Worldwide Dissemination of the blaOXA-23 Carbapenemase Gene of Acinetobacter baumannii1

Controlling the spread of this gene will be difficult.

A cinetobacter baumannii is a gram-negative organism that is increasingly recognized as a major pathogen causing nosocomial infections, including bacteremia and ventilator-associated pneumonia, particularly in patients admitted to intensive care units (1). Several studies have shown the geographically widespread occurrence of multidrug-resistant A. baumannii strains, which suggested a clonal relatedness of these strains. Three international A. baumannii clones associated with multidrug resistance (European clones I, II, and III) have been reported (2).
Increasing resistance to carbapenems has been observed worldwide in the past decade, frequently mediated by production of class D β-lactamases with carbapenemase activity. Three acquired class D β-lactamases with carbapenemase gene clusters have been described in A. baumannii, which correspond to bla OXA-23 -like, bla OXA-40 -like, and bla OXA-58 -like genes (3). The bla OXA-23 gene, fi rst characterized in Scotland (4), has been increasingly reported worldwide. A. radioresistens was recently identifi ed as the progenitor of the bla OXA-23 -like genes (5). Clonal outbreaks of carbapenem-resistant and OXA-23-producing A. baumannii have been reported in many countries, such as Bulgaria (6), People's Republic of China (7), Brazil (8), Iraq (9), Afghanistan (9), and French Polynesia (10).
Genetic acquisition of the bla OXA-23 gene was investigated and transposons Tn2006, Tn2007, and Tn2008 were identifi ed as genetic structures harboring this gene (10)(11)(12). In Tn2006, the bla OXA-23 gene is fl anked by 2 copies of the insertion sequence ISAba1, which are located in opposite orientations ( Figure 1). The functionality of Tn2006 has been recently demonstrated (13). Tn2008 is similar to Tn2006 but lacks the second copy of ISAba1 and the bla OXA-23 gene is associated with 1 copy of ISAba4 (which differs from ISAba1) in Tn2007 ( Figure 1) (11). As reported for strains from United Arab Emirates and Bahrain, the bla OXA-23 gene can be associated with only 1 copy of ISAba1 (14,15). We studied the clonal relationship and genomic environment of sequences surrounding the bla OXA-23 gene among a collection of OXA-23-producing isolates from 15 countries.

Clonal Relationships
Isolates were typed by using ApaI macrorestriction analysis and PFGE according to the manufacturer's recommendations (Bio-Rad, Marnes-la-Coquette, France). Bacteria were grown in a medium appropriate for the strain until an optical density of 0.8 to 1 at 600 nm was reached. One milliliter of cells was centrifuged, washed, and resuspended in 10 mmol/L Tris, pH 7.2, 20 mmol/L NaCl, 50 mmol/L EDTA. Immediately after resuspension, an equal volume of 2% low melting point InCert agarose (Bio-Rad) was added. Solid agarose plugs were lysed at 37°C for 2 h in 1 mL of lysis buffer (10 mmol/L Tris, pH 7.2, 50 mmol/L NaCl, 0.5% sodium laurylsarcosine, 0.2% sodium deoxycholate) supplemented with 20 mg/L of lysozyme. The plugs were then incubated at 55°C for 16 h with proteinase K buffer (100 mmol/L EDTA, pH 8, 0.2% sodium deoxycholate, 1% sodium laurylsarcosine) supplemented with 20 mg/L of proteinase K. Plugs were washed with Tris-EDTA buffer containing 1 mmol/L phenylmethylsulfonyl fl uoride (Sigma, St. Louis, MO, USA) and 3× with Tris-EDTA buffer at room temperature.
Whole-cell DNA of A. baumannii isolates was digested with ApaI overnight at room temperature (New England Biolabs, St. Quentin-en-Yvelines, France). Electrophoresis was performed on a 1% agarose gel with 0.5× Tris-borate-EDTA buffer by using a CHEF DRII apparatus (Bio-Rad). Samples were subjected to electrophoresis at 14°C, 6 volts/ cm, and a switch angle with 1 linear switch ramp of 3-8 s for 10.5 h, and then for 12-20 s for 10.5 h.
Identifi cation of PCR-based sequence groups was conducted by using 2 multiplex PCR assays designed to selectively amplify group 1 or group 2 alleles of the gene encoding outer-membrane protein A (ompA), the gene encoding part of a pilus assembly system required for biofi lm formation (csuE), and the gene encoding the intrinsic carbapenemase gene of A. baumannii) (bla OXA-51 ) ( (18). Sequencing of internal fragments was performed by using BigDye fl uorescent terminators and primers described (19). Sequences were compared with the A. baumannii database at the MLST Website (http://mlst.zoo.ox.ac.uk). To supplement epidemiologic results, we performed a second MLST typing using the scheme developed by Nemec et al. (20). Sequences of the 7 housekeeping genes were analyzed by using an A. baumannii database (www.pasteur.fr/recherche/genopole/ PF8/mlst/Abaumannii.html).

