Genetic diversity of carbapenem-hydrolyzing metallo-beta-lactamases from Chryseobacterium (Flavobacterium) indologenes

Abstract

The class B carbapenem-hydrolyzing beta-lactamase IND-1 has been characterized for Chryseobacterium indologenes strain 001. With internal primers for the bla gene for IND-1 (bla(IND-1)) and an internal bla(IND-1) probe, PCR amplifications failed, while hybridization results were positive when DNA from another C. indologenes isolate, strain CIP101026, was used as a template. Thus, a bla(IND)-related gene was cloned from this C. indologenes reference strain. Sequencing of the insert of a recombinant plasmid conferring resistance to carbapenems revealed an open reading frame with a G + C content of 39.9% and coding for a 243-amino-acid preprotein named IND-2. IND-2 shared 80% amino acid identity with IND-1 and had a similar broad-spectrum resistance profile, including resistance to carbapenems. It was classified in functional subgroup 3a of class B carbapenem-hydrolyzing beta-lactamases. IND-1 and IND-2, despite their genetic diversity, possessed similar kinetic parameters, except that ceftazidime was hydrolyzed less by IND-2. To obtain the entire bla(IND)-related gene sequences of eight other C. indologenes isolates, PCR was performed using internal and external primers, followed by inverse PCR techniques. The likely chromosome-mediated metallo-beta-lactamases of the 10 C. indologenes isolates were divided into several groups and subgroups. IND-1, IND-2, IND-2a, IND-3, and IND-4 shared 77 to 99% amino acid identity.

FIG. 1

Strategies of direct PCR and IPCR. Black bars represent the internal PCR product sequence obtained with primers 3 and 4. Grey bars represent known sequences of IPCR products. revA and revB (or revC and revD for C. indologenes 009) represent primers used for amplification of flanking regions. T3 and T7 primers were used for sequencing the IPCR products. Primers 5 and 6 were used for cloning the entire bla_IND-3 gene into pPCR-Script Cam SK(+) and into pBK-CMV. Primers 7 and 8 (same positions as primers 5 and 6, respectively) were used for cloning the bla_IND-4 gene. Double-headed arrows represent the entire bla_IND-related gene fragment.

FIG. 2

Amino acid sequence comparison of the IND-like β-lactamases of the 10 C. indologenes isolates. Broken lines indicate identical amino acids. IND-1 was from C. indologenes 001; IND-2 was from C. indologenes CIP101026, 002, and 003; IND-2a was from C. indologenes 004; IND-3 was from C. indologenes 005, 006, 007, and 008; and IND-4 was from C. indologenes 009. Shading correspond to conserved residues in CHβLs (25). Numbering is according to that for the IND-1 β-lactamase. The arrow indicates the peptide leader cleavage site determined for IND-2 by N-terminal sequence analysis of E. coli DH10B(pSO-2) cultures.