Non-antimicrobial strategies for the prevention and treatment of infections by multidrug-resistant gram-negative bacilli

Due to the increased of antimicrobial resistance rates and the difficulty of having effective treatment for infections caused by Gram-negative bacilli (GNB) such as Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli, it is necessary to develop non-antimicrobial therapeutic alternatives, which can be used together with the scarce and non-optimal antimicrobial available. In this doctoral thesis, and in order to stop the evolution of the bacterial infection, we aimed to evaluate two therapeutic alternatives: i) blocking the virulence factors of A. baumannii, P. aeruginosa and E. coli and ii) modulating the host immune system. Regarding the first therapeutic approach, we performed the design and evaluation of outer membrane protein A (OmpA) inhibitors on the interaction of A. baumannii, P. aeruginosa and E. coli with the host to block the mechanisms by which this protein produces the infection. Thus, we studied in vitro the effect of the lead peptide AOA-2 on the interaction between A. baumannii, P. aeruginosa and E. coli and human lung epithelial cells (A549) by adherence, immunofluorescence, fibronectin binding, and cell viability assays, and also we tested the effect of AOA-2 on biofilm formation by GNB. In vivo, the therapeutic efficacy of AOA-2 against A. baumannii, P. aeruginosa and E. coli was evaluated. Moreover, the synergy between AOA-2 and colistin by microdilution assay and time-kill curves was studied, and the outer membrane proteins profile in the presence or absence of the AOA-2 was analyzed. Consequently, we constructed an A. baumannii knockout deficient in the omp25 gene and its complemented strain to perform the same experiments. Finally, we evaluated in vivo the therapeutic efficacy of AOA-2 in combination with sub-optimal doses of colistin in peritoneal sepsis caused by A. baumannii. We found that AOA-2 prevented the adhesion of A. baumannii, P. aeruginosa and E. coli to A549 cells protecting them from death, and also reduced biofilm formation by these pathogens. In addition, in the murine peritoneal sepsis model caused by A. baumannii, P. aeruginosa and E. coli, AOA-2 treatment significantly reduced bacteremia, bacterial load in spleen and lung, and mice mortality rates. Regarding the combination of AOA-2 with colistin, the presence of AOA-2 increases the susceptibility of colistin MICs for colistin-susceptible and colistin-resistant A. baumannii strains. Time-kill curves showed a synergistic activity between AOA-2 and colistin, and the protein profile exhibited an overexpression of the Omp25 protein in the strains treated with AOA-2. In vivo, AOA-2 in combination with colistin reduced bacterial spleen and lung loads, bacteremia and mortality rates, in comparison with the monotherapy with colistin. Regarding the second therapeutic approach, we evaluated the effect of lysophosphatidylcholine (LPC), a chemoattractant immunomodulatory factor which stimulates cells from the immune system, as an adjuvant treatment with antimicrobials to treat infections caused by susceptible and MDR strains of A. baumannii and P. aeruginosa in experimental murine models of peritoneal sepsis and pneumonia. Pharmacokinetics and pharmacodynamics parameters of colistin, tigecycline, imipenem and ceftazidime, and minimal lethal dose (MLD) of each strain were determined. In murine experimental models of peritoneal sepsis and pneumonia, mice were pretreated with LPC, infected with MLD of the correspondence strain, and treated or not with antimicrobials. We found that LPC in combination with colistin, tigecycline or imipenem shows beneficial effects in infections caused by susceptible and MDR A. baumannii. Furthermore, the combination of LPC with ceftazidime or imipenem improves the prognosis of infections caused by MDR P. aeruginosa. The data of this doctoral thesis indicate that both the blocking of bacterial virulence factors and the stimulation of the immune system with AOA-2 and LPC, respectively, alone or in combination with antimicrobials, could protect against infections by GNB.