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Proteomic studies have improved our understanding of the microbial world. The most recent advances in this field have helped us to explore aspects beyond genomics. For example, by studying proteins and their regulation, researchers now understand how some pathogenic bacteria have adapted to the lethal actions of antibiotics. Proteomics has also advanced our knowledge of mechanisms of bacterial virulence and some important aspects of how bacteria interact with human cells and, thus, of the pathogenesis of infectious diseases. This review article addresses these issues in some of the most important human pathogens. It also reports some applications of MALDI-TOF mass spectrometry that may be important for the diagnosis of bacterial resistance in clinical laboratories in the future. The reported advances will enable new diagnostic and therapeutic strategies to be developed in the fight against some of the most lethal bacteria affecting humans.

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]