Abstract
Acinetobacter baumannii is an opportunistic pathogen that is an important source of nosocomial infections. Production of extended-spectrum β-lactamases (ESBLs) of the GES type in A. baumannii has been increasingly reported, and some of these GES-type enzymes possess some carbapenemase activity. Our aim was to analyze the resistance determinants and the clonal relationships of carbapenem-nonsusceptible A. baumannii clinical isolates recovered from hospitals in Kuwait. A total of 63 isolates were analyzed, and all were found to be positive for blaGES-type genes. One isolate harbored the blaGES-14 gene encoding an ESBL with significant carbapenemase activity, whereas the other isolates harbored the blaGES-11 ESBL gene. Thirty-three isolates coharbored the blaOXA-23 and blaGES-11 genes. Analyses of the genetic locations indicated that the blaGES-11/-14 genes were plasmid located. It is noteworthy that the blaOXA-23 and blaGES-11 genes were colocated onto a single plasmid. Nine different pulsotypes were observed among the 63 isolates. This study showed the emergence of GES-type ESBLs in A. baumannii in Kuwait, further suggesting that the Middle East region might be a reservoir for carbapenemase-producing A. baumannii.
INTRODUCTION
Acinetobacter baumannii is an opportunistic pathogen that is an important causative agent of nosocomial infections, such as pneumonia, septicemia, urinary tract infections, and wound infections (1). Multidrug-resistant (MDR) A. baumannii isolates are increasingly reported worldwide and are often a source of nosocomial infections. Treatment of infections due to this microorganism is becoming a serious clinical concern, since A. baumannii is very often resistant to multiple antibiotics (1). One of the main mechanisms of resistance to β-lactam molecules in this species is the production of β-lactamases (2). Resistance to carbapenems is mostly related to the production of carbapenem-hydrolyzing class D β-lactamases (CHDLs) and, to a lesser extent, of metallo-β-lactamases (MBLs). Among these CHDLs, OXA-23 is the most commonly identified CHDL worldwide (2), and its corresponding gene is located on either a plasmid or a chromosome and at the origin of its acquisition is associated with the insertion sequence ISAba1 or ISAba4 (3). Although resistance to carbapenems is mostly related to the production of CHDLs that do not include broad-spectrum cephalosporins in their hydrolytic spectrum, resistance to broad-spectrum cephalosporin molecules in A. baumannii usually results from overexpression of the natural AmpC-type enzyme but also from the acquisition of extended-spectrum β-lactamases (ESBLs) (2). Those ESBLs may correspond to TEM or SHV derivatives but mostly correspond to Ambler class A β-lactamases of the VEB, PER, and GES types (2). ESBLs of the GES type are being reported increasingly in Gram-negative rods, including Pseudomonas aeruginosa, Enterobacter cloacae, and Klebsiella pneumoniae (4) and were recently reported in A. baumannii (5–7). While the hydrolysis profile of GES-1 is similar to that of other ESBLs (8), including penicillins and broad-spectrum cephalosporins, GES-1 nonetheless does not hydrolyze monobactams, and some GES variants possess significant carbapenemase activity. A Gly170Ser substitution, located inside the omega loop of the catalytic site, was identified in GES-4, GES-5, and GES-6, enzymes that hydrolyze carbapenems and cephamycins (4). GES-11, identified in A. baumannii and differing from GES-1 by a single amino acid substitution (a Gly243Ala change), possesses increased activity against aztreonam (7). GES-14, also identified in A. baumannii and differing from GES-11 by a Gly170Ser amino acid substitution, possesses an extended spectrum of activity toward carbapenems and cephamycins, in addition to its ability to compromise monobactams, giving rise to a very broad-spectrum enzyme that is active against all β-lactams (6).
The aim of this study was to analyze the resistance determinants and the clonal relationships of a collection of carbapenem-nonsusceptible A. baumannii clinical isolates recovered from different hospitals in Kuwait.
MATERIALS AND METHODS
Bacterial isolates and susceptibility testing.
