Respuesta inmunitaria celular y eficacia del tratamiento con células de memoria e IgM en la neumonía experimental por Pseudomonas aeruginosa y Acinetobacter baumannii

Acinetobacter baumannii y Pseudomonas aeruginosa son bacilos Gram-negativos (BGN) no fermentadores, y paradigmas de las resistencias microbianas, encontrándose dentro del grupo de microorganismos denominados ESKAPE por la Organización Mundial de la Salud (OMS) (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, P. aeruginosa, y especies de Enterobacter). Las infecciones causadas por estos patógenos son comunes en las Unidades de Cuidados Intensivos (UCI), así como en pacientes con alteraciones anatómicas y/o funcionales de órganos en el caso de la P. aeruginosa, y están asociadas con elevadas tasas de morbi-mortalidad, estancias hospitalarias prolongadas, así como costes elevados. El aumento exponencial de las resistencias microbianas al que nos enfrentamos mundialmente, lleva en ocasiones, a que no existan alternativas terapéuticas óptimas en el tratamiento de estas infecciones. En este contexto, la OMS publicó en 2017 una alerta mundial sobre la necesidad urgente de búsqueda de nuevas alternativas terapéuticas para la prevención, diagnóstico y tratamiento de este tipo de infecciones. En este trabajo, nos centramos en una de las alternativas no antimicrobianas a explorar, en concreto en la inmunoterapia como coadyuvante a antimicrobianos, ya sea mediante linfocitos de memoria o mediante inmunoglobulinas enriquecidas en IgM. La modulación de la respuesta inmune se ha demostrado como una estrategia terapéutica ventajosa en la prevención y tratamiento de infecciones, la supresión de respuestas autoinmunes e inflamatorias y/o la estimulación de la inmunidad antitumoral en pacientes con cáncer. En este trabajo se describe la caracterización de modelos murinos de neumonía no letales por cepas clínicas multirresistentes de A. baumannii y P. aeruginosa con diferentes patrones de resistencia a colistina. Además, se describe la respuesta inmune celular desencadenada por este tipo de infecciones, con la finalidad última de ser capaces de modular la respuesta inmune del huésped para diseñar una terapia coadyuvante a los antimicrobianos en el tratamiento de este tipo de infecciones. En este trabajo, observamos que neutrófilos y macrófagos son capaces de controlar y resolver de manera precoz la infección. Además de describir el efecto de la mucina porcina en la respuesta celular inmune del huésped, provocando daños tisulares y reclutamiento celular. Asimismo, en este trabajo, evaluamos la eficacia del tratamiento con linfocitos de memoria en modelos murinos letales de neumonía causada por BGN no fermentadores con distinta susceptibilidad a colistina, siendo eficaz en la reducción de la concentración bacteriana en pulmón y sangre, así como en la disminución del porcentaje de mortalidad y bacteriemia. Finalmente, evaluamos en un modelo murino letal de neumonía causada por cepas clínicas multirresistentes de P. aeruginosa, con diferente patrón de resistencia a colistina, la eficacia del tratamiento con inmunoglobulinas intravenosa enriquecida en IgM en combinación con antimicrobianos, la cual es eficaz en la reducción de la concentración bacteriana en pulmón y sangre así como en la disminución del porcentaje de mortalidad y bacteriemia. En conclusión, y con la cautela necesaria que en todo resultado obtenido de estudios en modelos animales debe tenerse a la hora de extrapolar conclusiones al ser humano, los resultados in vivo del presente estudio sugieren que la inmunoterapia con linfocitos de memoria y/o inmunoglobulinas intravenosas enriquecida en IgM, pueden ser una opción terapéutica, en combinación con antimicrobianos, para el tratamiento de neumonías causadas por cepas de A. baumannii y P. aeruginosa, independientemente de su susceptibilidad a colistina

In vitro Activity of Pentamidine Alone and in Combination With Aminoglycosides, Tigecycline, Rifampicin, and Doripenem Against Clinical Strains of Carbapenemase-Producing and/or Colistin-Resistant Enterobacteriaceae

