ABSTRACT
During a PCR-based surveillance study of β-lactam resistance, 125 multidrug-resistant (MDR) Acinetobacter baumannii isolates were obtained from 18 hospitals in Belgium from January 2008 to December 2009. Nine GES-positive A. baumannii isolates were detected at 6 Belgian hospitals. DNA sequencing of the blaGES genes identified GES-11, GES-12, and a novel variant GES-14, which differs from GES-11 by a single amino acid substitution (Gly170Ser). All index isolates were travel associated and originated from patients transferred from Turkey (n = 2), Egypt (n = 2), and Palestinian territories (Gaza) (n = 2). A nosocomial outbreak involving three additional patients occurred in a burn unit at a single hospital. No clonal relatedness could be established between the 6 index isolates by pulsed-field gel electrophoresis (PFGE) analysis. Three different alleles (the plasmid-located blaGES-11 and blaGES-12 and a likely chromosomally located novel variant blaGES-14) were detected as part of a class 1 integron, also including the aac6′Ib and dfrA7 genes. Restriction analysis of plasmids suggests a common origin for the plasmids bearing blaGES-11 and blaGES-12. Cloning of the blaGES genes in Escherichiacoli identified GES-14 as hydrolyzing imipenem, while GES-12 showed the highest specific activity against ceftazidime. This report highlights the emergence of various blaGES-like genes, especially those conferring carbapenem resistance in A. baumannii and its importation in Western Europe from Middle Eastern countries.
Acinetobacter baumannii has been recognized over the last 2 decades as an emerging opportunistic pathogen that is associated mostly with life-threatening infections in highly debilitated patients and nosocomial outbreaks (5, 26, 29). In addition to their ability to survive for long-term periods in adverse environmental conditions, these organisms display numerous intrinsic and acquired antimicrobial mechanisms, such as changes in outer membrane proteins, overexpression of endogenous efflux pumps, chromosomal and plasmid-acquired β-lactamases, aminoglycoside-modifying enzymes, and fluoroquinolone resistance, among others (5). The genetic flexibility and high adaptability of this organism have resulted in the rapid and global emergence over the last few years of multidrug-resistant (MDR) A. baumannii strains resistant to most classes of antimicrobial drugs, including carbapenems (10). Carbapenem resistance in A. baumannii is most often mediated by acquired β-lactamases of class D (OXA-23-like, OXA-40-like, and OXA-58-like) and less frequently by metallo-β-lactamases (MBLs) (24). In addition, the low outer membrane permeability and/or the upregulation of intrinsic efflux systems are also contributory factors toward carbapenem resistance (24). Very recently the Ambler class A of the GES and KPC (carbapenemase) types have also been reported for A. baumannii (14, 25).
b la GES genes are usually carried on integrons found in various species, predominantly Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, and these resistance determinants have been reported in several countries in Europe, Asia, South America, and South Africa (18). Sixteen GES variants differing by one to three amino acid residues have been reported (18) (http://lahey.org/studies/other.asp ). GES β-lactamases exhibit extended-spectrum properties, hydrolyzing oxyimino-cephalosporins. However, differences in the substrate spectra have been observed. Variants with either Gly to Asn (GES-2) or Gly to Ser (GES-4, -5, and -6) at Ambler position 170 are of special clinical importance since they exhibit increased carbapenemase activity compared to other GES enzymes (18). In addition GES-4, -5, and -6 also hydrolyze cefoxitin. Also Gly243Ser substitutions as observed with GES-9 expand the hydrolysis spectrum to aztreonam (21). Following the recent report of GES-11 β-lactamase in one A. baumannii isolate in France presenting a Gly243Ala substitution, we describe in this report the emergence of three different variants, including a novel GES variant, GES-14, that were identified during a screening for acquired carbapenemase genes in multidrug-resistant A. baumannii isolates collected from January 2008 to December 2009 from 18 Belgian hospitals.
MATERIALS AND METHODS
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 blaOXA-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 J53AzR 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 blaOXA-51-like, blaOXA-23-like, blaOXA-40-like, and blaOXA-58-like carbapenemases and the MBLs blaIMP and blaVIM as previously described (2). The presence of the insertion element ISAba1 upstream of blaOXA-51-like and blaADC 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 blaGES 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 blaGES 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 blaGES-11, blaGES-12, and blaGES-14 have been assigned to the GenBank nucleotide database under the accession numbers HM622144 (GES-11), HM622145 (GES-12), and GU207844 (GES-14).
RESULTS
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.
MICs of selected antibiotics of GES-positive A. baumannii isolates (A to I) and recipient/electroporant A. baumannii strains
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).
Case history and characteristics of A. baumannii clinical isolates
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).
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 blaGES-like genes and for blaOXA-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 blaOXA genes (OXA-23, OXA-40, and OXA-58 groups) as well as for blaIMP and blaVIM genes. ISAba1 was detected upstream of the blaADC gene in seven isolates but never upstream of the blaOXA-51-like genes (Table 2).
Sequencing of blaGES 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 blaGES genes.Sequence analysis of the close genetic context of blaGES 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 blaGES alleles. Plasmid extraction experiments identified plasmids in blaGES-11- and blaGES-12-expressing A. baumannii (data not shown) but not in blaGES-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 blaGES-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 blaGES-11 and of the blaGES-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.
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.
Resistance profile and specific activity of cloned GES alleles expressed in E. coli TOP10a
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.
