Resumen de Mechanisms of fluoroquinolone resistance in Escherichia coli, Salmonella Typhimurium and Yersinia enterocolitica. Influence on expression of virulence factors.
INTRODUCTION Quinolones are potent and broad spectrum antibacterial agents and, among them, several fluoroquinolones, such as ciprofloxacin, are the treatment of choice against several human infections. Resistance to quinolones is steadily increasing and can be explained by different mechanisms: i) mutations in the target genes, ii) mutations leading to decreased intracellular accumulation of the antibiotic (either by overexpression of efflux pumps or diminished production of porins, the entrance channels for quinolones), and iii) aquisition of plasmid-encoded genes. Acquisition of quinolone resistance is a stepwise process. The first mutation usually leads to nalidixic acid resistance whereas further mutations trigger resistance to fluoroquinolones such as ciprofloxacin. WORK JUSTIFICATION Nalidixic acid resistance is an increasing trend among Escherichia coli, Salmonella Typhimurium and Yersinia enterocolitica clinical isolates thereby compromising the success of antimicrobial therapies. However, ciprofloxacin resistance is not increasing to the same extent in S. Typhimuium and Y. Enterocolitica strains. These results suggest that ciprofloxacin resistance acquisition can somehow impair the bacterial ability in causing disease. Moreover, an in depth study of the mechanisms of fluoroquinolone resistance may lead to the development of new strategies for either combating or reducing the emergence or potential emergence of fluoroquinolone resistance or limiting this effect. OBJECTIVES The main objective of this thesis was to study the molecular mechanisms leading to fluoroquinolone resistance in several pathogens of clinical importance: uropathogenic E. coli and enteric S. Typhimurium and Y. enterocolitica isoaltes. Futhermore, in parallel, the effect of fluoroquinolone resistance acquisition on the expression of several virulence factors (e.g., those leading to invasion and biofilm production) in the two enteric pathogens was evaluated. RESULTS The results obtained in this thesis have led to the publication of four original articles, two manuscripts pending of acceptance in scientific journals and two blocks of additional results. • E. coli: The mechanisms of resistance to fluoroquinolones were studied in two strains: PS5, a susceptible clinical isolate, and NorE5, a fluoroquinolone resistant mutant obtained in vitro (norfloxacin MIC of 32 microg/mL). NorE5 was shown to overexpress the efflux pump AcrAB-TolC attributable to increased SoxS expression likely associated to a mutation within SoxR. Additionally, the mdtG and ompN genes were reported to belong to the soxS regulon. Particularly, the ompN gene is cotranscribed with the upstream gene ydbK. • S. Typhimurium: The prevalence of quinolone resistance mechanisms was evaluated in a collection of Salmonella spp clinical isolates. Almost half of the strains (41.5%) were resistant to nalidixic acid due to a mutation in the target gene gyrA and efflux pump overexpression. Moreover, two S. Typhimurium clinical isolates susceptible to nalidixic acid were selected to obtain in vitro ciprofloxacin resistant mutants (50-64, ciprofloxacin MIC of 64 microg/mL, and 59-64, ciprofloxacin MIC of 256 microg/mL). Strain 50-64 acquired three target gene mutations, overexpressed AcrAB-TolC but showed a reduced fitness as well as invasion on cell culture. Strain 59-64 acquired five target gene mutations, overexpressed the regulator ramA leading to increased efflux independently of AcrAB, which was inactivated in both the clinical and the resistant strain. • Y. enterocolitica: A nalidixic acid-susceptible Y. enterocolitica clinical isolate was selected to obtain in vitro a ciprofloxacin resistant mutant (Y.83-64, ciprofloxacin MIC of 64 microg/mL). The resistant strain acquired four target gene mutations, overexpressed AcrAB-TolC attributable to a mutation in the promoter of the marA homolog (marAYe) present in Yersinia. Moreover, strain Y.83-64 showed reduced invasion. In parallel, the heterogeneity in the selection of target gene mutations was assessed in an intermediate mutant (ciprofloxacin MIC of 4 microg/mL).
