Phage-antibiotic synergy against daptomycin-nonsusceptible MRSA in an ex vivo simulated endocardial pharmacokinetic/pharmacodynamic model.

Phage-antibiotic combinations (PAC) offer a potential solution for treating refractory daptomycin-nonsusceptible (DNS) methicillin-resistant Staphylococcus aureus (MRSA) infections. We examined PAC activity against two well-characterized DNS MRSA strains (C4 and C37) in vitro and ex vivo. PACs comprising daptomycin (DAP) ± ceftaroline (CPT) and a two-phage cocktail (Intesti13 + Sb-1) were evaluated for phage-antibiotic synergy (PAS) against high MRSA inoculum (109 CFU/mL) using (i) modified checkerboards (CB), (ii) 24-h time-kill assays (TKA), and (iii) 168-h ex vivo simulated endocardial vegetation (SEV) models. PAS was defined as a fractional inhibitory concentration ≤0.5 in CB minimum inhibitory concentration (MIC) or a ≥2 log10 CFU/mL reduction compared to the next best regimen in time-kill assays and SEV models. Significant differences between regimens were assessed by analysis of variance with Tukey's post hoc modification (α = 0.05). CB assays revealed PAS with Intesti13 + Sb-1 + DAP ± CPT. In 24-h time-kill assays against C4, Intesti13 + Sb-1 + DAP ± CPT demonstrated synergistic activity (-Δ7.21 and -Δ7.39 log10 CFU/mL, respectively) (P < 0.05 each). Against C37, Intesti13 + Sb-1 + CPT ± DAP was equally effective (-Δ7.14 log10 CFU/mL each) and not significantly different from DAP + Intesti13 + Sb-1 (-Δ6.65 log10 CFU/mL). In 168-h SEV models against C4 and C37, DAP ± CPT + the phage cocktail exerted synergistic activities, significantly reducing bio-burdens to the detection limit [2 log10 CFU/g (-Δ7.07 and -Δ7.11 log10 CFU/g, respectively)] (P < 0.001). At 168 h, both models maintained stable MICs, and no treatment-emergent phage resistance occurred with DAP or DAP + CPT regimens. The two-phage cocktail demonstrated synergistic activity against two DNS MRSA isolates in combination with DAP + CPT in vitro and ex vivo. Further in vivo PAC investigations are needed.

Synergistic bactericidal effects of phage-enhanced antibiotic therapy against MRSA biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) causes biofilm-related medical device infections. Phage-antibiotic combinations offer potential therapy due to proven in vitro antibiofilm efficacy. We evaluated phage-antibiotic synergy against biofilms using modified checkerboard and 24-h time-kill assays. Humanized-simulated daptomycin (DAP) (10, 8, and 6 mg/kg q24h) and ceftaroline (CPT) (600 mg q12h) were combined with Intesti13, Sb-1, and Romulus phages (tMOI 1, q12h). Assays were conducted in 168-h biofilm reactor models against DAP non-susceptible (DNS) vancomycin intermediate S. aureus (VISA) MRSA D712 and DAP-susceptible MRSA 8014. Synergistic activity and bactericidal activity were defined as ≥2log10 CFU/mL reduction from antibiotic-only regimens and ≥3log10 CFU/mL decrease from baseline at 24 h. Differences were analyzed by one-way analysis of variance with Tukey's post hoc test (P ≤ 0.05 is considered significant). Surviving bacteria were examined for antibiotic minimum biofilm inhibitory concentration (MBIC) changes and phage susceptibility. In 168-h biofilm models, humanized DAP 10 mg/kg + CPT, combined with a 2-phage cocktail (Intesti13 + Sb-1) against D712, and a 3-phage cocktail (Intesti13 + Sb-1 + Romulus) against 8014, demonstrated synergistic bactericidal activity. At 168 h, bacteria were minimally detectable [2log10 CFU/cm2 (-Δ4.23 and -Δ4.42 log10 CFU/cm2; both P < 0.001)]. Antibiotic MBIC remained unchanged compared to baseline across various time points. None of the tested bacteria at 168 h exhibited complete phage resistance. This study reveals bactericidal efficacy of DAP + CPT with 2-phage and 3-phage cocktails against DNS VISA and MRSA isolates (D712 and 8014) in biofilm models, maintaining susceptibility. Further research is needed for diverse strains and durations, aligning with infection care. IMPORTANCE: The prevalence of biofilm-associated medical device infections caused by methicillin-resistant Staphylococcus aureus (MRSA) presents a pressing medical challenge. The latest research demonstrates the potential of phage-antibiotic combinations (PACs) as a promising solution, notably in vitro antibiofilm efficacy. By adopting modified checkerboard and 24-h time-kill assays, the study investigated the synergistic action of phages combined with humanized-simulated doses of daptomycin (DAP) and ceftaroline (CPT). The results were promising: a combination of DAP, CPT, and either a 2-phage or 3-phage cocktail effectively exhibited bactericidal activity against both DAP non-susceptible vancomycin intermediate S. aureus MRSA and DAP-susceptible MRSA strains within 168-h biofilm models. Moreover, post-treatment evaluations revealed no discernible rise in antibiotic resistance or complete phage resistance. This pioneering work suggests the potential of PACs in addressing MRSA biofilm infections, setting the stage for further expansive research tailored to diverse bacterial strains and treatment durations.

