Epigenetics of Antimicrobial Resistance in Gram-Negative Bacteria

Resumen

Resistance to antimicrobials is a well-known phenomenon leading to difficulties in the treatment of infectious diseases. The genetic determinants of such resistance are in general well understood: plasmids, transposons, insertion sequences and integrons are the most frequently related genetic elements. The word epigenetics refers to changes in the phenotype or in the gene expression caused by mechanisms other than underlying DNA sequence. In some cases these changes can remain for generations. Serratia marcescens is an enterobacterium characterized by its natural (intrinsic) resistance to most antibiotics. It is also a relevant opportunistic pathogen which has been involved in several pathologies such as urinary tract infections, prostheses infections, cellulitis, bacteremia and others. P. aeruginosa is a Gram-negative bacterium considered one of the major nosocomial pathogens worldwide. It causes several infections such as wound and burn infections as well as respiratory tract infections mostly affecting cystic fibrosis patients. An increasing prevalence of infections caused by multidrug-resistant (MDR) isolates has been reported in many countries and is actually a cause of concern. Both, P. aeruginosa and S. marcescens are relevant nosocomial pathogens. Some of the classic antimicrobials used to treat these pathogens are out-of-date and several of the new drugs available have already become targets for bacterial mechanism of resistance. Environmental conditions exert high pressure not only in the selection of genes encoding resistance to antibiotics or integron fixation in bacterial genomes or plasmids and other mobile elements transmission, but also in the expression of these potentialities that leads to resistance. Thus the role of epigenetics remains to be investigated. In addition it is well known that bacteria causing infections are naturally forming part of biofilms instead the planktonic way of life normally assumed to be in laboratory conditions. The aim of this thesis was the study of unconventional mechanisms of antimicrobial resistance contributing to MDR phenotypes in both S.marcescens and P. aeruginosa. Also the exploration of changes in antimicrobial susceptibilities of Serratia marcescens in the last 50 years by comparing isolates collected between 1945 and 1950, and current isolates. ¬The main conclusions obtained from this study are: 1. The resistome of Serratia marcescens did not change significantly during the antibiotic era. 2. Antibiotic withdrawing tends to restore original susceptible phenotypes, irrespective to the molecular mechanism involved in resistance. 3. None of Serratia strains studied presented integrons, any extended spectrum ß-lactamases. 4. Phenotypically determination of susceptibilities of old strains inactive during the last 60 years have confirmed results obtained by metagenomics i.e. the genes of resistance already existed before antibiotics discovery and use. 5. Multiresistant Pseudomonas aeruginosa harbored class 1 integrons containing a cassette encoding aminoglycoside adenylyltransferase (aadB). 6. Multiresistant Pseudomonas aeruginosa overexpressed MexAB-OprM and MexXY efflux machinery. 7. CCCP use should be avoided in experiments performed with P. aeruginosa and probably in other aerobic bacteria. 8. Meropenem induces the formation of aberrant long rods which can survive, accumulate less antibiotic than normal bacteria, and can revert to normal forma when antibiotic pressure disappears. 9. Colistin, the last therapeutic option to fight against Pseudomonas infections in cystic fibrosis patients, is normally active although cases of resistance have arisen recently. 10. Resistance to colistin seems to be mediated by lipopolysaccharide singular properties. 11. Colistin induces injuries in lipid bilayers, which can be studied by means of planar black lipid bilayer techniques. Preliminary results showed the ability of colistin to induce transient channels in the bilayers, with some dependence to voltage. 12. Recovery of susceptibility to imipenem is slower than acquision of resistance, since the selective advantage conferred by imipenem resistance in the presence of the antimicrobial is strong whereas OprD expression is likely evolutionarily advantageous only under certain and unknown environmental conditions.