Enterobacteriaceae cause different types of community- and hospital-acquired infections. Moreover, the spread of multidrug-resistant Enterobacteriaceae is a public health problem and the World Health Organization pointed them among the pathogens in which the search of new antibiotics is critical. The objective of this study was to analyze the in vitro activity of pentamidine alone and in combination with gentamicin, tobramycin, amikacin, tigecycline, rifampicin, or doripenem against eight clinical strains of carbapenemase-producing and/or colistin-resistant Enterobacteriaceae: five carbapenemase-producing Klebsiella pneumoniae, one carbapenemase-producing Escherichia coli, and two colistin-resistant Enterobacter cloacae. MIC and MBC were determined following standard protocols. MIC results were interpreted for all the antibiotics according to the EUCAST breakpoints but for rifampicin in which the French FSM breakpoint was used. Bactericidal and synergistic activity of pentamidine alone and in combination with antibiotics at concentrations of 1xMIC was measured by time-kill curves. For one selected strain, K. pneumoniae OXA-48/CTX-M-15 time-kill curves were performed also at 1/2xMIC of pentamidine. All studies were performed in triplicate. Pentamidine MIC range was 200-800 μg/mL. The 50, 12.5, 62.5, 87.5, and 62.5% of the strains were susceptible to gentamicin, tobramycin, amikacin, tigecycline, and doripenem, respectively. Only the two E. cloacae strains were susceptible to rifampicin. Pentamidine alone at 1xMIC showed bactericidal activity against all strains, except for the E. cloacae 32 strain. The bactericidal activity of pentamidine alone was also observed in combination. The combinations of pentamidine were synergistic against E. cloacae 32 with amikacin and tobramycin at 24 h and with tigecycline at 8 h. Pentamidine plus rifampicin was the combination that showed synergistic activity against more strains (five out of eight). Pentamidine plus doripenem did not show synergy against any strain. At 1/2xMIC, pentamidine was synergistic with all the studied combinations against the K. pneumoniae OXA-48/CTX-M-15 strain. In summary, pentamidine alone and in combination shows in vitro activity against carbapenemase-producing and/or colistin-resistant Enterobacteriaceae. Pentamidine appears to be a promising option to treat infections caused by these pathogens.Plan Nacional de I+D+I 2013-2016 REIPI RD16/0016/0009Instituto de Salud Carlos III. Subdirección General de Redes y Centros de Investigación Cooperativa REIPI RD16/0016/0009Ministerio de Economía, Industria y Competitividad REIPI RD16/0016/0009Spanish Network for Research in Infectious Diseases REIPI RD16/0016/0009European Development Regional Fun

In vitro activity of piperazine derivates against multidrug-resistance bacteria

Motivation: The increasing prevalence of multidrug-resistance represent a serious challenge for clinical management and public health. Multidrug-resistant (MDR) bacteria is a common cause of infections, especially in inmunocompromised patient. Nowadays, colistin has re-emerged as one of the last therapeutic option against these kinds of infections, but colistin resistant strains have been reported, leaving no alternative of treatment. The aim of this work is to study in vitro the activity of piperazine derivates against MDR and colistin resistant bacteria. Methods: Clinical and standard strains: MDR: Acinetobacter baumannii (Ab; n=1), Klebsiella pneumoniae carbapenemases producing (n=4), Pseudomonas aeruginosa (Pa; n=2), Escherichia coli ATCC 25922 (n=1), colistin resistant A. baumannii (n=13). Piperazine derivatives: four different families were tested: 1, 2, 3, and 4. The derivivates were synthesized in the Pharmacy Faculty of Seville. A) Inhibition screening: all strains at a concentration of 5x105 CFU/mL were tested at 50 µM of each derivative. B) Minimal Inhibitory Concentration (MIC): were calculated for the derivates that inhibit the bacterial growth. C) Time-kill curves: were performed for six derivates against two colistin resistant A. baumannii clinical strains.  Results: A) Inhibition was observed only in colistin resistant A. baumannii clinical strains. B) Family 1, inhibited the growth of 46 % (6/13) of the strains. Family 2, inhibited the growth of 30% (4/13) of the strains. Family 3, inhibited the growth of 30% (4/13) of the strains. Family 4, inhibited the growth of 38% (5/13) of the strains. C) Family 1: MIC range was 50-3.12 µM. Family 2: MIC range was 50-6.25 µM. Family 3: MIC range was 50-1.56 µM. Family 4: MIC range was 50-3.12 µM. D) One piperazine derivates presented bactericidal activity at 24 hours against one of the tested strains.  Conclusions: Piperazine derivatives showed in vitro activity against colistin resistant A. baumannii clinical strains. Further studies, in vitro and in vivo need to be performed in order to confirm the activity of the piperazine derivates against infections due to these kinds of infections

