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Antimicrob Agents Chemother. Aug 2008; 52(8): 2955–2958.
Published online May 27, 2008. doi:  10.1128/AAC.00072-08
PMCID: PMC2493133

Characterization of the Carbapenem-Hydrolyzing Oxacillinase Oxa-58 in an Acinetobacter Genospecies 3 Clinical Isolate[down-pointing small open triangle]

Abstract

Based on imipenem resistance in an Acinetobacter genospecies 3 clinical isolate, we were able to identify, for the first time in this genomic species, a plasmid-encoded blaOXA-58 gene that was 100% homologous to the same gene in Acinetobacter baumannii.

Since 1986 members of the genus Acinetobacter are determined by DNA-DNA hybridization. Genospecies 1 (Acinetobacter calcoaceticus), 2 (A. baumannii), 3, and 13TU are genetically closely related and are commonly known as the A. calcoaceticus-A. baumannii complex. With the exception of genospecies 1, the other members of this complex have been involved in nosocomial infections and have the ability to spread in hospitals (3, 9, 19, 23, 25, 26). Treatment of these nosocomial infections is becoming a problem because increasing resistance to antibiotics, especially in the case of A. baumannii. In the last decade, carbapenem resistance in Acinetobacter spp. has been reported worldwide (3, 16, 23), mostly associated with the synthesis of carbapenem-hydrolyzing β-lactamases, reduced outer membrane permeability and, occasionally, modification of penicillin-binding proteins (7, 16, 22, 27). The most prevalent carbapenemases in Acinetobacter spp. are the carbapenem-hydrolyzing class D β-lactamases, which are divided into four phylogenetic subgroups: OXA-23, OXA-24, and OXA-58 with all their variants and the OXA-51 family, which is intrinsic to A. baumannii (16, 27).

OXA-58 confers reduced susceptibility to carbapenems, but it produces high-level resistance to carbapenems when additional efflux mechanisms are expressed (12, 15). It was first identified in France in 2003 and, at present, is found worldwide in A. baumannii isolates (13, 16, 20, 21), as well as in A. junii isolates from Romania and Australia (12, 14).

The clinical isolates Ac057 (Acinetobacter sp. strain G3) and Ac058 (A. baumannii) were obtained from the same hospital in November 2000 and were identified by amplified ribosomal DNA restriction analysis (ARDRA) (6, 24). The epidemiological difference was corroborated by pulsed-field gel electrophoresis (PFGE) with ApaI (Promega, Madrid, Spain) under conditions described elsewhere (11).

Antimicrobial susceptibility analysis was performed by Etest according to the manufacturer's instructions (AB Biodisk, Sölna, Sweden) and determined that both strains had an imipenem MIC of >32 μg/ml (Table (Table1)1) . The breakpoints for imipenem were those proposed by the Clinical and Laboratory Standards Institute (5).

TABLE 1.
MICs for the clinical isolates used in this study

PCR analysis with specific primers for all class D β-lactamases (Table (Table2)2) determined the presence of the blaOXA-58 gene in both strains; A. baumannii strain Ac058 was also positive for the blaOXA-51 gene. Additional primers were designed at the beginning and end of the blaOXA-58 gene (Table (Table2)2) to amplify the whole fragment. This gene presented 100% homology with the blaOXA-58 gene from A. baumannii listed in GenBank.

