A permeability-increasing drug synergizes with bacterial efux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains

Resistance to antibiotics poses a major global threat according to the World Health Organization. Restoring the activity of existing drugs is an attractive alternative to address this challenge. One of the most efficient mechanisms of bacterial resistance involves the expression of efflux pump systems capable of expelling antibiotics from the cell. Although there are efflux pump inhibitors (EPIs) available, these molecules are toxic for humans. We hypothesized that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of EPI necessary to sensitize bacteria to antibiotics that are efflux substrates. To test this hypothesis, we measured the ability of polymyxin B nonapeptide (PMBN), to synergize with antibiotics in the presence of EPIs. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa strains overexpressing the MexAB-OprM efflux system. Synergy between PMBN and EPIs boosted azithromycin activity by a factor of 2,133 and sensitized P. aeruginosa to all tested antibiotics. This reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitization. The selected antibiotic-EPI-PMBN combination caused a 10 million-fold reduction in the viability of biofilm forming cells. We proved that AMPs can synergize with EPIs and that this phenomenon can be exploited to sensitize bacteria to antibiotics

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

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

Permeability enhancers sensitize β-lactamase-expressing Enterobacteriaceae and Pseudomonas aeruginosa to β-lactamase inhibitors, thereby restoring their β-lactam susceptibility

Objectives: β-lactamases are the major resistance determinant for β-lactam antibiotics in Gram-negative bacteria. Although there are β-lactamase inhibitors (BLIs) available, β-lactam-BLI combinations are increasingly being neutralised by diverse mechanisms of bacterial resistance. This study hypothesised that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of BLIs necessary to sensitise bacteria to antibiotics that are β-lactamase substrates. Methods: To test this hypothesis, checkerboard assays were performed to measure the ability of several AMPs to synergise with piperacillin, ticarcillin, amoxicillin, ampicillin, and ceftazidime in the presence of either tazobactam, clavulanic acid, sulbactam, aztreonam, phenylboronic acid (PBA), or oxacillin. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae overexpressing β-lactamases. Results: Synergy between polymyxin B nonapeptide (PMBN) and tazobactam boosted piperacillin activity by a factor of 128 in Escherichia coli (from 256 to 2 mg/L, fractional inhibitory concentration index (FICI) ≤ 0.02) and by a factor of at least 64 in Klebsiella pneumoniae (from 1024 mg/L to 16 mg/L, FICI ≤ 0.05). Synergy between PMBN and PBA enhanced ceftazidime activity 133 times in Pseudomonas aeruginosa (from 16 mg/L to 0.12 mg/L, FICI ≤ 0.03). As a consequence, MICs of all the tested antibiotics were brought down to therapeutic range. In addition, the combinations also reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitisation. Ceftazidime/PBA/PMBN at 50 times the planktonic MIC caused a 10 million-fold reduction in the viability of mature biofilms. Conclusion: This study proved that AMPs can synergise with BLIs and that this phenomenon can be exploited to sensitise bacteria to antibiotics

A permeability-increasing drug synergizes with bacterial efux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains

Resistance to antibiotics poses a major global threat according to the World Health Organization. Restoring the activity of existing drugs is an attractive alternative to address this challenge. One of the most efficient mechanisms of bacterial resistance involves the expression of efflux pump systems capable of expelling antibiotics from the cell. Although there are efflux pump inhibitors (EPIs) available, these molecules are toxic for humans. We hypothesized that permeability-increasing antimicrobial peptides (AMPs) could lower the amount of EPI necessary to sensitize bacteria to antibiotics that are efflux substrates. To test this hypothesis, we measured the ability of polymyxin B nonapeptide (PMBN), to synergize with antibiotics in the presence of EPIs. Assays were performed using planktonic and biofilm-forming cells of Pseudomonas aeruginosa strains overexpressing the MexAB-OprM efflux system. Synergy between PMBN and EPIs boosted azithromycin activity by a factor of 2,133 and sensitized P. aeruginosa to all tested antibiotics. This reduced several orders of magnitude the amount of inhibitor needed for antibiotic sensitization. The selected antibiotic-EPI-PMBN combination caused a 10 million-fold reduction in the viability of biofilm forming cells. We proved that AMPs can synergize with EPIs and that this phenomenon can be exploited to sensitize bacteria to antibiotics

A comparison between SARS-CoV-2 and Gram-negative bacte- 1 ria-induced hyperinflammation and sepsis

Sepsis is a life-threatening condition caused by the body's overwhelming response to an infection, such as pneumonia or urinary tract infection. It occurs when the immune system releases cytokines into the bloodstream, triggering widespread inflammation. If not treated, it can lead to organ failure and death. Unfortunately, sepsis has a high mortality rate, with studies reporting rates ranging from 20% to over 50%, depending on the severity and promptness of treatment. According to the World Health Organization (WHO), the annual death toll in the world is about 11 million. One of the main toxins responsible for inflammation induction are lipopolysaccharides (LPS, endotoxin) from Gram-negative bacteria, which rank among the most potent immunostimulants found in nature. Antibiotics are consistently prescribed as a part of anti-sepsis-therapy. However, antibiotic therapy (i) is increasingly ineffective due to resistance development and (ii) most antibiotics are unable to bind and neutralize LPS, a prerequisite to inhibit the interaction of endotoxin with its cellular receptor complex, namely Toll-like receptor 4 (TLR4)/MD-2, responsible for the intracellular cascade leading to pro-inflammatory cytokine secretion. The pandemic virus SARS-CoV-2 has infected hundreds of millions of humans worldwide since its emergence in 2019. The COVID-19 (Coronavirus disease-19) caused by this virus is associated with high lethality, particularly for elderly and immunocompromised people. As of August 2023, nearly 7 million deaths were reported worldwide due to this disease. According to some reported studies, upregulation of TLR4 and the subsequent inflammatory signaling detected in COVID-19 patients "mimics bacterial sepsis". Furthermore, the immune response to SARS-CoV-2 was described by others as "mirror image of sepsis". Similarly, the cytokine profile in sera from severe COVID-19 patients was very similar to those suffering from the acute respiratory distress syndrome (ARDS) and sepsis. Finally, the severe COVID-19 infection is frequently accompanied by bacterial co-infections, as well as by the presence of significant LPS concentrations. In the present review, we will analyze similarities and differences between COVID-19 and sepsis at the pathophysiological, epidemiological, and molecular levels