High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution

Abstract

A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla(CTX-M-32)) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis

FIG. 1.

Nucleotide sequence of a 2,326-bp DNA fragment of the pMC-2 plasmid. The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence. Stop codons are indicated by asterisks. The −35 and −10 promoter sequences of the bla_CTX-M-32 gene and the IRR sequence of ISEcp1 are underlined and indicated by bold letters, as is the +1 position of the transcriptional start of the bla_CTX-M-32 gene (10). The CTX-M-32 and transposase of IS5 proteins are indicated by arrows. Bold amino acids are those conserved in class A β-lactamases (15). Oligonucleotides used for sequencing are indicated by arrows, and KpnI restriction sites delimiting the 4-kbp insert are also underlined.

FIG. 1.

Nucleotide sequence of a 2,326-bp DNA fragment of the pMC-2 plasmid. The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence. Stop codons are indicated by asterisks. The −35 and −10 promoter sequences of the bla_CTX-M-32 gene and the IRR sequence of ISEcp1 are underlined and indicated by bold letters, as is the +1 position of the transcriptional start of the bla_CTX-M-32 gene (10). The CTX-M-32 and transposase of IS5 proteins are indicated by arrows. Bold amino acids are those conserved in class A β-lactamases (15). Oligonucleotides used for sequencing are indicated by arrows, and KpnI restriction sites delimiting the 4-kbp insert are also underlined.

FIG. 1.

Nucleotide sequence of a 2,326-bp DNA fragment of the pMC-2 plasmid. The deduced amino acid sequence is indicated in single-letter code below the nucleotide sequence. Stop codons are indicated by asterisks. The −35 and −10 promoter sequences of the bla_CTX-M-32 gene and the IRR sequence of ISEcp1 are underlined and indicated by bold letters, as is the +1 position of the transcriptional start of the bla_CTX-M-32 gene (10). The CTX-M-32 and transposase of IS5 proteins are indicated by arrows. Bold amino acids are those conserved in class A β-lactamases (15). Oligonucleotides used for sequencing are indicated by arrows, and KpnI restriction sites delimiting the 4-kbp insert are also underlined.

FIG. 2.

Electrophoresis analysis of CTX-M-32 and CTX-M-1 purified extracts in a sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis gel stained with Coomassie brilliant blue R-250. Lanes: 1, protein molecular markers; 2, purified CTX-M-1 protein used in kinetic experiments; 3, purified CTX-M-32 protein used in kinetic experiments.