Targeting Dalbavancin Inoculum Effect: Adjunctive Single Dose of Daptomycin.

INTRODUCTION: Daptomycin (DAP) has proven to be a viable alternative amid vancomycin resistance; however, the use of DAP post vancomycin treatment has led to the development of DAP non-susceptible (DNS) strains. Dalbavancin (DAL), a novel single-dosed lipoglycopeptide, has shown enhanced activity against highly resistant Staphylococcus aureus strains. However, on the basis of previous reports and our observations, DAL does not demonstrate similar activity at high versus low inoculum levels. Therefore, we hypothesized that addition of DAP even at minimal concentrations (single dose on day 1) will lower the inoculum to the level that can be cleared by dalbavancin. METHODS: Isolates from methicillin-resistant S. aureus (MRSA)-infected patients with varying susceptibility profiles were evaluated using broth microdilution methods. Two DNS-VISA strains (vancomycin intermediate resistant S. aureus) and one MRSA strain were further evaluated in a one-compartment PK/PD model using a high starting initial inoculum of 109 CFU/mL as well as low initial inoculum of 107 CFU/mL over 168 h to assess the activity of DAL and DAP monotherapy and in combination. RESULTS: Single therapies were not bactericidal when evaluated in the 168 h in vitro one-compartment model with an initial inoculum of 109; however, the combination of DAL plus single dose of DAP resulted in enhanced killing at the end of the 168-h exposure. DAL single therapy caused reduction in colony counts down to detection limit (2 log10 CFU/ml) at a lower inoculum but did not show enhancement (< 2 log10 CFU/ml) at higher initial inoculums (P < 0.01) for all three strains. Similarly, DAP caused initial bacterial reduction up to 4 log10 CFU/ml with regrowth at about 32 h of exposure, which stayed at initial inoculum levels for the duration of the model for all three strains. CONCLUSIONS: Dalbavancin inoculum effect is a major issue in bacterial infections with high bacterial loads and the combination of DAL plus single dose of DAP showed promise in eradicating resistant S. aureus strains at high inoculums.

Phage-antibiotic combinations against multidrug-resistant Pseudomonas aeruginosa in in vitro static and dynamic biofilm models.

Biofilm-producing Pseudomonas aeruginosa infections pose a severe threat to public health and are responsible for high morbidity and mortality. Phage-antibiotic combinations (PACs) are a promising strategy for combatting multidrug-resistant (MDR), extensively drug-resistant (XDR), and difficult-to-treat P. aeruginosa infections. Ten MDR/XDR P. aeruginosa strains and five P. aeruginosa-specific phages were genetically characterized and evaluated based upon their antibiotic susceptibilities and phage sensitivities. Two selected strains, AR351 (XDR) and I0003-1 (MDR), were treated singly and in combination with either a broad-spectrum or narrow-spectrum phage, phage EM-T3762627-2_AH (EM), or 14207, respectively, and bactericidal antibiotics of five classes in biofilm time-kill analyses. Synergy and/or bactericidal activity was demonstrated with all PACs against one or both drug-resistant P. aeruginosa strains (average reduction: -Δ3.32 log10 CFU/cm2). Slightly improved ciprofloxacin susceptibility was observed in both strains after exposure to phages (EM and 14207) in combination with ciprofloxacin and colistin. Based on phage cocktail optimization with four phages (EM, 14207, E20050-C (EC), and 109), we identified several effective phage-antibiotic cocktails for further analysis in a 4-day pharmacokinetic/pharmacodynamic in vitro biofilm model. Three-phage cocktail, EM + EC + 109, in combination with ciprofloxacin demonstrated the greatest biofilm reduction against AR351 (-Δ4.70 log10 CFU/cm2 from baseline). Of remarkable interest, the addition of phage 109 prevented phage resistance development to EM and EC in the biofilm model. PACs can demonstrate synergy and offer enhanced eradication of biofilm against drug-resistant P. aeruginosa while preventing the emergence of resistance.

Evaluation of Omadacycline Alone and in Combination with Rifampin against Staphylococcus aureus and Staphylococcus epidermidis in an In Vitro Pharmacokinetic/Pharmacodynamic Biofilm Model.