In vitro activity of pentamidine alone and in combination with antibiotics against multidrug-resistant clinical Pseudomonas aeruginosa strains

Multidrug-resistant (MDR) Pseudomonas aeruginosa is a public health problem causing both community and hospital-acquired infections, and thus the development of new therapies for these infections is critical. The objective of this study was to analyze in vitro the activity of pentamidine as adjuvant in combinations to antibiotics against seven clinical P. aeruginosa strains. The Minimum Inhibitory Concentration (MIC) was determined following standard protocols, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; however, the gentamicin activity was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The bactericidal in vitro activity was studied at 1×MIC concentrations by time–kill curves, and also performed in three selected strains at 1/2×MIC of pentamidine. All studies were performed in triplicate. The pentamidine MIC range was 400–1600 µg/mL. Four of the strains were MDR, and the other three were resistant to two antibiotic families. The combinations of pentamidine at 1×MIC showed synergistic activity against all the tested strains, except for pentamidine plus colistin. Pentamidine plus imipenem and meropenem were the combinations that showed synergistic activity against the most strains. At 1/2×MIC, pentamidine plus antibiotics were synergistic with all three analyzed strains. In summary, pentamidine in combination with antibiotics showed in vitro synergy against multidrug-resistant P. aeruginosa clinical strains, which suggests its possible use as adjuvant to antibiotics for the therapy of infections from MDR P. aeruginosa.Instituto de Salud Carlos III Proyectos de Investigación en Salud PI18-01842Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, Red Española de Investigación en Enfermedades Infecciosas REIPI RD16 / 0016/0009Fondo Regional de Desarrollo Europeo Una forma de lograr Europa, Operativa programa Crecimiento inteligente 2014-2020. T.CUniversidad de Sevilla. Servicio Andaluz de Salud, Junta de Andalucía C1-0038-2019Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanciado por la Unión Fondo Regional de Desarrollo RD16 / 0016/0009Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanciado por European Development Regional Fund (A Way to Achieve Europe) y por la Red Española de Investigación en Enfermedades Infecciosas JR17 / 0002

Efficacy of Colistin and Its Combination With Rifampin in Vitro and in Experimental Models of Infection Caused by Carbapenemase-Producing Clinical Isolates of Klebsiella pneumoniae.

Despite the relevance of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) infections there are a scarce number of studies to evaluate in vivo the efficacy of combinations therapies. The bactericidal activity of colistin, rifampin, and its combination was studied (time-kill curves) against four clonally unrelated clinical isolates of CP-Kp, producing VIM-1, VIM-1 plus DHA-1(acquired AmpC β-lactamase), OXA-48 plus CTX-M-15 (extended spectrum β-lactamase) and KPC-3, respectively, with colistin MICs of 0.5, 64, 0.5, and 32 mg/L, respectively. The efficacies of antimicrobials in monotherapy and in combination were tested in a murine peritoneal sepsis model, against all the CP-Kp. Their efficacies were tested in the pneumonia model against the OXA-48 plus CTX-M-15 producers. The development of colistin-resistance was analyzed for the colistin-susceptible strains in vitro and in vivo. In vitro, colistin plus rifampin was synergistic against all the strains at 24 h. In vivo, compared to the controls, rifampin alone reduced tissue bacterial concentrations against VIM-1 and OXA-48 plus CTX-M-15 strains; CMS plus rifampin reduced tissue bacterial concentrations of these two CP-Kp and of the KPC-3 strain. Rifampin and the combination increased the survival against the KPC-3 strain; in the pneumonia model, the combination also improved the survival. No resistant mutants appeared with the combination. In conclusion, CMS plus rifampin had a low and heterogeneous efficacy in the treatment of severe peritoneal sepsis model due to CP-Kp producing different carbapenemases, increasing survival only against the KPC-3 strain. The combination showed efficacy in the less severe pneumonia model. The combination prevented in vitro and in vivo the development of colistin resistant mutants.España, Consejería de Salud Junta de Andalucía PI-0622-201

