Identification of a Stable Chromosomal Tandem Multicopy of , a New Carbapenemase.

This study characterizes a new genetic structure containing a multicopy of a variant with an A676C substitution, . 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 gene was inserted between 42-type sequences, flanked by an sequence, nearby , and . On the C. freundii chromosome, the gene was inserted into a Tn6230-like transposon as a stable five-tandem-repeat multimer, flanked by the same as in P. aeruginosa. This structure was stable across subcultures and did not change in the presence of carbapenems. The gene was cloned into the pCR-Blunt plasmid to study antimicrobial susceptibility patterns and into pET29a for kinetic activity analysis. VIM-63 showed higher values than VIM-2 for ceftazidime and cefepime and higher 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. VIM group metallo-β-lactamases are usually captured by 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 gene would indicate that the capture of this gene by 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.