JAC Advance Access originally published online on December 8, 2004
Journal of Antimicrobial Chemotherapy 2005 55(1):115-118; doi:10.1093/jac/dkh500
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JAC vol.55 no.1 © The British Society for Antimicrobial Chemotherapy 2004; all rights reserved
A nosocomial outbreak of Acinetobacter baumannii isolates expressing the carbapenem-hydrolysing oxacillinase OXA-58
1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Faculté de Médecine Paris-Sud, Université Paris XI, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex; 2 Laboratoire de Bactériologie-Hygiène, CHU Rangueil, 31403 Toulouse cedex 4, France
Received 9 September 2004; returned 16 September 2004; revised 11 October 2004; accepted 11 October 2004
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Abstract |
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Objective: The aim of this study was to analyse the spread of the blaOXA-58 gene as a source of carbapenem resistance in Acinetobacter baumannii in the burns unit of a university hospital in Toulouse in 2003–2004.
Methods: Six carbapenem-resistant A. baumannii isolates from six patients, and a carbapenem-resistant environmental A. baumannii isolate were collected in the burns unit of the Rangueil hospital (Toulouse). Susceptibility tests were carried out by disc diffusion and agar dilution methods. The detection of the blaOXA-58 gene was conducted by PCR followed by sequence analysis. Plasmids were extracted and hybridized with a probe specific for blaOXA-58. DNA fingerprints were obtained by pulsed-field gel electrophoresis of ApaI- or SmaI-digested chromosomal DNA of the tested strains.
Results: The multidrug-resistant clinical isolates had a similar 30 kb plasmid that encoded the carbapenem-hydrolysing ß-lactamase OXA-58. These isolates were clonally related. The unrelated environmental carbapenem-resistant A. baumannii isolate had a similar blaOXA-58-carrying plasmid, suggesting spread of this gene.
Conclusions: A novel oxacillinase was the source of carbapenem resistance in the A. baumannii isolates. Its gene was plasmid-, but not integron-borne.
Keywords: A. baumannii , carbapenem resistance , nosocomial infections
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Acinetobacter baumannii is an important cause of nosocomial infections such as pneumonia, septicaemia, urinary tract infections and wound infections.1 It may become resistant to a wide range of antibiotics, thus complicating the treatment of nosocomial infections.1 Carbapenems are drugs of choice for treating Acinetobacter infections, but their effect may be compromised by carbapenem-hydrolysing ß-lactamases.1 Acquisition of IMP-like and VIM-like metallo-ß-lactamases (Ambler class B) has been reported worldwide in A. baumannii.2
In addition, seven oxacillinases (Ambler class D) with carbapenem-hydrolysing activity have been characterized previously in A. baumannii and were involved in outbreaks in several cases.3–7 Oxacillinases OXA-23, OXA-27,3,6 and OXA-49 (GenBank AY288523) have 99% amino acid identity, but they share only 60% identity with a second group of oxacillinases consisting of OXA-24, -25, -26 and -40; these latter enzymes differ by a few amino acid substitutions.3–5 Outbreaks of OXA-type carbapenemase-producing A. baumannii have been reported: OXA-24 in Madrid, Spain;4 OXA-23 in Brazil;6 and endemic carbapenem resistance in A. baumannii in Bilbao, Spain, was associated with OXA-40.7
Recently, we characterized a novel carbapenem-hydrolysing oxacillinase, OXA-58 from A. baumannii, from Toulouse, which belongs to a novel group of oxacillinases.8
The aim of this study was to analyse the spread of blaOXA-58 as a source of carbapenem-resistance in A. baumannii in the burns unit of a hospital in Toulouse in 2003–2004.
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Materials and methods |
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Bacterial isolates
A. baumannii clinical and environmental isolates (strains 1–8) were isolated from August 2003 to March 2004 and were identified using the API20NE system (bioMérieux, Marcy-l'Étoile, France) and by sequencing of 16S rRNA genes.5 E. coli NCTC 50192 harbouring four plasmids of 154, 66, 38 and 7 kb was used as a size marker for plasmids. Reference strain A. baumannii CIP7010T (Institut Pasteur Strain Collection, Paris, France) was used as the host in conjugation experiments.
