Antimicrobial Blue Light Inactivation of Polymicrobial Biofilms

Polymicrobial biofilms, in which mixed microbial species are present, play a significant role in persistent infections. Furthermore, polymicrobial biofilms promote antibiotic resistance by allowing interspecies transfer of antibiotic resistance genes. In the present study, we investigated the effectiveness of antimicrobial blue light (aBL; 405 nm), an innovative non-antibiotic approach, for the inactivation of polymicrobial biofilms. Dual-species biofilms with Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) as well as with P. aeruginosa and Candida albicans were reproducibly grown in 96-well microtiter plates or in the CDC biofilm reactor for 24 or 48 h. The effectiveness of aBL inactivation of polymicrobial biofilms was determined through colony forming assay and compared with that of monomicrobial biofilms of each species. aBL-induced morphological changes of biofilms were analyzed with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). For 24-h old monomicrobial biofilms formed in 96-well microtiter plates, 6.30-log10 CFU inactivation of P. aeruginosa, 2.33-log10 CFU inactivation of C. albicans and 3.48-log10 CFU inactivation of MRSA were observed after an aBL exposure of 500 J/cm2. Under the same aBL exposure, 6.34-log10 CFU inactivation of P. aeruginosa and 3.11-log10 CFU inactivation of C. albicans were observed, respectively, in dual-species biofilms. In addition, 2.37- and 3.40-log10 CFU inactivation were obtained in MRSA and P. aeruginosa, dual-species biofilms. The same aBL treatment of the biofilms developed in the CDC-biofilm reactor for 48 h significantly decreased the viability of P. aeruginosa monomicrobial and polymicrobial biofilm when cocultured with MRSA (3.70- and 3.56-log10 CFU inactivation, respectively). 2.58-log10 CFU inactivation and 0.86-log10 CFU inactivation was detected in MRSA monomicrobial and polymicrobial biofilm when cocultured with P. aeruginosa. These findings were further supported by the CLSM and SEM experiments. Phototoxicity studies revealed a no statistically significant loss of viability in human keratinocytes after an exposure to 216 J/cm2 and a statistically significant loss of viability after 500 J/cm2. aBL is potentially an alternative treatment against polymicrobial biofilm-related infections. Future studies will aim to improve the efficacy of aBL and to investigate aBL treatment of polymicrobial biofilm-related infections in vivo

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

Twitter as a Tool for Teaching and Communicating Microbiology: The #microMOOCSEM Initiative

Online social networks are increasingly used by the population on a daily basis. They are considered a powerful tool for science communication and their potential as educational tools is emerging. However, their usefulness in academic practice is still a matter of debate. Here, we present the results of our pioneering experience teaching a full Basic Microbiology course via Twitter (#microMOOCSEM), consisting of 28 lessons of 40-45 minutes duration each, at a tweet per minute rate during 10 weeks. Lessons were prepared by 30 different lecturers, covering most basic areas in Microbiology and some monographic topics of general interest (malaria, HIV, tuberculosis, etc.). Data analysis on the impact and acceptance of the course were largely affirmative, promoting a 330% enhancement in the followers and a >350-fold increase of the number of visits per month to the Twitter account of the host institution, the Spanish Society for Microbiology. Almost one third of the course followers were located overseas. Our study indicates that Massive Online Open Courses (MOOC) via Twitter are highly dynamic, interactive, and accessible to great audiences, providing a valuable tool for social learning and communicating science. This strategy attracts the interest of students towards particular topics in the field, efficiently complementing customary academic activities, especially in multidisciplinary areas like Microbiology.Versión del edito

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

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

Abstract 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

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

Ibiza: Life cycle and Evolution as a Tourist destination

The island of Ibiza is a second generation tourist destination that developed in the second half of the 20th century. According to previous studies, Ibiza would be in the stagnation or decline phase of the Tourism Area Life Cycle, because it combines elements of stagnation and decline. The objective of this paper is to carry out an exploratory study among businessmen and workers in the service sector of Ibiza on their perception in relation to various characteristics and changes that occurred in tourism during the years 2014 to 2018, characterized by record numbers of tourist arrivals and the development of a luxury supply with very high prices. A convenience sampling with quotas by sector (hotels, restaurants and other services) and municipality (Eivissa, Sant Antoni de Portmany and Santa Eulària des Riu) was carried out in December 2018, obtaining a total sample of 90 people. The perception of the changes that have taken place is mostly negative: there is a nostalgia for the past and they consider that the island has worsened in the last 50 years; the lack of connections in winter is a serious problem in the workplace; the behavior of tourists has changed a lot in the last 5 years; the price-quality ratio of the supply is not adequate; and it is not clear whether luxury tourism and the increase in 5-star hotels is positive.La isla de Ibiza es un destino turístico de segunda generación que se desarrolló en la segunda mitad del siglo XX. Según estudios previos, Ibiza estaría en la fase de estancamiento o declive del Ciclo de Vida del Destino Turístico, debido a que combina elementos de estancamiento y declive. El objetivo de este trabajo es realizar un estudio exploratorio entre empresarios y trabajadores del sector servicios de Ibiza sobre su percepción en relación a diversas características y cambios acaecidos en el turismo durante los años 2014 a 2018, caracterizados por cifras record de llegadas de turistas y el desarrollo de una oferta de lujo y con precios muy elevados. Se realizó un muestreo de conveniencia con cuotas por sector (hoteles, restaurantes y otros servicios) y municipio (Eivissa, Sant Antoni de Portmany y Santa Eulària des Riu) en diciembre de 2018, obteniendo una muestra total de 90 personas. La percepción de los cambios sucedidos es mayoritariamente negativa: hay una nostalgia del pasado y consideran que la isla ha empeorado en los últimos 50 años; la falta de conexiones en invierno es un grave problema en el ámbito laboral; el comportamiento de los turistas ha cambiado mucho en los últimos 5 años; la relación calidad precio de la oferta no es la adecuada; y no está claro si el turismo de lujo y el aumento de los hoteles de 5 estrellas es positivo

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