Isolation of Genetically Indistinguishable Carbapenem-Resistant and -Susceptible Acinetobacter baumannii Isolates from a Single Patient

LETTER

The efficacy of carbapenems has been threatened by the increasing frequency of carbapenem-resistant bacteria. Acinetobacter baumannii carrying the bla_OXA-23 carbapenemase gene has spread worldwide (1). We isolated carbapenem-resistant and then -susceptible A. baumannii from the sputum of a 74-year-old male patient. Here we describe the characterization of these isolates and discuss the implications of these findings and their clinical importance.

The first isolate (MRY12-278) was obtained 3 days after treatment with meropenem (MEPM; 1,500 mg/day) for suspected pneumonia. The isolate was resistant to MEPM (MIC, >8 μg/ml), imipenem (IPM; MIC, >8 μg/ml), amikacin (MIC, >32 μg/ml), gentamicin (MIC, >8 μg/ml), ciprofloxacin (MIC, >2 μg/ml), and levofloxacin (MIC, >4 μg/ml) by the broth microdilution method of the Clinical and Laboratory Standards Institute (2). MEPM was discontinued immediately, as the responsible physicians felt that the clinical scenario was more consistent with colonization rather than infection. Seven days after MEPM discontinuation, the other isolate (MRY12-281) was obtained; its susceptibility profile was identical except that it was susceptible to MEPM and IPM (MICs, <4 μg/ml).

Both isolates yielded identical banding profiles by pulsed-field gel electrophoresis (PFGE) using ApaI (3) (Fig. 1), suggesting their derivation from a single clone. When tested, the isolates were negative for the production of metallo-β-lactamases by a double-disc synergy test (4). OXA-type carbapenemase-encoding genes and the ISAba element, which contains the promoter sequence upstream of the bla_OXA genes, were screened by PCR with primers specific for bla_OXA-51-like, bla_OXA-23-like, bla_OXA-58-like, bla_OXA-24-like, and ISAba1 (5, 6). bla_OXA-51-like was detected in both isolates, but ISAba1 was not found upstream of the gene. bla_OXA-58-like and bla_OXA-24-like were not detected in either isolate. bla_OXA-23-like and the upstream ISAba1 element were detected in MRY12-278 but not in MRY12-281. The nucleotide sequence of bla_OXA-23-like was identical to that of the published bla_OXA-23 gene (GenBank accession no. AJ132105). These findings indicate that MRY12-278 carbapenem resistance was conferred by bla_OXA-23. To determine whether bla_OXA-23 was located on a plasmid, S1 nuclease-digested DNA was separated by PFGE and hybridized with a digoxigenin (DIG)-labeled probe (Roche Applied Science, Mannheim, Germany) specific for bla_OXA-23 (Fig. 2) (7). The resistant isolate harbored an ∼60-kbp plasmid containing bla_OXA-23. Thus, MRY12-278 and MRY12-281 are clonal except for the presence of the plasmid. We believe that the carbapenem-susceptible isolate MRY12-281 was derived from resistant isolate MRY12-278 by loss of the plasmid carrying bla_OXA-23. Both isolates were grouped into sequence type 92 (ST92) according to the Bartual et al. multilocus sequence typing scheme (8). ST92 is of the most epidemic clonal lineage (international clone II lineage, which frequently causes outbreaks worldwide).

Fig 1.

Pulsed-field gel electrophoresis (PFGE) of ApaI-digested DNA from A. baumannii isolates. Lane 1, MRY12-278; lane 2, MRY12-281; lane M, contour-clamped homogeneous electric field (CHEF) DNA size standard lambda ladder (Bio-Rad).

Fig 2.

Localization of the bla_OXA-23 gene in A. baumannii isolates MRY12-278 and -281. S1 nuclease-digested DNA was separated by PFGE. (A) Ethidium bromide-stained image; (B) hybridization with a probe specific for the bla_OXA-23 gene. Lane 1, MRY12-278; lane 2, MRY12-281; lane M, low-range PFG marker (New England BioLabs).

Infections caused by resistant A. baumannii strains are a major concern for clinicians, as available treatment options are limited. Our results indicated that transformation of a carbapenem-resistant strain into a susceptible strain indeed occurs in clinical settings. When susceptible strains predominate, resistant strains might be undetectable, and vice versa. It is important to consider such situations carefully during antibiotic therapy.