Biofilm-associated infections lead to substantial morbidity. Omadacycline (OMC) is a novel aminomethylcycline with potent in vitro activity against Staphylococcus aureus and Staphylococcus epidermidis, but data surrounding its use in biofilm-associated infections are lacking. We investigated the activity of OMC alone and in combination with rifampin (RIF) against 20 clinical strains of staphylococci in multiple in vitro biofilm analyses, including an in vitro pharmacokinetic/pharmacodynamic (PK/PD) CDC biofilm reactor (CBR) model (simulating human exposures). The observed MICs for OMC demonstrated potent activity against the evaluated strains (0.125 to 1 mg/L), with an increase of MICs generally observed in the presence of biofilm (0.25 to >64 mg/L). Furthermore, RIF was shown to reduce OMC biofilm MICs (bMICs) in 90% of strains, and OMC plus RIF combination in biofilm time-kill analyses (TKAs) exhibited synergistic activity in most of the strains. Within the PK/PD CBR model, OMC monotherapy primarily displayed bacteriostatic activity, while RIF monotherapy generally exhibited initial bacterial eradication, followed by rapid regrowth likely due to the emergence of RIF resistance (RIF bMIC, >64 mg/L). However, the combination of OMC plus RIF produced rapid and sustained bactericidal activity in nearly all the strains (3.76 to 4.03 log10 CFU/cm2 reductions from starting inoculum in strains in which bactericidal activity was reached). Furthermore, OMC was shown to prevent the emergence of RIF resistance. Our data provide preliminary evidence that OMC in combination with RIF could be a viable option for biofilm-associated infections with S. aureus and S. epidermidis. Further research involving OMC in biofilm-associated infections is warranted.

Bacteriophage-antibiotic combination therapy for multidrug-resistant Pseudomonas aeruginosa: In vitro synergy testing.

AIMS: Here, we investigate the impact of phage-antibiotic combinations (PAC) on bacterial killing, resistance development and outer membrane vesicle (OMV) production in multidrug-resistant (MDR) P. aeruginosa. METHODS AND RESULTS: After screening 10 well-characterized MDR P. aeruginosa strains against three P. aeruginosa phages, representative strains, R10266 and R9316, were selected for synergy testing based on high phage sensitivity and substantial antibiotic resistance patterns, while phage EM was chosen based on host range. To understand the impact of phage-antibiotic combinations (PAC) against MDR P. aeruginosa, time-kill analyses, OMV quantification and phage/antibiotic resistance testing were performed. Phage and meropenem demonstrated synergistic activity against both MDR strains. Triple combination regimens, phage-meropenem-colistin and phage-ciprofloxacin-colistin, resulted in the greatest CFU reduction for strains R9316 (3.50 log10 CFU ml-1 ) and R10266 (4.50 log10 CFU ml-1 ) respectively. PAC resulted in regained and improved antibiotic susceptibility to ciprofloxacin (MIC 2 to 0.0625) and meropenem (MIC 32 to 16), respectively, in R9316. Phage resistance was prevented or reduced in the presence of several classes of antibiotics and OMV production was reduced in the presence of phage for both strains, which was associated with significantly improved bacterial eradication. CONCLUSIONS: These findings support the potential of phage-antibiotic synergy (PAS) to augment killing of MDR P. aeruginosa. Systematic in vitro and in vivo studies are needed to better understand phage interactions with antipseudomonal antibiotics, to define the role of OMV production in P. aeruginosa PAC therapy and to outline pharmacokinetic and pharmacodynamic parameters conducive to PAS. SIGNIFICANCE AND IMPACT OF STUDY: This study identifies novel bactericidal phage-antibiotic combinations capable of thwarting resistance development in MDR and XDR P. aeruginosa strains. Furthermore, phage-mediated OMV reduction is identified as a potential mechanism through which PAC potentiates bacterial killing.

Evaluation of Bacteriophage-Antibiotic Combination Therapy for Biofilm-Embedded MDR Enterococcus faecium.

Multidrug-resistant (MDR) Enterococcus faecium is a challenging pathogen known to cause biofilm-mediated infections with limited effective therapeutic options. Lytic bacteriophages target, infect, and lyse specific bacterial cells and have anti-biofilm activity, making them a possible treatment option. Here, we examine two biofilm-producing clinical E. faecium strains, daptomycin (DAP)-resistant R497 and DAP-susceptible dose-dependent (SDD) HOU503, with initial susceptibility to E. faecium bacteriophage 113 (ATCC 19950-B1). An initial synergy screening was performed with modified checkerboard MIC assays developed by our laboratory to efficiently screen for antibiotic and phage synergy, including at very low phage multiplicity of infection (MOI). The data were compared by one-way ANOVA and Tukey (HSD) tests. In 24 h time kill analyses (TKA), combinations with phage-DAP-ampicillin (AMP), phage-DAP-ceftaroline (CPT), and phage-DAP-ertapenem (ERT) were synergistic and bactericidal compared to any single agent (ANOVA range of mean differences 3.34 to 3.84 log10 CFU/mL; p < 0.001). Furthermore, phage-DAP-AMP and phage-DAP-CPT prevented the emergence of DAP and phage resistance. With HOU503, the combination of phage-DAP-AMP showed the best killing effect, followed closely by phage-DAP-CPT; both showed bactericidal and synergistic effects compared to any single agent (ANOVA range of mean differences 3.99 to 4.08 log10 CFU/mL; p < 0.001).

