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Antimicrobial Agents and Chemotherapy, July 2007, p. 2324-2328, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01502-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Multicopy blaOXA-58 Gene as a Source of High-Level Resistance to Carbapenems in Acinetobacter baumannii{triangledown}

Alessia Bertini,1 Laurent Poirel,2 Sandrine Bernabeu,2 Daniela Fortini,1 Laura Villa,1 Patrice Nordmann,2 and Alessandra Carattoli1*

Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanitá, Rome Italy,1 Service de Bactériologie-Virologie, Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Université Paris XI, K.-Bicêtre, France2

Received 29 November 2006/ Returned for modification 20 January 2007/ Accepted 11 April 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the blaOXA-58 gene showed genetic variability, with the presence in several isolates of multiple copies of the blaOXA-58 gene; this extra copy number was likely related to an IS26-mediated transposition or recombination process.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Acinetobacter baumannii is an important opportunistic pathogen responsible for a variety of nosocomial infections particularly affecting critically ill patients in intensive care units, where it is associated with significantly increased mortality (12, 18). In recent years, several outbreaks of nosocomial infections caused by A. baumannii strains resistant to a wide range of antibiotics, including broad-spectrum β-lactams, aminoglycosides, and fluoroquinolones, have been documented (5, 19, 30). While carbapenems are the drugs of choice for treating Acinetobacter infections, carbapenem resistance is now increasing (6, 9, 10, 16, 19, 27, 28, 34).

Acinetobacter spp. may develop resistance to carbapenems through various mechanisms, including decreased permeability (mostly related to porin modifications), overexpression of efflux pumps, and production of carbapenemases (25, 32). The carbapenemases may be metallo-β-lactamases or carbapenem-hydrolyzing oxacillinases (33). Four groups of carbapenem-hydrolyzing oxacillinases have been identified in A. baumannii, represented by the OXA-23, OXA-24, OXA-51, and OXA-58 enzymes (4, 15, 24, 26). The blaOXA-51-like genes occur naturally in that species (15) and are usually weakly expressed. However, insertion sequence ISAba1 may enhance the expression of blaOXA-51-like genes by providing promoter sequences, indicating that OXA-51-like enzymes might play a role in reduced susceptibility to carbapenems (29). Plasmid-borne blaOXA-23 and blaOXA-58 genes have been shown to contribute significantly to carbapenem resistance in A. baumannii (15). The blaOXA-58 gene has been detected in carbapenem-resistant A. baumannii isolates from different parts of the world (1, 4, 13, 23, 26). It has been shown that the blaOXA-58 gene is often associated with insertion sequences (ISAba1, ISAba2, ISAba3, or IS18) involved in its expression, and likely in its acquisition in the case of ISAba3 (23). In addition, it has been shown that a homologous recombination process was at the origin of blaOXA-58 acquisition in a French A. baumannii isolate (22). Recently, the blaOXA-58 gene was identified in six clonally related and carbapenem-resistant A. baumannii isolates recovered in 2005 at the University Hospital Policlinico Umberto I in Rome, Italy (1). These isolates carried a plasmid-borne blaOXA-58 gene (the plasmid was designated pOUR, for OXA-58 Umberto I Rome) and were resistant to carbapenems, according to the current CLSI criteria, with variable MICs of imipenem and meropenem. Thus, our goal was to elucidate the mechanism(s) at the origin of this resistance level heterogeneity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Bacterial strains and MIC determinations. Three carbapenem-resistant A. baumannii isolates were analyzed in this study. They were recovered in 2005 from patients hospitalized in the intensive care unit of the Policlinico Umberto I, a large university hospital in Rome, Italy (Table 1) (1). They showed identical pulsed-field gel electrophoresis (PFGE) patterns and were positive for the blaOXA-58 gene. A. baumannii MAD, recovered from France and carrying the blaOXA-58 gene, was used as the reference strain (24). Imipenem MICs of >32 µg/ml have been evaluated by the dilution technique on solid media according to the CLSI guidelines (3); the other MICs were the result of an Etest interpretation (31).


