Mechanisms of Resistance

Detection and Characterization of VIM-31, a New Variant of VIM-2 with Tyr224His and His252Arg Mutations, in a Clinical Isolate of Enterobacter cloacae

Pierre Bogaerts, Carine Bebrone, Te-Din Huang, Warda Bouchahrouf, Yves DeGheldre, Ariane Deplano, Kurt Hoffmann, Youri Glupczynski
DOI: 10.1128/AAC.06249-11

ABSTRACT

We report the first description of the metallo-β-lactamase VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in Enterobacter cloacae 11236, which was isolated from blood specimens of a patient with colonic adenocarcinoma in Belgium. blaVIM-31 was found on a class 1 integron located on a self-transferable but not typeable 42-kb plasmid. Compared to values published elsewhere for VIM-2, the purified VIM-31 enzyme showed weaker catalytic efficiency against all the tested beta-lactam agents (except for ertapenem), resulting from lower kcat (except for ertapenem) and higher Km values for VIM-31.

INTRODUCTION

The worldwide spread of metallo-β-lactamases (i.e., VIM, IMP, and NDM enzymes) in carbapenem-resistant Gram-negative bacteria presently constitutes a major public health issue.

So far, 33 distinct blaVIM alleles have been described (www.lahey.org/Studies) for a variety of Gram-negative opportunistic pathogens, but only VIM-1, VIM-2, VIM-4, VIM-7, and VIM-27 enzymes (7, 12, 15, 22, 31) have been characterized biochemically and structurally. While VIM-1-derived enzymes have been reported largely for Enterobacteriaceae from around the world, especially from Greece but also recently from Belgium (4), VIM-2 has been associated mostly with Pseudomonas aeruginosa (32). The occurrence of VIM-2 has nevertheless also been reported for clinical isolates of Enterobacteriaceae from Asia (17, 24, 39), Mexico (28), Argentina (16), and Tunisia (10). Moreover, a recent report showed the presence of VIM-2 in Enterobacter ludwigii isolated from sewage water of a hospital, highlighting the involvement of environmental bacteria as potential reservoirs for the dissemination of clinically relevant metallo-β-lactamase resistance genes (37). In many subclass B1 (e.g., IMP and NDM) and B2 (e.g., CphA) metallo-β-lactamases, the amino acid at position 224 (a conserved Lys residue), close to the active site, plays an important role in substrate binding (2, 27). In the case of VIM enzymes, Lys224 is not present and is replaced by His224 in VIM-1 and by Tyr224 in VIM-2. The side chains of histidine and tyrosine residues are much shorter than that of a lysine residue, thereby preventing the interaction of these two amino acids with the carboxylate moiety (C4 or C3) of the substrate. For VIM enzymes, it is hypothesized that Arg228, with its long side chain, may replace Lys224 in interacting with the substrate (15, 22).

We report here the detection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in an Enterobacter cloacae clinical isolate recovered from an elderly patient in Belgium.

MATERIALS AND METHODS

Bacterial strains and antimicrobial susceptibility.Enterobacter cloacae 11236 was recovered in June 2011 from blood culture specimens from a patient hospitalized in Brussels, Belgium. The isolate was identified to the species level by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) with a Microflex LT mass spectrometer (Bruker Daltonik GmbH, Leipzig, Germany). Susceptibility to antimicrobials was determined by the disc diffusion method, and MICs of antimicrobial agents were determined by Etest (bioMérieux, Marcy l'Etoile, France) or broth microdilution (Sensititre; Trek Diagnostic Systems, Cleveland, OH) according to CLSI guidelines (11). EDTA-disc synergy tests were performed for the screening of metallo-β-lactamase production (4).