TABLE 2.
Oligonucleotide sequences used in this study

Plasmid DNA identification was attempted by using genomic mapping with I-CeuI (10) and by digestion with the S1 nuclease (1). I-CeuI cuts a 26-bp site in the rrl gene (23S rRNA), shearing the bacterial genome into an analyzable number of fragments (10). The S1 nuclease transforms supercoiled plasmids into linear molecules (1). Digested genomic DNA and plasmids were sepa-rated by PFGE (Fig. (Fig.1).1). Probes were marked with the PCR DIG probe synthesis kit (Roche, Barcelona, Spain), and detection was performed with anti-digoxigenin antibody conjugated to alkaline phosphatase and the color substrates NBT/BCIP (Roche) according to the manufacturer's instructions. In Fig. Fig.1a,1a, the most intense bands would represent fragments of genomic DNA, and the faded bands represent plasmid DNA. Hybridization with probes for the blaOXA-58 gene (Fig. (Fig.1c)1c) and the 23S rRNA gene (Fig. (Fig.1b)1b) suggest that in both isolates the blaOXA-58 gene is present in a plasmid. With the S1 nuclease (Fig. (Fig.2a),2a), the highest band would be the genomic DNA and the remaining bands would be linear plasmids. Hybridization with the probe for the OXA-58 gene (Fig. (Fig.2c)2c) gives the same pattern as obtained with I-CeuI. The hybridization signal with the probe for the 23S rRNA gene was only observed in the undigested genomic DNA (Fig. (Fig.2b).2b). Although conjugation experiments did not show any plasmid transfer between strains, Southern blot analysis suggests that the blaOXA-58 gene could be present in a plasmid in both strains, and the plasmid from A. baumannii is possibly different from the plasmid in the Acinetobacter genospecies 3 isolate.

FIG. 1.
Plasmid identification by genomic mapping with I-CeuI. (a) PFGE gel. (b) Hybridization with probe for the 23S rRNA gene. (c) Hybridization with probe for the OXA-58. Lane 1, A. baumannii strain Ac058; lane 2, Acinetobacter genospecies 3 strain Ac057.
FIG. 2.
Plasmid identification by digestion with S1 nuclease. (a) PFGE gel (b). Hybridization with probe for the 23S rRNA gene. (c) Hybridization with probe for the OXA-58. Lane 1, A. baumannii strain Ac058; lane 2. Acinetobacter genospecies 3 strain Ac057.

In order to determine the genetic structure surrounding of the blaOXA-58 gene, DNA from both isolates was digested with MspI “C*CGG” (Promega). The fragments obtained were autoligated overnight at 16°C using a T4 DNA ligase (Promega). The fragment of DNA containing the blaOXA-58 gene was used as a template for a PCR with inverse primers designed from the blaOXA-58 gene sequence (Table (Table2).2). All PCR fragments were sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Warrington, United Kingdom) and analyzed in an automatic DNA sequencer (3100 Genetic Analyzer; Applied Biosystems).

Analysis of the genetic surrounding confirms that both plasmids are different (Fig. (Fig.3).3). In Ac057, the blaOXA-58 gene is surrounded by two copies of the Insertion Sequence ISAba3; the copy downstream has the same direction as the blaOXA-58 gene, and the upstream copy has the opposite direction (Fig. (Fig.3).3). This structure has already been described in A. baumannii by Poirel et al. (17, 18).

FIG. 3.
Structure of the genetic surrounding in Acinetobacter genospecies 3 strain Ac057, which is structurally identical to the one described by Poirel et al. (17).

The presence of the OXA-58 alone does not account for the level of resistance to imipenem of these isolates (MIC of >32 μg/ml). Further work is needed to determine whether additional efflux pumps or porin modifications are involved.

A. baumannii is certainly the most frequently isolated species in hospitals and also the microorganism of greatest clinical interest in this genus. However, Acinetobacter genospecies 3 and 13 are also nosocomial pathogens, and they should be considered in hospital settings. Previous studies in Acinetobacter genospecies 3 have revealed the presence of AmpC (2), IMP-4 (4), and blaVIM-2 (28). In addition to these previously described enzymes, we report here, for the first time, the presence of the blaOXA-58 in this microorganism. The main reason for the lack of interest on non-baumannii Acinetobacter isolates is probably their susceptibility to antimicrobial agents (9). However, as suggested by Horrevorts et al. (8), the clinical significance of genospecies 3 can be underestimated because the resistant strains can be erroneously classified as A. baumannii.

Nucleotide sequence accession number.

The GenBank accession number for the blaOXA-58 in Acinetobacter genospecies 3 is EU642594.

Acknowledgments

This study has been supported by grant SGR050444 from the Departament d'Universitats, Recerca I Societat de la Informació de la Generalitat de Catalunya, Spain, and by the Spanish Ministry of Health (FIS 04/0068 to J.V.). This study was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008), as well.

Footnotes

[down-pointing small open triangle]Published ahead of print on 27 May 2008.

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