Integration of omics and bioinformatics to identify new therapeutic targets for Acinetobacter baumannii. Virulence role of CarO in Acinetobacter baumannii infections

Acinetobacter baumannii remains a significant and difficult-to-treat pathogen that causes a range of interactions with the human host from asymptomatic colonization and carriage in the skin, intestinal tract, and respiratory tract to invasive infection, such as nosocomial pneumonia or bacteraemia. Especially, this pathogen affects critically-ill and immunocompromised patients admitted to Intensive Care Units (ICUs), causing them severe infections, which are associated with long hospital stay and high mortality rates. The success of this bacterium is due to a combination of several factors, highlighting its extraordinary ability to develop antimicrobial resistance that results in the rapid nosocomial spread of strains resistant to almost all known antimicrobials, including including the last reservoirs of our antimicrobial arsenal such as the carbapenems, worldwide. Thus, in the recent global priority list of antimicrobial-resistant bacteria of the World Health Organization, carbapenem-resistant A. baumannii (CRAB) is considered as a "critical priority" for the development of new antimicrobials, due to the lack of therapeutic options. This situation has promoted the search of new therapeutic strategies to deal with multidrug-resistant (MDR) A. baumannii strains displaying additional carbapenem-resistance, and non-antimicrobial approaches aimed at bacterial virulence factors may represent a promising alternative. Nevertheless, our knowledge on A. baumannii pathogenesis and virulence traits is still relatively scarce. In this Doctoral Thesis, and in order to stop the evolution of A. baumannii infections, we aimed to study more deeply the pathogenesis of CRAB infections using omics and bioinformatics to ultimately discover new therapeutic targets for anti-A. baumannii drugs. Firstly, we studied MDR/CRAB isolates from tracheobronchial aspirate samples of ICU adult patients who suffered A. baumannii bacteraemic ventilator-associated pneumonia (VAP) or remained exclusively colonised by this pathogen. We analysed the in vitro and in vivo virulence of these isolates, in order to know if the invasive isolates exhibited higher virulence than the colonising ones, but no differences were found. Moreover, when the whole-genome sequencing (WGS) data of these isolates were analysed following different approaches, again we did not find any difference between both phenotypes regarding their clonal relationship, antimicrobial resistance mechanisms, or known virulence determinants. However, the invasive A. baumannii isolates exhibited higher levels of expression of the outer membrane protein (OMP) OmpA than the colonising ones, suggesting that those phenotypes depend on the regulation of already-known or still unknown virulence factors, instead of on the genomic content. Secondly, six bacteraemic MDR/CRAB clinical isolates obtained from six clinically homogeneous ICU adult patients with bacteraemia secondary to VAP, who were subjected to optimal colistin treatment but with radically different clinical outcomes, were comparatively analyzed by WGS complemented with proteomic and immunoblot techniques. These analyses indicated that the carO gene, encoding for the second most abundant β-barrel protein of the A. baumannii outer membrane (OM), CarO, was interrupted by different disruptive events in the isolates from patients who recovered from infection, while it was intact in the isolates from patients who did not survive. When the virulence role of A. baumannii CarO was analyzed in model systems, an isogenic mutant lacking carO (ATCC 17978 ΔcarO) showed lower ability in vitro to adhere and invade cultured human lung epithelial cells, and exhibited a higher minimum lethal dose and a lower dissemination potential into essential organs and fluids in a murine model of peritoneal sepsis. All of the above deficiencies were reverted in the ATCC 17978 ΔcarO mutant transformed with a carO expression plasmid restoring OM CarO levels. Thus, the results presented here reveal a previously unnoticed virulence role for the A. baumannii OMP CarO, which may be responsible of the poor clinical outcome and therefore a potential target for the development of novel anti-A. baumannii drugs.