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Plasmid-mediated resistance to third-generation cephalosporins caused by point mutations in TEM-type penicillinase
W Sougakoff1, S Goussard, G Gerbaud
1Unité des Agents Antibactériens, Institut Pasteur, Paris, France.
Abstract:
Infections due to strains of Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii resistant to third-generation cephalosporins have been observed recently in France and the Federal Republic of Germany. This resistance phenotype is due to the production of new plasmid-mediated, broad-substrate-range beta-lactamases designated TEM-3 to TEM-7. DNA-DNA hybridization analysis with a probe specific for TEM-1 indicated that the corresponding genes blaT-3 to blaT-7 were variants of the structural genes for TEM-type beta-lactamases. In the present studies, a 2.5-kilobase BamHI plasmid DNA fragment encoding TEM-3 was cloned in E. coli, and the entire nucleotide sequence of blaT-3 was determined. The deduced amino acid sequence of TEM-3 differed in two positions from that of the TEM-2 enzyme: lysine (TEM-3) was substituted for glutamic acid (TEM-2) at residue 104 and serine (TEM-3) for glycine (TEM-2) at residue 238 in the numbering system of Ambler. Spontaneous mutants of TEM penicillinases with increased activity against third-generation cephalosporins were obtained in vitro by selection on cefotaxime or ceftazidime. It therefore appears that mutations in TEM-type beta-lactamases contribute to resistance to new-generation cephalosporins.
Insights
New beta-lactamases, TEM-3 to TEM-7, cause resistance to third-generation cephalosporins in bacteria like Klebsiella pneumoniae. Mutations in TEM-type beta-lactamases also contribute to this emerging antibiotic resistance.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Emerging resistance to third-generation cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii has been reported.
- This resistance is linked to plasmid-mediated, broad-substrate-range beta-lactamases.
Purpose of the Study:
- To investigate the genetic basis of resistance to third-generation cephalosporins.
- To characterize the novel beta-lactamases responsible for this resistance phenotype.
Main Methods:
- DNA-DNA hybridization using a TEM-1 specific probe.
- Cloning of a DNA fragment encoding TEM-3 into E. coli.
- Nucleotide sequencing of the blaT-3 gene.
- Analysis of amino acid sequence variations.
- In vitro selection of spontaneous mutants.
Main Results:
- Genes blaT-3 to blaT-7 are variants of TEM-type beta-lactamase genes.
- The nucleotide sequence of blaT-3 was determined, revealing two amino acid differences compared to TEM-2.
- Spontaneous mutants with enhanced cephalosporin activity were generated in vitro.
Conclusions:
- Mutations in TEM-type beta-lactamases are a significant factor in bacterial resistance to new-generation cephalosporins.
- Understanding these genetic mechanisms is crucial for combating antibiotic resistance.