Potenciación de antibióticos, inhibidores de betalactamasas y bombas de expulsión mediante péptidos antimicrobianos en bacterias gramnegativas multiresistentes

Abstract

Resistance to antibiotics poses a “major global threat” to public health according to World Health Organization. The increasing emergence of bacterial clones insensitive to these drugs greatly limits the therapeutic options for infectious diseases and highlights the urgent need to develop novel treatments effective against these organisms. In the present work, we demonstrated that subinhibitory concentrations of certain antimicrobial peptides can neutralize several antibiotic resistance mechanisms expressed by Gramnegative multi-drug resistant pathogens such as Klebsiella pneumoniae and Pseudomonas aeruginosa (“ESKAPE” pathogens) and Escherichia coli. This enhancement of antibiotic activity resulted in the sensitization of these organisms to several antibiotic classes. We hypothesized that antimicrobial peptides could potentiate the activity of inhibitors of either β-lactamases or antibiotic efflux pump systems and sensitize bacteria to antibiotics substrate of those resistance mechanisms. To test this hypothesis we measured the ability of peptides to synergize with those antibiotics in the presence of selected inhibitors of those systems. As peptides, we used the nonapeptides of polymyxin B and polymyxin E (PMBN and PMEN), as well as a peptide library derived from human lactoferricin with improved bacterial permeabilizing activity and very low toxicity towards human cells. To characterize the antimicrobial efficiency of the combinations, we used an array of techniques including conventional MIC/MBC testing, checkerboard analysis, growth kinetics, killing curves, and anti-biofilm activity against biofilms measured by confocal microscopy and viable counts on biofilms grown under static (on microplates) and dynamic (in a CDC-reactor) flow regimes. Using planktonic cultures, we demonstrated that, PMBN was able to greatly enhance the activity of several (i) β-lactamase inhibitors in a β-lactamase AmpC overproducing P. aeruginosa strain (potentiating the activity of amoxicillin, ampicillin, ticarcillin, piperacillin and ceftazidime), (ii) β-lactamase inhibitors in ESBL-producing Enterobacteriaceae strains (sensitizing them to ampicillin, amoxicillin, ticarcillin and piperacillin) and (iii) efflux pump inhibitors in a MexAB-OprM pump P. aeruginosa overproducing strain (enhancing the activity of aztreonam, ceftazidime, doxycycline, levofloxacin, piperacillin and azithromycin). In addition, all the triple combinations selected were able to cause a 10- 100 million fold reduction in the viability of biofilm forming cells. Finally, we showed that these antimicrobial peptides can potentiate not only resistance mechanism inhibitors (β-lactamases and efflux pumps), but they can also enhance the activity of several antibiotics that specifically target Gram-positive bacteria (i.e. vancomycin), sensitizing P. aeruginosa, E. coli and K. pneumoniae to them. This strategy allows the use of these combinations as empirical therapy with a broad spectrum of activity.