Identification of a Stable Chromosomal Tandem Multicopy of blaVIM-63, a New blaVIM-2 Carbapenemase

Abstract

This study characterizes a new genetic structure containing a multicopy of a bla_VIM-2 variant with an A676C substitution, bla_VIM-63. This gene was detected on the chromosome of two carbapenem-resistant clinical strains of Citrobacter freundii ST22 recovered from two patients, separated by a 6-month period, and previously in Pseudomonas aeruginosa ST2242 from the same hospital unit. Short-read sequencing was used to characterize the new variant in both species, and long-read sequencing was used to characterize the genome of C. freundii. On the P. aeruginosa chromosome, the bla_VIM-63 gene was inserted between ISPsy 42-type sequences, flanked by an intl1 sequence, nearby aph(3')-VI, and sul1. On the C. freundii chromosome, the bla_VIM-63 gene was inserted into a Tn6230-like transposon as a stable five-tandem-repeat multimer, flanked by the same intl1 as in P. aeruginosa. This structure was stable across subcultures and did not change in the presence of carbapenems. The bla_VIM-63 gene was cloned into the pCR-Blunt plasmid to study antimicrobial susceptibility patterns and into pET29a for kinetic activity analysis. VIM-63 showed higher K_m values than VIM-2 for ceftazidime and cefepime and higher k_cat values for cefotaxime, ceftazidime, imipenem, and ertapenem, without differences in MIC values. This is the first study to describe this new variant, VIM-63, in two different species with a chromosomal location integrated into different mobile elements and the first to describe a stable multimer of a metallo-β-lactamase. Despite the amino acid substitution, the susceptibility pattern of the new variant was similar to that of VIM-2. IMPORTANCE VIM group metallo-β-lactamases are usually captured by IntI1 integrases. This work describes the detection for the first time of a novel, previously unknown variant of VIM-2, VIM-63. This carbapenemase has been found on the chromosome of two different species, Citrobacter freundii and Pseudomonas aeruginosa, from the same hospital. The adjacent genetic environment of the bla_VIM-63 gene would indicate that the capture of this gene by IntI1 has occurred in two different genetic events in each of the species, and in one there has been a stable integration of tandem copies of this gene.

Keywords: VIM-2 variant; VIM-63; WGS; carbapenemase; multimer.

FIG 1

Schematic map of the two genetic environments of bla_VIM-63. (A) Homology between the genome of Pseudomonas aeruginosa 20190031 and Citrobacter freundii 20200334, obtained using EasyFig v2.2.5. The bla_VIM-63 gene is shown in red, intl1 is shown in green, the resistance genes are shown in light blue, genes associated with resistance to metals are shown in dark blue, the integration sequences are shown in pink, and the antitoxin-toxin system is shown in yellow. (B) Comparison of the promoter region. Pc, P2, and PintI1 promoters are indicated by horizontal brackets, and the −35 and −10 promoter elements are boxed. The integrase and the bla_VIM-63 start codons are bold and colored in green and red, respectively.

FIG 2

Three-dimensional structure of VIM proteins obtained using the Swiss-Model platform (34, 35).