First isolation of the blaOXA-23 carbapenemase gene from an environmental Acinetobacter baumannii isolate.

Acinetobacter baumannii is frequently associated with nosocomial infections, and its increasing resistance to carbapenems may significantly reduce the choice of effective antibiotics (2). Since the first description of a carbapenem-hydrolyzing class D -lactamase (CHDL), ARI-1 (renamed OXA-23), from a clinical isolate of A. baumannii in Scotland in 1995 (11), the corresponding blaOXA-23 gene has been detected in many A. baumannii clinical isolates worldwide (Brazil, Spain, Belgium, Singapore, Portugal, and France) and once in Proteus mirabilis in France (1). The blaOXA-23 gene can be plasmid or chromosome borne (12). Three main groups of oxacillinases (OXA-23, -40, and -58) possessing weak carbapenemase activity have been characterized for A. baumannii (6). All CHDL-positive isolates were found in hospital settings. In this report, we describe the first A. baumannii strain isolated in the environment and producing such acquired -lactamase. A. baumannii B9 was recovered from water of the Seine river in downtown Paris as a result of screening for multidrug-resistant isolates. Samples were collected and processed on the day of their collection. The sampling procedure consisted of filtrating 100 ml water through nitrocellulose membranes (0.45 m; Millipore), resuspending the filters in 1 ml of sterile water, and plating 100l aliquots on imipenem (2 g/ml)-containing MacConkey agar plates. Isolate B9 was identified by the API 32GN system (bioMérieux, Marcy l’Etoile, France) and by sequencing of the 16S rRNA genes. MICs were determined by Etest (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar plates at 37°C. Isolate B9 was resistant to all -lactams including carbapenems (Table 1). It was also resistant to quinolones and fluoroquinolones, chloramphenicol, tetracycline, and tigecycline and susceptible to aminoglycosides (tobramycin, amikacin, netilmicin, kanamycin, and gentamicin). PCR experiments, performed as previously described for screening of the presence of the blaOXA-23, blaOXA-40, and blaOXA-58 genes (6), followed by sequencing, showed that this isolate possessed the blaOXA-23 gene. Repeated attempts to transfer the OXA-23 determinant from A. baumannii B9 to azide-resistant Escherichia coli J53 by mating-out assay (9) and to A. baumannii CIP7010 by electroporation (6) failed. In addition, plasmid extraction from A. baumannii B9 performed by the Kieser technique (7) did not yield any plasmid. These results suggested the chromosomal location of the blaOXA-23 gene, which was confirmed by a total DNA digest with the I-CeuI enzyme, its separation by pulsedfield gel electrophoresis, and hybridization with the blaOXA-23 and rRNA probes, as described previously (1). PCR amplification with primers for ISAba1 alone and in combination with the blaOXA-23 gene revealed a structure in which a single copy of this insertion sequence is located upstream of the blaOXA-23 gene but is absent on its downstream extremity as opposed to what has been observed for the composite transposon Tn2006 (4). This is the first isolation of an environmental A. baumannii strain producing an acquired CHDL. The progenitor of the blaOXA-23 gene had been identified as being Acinetobacter radioresistens, in which the blaOXA-23 gene is not expressed or is poorly expressed (10). That species is rarely involved in human infections but is known to be present in the environment. Therefore, it is hypothesized that genetic exchanges between the two Acinetobacter species may lead to acquisition and expression of the blaOXA-23 gene in A. baumannii. This might occur in aquatic environments, where A. baumannii and A. radioresistens could be in close contact. The A. baumannii B9 isolate was not recovered in the immediate vicinity of a hospital wastewater discharge site. Pulsed-field gel electrophoresis (PFGE) analysis of ApaI-restricted DNA from A. baumannii B9 and from clinical A. baumannii isolates from different geographical origins showed that A. baumannii B9 was clonally related to a human A. baumannii isolate previously identified in New Caledonia in June 2004 (8). Interestingly, Acinetobacter spp. have been previously used as an indicator of antimicrobial resistance in aquatic environments (13). This may indicate the presence of some selective pressure in the Seine river, although environmental Acinetobacter sp. strains are usually of the wildtype phenotype of resistance. Our study suggests that the ongoing spread of CHDL-producing A. baumannii is currently occurring simultaneously in different environments and seems not to be restricted to the hospital setting. Although it does not provide evidence for the direct transfer of resistance elements from the soil resistome to pathogenic bacteria, this finding emphasizes the importance of a survey of environmental strains that may act as a source and/or reservoir of resistance genes with clinical relevance, as exemplified by D’Costa et al. (5). Additionally, Shewanella