Unusual Detection of an Acinetobacter Class D Carbapenemase Gene, bla_OXA-23, in a Clinical Escherichia coli Isolate

LETTER

Carbapenem resistance in Acinetobacter baumannii is typically mediated by the acquisition of class D β-lactamases belonging to gene clusters of bla_OXA-23-like, bla_OXA-40-like, and bla_OXA-58-like (1). In the Enterobacteriaceae, carbapenem-hydrolyzing class D β-lactamases are encoded by a dissimilar set of genes, the bla_OXA-48-like genes (2). The presence of bla_OXA-23 in Enterobacteriaceae is exceptional and has been described once in Proteus mirabilis, where it was chromosomally encoded (3).

As a result of national surveillance efforts to characterize non-carbapenem-susceptible Enterobacteriaceae isolates, we detected a case of bla_OXA-23 in Escherichia coli, which to the best of our knowledge is the first description of plasmid-borne bla_OXA-23 in E. coli.

The isolate was recovered from the urine sample of an elderly woman. However, the isolate was not likely to be a cause of infection, as urine microscopy did not show an increased number of white blood cells. The isolate exhibited resistance to β-lactams, fluoroquinolones, and chloramphenicol. Sensitivity to tigecycline, colistin, and aminoglycosides was observed (Table 1).

TABLE 1.

MICs of antibiotics for the Escherichia coli isolate harboring bla_OXA-23

Antibiotic	MIC (mg/liter)
Aztreonam	32
Piperacillin-tazobactam	>256
Cefoxitin	128
Cefotaxime	32
Ceftazidime	16
Imipenem	3
Meropenem	4
Ertapenem	6
Tigecycline	0.125
Tetracycline	86
Levofloxacin	>32
Ciprofloxacin	>32
Amikacin	0.125
Gentamicin	2
Chloramphenicol	16
Colistin	0.125

Comprehensive PCR screening and sequencing for β-lactamase genes was performed (4, 5). The isolate carried bla_OXA-23, which had 100% identity with A. baumannii bla_OXA-23. It was also positive for TEM-1, OXA-1, and CMY-2 but was negative for other carbapenemases and extended spectrum β-lactamases. The isolate had a multilocus sequence type of ST471. (http://www.pasteur.fr/recherche/genopole/PF8/mlst/EColi.html).

Two large plasmids of approximately 50 and 100 kb were detected (6), with Southern blot hybridization localizing bla_OXA-23 to the 50-kb plasmid.

Solid-medium conjugation assays were performed to assess the transferability of bla_OXA-23 from the clinical isolate to the azide-resistant recipient E. coli J53 and to a spontaneous rifampin-resistant mutant of A. baumannii, ATCC 17978. However, we did not manage to isolate any transconjugants, suggesting that bla_OXA-23 was harbored on a non-self-conjugative plasmid.

The immediate nucleotide sequences flanking bla_OXA-23 were determined by inverse PCR and the primer walking approach (7). E. coli bla_OXA-23 is flanked by two copies of insertion sequence IS1 (Fig. 1). In contrast, in A. baumannii, bla_OXA-23 is found as part of a transposon (Tn2006, Tn2007, Tn2008) associated with insertion sequence ISAba1 or ISAba4 (8, 9) (Fig. 1).

FIG 1.

Schematic representation of the genetic organization of bla_OXA-23. (A) In Acinetobacter baumannii, ISAba insertion sequence elements are typically found immediately upstream of bla_OXA-23 (8, 9). (B) In the clinical Escherichia coli isolate, bla_OXA-23 is flanked by two copies of Enterobacteriaceae insertion sequence IS1. The terminal left inverted repeat (IRL) of IS1 is indicated by the boldface nucleotides with gray shading. The putative −35 promoter of the IRL is underlined and may form hybrid promoters with existing −10 promoters, enabling transcription (11). The ISAba1-associated −35 and −10 promoters are underlined (10). The ATG start codon of OXA-23 is indicated by the vertical arrow (↓). Gray checked regions represent 100% identity between E. coli and A. baumannii. ATPase, gene encoding putative AAA ATPase; DEAD, gene encoding the putative DEAD (Asp-Glu-Ala-Asp) helicase.

One hundred eighty-three bases upstream, the E. coli bla_OXA-23 start codon was completely identical to ISAba1 (GenBank accession number GQ861438.1). Within this region, −35 and −10 ISAba1-associated promoters were identified and likely to be functional (10) (Fig. 1). The 764-bp stretch after the bla_OXA-23 stop codon comprised a partially truncated putative AAA ATPase identical to that in A. baumannii Tn2006, Tn2007, and Tn2008 (Fig. 1).

We hypothesized that E. coli bla_OXA-23 may be carried within a transposon, not unlike those identified in A. baumannii but interrupted by IS1. However, attempts to PCR map the region using both ISAba1 and ISAba4 as reference sequences did not yield amplicons, suggesting that it was in a genetic configuration different from that found in A. baumannii.

In summary, we describe the novel detection of an E. coli isolate carrying an Acinetobacter bla_OXA-23 gene associated with Enterobacteriaceae IS1 on a 50-kb non-self-conjugative plasmid. The discovery highlights the potential for the tremendous spread of carbapenemases.

Nucleotide sequence accession number.

The bla_OXA-23 sequence from the clinical E. coli isolate has been deposited into GenBank under accession number KJ716226.