Non-antimicrobial strategies for the prevention and treatment of infections by multidrug-resistant gram-negative bacilli

Due to the increased of antimicrobial resistance rates and the difficulty of having effective treatment for infections caused by Gram-negative bacilli (GNB) such as Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli, it is necessary to develop non-antimicrobial therapeutic alternatives, which can be used together with the scarce and non-optimal antimicrobial available. In this doctoral thesis, and in order to stop the evolution of the bacterial infection, we aimed to evaluate two therapeutic alternatives: i) blocking the virulence factors of A. baumannii, P. aeruginosa and E. coli and ii) modulating the host immune system. Regarding the first therapeutic approach, we performed the design and evaluation of outer membrane protein A (OmpA) inhibitors on the interaction of A. baumannii, P. aeruginosa and E. coli with the host to block the mechanisms by which this protein produces the infection. Thus, we studied in vitro the effect of the lead peptide AOA-2 on the interaction between A. baumannii, P. aeruginosa and E. coli and human lung epithelial cells (A549) by adherence, immunofluorescence, fibronectin binding, and cell viability assays, and also we tested the effect of AOA-2 on biofilm formation by GNB. In vivo, the therapeutic efficacy of AOA-2 against A. baumannii, P. aeruginosa and E. coli was evaluated. Moreover, the synergy between AOA-2 and colistin by microdilution assay and time-kill curves was studied, and the outer membrane proteins profile in the presence or absence of the AOA-2 was analyzed. Consequently, we constructed an A. baumannii knockout deficient in the omp25 gene and its complemented strain to perform the same experiments. Finally, we evaluated in vivo the therapeutic efficacy of AOA-2 in combination with sub-optimal doses of colistin in peritoneal sepsis caused by A. baumannii. We found that AOA-2 prevented the adhesion of A. baumannii, P. aeruginosa and E. coli to A549 cells protecting them from death, and also reduced biofilm formation by these pathogens. In addition, in the murine peritoneal sepsis model caused by A. baumannii, P. aeruginosa and E. coli, AOA-2 treatment significantly reduced bacteremia, bacterial load in spleen and lung, and mice mortality rates. Regarding the combination of AOA-2 with colistin, the presence of AOA-2 increases the susceptibility of colistin MICs for colistin-susceptible and colistin-resistant A. baumannii strains. Time-kill curves showed a synergistic activity between AOA-2 and colistin, and the protein profile exhibited an overexpression of the Omp25 protein in the strains treated with AOA-2. In vivo, AOA-2 in combination with colistin reduced bacterial spleen and lung loads, bacteremia and mortality rates, in comparison with the monotherapy with colistin. Regarding the second therapeutic approach, we evaluated the effect of lysophosphatidylcholine (LPC), a chemoattractant immunomodulatory factor which stimulates cells from the immune system, as an adjuvant treatment with antimicrobials to treat infections caused by susceptible and MDR strains of A. baumannii and P. aeruginosa in experimental murine models of peritoneal sepsis and pneumonia. Pharmacokinetics and pharmacodynamics parameters of colistin, tigecycline, imipenem and ceftazidime, and minimal lethal dose (MLD) of each strain were determined. In murine experimental models of peritoneal sepsis and pneumonia, mice were pretreated with LPC, infected with MLD of the correspondence strain, and treated or not with antimicrobials. We found that LPC in combination with colistin, tigecycline or imipenem shows beneficial effects in infections caused by susceptible and MDR A. baumannii. Furthermore, the combination of LPC with ceftazidime or imipenem improves the prognosis of infections caused by MDR P. aeruginosa. The data of this doctoral thesis indicate that both the blocking of bacterial virulence factors and the stimulation of the immune system with AOA-2 and LPC, respectively, alone or in combination with antimicrobials, could protect against infections by GNB.Premio Extraordinario de Doctorado U

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

Papel del Factor de Transcripción EB (TFEB) en la entrada de Acinetobacter baumannii en el huésped