Eradication of Biofilm-Mediated Methicillin-Resistant Staphylococcus aureus Infections In Vitro: Bacteriophage-Antibiotic Combination.

Bacterial biofilms are difficult to eradicate and can complicate many infections by forming on tissues and medical devices. Phage+antibiotic combinations (PAC) may be more active on biofilms than either type of agent alone, but it is difficult to predict which PAC regimens will be reliably effective. To establish a method for screening PAC combinations against Staphylococcus aureus biofilms, we conducted biofilm time-kill analyses (TKA) using various combinations of phage Sb-1 with clinically relevant antibiotics. We determined the activity of PAC against biofilm versus planktonic bacteria and investigated the emergence of resistance during (24 h) exposure to PAC. As expected, fewer treatment regimens were effective against biofilm than planktonic bacteria. In experiments with isogenic strain pairs, we also saw less activity of PACs against DNS-VISA mutants versus their respective parentals. The most effective treatment against both biofilm and planktonic bacteria was the phage+daptomycin+ceftaroline regimen, which met our stringent definition of bactericidal activity (>3 log10 CFU/mL reduction). With the VISA-DNS strain 8015 and DNS strain 684, we detected anti-biofilm synergy between Sb-1 and DAP in the phage+daptomycin regimen (>2 log10 CFU/mL reduction versus best single agent). We did not observe any bacterial resensitization to antibiotics following treatment, but phage resistance was avoided after exposure to PAC regimens for all tested strains. The release of bacterial membrane vesicles tended to be either unaffected or reduced by the various treatment regimens. Interestingly, phage yields from certain biofilm experiments were greater than from similar planktonic experiments, suggesting that Sb-1 might be more efficiently propagated on biofilm. IMPORTANCE Biofilm-associated multidrug-resistant infections pose significant challenges for antibiotic therapy. The extracellular polymeric matrix of biofilms presents an impediment for antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. Some bacteriophages (phages) can move across the biofilm matrix, degrade it, and support antibiotic penetration. However, little is known about how phages and their hosts interact in the biofilm environment or how different phage+antibiotic combinations (PACs) impact biofilms in comparison to the planktonic state of bacteria, though scattered data suggest that phage+antibiotic synergy occurs more readily under biofilm-like conditions. Our results demonstrated that phage Sb-1 can infect MRSA strains both in biofilm and planktonic states and suggested PAC regimens worthy of further investigation as adjuncts to antibiotics.

Pseudouridine synthase 7 is an opportunistic enzyme that binds and modifies substrates with diverse sequences and structures.

Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.

Evaluation of Bacteriophage Cocktails Alone and in Combination with Daptomycin against Daptomycin-Nonsusceptible Enterococcus faecium.

Enterococcus faecium is a significant multidrug-resistant pathogen. Bacteriophage cocktails are being proposed to complement antibiotic therapy. After a screen of 8 E. faecium strains against 4 phages, 2 phages (113 and 9184) with the broadest host ranges were chosen for further experiments. Transmission electron microscopy, whole-genome sequencing, comparative genome analyses, and time-kill analyses were performed. Daptomycin (DAP) plus the phage cocktail (113 [myophage] and 9184 [siphopage]) showed bactericidal activity in most regimens, while DAP addition prevented phage 9184 resistance against daptomycin-nonsusceptible E. faecium.

Exebacase in Addition to Daptomycin against MRSA.

Exebacase is a lysin (cell wall hydrolase) with direct lytic activity against Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). Time-kill analysis experiments illustrated bactericidal activity of exebacase-daptomycin against MRSA strains MW2 and 494. Furthermore, exebacase in addition to daptomycin (10, 6, and 4 mg/kg/day) in a two-compartment ex vivo pharmacokinetic/pharmacodynamic simulated endocardial vegetation model with humanized doses resulted in reductions of 6.01, 4.99, and 2.81 log10 CFU/g (from initial inoculum) against MRSA strain MW2.