Southern Blot Analysis and Location of bla OXA-23 Gene
Southern blot analysis was performed by using total genomic DNA digested with EcoRI, separated by electro- phoresis on 0.8% agarose gels, transferred onto Hybond N+ membranes, and hybridized with enhanced chemiluminescence labeled probes overnight at 42°C. The membranes were developed according to the manufacturer's instructions (GE Healthcare, Saclay, France). Chromosomal or plasmid locations of the β-lactamase gene were assessed by hybridization of I-CeuI-digested genomic DNA with bla OXA-23 and 16S rDNA probes and electrophoresis (20-120 s for 9 h and 60-100 s for 11 h at 14°C and 5 V/ cm 2 ) (21). DNA was transferred from an agarose gel to a nylon membrane by capillary transfer. Hybridization, labeling, and detection were conducted as described above.
Mating-out assays were performed by using isolates that had plasmid-borne bla OXA-23 as donors and rifampin-resistant A. baumannii BM4547 as recipients as described (22). Transconjugants were selected on trypticase soy agar plates containing ticarcillin (50 mg/L) and rifampin (50 mg/L).

Cloning Experiments
To identify entire transposon structures containing the bla OXA-23 gene in different isolates and determine their location in the target DNA, a cloning procedure was used. Some data had been reported for 6 of 20 isolates (11). Total DNA was digested with either SacI or SalI, ligated into the SacI or SalI sites of plasmid pBK-CMV (kanamycin-resistant cloning vector), and the recombinant plasmids were transformed into Escherichia coli TOP10, as described (14). Recombinant plasmids were selected on trypticase soy agar plates containing amoxicillin (50 mg/L) and kanamycin (30 mg/L). Cloned DNA fragments of several recombinants plasmids were sequenced on both strands by primer walking as described (11).

Clonal Relatedness of the Isolates
Twenty carbapenem-resistant A. baumannii isolates were obtained from 15 countries (Table). All isolates were highly resistant to ticarcillin (MIC >256 mg/L) and showed a high level of resistance to ceftazidime (MIC >256 mg/L), except isolates Ab14 (MIC 4 mg/L) 861 and DOS (MIC 8 mg/L). All isolates were resistant to imipenem and meropenem (MIC >16 mg/L) (Table).

Location and Transferability of the bla OXA-23 Gene
Location of the bla OXA-23 gene was evaluated by using the I-CeuI method. Eleven isolates had the bla OXA-23 gene on the chromosome, with a hybridization signal for an ≈40kb band for isolate AS1 and an ≈200-kb band for 10 isolates (Table). Nine isolates carried the bla OXA-23 gene on a plasmid and 1 isolate had 2 copies of the bla OXA-23 gene, 1 on the chromosome and 1 on a 7-kb plasmid (Table).
To examine the copy number of the bla OXA-23 gene in different A. baumannii genomes, we performed Southern blot hybridization on EcoRI-digested DNA fragments using a 589-bp DNA probe specifi c for the bla OXA-23 gene. Sixteen isolates showed only 1 copy of the bla OXA-23 gene. Isolates BEL, Ab14, and DOS had 2 copies of the bla OXA-23 gene on different plasmids, and Ab13 had 1 copy on the chromosome and 1 copy on a plasmid according to results of the I-Ceu1 technique.
Mating-out assays were performed by using the 10 plasmid-positive strains as donor strains and rifampin-resistant A. baumannii BM4547 as the recipient strain. Five transconjugants were obtained; all had a 130-kb plasmid that did not provide additional antimicrobial drug resistance to the A. baumannii recipient strain, except in 1 case (co-resistance to kanamycin and amikacin on a bla OXA-23 -carrying plasmid that originated from isolate 1190). Plasmids carrying the bla OXA-23 gene in isolates Ab14, DOS, BEL, and 877 were not self-transferable (Table) (24).

