Sequence of the Gene Encoding a Plasmid-Mediated Cefotaxime-Hydrolyzing Class A β-Lactamase (CTX-M-4): Involvement of Serine 237 in Cephalosporin Hydrolysis

Abstract

The sequence of the gene encoding a novel cefotaxime-hydrolyzing β-lactamase (CTX-M-4) was determined. It was located in a plasmid harbored by a Salmonella typhimurium strain. CTX-M-4 was similar to the plasmidic cefotaxime-hydrolyzing β-lactamases CTX-M-2 and Toho-1 and related to the chromosomal β-lactamase ofKlebsiella oxytoca. A Ser-237→Ala substitution, introduced by site-directed mutagenesis, caused minor alterations in the interaction of CTX-M-4 with β-lactams, reducing slightly the relative hydrolytic activity against cefotaxime and the susceptibility to inhibition by clavulanate.

Two novel plasmid-mediated cefotaxime-hydrolyzing β-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium

“…More e¤cient inhibition by tazobactam, as compared with clavulanic acid, has also been observed for other CTX-M-type L-lactamases [6,12]. Both enzymes were inhibited by low concentrations of tazobactam (IC SH of 1 WM) while clavulanate and sulbactam were less active (both IC SH s approximately 16 WM).…”

CTX-M Enzymes: Origin and Diffusion

“…Two attractive issues are raised in relation to the capacities of CTX-Ms to confer resistance to non-oxyimino-β-lactam antibiotics, including β-lactam-β-lactamase inhibitor combinations (IR-CTX-M) and/or carbapenems (Girlich et al, 2008; Ripoll et al, 2011), as these variants have not yet been found in nature. In laboratory conditions, IR-CTX-M mutants carrying the S130G, K234R, and S237A mutations have been described (Gazouli et al, 1998; Aumeran et al, 2003; Ripoll et al, 2011). The selection of these mutants conferring resistance to β-lactam plus β-lactamase inhibitors showed an antagonistic pleiotropic effect with a simultaneous loss of the hydrolytic activity against cefotaxime and ceftazidime.…”

Structural Insights into Substrate Recognition and Product Expulsion in CTX-M Enzymes

“…16 Accordingly, CTX-M enzymes have unique amino acid sequences, with 70% or higher identity within the subgroup, yet exhibit only 40% or less identity with other class A β-lactamases, indicating that their ability to hydrolyze extended-spectrum cephalosporins is an intrinsic enzymatic property of the subgroup and not the result of point mutations. 16 CTX-M enzymes have been studied by site-directed mutagenesis, [38][39][40][41][42][43] which revealed the involvement of both residues Ser237 and Arg276 in extended-spectrum activity. An important step in the understanding of catalytic mechanism was the determination of the crystal structures of CTX-M-9, CTX-M-14, CTX-M-16, CTX-M-27, and CTX-M-44 (also designated as Toho-1).…”