Differences in phenotypic and genotypic traits against antimicrobial agents between Acinetobacter baumannii and Acinetobacter genomic species 13TU

Journal of Antimicrobial Chemotherapy (2007) 59, 633 639 doi:10.1093/jac/dkm007 Advance Access publication 6 March 2007 Differences in phenotypic and genotypic traits against antimicrobial agents between Acinetobacter baumannii and Acinetobacter genomic species 13TU Jung Hoon Lee 1, Chul Hee Choi 1, Hee Young Kang 1, Ji Young Lee 1, Jungmin Kim 1, Yoo Chul Lee 1, Sung Yong Seol 1, Dong Taek Cho 1, Kun Woo Kim 2, Do Young Song 3 and Je Chul Lee 1 * 1 Department of Microbiology, Kyungpook National University School of Medicine, Daegu 700-422, Republic of Korea; 2 Department of Internal Medicine, Daegu Fatima Hospital, Daegu 701-600, Republic of Korea; 3 Department of Clinical Pathology, Daegu Fatima Hospital, Daegu 701-600, Republic of Korea Received 19 October 2006; returned 15 December 2006; revised 11 January 2007; accepted 12 January 2007 Objectives: To investigate the differences in antimicrobial susceptibility and resistance mechanisms against imipenem between Acinetobacter baumannii and Acinetobacter genomic species 13TU. Methods: A total of 232 non-duplicate Acinetobacter species were consecutively collected from two Korean hospitals in Daegu, Republic of Korea, between November 2004 and November 2005. Antimicrobial susceptibility was determined by agar dilution methods. Resistance to imipenem was characterized by a carbapenemase activity test and PCR amplification. PFGE was performed to determine the clonal relatedness of imipenem-resistant Acinetobacter species. Results: A. baumannii was the most prevalent species (61.2%), followed by Acinetobacter genomic species 13TU (25.9%). The resistance rates of A. baumannii to most antimicrobial agents were higher than those of other Acinetobacter species, while the resistance rate to imipenem was the highest in Acinetobacter genomic species 13TU. Imipenem-resistant Acinetobacter genomic species 13TU isolates produced VIM-2 metallo-b-lactamase, while imipenem-resistant A. baumannii isolates produced OXA-23 and/or OXA-51 b-lactamase. Imipenem-resistant Acinetobacter strains originated from different clones in each hospital. Conclusions: Two prevalent Acinetobacter species, A. baumannii and Acinetobacter genomic species 13TU, possess distinct phenotypic and genotypic traits against antimicrobials. Keywords: carbapenem resistance, metallo-b-lactamases, b-lactamases, A. baumannii Introduction Bacteria of the genus Acinetobacter are important opportunistic pathogens that are responsible for nosocomial infections. Acinetobacter species are highly resistant to commonly used antibiotics such as penicillins, cephalosporins, aminoglycosides and fluoroquinolones by intrinsic and acquired mechanisms. Carbapenems have been used effectively to treat multiresistant Acinetobacter infections in many centres, but carbapenemresistant Acinetobacter species have been reported worldwide 1 and they have given rise to serious therapeutic problems. Acinetobacter baumannii carries intrinsic carbapenemhydrolysing class D b-lactamases (OXA-51/69 variants), but acquisition of further class B and D carbapenem-hydrolysing b-lactamases is an important cause of carbapenem resistance in Acinetobacter species. Class B metallo-b-lactamases, including IMP, VIM and SIM-1 enzymes, have been found, but most Acinetobacter species produce class D b-lactamases, including OXA-23-like, OXA-24-like and OXA-58 enzymes. 