GES Extended-Spectrum β-Lactamases in Acinetobacter baumannii Isolates in Belgium

Bacterial strains.A phenotypic and genotypic surveillance study of β-lactam resistance was conducted in Belgium between January 2008 and December 2009. The participating hospitals (n = 90) were requested, on a voluntary basis, to send to the reference bacteriology laboratory of UCL Mont-Godinne University hospital all consecutive clinical isolates of multidrug-resistant Acinetobacter species (resistance to at least three classes of antibiotics among penicillins, carbapenems, aminoglycosides, and/or fluoroquinolones) with their corresponding susceptibility report and basic epidemiologic information. Identification of the isolates as Acinetobacter baumannii was initially determined by VITEK2 Gram-negative identification cards and confirmed by mass spectrometry using MALDI-TOF and MALDI Biotyper version 2.0 software (Bruker Daltonics) and the specific PCR targeting the bla_OXA-51 gene (27).

K. pneumoniae ORI-1 (23), Enterobactercloacae CHE-1 (22), and P. aeruginosa DEJ (21), producing GES-1, GES-5, and GES-9, respectively, were used in this study. Electro-competent E. coli TOP10 (Invitrogen, Eragny, France) and A. baumannii CIP7010 (Pasteur Institut, Paris, France) and N9040364 (wild-type clinical isolate) were used as recipients in electroporation experiments (16). Sodium azide E. coli J53Az^R and rifampin-resistant A. baumannii CIP7010 strains were used for conjugation experiments. E. coli 50192 was used as a reference strain for plasmid extraction (16). The PCR-Script Cam cloning vector was used for PCR cloning experiments (Stratagene, Massy, France).

Antimicrobial agents and MIC determinations.Antibiograms were determined by the disc diffusion method against a panel of 16 antimicrobial agents and MICs of aztreonam, ceftazidime, cefepime, piperacillin-tazobactam, imipenem, meropenem, doripenem, ciprofloxacin, amikacin, gentamicin, tobramycin, and colistin were determined by Etest (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar (bioMérieux, Marcy-L'Etoile, France) and interpreted as recommended by the Clinical and Laboratory Standards Institute (CLSI) and by the European Committee for Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/ ). All plates were incubated at 37°C for 18 h.

PCR detection of bla genes.To detect the presence of the most common carbapenemases, multiplex PCR was performed with specific primers targeting bla_OXA-51-like, bla_OXA-23-like, bla_OXA-40-like, and bla_OXA-58-like carbapenemases and the MBLs bla_IMP and bla_VIM as previously described (2). The presence of the insertion element ISAba1 upstream of bla_OXA-51-like and bla_ADC was investigated by PCR (1). PCR screening with family-specific β-lactamase primers for GES600FW (5′-CTG GCA GGG ATC GCT CAC TC-3′), GES600RV (5′-TTC CGA TCA GCC ACC TCT CA-3′), KPC, OXA-48, VEB, and PER was also performed as previously described (8, 17). The genetic context of GES was obtained with a primer located at the 5′ end inside the integrase I coding gene (primer intI, 5′-CAG TGG ACA TAA GCC TGT TC-3′) and with primer CSRV (12) combined with the primers GESFW600 and GESRV600. Selected PCR products were sequenced by external service (Macrogen Inc., Seoul, South Korea), and sequence alignment and analysis were done online by using the BLAST program at www.ncbi.nlm.nih.gov

PFGE.Pulsed-field gel electrophoresis (PFGE) was performed on all A. baumannii isolates as previously described, except that DNA was digested with ApaI (Invitrogen, Merelbeke, Belgium) and PFGE separation conditions were 1 to 10 s for 13 h and then 10 to 15 s for 10 h (3). PFGE patterns were analyzed using BioNumerics software (Applied Maths, Kortrijk, Belgium), and clonal relatedness was determined following the classification criteria previously described (3). PFGE types included profiles showing 0 to 6 DNA fragment differences corresponding generally to a ≥80% level of Dice similarity and were designated by numerals.

Plasmid content, mating out, and electroporation experiments.Direct transfer of resistance into azide-resistant E. coli J53 and rifampin-resistant A. baumannii CIP7010 was attempted as previously reported (9, 19). Plasmids were introduced by electroporation into E. coli TOP10 and A. baumannii CIP7010 and N9040364 (9) using a Gene Pulser II (Bio-Rad, Marnes-la-Coquette, France), as previously described. Electrotransformants were selected on brain heart infusion (BHI) agar medium containing ticarcillin at 100 μg/ml.

Natural plasmids were extracted using the Kieser extraction method (11) or with a Qiagen plasmid DNA maxi kit. Plasmid extracts were subsequently analyzed by electrophoresis on a 0.7% agarose gel. One microgram of natural plasmids was digested with 10 U of HindIII and 10 U of EcoRI (New England Biolabs, Hitchin, United Kingdom) at 37°C for 1 h. Restricted fragments were separated on a 0.8% agarose gel in 0.5× Tris-borate-EDTA (TBE) (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA, pH 8.3) buffer containing 0.5 μg/ml of ethidium bromide for 1 h at 150 V and visualized under UV light.