Motivación:El sistema endosoma/lisosoma está implicado en procesos fundamentales tales como la secreción, reparación de la membrana plasmática, señalización y en el metabolismo energético(1).El factor de transcripción EB (TFEB) forma parte de este sistema (2) y podría estar implicado en la entrada y la persistencia de Acinetobacter baumannii en el huésped mediante la activación del mismo. A.baumannii es un agente infeccioso de gran importancia clínica que está asociado con las infecciones nosocomiales (3). Hasta el momento no hay trabajos realizados para observar dicho proceso. El objetivo de este estudio es determinar el papel del TFEB en la entrada y la persistencia de A.baumannii en el huésped mediante el silenciamiento del ARN del TFEB y su sobreexpresión in vitro. Métodos: Se utilizaron células epiteliales de pulmón humano (A549) que fueron infectadas con la cepa A.baumannii (ATCC 17978 con 108 ufc/ml) para observar los diferentes objetivos del proyecto. Para todos los ensayos en primer lugar se cultivaron las células A549 en placas con medio de cultivo completo durante 24 h para realizar ensayos de silenciamiento, expresión y sobreexpresión del TFEB. Para efectuar el silenciamiento del ARN del TFEB o su sobreexpresión se realizó una transfección con el siRNA del TFEB o con un plásmido pEGFP-N1-TFEB durante 48 o 24 h, respectivamente, y luego realizamos ensayos de adherencia e invasión bacteriana en las células A549 durante 2 h. La expresión del TFEB se determinó por Westerm Blot. También se llevó a cabo ensayos de inmunomarcaje con diferentes anticuerpos para observar las diferencias de expresión del TFEB entre células infectadas y no infectadas durante 30 y 120 min.Resultados: Se observó que el silenciamiento del ARN del TFEB disminuye la entrada bacteriana en las células epiteliales (64% vs. control). Todo lo contrario en el caso de la sobreexpresión del TFEB que vimos que hay una mayor invasión bacteriana (266% vs. control). Referente a los ensayos de expresión del TFEB vemos un aumento progresivo de la expresión del TFEB tras 30 y 120 min de la infección con respecto a las células que no han sido infectadas.Conclusiones: Los resultados obtenidos en este estudio permiten concluir que el TFEB está implicado en la entrada de A.baumannii en el huésped. Esto puede abrir nuevos caminos para encontrar inhibidores de estas vías y así poder evitar las infecciones causadas por este tipo de bacterias, puesto que hoy en día es un patógeno resistente

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

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

Risk categories in COVID-19 based on degrees of inflammation: data on more than 17,000 patients from the Spanish SEMI-COVID-19 registry

Background: the inflammation or cytokine storm that accompanies COVID-19 marks the prognosis. This study aimed to identify three risk categories based on inflammatory parameters on admission. Methods: retrospective cohort study of patients diagnosed with COVID-19, collected and followed-up from 1 March to 31 July 2020, from the nationwide Spanish SEMI-COVID-19 Registry. The three categories of low, intermediate, and high risk were determined by taking into consideration the terciles of the total lymphocyte count and the values of C-reactive protein, lactate dehydrogenase, ferritin, and D-dimer taken at the time of admission. Results: a total of 17,122 patients were included in the study. The high-risk group was older (57.9 vs. 64.2 vs. 70.4 years; p < 0.001) and predominantly male (37.5% vs. 46.9% vs. 60.1%; p < 0.001). They had a higher degree of dependence in daily tasks prior to admission (moderate-severe dependency in 10.8% vs. 14.1% vs. 17%; p < 0.001), arterial hypertension (36.9% vs. 45.2% vs. 52.8%; p < 0.001), dyslipidemia (28.4% vs. 37% vs. 40.6%; p < 0.001), diabetes mellitus (11.9% vs. 17.1% vs. 20.5%; p < 0.001), ischemic heart disease (3.7% vs. 6.5% vs. 8.4%; p < 0.001), heart failure (3.4% vs. 5.2% vs. 7.6%; p < 0.001), liver disease (1.1% vs. 3% vs. 3.9%; p = 0.002), chronic renal failure (2.3% vs. 3.6% vs. 6.7%; p < 0.001), cancer (6.5% vs. 7.2% vs. 11.1%; p < 0.001), and chronic obstructive pulmonary disease (5.7% vs. 5.4% vs. 7.1%; p < 0.001). They presented more frequently with fever, dyspnea, and vomiting. These patients more frequently required high flow nasal cannula (3.1% vs. 4.4% vs. 9.7%; p < 0.001), non-invasive mechanical ventilation (0.9% vs. 3% vs. 6.3%; p < 0.001), invasive mechanical ventilation (0.6% vs. 2.7% vs. 8.7%; p < 0.001), and ICU admission (0.9% vs. 3.6% vs. 10.6%; p < 0.001), and had a higher percentage of in-hospital mortality (2.3% vs. 6.2% vs. 23.9%; p < 0.001). The three risk categories proved to be an independent risk factor in multivariate analyses. Conclusion: the present study identifies three risk categories for the requirement of high flow nasal cannula, mechanical ventilation, ICU admission, and in-hospital mortality based on lymphopenia and inflammatory parameters