1 Recently, isolates of Acinetobacter species that are resistant to all antibiotics routinely tested, and susceptible only to colistin, have been detected in Taiwan, 2 Italy 3 and Greece. 4 Polymyxins and tigecycline are becoming an important option for the treatment of multiresistant Acinetobacter species. The A. calcoaceticus A. baumannii complex (Acb complex) is composed of A. calcoaceticus, A. baumannii and genomic species 3 and 13TU. These are genetically closely related and include the most important acinetobacters in a clinical setting;... *Corresponding author. Tel: þ82-53-420-4844; Fax: þ82-53-427-5664; E-mail: leejc@knu.ac.kr... 633 # The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Lee et al. A. baumannii and genomic species 3 and 13TU are commonly associated with nosocomial infections and are usually multiresistant. As only limited data are available, we analysed the antimicrobial susceptibilities and imipenem resistance mechanisms of clinical Acinetobacter isolates belonging to different genomic species. Materials and methods Bacterial strains A total of 232 Acinetobacter species were consecutively collected from a secondary hospital and a tertiary teaching hospital in Daegu, Republic of Korea, between November 2004 and November 2005. Only the first isolate from each patient was included; multiple isolates from a single patient were included only if they represented different amplified ribosomal DNA restriction analysis (ARDRA) profiles. Organisms were identified to the genus Acinetobacter using API20NE (biomérieux, Marcy l Étoile, France). Genomic species were identified by ARDRA as previously described, 5 using restriction endonucleases (CfoI, AluI, MboI, MspI, RsaI, BfaI and BsmAI). Antimicrobial susceptibility testing An antimicrobial susceptibility test was performed by agar dilution in Mueller Hinton agar (Difco Laboratories, Detroit, MI, USA) according to the guidelines of the CLSI (formerly NCCLS). 6 Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. The final concentrations of antimicrobial agents ranged from 0.25 to 1024 mg/l, and the agents tested included ampicillin/ sulbactam, piperacillin, cefoperazone, cefotaxime, ceftazidime, cefepime, imipenem, aztreonam, amikacin, gentamicin, tobramycin, ciprofloxacin, trimethoprim/sulfamethoxazole and colistin. Screening of carbapenemase activity In order to analyse the production of class B and D carbapenemases, imipenem-resistant isolates were first screened by a modified Hodge test. 7 The surface of a Mueller Hinton agar plate was inoculated with an overnight culture suspension of E. coli ATCC 25922. An imipenem disc was placed at the centre of the plate and imipenem-resistant Acinetobacter was streaked heavily from the edge of the disc to the periphery of the plate. The presence of a distorted inhibition zone, after overnight incubation, was interpreted as a positive result. An imipenem-edta double disc synergy test was performed to screen for the production of metallo-b-lactamases. An overnight culture of the modified Hodge test positive strains was inoculated on a Mueller Hinton agar plate. The imipenem disc (30 mg) and a blank filter paper disc were placed 15 mm apart from edge to edge, and 10 ml of 0.5 M EDTA solution was applied to the blank disc. After overnight incubation, the presence of an enlarged zone of inhibition was interpreted as a positive result, which shows the inactivation of class B metallo-b-lactamase activity by EDTA. PCR amplification and sequencing PCR was performed in a 20 ml volume containing: 2 ml of boiled bacterial suspensions, 20 pm of each primer, 250 mm dntps, 10 mm Tris HCl (ph 8.3), 50 mm KCl, 2.5 mm MgCl 2 and 1.5 U of Taq DNA polymerase (Bioneer, Daejeon, Republic of Korea). Genes coding for carbapenemases were sought by PCR using primers specific for the genes bla VIM-2, 8 bla IMP-1, 8 bla SIM-1, 9 bla OXA-24, 10 bla OXA-51 -type, 11 11 bla OXA-58 and bla OXA-23 (OXA-23-sense: 5 0 -CCTCAGGTGTGCTGGTTATT-3 0 and OXA-23-antisense: 5 0 -CCCAACCAGTCTTTCCAAA-3 0 ). The presence of ISAba1 inserted upstream of bla OXA-51 was sought by PCR as previously described. 