Sixty-three nonduplicate and carbapenem-nonsusceptible A. baumannii clinical isolates were included in this study. These isolates were identified by using the API 20 NE system (bioMérieux, Marcy l'Etoile, France), 16S rRNA gene sequencing, and culture at 44°C, as described previously (9). The antibiotic susceptibilities of the isolates were determined by the disc diffusion technique on Mueller-Hinton agar. MICs were determined by using Etest strips (AB bioMérieux, La Balme-les-Grottes, France) and interpreted according to CLSI guidelines (10). The production of MBLs was evaluated using Etest strips as recommended by the manufacturer (AB bioMérieux) and by the combined-disc test as described previously (11).
PCR amplification and sequencing.
PCR experiments were performed using standard conditions to search for β-lactamase genes that have been identified previously in A. baumannii, i.e., narrow-spectrum β-lactamase genes blaSCO-1 and blaRTG-3, ESBL genes blaPER, blaGES, and blaVEB, MBL genes blaNDM, blaVIM, blaIMP, and blaSIM, acquired CHDL genes blaOXA-23, blaOXA-40, blaOXA-58, and blaOXA-143, and the intrinsic blaOXA-51-like CHDL gene (see Table S1 in the supplemental material). Detection of the ISAba1 element upstream of the blaADC and blaOXA-51 genes was also performed. The primers used in this study are listed in Table S1. Detection of the 16S RNA methylase genes was also performed as described previously (12). Amplified DNA fragments were purified with the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France). Both strands of the amplification products obtained were sequenced with an ABI 3100 sequencer (Applied Biosystems, Foster City, CA). The nucleotide and deduced protein sequences were analyzed with software available over the Internet at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).
Acinetobacter PCR-based replicon typing (AB-PBRT) has been developed to type the plasmids circulating in A. baumannii. In total, 19 PCR amplifications were used as described previously (13).
Analysis of the genetic support of β-lactamase genes.
In order to determine the genetic locations of carbapenemase and the ESBL genes, conjugation assays were performed using A. baumannii BM4547 (rifampin resistant) and azide-resistant Escherichia coli J53 (Invitrogen, Cergy-Pontoise, France) as recipient strains. Plasmid DNA was extracted by using the Kieser method (14), and electroporation was performed using A. baumannii CIP70.10 as the donor. Selection was based on ticarcillin (50 μg/ml) and sodium azide (100 μg/ml) or rifampin (100 μg/ml), depending on the recipient strain used.
DNA-DNA hybridization analyses were performed by using total plasmid DNA extracted as described above, separated by electrophoresis on 0.8% agarose gels, transferred onto Hybond-N+ membranes, and hybridized with enhanced chemiluminescence-labeled probes overnight at 42°C. Plasmid locations of the β-lactamase genes were assessed by the hybridization of plasmid DNA with DNA probes specific for the blaOXA-23 or blaGES genes.
Molecular typing and clonal relationships.
Isolates were typed by using ApaI macrorestriction analyses and pulsed-field gel electrophoresis (PFGE) according to the manufacturer's recommendations (Bio-Rad, Marnes-la-Coquette, France). Whole-cell DNA of A. baumannii isolates was digested with ApaI for 3 h at 37°C (Fermentas, St. Rémy-Les-Chevreuses, France) as described previously (3). Electrophoresis was performed on an agarose gel using a CHEF-DR II apparatus (Bio-Rad). Isolates were also typed by randomly amplified polymorphic DNA (RAPD) analyses using DAF4 and M13 primers as described previously (15).
The identification of PCR-based sequence groups was conducted by using 2 multiplex PCR assays designed to identify the worldwide clones as described previously (16). Clonal relationships were established by multilocus sequence typing (MLST) by using 7 standard housekeeping loci as described previously (17). Sequences of the 7 housekeeping genes were analyzed by using an A. baumannii database (www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii.html).
RESULTS AND DISCUSSION
Antimicrobial susceptibilities.