Clinical Pharmacology of Bacteriophage Therapy: A Focus on Multidrug-Resistant Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the most common causes of healthcare-associated diseases and is among the top three priority pathogens listed by the World Health Organization (WHO). This Gram-negative pathogen is especially difficult to eradicate because it displays high intrinsic and acquired resistance to many antibiotics. In addition, growing concerns regarding the scarcity of antibiotics against multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. aeruginosa infections necessitate alternative therapies. Bacteriophages, or phages, are viruses that target and infect bacterial cells, and they represent a promising candidate for combatting MDR infections. The aim of this review was to highlight the clinical pharmacology considerations of phage therapy, such as pharmacokinetics, formulation, and dosing, while addressing several challenges associated with phage therapeutics for MDR P. aeruginosa infections. Further studies assessing phage pharmacokinetics and pharmacodynamics will help to guide interested clinicians and phage researchers towards greater success with phage therapy for MDR P. aeruginosa infections.

Dalbavancin, Vancomycin and Daptomycin Alone and in Combination with Cefazolin against Resistant Phenotypes of Staphylococcus aureus in a Pharmacokinetic/Pharmacodynamic Model.

The most efficacious antimicrobial therapy to aid in the successful elimination of resistant S. aureus infections is unknown. In this study, we evaluated varying phenotypes of S. aureus against dalbavancin (DAL), vancomycin (VAN), and daptomycin (DAP) alone and in combination with cefazolin (CFZ). The objective of this study was to observe whether there was a therapeutic improvement in adding a beta-lactam to a glycopeptide, lipopeptide, or a lipoglycopeptide. We completed a series of in vitro tests including minimum inhibitory concentration testing (MIC) of the antimicrobials in combination, time-kill analysis (TKA), and a 168 h (7-day) one-compartment pharmacokinetic/pharmacodynamic (PK/PD) model on two daptomycin non-susceptible (DNS), vancomycin intermediate S. aureus strains (VISA), D712 and 6913. Results from our MIC testing demonstrated a minimum 2-fold and a maximum 32-fold reduction in MIC values for DAL, VAN, and DAP in combination with CFZ, in contrast to either agent used alone. The TKAs completed on four strains paralleled the enhanced activity demonstrated via the combination MICs. In the one-compartment PK/PD models, the combination of DAP plus CFZ or VAN plus CFZ resulted in a significant (p < 0.001) improvement in bactericidal activity and overall reduction in CFU/ml over the 7-day period. While the addition of CFZ to DAL improved time to bactericidal activity, DAL alone demonstrated equal and more sustained overall activity compared to all other treatments. The use of DAL alone, with or without CFZ and the combinations of VAN or DAP with CFZ appear to result in increased bactericidal activity against various recalcitrant S. aureus phenotypes.

Bacteriophage AB-SA01 Cocktail in Combination with Antibiotics against MRSA-VISA Strain in an In Vitro Pharmacokinetic/Pharmacodynamic Model.

This study aimed to test the efficacy of bacteriophage-antibiotic combinations (BACs) in vitro in 24-h time-kill settings and in ex vivo simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic models for 96 h. BACs prevented the development of bacteriophage resistance, while some bacteriophage resistance emerged in bacteriophage-alone treatments. In addition, BACs resulted in an enhancement of bacterial eradication in SEV models. Our findings support the potential activity of BAC therapy for combating serious methicillin-resistant Staphylococcus aureus (MRSA) infections.

Bacteriophage-Antibiotic Combinations for Enterococcus faecium with Varying Bacteriophage and Daptomycin Susceptibilities.

Concerns regarding increased prevalence of daptomycin (DAP)-resistant strains necessitate novel therapies for Enterococcus faecium infections. Obligately lytic bacteriophages are viruses that target, infect, and kill bacterial cells. Limited studies have evaluated phage-antibiotic combinations against E. faecium After an initial screen of eight E. faecium strains, three strains with varying DAP/phage susceptibilities were selected for further experiments. Phage-to-strain specificity contributed to synergy with antibiotics by time-kill analyses and was associated with lower development of phage resistance.

Bacteriophage-Antibiotic Combination Strategy: an Alternative against Methicillin-Resistant Phenotypes of Staphylococcus aureus.

Comparative time-kill experiments with Staphylococcus aureus bacteriophage (phage) Sb-1 alone and phage-antibiotic combinations (PACs) against two methicillin-resistant S. aureus (MRSA) strains have shown synergy with both daptomycin-phage and vancomycin-phage combinations. PACs prevented development of phage resistance and demonstrated bactericidal activity for all triple combinations. In addition, the extracellular membrane vesicle (MV) formation and the potential impact of phage on MV suppression were examined. Our results demonstrate the potential of PAC for combating MRSA infections.

Impact of Daptomycin Dose Exposure Alone or in Combination with ß-Lactams or Rifampin against Vancomycin-Resistant Enterococci in an In Vitro Biofilm Model.