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  		TABLE 1. Characteristics of the clonally related A. baumannii clinical isolates and their transformants

 
β-Lactamase activity. Crude enzymatic extracts were obtained by sonication as described previously. The hydrolytic activities of the β-lactamase extracts against imipenem and ceftazidime were determined by UV spectrophotometry as described previously and were expressed in units (1 unit was the amount of enzyme hydrolyzing 1 µmol of substrate per min per mg of protein) (20).

Isoelectric focusing analysis was performed with a pH 3.5 to 9.5 ampholin polyacrylamide gel (GE Healthcare, Saclay, France) for 2.5 h at 25 W, 25 mA, and 2,000 V in a flatbed Multiphor II apparatus with culture extracts of the A. baumannii isolates obtained as mentioned above (14).

PCR experiments. Total DNAs of A. baumannii isolates were extracted by using the Wizard Genomic DNA purification kit (Promega, Milan, Italy) according to the manufacturer's procedure. The DNAs were used as templates under standard PCR conditions with a series of primers. Amplification of the blaOXA-58 gene was performed with primers OXA-58A-OXA-58B (15) and that of the naturally occurring blaOXA-51/69 gene with primers OXA-69A-OXA-69B (15). The blaOXA-20 gene was identified using the primers OXA-20F (5'-AGA ATA GCA CGC GCA ATT GC-3') and OXA-20B (5'-CTG TTG TAC TTG TCT CTC TTG G-3'). The putative colinearity between insertion sequence ISAba1 and the naturally occurring blaOXA-51-like gene was searched for by using primers preABprom+ and OXA-69B, as reported previously (14, 15).

Plasmid analysis. Plasmids were purified using the PureLink Hipure Plasmid Midiprep Kit (Invitrogen, Milan, Italy). SacI- and HindIII-generated fragments were separated on 1% agarose gels, transferred onto nylon membranes (Nylon Membranes Positively Charged; Roche Diagnostics, Monza, Italy), and hybridized with PCR-generated probes specific for blaOXA-58 labeled with digoxigenin (DIG DNA Labeling and Detection kit; Roche Diagnostics, Monza, Italy).

Cloning and sequencing. PCR products were ligated into the pCR2.1 vector (Invitrogen, Milan, Italy) and used to transform competent Escherichia coli INV{alpha}F' cells (INV{alpha}F' Chemically Competent Escherichia coli; Invitrogen, Milan, Italy), and selection of the transformants was performed on LB agar plates containing ampicillin (100 µg/ml).

SacI libraries were obtained from isolates 183 and 186, and EcoRI libraries were obtained from isolates 183, 186, and 193 in the pZErO-2 vector (Invitrogen, Milan, Italy). The libraries were used to transform competent E. coli DH5{alpha} cells (MAX Efficiency DH5{alpha} Chemically Competent Cells; Invitrogen, Milan, Italy), and selection of the transformants was performed on LB agar plates containing kanamycin (40 µg/ml), ampicillin (20 µg/ml), and 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). The nucleotide sequences of the SacI-generated inserts were fully determined on both strands with an ABI Prism 377 sequencer (Applied Biosystems) with universal and internal primers. The EcoRI-generated inserts were partially sequenced using universal and internal primers.

The EcoRI restriction fragments of 17, 13.5, and 10 kb from the 186, 193, and 183 plasmids, respectively, were separated by and eluted from agarose gels with the QIAGEN Gel Extraction Kit (QIAGEN, Courtaboeuf, France); 40 ng of each eluted band was self-ligated in 50 µl of reaction mixture with T4 DNA ligase (Roche Diagnostic, Meylan, France). Ligation mixtures were electrotransformed into A. baumannii strain BM4547 by electroporation (14), and transformants were selected on tircarcillin-containing plates (50 µg/ml).