A total of 63 consecutive nonrepetitive carbapenem-nonsusceptible A. baumannii clinical isolates, including 59 invasive strains and 4 colonizers, were obtained from clinical specimens taken from patients on admission to one of six hospitals located in different parts of Kuwait between December 2007 and June 2008. Of these 63 isolates, 32 (50.8%) were from patients with proven lower respiratory tract infections, 11 (17.5%) from blood cultures, 7 (11.1%) from urine, 1 (1.6%) from body fluid, and 4 (6.4%) from other sites. Among these 63 isolates, all were resistant to broad-spectrum cephalosporins, and 54% (34/63) were resistant to carbapenems (Table 1). All isolates were resistant to ciprofloxacin, chloramphenicol, and sulfonamides, and 92% and 46% were resistant to amikacin and gentamicin, respectively. All the isolates remained susceptible to colistin and rifampin. Four isolates were resistant to tigecycline (MIC, 1.5 to 3 μg/ml) according to guidelines described by Jones et al. (18), and eight isolates had reduced susceptibilities to tigecycline (MICs, 1 μg/ml).
Table 1.
β-Lactams for A. baumannii clinical isolates K22, K31, and K78, A. baumannii BM4547(pK22), BM4547(pK31), and BM4547(pK78) (transconjugant), and the A. baumannii BM4547 reference strain
| β-Lactam(s)a | MIC (μg/ml) of: |
||||||
|---|---|---|---|---|---|---|---|
| A. baumannii isolate K22 (GES-11) | A. baumannii isolate K31 (GES-14) | A. baumannii isolate K78 (GES-11, OXA-23) | A. baumannii BM4547(pK22) (GES-11) | A. baumannii BM4547(pK31) (GES-14) | A. baumannii BM4547(pK78) (GES-11, OXA-23) | A. baumannii BM4547b | |
| Ticarcillin | >256 | >256 | >256 | >256 | >256 | >256 | 8 |
| Ticarcillin + CLA | 256 | 256 | >256 | 256 | 128 | >256 | 8 |
| Piperacillin | >256 | >256 | >256 | >256 | >256 | >256 | 4 |
| Piperacillin + TZB | 256 | 256 | >256 | 256 | 128 | >256 | 4 |
| Cefoxitin | >256 | >256 | >256 | >256 | >256 | >256 | 256 |
| Cefotaxime | >64 | >64 | >64 | >64 | >64 | >64 | >32 |
| Cefotaxime + CLA | >1 | >1 | >1 | >1 | >1 | >1 | ND |
| Ceftazidime | >64 | >64 | >64 | >64 | >64 | >64 | 4 |
| Ceftazidime + CLA | >1 | >1 | >1 | >1 | >1 | >1 | ND |
| Cefepime | >64 | >64 | >64 | 32 | 32 | 32 | 16 |
| Aztreonam | >64 | >64 | >64 | 64 | >64 | >64 | 32 |
| Meropenem | 4 | 16 | 32 | 2 | 12 | 24 | 0.5 |
| Doripenem | 2 | 16 | 32 | 2 | 12 | 24 | 0.25 |
| Imipenem | 2 | 24 | 32 | 1 | 24 | 24 | 0.25 |
CLA, clavulanic acid (4 μg/ml); TZB, tazobactam (4 μg/ml).
ND, not determined.
Characterization of β-lactamase genes.
Phenotypic assays showed that all the isolates remained negative for MBL production. PCR experiments for the detection of the blaNDM-1, blaIMP-type, blaVIM-type, and blaSIM-1 genes gave negative results. Synergy tests with discs containing ceftazidime and ticarcillin-clavulanic acid, using cloxacillin-containing Mueller-Hinton agar plates as described previously (6), gave positive results. PCR followed by sequencing identified the blaGES ESBL genes in all the isolates (Table 2). The blaVEB-1 and blaPER-1 genes were not identified. Additionally, PCR experiments followed by sequence analyses led to the identification of the blaOXA-23 gene in 33 isolates, in addition to a natural blaOXA-51-like gene identified in all the isolates (Table 2). Sequencing of the blaOXA-51-like gene revealed several variants corresponding to blaOXA-66, blaOXA-64, blaOXA-98, and blaOXA-71 (19).
