Uso de fármacos, con mecanismo de acción sobre la pared bacteriana, como adyuvante al tratamiento antimicrobiano para las infecciones causadas por bacilos Gram-negativos multirresistentes

El aumento de las tasas de resistencia antimicrobiana ha dibujado un escenario global donde la dificultad de encontrar un tratamiento eficaz para las infecciones causadas por bacilos gramnegativos (BGN), tales como Acinetobacter baumannii, Pseudomonas aeruginosa y especies de Enterobacteriales como Escherichia coli o Klebsiella pneumoniae, hace necesario tanto la investigación como el desarrollo de alternativas terapéuticas no antimicrobianas, que puedan ser utilizadas junto a las escasas opciones antibióticas disponibles. En esta Tesis Doctoral, con el propósito de indagar en estas alternativas, se tuvo como objetivo el estudio de dos tipos de opciones: i) la mejora de un agente que bloquea uno de los principales factores de virulencia de estos BGN y ii) reposicionar fármacos con el fin de darle un nuevo uso terapéutico, en este caso como antimicrobiano. Previamente a la mejora del compuesto, se quiso estudiar el mecanismo de inhibición por el que inhibía a la proteína de membrana externa A (OmpA). En relación al primer enfoque terapéutico, se realizó el desarrollo de una librería de péptidops derivados del AOA-2, un compuesto inhibidor de la OmpA, que ya anteriormente se había descrito su actividad tanto in vitro contra A. baumannii, P. aeruginosa y E. coli como in vivo en un modelo experimental murino de infección por A. baumannii. En este contexto, se obtuvo una librería de diez compuestos derivados candidatos a mejorar la actividad in vitro de la molécula madre. Mediante los mismos ensayos in vitro descritos en el estudio donde se demostró la actividad de AOA-2, se realizaron diferentes ensayos de viabilidad celular y adherencia bacteriana que sirvieron de cribado para la evaluación de los mejores péptidos optimizados. Esto conllevó a que dos péptidos derivados, RW01 y RW06, mostraran los mejores resultados en los ensayos realizados contra los diferentes patógenos BGN, mejorando la actividad mostrada por AOA-2. Además, se llevó a cabo el estudio para comprobar si el tratamiento con AOA-2 afectaba de alguna manera a la respuesta del sistema inmune, tanto innato como adaptativo, del huésped. Los datos obtenidos no pudieron reflejar que este compuesto provocase algún hecho importante que tuviera que ver con el sistema inmune. En cuanto al segundo enfoque terapéutico, se quiso evaluar dos compuestos pertenecientes a la familia de los antihelmínticos como candidatos a ser reposicionados como antimicrobianos o adyuvantes al tratamiento antimicrobiano. Por una parte, se demostró que la niclosamida es capaz de potenciar el efecto de la colistina contra las cepas sensibles a colistina (Col-S) y resistentes a colistina (Col-R) de A. baumannii y K. pneumoniae, debido a la alteración de la proporción de carga superficial negativa en la membrana externa de estas cepas. Por otra parte, la oxiclozanida potenció el efecto de la colistina en cepas Col-S y Col-R de A. baumannii, P. aeruginosa y K. pneumoniae. Este efecto podría deberse a una interrupción estructural de su envoltura celular. Estos resultados proporcionan nuevos conocimientos sobre el uso de estos antihelmínticos en combinación con colistina como una nueva alternativa para el tratamiento de las infecciones causadas por BGN. En conjunto, los datos de esta tesis doctoral indican que tanto el bloqueo de los factores de virulencia bacteriana mediante péptidos inhibidores derivados de AOA-2 como el reposicionamiento de fármacos como la niclosamida o la oxiclozanida, podrían tratarse de opciones adyuvantes con las que poder hacer frente a las infecciones por BGN multirresistentes

Drug Repurposing for the Treatment of Bacterial and Fungal Infections

Multidrug-resistant (MDR) pathogens pose a well-recognized global health threat that demands effective solutions; the situation is deemed a global priority by the World Health Organization and the European Centre for Disease Prevention and Control. Therefore, the development of new antimicrobial therapeutic strategies requires immediate attention to avoid the ten million deaths predicted to occur by 2050 as a result of MDR bacteria. The repurposing of drugs as therapeutic alternatives for infections has recently gained renewed interest. As drugs approved by the United States Food and Drug Administration, information about their pharmacological characteristics in preclinical and clinical trials is available. Therefore, the time and economic costs required to evaluate these drugs for other therapeutic applications, such as the treatment of bacterial and fungal infections, are mitigated. The goal of this review is to provide an overview of the scientific evidence on potential non-antimicrobial drugs targeting bacteria and fungi. In particular, we aim to: (i) list the approved drugs identified in drug screens as potential alternative treatments for infections caused by MDR pathogens; (ii) review their mechanisms of action against bacteria and fungi; and (iii) summarize the outcome of preclinical and clinical trials investigating approved drugs that target these pathogens