Quantification of the blaOXA-58 gene by real-time PCR amplification. Real-time experiments were performed with three independent 10-fold dilution series for each strain tested. They were repeated at least three times in different PCR runs. The repAci1 gene was chosen as the reference point to control the plasmid copy number. The PCR assay for the repAci1 gene was performed with primers repAFW (5'-GAG AGA TTT AGT TGT AAA GGA CAA TGC-3') and repARV (5'-CGA CTC ATA ACA TTT CGG ATA TTC CCA TTA-3').

The quantity of each target in each isolate was expressed as the difference in threshold cycle values between the repAci1 and the blaOXA-58 genes. PCR efficiency was approximately 90% for both targets. Real-time PCR was performed by an initial denaturation at 95°C for 10 s, 60°C for 10 s, and 72°C for 20 s, using primers OXA-58A and OXA-58B plus repAFW and repARV. The real-time PCR was performed in a volume of 20 µl containing 20 ng of purified genomic DNA, 2 µl of reaction buffer (Faststart DNA Master SYBER Green I; Roche Diagnostics, Monza, Italy), 4 mM MgCl2, 0.25 µM of each primer. Data were analyzed with LightCycler software, version 4.2.

Nucleotide sequence accession numbers. The nucleotide sequences obtained in the study for isolates 183 and 186 have been assigned accession numbers EF138631 and EF138630, respectively, in the EMBL-GenBank database.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Variability of the MICs. MICs of imipenem, meropenem, and ceftazidime for A. baumannii isolates are presented in Table 1. These isolates were previously demonstrated to be blaOXA-58 positive, but isoelectric focusing analysis revealed the production of additional β-lactamases. A band at pI 7.2 corresponded to OXA-58, a band at pI 7.4 to the narrow-spectrum oxacillinase OXA-20 (whose gene was further identified by PCR), and a band at pI 9.2 to the natural AmpC (data not shown). The three A. baumannii isolates included in this study showed variable MICs of carbapenems and variable hydrolytic specific activities using imipenem as a substrate (Table 1). Isolate 183 showed lower MICs of imipenem and meropenem than isolates 186 and 193. In addition, compared to isolate 183, imipenem hydrolysis rates were 1.6-fold and 1.3-fold higher for isolates 186 and 193, respectively. For comparison, specific hydrolytic activities for ceftazidime (a β-lactam that is not a substrate of OXA-58) were almost identical, ruling out possible differences in AmpC expression in some isolates. PCR assays were performed (14) and showed the absence of colinearity between ISAba1 and the blaOXA-51-like gene in all the isolates tested (data not shown), likely ruling out any involvement of that naturally occurring carbapenem-hydrolyzing oxacillinase as a source of variable phenotypes.

Detailed analysis of the plasmids carrying the blaOXA-58 gene. Restriction by SacI and HindIII enzymes revealed that plasmids from isolates 186 and 193 showed similar restriction patterns, differing slightly from a plasmid of isolate 183 (Fig. 1). Hybridization with a blaOXA-58-specific probe performed on SacI-restricted fragments gave an approximately 6.8-kb positive signal and also an additional 4.5-kb positive signal for plasmids from isolates 186 and 193, whereas the latter signal was not detected in a plasmid from isolate 183. Due to a HindIII site within the blaOXA-58 coding sequence, the HindIII restriction generated two fragments for each blaOXA-58 gene copy. The results from the blaOXA-58-specific hybridization performed on the HindIII-restricted plasmids identified a probable single gene copy (two positive signals) for plasmid 183, whereas multiple blaOXA-58 copies (three HindIII- positive fragments) were identified with plasmids from isolates 186 and 193.


Figure 1
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  		FIG. 1. Plasmid localization of the blaOXA-58 gene. Shown is agarose (1%) gel electrophoresis in 1x Tris-acetate-EDTA buffer of SacI- or HindIII-digested plasmids from A. baumannii isolates, stained with ethidium bromide and visualized under UV light, and Southern blot hybridization with the blaOXA-58 probe. M is a 1-kb Plus Ladder, and M1 is the HindIII-digested lambda DNA (Invitrogen, Milan, Italy).