DISCUSSION
Whereas acquired resistance to carbapenem in A. baumannii was reported mainly as a consequence of acquired class D carbapenem hydrolyzing β-lactamases and to a lesser extent to class B MBLs (24), class A β-lactamase, such as KPC and GES, were until now essentially detected in Enterobacteriaceae and in P. aeruginosa (13). Very recently, a new GES-11 isoform was detected for the first time in one multidrug-resistant and carbapenem-resistant A. baumannii isolate recovered in France (14). Our data further point out the fact that several GES-expressing A. baumannii strains might now be emerging independently in different areas in the world and indicate that, along with KPC-type β-lactamases (that were also recently reported in an Acinetobacter isolate from Puerto Rico [25]), class A β-lactamases may now account as an additional mechanism of resistance to carbapenems in A. baumannii.
Three different GES isoforms were detected in our study: GES-11, -12, and -14, with GES-12 and GES-14 differing from GES-11 by a single amino acid substitution. The GES-11- and GES-12-associated plasmids, although displaying very similar banding patterns by restriction endonuclease analysis, were found in isolates with different PFGE types, suggesting horizontal plasmid transfer between different A. baumannii isolates. While GES-producing isolates were already discovered in Enterobacteriaceae isolates in Belgian hospitals (P. Bogaerts, unpublished data), their genetic contexts (aacA4blaGES-likesmrdhfrIaadA1), as well as the GES isoforms of the A. baumannii isolates found here, were clearly different from those previously observed with Enterobacteriaceae isolates, thus ruling out the horizontal transfer of blaGES-harboring plasmids from the Enterobacteriaceae to A. baumannii occurring in Belgian hospitals.
Moreover, the fact that all GES-producing A. baumannii isolates were recovered over a short period of time from patients transferred from different countries suggests that such isolates might be more widespread than it is currently appreciated.
GES-11, GES-9, and GES-1 differ by one single amino acid substitution at Ambler position 243 (Gly243Ser for GES-9 or Gly243Ala in GES-11), a position known to be involved in the extension of the hydrolysis spectrum to aztreonam (21). GES-11 and GES-9 display a higher hydrolytic activity against ceftazidime and aztreonam than GES-1, but they do not hydrolyze cefoxitin or imipenem, confirming the involvement of Ser and Ala243 in the hydrolysis of aztreonam and ceftazidime.
GES-12 differs from GES-11 by a single Thr237Ala substitution, increasing significantly the levels of hydrolysis of ceftazidime and of aztreonam and hence rendering GES-12 a very potent ceftazidimase. This mutation was observed for the first time with the GES-type enzyme. Nevertheless in another class A β-lactamase type (CTX-M-4), the position 237 was described as modifying the hydrolysis spectrum of the enzyme (7) and hence is suspected to participate in the shape of the catalytic pocket of the class A enzyme. Although both GES-11 and GES-12 possess only a very weak hydrolytic activity against imipenem (with a level of hydrolysis comparable to that of GES-1 and of GES-9), it seems sufficient to alter the carbapenem susceptibility in A. baumannii as observed with the transformants, and it may represent an additional mechanism of resistance in this organism.
Finally, GES-14 also differs from GES-11 by a single mutation, Gly170Ser, which results in a marked (almost 10-fold) increase in the level of hydrolysis of imipenem and of cefoxitin, making GES-14 the first true GES carbapenemase recovered in A. baumannii. A single Gly170Ser substitution was previously shown to be responsible for the expansion of the breadth of hydrolytic activity to carbapenem and to cefoxitin in GES-5 (28). It was recently demonstrated by Frase et al. that this Gly170 substitution in GES enzymes had a major impact on the turnover of imipenem during the catalytic cycle through a more than 5,000-fold enhancement in the rate of acetylation (6).
The expected increase of aztreonam hydrolysis consecutively to mutation Gly243Ala at position 243 is not observed in GES-14 possibly because of the counter effect of Ser170, which also diminishes the hydrolysis efficiency of GES-5 and GES-14 against ceftazidime.
The analysis of hydrolysis activity of the GES-expressing E. coli extracts confirmed that only GES-14 harbored a carbapenemase activity. For clinical isolates expressing GES-11 and GES-12, the observed resistance to carbapenem is probably due to additional mechanisms, such as impermeability (5) combined with ADC overexpression due to ISAba1. This hypothesis is further suggested by the observation that the GES-11 expressing isolates harboring the insertion sequence ISAba1 upstream of blaADC (isolates from patients A and C) present a higher level of resistance to carbapenems than those which do not overexpress ADC (isolates from patients B and I). The involvement of OXA-51-like enzymes in carbapenem-resistance could be ruled out since ISAba1 was not detected upstream to these genes.
In conclusion, our results suggest that GES β-lactamases might now be emerging in A. baumannii. The emergence of carbapenem-hydrolyzing enzymes, such as GES-14, highlights the propensity of A. baumannii to acquire new resistance mechanisms, and it further underlines the absolute necessity to control inpatients transferred from hospitals in foreign countries known to have a high prevalence of resistance mechanisms.
ACKNOWLEDGMENTS
This work was funded by INSERM, by a grant from the Ministère de l'Education Nationale et de la Recherche (UPRES-EA3539), Université Paris XI, Paris, by the Assistance Publique-Hôpitaux de Paris, France, and by the European Community (7th PCRD, TROCAR contract HEALTH-F3-2008-223031, and 7th PRCD TEMPOtest-QC contract HEALTH-2009-2.3.1.3 no.° 241742).
We thank R. Cartuyvels (Virga Jesse Hospital, Hasselt), H. Rodriguez-Villalobos (Erasme University hospital, Brussels), and A. de Moreau (Bracops hospital, Brussels) for providing strains and Warda Bouchahrouf for excellent technical assistance.
FOOTNOTES
- Received 25 June 2010.
- Returned for modification 6 August 2010.
- Accepted 20 August 2010.
- Copyright © 2010 American Society for Microbiology