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Objectives In order to assess whether or not the Arg-276 of CTX-M-type enzymes is equivalent to the Arg-244 of IRT-TEM-derivative enzymes, we replaced the former with six different amino acids, some of them previously described as involved in resistance to β-lactamase inhibitors in TEM-IRT derivatives. We also investigated the role of Arg276 in cefotaxime hydrolysis. Methods By site-directed mutagenesis and by use of the blaCTX-M-1 gene as template, Arg-276 was replaced with six different amino acids (Trp, His, Cys, Asn, Gly and Ser). MICs of β-lactams alone and in combination with β-lactamase inhibitors were established. The seven enzymes (CTX-M-1 wild-type and six derived mutants) were purified by affinity chromatography, and kinetic parameters (kcat, Km, kcat/Km) towards cefalotin and cefotaxime were determined. Clavulanic acid IC50 values were also assessed with all enzymes. Results No increase in MICs of β-lactam/β-lactamase inhibitor combination was detected with any of the six CTX-M-1-derived mutants, in agreement with the clavulanic acid IC50 values. The MICs of cefotaxime were clearly lower for the Escherichia coli harbouring the Trp, Cys, Ser and Gly CTX-M-1 mutant enzymes than for CTX-M-1, and these enzymes showed a clearly reduced catalytic efficiency towards cefotaxime. As regards cefalotin, there was a moderate reduction in catalytic efficiency for Cys and His. Conclusions Arg-276 in CTX-M-type β-lactamases is not equivalent to Arg-244 in IRT-type enzymes. Position Arg-276 appears to be important for cefotaxime hydrolysis in CTX-M-type enzymes, although different effects were obtained regarding the replaced amino acid. [ABSTRACT FROM AUTHOR]

Objectives The main objective of the present study was to demonstrate the presence of a β-lactamase ampC gene in the chromosome of the non-pathogenic bacterium Acinetobacter baylyi ADP1. Methods β-Lactam MICs were determined by Etest. The ampC gene was amplified by PCR, with specific oligonucleotides, then cloned into pBGS18 and pAT-RA plasmids and transformed into Escherichia coli TG1 and parental A. baylyi as hosts. The gene was sequenced and analysed. The AmpC protein was expressed, purified by affinity chromatography and the kinetic parameters determined. Results An ampC gene was amplified from the ADP1 genome. Sequencing of the gene showed typical SVSK and KTG domains and the typical YXN Class C motif. The amplified gene showed significant identity (48.5% to 49.3%) with the AmpC enzymes of Acinetobacter baumannii and AG3 strains, which have recently been renamed ADC-1 to ADC-7. MIC analysis revealed a cephalosporinase profile for the E. coli TG1 clone as well as for the parental A. baylyi strain that overexpressed the ampC gene cloned under the control of an external promoter. Analysis of kinetic parameters of the purified enzyme showed higher catalytic efficiency for cefalotin than for ampicillin. Conclusions This study represents the first report of an AmpC β-lactamase in A. baylyi, which was shown by biochemical and microbiological experiments to have a typical cephalosporinase profile. The presence of the respective gene in the chromosome of A. baylyi ADP1 suggested that this ampC gene is the naturally occurring cephalosporinase in this species, as previously reported for other Acinetobacter spp. We tentatively named the enzyme ADC-8. [ABSTRACT FROM AUTHOR]

Objectives To characterize the extended-spectrum β-lactamases (ESBLs) as well as their genetic environment in different isolates of Enterobacteriaceae from a patient with repeated urinary tract infections. Methods Two isolates of Escherichia coli and one Proteus mirabilis, all with ESBL phenotypes, were studied. Conjugation experiments and restriction fragment length polymorphisms (RFLPs) were performed. Cloning of the bla genes was by plasmid restriction and fragments ligation. Antibiotic susceptibility testing was by Etest. The genetic environment was analysed by direct sequencing of the DNA surrounding the bla gene. RT–PCR was performed to study the differences in the blaCTX-M gene expression. Results The bla gene was transferred by conjugation from the three clinical isolates, which by RFLP showed the same plasmid. The bla gene and surrounding sequences were cloned, an ∼9 kbp AccI fragment was sequenced and the blaCTX-M-32 gene was identified. The MICs of ceftazidime for transconjugants and transformants bearing the blaCTX-M-32 gene were lower than those previously reported. Analysis of the DNA surrounding the ESBL gene revealed a new genetic structure with two insertion sequences, IS5 and IS1, located immediately upstream of the blaCTX-M-32 gene; IS1 was located between the bla gene and IS5, and within the −10 and −35 promoter boxes of the blaCTX-M-32 gene. Microbiological and biochemical studies revealed lower blaCTX-M-32 gene expression in bacterial isolates with IS1 between the promoter boxes. Conclusions Data suggest putative in vivo horizontal blaCTX-M-32 gene transfer between two different genera of Enterobacteriaceae. A new complex structure, IS5–IS1, was detected upstream of the bla gene and IS1 negatively modulated expression of the blaCTX-M-32 gene because its location modified the bla promoter region. [ABSTRACT FROM AUTHOR]