Susceptibility testing
The antimicrobial agents and their sources have been referenced elsewhere.5 Antibiotic-containing discs were used for detection of antibiotic susceptibility with Mueller–Hinton agar plates and a disc diffusion assay (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) (www.sfm.fr). MICs of ß-lactams were determined by an agar dilution technique as previously reported,5 and results were interpreted according to the guidelines of the National Committee for Clinical Laboratory Standards.9
PCR amplification
PCR experiments were carried out using primers specific for the blaOXA-58 gene. Primers preOXA-58prom+ (5'-TTATCAAAATCCAATCGGC-3') and preOXA-58B (5'-TAACCTCAAACTTCTAATTC-3'), able to amplify a 934 bp external fragment of the blaOXA-58 gene, were used to amplify the entire blaOXA-58 gene followed by sequencing with an Applied Biosystems sequencer (ABI 300). Primers OXA-58A (5'-CGATCAGAATGTTCAAGCGC-3') and OXA-58B (5'-ACGATTCTCCCCTCTGCGC-3'), amplifying a 528 bp internal fragment of the blaOXA-58 gene, were used to generate a probe for Southern analysis.
Plasmid analysis and hybridization
Plasmid extraction was performed using the Kieser method designed to isolate large-sized plasmids.10 The plasmid extraction of each isolate was submitted to digestion with EcoRI restriction enzyme and restriction patterns of these plasmids were compared.
Southern transfer of the plasmid extract was carried out on a Nylon membrane (Hybond N+; Amersham Pharmacia Biotech, Orsay, France), as described previously.5 The membrane was hybridized with a probe specific for blaOXA-58. Southern hybridization was carried out as described by the manufacturer using the ECL non-radioactive labelling and detection kit (Amersham Pharmacia Biotech).
Conjugation and transformation experiments
Direct transfer of ß-lactam resistance markers into rifampicin-resistant A. baumannii CIP7010T was attempted by liquid and solid mating-out assays. Transconjugants were selected on trypticase soy (TS) agar plates containing rifampicin (50 mg/L) plus ticarcillin (50 mg/L).
The plasmid extract of A. baumannii isolate 2 was also used for electrotransformation experiments in A. baumannii CIP7010T. Electroporation products were selected on ticarcillin (50 mg/L)-containing plates.
Pulsed-field gel electrophoresis
PFGE analysis was performed according to the manufacturer's instructions (Bio-Rad). In brief, whole-cell DNA of the A. baumannii isolates was digested with ApaI overnight at 25°C (New England Biolabs, St Quentin en Yvelines, France), or with SmaI overnight at 37°C (Amersham Pharmacia Biotech). Electrophoresis was carried out with a CHEF DRII apparatus (Bio-Rad) through a 0.8% agarose gel in 0.5x Tris/borate/EDTA buffer. Migration conditions were as follows: temperature, 14°C; voltage, 6 V/cm; and switch angle, 120°, with one linear switch ramp of 3 to 20 s for 10 h, then 12 to 20 s for 10 h.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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Bacterial identification and susceptibility tests
From August 2003 to March 2004, seven carbapenem-resistant A. baumannii isolates were collected (isolates 2 to 8). Six strains (2 to 7) were from six patients hospitalized in the burns unit and one was an environmental isolate (isolate 8), collected during the same period of time in the surgical room of the burns unit during systematic air sampling (Table 1). A carbapenem-susceptible A. baumannii isolate (isolate 1) collected from one of the six patients was also retained in our study, in order to compare this isolate with carbapenem-resistant isolates.
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The age of the patients ranged from 20 to 91 years and all of them except patient F had been treated with imipenem previously (Table 1). Infection with multidrug-resistant A. baumannii was considered to have contributed to the death of two out of the four patients who died (patients B and F). Two patients survived after being treated with imipenem and amikacin; one was considered as being colonized whereas the other was infected (Table 1).
A. baumannii isolates 2 to 8 were resistant to carbapenems, MICs of imipenem and meropenem being 
32 mg/L (Table 2). Isolate 1 was susceptible to carbapenems with MICs of imipenem and meropenem of 0.5 mg/L (Table 2). The carbapenem-resistant isolates were resistant to all aminoglycosides, fluoroquinolones and most ß-lactams, but remained susceptible to rifampicin and colistin (Table 2). Sulbactam retained activity against carbapenem-resistant A. baumannii isolates 2 to 8, but these isolates remained resistant to the combination of ampicillin and sulbactam (MICs, 32 mg/L; Table 2).
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PCR amplification and genetic location of the blaOXA-58 gene
PCR experiments using primers specific for the blaOXA-58 gene gave positive results for the seven carbapenem-resistant A. baumannii isolates. The carbapenem-susceptible A. baumannii isolate was blaOXA-58-negative.
The carbapenem-resistant A. baumannii isolates had an 
30 kb plasmid whereas no plasmid was detected in the carbapenem-susceptible A. baumannii isolate (data not shown). An additional 17 kb plasmid was found in A. baumannii isolate 8.
A Southern membrane was hybridized with a 528 bp PCR-generated probe specific for the blaOXA-58 gene giving a positive signal with the 30 kb plasmid for the seven carbapenem-resistant A. baumannii isolates.