12 PCR products were sequenced with an automated sequencer (ABI 3100; Applied Biosystems, Foster City, CA, USA). Pulsed-field gel electrophoresis Genomic DNA was digested with ApaI (Roche Diagnostics, Mannheim, Germany) and separated on a 1.0% agarose gel using a contour-clamped homogeneous-field apparatus (CHEF DRIII systems, Bio-Rad Laboratories, Hercules, CA, USA) in 0.5 TBE buffer. 13 The banding patterns were analysed with GelCompar II software (Applied Maths, Kortrijk, Belgium). Results Identification of Acinetobacter genomic species Of the 232 non-duplicate Acinetobacter species, 224 isolates were assigned to seven genomic species based on their ARDRA profiles: A. baumannii (n ¼ 142), Acinetobacter genomic species 13TU (n ¼ 60), Acinetobacter genomic species 3 (n ¼ 14), Acinetobacter genomic species 10 (n ¼ 4), A. junii (n ¼ 2), A. johnsonii (n ¼ 1) and Acinetobacter genomic species 14BJ (n ¼ 1). The remaining eight isolates, which were not classified to any genomic species by their ARDRA profiles, were assigned to unclassified Acinetobacter species. Antimicrobial susceptibility of Acinetobacter species The MIC distributions for the Acinetobacter species are shown in Table 1. Over 65% of the isolates were resistant to cefoperazone and aztreonam, and over 30% were resistant to ampicillin/ sulbactam, piperacillin, cefotaxime, ceftazidime, cefepime, amikacin, gentamicin, tobramycin, ciprofloxacin and trimethoprim/ sulfamethoxazole. Resistance to colistin (MIC 8 mg/l) was found in two isolates of A. johnsonii and Acinetobacter genomic species 10. Only one isolate, A. baumannii isolate 05P1226, was susceptible only to colistin (Table 2). Twenty-one isolates, including Acinetobacter genomic species 13TU (n ¼ 10), 3 (n ¼ 8), A. junii (n ¼ 2) and 14BJ (n ¼ 1), were susceptible to all antimicrobial agents tested. The resistance rates of A. baumannii were higher than those of other Acinetobacter species to ampicillin-sulbactam, piperacillin, cefotaxime, ceftazidime, cefepime, aztreonam, amikacin, gentamicin, tobramycin, ciprofloxacin and trimethoprim/sulfamethoxazole. However, the resistance rate for imipenem was higher for Acinetobacter genomic species 13TU than for other Acinetobacter species. Imipenem resistance and carbapenem-hydrolysing b-lactamases The overall incidence of resistance to imipenem was 13.8% (32/232 isolates) and was found only in the Acb complex: 23.3% (14/60) in Acinetobacter genomic species 13TU, 12% 634

Table 1. Antimicrobial susceptibility of Acinetobacter species isolated from clinical specimens in two hospitals in Daegu, Korea 635 MIC (mg/l) of antimicrobial agent Genomic species SAM PIP CFP CTX CAZ FEP IPM ATM AMK GEN TOB CIP SXT COL A. baumannii MIC 50 32 1024 1024 512 128 32 2 32 1024 1024 1024 32 32 1 (n ¼ 142) MIC 90 32 1024 1024 1024 256 128 16 128 1024 1024 1024 64 128 1 range 1 256 8 16 4 1 0.5 512,0.25 128 4 0.5 0.5 0.5 0.25 128 0.25 0.5 2 1024 1024 1024 1024 1024 1024 1024 1024 512 13TU MIC 50 1 16 64 16 8 4,0.25 32 4 4,1,0.25 1 2 (n ¼ 60) MIC 90 8 128 512 128 32 16 16 64 64 256 64 1 8 2 range,0.25 16 4 256 32 512 2 256 1,0.25 16,0.25 32 2 2 128 0.5 256,0.25 64 0.25 