 
Cloning and characterization of the blaOXA-58 surrounding regions. A SacI library was constructed from isolates 183 and 186, and SacI inserts containing the blaOXA-58 gene were cloned and fully sequenced (Fig. 2). An identical 6,860-bp SacI fragment was obtained from plasmids from isolates 186 and 183 (giving rise to recombinant plasmids p186-6.8 and p183-6.8 [Fig. 2]) and showed 99% nucleotide identity with the sequence identified in A. baumannii MAD (positions 2051 to 5257; GenBank no. AY665723) (22), containing ISAba2, blaOXA-58, and ISAba3. The ISAba3 element was followed by insertion sequence IS26, a structure differing from that identified in A. baumannii MAD, and then by a second ISAba2-like element, whose InsB transposase unit exhibited 93% amino acid identity with that of ISAba2, presenting a stop codon in its center region as a result of a 1-bp insertion.


Figure 2
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  		FIG. 2. Consensus map of the blaOXA-58 gene region. The insertion sequences identified within the resistance regions are shown as boxes, and the arrows indicate the directions of transcription of the genes. The blaOXA-58 gene is represented by the heavy black arrow. Restriction sites for EcoRI, HindIII, and SacI are indicated. The bars below the map represent the positions of the constructs obtained from the libraries and sequenced. The dashed lines show the insertion site of the second copy of the IS26-ISAba2-blaOXA-58-ISAba3 module in isolate 193 with respect to isolate 183. The brackets indicate the presence of two IS26-ISAba2-blaOXA-58-ISAba3 modules in isolate 186.

 
Recombinant plasmid p186-4.5, containing a 4,542-bp SacI fragment, was obtained from plasmid DNA from isolate 186, which possessed a second copy of the blaOXA-58 gene flanked by the ISAba2, ISAba3, and IS26 elements. This sequence revealed a perfect duplication of the IS26-ISAba2-blaOXA-58-ISAba3 structure. Recombinant plasmids p186EcoRI, p193EcoRI, and p183EcoRI carrying EcoRI fragments of 17, 13.5, and 10 kb were obtained from isolates 186, 193, and 183, respectively. DNA sequencing and PCR mapping demonstrated the colinearity of the two blaOXA-58 copies in a head-to-tail orientation (Fig. 2).

The presence of IS26 elements in these duplicated regions indicates that the insertion sequences could have mediated the duplication of the blaOXA-58 locus by imperfect duplicative transposition, starting with the cointegrate fusion and duplication of the elements but lacking the resolution of the cointegrate (7). A very similar IS26-mediated amplification of resistance genes was described in VIM-1-producing Klebsiella pneumoniae strains isolated in Greek hospitals (17). In that case, the K. pneumoniae isolates exhibited highly similar pulsed-field gel electrophoresis patterns but MICs of imipenem varying from 2 to 64 µg/ml. It was shown that the IncN plasmid mediating the higher MIC of imipenem carried two copies of the In-541 integron carrying the blaVIM-1 gene flanked by IS26 elements (17). Also, a relationship between the IS26-blaSHV gene copy number and levels of resistance to ceftazidime, cefotaxime, and aztreonam was observed for a collection of K. pneumoniae clinical isolates from Brisbane, Australia (11).

Identification of a novel replicon on blaOXA-58 positive plasmids. Sequence analysis of the EcoRI-made recombinant plasmids obtained from isolates 186, 193, and 183 identified an open reading frame whose corresponding gene showed 63% nucleotide identity with the repM gene of plasmid pMAC, a 9.5-kb plasmid from A. baumannii (EMBL no. AAT09649) harboring genes involved in organic peroxide resistance (8). This rep gene, designated repAci1, was flanked at its 5' end by four 23-bp-long direct repeats (5'-ATATGTCCACGTTTACCTTGCA-3'), namely, the iterons controlling the plasmid replication rate. Further upstream, the origin of plasmid replication (oriV) was also identified. A quantitative determination of the blaOXA-58 gene copies was performed for the repAci1 gene, assumed to be one copy per plasmid. This experiment confirmed that isolate 186 possessed three copies, isolate 193 two copies, and isolate 183 only one copy of the blaOXA-58 gene (Fig. 3).