Table 2.
Clinical features, β-lactamase detection, and genotyping of A. baumannii clinical isolates
| Isolate | Date of isolation (mo-day-yr) | Hospital | Hospital unita | Specimen sourceb | Acquired carbapenemase | OXA-51-like | ISAba1-ampC | ESBL | EC/STc | RAPD pattern (group) | PFGE pattern (clone name) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| K35 | 12-30-07 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K36 | 12-30-07 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K40 | 12-22-07 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K45 | 12-30-07 | Al Jahra | Medical | Urine culture | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K46 | 12-24-07 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K50 | 01-13-08 | Al Jahra | ICU | Urine culture | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K66 | 01-06-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K68 | 01-06-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K72 | 01-09-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K73 | 01-09-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K77 | 01-20-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K78 | 01-22-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K79 | 01-20-08 | Al Jahra | ICU | Sputum | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K80 | 01-22-08 | Al Jahra | Surgical ward | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K81 | 02-07-08 | Al Jahra | ICU | Leg | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K82 | 02-18-08 | Al Jahra | ICU | Bed swab | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K86 | 01-14-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K88 | 01-16-08 | Al Jahra | ICU | Foot wound | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K102 | 02-19-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K103 | 02-19-08 | Al Jahra | Medical ward | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K104 | 02-25-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K105 | 02-24-08 | Al Jahra | Medical ward | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K106 | 02-24-08 | Al Jahra | Surgical ward | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K109 | 03-02-08 | Al Jahra | Medical ward | Blood culture | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K112 | 02-29-08 | Al Jahra | ICU | Endotracheal tube | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K118 | 02-07-08 | Al Jahra | Medical ward | BAL | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K144 | 03-23-08 | Al Jahra | ICU | Blood culture | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K162 | 04-26-08 | Al Jahra | Medical ward | Sputum | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K219 | 05-18-08 | Al Jahra | Surgical ward | Urine culture | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K221 | 05-07-08 | Al Jahra | Medical ward | Urine culture | OXA-23 | OXA-66 | − | GES-11 | nt/ST158 | 1 | A |
| K5 | 12-04-07 | Mubarak Al Kabeer | Medical ward | Blood culture | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K21 | 12-12-07 | Al Adan | Neonatal ICU | Blood culture | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K22 | 12-12-07 | Al Adan | Neonatal ICU | Blood culture | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K75 | 02-10-08 | Mubarak Al Kabeer | ICU | Endotracheal secretions | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K107 | 03-02-08 | Mubarak Al Kabeer | ICU | Blood culture | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K120 | 02-10-08 | Al Adan | Neonatal ICU | Blood culture | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K130 | 03-19-08 | Al Adan | Medical ward | Endotracheal tube | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K140 | 03-19-08 | Mubarak Al Kabeer | Medical ward | Pus | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K147 | 04-02-08 | Al Babtain | Medical ward | Urine culture | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K176 | 01-19-08 | Al Adan | Neonatal ICU | Endotracheal sample | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B1 |
| K198 | 05-25-08 | Mubarak Al Kabeer | Nephrology ward | Dialysis tip | − | OXA-98 | − | GES-11 | nt/ST49 | 4 | B1 |
| K202 | 02-22-08 | Al Adan | Neonatal ICU | Endotracheal tube | − | OXA-98 | − | GES-11 | nt/ST49 | 4 | B1 |