0.25 0.5 4 1024 1024 128 16 3 MIC 50 2 16 64 8 4 2,0.25 16 2 0.5 0.5,0.25 1 0.5 (n ¼ 14) MIC 90 16 128 256 128 512 32 0.5 1024 64 256 64 32 16 1 range 1 16 4 256 16 4 128 1 0.5 64,0.25 4 1 256 0.5 256,0.25,0.25 32,0.25 512 0.5 2 1024 512 16 1024 64 Other a MIC 50 2 32 64 8 8 2,0.25 32 2 2,0.5,0.25 0.5 2 (n ¼ 16) MIC 90 4 128 128 16 16 8 1 64 4 8 4 16 4 4 range,0.25 4 4 256 8 128 2 32 2 16 0.5 8,0.25 8 8 64 0.5 4 0.5 64,0.25 16 0.25 64,0.25 32 1 32 SAM, ampicillin/sulbactam; PIP, piperacillin; CFP, cefoperazone; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem; ATM, aztreonam; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; SXT, trimethoprim/sulfamethoxazole; COL, colistin. a Acinetobacter genomic species 10 (n ¼ 4), A. junii (n ¼ 2), A. johnsonii (n ¼ 1), Acinetobacter genomic species 14BJ (n ¼ 1) and unclassified Acinetobacter species (n ¼ 8). Antimicrobial susceptibility in Acinetobacter species Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 2014

Table 2. Characterization of imipenem-resistant Acinetobacter species Species Isolate Isolation date Ward/ Source MIC (mg/l) hospital a of imipenem Modified Hodge test EDTA disc synergy test Carbapenemase PFGE pattern b 636 Acinetobacter 04P053 12 November 2004 IG/S sputum 16 þ þ VIM-2 A 13TU 04P175 22 November 2004 IN/S sputum 16 þ þ VIM-2 B 05P092 01 January 2005 NS/S sputum 16 þ þ VIM-2 B 05P096 01 January 2005 IA/S sputum 16 þ þ VIM-2 B 05P145 06 January 2005 IP/S sputum 16 þ þ VIM-2 A 05P162 07 January 2005 IP/S sputum 16 þ þ VIM-2 A 05P724b 08 March 2005 NS/S sputum 16 þ þ VIM-2 B 05P808 19 March 2005 IA/S sputum 16 þ þ VIM-2 B 05P893 31 March 2005 GS/S sputum 16 þ þ VIM-2 B 05P976 09 April 2005 GS/S sputum 16 þ þ VIM-2 A 05P1126 28 April 2005 IC/S sputum 16 þ þ VIM-2 C 05P1130 28 April 2005 NS/S sputum 16 þ þ VIM-2 A 05P1145 29 April 2005 IP/S sputum 32 þ þ VIM-2 A 05KA161 21 November 2005 IC/T urine 16 þ þ VIM-2 C A. baumannii 04KA017 23 Deember 2004 NS/T wound 32 þ 2 OXA-23 and -51 c 05KA026 14 February 2005 CS/T sputum 32 þ 2 OXA-23 and -51 c 05KA119 29 August 2005 OS/T wound 32 þ 2 OXA-23 and -51 c 05KA130 05 September 2005 ENT/T sputum 32 þ 2 OXA-23 and -51 e 05KA131 02 September 2005 IR/T blood 128 þ 2 OXA-23 and -51 e 05KA144 10 October 2005 OS/T wound 64 þ 2 OXA-23 and -51 e 05KA171 16 December 2005 CS/T wound 16 þ 2 OXA-51 only b 05P997 15 April 2005 IP/S sputum 32 þ 2 OXA-23 and -51 d 05P1067 23 April 2005 IP/S sputum 16 þ 2 OXA-23 and -51 d 05P1078 26 April 2005 IA/S sputum 32 þ 2 OXA-23 and -51 d 05P1108 26 April 2005 IP/S sputum 32 þ 2 OXA-23 and -51 d 05P1165 01 May 2005 IP/S sputum 32 þ 2 OXA-23 and -51 d 05P1183 03 May 2005 IP/S sputum 32 þ 2 OXA-23 and -51 d 05P1226 09 May 2005 IP/S sputum 32 þ 2 OXA-23 and -51 d 05P2140 02 October 2005 GS/S wound 16 þ 2 OXA-51 only a 05P2164 18 June 2005 PD/S throat 16 þ 2 OXA-51 only a 05P2165 19 June 2005 IH/S sputum 16 þ 2 OXA-51 only a Lee et al. Acinetobacter 3 05KA103 20 September 2005 IH/T sputum 16 þ 2 OXA-23 2 a Wards: IG, gastroenterological internal medicine; IN, endocrinological internal medicine; NS, neurosurgery; IA, allergic internal medicine; IP, pulmonary internal medicine; GS, general surgery; IC, circulatory internal medicine; NS, neurosurgery; CS, cardiac surgery; OS, orthopaedic surgery; ENT, otorhinolaryngology; IR, rheumatological internal medicine; PD, paediatrics; IH, haematological internal medicine. Hospitals: S, secondary hospital; T, tertiary hospital. b PFGE profiles were arbitrarily classified, as shown in Figure 1. Downloaded from http://jac.oxfordjournals.org/ at Pennsylvania State University on March 3, 2014

