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 effect of ceftazidime-avibactam pressure on ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae clinical isolates.

Motivation: Infections caused by KPC-producing Klebsiella pneumoniae represent a challenge due to the limited available treatement choices. In this context, ceftazidime-avibactam (CAZ-AVI) is postulated as an alternative treatment effective against class A beta-lactamases such as KPC [1]. But, recent data reported the failure of CAZ-AVI treatment of infections by KPC-producing K. pneumoniae due to the development of CAZ-AVI resistance [2]. However, little is known concernig the CAZ-AVI resistance developement by CAZ-AVI selective pressure. Here, we aimed to determinate in vitro whether the exposure of KPC-producing K. pneumoniae clinical isolates to CAZ-AVI subinhibitory concentrations could lead the selection of CAZ-AVI resistant isolates. Methods: Seventeen KPC-producing K. pneumoniae clinical isolates (7 KPC-2, 9 KPC-3 and 1 KPC-11) were analyzed. Minimum inhibitory concentrations (MICs) of CAZ-AVI were determined by broth microdilution using a fixed AVI cocentration of 4 mg/L [3]. Moreover, these isolates were further exposed to increasing concentrations of CAZ and fixed 4 mg/L of AVI, from a sub-MIC up to 256/4 mg/L of CAZ-AVI (or the concentration able to kill the bacterial isolate) at 37ºC with shaking during 24h. New MICs to CAZ-AVI were determined in each condition and after 15 days without CAZ-AVI pressure. Therefore, in order to demonstrated that blaKPC gene is responsible for acquisition of CAZ-AVI resistance in KPC-producing K. pneumoniae, blaKPC-2 and blaKPC-3 were cloned into a reference K. pneumoniae CECT 997 strain. Resistance or susceptibility were determined according to EUCAST criteria [3]. Results: All (17/17, 100%) KPC-producing K. pneumoniae isolates were able to grow at high concentrations of CAZ-AVI (≥64/4 mg/L), increasing their resistance to CAZ-AVI ≥8-fold. Likewise, fifteen of the 17 (88.2%) resistant isolates maintained the acquired CAZ-AVI resistance 15 days after without CAZ-AVI pressure. In addition, the CECT 997 mutants with blaKPC-2 or blaKPC-3 were able to grow up to 256/4 mg/L of CAZ-AVI, displaying and maintaining CAZ-AVI MIC shift from <0.01/4 mg/L (susceptible) to 512/4 mg/L (resistant). Conclusions: These data suggest that exposure of KPC-producing K. pneumoniae to subinhibitory CAZ-AVI concentrations could lead to the selection of CAZ-AVI resistance and this resistance is stable over the time

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

In vitro and in vivo evaluation of two combined β-lactamase inhibitors against carbapenem-resistant Acinetobacter baumannii

The objective of this study was to evaluate the in vitro and in vivo efficacy of clavulanic acid (C/A) in combination with tazobactam against clinical strains of carbapenem-resistant Acinetobacter baumannii. The MIC of 24 clinical strains of A. baumannii was determined, and a checkerboard assay and time-kill curve analysis were performed in selected strains to determine the synergy between C/A and tazobactam. The efficacy of C/A in monotherapy and in combination with tazobactam was evaluated in vitro in cell culture experiments and in a murine peritoneal sepsis model. The C/A and C/A plus tazobactam MIC50 were 128 and <1 mg/L, respectively. The checkerboard assay showed that tazobactam (4 and 8 mg/L) demonstrated synergy with C/A against A. baumannii Ab40, an OXA-24 producer strain, and Ab293, a lacking OXA β-lactamase strain. The time-kill curve assay showed both bactericidal and synergistic effects against Ab40 and Ab293, with C/A 1xMIC and tazobactam (4 and 8 mg/L) at 24 h. In the murine peritoneal sepsis model with Ab293 strain, the combination of C/A and tazobactam reduced bacterial loads in tissues and blood by 2 and 4 log10 CFU/g or mL compared with C/A alone. Combining C/A with tazobactam could be considered as a potential alternative strategy to treat A. baumannii in some cases, and future work with more strains is needed to confirm this possibility.Centro Andaluz de Biología del DesarrolloUniversidad Pablo de Olavid

Drugs Repurposing for Multi-Drug Resistant Bacterial Infections

Different institutions recognized that antimicrobial resistance is a global health threat that has compounded by the reduction in the discovery and development of new antimicrobial agents. Therefore, the development of new antimicrobial therapeutic strategies requires immediate attention to avoid the 10 million deaths predicted to occur by 2050 as a result of multidrug-resistant (MDR) bacteria. Despite the great interest in the development of repurposing drugs, only few repurposing drugs are under clinical development against Gram-negative critical-priority pathogens. In this chapter, we aim: (i) to discuss the therapeutic potential of the repurposing drugs for treating MDR bacterial infections, (ii) to summarize their mechanism of action, and (iii) to provide an overview for their preclinical and clinical development against these critical-priority pathogens

Phylogeny, Resistome, and Virulome of Escherichia coli Causing Biliary Tract Infections

Escherichia coli is the most frequent Gram-negative bacilli involved in intra-abdominal infections. However, despite high mortality rates associated with biliary tract infections due to E. coli, there is no study focusing on this pathogen. In this study, we have characterized a group of 15 E. coli isolates obtained from 12 patients with biliary tract infections. Demographic and clinical data of the patients were recovered. Phylogeny, resistome, and virulome analysis through whole genome sequencing and biofilm formation were investigated. Among the 15 E. coli isolates, no predominant sequence type (ST) was identified, although 3 of them belonged to unknown STs (20%). Resistance to ampicillin, amoxicillin/clavulanic acid, cotrimoxazole, and quinolones was more present in these isolates; whereas, third and fourth generation cephalosporins, carbapenems, amikacin, tigecycline, and colistin were highly active. Moreover, high diversity of virulence factors has been found, with sfa, fimH, and gad the most frequently detected genes. Interestingly, 26.6% of the E. coli isolates were high biofilm-producers. Altogether, our data characterized for the first time E. coli isolates associated with biliary tract infections in terms of genomic relationship, resistome, and virulome.España, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades (CP15/00132)España, 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 (RD16/0016/0009

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

Perspectives for clinical use of engineered human host defense antimicrobial peptides.

Infectious diseases caused by bacteria, viruses or fungi are among the leading causes of death worldwide. The emergence of drug-resistance mechanisms, especially among bacteria, threatens the efficacy of all current antimicrobial agents, some of them already ineffective. As a result, there is an urgent need for new antimicrobial drugs. Host defense antimicrobial peptides (HDPs) are natural occurring and well-conserved peptides of innate immunity, broadly active against Gram-negative and Gram-positive bacteria, viruses and fungi. They also are able to exert immunomodulatory and adjuvant functions by acting as chemotactic for immune cells, and inducing cytokines and chemokines secretion. Moreover, they show low propensity to elicit microbial adaptation, probably because of their non-specific mechanism of action, and are able to neutralize exotoxins and endotoxins. HDPs have the potential to be a great source of novel antimicrobial agents. The goal of this review is to provide an overview of the advances made in the development of human defensins as well as the cathelicidin LL-37 and their derivatives as antimicrobial agents against bacteria, viruses and fungi for clinical use

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