Tesis doctoral de Alicia Fajardo Lubian
In hospitals, the capability of bacteria to produce infection relies, not just in their virulence, but in their resistance to antibiotics also. The hypothesis of the current work is the existence of networks, which interconnect antibiotic resistance and virulence of bacterial pathogens. To analyze this potential crosstalk, we have searched genes simultaneously involved in both processes using as a model pseudomonas aeruginosa, a relevant intrinsically-resistant opportunistic pathogen. To that goal, we have screened two transposon-tagged libraries obtained in two different p. aeruginosa strains: 59.20 and tb, looking for mutants simultaneously affected in virulence and antibiotic susceptibility. Altered susceptibility to one or more antibiotics was observed in 222 of the 5952 tested mutants (3,7%). The position of the transposon was determined by inverse polymerase chain reaction, sequencing of the amplicons, and comparison to the available sequence of p. Aeruginosa. After filtering insertions in the same gene, we have detected 118 loci in the genome of p. Aeruginosa that contribute to its characteristic phenotype of antibiotic susceptibility. Since the genome of p. Aeruginosa contains 5570 genes, our results indicate that at least 1.8% of its genome contributes to its characteristic phenotype of susceptibility to antibiotics. in the present work, we have identified genes which inactivation increases p. Aeruginosa susceptibility to several antimicrobials. Those genes define novel targets for the development of anti-resistance drugs. Mutations in other genes make p. Aeruginosa more resistant and therefore define novel mechanisms for mutation-driven acquisition of antibiotic resistance, opening a new research field based in the prediction of resistance before it emerges in clinical environments. noteworthy, most mutants had altered susceptibility to multiple antibiotics, indicating that the development of resistance is not specific to a given antibiotic. To evaluate the virulence of the selected mutants, we have studied changes in their cytotoxicity against a macrophage cell line. We obtained that 51% of 222 mutants with changes in susceptibility presented altered cytotoxicity too. there was not an inverse correlation between cytotoxicity and antibiotic resistance. These results point out that acquiring resistance is not always associated with a lower virulence (fitness cost), so the effects of resistance are specific and depending on the type of involved mechanism. After filtering insertion occurring in the same gene, we have detected 73 loci in the genome of p. aeruginosa that contribute to its virulence and antibiotic susceptibility simultaneously. Among these loci, only few genes have been previously annotated as antibiotic resistance or virulence genes. although a few others, are predicted to play a role in these processes in p. Aeruginosa, the large majority of genes emerging from our screen were not previously thought to play any role in the antibiotic susceptibility or cytotoxicity of this bacterial species. Thus, our work supports the idea that in p. Aeruginosa resistance and virulence are the consequence of a complex network of elements, including non-classical antibiotic resistance and virulence genes. we present in detail a study of four of these mutants that present the insertion in genes belonging to different functional categories and could be involved in resistance/virulence crosstalk at different levels. On one hand, we selected two mutants with insertion in genes similar to classical antibiotic resistance determinants. One of them is a putative betalactamase that could be probably involved in peptidoglycan synthesis and the other one is a rnd efflux transporter that could be related in cell to cell signalling mediated by qs. On the other hand, we analized two mutants in probable sensor/response regulators. One gene belongs to a system that controls the intracelular levels of digmpc, a molecule known to regulate biofilm formation and virulence. The last gene encoded a putative hybrid sensor response regulator that could be involed in signalling networks mediated by small rnas. the results of our work validate the hypothesis on the existence of a crosstalk between resistance and virulence and present data for beginning to understand such crosstalk.
Datos académicos de la tesis doctoral «Busqueda de genes implicados simultaneamente en la resistencia a los antibioticos y la virulencia de pseudomonas aeruginosa«
- Título de la tesis: Busqueda de genes implicados simultaneamente en la resistencia a los antibioticos y la virulencia de pseudomonas aeruginosa
- Autor: Alicia Fajardo Lubian
- Universidad: Autónoma de Madrid
- Fecha de lectura de la tesis: 27/11/2009
Dirección y tribunal
- Director de la tesis
- José Luís Martínez Menéndez
- Tribunal
- Presidente del tribunal: josé Berenguer Carlos
- gracia Morales kucharski (vocal)
- esther Culebras lopez (vocal)
- renata Moreno albiger (vocal)