Characterization of VCC-1, a Novel Ambler Class A Carbapenemase from Vibrio cholerae Isolated from Imported Retail Shrimp Sold in Canada

Abstract

One of the core goals of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) is to monitor major meat commodities for antimicrobial resistance. Targeted studies with methodologies based on core surveillance protocols are used to examine other foods, e.g., seafood, for antimicrobial resistance to detect resistances of concern to public health. Here we report the discovery of a novel Ambler class A carbapenemase that was identified in a nontoxigenic strain of Vibrio cholerae (N14-02106) isolated from shrimp that was sold for human consumption in Canada. V. cholerae N14-02106 was resistant to penicillins, carbapenems, and monobactam antibiotics; however, PCR did not detect common β-lactamases. Bioinformatic analysis of the whole-genome sequence of V. cholerae N14-02106 revealed on the large chromosome a novel carbapenemase (referred to here as VCC-1, for Vibrio cholerae carbapenemase 1) with sequence similarity to class A enzymes. Two copies of blaVCC-1 separated and flanked by ISVch9 (i.e., 3 copies of ISVch9) were found in an acquired 8.5-kb region inserted into a VrgG family protein gene. Cloned blaVCC-1 conferred a β-lactam resistance profile similar to that in V. cholerae N14-02106 when it was transformed into a susceptible laboratory strain of Escherichia coli. Purified VCC-1 was found to hydrolyze penicillins, 1st-generation cephalosporins, aztreonam, and carbapenems, whereas 2nd- and 3rd-generation cephalosporins were poor substrates. Using nitrocefin as a reporter substrate, VCC-1 was moderately inhibited by clavulanic acid and tazobactam but not EDTA. In this report, we present the discovery of a novel class A carbapenemase from the food supply.

FIG 1

Schematic map of the region containing bla_VCC-1 on the chromosome of V. cholerae N14-02106 (GenBank accession number KT818596). Block arrows, the direction of transcription of the genes; H, hypothetical protein. The sequences of the 5 bp immediately flanking each terminal inverted repeat of the three copies of ISVch9 are shown. The putative direct repeats of TAA flanking the acquired region are indicated by small arrows within the 5-bp sequences.

FIG 2

Dendrogram of 18 representative class A β-lactamases using precursor amino acid sequences. The alignment and neighbor-joining tree were produced using the Clustal Omega program from EMBL-EBI bioinformatics services (http://www.ebi.ac.uk/Tools/msa/clustalo/), and the tree was visualized using the FigTree (v1.4.2) viewer (http://tree.bio.ed.ac.uk/software/figtree/). Branch lengths are scaled to the number of amino acid changes per site. The β-lactamases (GenBank accession numbers) are as follows: BEL-1 (DQ089809), BES-1 (AF234999), BIC-1 (GQ260093), BKC-1 (KP689347), CARB-1 (D86225), CTX-M-1 (X92506), FRI-1 (KT192551) GES-1 (AF156486), IMI-1 (U50278), KPC-2 (AY034847), LAP-1 (EF026092), NMC-A (Z21956), PER-1 (Z21957), SFC-1 (AY354402), SHV-1 (AF148850), SME-1 (Z28968), TEM-1 (AY458016), VEB-1 (AF010416), and VCC-1 (KT818596).

FIG 3

Amino acid sequence alignment of VCC-1 with the sequences of previously described carbapenemases. Signature motifs of β-lactamases are indicated in bold. The sequence numbering follows the Ambler scheme for class A β-lactamases. GenBank accession numbers are listed in the legend to Fig. 2.