Ciprofloxacin in combination with bacteriophage cocktails against multi-drug resistant Pseudomonas aeruginosa in ex vivo simulated endocardial vegetation models.

Pseudomonas aeruginosa-associated infective endocarditis represents difficult-to-treat, deep-seated infections. Phage-antibiotic combinations have shown to eradicate multi-drug resistant (MDR) P. aeruginosa, limit the development of phage resistance, and restore antibiotic sensitivity. The objective of this study was to evaluate the activity of phage-ciprofloxacin (CIP) combinations in 4-day ex vivo simulated endocardial vegetation (SEV) models against drug-resistant P. aeruginosa isolates. Two P. aeruginosa isolates, extensively drug-resistant AR351 and MDR I0003-1, were selected for their drug resistance and sensitivity to phage. Three phages [LL-5504721-AH (LL), E2005-C (EC), and 109] and CIP were evaluated alone and in combination for their activity and influence on drug and phage resistance using 24-h time-kill analysis. The three-phage cocktail (q24h) in combination with CIP (400 mg q12h) was then tested in dynamic 4-day ex vivo SEV models, with reduction of log10 CFU/mL compared using ANOVA with Bonferroni analysis. Compared to other combinations, CIP-LL-EC-109 demonstrated synergistic and bactericidal activity from starting CFU/g against AR351 and I0003-1 (-Δ5.65 and 6.60 log10 CFU/g, respectively; P < 0.001). Additionally, CIP-LL-EC-109 mitigated phage resistance, while all other therapies had a high degree of resistance to >1 phages, and all phage-containing regimens prevented CIP mean inhibitory concentration increases compared to CIP alone for both AR351 and I0003-1 at 96 h.

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.

Phage-Antibiotic Cocktail Rescues Daptomycin and Phage Susceptibility against Daptomycin-Nonsusceptible Enterococcus faecium in a Simulated Endocardial Vegetation Ex Vivo Model.