Figure 3
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  		FIG. 3. Determination of the blaOXA-58 gene copy number by real-time PCR amplification. Shown are amplification curves obtained in quantitative real-time PCR experiments performed on the repAci1 gene (one copy per plasmid) and the blaOXA-58 gene measured for the isolates 183 (one copy per plasmid), 193 (two copies per plasmid), and 186 (three copies per plasmid).

 
The increase in the carbapenem resistance level is linked to increased production of OXA-58. Although the amount of β-lactamase production can be critical in determining levels of resistance to its substrates, it is likely that the increase in the carbapenem resistance level was linked to an increased production of OXA-58 by the multiplication of the IS26-ISAba2-blaOXA-58-ISAba3 locus on plasmids carrying an identical replicase gene. To better assess the role of the reiterated copies of the IS26-ISAba2-blaOXA-58-ISAba3 structure in the levels of A. baumannii resistance to carbapenems, the EcoRI inserts from the 186, 193, and 183 plasmids, containing the blaOXA-58 locus but also the oriV and the repAci1 gene (and thus a potentially functional replicon), were self-circularized and transferred into a carbapenem-susceptible A. baumannii strain. The circularized EcoRI bands were detected extrachromosomally in the transformants, thus demonstrating the functional activity of the replication system within this plasmid locus. The MICs of imipenem, meropenem, and ceftazidime measured for the transformants obtained with the self-circularized EcoRI band are reported in Table 1. The MICs were consistent with the blaOXA-58 gene dosages in the three clones analyzed. The highest MICs of imipenem and meropenem were obtained for the A. baumannii transformants carrying the EcoRI insert with three blaOXA-58 gene copies (Table 1). Since the levels of resistance to several substrates (including carbapenems) were higher for original strains than for the transformants, it is clear that additional resistance mechanisms were expressed in the clinical isolates. Interestingly, the specific activity of imipenem was higher in these transformants than in the original strains. These results suggest a higher level of expression of the blaOXA-58 gene or an increased replication rate of the self-circularized EcoRI band in the A. baumannii BM4547 recipient strain compared to the original plasmids.

The identification of a novel replication system associated with the plasmids carrying the blaOXA-58 gene provides an important source of information that may be used for further screening of blaOXA-58-positive plasmids of different origins. This screening can be useful to trace the spread of common plasmids carrying this relevant resistance gene in A. baumannii, as shown with enterobacterial isolates for other relevant carbapenem resistance determinants, such as metallo-β-lactamase VIM-1 (2).

Whereas it has already been demonstrated that increased resistance to carbapenems in A. baumannii could be due to porin modifications, efflux system overexpression, or acquisition of carbapenem-hydrolyzing β-lactamases and also increased expression of naturally occurring blaOXA-51-like genes (21), there is evidence that the presence of insertion sequences can mediate the amplification of the region containing the β-lactamase gene, significantly increasing the level of resistance in the clinical strain. This mechanism adds novel insights into the diversity of mechanisms at the origin of carbapenem resistance in A. baumannii.


   ACKNOWLEDGMENTS
 
We are grateful to A. Giordano and C. Mancini for encouraging this study.

This work was supported by the DRESP2 (6th PCRD, LSHM-CT-2005-018705) contract with the European Commission and the FIRB Project "Costruzione di un laboratorio nazionale per lo studio delle resistenze batteriche agli antibiotici" contract of the Italian Ministry of Research and University. L.P. is a researcher from the INSERM (Paris, France).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious, Parasitic, Immune-Mediated Diseases, Istituto Superiore di Sanitá, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39-06-49903128. Fax: 39-06-49387112. E-mail: alecara{at}iss.it Back

{triangledown} Published ahead of print on 16 April 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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Antimicrobial Agents and Chemotherapy, July 2007, p. 2324-2328, Vol. 51, No. 7
0066-4804/07/$08.00+0     doi:10.1128/AAC.01502-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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