Synergic effect of oxyclozanide in combination with colistin against colistin-resistant and colistin-susceptible clinical strains of Klebsiella pneumoniae

Motivation: Colistin is among the few antibiotics effective against Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant K. pneumoniae have been isolated (1). Therefore, combination therapies between colistin and old drug effective against these isolates are required. The objective of this study is to study in vitro the activity of oxyclozanide, an anthelmintic drug (2), in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant K. pneumoniae.Methods: Col-R (KPc21) and Col-S (CECT 997) K. pneumonia strains were used. Checkerboard assay with colistin and oxyclozanide to study the synergy between both drugs was performed. Time-kill assays using both strains at 6 log CFU/ml, colistin and oxyclozanide were tested alone and in combination with sub-minimal inhibitory concentration (MIC) of colistin (0.25 µg/ml for CECT 997 strain and 16 µg/ml for KPc21 strain) and oxyclozanide at 2 µg/ml. Analysis of KPc21 and CECT 997 strains cell walls in presence of 2 µg/ml oxyclozanide during 24 h by transmission electron microscopy (TEM) was performed. Permeability assays and outer membrane proteins (OMPs) profile analysis by SDS-PAGE of both strains were performed.Results: Checkerboard assay showed a synergic effect between colistin and oxyclozanide against the KPc21 strain (Fold change = 8), but not for CECT 997 strain (Fold change = 2). Time-kill assays showed a synergic effect between colistin and oxyclozanide against the KPc21 strain (decreasing the bacterial growth by 3.24 log CFU/mL) at 24 h, but not against the CECT 997 strain whose bacterial growth was reduced by 0.45 log CFU/mL. Incubation with oxyclozanide at 24 h did not cause change on the OMPs profile of both strains. Futhermore, the images from TEM showed that oxyclozanide disrupted the bacterial cell envelope affecting its permeability. The membrane permeabilization assay confirmed these data, in which the Col-R strain had higher membrane permeability.Conclusions: From these in vitro data, we concluded that oxyclozanide potentiates the bactericidal activity of colistin by disrupting the bacterial cell envelope. For this reason, oxyclozanide would be a good adjuvant for colistin to treating the infections caused by K. pneumoniae

Antibacterial activity of colloidal silver against Gram-negative and Gram-positive bacteria.

Motivation: Treatment of multidrug-resistant (MDR) bacteria represent a challenge for clinicians and public health authorities. Due to the emergence of resistance to a wide variety of antibiotics new alternative therapies are needed. Silver has been used to treat bacterial infections since antiquity due to its known antimicrobial properties [1]. The objective of this project was to study in vitro the activity of colloidal silver against Gram-negative and Gram-positive bacteria.Methods: Gram-negative bacteria [Acinetobacter baumannii (n=44), Pseudomonas aeruginosa (n=25) and Escherichia coli (n=79)] and Gram-positive bacteria [Staphylococcus aureus (n=34), Syaphylococcus epidermidis (n=14) and Enterococcus spp. (n=15)] were used. All strains were grown in a Mueller-Hinton Broth (MHB) at 37°C for 20-24 h. Minimal Inhibitory Concentration (MIC) was determined for all strains by using microdilution assay. To monitore the antibacterial activity, time-kill curve assays were performed on MHB at colloidal silver concentrations of 0.5x, 1x and 2x MIC with starting inoculum of 1x10^6 colony-forming units (cfu)/mL. Reactive Oxygen Species (ROS) production was measured at 6, 20 and 24 hours at colloidal silver concentrations of 0.25x, 0.5x and 1x MIC.Results:  Colloidal silver MIC range was from 4-8 mg/L for both Gram-negative and Gram-positive bacteria. Colloidal silver showed bactericidal activity against Gram-negative bacteria. However, it showed bacteriostatic activity against Gram-positive bacteria. For A. baumannii (Ab11 and ATCC 17978 strains), P. aeruginosa (Pa238 and Pa01 strains), and E. coli (mcr-1 positive strain) colloidal silver was bactericidal at 1x, and 2x MIC at 24h. However, at 24h, E. coli (ATCC 25922 strain) showed a regrowth at 0.5x, 1x and 2x MIC. Incubation of bacterial strains with colloidal silver led to a significant increase in ROS production at 24h in Gram-negative bacteria.Conclusions: Colloidal silver showed in vitro activity against these kind of pathogens, especially against Gram-negative bacteria. These results suggest that colloidal silver could be a new alternative for treatment of infections caused by MDR pathogens