Enterococcus faecium is a difficult-to-treat pathogen with emerging resistance to most clinically available antibiotics. Daptomycin (DAP) is the standard of care, but even high DAP doses (12 mg/kg body weight/day) failed to eradicate some vancomycin-resistant strains. Combination DAP-ceftaroline (CPT) may increase ß-lactam affinity for target penicillin binding proteins (PBP); however, in a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, DAP-CPT did not achieve therapeutic efficacy against a DAP-nonsusceptible (DNS) vancomycin-resistant E. faecium (VRE) isolate. Phage-antibiotic combinations (PAC) have been proposed for resistant high-inoculum infections. We aimed to identify PAC with maximum bactericidal activity and prevention/reversal of phage and antibiotic resistance in an SEV PK/PD model against DNS isolate R497. Phage-antibiotic synergy (PAS) was evaluated with modified checkerboard MIC and 24-h time-kill analyses (TKA). Human-simulated antibiotic doses of DAP and CPT with phages NV-497 and NV-503-01 were then evaluated in 96-h SEV PK/PD models against R497. Synergistic and bactericidal activity was identified with the PAC of DAP-CPT combined with phage cocktail NV-497-NV-503-01, demonstrating a significant reduction in viability down to 3-log10 CFU/g (-Δ, 5.77-log10 CFU/g; P < 0.001). This combination also demonstrated isolate resensitization to DAP. Evaluation of phage resistance post-SEV demonstrated prevention of phage resistance for PACs containing DAP-CPT. Our results provide novel data highlighting bactericidal and synergistic activity of PAC against a DNS E. faecium isolate in a high-inoculum ex vivo SEV PK/PD model with subsequent DAP resensitization and prevention of phage resistance. IMPORTANCE Our study supports the additional benefit of standard-of-care antibiotics combined with a phage cocktail compared to antibiotic alone against a daptomycin-nonsusceptible (DNS) E. faecium isolate in a high-inoculum simulated endocardial vegetation ex vivo PK/PD model. E. faecium is a leading cause of hospital-acquired infections and is associated with significant morbidity and mortality. Daptomycin is considered the first-line therapy for vancomycin-resistant E. faecium (VRE), but the highest published doses have failed to eradicate some VRE isolates. The addition of a ß-lactam to daptomycin may result in synergistic activity, but previous in vitro data demonstrate that daptomycin plus ceftaroline failed to eradicate a VRE isolate. Phage therapy as an adjunct to antibiotic therapy has been proposed as a salvage therapy for high-inoculum infections; however, pragmatic clinical comparison trials for endocarditis are lacking and difficult to design, reinforcing the timeliness of such analysis.

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.

Optimization of Phage-Antibiotic Combinations against Staphylococcus aureus Biofilms.

Phage therapy has gained attention due to the spread of antibiotic-resistant bacteria and narrow pipeline of novel antibiotics. Phage cocktails are hypothesized to slow the overall development of resistance by challenging the bacteria with more than one phage. Here, we have used a combination of plate-, planktonic-, and biofilm-based screening assays to try to identify phage-antibiotic combinations that will eradicate preformed biofilms of Staphylococcus aureus strains that are otherwise difficult to kill. We have focused on methicillin-resistant S aureus (MRSA) strains and their daptomycin-nonsusceptible vancomycin-intermediate (DNS-VISA) derivatives to understand whether the phage-antibiotic interactions are altered by the changes associated with evolution from MRSA to DNS-VISA (which is known to occur in patients receiving antibiotic therapy). We evaluated the host range and cross-resistance patterns of five obligately lytic S. aureus myophages to select a three-phage cocktail. We screened these phages for their activity against 24-h bead biofilms and found that biofilms of two strains, D712 (DNS-VISA) and 8014 (MRSA), were the most resistant to killing by single phages. Specifically, even initial phage concentrations of 107 PFU per well could not prevent visible regrowth of bacteria from the treated biofilms. However, when we treated biofilms of the same two strains with phage-antibiotic combinations, we prevented bacterial regrowth when using up to 4 orders of magnitude less phage and antibiotic concentrations that were lower than our measured minimum biofilm inhibitory concentration. We did not see a consistent association between phage activity and the evolution of DNS-VISA genotypes in this small number of bacterial strains. IMPORTANCE The extracellular polymeric matrix of biofilms presents an impediment to antibiotic diffusion, facilitating the emergence of multidrug-resistant populations. While most phage cocktails are designed for the planktonic state of bacteria, it is important to take the biofilm mode of growth (the predominant mode of bacterial growth in nature) into consideration, as it is unclear how interactions between any specific phage and its bacterial hosts will depend on the physical properties of the growth environment. In addition, the extent of bacterial sensitivity to any given phage may vary from the planktonic to the biofilm state. Therefore, phage-containing treatments targeting biofilm infections such as catheters and prosthetic joint material may not be merely based on host range characteristics. Our results open avenues to new questions regarding phage-antibiotic treatment efficiency in the eradication of topologically structured biofilm settings and the extent of eradication efficacy relative to the single agents in biofilm populations.

Phage Cocktails with Daptomycin and Ampicillin Eradicates Biofilm-Embedded Multidrug-Resistant Enterococcus faecium with Preserved Phage Susceptibility.

