult surgical intensive care units(ICUs), a pediatric ICU, a neurosurgery ICU, and a generalsurgery ICU at different locations in Split University Hospital(a 1,651-bed university teaching hospital with facilities at threesites). The hospital serves a pediatric and adult population ofca.500,000 and is also a referral hospital for much of southernCroatia (with a total population of ca. 1 million inhabitants).Isolates were initially recovered on blood agar plates fromroutine blood cultures, urine samples, wound exudates, cathe-ter tip specimens, and bronchial secretions. Conventional bio-chemical tests and the API 20NE system (bioMérieux, Marcy-l’Etoile, France) were used to presumptively identify theisolates as members of the Acinetobacter calcoaceticus-A. bau-mannii complex. Isolates were confirmed to be A. baumanniiby the identification of an OXA-51-type enzyme (10) (seebelow) and, for selected isolates, by tRNA spacer fingerprint-ing (2).Routine susceptibility testing used a disk diffusion method,while MICs were determined by broth microdilution withMueller-Hinton broth in 96-well microtiter plates (1). Resis-tance to imipenem and/or meropenem was confirmed usingEtests (AB Biodisk, Solna, Sweden). The isolates were alsotested for possible MBL production by using MBL Etests.Multidrug resistance was defined as resistance to three or moreantimicrobial classes.Crude bacterial DNA templates from bacterial cells wereprepared by boiling. Specific primers to detect the presence ofblaOXA23-like, blaOXA-24-like, blaOXA-51-like, and blaOXA-58-like genes (10) and ISAba1 (9) were used in PCR mixtures(50-l final volumes) containing 25 l of PCR master mix(Roche Diagnostics, Burgess Hill, United Kingdom), 20 l ofultrapure water, 1 l of each primer (50 M), and 2 l of theDNA template. Cycling conditions comprised 95°C for 5 min,followed by 30 cycles of 95°C for 1 min, 50°C for 1 min, and72°C for 2 min, with final elongation at 72°C for 10 min, exceptthat an annealing temperature of 58°C was used to investigatethe possible location of ISAba1 upstream of blaOXA-51-likegenes. Amplicons from selected isolates were purified usingthe QIAquick PCR purification kit (Qiagen, Hilden, Germany)and sequenced using an ABI Prism 377 genetic analyzer (Ap-plied Biosystems, Warrington, United Kingdom). A. bauman-nii sequence groups were determined as described by Turton etal. (8). Pulsed-field gel electrophoresis was performed follow-ing macrorestriction with ApaI (5), with subsequent clusteranalysis as described previously (5).All 106 isolates were multidrug resistant (Table 1), but sul-bactam and colistin nonsusceptibility was not detected. Theimipenem MICs for the isolates were 2 to 32 g/ml, and themeropenem MICs were 8 to 128 g/ml. Cumulative percent-ages of A. baumannii isolates inhibited by imipenem and mero-penem are shown in Table 2. All 106 isolates were negative forMBL production according to the results of MBL Etests, but* Corresponding author. Mailing address: Department of ClinicalMicrobiology, Split University Hospital, Spinciceva 1, 21 000 Split,Croatia. Phone: 385 21 556 201. Fax: 385 21 547 901. E-mail:ivanagoicbar@net.hr. Published ahead of print on 19 August 2009.3348PCR revealed that each isolate possessed a blaOXA-51-likegene. No other class D oxacillinase genes were detected byPCR. All isolates with high-level carbapenem resistance (thosefor which the imipenem or meropenem MIC was 8 g/ml[94% of isolates]) had ISAba1 inserted upstream of theblaOXA-51-like gene. Pulsed-field gel electrophoresis genotyp-ing revealed that the 106 isolates formed three related clusters(with 85% similarity) belonging to the sequence group 2(European clone 1) lineage (i.e., that including the Europeanclone 1 reference strain RUH 2037). Sequencing of theblaOXA-51-like genes (both strands) from six representative iso-lates for which meropenem MICs were elevated revealed thepresence of the unusual OXA-107 enzyme.The carbapenem-hydrolyzing class D -lactamases of A.baumannii form four phylogenetic subgroups: OXA-23-like,OXA-24 like, OXA-51-like, and OXA-58-like enzymes (6).Since the discovery of OXA-51 in 2004, at least 39 relatedenzymes in this group have been described (3). The first de-scription of carbapenem-resistant clinical isolates of A. bau-mannii in Croatia reported the presence of an OXA-69-likeoxacillinase (4). The unusual OXA-107 enzyme was first de-tected in A. baumannii isolates from Poland and Slovenia (3)and is closely related to OXA-69, with an amino acid change atposition 167 that replaces leucine with valine (3). OXA-107 isnot distinguished from OXA-69 by the multiplex PCR methodof Turton et al. (8). In the present study of isolates withblaOXA-107 as the sole detectable carbapenemase gene, imi-penem and/or meropenem resistance was associated withISAba1 in 94% of cases, supporting the suggestion of Turton etal. (9) that the presence of ISAba1 upstream of a blaOXA-51-like gene is