The plasmid extraction of each isolate was submitted to digestion with EcoRI restriction enzyme and restriction patterns of these plasmids were compared. Gel electrophoresis analysis identified an identical plasmid with a positive hybridization signal, a fragment of DNA of 11 kb, with the blaOXA-58 probe (Figure 1).
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Plasmid location of the blaOXA-58 gene was confirmed by electroporation of the 30 kb plasmid (pMAD) into A. baumannii CIP7010T giving rise to a A. baumannii CIP7010T transformant that had a ß-lactam resistance pattern consistent with the expression of ß-lactamase OXA-58 (Table 2). No other antibiotic resistance marker was co-transferred in A. baumannii CIP7010T. However, this plasmid was non-conjugative since all conjugation experiments failed using A. baumannii CIP7010T as recipient strain.
DNA fingerprinting
Genotypic comparison was carried out for evaluating clonality of the isolates. PFGE analysis after digestion of whole-cell DNAs with ApaI and SmaI restriction enzymes showed that six (isolates 2 to 7) out of the seven carbapenem-resistant isolates probably corresponded to a single strain according to the Tenover criteria (data not shown).11 The environmental isolate (isolate 8) was of a different genotype, but it contained the same blaOXA-58-carrying plasmid (Figure 1).
Identification of the same blaOXA-58-carrying plasmid in two different isolates of A. baumannii indicated the spread of this plasmid in clinical and environmental isolates. This study constitutes the second description of an outbreak of carbapenem-resistant A. baumannii producing a plasmid-encoded carbapenem-hydrolysing oxacillinase, after that of OXA-23-producing strains in Brazil.6
As observed for other genes that encode carbapenem-hydrolysing oxacillinase, blaOXA-58 was not present as a gene cassette in a class 1 integron.8 Thus, the spread of this ß-lactamase gene was not associated with class 1 integrons. This contrasts with the situation found with most other oxacillinase genes.12
The identification of blaOXA-40 in Bilbao,7 and now of blaOXA-58 in Toulouse indicates that distantly-related oxacillinases may be present in outbreak strains of carbapenem-resistant A. baumannii in hospitals located in southern Europe.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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This work was financed by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES, EA3539), Université Paris XI, France, and from the European Community, 6th PCRD, LSHM-CT-2003–503–335. L. P. is a researcher from the INSERM, France.
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Footnotes |
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* Corresponding author. Tel: +33-1-45-21-36-32; Fax: +33-1-45-21-63-40; Email: nordmann.patrice{at}bct.ap-hop-paris.fr
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionAcknowledgementsReferences |
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1 . Bergogne-Bérézin, E. & Towner, K. J. (1996). Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clinical Microbiology Reviews 9, 148–65.[Web of Science][Medline]
2 . Poirel, L. & Nordmann, P. (2002). Acquired carbapenem-hydrolyzing ß-lactamases and their genetic support. Current Pharmaceutical Biotechnology 3, 117–27.[CrossRef][Medline]
3
.
Afzal-Shah, M., Woodford, N. & Livermore, D. M. (2001). Characterization of OXA-25, OXA-26, and OXA-27, molecular class D ß-lactamases associated with carbapenem resistance in clinical isolates of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 45, 583–8.
4
.
Bou, G., Oliver, A. & Martinez-Beltran, J. (2000). OXA-24, a novel class D ß-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain. Antimicrobial Agents and Chemotherapy 44, 1556–61.
5
.
Héritier, C., Poirel, L., Aubert, D. et al. (2003). Genetic and functional analysis of the chromosome-encoded carbapenem-hydrolyzing oxacillinase OXA-40 of Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy 47, 268–73.
6
.
Dalla-Costa, L. B., Coelho, J. M., Souza, H. A. et al. (2003). Outbreak of carbapenem-resistant Acinetobacter baumannii producing the OXA-23 enzyme in Curitiba, Brazil. Journal of Clinical Microbiology 41, 3403–6.
7
.
Lopez-Otsoa, F., Gallego, L., Towner, K. J. et al. (2002). Endemic carbapenem resistance associated with OXA-40 carbapenemase among Acinetobacter baumannii isolates from a hospital in northern Spain. Journal of Clinical Microbiology 40, 4741–3.
8 . Poirel, L., Marqué, S., Héritier, C. et al. (2005). OXA-58, a novel ß-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrobial Agents and Chemotherapy, in press.
9 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A6. NCCLS, Wayne, PA, USA.
10 . Kieser, T. (1984). Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12, 19–36.[CrossRef][Web of Science][Medline]
11 . Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Web of Science][Medline]
12 . Naas, T. & Nordmann, P. (1999). OXA-type ß-lactamases. Current Pharmaceutical Design 5, 865–79.[Web of Science][Medline]
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