Cloning experiments of GES β-lactamase genes.Whole-cell DNAs were extracted as described previously (15). The bla_GES genes from K. pneumoniae ORI-1, E. cloacae CHE-1, P. aeruginosa DEJ, A. baumannii 09027, A. baumannii 09135, and A. baumannii 09534, producing GES-1, GES-5, GES-9, GES-11, GES-12, and GES-14, respectively, were PCR amplified using the Pfu thermostable polymerase (Stratagene, Massy, France). The two primers used were GES-CasA, which starts at the beginning of the ges gene cassette, thus including the native consensus ribosomal binding site (RBS) 5 bp upstream of the ATG start codon (5′-TTAGACGGGCGTACAAAGAT-3′), and GES-CasB, which matched to the end of the bla_GES gene (5′-CACCTGAGTTAAGCCGCGGT-3′). These PCR fragments were then cloned into pPCRScriptCam, downstream of the pLac promoter, yielding plasmids pGES-1, -5, -9, -11, -12, and -14 that contained the respective alleles. The sequences of the cloned PCR-generated DNA fragments were confirmed by complete resequencing on both strands.

Recombinant plasmids were transformed by electroporation into E. coli Top10. Antibiotic-resistant colonies were selected onto Trypticase soy (TS) agar plates containing amoxicillin (50 μg/ml) and chloramphenicol (30 μg/ml). Recombinant plasmid DNAs were extracted with a Qiagen plasmid DNA maxi kit (Qiagen, Antwerp, Belgium) and analyzed by restriction endonuclease digestions (Amersham Biosciences, Orsay, France) and agarose gel electrophoresis (Invitrogen, Cergy Pontoise, France).

Specific activity.β-Lactamase extracts were obtained by sonication as described previously (20). The specific β-lactamase activity (nmol/min/mg) of the extracts was measured by UV spectrophotometry (ULTROSPEC 2000 spectrophotometer; Amersham Pharmacia Biotech, Orsay, France) as described previously (20). The specific β-lactamase activities were obtained with 100 μM aztreonam, ceftazidime, cefoxitin, and imipenem as substrates (21). The total protein content was measured with the Bio-Rad DC protein assay kit (Bio-Rad, Marnes-la-Coquette, France).

Nucleotide sequence accession numbers.The nucleotide sequences of bla_GES-11, bla_GES-12, and bla_GES-14 have been assigned to the GenBank nucleotide database under the accession numbers HM622144 (GES-11), HM622145 (GES-12), and GU207844 (GES-14).

Origin of A. baumannii isolates and antimicrobial susceptibility.From a total of 125 multidrug-resistant Acinetobacter isolates originating from 18 Belgian hospitals (20% of the participating hospitals) between January 2008 to December 2009, 9 (7.2%) were identified as GES positive by PCR. These isolates were detected in 6 different hospitals (4 located in Brussels, 1 in Flanders, and 1 in the Wallonia region of Belgium).

The susceptibility tests showed that colistin had the lowest MICs of the tested antibiotics and was the only antimicrobial agent retaining in vitro activity against these strains (Table 1). High-level resistance to fluoroquinolones, aminoglycosides, and to the β-lactam antibiotics, including carbapenem, was observed.

Clinical data.The patient's pertinent clinical information is shown in Table 2. There were six male and three female patients, with an average age of 45 years (ranging from 5 to 79 years). Eight isolates were identified from patients in the intensive care units or in burn units (4 patients each), and one isolate was identified from a patient in a general surgical ward; isolates were either from wounds or abscesses (five isolates), endotracheal aspirates (two isolates), or pleural fluid or blood (one isolate each). The average hospital length of stay was 37 days (range, 9 to 78 days).

Six patients had skin and soft tissue infections with or without sepsis, two patients had ventilator-associated pneumonia (one with empyema), and one was colonized only (asymptomatic intestinal carriage). All patients had significant comorbidity conditions, such as chronic lung disease, cardiovascular disease, severe burns, or traumatic war injuries. Six patients had been transferred from hospitals in countries abroad (Turkey [n = 2], Egypt [n = 2], and Palestinian territories [Gaza] [n = 2]) (Table 2). At one center (CHA) a nosocomial outbreak occurred in a burn unit following the transfer of a patient with extensive burn wounds who had been transferred from a hospital in Cairo, Egypt. The crude mortality rate for the nine patients was 33%. All but one patient (the colonized patient F) had been treated with multiple antibiotic courses before their transfer or during hospitalization (data not shown). Four patients were not treated, two patients were treated with colistin in association with a carbapenem (one of them also received amikacin), one patient was treated with an association of meropenem and ciprofloxacin, and the two remaining patients were treated with amoxicillin plus clavulanate.