| K228 | 05-27-08 | Al Adan | Surgical ward | Urine culture | − | OXA-98 | − | GES-11 | nt/ST49 | 4 | B1 |
| K238 | 06-26-08 | Al Adan | Neonatal ICU | Endotracheal tube | − | OXA-98 | − | GES-11 | nt/ST49 | 4 | B1 |
| K239 | 06-25-08 | Al Adan | Neonatal ICU | Endotracheal tube | − | OXA-98 | − | GES-11 | nt/ST49 | 4 | B1 |
| K248 | 04-01-08 | Al Adan | Neonatal ICU | Respiratory sample | − | OXA-98 | − | GES-11 | nt/ST49 | 4 | B1 |
| K63 | 01-08-08 | Al Razi | Medical ward | Urine | − | OXA-98 | − | GES-11 | nt/ST49 | 2 | B2 |
| K65 | 11-23-08 | Al Jahra | ICU | Tracheal sample | OXA-23 | OXA-66 | + | GES-11 | II/ST104 | 2 | D |
| K191 | 06-12-08 | Al Sabah | Medical ward | Wound | OXA-23 | OXA-66 | − | GES-11 | II/ST2 | 3 | E |
| K55 | 01-20-08 | Mubarak Al Kabeer | ICU | Endotracheal sample | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | C |
| K70 | 02-27-08 | Mubarak Al Kabeer | ICU | Pus | OXA-23 | OXA-64 | − | GES-11 | nt/ST113 | 1 | C |
| K108 | 03-02-08 | Mubarak Al Kabeer | CCU | Blood culture | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K126 | 03-09-08 | Mubarak Al Kabeer | CCU | Ventilator swab | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K127 | 03-12-08 | Mubarak Al Kabeer | ICU | Pus | − | OXA-64 | + | GES-11 | nt/ST113 | 1 | F |
| K131 | 04-01-08 | Al Razi | Medical ward | Urine culture | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K138 | 02-22-08 | Al Adan | Neonatal ICU | Endotracheal tube | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K145 | 04-02-08 | Mubarak Al Kabeer | ICU | Blood culture | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K152 | 04-01-08 | Mubarak Al Kabeer | ICU | Blood culture | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K167 | 05-08-08 | Mubarak Al Kabeer | ICU | Blood culture | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K250 | 04-01-08 | Mubarak Al Kabeer | ICU | Rectal swab | − | OXA-64 | − | GES-11 | nt/ST113 | 1 | F |
| K89 | 02-10-08 | Mubarak Al Kabeer | CCU | Endotracheal sample | − | OXA-64 | − | GES-11 | nt/ST178 | 1 | G |
| K121 | 02-21-08 | Al Adan | Medical ward | Tracheal sample | − | OXA-71 | − | GES-11 | III/ST3 | 1 | H |
| K31 | 04-01-08 | Al Adan | ICU | Bed rail | GES-14 | OXA-71 | + | GES-14 | III/ST3 | 1 | I |
Genetic support of the blaGES-14 gene and transfer of β-lactam resistance.
Plasmid DNA analyses showed that all A. baumannii clinical isolates harbored plasmids of ca. 90 to 100 kb in size (data not shown). Mating-out assays revealed A. baumannii BM4547 transconjugants harboring either the blaGES-14 or the blaGES-11 gene. All transconjugants obtained using the OXA-23-producing A. baumannii isolates as donors coharbored the blaOXA-23 gene. The blaGES-11-carrying transconjugants exhibited an ESBL phenotype but also showed reduced susceptibilities to carbapenems, suggesting the presence of weak hydrolysis of carbapenems by GES-11 potentiated by the efflux overproduction in A. baumannii BM4547. The A. baumannii transconjugants harboring the blaGES-14-positive plasmid showed resistance to carbapenems, confirming that the expression of GES-14 led to resistance to carbapenems, as reported previously (6). Finally, all A. baumannii transconjugants expressing both OXA-23 and GES-11 and actually harboring a single plasmid bearing both the blaGES-11 and blaOXA-23 genes were resistant to all β-lactams, including carbapenems. All transconjugants showed acquired resistance to chloramphenicol, tetracycline, and aminoglycosides. Southern hybridizations performed with blaGES- and blaOXA-23-specific probes confirmed that these two genes were colocated onto a ca. 95-kb plasmid (data not shown).
Attempts to transfer these natural plasmids by conjugation and electroporation into E. coli TOP10 as a recipient strain failed. This result suggests that those plasmids possessed a replication module that does not allow replication in E. coli, as previously suggested (6). The typing of these plasmids by A. baumannii PCR-based replicon typing (AB-PBRT) indicated that all plasmids belonged to the plasmid group Gr6. This group of plasmids has been shown to be highly prevalent in A. baumannii and was previously found to be associated with the blaOXA-23, blaOXA-40, and blaOXA-58 CHDL genes (20).
