New carbenicillin-hydrolyzing beta-lactamase (CARB-7) from Vibrio cholerae non-O1, non-O139 strains encoded by the VCR region of the V. cholerae genome

Abstract

In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a beta-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for bla(TEM) or primers for bla(CARB) gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the beta-lactamase was determined by bidirectional sequencing of a Sau3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this beta-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the bla(CARB-7) gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the bla(CARB-7) gene shared 93% identity with a locus situated inside V. cholerae's chromosome 2. These results strongly suggest the chromosomal location of the bla(CARB-7) gene, making this the first communication of a beta-lactamase gene located on the VCR island of the V. cholerae genome.

FIG. 1.

Strategy used for sequencing the bla_CARB-7 gene and the DNA flanking regions. (A) Restriction map of the Sau3AI fragment from the ME11762 strain (black box). Only the ending Sau3AI sites are indicated. Grey boxes indicate DNA of pACYC184 vector. (B) The insert of pGMPS1 was liberated by digestion with SalI and EcoRV and was subcloned into SmaI-SalI sites of pBGS19 vector (white boxes), rendering pGMPS2; grey boxes represent approximately 500 bp that belong to pACYC184 vector. Inserts of plasmids derived of pGMPS2 were sequenced. (C) bla_CARB-7 gene and its flanking DNA regions. The sense, length, and primers used for each sequence reaction are indicated by the black arrows. The grey box represents the bla_CARB-7 gene, the white arrow represent its translational orientation, the flanking fragments are depicted as hatched boxes, and VCR are depicted as open boxes. Restriction sites used for subcloning into pBGS19 vector are indicated. Pf and Pr, universal primers (forward and reverse, respectively).

FIG. 2.

Nucleotide sequence of the 2,214-bp fragment of pGMPS2 containing the CARB-7 β-lactamase coding region. The deduced amino acid sequence of the ORF proposed to encode CARB-7 β-lactamase is represented in one-letter code. VCR on the flanking sequences of bla_CARB-7 gene are underlined. The fragment showing high homology with the V. cholerae chromosome is indicated by the grey box (GenBank accession number AE004376 [nucleotides 7944 to 8706]). Restriction sites used for subcloning of bla_CARB-7 gene are double underlined.

FIG. 3.

Alignment of the deduced amino acid sequence of CARB-7 with those of the closely related class A β-lactamases PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4, P. mirabilis N29, and CARB-6. The shadowed boxes (I to VII) correspond to amino acid boxes conserved in all penicillin-recognizing enzymes (12). The arrow indicates amino acid position 139, with the change G→D. Asterisks indicate identical residues. Dots indicate conserved amino acids. Sequences are numbered as described by Ambler et al. (1).

FIG. 4.

Nucleotide sequence of the 123-bp element. (A) Multiple alignment of nucleotide sequence of the three VCR found on the flanking regions of the bla_CARB-7 gene with a proposed VCR consensus sequence (4). Identical bases are indicated by an asterisk. The alignment was generated with the multiple-alignment program CLUSTAL. (B) Proposed secondary structure for each VCR sequence, determined with DNASIS software (version 2.5).