PCR detection of bla genes and sequencing.Detection of extended-spectrum β-lactamase (ESBL) genes (blaTEM,SHV, blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-9-like, and blaCTX-M-8/25/26-like) and of carbapenemase genes (blaVIM, blaIMP, blaNDM-1, blaOXA-48, and blaKPC) was performed by DNA microarray analysis (Check-MDR CT102; Check-Points, Wageningen, The Netherlands) according to a previously described protocol (30). blaOXA-1-like, blaOXA-2-like, and blaOXA-10-like genes, genes encoding class A ESBL enzymes (BEL, GES, VEB, and PER), and qnr genes, involved in quinolone resistance, were detected by PCR as previously described (3, 6). The genetic context of blaVIM-31 was assessed by PCR amplification and sequencing of the variable region of the integron by using primers designed on the basis of the 5′ and 3′ conserved segments of the class 1 integron (25), using an external sequencing service (Macrogen, Seoul, South Korea). Nucleotide sequences were analyzed with the BLASTN algorithm, available from the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/).

Molecular typing.Pulsed-field gel electrophoresis (PFGE) analysis of XbaI-digested genomic DNA was used to examine the genetic relatedness of E. cloacae 11236 to other E. cloacae isolates known to be epidemic in several Belgian hospitals (A. Deplano, personal data). PFGE patterns were analyzed with BioNumerics software (Applied Maths, Kortrijk, Belgium), using the Dice similarity coefficient and the unweighted-pair group method using average linkages (UPGMA) as previously described (5). The PFGE classification criteria were described previously, and similar PFGE types included patterns showing differences of 0 to 6 DNA fragments (38).

Plasmid analysis, mating assay, and electroporation experiments.Plasmid DNA was extracted by the Kieser method (18) or by use of a QIAfilter Plasmid Midi kit (Qiagen, Venlo, The Netherlands). Escherichia coli NCTC50192, harboring four plasmids, of 154, 66, 48, and 7 kb, was used as a plasmid size marker. Plasmid DNA was analyzed by 0.7% agarose gel electrophoresis. Determination of the incompatibility groups of plasmids was done as described by Carattoli et al. (8). Transfer of the β-lactamase genes from E. cloacae 11236 to Escherichia coli J53 (azide resistant) and Pseudomonas aeruginosa PU21 (rifampin resistant) was attempted by solid and liquid mating assays at 37°C (13). Selection was performed on brain heart infusion (BHI) agar plates supplemented with ticarcillin at 100 μg/ml and azide at 150 μg/ml for E. coli and with ticarcillin at 100 μg/ml and rifampin at 100 μg/ml for P. aeruginosa. Moreover, plasmid DNA extracts from E. cloacae 11236 were electroporated into E. coli TOP10 cells (Invitrogen, Merelbeke, Belgium) by use of a Gene Pulser II apparatus (Bio-Rad, Marnes-la-Coquette, France) as previously described (1, 9), and transformants were selected on BHI agar plates containing 100 μg/ml of ticarcillin. Electroporation of a plasmidic extract of E. cloacae 11236 into Pseudomonas aeruginosa PAO1 was also performed as previously described (13), and transformants were selected on BHI agar plates containing 100 μg/ml of ticarcillin or 20 μg/ml of tobramycin.

Cloning of blaVIM-31.The blaVIM gene was amplified with HotStarTaq DNA polymerase (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions, using primers 5′-CATGCCATGGTCAAACTTTTGAGTAAGTTATTG-3′ and 5′-CTGAATTCCTACTCAACGACTGAGCGATTTGTG-3′, designed to add NcoI and EcoRI restriction sites (underlined in the sequences) upstream and downstream of the VIM-31 gene, respectively. The PCR product was then cloned into pET-28a (Novagen, Madison, WI), and the expression vector construct, named pET-28a/VIM-31, was introduced into E. coli BL21(DE3) (New England BioLabs, Ipswich, MA) by electroporation. The construct was verified by PCR and sequencing with primers targeting the T7 RNA polymerase promoter and terminator.

Production and purification.pET-28a/VIM-31-expressing E. coli was grown overnight at 37°C with shaking in 50 ml LB medium supplemented with 50 μg/ml kanamycin. The bacterial suspension was diluted 100-fold in a total of 2 liters of Terrific Broth supplemented with kanamycin (50 μg/ml), and the expression of VIM-31 was induced with isopropyl-β-d-thiogalactopyranoside (final concentration, 1 mM) when the culture reached an A600 of 0.6. The induced culture was incubated overnight at 28°C with shaking.