Genetic environment of the blaGES genes.
PCR mapping was performed to identify the genetic structure bracketing the blaGES genes. Downstream of the blaGES-14 gene, the aacA4 gene cassette encoding the AAC(6′)-Ib aminoglycoside acetyltransferase was identified. It was followed by the dfrA7 gene cassette, which encodes resistance to trimethoprim, and then the 3′ extremity of class 1 integrons as observed previously (6). Also as previously observed, the integrase gene upstream of the blaGES genes was truncated in its 5′ extremity. The complete In125 integron, composed of two class 1 integrons, was identified for only the isolate possessing the blaGES-14 gene. For the isolates carrying the blaGES-11 gene, the 5′ extremity of the In125 integron was not identified by PCR mapping as observed previously (7).
Genetic environment of the blaOXA-23 gene.
The insertion sequence ISAba1 was detected upstream of the blaOXA-23 gene in all positive isolates, but no ISAba1 was detected downstream of the blaOXA-23 gene, ruling out the hypothesis that this gene could be part of the composite transposon Tn2006. Sequence analysis of the region separating ISAba1 from the blaOXA-23 gene revealed a 7-bp deletion corresponding to the sequence previously identified in Tn2008 carrying blaOXA-23 in A. baumannii isolates from the United States, Romania, and China (21–23).
Genotyping of clinical isolates.
Genotypic comparisons were performed by using different techniques. RAPD analysis clustered the collection into four groups with a major clonal group (group 1) (Table 2). PFGE analysis showed that the 63 isolates were grouped into nine distinct clones named A to I (Table 2). Among these pulsotypes, pulsotype A, including 30 isolates, was the main group, pulsotype B included 15 isolates, and pulsotype F included 9 isolates. The other groups included either 1 or 2 isolates each. Pulsotypes A, C, F, G, H, and I corresponded to RAPD group 1, pulsotype B corresponded to RAPD groups 2 and 4, and pulsotype E corresponded to RAPD group 3. MLST analyses showed that pulsotype A belonged to ST158 (41-42-13-1-5-4-14), a recently identified sequence type (ST) (http://www.pasteur.fr/cgi-bin/genopole/PF8/mlstdbnet.pl?file=acin_isolates.xml). All isolates belonging to ST158 were isolated in the same hospital but different wards. Therefore, this clone appears to be endemic in the Al Jahrah hospital (Table 2). This clone coproduced the two carbapenemases GES-11 and OXA-23 and showed high levels of resistance to carbapenems (Table 1). Pulsotype B belonged to ST49 (3-3-6-2-3-1-5), which is an ST that has already been identified in the United States and the Netherlands (15, 21). This ST is actually a double-locus variant of ST3 (3-3-2-2-3-1-3), which has been widely identified throughout the world and corresponds to the worldwide clone III (24, 25). Isolates corresponding to pulsotypes G and H belonged to ST3, confirming that this ST is prevalent in Kuwait. The third main sequence type corresponded to ST113 (3-3-3-4-7-4-4), which was recently described in Saudi Arabia among GES-producing A. baumannii (26), or to ST178 (3-1-3-4-7-4-4), which is a single-locus variant of ST113. The remaining clones belonged to worldwide clone II derivatives, either ST2 (2-2-2-2-2-2-2) or ST104 (2-2-2-2-2-2-14), the latter of which is a single-locus variant of ST2. This clone has also been shown to be widely distributed throughout the world and is associated with the production of CHDLs (1, 17, 21, 24). Only two isolates belonging to clonal complex II were identified among our isolates. Overall, clonal lineages identified in Kuwait were significantly different from those reported in Europe, where ST1 and ST2 have been the major clones recovered (16, 17, 24). We identified only a few isolates belonging to ST2 and found none belonging to ST1. These results may be explained by the fact that our collection was recovered during the 2007-2008 time period.
Conclusion.
