Twenty-two MDR A. baumannii isolates were collected and characterized [5]. Table 1 shows the minimal inhibitory concentrations (MICs) and antimicrobial resistance determinants of the isolates characterized in this study. These isolates were collected from different patients at a single university hospital in Daejeon, Korea during the period between 2007 and 2011. Seven additional strains that were susceptible to carbapenem but resistant to many other antimicrobial agents were also included in this study. Piperacillin, piperacillin/tazobactam, cefepime, ceftazidime, meropenem, and ticarcillin susceptibility testing was performed using the Vitek 2 system (bioMérieux, Marcy l'Eltoile, France). The MICs of imipenem, amikacin, gentamicin, and ciprofloxacin were determined using the Etest (AB Biodisk, Solna, Sweden). Interpretation was performed according to the criteria approved by the CLSI guidelines [6]. Escherichia coli ATCC 25922 was used as a reference strain. Clinical isolates of A. baumannii were identified by rpoB gene analysis and by the presence of the bla_oxa-51-like gene [7].

The MDR phenotype was defined as resistance to representative antimicrobial agents of at least 3 different classes of drugs: aminoglycosides (gentamicin, amikacin), antipseudomonal penicillins (ticarcillin, piperacillin, piperacillin/tazobactam), carbapenems (imipenem, meropenem), antipseudomonal cephalosporins (ceftazidime, cefepime), and fluoroquinolones (ciprofloxacin) [8].

Whole-cell (genomic) DNA was obtained from each target strain using a genomic DNA purification kit (SolGent, Daejeon, Korea) according to the manufacturer's instructions. PCR was performed using 50 ng of genomic DNA, 2.5 µL of 10× Taq buffer, 0.5 µL of 10 mM dNTP mix, 20 pmol of each primer, and 0.7 U of Taq DNA polymerase (SolGent) in a total volume of 25 µL. Each target gene was amplified in a GeneAmp PCR System 9600 thermal cycler (Perkin-Elmer Cetus Corp., Norwalk, CT, USA). Thermal cycling conditions consisted of an initial denaturation cycle at 95℃ for 5 min, followed by 30 cycles of 95℃ for 30 sec, 52℃ for 40 sec, and 72℃ for 30 sec, with a final extension at 72℃ for 5 min. The annealing temperature was 52℃, unless otherwise specified. The amplified products were separated via electrophoresis on 1.5% (w/v) agarose gels containing ethidium bromide, and visualized using a BioDoc-14TM Imaging system (UVP, Cambridge, UK). For sequencing, PCR products were purified with a PCR purification kit (SolGent) according to the manufacturer's protocols.

The 2 multiplex PCR assays were performed as previously described [9] to identify members of the EU clone I and EU clone II lineages. Epidemiological typing of the isolates was performed by repetitive extragenic palindromic sequence (REP) PCR [10]. The Oxford multilocus sequence typing (MLST) scheme [11], which uses 7 housekeeping genes (gltA, gyrB, gdhB, recA, cpn60, gpi, and rpoD24) was used to determine the sequence types (STs). A ST number was assigned by comparing the allele sequences to those on the MLST site (http://pubmlst.org/abaumannii/). In addition, class 1 integrons were detected and sequenced using PCR conditions and a primer described previously [5].

The genes associated with the AbaR islands were detected by PCR using published primers (Table 2). The amplified regions are shown in Fig. 1 [4]. To investigate the structure of the AbaR backbone, PCR mapping experiments were performed as described previously [4]. The amplicons were purified and sequenced using a BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 3730XL DNA analyzer (PE Applied Biosystems). DNA fragments (up to 1 kb in size) were sequenced using the overlapping PCR technique. The various DNA sequences were confirmed using the Basic Local Alignment Search Tool (BLAST) program (http://blast.ncbi.nlm.gov).

The 29 MDR A. baumannii strains were tested to determine their association with the EU clone lineages using multiplex PCRs. Twenty-seven isolates belonged to the EU clone II lineage and carried allele 66 of the intrinsic bla_OXA-51-like genes, which corresponds to their assignment to the EU clone II lineage. MLST analysis of the EU clone II isolates revealed 5 STs (75, 92, 137, 138, and 357) (Table 3). In particular, ST357 (1-12-3-2-2-145-3) was confirmed for the first time in Korea. The strains identified as ST357 were susceptible to imipenem although they were MDR strains (Table 1). Among the 29 MDR A. baumannii strains, only 2 isolates belonged to the EU clone I lineage and contained allele 69 of the intrinsic bla_OXA-51-like genes, which corresponds to their assignment to the EU clone I lineage. The 2 isolates were confirmed as ST109 (10-12-4-11-4-9-5) by MLST analysis.

Most of the MDR A. baumannii isolates (93.1%) contained 2.5-kb class 1 integrons, and the gene cassette arrays were divided into 2 types by nucleotide sequencing. The gene cassette array aacA4-catB8-aadA1 was detected in only the EU clone II isolates, whereas all EU clone I isolates only carried the gene cassette array aacC1-orfP-orfQ-aadA1.

To determine the clonality, REP-PCR was performed; the 29 MDR A. baumannii isolates displayed only 2 REP-PCR patterns, designated type I and type II (Fig. 2). The 2 EU clone I isolates exhibited the type I pattern, while the 27 EU clone II isolates exhibited the type II pattern.

The comM gene was not detected in any of our isolates, but was amplified from the Acinetobacter calcoaceticus control strain.

Segments J1 and J2, which form the boundaries of the AbaR backbone transposon Tn6019, were amplified from all 29 isolates (both EU clone I and EU clone II), whereas Tn6018 and an interrupted uspA gene were only present in the EU clone I isolates. These results indicated that an AbaR was only present in the EU clone I isolates. However, it is important to note that J4 (the junction of Tn6018-R with Tn6019) was amplified, but J3 (the junction of Tn6018-L with Tn6019) was not amplified in the EU clone I isolates. In addition, only J6 of the segments J5 and J6 (the junctions of multiple-antibiotic resistance region [MARR] with Tn6018) was amplified in these isolates.

To characterize the AbaRs contained in the EU clone I isolates, we mapped the continuous regions of J1, J2, J4, and J6 by overlapping PCRs and sequencing. The 22-kb AbaR7 (GenBank accession no. GQ406246) was identified in the genome of the MDR A. baumannii isolates that belonged to the EU clone I lineage and were confirmed as ST109 (Fig. 3). The MARR was located between J1 and J6, and harbored a class 1 integron-containing aacC1-orfP-orfQ-aadA1 gene cassette array. The aacC1 gene conferred resistance to gentamicin, and the aadA1 gene conferred resistance to streptomycin and spectinomycin. The aphA1 gene, the kanamycin and neomycin resistance gene, was flanked by directly oriented copies of IS26.