VIM-31 was purified by ion-exchange chromatography and gel filtration. Briefly, the bacterial suspension was pelleted, resuspended in 60 ml of buffer A (50 mM HEPES, pH 7.5, 50 μM ZnCl2), disrupted by sonication, and cleared by ultracentrifugation. The supernatant was then dialyzed against buffer A and loaded onto a Q-Sepharose FF column equilibrated with the same buffer. The proteins were eluted with a linear NaCl gradient (0 to 1 M). Fractions were analyzed by SDS-PAGE and by the ability to hydrolyze nitrocefin. Those fractions containing β-lactamase activity were pooled and dialyzed against buffer A overnight at 4°C. This partially purified enzyme was loaded onto a MonoQ column (GE Healthcare, Diegem, Belgium) equilibrated with the same buffer. The proteins were eluted with a linear gradient of NaCl (0 to 0.5 M). The fractions containing β-lactamase activity were pooled and concentrated to a volume of 2 ml. Size-exclusion chromatography was performed on a Hiload 16/60 Superdex 75 preparation-grade column (GE Healthcare, Diegem, Belgium) equilibrated with buffer A supplemented with 250 mM NaCl. Finally, fractions containing the highest β-lactamase activities were pooled and subsequently dialyzed overnight at 4°C against buffer A. The VIM-31 enzyme was >95% pure as judged by SDS-PAGE (data not shown).

Determination of kinetic parameters.All experiments were performed at 30°C in buffer A in the presence of 50 μM Zn2+ (22). Imipenem and ertapenem were obtained from Merck Sharp and Dohme Research Laboratories (Rahway, NJ). Ampicillin, benzylpenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, cefepime, meropenem, and aztreonam were obtained from Sigma-Aldrich (Steinheim, Germany). Nitrocefin was a gift from ProGenosis (Liége, Belgium). Hydrolysis of the antibiotics was monitored by following the absorbance variation resulting from the opening of the β-lactam ring, using a Uvikon 943 spectrophotometer equipped with thermostatically controlled cells. The wavelengths and extinction coefficients used were those previously reported (21, 33) for all drugs tested except for ertapenem (36). The kcat and Km parameters were determined under initial rate conditions, using the Hanes linearization of the Henri-Michaelis-Menten equation.

Homology model.A homology model of VIM-31 was constructed using the EsyPred3D server (www.fundp.ac.be/urbm/bioinfo/esypred), based on the structure of reduced VIM-2 (Protein Data Bank [PDB] accession number 1KO3).

Nucleotide sequence accession number.The nucleotide sequence data reported in this paper have been assigned to the EMBL/GenBank nucleotide sequence database under accession number JN982330. VIM numbering was assigned according to the method of the Lahey clinic.

RESULTS AND DISCUSSION

Clinical isolate, susceptibility profile, and patient outcome.In June 2011, an 87-year-old female underwent a left hemicolectomy for intestinal adenocarcinoma. Her immediate postoperative status was complicated by the occurrence of a deep surgical site infection, for which she received a 2-week course of antimicrobial treatment, first comprising piperacillin-tazobactam and then comprising meropenem. Six days after the end therapy, she developed a peritonitis caused by a carbapenem-resistant Enterobacter cloacae isolate (E. cloacae 11236) as suspected by Vitek2 (meropenem MIC of >16 mg/liter and imipenem MIC of 4 mg/liter [data not shown]).

Etest MIC determination (Table 1) confirmed that E. cloacae 11236 was intermediately susceptible to imipenem (MIC = 3 μg/ml) and resistant to ertapenem (MIC = 6 μg/ml), but it was susceptible to meropenem (MIC = 0.5 μg/ml) and also to aztreonam, tigecycline, amikacin, and ciprofloxacin. The presence of a metallo-β-lactamase in this isolate was suspected by a positive double-disc synergy test with imipenem and EDTA.

View this table:
Table 1

Etest or microdilution MICs of E. cloacae 11236, E. coli J53 (ECJ53) and E. coli BL21 (ECBL21) acceptor strains, a transconjugant, and VIM-2- and VIM-31-expressing clones

The patient gradually improved following a 9-day course of treatment with ciprofloxacin (400 mg twice daily [BID] intravenously for 3 days, followed by 500 mg BID orally for 6 days), but she remained persistently colonized with E. cloacae 11236 in the urine for several weeks and returned home.

