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