Multidrug-resistant (MDR) Enterococcus faecium is a challenging nosocomial pathogen known to colonize medical device surfaces and form biofilms. Bacterio (phages) may constitute an emerging anti-infective option for refractory, biofilm-mediated infections. This study evaluates eight MDR E. faecium strains for biofilm production and phage susceptibility against nine phages. Two E. faecium strains isolated from patients with bacteremia and identified to be biofilm producers, R497 (daptomycin (DAP)-resistant) and HOU503 (DAP-susceptible dose-dependent (SDD), in addition to four phages with the broadest host ranges (ATCC 113, NV-497, NV-503-01, NV-503-02) were selected for further experiments. Preliminary phage-antibiotic screening was performed with modified checkerboard minimum biofilm inhibitory concentration (MBIC) assays to efficiently screen for bacterial killing and phage-antibiotic synergy (PAS). Data were compared by one-way ANOVA and Tukey (HSD) tests. Time kill analyses (TKA) were performed against R497 and HOU503 with DAP at 0.5× MBIC, ampicillin (AMP) at free peak = 72 µg/mL, and phage at a multiplicity of infection (MOI) of 0.01. In 24 h TKA against R497, phage-antibiotic combinations (PAC) with DAP, AMP, or DAP + AMP combined with 3- or 4-phage cocktails demonstrated significant killing compared to the most effective double combination (ANOVA range of mean differences 2.998 to 3.102 log10 colony forming units (CFU)/mL; p = 0.011, 2.548 to 2.868 log10 colony forming units (CFU)/mL; p = 0.023, and 2.006 to 2.329 log10 colony forming units (CFU)/mL; p = 0.039, respectively), with preserved phage susceptibility identified in regimens with 3-phage cocktails containing NV-497 and the 4-phage cocktail. Against HOU503, AMP combined with any 3- or 4-phage cocktail and DAP + AMP combined with the 3-phage cocktail ATCC 113 + NV-497 + NV-503-01 demonstrated significant PAS and bactericidal activity (ANOVA range of mean differences 2.251 to 2.466 log10 colony forming units (CFU)/mL; p = 0.044 and 2.119 to 2.350 log10 colony forming units (CFU)/mL; p = 0.028, respectively), however, only PAC with DAP + AMP maintained phage susceptibility at the end of 24 h TKA. R497 and HOU503 exposure to DAP, AMP, or DAP + AMP in the presence of single phage or phage cocktail resulted in antibiotic resistance stabilization (i.e., no antibiotic MBIC elevation compared to baseline) without identified antibiotic MBIC reversion (i.e., lowering of antibiotic MBIC compared to baseline in DAP-resistant and DAP-SDD isolates) at the end of 24 h TKA. In conclusion, against DAP-resistant R497 and DAP-SDD HOU503 E. faecium clinical blood isolates, the use of DAP + AMP combined with 3- and 4-phage cocktails effectively eradicated biofilm-embedded MDR E. faecium without altering antibiotic MBIC or phage susceptibility compared to baseline.

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.

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.

In Vitro Synergy of Colistin in Combination with Meropenem or Tigecycline against Carbapenem-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is currently classified as one of six pathogens that contribute to increased patient mortality. Thus, exploratory studies navigating alternative treatment strategies are of supreme interest. Herein, we completed minimum inhibitory concentration (MIC) testing, and time-kill analyses (TKA) on 50 carbapenem-resistant Acinetobacterbaumannii isolates including 28 colistin-resistant isolates. Upon testing of MEM or TGC in the presence of sub-inhibitory COL against the 50 isolates, there was a median 2-fold reduction in MEM and TGC MICs. In the TKAs, the COL+MEM combination was synergistic in 45 (90%) isolates and bactericidal in 43 (86%) isolates at 24 hours, whereas the COL+TGC combination TKAs demonstrated synergy in 32 (64%) isolates and bactericidal activity was shown in 28 (56%) isolates. Additionally, sulbactam (SUL) and TGC were added to the COL+MEM dual therapy regimen to assess the possible utility of a triple therapy regimen against five non-responsive isolates. The COL+MEM+SUL and COL+MEM+TGC regimens effectively restored synergy in (5/5) 100% of the isolates. The results of this study demonstrate the potential utility of COL combinations in the treatment of carbapenem-resistant isolates.

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.