β-Lactamase genes, genetic support transfer, and typing.Analysis of β-lactamase-encoding genes detected the presence of blaVIM in addition to blaCTX-M of group 9. PCR amplification and sequencing of the blaVIM-bearing integron (In669 [GenBank accession no. JN982330]; the integron number was assigned by INTEGRALL, the integron database [29]) revealed the presence of a new blaVIM-31 allele located upstream of the aacA4 allele. The blaVIM/aacA4 structure of In669 was found to match almost perfectly with a VIM-2-harboring integron described for an Acinetobacter sp. isolate in Korea (GenBank accession no. EF125009), except for three single nucleotide polymorphisms (one upstream and two within blaVIM-2), as well as with the first 2 cassettes of In71 in P. aeruginosa TS-832347 (GenBank accession no. AM180753) (20), recovered in Italy. blaVIM-31 differs from blaVIM-2 by two single nucleotide polymorphisms, resulting in Tyr224His and His252Arg mutations according to the class B standard numbering of Garau et al. (14). Additional PCR amplification and sequencing revealed the presence of blaCTX-M-9 and qnrA1 in E. cloacae 11236. Plasmid extracts showed the presence of three plasmids, of ca. 290, 136, and 42 kb, with the latter (p11236) being a blaVIM-31-harboring nontypeable plasmid (8) which could be transferred by electroporation into E. coli TOP10 (not shown) and also by mating experiments into E. coli J53 (Table 1). Despite several attempts, p11236 could not be transferred to P. aeruginosa PAO1 or PU21, suggesting that it was not self-replicative in P. aeruginosa. Molecular detection of the resistance genes in the E. coli transformant (E. coli TOP10/p11236) and transconjugant (E. coli J53/p11236) confirmed that blaVIM-31 was not located on the same plasmid as the qnrA1 and blaCTX-M-9 genes (data not shown). The VIM-31-expressing E. coli J53 transconjugant displayed lower MICs, especially for cephalosporins, monobactam, and carbapenems (Table 1). Regarding carbapenems, this suggests that reduced outer membrane permeability could also contribute to decreased susceptibility or to resistance to carbapenems in this clinical isolate, as already demonstrated (23). According to PFGE analysis, E. cloacae 11236 corresponds to a sporadic isolate and does not cluster with any of the major CTX-M-9-producing E. cloacae epidemic clones identified previously in Belgian hospitals (35; data not shown), suggesting the independent acquisition of VIM-31 by E. cloacae 11236.

VIM-expressing clones and kinetics.MICs of VIM-31- and VIM-2/pET-28a-expressing E. coli BL21 (Table 1) for various antibiotics were not significantly different from each other, with the single exception of ceftazidime (MIC = 2 μg/ml for E. coli BL21/pET-28a/VIM-2 and MIC = 0.5 μg/ml for E. coli BL21/pET-28a/VIM-31).

Under the experimental conditions employed, purified VIM-31 (calculated pI = 5.00; molecular mass = 25,069 Da) hydrolyzed all compounds tested except for aztreonam. The individual kinetic parameters (kcat and Km) of VIM-31 with several β-lactam substrates and a comparison with those of VIM-2 are reported in Table 2.

View this table:
Table 2

Kinetic parameters of VIM-31 and VIM-2 enzymes

The highest catalytic efficiencies for the VIM-31 enzyme were observed for cefotaxime, cephalothin, nitrocefin, and imipenem, while ceftazidime, cefepime, and cefoxitin were rather poor substrates. With carbapenems, the kcat/Km ratios ranged from 67 mM−1 s−1 (ertapenem) to 2,400 mM−1 s−1 (imipenem), resulting from a combination of low Km values and very low turnover rates, a characteristic behavior of the VIM-type enzymes. Of the three carbapenems tested, VIM-31 showed greater hydrolytic efficiency against imipenem (Table 2), a result also reported for VIM-2 by Docquier et al. (12) and Samuelsen et al. (36).