Variability of Genetic Structures Flanking the bla OXA-23 Gene
The 10 isolates that belonged to European clone II had a bla OXA-23 gene that was part of Tn2006. The 9-bp direct repeat (DR) that corresponded to duplication of the Tn2006 target site, which was consistent with a transposition event, was identifi ed in the 9 ST22/ST2 isolates. Tn2006 was inserted in different locations on the chromosomes of those isolates (Table). For isolates 240, 512, 810, 859, 883, and Aus, the insertion occurred between 2 genes encoding hypothetical proteins (DR: GTCATTTAA) (Figure 1). In iso-late 761, transposon Tn2006 was located between a gene encoding a hypothetical protein and a gene encoding an isoleucyl tRNA synthase (DR: ATTCGCGGG). In isolate 863, Tn2006 was identifi ed between a gene encoding a cytochrome D terminale oxidase and a putative transposase (DR: ATAATTATT). In isolate 585, Tn2006 was located between a gene encoding a hypothetical protein and a sul1 gene (DR: ATTCGCGGG). The plasmid-borne bla OXA-23 gene identifi ed in isolate Ab13 was also part of Tn2006 but was inserted into the sul gene that encoded a putative sulfonamide resistance determinant (DR: ATTCGCGGG).
Isolates that belonged to European clone I had diverse genetic structures at the origin of bla OXA-23 acquisition. Two isolates had transposon Tn2006: one on the chromosome (AS1) and 1 on a plasmid (910). Transposon Tn2007 was identifi ed in 3 isolates; it was specifi c for the same open reading frame in 2 isolates (BEL and Ab14) (Figure 2).  Only 1 copy of ISAba1 was identifi ed upstream of the bla OXA-23 gene in isolates AS3, 1190, 861, and 877. Transposon Tn2008 was identifi ed only in isolate 614 ( Figure 1). Sequences of these specifi c genetic structures have been deposited in Genbank (accession nos. EF127491, EF059914, GQ861438, and GQ861439).

Discussion
This study was conducted to defi ne which features may explain the worldwide dissemination of the bla OXA-23 gene in A. baumannii. Isolates were from the Middle East, Europe, and Asia; there were no isolates from North America. Except for 2 isolates, the isolates investigated in this study belonged to European clones I or II. Clustering of A. baumannii isolates was determined by MLST and PFGE; our collection was composed of 13 PFGE types corresponding to 9 STs. Eight STs were identifi ed among the OXA-23-producing A. baumannii; the most common STs were ST22/ST2 found in France (n = 2), Vietnam, New Caledonia, Thailand, Australia, Reunion, South Africa, and Tahiti. Spread of bla OXA-23 -positive A. baumannii isolates that belong to clone ST22 has been demonstrated in South Korea (25). Analysis of the target site of bla OXA-23 acquisition showed that in the same clone, such as ST22, acquisition of the Tn2006 composite transposon had occurred at different positions in the A. baumannii genome, which suggested that Tn2006-mediated acquisition of bla OXA-23 may occur as independent events, or that Tn2006 is a structure that is mobile in a given genome. A single clone could have different genetic structures at the origin of the bla OXA-23 acquisition.
We showed that the bla OXA-23 gene associated with Tn2006 could be located on the chromosome or a plasmid. This result agrees with our recent fi ndings, which showed that Tn2006 is capable of transposition (13). We have also observed that 5 isolates with different sequence types (ST-New1, ST25) harbored a similar 130-kb plasmid. The same strains with the same genetic structure were identifi ed in 8 countries in different parts of the world.
In conclusion, the current worldwide dissemination of the bla OXA-23 gene is driven by >7 MLST types associated with different genetic structures and plasmids. We have identifi ed complex and dynamic spreading of bla OXA-23 that will be diffi cult to control because this spread is not associated with a single entity.  (20). Lane M, molecular size markers (48.5 kb).