VIM-47, a New Variant of the Autochthonous Metallo-β-Lactamase VIM-13 from the Balearic Islands in Spain

LETTER

Carbapenemase-producing Pseudomonas aeruginosa is increasing worldwide, linked to wide-spread international high-risk clones (such as ST235, ST111, or ST175), thus representing a major epidemiological and therapeutic challenge (1, 2). Active surveillance of multidrug-resistant (MDR) pathogens, including carbapenemase-producing P. aeruginosa, is routinely performed in the intensive care unit (ICU) of Hospital Son Llàtzer (Palma de Mallorca, Balearic Islands, Spain).

Carbapenemase-producing P. aeruginosa isolates were detected in around 8% of all patients showing P. aeruginosa-positive cultures in 2015 and typically involved either the globally spread metallo-β-lactamase (MBL) VIM-2 or the autochthonous MBL VIM-13. VIM-13 belongs to the VIM-1 cluster, although it shows considerable divergence (93% similarity at the amino acid level) from this enzyme (3). Interestingly, VIM-13-producing P. aeruginosa has been detected in several hospitals on Mallorca, linked to high-risk clone ST235, but it has never been documented outside the Balearic Islands (3, 4). Moreover, analysis of the sewage water of Hospital Son Llàtzer revealed the presence of VIM-13 in multiple environmental species, including Brevundimonas diminuta, Rhizobium radiobacter, Pseudomonas monteilii, and Ochrobactrum anthropi (5).

However, in May 2015, two MDR isolates that were recovered from urine samples and rectal swabs of two ICU patients and were nonsusceptible, according to EUCAST breakpoints (http://eucast.org/), to all of the antibiotics tested (ticarcillin, piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, gentamicin, tobramycin, amikacin, and ciprofloxacin) except colistin showed a positive MBL phenotype (positive double-disk synergy test with carbapenems and EDTA) but were negative in PCR assays directed against the bla_VIM-1 and bla_VIM-2 clusters (6). However, a multiplex carbapenemase PCR (7) yielded a positive result for bla_VIM-like genes. Thus, we performed a class 1 integron PCR assay, followed by DNA sequencing (6), to detect potential VIM gene cassettes, and the results are shown in Fig. 1. Indeed, a class 1 integron containing a bla gene 99% similar to bla_VIM-13 was detected in both cases. However, the gene differed from bla_VIM-13 at two nucleotides (A6C and A13G), both leading to amino acid replacements (L2F and I5V, respectively) of residues conserved in the VIM-1 cluster. Moreover, both nucleotides fell within the forward primer sequence used for amplification of the bla_VIM-1 cluster, and this fact should explain the negative VIM-1 PCR results. The new variant was designated VIM-47. Interestingly, analysis by multilocus sequence typing according to established protocols (http://pubmlst.org/) revealed that one of the isolates belonged to high-risk clone ST235 (like the original VIM-13-producing isolate) (4) but the other one belonged to a different widespread high-risk clone, ST175 (Fig. 1).

FIG 1.

Structures of the class 1 integrons carrying bla_VIM-47 in P. aeruginosa clinical isolates. The VIM-13-producing PA-SL2 strain was characterized previously (3, 4).

Moreover, integron structures differed between the two VIM-47-producing isolates and between either of them and the previously described VIM-13-producing isolates (Fig. 1). To investigate the location of this resistance element (plasmid or chromosome), genomic DNA of both isolates harboring bla_VIM-47 was digested with I-Ceu1, separated by pulsed-field gel electrophoresis, and hybridized with bla_VIM-47 and rRNA probes as described previously (8). The results suggested a chromosomal location of the bla_VIM-47 genes in both strains, given the cohybridization of the MBL and rRNA gene probes (data not shown). Whether the emergence of this new variant is a consequence of random mutation or natural selection remains to be elucidated. Moreover, since both amino acid substitutions are located within the signal peptide (3), the catalytic properties are not expected to be modified. However, the modification of the signal peptide has been shown to significantly affect the fitness of certain β-lactamases (9). Regardless of the effect (if any) of these amino acid replacements, this report illustrates the diagnostic challenge of the continuous evolution of troubling MBLs, as well as their local linkage to widespread international high-risk clones.

Nucleotide sequence accession number.

The sequence of the VIM-47 variant determined in this study has been deposited in GenBank and assigned accession no. KT954134.1.