Detection of VIM-34, a novel VIM-1 variant identified in the intercontinental ST15 Klebsiella pneumoniae clone.

Sir,

Enterobacteriaceae producing metallo-β-lactamases (MBLs), and particularly VIM-type MBLs, have frequently been implicated in hospital outbreaks across Europe,[1]blaVIM genes having been linked to Tn402 derivatives, epidemic plasmids (IncN, IncI1, IncHI2) and occasionally with particular Enterobacteriaceae clones.[1-4] VIM enzymes have been classified in three clusters (VIM-1, VIM-2 and VIM-7) according to their amino acid sequences (http://www.lahey.org/studies), VIM-1 and VIM-2 being the most widespread variants.[5] In this study, we report the molecular epidemiology and the antibiotic susceptibility profiles of Klebsiella pneumoniae clinical isolates producing VIM-34, a novel VIM-1 variant identified in Portugal.

In October 2011 and October 2012, two K. pneumoniae isolates (strains K43 and K47, respectively) showing reduced susceptibility to carbapenems (MICs 0.38–1.0 mg/L) were recovered from urine samples of hospitalized patients in a general hospital in northern Portugal (Hospital Pedro Hispano). They are the only carbapenemase-producing Enterobacteriaceae isolates identified in this hospital since the beginning of 2011, when reference protocols for carbapenemase detection were adopted.

Antimicrobial susceptibility tests were performed using the Etest for β-lactams and disc diffusion for all other antimicrobial agents. These showed that all isolates were resistant to diverse cephalosporins, aztreonam, β-lactam/β-lactamase inhibitor combinations (Table 1), nalidixic acid, ciprofloxacin, chloramphenicol, gentamicin, kanamycin, netilmicin, streptomycin, tobramycin and sulphonamides, but susceptible to trimethoprim and amikacin (http://www.eucast.org/).[6] Standard disc diffusion phenotypic tests using different β-lactams and β-lactamase inhibitors (cefotaxime, ceftazidime, imipenem; 0.2 mM EDTA, clavulanic acid),[6] isoelectric focusing, PCR and sequencing[7] demonstrated the production of VIM-34 (pI = 5.4) (GenBank accession number JX013656), a novel VIM-type enzyme differing from VIM-1 by one amino acid change (V113I, according to MBL standard numbering scheme) and co-production of SHV-1 (pI = 7.6) and SHV-12 (pI = 8.2) extended-spectrum β-lactamase. We could not identify the origin of these isolates but as both patients had multiple previous hospitalizations (including in other hospitals) and carried the same novel blaVIM type, a common nosocomial source seems more plausible than community acquisition.

Table 1.

MICs of different β-lactam antibiotics for VIM-34-producing wild-type isolates and recombinant strains encoding VIM-34 or VIM-1

Antibiotic	MIC (mg/L)
	Klebsiella pneumoniae K43 (VIM-34)a	E. coli DH5α
		pBGS18	pBGS18/VIM-1	pBGS18/VIM-34
Amoxicillin/clavulanate	24	2	24	12
Ticarcillin/clavulanate	>256	2	>256	>256
Piperacillin/tazobactam	>256	0.75	6	6
Cefalotin	>256	2	64	32
Ceftazidime	>256	0.19	12	8
Cefotaxime	32	0.125	2	2
Cefepime	12	0.016	0.75	0.5
Cefpirome	32	0.032	1.5	2
Cefoxitin	32	4	12	4
Aztreonam	32	0.023	0.023	0.016
Ertapenem	0.38b	0.006	0.008	0.008
Imipenem	1.0b	0.125	0.38	0.38
Meropenem	0.5b	0.016	0.032	0.023

aK47 isolate exhibited identical antibiotic susceptibility profiles.

bMIC values interpreted as susceptible by both EUCAST and CLSI guidelines, but above the epidemiological cut-off values defined for K. pneumoniae (http://www.eucast.org).[6]

