Inducible and acquired antibiotic resistance in stenotrophomonas maltophilia

Resumen

Antibiotic resistance is a global public health problem, which is aggravated by the emergence and spread of multidrug resistant (MDR) bacteria, especially Gram-negative bacterial species. Bacteria can present intrinsically low susceptibility to antibiotics as a result of the presence of resistance determinants encoded in their genomes. Besides, they can also become resistant through the acquisition of resistance genes through horizontal gene transfer, as well as by the selection of genetic mutations. Transient resistance can also be achieved as a consequence of a temporal induction of the expression of some of these resistance mechanisms, such as MDR efflux pumps. Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen with an environmental origin and associated with infections in compromised patients, including those with cystic fibrosis and with other underlying pathologies. This bacterium exhibits an intrinsic low susceptibility to multiple antimicrobial compounds. Particularly, MDR efflux pumps from the resistance nodulation division (RND) family are among the most relevant determinants contributing to the intrinsic and acquired antibiotic resistance of S. maltophilia. The role of RND efflux pumps in transient resistance has not been studied in S. maltophilia in detail. Hence, in this thesis, we aim to analyse the contribution of two S. maltophilia RND efflux pumps in transient resistance to antibiotics. To that goal, two screenings using fluorescence-based strains and a broad variety of compounds have been performed in order to find inducers of the expression of SmeVWX and SmeYZ efflux pumps. Inducer compounds were identified for both efflux systems, pointing that smeVWX expression is likely induced by the thiol-reactivity of the compounds, while smeYZ is induced by molecules that inhibit protein synthesis. The role of these identified inducers in transient resistance to antibiotics was also confirmed. With the aim of defining new mechanisms involved in the acquisition of mutation-driven resistance to antibiotics, as well as to decipher the evolutionary trajectories towards such resistance, S. maltophilia was submitted to experimental evolution in the presence of increasing concentrations of the antibiotics ceftazidime or tigecycline. Whole-genome sequencing of the final-step populations revealed that SmeH, the transporter protein of the SmeGH efflux pump, is an important contributor towards ceftazidime resistance acquisition. Amino acid substitutions in this efflux protein do not give rise to a fitness burden. However, they modify the susceptibility against other antimicrobials, possibly by producing changes in the access and binding of the substrate. Conversely, the first step towards the acquisition of tigecycline resistance is the overexpression of the SmeDEF efflux pump through mutations in the smeT gene, which encodes the transcriptional repressor of this efflux system. Besides, mutations in genes related to the ribosome (the tigecycline target), and to the lipopolysaccharide biosynthesis and membrane homeostasis, were also found mutated along the evolution period in the different evolved lineages. These mutations, which lead to cross-resistance to several antibiotics and collateral susceptibility to fosfomycin, impose a fitness cost for the S. maltophilia populations. Overall, the presented results highlight the relevance of the S. maltophilia RND efflux pumps, since they play a fundamental role not only in intrinsic resistance, but also in the acquisition of resistance through mutations and in transient resistance through their overexpression under specific stress conditions.