Antimicrobial susceptibility in Acinetobacter species (17/142) in A. baumannii and 7.1% (1/14) in Acinetobacter genomic species 3. All imipenem-resistant Acinetobacter genomic species 13TU were positive in the modified Hodge test and imipenem-edta double disc synergy test, indicating the production of class B metallo-b-lactamases (Table 2). Seventeen imipenem-resistant A. baumannii isolates were positive in the modified Hodge test, but negative in the imipenem/edta double disc synergy test, indicating the production of class D or another type of carbapenemase. One imipenem-resistant Acinetobacter genomic species 3 isolate was also positive only in the modified Hodge test. By PCR and sequencing, all imipenem-resistant isolates of Acinetobacter genomic species 13TU were shown to carry bla VIM-2, 13 imipenem-resistant A. baumannii carried bla OXA-23 and bla OXA-51, and one Acinetobacter 3 species carried bla OXA-23 only. Four imipenem-resistant A. baumannii isolates carried bla OXA-51 as the sole carbapenemase gene detected. Since high carbapenem MICs for such isolates may result from enhanced transcription of bla OXA-51 from promoter sequences in ISAba1, the position of ISAba1 relative to bla OXA-51 was determined by PCR; ISAba1 was inserted upstream of bla OXA-51 in all four A. baumannii isolates. Clonal relatedness of imipenem-resistant Acinetobacter species PFGE was performed to determine the clonal relatedness of imipenem-resistant Acinetobacter species. Fourteen bla VIM-2 - carrying Acinetobacter genomic species 13TU isolates were (a) Dice (Opt:1.10%) (Tol 1.1 1.1%) (H > 0.0 S > 0.0%) [0.0% 100.0%] 70 75 80 85 90 95 100 A B C (b) Dice (Opt:1.10%) (Tol 1.1 1.1%) (H > 0.0 S > 0.0%) [0.0% 100.0%] 70 75 80 85 90 95 100 a b c d e Acinetobacter 13TU 05P162 Acinetobacter 13TU 05P1130 Acinetobacter 13TU 04P053 Acinetobacter 13TU 05P145 Acinetobacter 13TU 05P1145 Acinetobacter 13TU 05P976 Acinetobacter 13TU 05P092 Acinetobacter 13TU 05P096 Acinetobacter 13TU 05P724b Acinetobacter 13TU 05P808 Acinetobacter 13TU 05P893 Acinetobacter 13TU 04P175 Acinetobacter 13TU 05P1126 Acinetobacter 13TU 05KA161 A. baumannii 05P2140 A. baumannii 05P2165 A. baumannii 05P2164 A. baumannii 04KA171 A. baumannii 05KA079 A. baumannii 05KA119 A. baumannii 05KA026 A. baumannii 05P1067 A. baumannii 05P1078 A. baumannii 05P1108 A. baumannii 05P1165 A. baumannii 05P997 A. baumannii 05P1183 A. baumannii 05P1226 A. baumannii 05KA130 A. baumannii 05KA131 A. baumannii 05KA144 Figure 1. PFGE patterns of imipenem-resistant Acinetobacter genomic species 13TU (a) and A. baumannii isolates (b). 637

Lee et al. classified into three PFGE groups at a similarity value of 0.9 (Figure 1a). However, PFGE profiles A and B show very similar band patterns, suggesting that they originated from an identical clone. This clone accounted for 85.7% (12/14) of imipenemresistant Acinetobacter genomic species 13TU in a secondary hospital (Table 2). Seventeen imipenem-resistant A. baumannii isolates were classified into five groups at a similarity value of 0.9 (Figure 1b). PFGE profiles a and d appeared in 10 A. baumannii isolates from a secondary hospital, while PFGE profiles b, c and e appeared in seven A. baumannii isolates from a tertiary hospital (Table 2). These findings suggest that imipenem-resistant Acinetobacter strains originated from different clones in each hospital. Discussion The present study demonstrated differences in antimicrobial susceptibility and resistance mechanisms against imipenem between A. baumannii and Acinetobacter genomic species 13TU. Resistance rates and