Healthcare workers hospitalized due to COVID-19 have no higher risk of death than general population. Data from the Spanish SEMI-COVID-19 Registry

Aim To determine whether healthcare workers (HCW) hospitalized in Spain due to COVID-19 have a worse prognosis than non-healthcare workers (NHCW). Methods Observational cohort study based on the SEMI-COVID-19 Registry, a nationwide registry that collects sociodemographic, clinical, laboratory, and treatment data on patients hospitalised with COVID-19 in Spain. Patients aged 20-65 years were selected. A multivariate logistic regression model was performed to identify factors associated with mortality. Results As of 22 May 2020, 4393 patients were included, of whom 419 (9.5%) were HCW. Median (interquartile range) age of HCW was 52 (15) years and 62.4% were women. Prevalence of comorbidities and severe radiological findings upon admission were less frequent in HCW. There were no difference in need of respiratory support and admission to intensive care unit, but occurrence of sepsis and in-hospital mortality was lower in HCW (1.7% vs. 3.9%; p = 0.024 and 0.7% vs. 4.8%; p<0.001 respectively). Age, male sex and comorbidity, were independently associated with higher in-hospital mortality and healthcare working with lower mortality (OR 0.211, 95%CI 0.067-0.667, p = 0.008). 30-days survival was higher in HCW (0.968 vs. 0.851 p<0.001). Conclusions Hospitalized COVID-19 HCW had fewer comorbidities and a better prognosis than NHCW. Our results suggest that professional exposure to COVID-19 in HCW does not carry more clinical severity nor mortality

AOA-2 Derivatives as Outer Membrane Protein A Inhibitors for Treatment of Gram-Negative Bacilli Infections

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (Grants PI15/01358, 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, Inno-vación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by the European Development Regional Fund “A way to achieve Europe,” Operative program Intelligent Growth 2014–2020. YS was supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132).Ye

AOA-2 Derivatives as Outer Membrane Protein A Inhibitors for Treatment of Gram-Negative Bacilli Infections

Previously, we identified that a cyclic hexapeptide AOA-2 inhibited the interaction of Gram-negative bacilli (GNB) like Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli to host cells thereby preventing the development of infection in vitro and in a murine sepsis peritoneal model. In this work, we aimed to evaluate in vitro a library of AOA-2 derivatives in order to improve the effect of AOA-2 against GNB infections. Ten AOA-2 derivatives were synthetized for the in vitro assays. Their toxicities to human lung epithelial cells (A549 cells) for 24 h were evaluated by determining the A549 cells viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The effect of these peptide derivatives and AOA-2 at 250, 125, 62.5, and 31.25 μg/mL on the attachment of A. baumannii ATCC 17978, P. aeruginosa PAO1 and E. coli ATCC 25922 strains to A549 cells was characterized by adherence and viability assays. None of the 10 derivatives showed toxicity to A549 cells. RW01 and RW06 have reduced more the adherence of ATCC 17978, PAO1 and ATCC 2599 strains to A549 cells when compared with the original compound AOA-2. Moreover, both peptides have increased slightly the viability of infected A549 cells by PAO1 and ATCC 25922 than those observed with AOA-2. Finally, RW01 and RW06 have potentiated the activity of colistin against ATCC 17978 strain in the same level with AOA-2. The optimization program of AOA-2 has generated two derivatives (RW01 and RW06) with best effect against interaction of GNB with host cells, specifically against P. aeruginosa and E. coli.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (Grants PI15/01358 and PI16/01378) 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, Inno-vación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co-financed by the European Development Regional Fund “A way to achieve Europe,” Operative program Intelligent Growth 2014–2020. YS was supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132)