The blaVIM-34 from the K47 isolate was cloned in the pBGS18 (kanamycin resistance) plasmid using primers VIM-EcoRI (5′-GGGAATTCGCAGTCGCCCTAAAACAAAG-3′) and VIM-PstI (5′-AACTGCAGCCGCTCCAACGATTTGTTAT-3′) (restriction sites are underlined), and the expression vector (pBGS18/VIM-34) was further introduced into Escherichia coli DH5α, as previously reported.[8] MICs of different β-lactams were determined using the Etest (in triplicate) and compared with those corresponding to a blaVIM-1-carrying clone obtained in the same conditions (Table 1). The VIM-34-producing E. coli recombinant yielded β-lactam MIC values similar to those observed in the VIM-1-encoding transformant (with the exception of cefoxitin; Table 1). Because our experiments were performed in an isogenic context and identical standard experimental conditions, we are able to hypothesize that the substitution V113I has a low influence on the MICs of carbapenems, although further studies of enzymatic activity are required to confirm this observation.

The isolates exhibited identical XbaI-PFGE profiles and clonal identification by multilocus sequence typing (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) revealed that they belong to the intercontinental ST15 K. pneumoniae clone, widely disseminated in different European countries and associated with the spread of extended-spectrum β-lactamases (CTX-M-15; diverse SHV types) and/or MBLs (VIM-1, NDM-1).[2,3,7,9,10] Conjugation assays performed by broth and/or filter mating methods using E. coli HB101 (azide and kanamycin resistant, Lac-, plasmid free) as recipient at 22°C and 37°C (selection of transconjugants in MacConkey agar with 2 mg/L of ceftazidime and 130 mg/L of azide)[7] failed to yield transconjugants either for blaVIM-34 or blaSHV-12. The location of bla (blaVIM-34, blaSHV-12) genes and plasmid characterization were accomplished by S1- and I-CeuI-PFGE, and identification of incompatibility groups.[7] In both isolates, blaVIM-34, blaSHV-12 and repHI2 probes hybridized in the same chromosomal band (I-CeuI-PFGE) whereas no signals were observed in the S1 gel, suggesting the acquisition of both bla genes by an IncHI2 plasmid and subsequent plasmid (whole or in part) integration. A chromosomal location for bla genes, including blaVIM, has been occasionally observed in different Enterobacteriaceae species.[1]

The linkage of blaVIM-34 to class 1 integrons and Tn402 derivatives was investigated by PCR (intI1, 5′CS-3′CS region, orf5, orf6, IS1326, IS1353, IS6100) and sequencing.[4,11]blaVIM-34 was located within an ∼6 kb class 1 integron named In817 by INTEGRALL (http://integrall.bio.ua.pt/) (GenBank accession number JX185132), with an original array of gene cassettes comprising blaVIM-34, aacA4′, aphA15, aadA1b and catB2 (Figure S1; available as Supplementary data at ). The absence of tni402 sequences and the high similarity detected with In70 and In113, identified in VIM-1-producing Achromobacter xylosoxidans, K. pneumoniae and E. coli isolates, suggests that the In817 integron might have arisen by both recombination and in vivo evolution events (Figure S1; available as Supplementary data at ).[4]

In summary, we present the first report of VIM-34, a VIM-1-like variant embedded in the novel integron type In817 on the chromosome of the intercontinental ST15 K. pneumoniae clone, associated with carbapenem susceptibility profiles similar to those observed for VIM-1. This study highlights the risk of further dissemination of the multidrug-resistant ST15 K. pneumoniae clone and genetic backgrounds containing metallo-β-lactamase genes in our country, which deserves future monitoring.

Funding

This study was supported by Fundação para a Ciência e Tecnologia, which is part of the Ministry of Science, Technology and Innovation of Portugal (through grants no. PEst-C/EQB/LA0006/2011, PTDC/AAC-AMB/103386/2008, EXPL/DTP-EPI/0196/2012 and FCOMP-01-0124-FEDER-027745), Fundação Ensino e Cultura Fernando Pessoa, and an ESCMID Research Grant 2012 awarded to Ângela Novais. Carla Rodrigues was supported by Fundação para a Ciência e Tecnologia through grant no. SFRH/BD/84341/2012. Ângela Novais was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (PIEF-GA-2009-255512).

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