Prevalence and clinical impact of Streptococcus pneumoniae nasopharyngeal carriage in solid organ transplant recipients

Background: S. pneumoniae is the leading cause of community-acquired pneumonia in the solid organ transplant recipient (SOTR); nevertheless, the prevalence of colonization and of the colonizing/infecting serotypes has not been studied in this population. In this context, the aim of the present study was to describe the rate, characteristics, and clinical impact of S. pneumoniae nasopharyngeal carriage. Methods: A prospective observational cohort of Solid Organ Transplant recipients (SOTR) was held at the University Hospital Virgen del Rocío, Seville, Spain with the aim to evaluate the S. pneumoniae colonization and the serotype prevalence in SOTR. Two different pharyngeal swabs samples from 500 patients were included in two different seasonal periods winter and spring/summer. Optochin and bile solubility tests were performed for the isolation of thew strains. Antimicrobial susceptibility studies (MICs, mg/l) of levofloxacin, trimethoprim-sulfamethoxazole, penicillin, amoxicillin, cefotaxime, ceftriaxone, erythromycin, azithromycin and vancomycin for each isolate were determined by E-test strips. Capsular typing was done by sequential multiplex PCR reactions. A multivariate logistic regression analysis of factors potentially associated with pneumococcal nasopharyngeal carriage and disease was performed. Results: Twenty-six (5.6%) and fifteen (3.2%) patients were colonized in winter and spring/summer periods, respectively. Colonized SOT recipients compared to non-colonized patients were more frequently men (79.5% vs. 63.1%, P < 0.05) and cohabitated regularly with children (59% vs. 32.2%, P < 0.001). The most prevalent serotype in both studied periods was 35B. Forty-five percent of total isolates were included in the pneumococcal vaccine PPV23. Trimethoprim-sulfamethoxazole and macrolides were the less active antibiotics. Three patients had non- bacteremic pneumococcal pneumonia, and two of them died. Conclusions: Pneumococcal colonization in SOTR is low with the most colonizing serotypes not included in the pneumococcal vaccines.Pfizer, 2014 ASPIRE Awards in Vaccine Research in Europe (Pfizer Reference # WI191483)Plan Nacional de I + D + i 2013–2016 , Instituto de Salud Carlos III, Ministerio de Economía, Industria y Competitividad REIPI RD16/0016/0009 Fondo Regional de Desarrollo Europeo "Una forma de alcanzar Europa", Programa operativo Crecimiento inteligente 2014–2020

Efficacy of dual carbapenem treatment in a murine sepsis model of infection due to carbapenemase-producing Acinetobacter baumannii

[Objectives] To evaluate the in vivo efficacy of a dual carbapenem combination containing imipenem plus meropenem against carbapenem-resistant Acinetobacter baumannii producing carbapenemases OXA-23 or OXA-58.[Methods] An experimental model of peritonitis using C57BL/6J female mice was developed and the minimum lethal doses were calculated for infections due to OXA-23 or OXA-58 producers of A. baumannii clinical isolates. The efficacies of the carbapenems in monotherapy and in combination were tested.[Results] Meropenem was better than imipenem in mice infected with either of the carbapenem-resistant A. baumannii (CRAb) strains. The combination of meropenem plus imipenem significantly improved the clearance of CRAbs from spleen compared with non-treated groups. The carbapenem-containing combination was better than imipenem for treating mice infected with both carbapenemase producers. In blood, the carbapenem combination significantly decreased the bacterial load of the OXA-23 producers compared with imipenem or meropenem used in monotherapy.[Conclusions] These results suggest that dual carbapenem combination could be an option for the treatment of infections due to carbapenemase-producing A. baumannii such as OXA-23 and OXA-58 producers.This study was carried out as part of our routine work. Authors from the Institute of Biomedicine of Seville, Seville, Spain, are supported 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 Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by European Development Regional Fund A way to achieve Europe, Operative programme Intelligent Growth 2014–2020. T.C.C. is supported by the V Plan Propio of the University of Seville (USE-19631-N). M.E.P.I. is supported by the programme ‘Nicolás Monardes’ (C1-0038–2019), Servicio Andaluz de Salud, Junta de Andalucía, Spain.Peer reviewe

Discovery of niclosamide and its O-alkylamino-tethered derivatives as potent antibacterial agents against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates

