Novel cefotaximase (CTX-M-16) with increased catalytic efficiency due to substitution Asp-240-->Gly

Abstract

Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla(CTX-M) genes encoding beta-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla(CTX-M-9) gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla(CTX-M-16), observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240-->Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 microg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 microg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher k(cat) for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla(CTX-M-9) genes and the bla(CTX-M-16) gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M beta-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum beta-lactamases in Brazil.

FIG. 1

(A) Agarose (0.7%) electrophoresis of plasmid DNA from the three clinical strains, Rio-6, Rio-7, and Rio-9, and the E. coli transconjugants. (B) Hybridization of plasmid content with a CTX-M probe. Lanes: 1 to 4, reference plasmids RSa (39 kb), TP114 (61 kb), pCFF04 (85 kb), and pCFF14 (180 kb); 5, E. coli Rio-6; 6, E. coli Rio-7; 7, E. cloacae Rio-9; 8, E. coli DH5α(pRio-6); 9, E. coli DH5α(pRio-9).

FIG. 2

Alignments of the CTX-M-16 amino acid sequence with those of CTX-M-1, CTX-M-2, CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-6, CTX-M-7, CTX-M-8, CTX-M-9, Toho-1, and Toho-2. Dots indicate amino acids identical to those of CTX-M-16. Italic letters represent the peptide signal of CTX-M-16, as determined by hydropathy plotting. The peptide signals determined by amino acid sequencing are boxed. The amino acids are numbered according to the standard numbering scheme for the class A β-lactamases of Ambler et al. (1).

FIG. 3

Dendrogram of CTX-M family. Branch lengths are scaled according to the amino acid changes. The scale indicates the distance measured for protein sequences based on the Dayhoff PAM matrix (13). The percentages at the branch points refer to the number of times a particular nod was found in 100 bootstrap replications. The distance along the vertical axis has no significance.

FIG. 4

Electrophoresis analysis of CTX-M-9 and CTX-M-16 purified extracts. (A) SDS-PAGE stained with Coomassie brillant blue R-250. (B) Zymogram detection of β-lactamase activity with nitrocefin after renaturation treatment and SDS-PAGE. Lanes: 1, protein molecular mass reference; 2, clarified extract of β-lactamase CTX-M-16; 3, fraction with β-lactamase activity eluted from ionic exchange column; 4, purified extract of β-lactamase CTX-M-16.