Desarrollo de nuevos procedimientos rápidos y eficaces para evaluar in situ la sensibilidad o resistencia bacteriana a distintos tipos de antibióticos

Abstract

Antibiotic resistance is a serious global public health problem. Classical antibiograms usually require 18-24 hours, but in critical situations a rapid and accurate characterisation of the bacterial strain can be decisive.This thesis develops procedures to determine antibiotic sensitivity/resistance in highly pathogenic bacteria in a simple, fast and efficient way. All of them have in common the immobilisation of the bacteria in a microgel for observation under fluorescence microscopy.For each type of antibiotic, a phenotypic parameter was established and validated to discriminate between resistant and sensitive strains. Quinolones, which induce DNA breaks, were evaluated by direct visualisation of nucleoid fragmentation. For antibiotics that inhibit peptidoglycan synthesis, bacterial wall damage was determined by promoting nucleoid release or cell elongation. Colistin, which acts on the plasma membrane, showed secondary involvement in DNA and cell wall. Antibiotics that inhibit protein synthesis were evaluated for the prevention of autolytic processes induced in Gram-positives and elongation due to SOS response caused by mitomycin C in Gram-negatives.The results were obtained between 40-210 minutes, in close agreement with standard antibiograms.