Genetic, biochemical characterization and mutagenesis of the chromosomal class A β-lactamase of Raoultella (formerly Klebsiella) terrigena

Abstract

Background

Chromosomal class A β-lactamases have been characterized in Raoultella ornithinolytica and Raoultella planticola. The purpose of this study was to characterize that of Raoultella terrigena.

Materials and methods

The bla_TER-1 gene of R. terrigena strain ATCC33257^T was cloned (pACter-1) and sequenced. It was then used to detect the bla gene of strains BM 85 01 095 and SB2796. The hypermutable Escherichia coli strain AB1157 mutS::Tn10 was transformed with pACter-1 and mutants growing on plates containing > 2 mg/L ceftazidime were studied. Notably, the impact of mutations only observed in the promoter region on β-lactam resistance was assessed by site-directed mutagenesis experiments.

Results

R. terrigena strains ATCC33257^T and BM 85 01 095 had the same bla gene and deduced protein (TER-1) whereas there were 3 substitutions in those of strain SB2796 (TER-2). Class A β-lactamases TER showed 78%, 69.9% and 38.7% identity with PLA or ORN, TEM-1 and KOXY, respectively. Compared with TEM-1, TER-1 and TER-2 showed 2 particular substitutions, Leu75Pro and Glu240Asn demonstrated to be involved in the inherent β-lactam resistance profile of R. terrigena. TER-1 (pI of 7.6) had a high activity against penicillin G and a significantly low one against amoxicillin. Substitution G/T observed in the -35 region of the bla_TER gene harbored by strains growing in the presence of ≥ 2 mg/L ceftazidime was shown to be responsible for this growth.

Conclusion

TER is a new class A β-lactamase belonging to functional group 2b.