Drug resistance in the enterobacteriaceae familyepidemiological, biochemical and molecular studies on beta-lactamases

Resumo

The main goal of the present work was to contribute for the elucidation of drug-resistance mechanisms in Enterobacteriaceae. The studies were directed toward the epidemiological characterization of Enterobacteriaceae isolates expressing of extended-spectrum ß-lactamase enzymes (EBSL) among clinical isolates, by biochemical and molecular techniques. All strains were isolated from human patients from two hospital centers and from one ambulatory center located in the North of Portugal, namely in the region of Porto, Viana do Castelo and Ponte de Lima. The strains were collected from several infectious episode types, namely from bloodstream infections (BSI), urinary tract-infections (UTI), low respiratory-tract infection (LRTI) and skin and soft tissue infections (SSTI). Enterobacteriaceae members isolated and suspected to harbor an ESBL enzyme were confirmed by antimicrobial susceptibility testing, drug inactivation, electrophoretic methods and molecular genetic based-methods such as PCR, cloning, sequencing and sequence alignment. Molecular fingerprinting methods were also used in order to understand and identify nosocomial outbreaks. In the present work among the several methods that may be used for this purpose a PCR-based method for conserved among Enterobacteriaceae species regions named enterobacteria repetitive intragenic consensus (ERIC) regions was selected. ERIC fingerprinting analysis was enhanced by an automated bioinformatic gel analyzer, FPQuest II from Bio-Rad Laboratories. Our main results point to i) an high diversity of molecular types of EBSL disseminated in the North of Portugal; ii) reduced susceptibility to all cephalosporins including to the fourth-generation cephalosporin (cefepime); iii) reduced susceptibility to other non-ß-lactams, in particular to quinolones such as ciprofloxacin but also to aminoglycosides (gentamicin) and to suphonamides (trimethoprim/sulphamethoxazol). Therefore understanding in detail, for each pathogen agent, the mechanisms of antimicrobial resistance in the population is even beyond academic importance. For physicians, these mechanisms are important for making well-informed decisions about the design of treatment protocols, the choice of antibiotics and doses for particular indications. For policymakers, these issues have direct importance on the design of campaigns to encourage more rational antibiotic use and on the priorities in regulating the use of antimicrobial agents for human and animal use. For population and patients the correct choice of the antibiotics may result in a better treatment, in some cases less pain, in some cases greater life expectancy and in all cases a more joust antimicrobial weapons arsenal available in this fight against microbes.