In Vitro Antibacterial Activity of Cefiderocol against Multidrug-Resistant Acinetobacter baumannii.

Cefiderocol (CFDC), a novel siderophore cephalosporin, demonstrates strong activity against multidrug-resistant (MDR) Acinetobacter baumannii. Limited studies have evaluated CFDC alone and in combination with other Gram-negative antibiotics against MDR A. baumannii isolates. Susceptibility testing revealed lower CFDC MIC values (87% of MICs ≤ 4mg/liter) than the comparator Gram-negative agents. Six isolates, with elevated CFDC MICs (16 to 32 mg/liter) were selected for further experiments. Time-kill analyses presented with synergistic activity and beta-lactamase inhibitors increased CFDC susceptibility in each of the isolates.

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.

Mechanistic Insights Into the Differential Efficacy of Daptomycin Plus ß-Lactam Combinations Against Daptomycin-Resistant Enterococcus faecium.

BACKGROUND: The combination of daptomycin (DAP) plus ampicillin (AMP), ertapenem (ERT), or ceftaroline has been demonstrated to be efficacious against a DAP-tolerant Enterococcus faecium strain (HOU503). However, the mechanism for the efficacy of these combinations against DAP-resistant (DAP-R) E. faecium strains is unknown. METHODS: We investigated the efficacy of DAP in combination with AMP, ERT, ceftaroline, ceftriaxone, or amoxicillin against DAP-R E. faecium R497 using established in vitro and in vivo models. We evaluated pbp expression, levels of penicillin-binding protein (PBP) 5 (PBP5) and ß-lactam binding affinity in HOU503 versus R497. RESULTS: DAP plus AMP was the only efficacious regimen against DAP-R R497 and prevented emergence of resistance. DAP at 8, 6, and 4 mg/kg in combination with AMP was efficacious but showed delayed killing compared with 10 mg/kg. PBP5 of HOU503 exhibited amino acid substitutions in the penicillin-binding domain relative to R497. No difference in pbp mRNA or PBP5 levels was detected between HOU503 and R497. labeling of PBPs with Bocillin FL, a fluorescent penicillin derivative, showed increased ß-lactam binding affinity of PBP5 of HOU503 compared with that of R497. CONCLUSIONS: Only DAP (10 mg/kg) plus AMP or amoxicillin was efficacious against a DAP-R E. faecium strain, and pbp5 alleles may be important contributors to efficacy of DAP plus ß-lactam therapy.

Combinations of (lipo)glycopeptides with ß-lactams against MRSA: susceptibility insights.

BACKGROUND: Increasing application of vancomycin due to the high prevalence of MRSA infections has led to the emergence of vancomycin intermediate-resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA). Consequently, the need for alternative therapies that target MRSA has become evident. OBJECTIVES: To evaluate the synergy between (lipo)glycopeptides (LGP/GPs) (vancomycin, teicoplanin, telavancin, dalbavancin and oritavancin) and ß-lactams (ceftaroline, cefepime, cefazolin and oxacillin) against MRSA, hVISA, VISA and daptomycin non-susceptible (DNS) phenotypes. METHODS: Twenty randomly selected clinical MRSA strains (i.e. 5 MRSA, 5 hVISA, 5 VISA and 5 DNS) were assessed versus LGP/GPs alone and LGP/GPs in combination with ß-lactams for MICs. Although verification of antibiotic potency against bacterial strains is assessed by the microbroth dilution (MBD) MIC method recommended by the CLSI, some antibiotics need modified assay conditions in order to demonstrate their optimal activity. RESULTS: Addition of ß-lactams reduced MIC values of LGP/GPs against all strains (up to 160-fold reduction). In general, LGPs (dalbavancin, oritavancin and telavancin) were more active (significant differences in MIC values, up to 8-fold) compared with vancomycin and teicoplanin. The majority of these combinations were bactericidal and superior to any single agent. CONCLUSIONS: This report has examined the susceptibility patterns of LGP/GPs and their combination with ß-lactams. Of interest, the impact of susceptibility tests (in terms of MIC plates and their surface area) on the synergistic activity in 24 h time-kill experiments was apparent for LGPs. Further clinical research is required to investigate synergy with LGP/GPs and ß-lactams against these Staphylococcus strains.

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.