MICs of A. baumannii to most antimicrobial agents were higher than those of other Acinetobacter species. Over 50% of A. baumannii isolates were resistant to currently used antimicrobial agents, such as ampicillinsulbactam, cefepime and ciprofloxacin, while less than 5% of Acinetobacter genomic species 13TU were resistant to these agents. Older antimicrobial agents, such as polymyxins, have been re-evaluated therapeutically due to the emergence of multiresistant Gram-negative bacteria, including P. aeruginosa, Klebsiella pneumoniae and Acinetobacter species. 4 The reuse of polymyxins in multiresistant bacterial infections contributed to the emergence of bacteria resistant to all classes of available antimicrobial agents. Such pan-drug-resistant bacteria have been recently reported among P. aeruginosa and K. pneumoniae, 4,14 but pan-drug-resistant Acinetobacter species have not been reported yet. In the current study, a single A. baumannii isolate was found that was susceptible only to colistin. The intensive use of carbapenems may have facilitated the rapid emergence of resistance in clinical Acinetobacter species. The acquisition of class B and D carbapenemases is typically responsible for resistance to carbapenems in many clinical isolates, although resistance has also been associated with reduced drug uptake through a porin, reduced affinity of penicillinbinding proteins, and with drug efflux pumps. All imipenemresistant Acinetobacter species tested here carried at least one carbapenemase. Although class B metallo-b-lactamases have been identified in a wide variety of Gram-negative bacteria, metallo-b-lactamase-producing A. baumannii have not been commonly detected anywhere in the world. The most widespread carbapenemases in Acinetobacter species are class D b-lactamases. 1 In the current study, we demonstrated major differences in the imipenem resistance mechanisms of isolates of A. baumannii and Acinetobacter genomic species 13TU in Daegu, Republic of Korea: OXA-23 and OXA-51 b-lactamases were responsible for the resistance of A. baumannii, while VIM-2 metallo-b-lactamase was responsible for the resistance of Acinetobacter genomic species 13TU. There are two recent reports regarding carbapenemase-producing A. baumannii in Korea: VIM-2-producing A. baumannii was isolated from a tertiary hospital in Seoul 15 and OXA-23 b-lactamase was found in outbreak-associated carbapenem-resistant A. baumannii in Busan. 8 However, the exact genomic species in these reports was unclear because phenotypic identification systems were used. The distribution of carbapenemases should be clarified according to Acinetobacter genomic species. In conclusion, A. baumannii and Acinetobacter genomic species 13TU were the two most prevalent Acinetobacter species in Daegu, Republic of Korea, and they showed different microbiological characteristics in terms of antimicrobial susceptibility and resistance mechanisms against carbapenems. It must be determined whether these differences result from genetic characteristics of the individual species, or whether they are influenced by antibiotic selective pressure in clinical settings. Acknowledgements We thank Kyung Min Jeong for her assistance with laboratory work. This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH03-0002), and a grant from Eulji University (2002). Transparency declarations None to declare. References 1. Poirel L, Nordmann P. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin Microbiol Infect 2006; 12: 826 36. 2. Hsueh PR, Teng LJ, Chen CY et al. 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