Enterococcus faecium strains are commonly resistant to vancomycin and ß-lactams. In addition, E. faecium often causes biofilm-associated infections and these infections are difficult to treat. In this context, we investigated the activity of dosing regimens using daptomycin (DAP) (8, 10, 12, and 14 mg/kg of body weight/day) alone and in combination with ceftaroline (CPT), ampicillin (AMP), ertapenem (ERT), and rifampin (RIF) against 2 clinical strains of biofilm-producing vancomycin-resistant Enterococcus faecium (VREfm), namely, strains S447 and HOU503, in an in vitro biofilm model. HOU503 harbors common LiaS and LiaR substitutions, whereas S447 lacks mutations associated with the LiaFSR pathway. MIC results demonstrated that both strains were susceptible to DAP and resistant to CPT, AMP, ERT, and RIF. The 168-h pharmacokinetic/pharmacodynamic (PK/PD) CDC biofilm reactor models (simulating human antibiotic exposures) were used with titanium and polyurethane coupons to evaluate the efficacy of antibiotic combinations. DAP 12 and 14 achieved bactericidal activity against S447 but lacked such effect against HOU503. Addition of ERT and RIF enhanced DAP activity, allowing DAP 8 and 10 plus ERT or RIF to produce bactericidal activity against both strains at 168 h. While DAP 8 and 10 plus CPT improved killing, they did not reach bactericidal reduction against S447. Combination of AMP, CPT, ERT, or RIF resulted in enhanced and bactericidal activity for DAP against HOU503 at 168 h. Our data provide further support for the use of combinations of DAP with AMP, ERT, CPT, and RIF in infections caused by biofilm producing VREfm. Further research involving DAP combinations against biofilm-producing enterococci is warranted.

Bacteriophage Therapeutics: A Primer for Clinicians on Phage-Antibiotic Combinations.

Multidrug-resistant organisms have caused a marked depletion of effective antimicrobials, and the narrow pipeline of antibiotics has demanded the need to find novel therapeutic alternatives including nonantibiotic agents. Bacteriophages (phages) are viruses that use the bacterial machinery to infect, replicate, and kill bacterial cells. Although a marked decline in their use was driven by the discovery of antibiotics, the era of antibiotic resistance has led to a resurgence of phage therapy into clinical practice. The term phage-antibiotic synergy (PAS) was coined just over a decade ago and described that sublethal concentrations of antibiotics could stimulate phage production by bacterial cells. Recent literature has described PAS and other encouraging interactions with various phage and antibiotic combinations against a variety of bacterial strains. The primary objective of this review is to discuss the positive interactions between phage and antibiotic combinations, with an emphasis on PAS, reductions in bacterial growth or minimum inhibitory concentrations, enhanced biofilm eradication, and alterations in the emergence of bacterial resistance. A peer-reviewed literature search was conducted (1890-2019) using the PubMed, Medline, and Google Scholar databases. Although more investigation is certainly needed, the combination of bacteriophages with antibiotics is a promising strategy to target organisms with limited or no therapeutic options. This approach may also foster the ability to lower the antibiotic dose and may reduce the potential for antibiotic resistance emergence during therapy.

Discovery of Mcl-1 inhibitors from integrated high throughput and virtual screening.

Protein-protein interactions (PPIs) represent important and promising therapeutic targets that are associated with the regulation of various molecular pathways, particularly in cancer. Although they were once considered "undruggable," the recent advances in screening strategies, structure-based design, and elucidating the nature of hot spots on PPI interfaces, have led to the discovery and development of successful small-molecule inhibitors. In this report, we are describing an integrated high-throughput and computational screening approach to enable the discovery of small-molecule PPI inhibitors of the anti-apoptotic protein, Mcl-1. Applying this strategy, followed by biochemical, biophysical, and biological characterization, nineteen new chemical scaffolds were discovered and validated as Mcl-1 inhibitors. A novel series of Mcl-1 inhibitors was designed and synthesized based on the identified difuryl-triazine core scaffold and structure-activity studies were undertaken to improve the binding affinity to Mcl-1. Compounds with improved in vitro binding potency demonstrated on-target activity in cell-based studies. The obtained results demonstrate that structure-based analysis complements the experimental high-throughput screening in identifying novel PPI inhibitor scaffolds and guides follow-up medicinal chemistry efforts. Furthermore, our work provides an example that can be applied to the analysis of available screening data against numerous targets in the PubChem BioAssay Database, leading to the identification of promising lead compounds, fuelling drug discovery pipelines.