Carbapenemase-producing Enterobacteriaceae (CPE) represents the most worrisome evolution of the antibiotic resistance crisis, which is almost resistant to most of available antibiotics. This situation is getting even worse particularly due to the recent emergence of colistin resistance. Herein, niclosamide, an FDA-approved traditional drug, and its novel O-alkylamino-tethered derivatives were discovered as new and potent antibacterial agents against carbapenemase-producing and/or colistin resistant Enterobacteriaceae isolates. Among these molecules, compound 10 (HJC0431) with 4-aminobutyl moiety showed the broad antibacterial activities, effective against 6 strains. In vitro checkerboard and time-kill course studies demonstrated the synergistic effects of the screened compounds with colistin against the corresponding strains with various degrees.This work was supported by UTMB Technology Commercialization Program, and Sanofi Innovation Awards (iAwards), John Sealy Memorial Endowment Fund, and Institute for Translational Sciences (ITS) at UTMB, 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 Economía, Industria y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009), cofinanced by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 20142020, the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (PI15/00489) and Proyectos de Desarrollo Tecnológico en Salud (DTS17/00130), the Spanish Adenovirus Network (AdenoNet, BIO2015/68990-REDT), and the program “Nicolás Monardes” (C-0059-2018) Servicio Andaluz de Salud, Junta de Andalucía.Peer reviewe

In Vitro Activity of Pentamidine Alone and in Combination with Antibiotics against Multidrug-Resistant Clinical Pseudomonas aeruginosa Strains

© 2020 by the authors.Multidrug-resistant (MDR) Pseudomonas aeruginosa is a public health problem causing both community and hospital-acquired infections, and thus the development of new therapies for these infections is critical. The objective of this study was to analyze in vitro the activity of pentamidine as adjuvant in combinations to antibiotics against seven clinical P. aeruginosa strains. The Minimum Inhibitory Concentration (MIC) was determined following standard protocols, and the results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints; however, the gentamicin activity was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. The bactericidal in vitro activity was studied at 1×MIC concentrations by time–kill curves, and also performed in three selected strains at 1/2×MIC of pentamidine. All studies were performed in triplicate. The pentamidine MIC range was 400–1600 μg/mL. Four of the strains were MDR, and the other three were resistant to two antibiotic families. The combinations of pentamidine at 1×MIC showed synergistic activity against all the tested strains, except for pentamidine plus colistin. Pentamidine plus imipenem and meropenem were the combinations that showed synergistic activity against the most strains. At 1/2×MIC, pentamidine plus antibiotics were synergistic with all three analyzed strains. In summary, pentamidine in combination with antibiotics showed in vitro synergy against multidrug-resistant P. aeruginosa clinical strains, which suggests its possible use as adjuvant to antibiotics for the therapy of infections from MDR P. aeruginosa.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (PI18-01842), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía, Industria y Competitividad, 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. T.C.-C. is supported by the V Plan Propio of the University of Seville with a postdoctoral contract as research personnel in training. M.E.P.-I. is a researcher belonging to the program “Nicolás Monardes” (C1-0038-2019), Servicio Andaluz de Salud, Junta de Andalucía, Spain. G.L.-H. has a grant from the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (A Way to Achieve Europe) and by the Spanish Network for Research in Infectious Diseases (grant REIPI RD16/0016/0009). R.Á.-M. has a grant Juan Rodes grant from the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, cofinanced by the European Development Regional Fund (A Way to Achieve Europe) and by the Spanish Network for Research in Infectious Diseases (JR17/00025)

Antimicrobial properties of clay-QACs nanocomposites

XXVII Reunión de la Sociedad Española de Arcillas, Madrid, 19 de Noviembre de 2021.Nanomaterials with inherent antimicrobial and/or biocidal activity are increasingly demanded in fields such as food packaging, medical devices and water purification systems. Clay minerals are nanostructured materials since one of their dimensions is within the range of 1-100 nm. They can be modified by anchoring biocidal molecules on their surface. Quaternary ammonium compounds (QACs) have been reported to have antimicrobial activity, that is dependent on their structure and size (Undabeytia, et al., 2014). In the current work, the bactericidal activity of a series of QACs-clay nanocomposites made of QACs differing in their structure was examinedThe authors acknowledge to Junta de Andalucía for financial support (grant P18-RT-5098)