VIM-2 had a higher catalytic efficiency than its His224-possessing VIM-31 variant for all substrates tested, with the exception of ertapenem, with the ratio of kcat/Km for VIM-2 to kcat/Km for VIM-31 ranging from 1.6 (ampicillin and imipenem) to 54 (cefoxitin) (Table 2). For each individual substrate, the kcat values determined for VIM-31 were lower than those for VIM-2 (only 1.1-fold lower for cefotaxime but up to 9-fold lower for cefoxitin). On the other hand, the Km values for VIM-31 were increased compared to those for VIM-2, with the exception of those for ampicillin, imipenem, and meropenem (12). VIM-31 was slightly more efficient than VIM-2 for the hydrolysis of ertapenem, with the kcat value determined for VIM-31 being 3-fold higher than that obtained for VIM-2 by Samuelsen et al. (36) and with their Km values remaining similar.

Positions of the mutations.A homology model of VIM-31 (Fig. 1) was constructed based on the structure of VIM-2 (PDB accession no. 1KO3). The two single mutations (Tyr224His and His252Arg) observed between VIM-31 and VIM-2 are both situated in the C-terminal part of the protein. The Tyr224His substitution, also present in VIM-1, VIM-4 (22), VIM-7 (7, 36), and VIM-12 (19), is located close to the active site and is situated on the L3 loop (Fig. 1). Tyr224 in VIM-2 was previously suggested to enhance the binding affinity of certain substrates, such as ceftazidime (12, 15), for which VIM-2 exhibits a lower Km value than that for VIM-1 (His224) (the Km values were 800 and 72 μM for VIM-1 and VIM-2, respectively). In line with these findings, VIM-2 also exhibits higher affinity than its His224-possessing variant for this substrate (the Km value of VIM-31 is increased 7-fold compared to that of VIM-2). The His252Arg substitution is situated on the α4 helix. This residue is distant from the active site of the VIM enzyme (more than 22 Å away), and its side chain points outside the protein surface, toward the external solvent. This renders unlikely any interaction of Arg252 with catalytically important residues of the protein or with the substrates of the enzyme. Nevertheless, we cannot totally exclude that the His252Arg mutation may also contribute to tuning the metallo-β-lactamase activity. Indeed, observations made previously for VIM-11 and VIM-19 indicate that amino acid substitutions located outside the active site (Asn165Ser compared to the VIM-2 sequence and Asn215Lys compared to the VIM-1 sequence, respectively) may also modulate the hydrolytic properties of the enzyme (26, 34).

Fig 1
Fig 1

(a) Superposition of VIM-2 and the homology model constructed for VIM-31. Positions showing variation between VIM-2 and VIM-31 (224 and 252) are labeled in gray for VIM-2 and in cyan for VIM-31. (b) VIM-2 active site. Zinc ions and water molecules are shown as gray and red spheres, respectively. Residues are numbered according to the BBL standard numbering scheme (14). Metal-ligand and hydrogen bonds are shown as dashed blue lines. The side chain of His224 in the VIM-31 homology model is shown with carbon atoms rendered in cyan. This figure was rendered using Pymol.

Conclusions.We have characterized VIM-31, a new VIM variant closely related to VIM-2 that was detected in a clinical isolate of E. cloacae in Belgium and located on a new integron, In669.

Comparison of the kinetic parameters of VIM-31 to those previously obtained by other groups for VIM-2 (12, 36) revealed that VIM-31 exhibits globally lower catalytic efficiencies than those of VIM-2 (because of lower kcat and higher Km values). This could be explained mostly by the single Tyr224His point mutation.

From an epidemiological point of view, the low level of resistance of E. cloacae 11236 to carbapenem drugs underscores the difficulties that may be associated with the detection of such resistance mechanisms in clinical practice.

ACKNOWLEDGMENTS

This work was supported by EU grant FP7-HEALTH-2009-SINGLE-STAGE TEMPOtest-QC, project 241742. C.B. is a fellow of the Alexander von Humboldt Foundation (Bonn, Germany).

FOOTNOTES

    • Received 29 November 2011.
    • Returned for modification 7 January 2012.
    • Accepted 19 February 2012.
    • Accepted manuscript posted online 5 March 2012.

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