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

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. The blaTER-1 gene of R. terrigena strain ATCC33257T 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>2mg/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. R. terrigena strains ATCC33257T 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 blaTER gene harbored by strains growing in the presence of≥2mg/L ceftazidime was shown to be responsible for this growth. TER is a new class A β-lactamase belonging to functional group 2b. Les β-lactamases chromosomiques de classe A de Raoultella ornithinolytica et Raoultella planticola ont été caractérisées. Le but de la présente étude était de caractériser celle de Raoultella terrigena. Le gène blaTER-1 de la souche de R. terrigena ATCC33257T a été cloné (pACter-1), séquencé, puis utilisé pour la détection du gène bla des souches de R. terrigena BM 85 01 095 et SB2796. La souche hypermutatrice de Escherichia coli AB1157 (mutS::Tn10) a été transformée avec le plasmide pACter-1 et les mutants cultivant en présence de>2mg/L de ceftazidime ont été étudiés. Les séquences du gène et de la protéine déduite (TER-1) sont identiques chez les souches ATCC33257T et BM 85 01 095 tandis que 3 substitutions sont observées dans la protéine déduite (TER-2) chez la souche SB2796. La β-lactamase TER de classe A présente 78 %, 69,9 % et 38,7 % d’identité avec les β-lactamases PLA ou ORN, TEM-1 et KOXY, respectivement. Comparées à TEM-1, TER-1 et TER-2 ont 2 substitutions particulières, Leu75Pro et Glu240Asn, dont