associated with high-level meropenem resistance.Sequence group 2 (European clone 1) is one of two majorlineages of multidrug-resistant A. baumannii that are wide-spread in Europe (7), including Bulgaria, Germany, Greece,The Netherlands, Norway, Poland, and Slovenia (7), but OXA-107 appears to be an uncommon enzyme and may represent amore recent evolutionary adaptation to antibiotic challengewith carbapenems.To our knowledge, this is the first extensive characterizationof clinical A. baumannii isolates producing a class D carbap-enemase in Croatia. Colistin and sulbactam are often the onlyremaining therapeutic options for the patients involved. Thefact that clonally related isolates were obtained over a numberof years suggests that these strains persisted unnoticed in thehospital’s ICUs, which then served as reservoirs for patientcolonization, followed by patient-to-patient transmission orcommon-source acquisition (e.g., through contaminated me-chanical ventilation equipment). Enhanced infection controlmeasures should be employed to limit the spread of A. bau-mannii strains within the hospital, and the consumption ofmeropenem should be restricted in order to reduce the selec-tion pressure in the hospital environment. Further studies inother hospitals in Croatia are needed to evaluate possibleinterhospital spread of multidrug-resistant A. baumanniistrains.Nucleotide sequence accession number. The blaOXA-51-likegene sequence determined in this study has been deposited inGenBank under accession number EF650033.We gratefully acknowledge helpful discussions with B. Evans (Uni-versity of Edinburgh).This work was supported, in part, by the UK Medical ResearchCouncil (grant no. RA0119) and the Croatian Ministry of Science,Education and Sport (grant no. 108-1080-0015). We otherwise declarethat we have no conflicting or dual interests in relation to this work.REFERENCES1. Clinical and Laboratory Standards Institute. 2007. Performance standardsfor antimicrobial susceptibility testing, 17th informational supplement,M100-S17. CLSI, Wayne, PA.2. Ehrenstein, B., A. T. Bernards, L. Dijkshoorn, P. Gerner-Smidt, K. J.Towner, P. J. Bouvet, F. D. Daschner, and H. Grundmann. 1996. Acineto-bacter species identification by using tRNA spacer fingerprinting. J. Clin.Microbiol. 34:2414–2420.3. Evans, B. A., A. Hamouda, K. J. Towner, and S. G. B. Amyes. 2008. OXA-51-like -lactamases and their association with particular epidemic lineagesof Acinetobacter baumannii. Clin. Microbiol. Infect. 14:268–275.4. Goic-Barisic, I., B. Bedenic, M. Tonkic, S. Katic, S. Kalenic, and V. Punda-Polic. 2007. First report of molecular characterization of carbapenem-resis-tant Acinetobacter baumannii in different intensive care units in UniversityHospital Split, Croatia. J. Chemother. 19:416–418.5. Gouby, A., M. J. Carles-Nurit, N. Bouziges, G. Bourg, R. Mesnard, and P. J.Bouvet. 1992. Use of pulsed-field gel electrophoresis for investigation ofhospital outbreak of Acinetobacter baumannii. J. Clin. Microbiol. 30:1588–1591.6. Poirel, L., and P. Nordmann. 2006. Carbapenem resistance in Acinetobacterbaumannii: mechanisms and epidemiology. Clin. Microbiol. Infect. 12:826–836.7. Towner, K. J., K. Levi, and M. Vlassialdi. 2008. Genetic diversity of carbap-enem-resistant isolates of Acinetobacter baumannii in Europe. Clin. Micro-biol. Infect. 14:161–167.8. Turton, J. F., S. N. Gabriel, C. Valderrey, M. E. Kaufmann, and T. L. Pitt.2007. Use of sequence-based typing and multiplex PCR to identify clonallineages of outbreak strains of Acinetobacter baumannii. Clin. Microbiol.Infect. 13:807–815.9. Turton, J. F., M. E. Ward, N. Woodford, M. E. Kaufmann, R. Pike, D. M.Livermore, and T. L. Pitt. 2006. The role of ISAba1 in expression of OXAcarbapenemase genes in Acinetobacter baumannii. FEMS Microbiol. Lett.258:72–77.10. Woodford, N., M. J. Ellington, J. M. Coelho, J. F. Turton, M. E. Ward, S.Brown, S. G. B. Amyes, and D. M. Livermoore. 2006. Multiplex PCR forgenes encoding prevalent OXA carbapenemases in Acinetobacter spp. Int. J.Antimicrob. Agents 27:351–353.TABLE 1. MICs for 106 A. baumannii isolates from SplitUniversity HospitalAntibioticMIC (g/ml)Range 50% 90%Imipenem 2–32 4 8Meropenem 8–128 8 32Ceftazidime 128–128 128 128Ceftriaxone 64–128 128 128Cefepime 32–128 64 128Ciprofloxacin 4–64 16 32Amikacin 32–128 64 128Gentamicin 8–128 128 128Piperacillin-tazobactam 64–128 128 128Ampicillin-sulbactam 1–4 1 2Colistin 0.19–0.50 0.19 0.38TABLE 2. Cumulative percentages of 106 A. baumannii isolatesfrom Split University Hospital that were inhibitedby imipenem and meropenemCarbapenem% Inhibited at antibiotic concn (g/ml) of:0.5 1 2 4 8 16 32 64 128Imipenem 31 71 85 93 100Meropenem 64 80 87 98 100VOL. 47, 2009 NOTES 3349

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Occurrence of OXA-107 and ISAba1 in carbapenem-resistant isolates of Acinetobacter baumannii from Croatia

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