TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli - PubMed (original) (raw)

TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli

J Silva et al. Antimicrob Agents Chemother. 2000 Apr.

Abstract

Escherichia coli R170, isolated from the urine of an infected patient, was resistant to expanded-spectrum cephalosporins, aztreonam, ciprofloxacin, and ofloxacin but was susceptible to amikacin, cefotetan, and imipenem. This particular strain contained three different plasmids that encoded two beta-lactamases with pIs of 7.0 and 9.0. Resistance to cefotaxime, ceftazidime, aztreonam, trimethoprim, and sulfamethoxazole was transferred by conjugation from E. coli R170 to E. coli J53-2. The transferred plasmid, RZA92, which encoded a single beta-lactamase, was 150 kb in length. The cefotaxime resistance gene that encodes the TLA-1 beta-lactamase (pI 9.0) was cloned from the transconjugant by transformation to E. coli DH5alpha. Sequencing of the bla(TLA-1) gene revealed an open reading frame of 906 bp, which corresponded to 301 amino acid residues, including motifs common to class A beta-lactamases: (70)SXXK, (130)SDN, and (234)KTG. The amino acid sequence of TLA-1 shared 50% identity with the CME-1 chromosomal class A beta-lactamase from Chryseobacterium (Flavobacterium) meningosepticum; 48.8% identity with the VEB-1 class A beta-lactamase from E. coli; 40 to 42% identity with CblA of Bacteroides uniformis, PER-1 of Pseudomonas aeruginosa, and PER-2 of Salmonella typhimurium; and 39% identity with CepA of Bacteroides fragilis. The partially purified TLA-1 beta-lactamase had a molecular mass of 31.4 kDa and a pI of 9.0 and preferentially hydrolyzed cephaloridine, cefotaxime, cephalothin, benzylpenicillin, and ceftazidime. The enzyme was markedly inhibited by sulbactam, tazobactam, and clavulanic acid. TLA-1 is a new extended-spectrum beta-lactamase of Ambler class A.

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Figures

FIG. 1

Nucleotide sequence showing the coding region for tla-1 gene. The deduced amino acid sequence of tla-1 is shown below the nucleotide triplets. A possible promoter and the ribosome-binding site are underlined and are presented as lowercase letters. Several proposed sites for _bla_TLA-1 after multiple sequence alignments with the most closely related β-lactamases are shown. ∗, 100% conserved residues in the more related class A β-lactamases; ↑1, signal sequence cleavage site for Pseudomonas aeruginosa; ↑2, signal sequence cleavage site for Proteus mirabilis; ↑#, catalytic serine; ↑ø, substrate binding; ‡, stop codon.

FIG. 2

Multiple alignment of the amino acid sequences of the closely related class A β-lactamases with that of TLA-1. Boxes I through VI correspond to those described by Joris et al. (19). The asterisks above the sequences indicate 100% conserved residues. Colons and periods indicate conserved and semiconserved residues, respectively. The lower trace represents the relative degree of conservation. TLA-1, E. coli (GenBank accession no. AF148067); CME-1, Chrysoebacterium meningosepticum (EMBL accession no. AJ006275); VEB-1, E. coli (EMBL accession no. O87489); PER-1, Pseudomonas aeruginosa (locus BLE1_PSEAE; SwissProt accession no. P37321); PER-2, Salmonella typhimurium (locus STBLAPER2; EMBL accession no. X93314); CBLA, Bacteroides uniformis (locus BLAC_BACUN; SwissProt accession no. P30898); CEPA, Bacteroides fragilis (GenBank accession no. L13472); CFXA, Bacteroides vulgatus (locus BLAC-BACVU; SwissProt accession no. P30899), TEM-3 (accession no. X64523). Gaps within the alignment are indicated by dashes.

FIG. 2

FIG. 3

Gel electrophoresis of the partially purified β-lactamase TLA-1. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide) with Coomassie blue staining. Lane 1, E. coli J53-2(pCA3000) cell extract; lane 2, β-lactamase partially purified by cation-exchange chromatography; lane 3, molecular mass standards proteins. The migration position and molecular mass of markers proteins are shown at the right.

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TLA-1: a new plasmid-mediated extended-spectrum beta-lactamase from Escherichia coli - PubMed (original) (raw)