Combating virulence of Gramnegative bacilli by OmpA inhibition

Preventing the adhesion of pathogens to host cells provides an innovative approach to tackling multidrug-resistant bacteria. In this regard, the identifcation of outer membrane protein A (OmpA) as a key bacterial virulence factor has been a major breakthrough. The use of virtual screening helped us to identify a cyclic hexapeptide AOA-2 that inhibits the adhesion of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli to host cells and the formation of bioflm, thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. Inhibition of OmpA ofers a strategy as monotherapy to address the urgent need for treatments for infections caused by Gram-negative bacilli.Instituto de Salud Carlos III, Ministerio de Economía y Competitividad REIPI RD12/0015/0001Unión Europea ES P201431400Ministerio de Economía y Competitividad CP15/01358Junta de Andalucía CTS-6173/12 y 2014LLAV-00064Junta de Andalucía PI12–006

Impacto de la modificación de la respuesta inmunitaria por la lisofosfatidilcolina en la eficacia de la terapia antibiótica en un modelo experimental de sepsis peritoneal y de neumonía por Pseudomonas aeruginosa

Introduction: Immune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. Methods: The imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime- resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF- and IL-10 levels were determined in murine mod- els of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. Results: In the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (−3.45 and −3.56 log10 CFU/g; P < 0.05) to a greater extent than ceftazidime monotherapy, while LPC + imipenem maintained the imipenem efficacy (−1.66 and −1.45 log10 CFU/g; P > 0.05). In the pneumonia model, LPC + ceftazidime or LPC + imipenem reduced the lung Pa238 concentrations (−2.37 log10 CFU/g, P = 0.1, or −1.35 log10 CFU/g, P = 0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC + imipenem and LPC+ceftazidime significantly decreased and increased the TNF- and IL-10 levels, respectively, in com- parison with the untreated controls and monotherapies. Conclusions: These results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.introducción: La estimulación de la respuesta inmunitaria podría ser adyuvante al tratamiento antimi- crobiano. En este estudio, hemos evaluado el impacto de la modificación de la respuesta inmunitaria por la lisofosfatidilcolina (LPC), combinada con imipenem ó ceftazidima, en modelos murinos de sepsis peritoneal (SP) y de neumonía por Pseudomonas aeruginosa (P. aeruginosa).Métodos: La cepa sensible a imipenem y ceftazidima (Pa39) y la cepa resistente a ambos antibióticos (Pa238) fueron usadas. Los parámetros farmacocinéticos/farmacodinámicos de ceftazidima fueron deter- minados. La eficacia terapéutica y los niveles de TNF- and IL-10 fueron determinados en los modelos murinos de SP y de neumonía por Pa39 y Pa238 y tratados con LPC, imipenem o ceftazidima, en monoter- apia ó en combinación. Resultados: En el modelo de SP, LPC + ceftazidima redujo la concentración de Pa238 en el bazo y el pulmón (–3,45 y –3,56 log10 UFC/g; p < 0,05) en comparación con ceftazidima, mientras LPC + impenem mantuvo la eficacia de imipenem (–1,66 y –1,45 log10 UFC/g; p > 0,05). En el modelo de neumonía, LPC + ceftazidima o LPC + imipenem redujo la concentración de Pa238 en pulmón (–2,37 log10 UFC/g, p = 0,1 o –1,35 log10 UFC/g, p = 0,75). Para Pa39, no se observó diferencia estadística significativa entre la terapia combinada y la monoterapia en los modelos de SP y de neumonía. Además, LPC + imipenem y LPC + ceftazidime redujeron y aumentaron los niveles de TNF- y IL-10, respectivamente, en comparación con los controles no tratados y las monoterapias. Conclusiones: Estos resultados demuestran el impacto de la modificación de la respuesta inmunitaria por LPC en combinación con antibióticos en el pronóstico de las infecciones por P. aeruginosa ceftazidima- resistente

Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

Acinetobacter baumannii is a significant human pathogen associated with hospital-acquired infections. While adhesion, an initial and important step in A. baumannii infection, is well characterized, the intracellular trafficking of this pathogen inside host cells remains poorly studied. Here, we demonstrate that transcription factor EB (TFEB) is activated after A. baumannii infection of human lung epithelial cells (A549). We also show that TFEB is required for the invasion and persistence inside A549 cells. Consequently, lysosomal biogenesis and autophagy activation were observed after TFEB activation which could increase the death of A549 cells. In addition, using the Caenorhabditis elegans infection model by A. baumannii, the TFEB orthologue HLH-30 was required for survival of the nematode to infection, although nuclear translocation of HLH-30 was not required. These results identify TFEB as a conserved key factor in the pathogenesis of A. baumannii.Consejería de Innovación, Ciencia y EmpresaInstituto de Salud Carlos IIIMinisterio de Economía, Industria y Competitividad. Subdirección General de Redes y Centros de Investigación CooperativaSpanish Ministry of Economy and Competitivenes

Synergistic Activity of Niclosamide in Combination With Colistin Against Colistin-Susceptible and Colistin-Resistant Acinetobacter baumannii and Klebsiella pneumoniae

Colistin is among the few antibiotics effective against multidrug-resistant Acinetobacter baumannii and Klebsiella pneumoniae clinical isolates. However, in the last few years, colistin-resistant A. baumannii and K. pneumoniae strains have emerged. Therefore, combination therapies, between colistin and other old drugs, restoring the activity of colistin are required. The main objective of this study was to analyse the activity of niclosamide, an anthelmintic drug, in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) A. baumannii and K. pneumoniae. The MIC were determined by microdilution assay and the time-kill curves were performed. The zeta potential of Col-S and Col-R of A. baumannii and K. pneumoniae in presence of niclosamide was assessed. Niclosamide in combination with colistin showed improved activity against Col-S and Col-R A. baumannii and K. pneumoniae. Time-killing curves showed synergic activity between niclosamide and colistin against Col-S and Col-R A. baumannii and K. pneumoniae, especially when niclosamide or colistin was added for second time at 4 h of the 24 h killing curve. Col-R A. baumannii and K. pneumoniae in presence of niclosamide exhibited a greater negative charge (−34.95 ± 0.35 mV and −38.85 ± 0.92 mV; P < 0.05) than Col-R A. baumannii and K. pneumoniae in absence of niclosamide (−26.85 ± 3.65 mV and −35.27 ± 0.72 mV). These data suggest that niclosamide might be combined with colistin, being a potential alternative for treatment of Col-R Gram-negative bacilli infections.Instituto de Salud Carlos IIIProyectos de Investigacion en Salud PI16/01378Ministerio de Economía y Competitividad CP15/0135

Corrigendum: Drug Repurposing for the Treatment of Bacterial and Fungal Infections.

[This corrects the article DOI: 10.3389/fmicb.2019.00041.]

Tamoxifen and its metabolites antibacterial activities

The development of new strategic therapies for multidrug-resistant bacteria, like the use of non-antimicrobial approaches and/or drugs repurposing to be used as monotherapies or in combination with clinically relevant antibiotics, has become an urgent need. A therapeutic alternative for infections by multidrug-resistant Gram-negative bacilli (MDR-GNB) is immune system modulation to improve the infection clearance. We showed that immunocompetent mice infected by Acinetobacter baumannii, Pseudomonas aeruginosa or Escherichia coli in peritoneal sepsis models and treated with tamoxifen at 80 mg/kg/d for three days reduced the release of MCP-1 and its signalling pathway IL-18 and phosphorylated ERK1/2. This reduction of MCP-1 induced the reduction of migration of inflammatory monocytes and neutrophils from bone marrow to blood. Indeed, the treatment with tamoxifen in murine peritoneal sepsis models reduced the bacterial load in tissues and blood; and increased the mice survival from 0% to 60-100%. Tamoxifen treatment of neutropenic mice infected by these pathogens increased mice survival up to 20-60%. Furthermore, susceptibility and time-kill assays showed that the metabolites of tamoxifen, N-desmethyltamoxifen, hydroxytamoxifen and endoxifen, the three together exhibited MIC90 values of 16 mg/L and were bactericidal against clinical isolates of A. baumannii and E. coli. This antimicrobial activity of tamoxifen metabolites parallels' an increased membrane permeability of A. baumannii and E. coli without affecting their outer membrane proteins profiles. Together, these data showed that tamoxifen present a therapeutic efficacy against MDR A. baumannii, P. aeruginosa and E. coli in experimental models of infections and can be repurposed as new treatment for GNB infections.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grants CP15/00132, PI16/01378 and PI19/01453) and by Plan Nacional de I+D+i 2013‐ 2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009) ‐ co‐ financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014‐2020. Younes Smani is supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132)