Molecular analysis of GES A. baumannii isolates.PFGE analysis revealed that the GES-positive A. baumannii isolates clustered into 6 different PFGE types (Table 2). All the A. baumannii isolates, except those outbreak related and recovered from the CHA center, displayed distinct PFGE types. Interestingly, the PFGE profile of the isolate recovered from patient A presented more than 80% similarity (but 9 DNA fragment differences) with the European clone I (4) (Fig. 1), and it displayed a PFGE type, 39, identical to that of the OXA-23-producing A. baumannii strains formerly recovered in a Belgian hospital in 2007 (1) (data not shown).

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FIG. 1.

Dendrogram of PFGE patterns of A. baumannii clinical isolates and reference strains of European clones I, II, and III. RUH0875, RUH0134, and LUH05875 correspond to European clones I, II, and III, respectively (4).

The PCR results obtained with family-specific ß-lactamase primers revealed that all isolates were positive for bla_GES-like genes and for bla_OXA-51-like groups (OXA-82 [four isolates], OXA-69 [two isolates], and OXA-51, OXA-66, OXA-94 [one isolate each]) but that they were negative for all acquired carbapenem-hydrolyzing bla_OXA genes (OXA-23, OXA-40, and OXA-58 groups) as well as for bla_IMP and bla_VIM genes. ISAba1 was detected upstream of the bla_ADC gene in seven isolates but never upstream of the bla_OXA-51-like genes (Table 2).

Sequencing of bla_GES genes and subsequent amino acid sequence deduction revealed the presence of novel GES variants. GES-11, described for one single isolate from France, was found in four isolates presenting a Gly243Ala substitution compared to GES-1 (14). GES-12, identified in all four A. baumannii isolates detected at CHA, presented two substitutions, Thr237Ala and Gly243Ala, compared to GES-1. Finally, GES-14 was identified in one A. baumannii isolate from patient H transferred from Turkey and presented two substitutions, Gly170Ser and Gly243Ala, compared to the GES-1 sequence.

Genetic environment of bla_GES genes.Sequence analysis of the close genetic context of bla_GES genes revealed that they were all part of a class 1 integron containing, downstream, the aac(6′)-Ib gene encoding an aminoglycoside, 6′-N-acetyltransferase, which modifies amikacin and tobramycin, and the dfrA7 resistance gene as described by Moubareck et al. (14) and differing only by the point mutations encountered in the bla_GES alleles. Plasmid extraction experiments identified plasmids in bla_GES-11- and bla_GES-12-expressing A. baumannii (data not shown) but not in bla_GES-14 isolates despite several attempts using different extraction methods. In addition, total DNA extracts of GES-14-expressing A. baumannii were repeatedly unable to transform a competent wild-type A. baumannii clinical isolate (no. N9040364), further suggesting that the bla_GES-14 gene was chromosomally located. On the other hand, GES-11- and GES-12-expressing transformants could be obtained by electroporation, confirming the plasmid location of the bla_GES-11 and of the bla_GES-12 genes.

The GES-11 and GES-12 A. baumannii electroporants were highly resistant to all β-lactam antibiotics, including the carbapenems (with imipenem, meropenem, and doripenem MICs ranging between 4 and 32 μg/ml), to trimethoprim, and to aminoglycosides but not to ciprofloxacin. Double digestion of plasmid extract (about 75 kb) from these transformants with EcoRI and HindIII restriction enzymes revealed a very similar banding pattern for pGES-11 and pGES-12 (4 and 5 band differences), suggesting a common origin for these plasmids. As an example, restriction profiles of pGES-11 and pGES-12 (originating from isolates 9027 and 9035, respectively) are shown in Fig. 2.

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FIG. 2.

Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from isolate 9135) extract digested with EcoRI and HindIII; lane 3, undigested pGES-11 crude extract; lane 4, undigested pGES-12 crude extract.

Biochemical properties.The resistance profile and specific activity of each GES-type-expressing E. coli clone (Table 3) show variable activity against ceftazidime, aztreonam, cefoxitin, and imipenem. GES-11 and GES-12 variants shared with GES-9 a high hydrolytic activity against ceftazidime, the specific activity of GES-12 (85.2 nmol/min/mg) being over 10 times higher than that of GES-1 (6.5 nmol/min/mg), GES-5 (activity too low to be measurable), and GES-14 (2.0 nmol/min/mg). Further, GES-11 and -12 variants did also hydrolyze aztreonam but had very weak hydrolytic activity against imipenem and no measurable activity against cefoxitin.

GES-14-expressing E. coli displayed a resistance profile (MIC values) similar to that of the GES-5-expressing clone (a carbapenem and cefoxitin hydrolyzing isoform) (28). It was able to hydrolyze imipenem and cefoxitin as efficiently as GES-5, while it displayed a very weak activity (like that of GES-5) against ceftazidime and aztreonam. Nevertheless, as observed for GES-5, E. coli expressing GES-14 remained susceptible to imipenem, suggesting the involvement of additional mechanisms in the carbapenem resistance observed in the GES-14-producing A. baumannii clinical isolate.