This study revealed a high prevalence of GES-producing A. baumannii strains in Kuwait that were not linked to a single clone, even though one clone was more commonly identified (47%) within the 63 isolates studied. Recently, a study performed in Belgium also reported a series of GES-producing A. baumannii isolates for which a link with different geographical origins, including Middle East countries, Palestinian territories, Turkey, and Egypt, was evidenced (5). Overall, those results strongly suggest that the Middle East may be an important reservoir of GES-type carbapenemases. Our results have shown that the dissemination of those genes was not linked to clonal strain dissemination but rather to plasmid dissemination. Noticeably, the plasmid either was carrying the blaGES carbapenemase gene alone or was coharboring the blaOXA-23 carbapenemase gene, making it able to more efficiently promote high-level resistance to carbapenems when acquired in A. baumannii.
Supplementary Material
ACKNOWLEDGMENTS
This work was partially funded by a grant from the INSERM (U914), UMR Université Paris-Sud, France, by grants from the European Community (R-GNOSIS, FP7/HEALTH-F3-2011-282512, MAGIC-BULLET, FP7/HEALTH-F3-2001-278232, and TEMPOtest-QC, FP7/HEALTH-2009-241742), and by a Kuwait University Research Administration grant (no. YM01/08).
Footnotes
Published ahead of print 22 October 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AAC.01384-12.
REFERENCES
- 1. Peleg AY, Seifert H, Paterson DL. 2008. Acinetobacter baumannii: emergence of a successful pathogen. Clin. Microbiol. Rev. 21:538–582 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Poirel L, Bonnin RA, Nordmann P. 2011. Genetic basis of antibiotic resistance in pathogenic Acinetobacter species. IUBMB Life 63:1061–1067 [DOI] [PubMed] [Google Scholar]
- 3. Mugnier PD, Poirel L, Naas T, Nordmann P. 2010. Worldwide dissemination of the blaOXA-23 carbapenemase gene of Acinetobacter baumannii. Emerg. Infect. Dis. 16:35–40 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Poirel L, Bonnin RA, Nordmann P. 2012. Genetic support and diversity of acquired extended-spectrum β-lactamases in Gram-negative rods. Infect. Genet. Evol. 12:883–893 [DOI] [PubMed] [Google Scholar]
- 5. Bogaerts P, Naas T, El Garch F, Cuzon G, Deplano A, Delaire T, Huang TD, Lissoir B, Nordmann P, Glupczynski Y. 2010. GES extended-spectrum β-lactamases in Acinetobacter baumannii isolates in Belgium. Antimicrob. Agents Chemother. 54:4872–4878 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Bonnin RA, Nordmann P, Potron A, Lecuyer H, Zahar JR, Poirel L. 2011. Carbapenem-hydrolyzing GES-type extended-spectrum β-lactamase in Acinetobacter baumannii. Antimicrob. Agents Chemother. 55:349–354 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Moubareck C, Bremont S, Conroy MC, Courvalin P, Lambert T. 2009. GES-11, a novel integron-associated GES variant in Acinetobacter baumannii. Antimicrob. Agents Chemother. 53:3579–3581 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Poirel L, Le Thomas I, Naas T, Karim A, Nordmann P. 2000. Biochemical sequence analyses of GES-1, a novel class A extended-spectrum β-lactamase, and the class 1 integron In52 from Klebsiella pneumoniae. Antimicrob. Agents Chemother. 44:622–632 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Ibrahim A, Gerner-Smidt P, Liesack W. 1997. Phylogenetic relationship of the twenty-one DNA groups of the genus Acinetobacter as revealed by 16S ribosomal DNA sequence analysis. Int. J. Syst. Bacteriol. 47:837–841 [DOI] [PubMed] [Google Scholar]
- 10. Clinical and Laboratory Standards Institute 2012. Performance standards for antimicrobial susceptibility testing (M100-S22). Clinical and Laboratory Standards Institute, Wayne, PA [Google Scholar]
- 11. Bonnin RA, Naas T, Poirel L, Nordmann P. 2012. Phenotypic, biochemical, and molecular techniques for detection of metallo-β-lactamase NDM in Acinetobacter baumannii. J. Clin. Microbiol. 50:1419–1421 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Bercot B, Poirel L, Nordmann P. 2011. Updated multiplex polymerase chain reaction for detection of 16S rRNA methylases: high prevalence among NDM-1 producers. Diagn. Microbiol. Infect. Dis. 71:442–445 [DOI] [PubMed] [Google Scholar]
- 13. Bertini A, Poirel L, Mugnier PD, Villa L, Nordmann P, Carattoli A. 2010. Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii. Antimicrob. Agents Chemother. 54:4168–4177 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Kieser T. 1984. Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid 12:19–36 [DOI] [PubMed] [Google Scholar]
- 15. Grundmann HJ, Towner KJ, Dijkshoorn L, Gerner-Smidt P, Maher M, Seifert H, Vaneechoutte M. 1997. Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp. J. Clin. Microbiol. 35:3071–3077 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Turton JF, Gabriel SN, Valderrey C, Kaufmann ME, Pitt TL. 2007. Use of sequence-based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii. Clin. Microbiol. Infect. 13:807–815 [DOI] [PubMed] [Google Scholar]
- 17. Diancourt L, Passet V, Nemec A, Dijkshoorn L, Brisse S. 2010. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One 5:e10034 doi:10.1371/journal.pone.0010034 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Jones RN, Ferraro MJ, Reller LB, Schreckenberger PC, Swenson JM, Sader HS. 2007. Multicenter studies of tigecycline disk diffusion susceptibility results for Acinetobacter spp. J. Clin. Microbiol. 45:227–230 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Zander E, Nemec A, Seifert H, Higgins PG. 2012. Association between β-lactamase-encoding blaOXA-51 variants and DiversiLab rep-PCR-based typing of Acinetobacter baumannii isolates. J. Clin. Microbiol. 50:1900–1904 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Towner KJ, Evans B, Villa L, Levi K, Hamouda A, Amyes SG, Carattoli A. 2011. Distribution of intrinsic plasmid replicase genes and their association with carbapenem-hydrolyzing class D β-lactamase genes in European clinical isolates of Acinetobacter baumannii. Antimicrob. Agents Chemother. 55:2154–2159 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Adams-Haduch JM, Paterson DL, Sidjabat HE, Pasculle AW, Potoski BA, Muto CA, Harrison LH, Doi Y. 2008. Genetic basis of multidrug resistance in Acinetobacter baumannii clinical isolates at a tertiary medical center in Pennsylvania. Antimicrob. Agents Chemother. 52:3837–3843 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Bonnin RA, Poirel L, Licker M, Nordmann P. 2011. Genetic diversity of carbapenem-hydrolysing β-lactamases in Acinetobacter baumannii from Romanian hospitals. Clin. Microbiol. Infect. 17:1524–1528 [DOI] [PubMed] [Google Scholar]
- 23. Wang X, Zong Z, Lu X. 2011. Tn2008 is a major vehicle carrying blaOXA-23 in Acinetobacter baumannii from China. Diagn. Microbiol. Infect. Dis. 69:218–222 [DOI] [PubMed] [Google Scholar]
- 24. Higgins PG, Dammhayn C, Hackel M, Seifert H. 2010. Global spread of carbapenem-resistant Acinetobacter baumannii. J. Antimicrob. Chemother. 65:233–238 [DOI] [PubMed] [Google Scholar]
- 25. van Dessel H, Dijkshoorn L, van der Reijden T, Bakker N, Paauw A, van den Broek P, Verhoef J, Brisse S. 2004. Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals. Res. Microbiol. 155:105–112 [DOI] [PubMed] [Google Scholar]
- 26. Ribeiro A, Al-Agamy MH, Shibl AM, Tawfik AF, Courvalin P, Jeannot K. 2012. Molecular epidemiology and mechanisms of carbapenem-resistant Acinetobacter baumannii in a Saudi Arabia hospital (P1256). Clin. Microbiol. Infect. 18:318 [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.