OBJECTIVES:Colistin is an essential antibiotic against carbapenem-resistant Gram-negative bacilli. Pharmacokinetic and pharmacodynamic limitations impact its efficacy, and susceptibility breakpoints equivocate regarding interpretation of MIC values. This post hoc analysis of the OVERCOME trial investigated the association between colistin MIC and outcomes. METHODS:OVERCOME, a randomized, double-blind, placebo-controlled trial, compared colistin monotherapy with colistin plus meropenem for treatment of carbapenem-resistant Gram-negative bacilli pneumonia and/or bloodstream infections. Outcomes were compared between participants whose infections were caused by pathogens with colistin MIC values ≤1 mg/L and those with MIC values of 2 mg/L. RESULTS:Among 369 included participants, the mean age was 67.7 ± 15.9 years, 246 (67%) were in the intensive care unit, 251 (68%) had pneumonia, and 286 (78%) were infected with Acinetobacter baumannii. Overall, no association between an infecting pathogen with an MIC of 2 mg/L and either clinical failure (adjusted odds ratio [aOR]: 1.59 [95% CI: 0.72-3.52]) or 28-day mortality (aOR: 1.62 [95% CI: 0.85-3.09]) was demonstrated. Among participants receiving monotherapy, an infecting pathogen with an MIC of 2 mg/L was independently associated with both clinical failure (aOR: 3.59 [95% CI: 1.10-11.77]) and 28-day mortality (aOR: 3.22 [95% CI: 1.32-7.84]). Among participants receiving combination therapy, no association was demonstrated between MIC and outcomes. CONCLUSIONS:When using colistin-based therapy for pathogens with an MIC of 2 mg/L, these findings support colistin and meropenem combination therapy over colistin monotherapy, particularly for pneumonia due to A. baumannii.

Background. Colistin, a last-line treatment for carbapenem-resistant Gram-negative bacilli (CRGNB), is frequently used in combination with meropenem because these agents often demonstrate in vitro synergy. Using data from the OVERCOME trial comparing colistin + meropenem to colistin + placebo for treatment of pneumonia or bloodstream infection due to CRGNB, we evaluated the impact of synergistic therapy on outcomes. Methods. In vitro synergy testing between colistin and meropenem was conducted using 24-hour time-kill analysis; synergy was defined as >2-log reduction in colony-forming units/mL compared to the most active single agent. Patients receiving synergistic combination therapy were compared to patients receiving functional colistin monotherapy (colistin alone or combination therapy without synergy). Outcomes included mortality, clinical failure, and microbiologic cure. Adjusted analyses controlled for variables on which randomization was stratified and confounders. Results. A total of 146 subjects receiving synergistic combination therapy and 261 subjects receiving functional monotherapy were included. Most had pneumonia (70%), CR Acinetobacter baumannii infection (79%) and were in intensive care (69%). Acinetobacter baumannii was more common in those receiving synergistic combination therapy than functional monotherapy (P < .001). Mortality rates were similar (38.3% and 41.4%, respectively). In adjusted analyses, synergistic combination therapy was associated with significantly lower clinical failure rates (55.3%, 64.3%, adjusted odds ratio [aOR] 0.62, P = .049), with consistent findings in pneumonia (62.6%, 71.8%, aOR 0.55, P = .04) and A. baumannii subgroups (57.4%, 69.4%, aOR 0.60, P = .06). Microbiologic cure rates were similar. Conclusions. Colistin-based, synergistic combination treatment with meropenem (compared to nonsynergistic colistin-based therapy) was associated with decreased clinical failure, particularly in people with pneumonia and A. baumannii.

ABSTRACTMethicillin-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.IMPORTANCEThe 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.

ABSTRACT 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 (10 9 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 log 10 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 log 10 CFU/mL, respectively) ( P < 0.05 each). Against C37, Intesti13 + Sb-1 + CPT ± DAP was equally effective (−Δ7.14 log 10 CFU/mL each) and not significantly different from DAP + Intesti13 + Sb-1 (−Δ6.65 log 10 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 log 10 CFU/g (−Δ7.07 and −Δ7.11 log 10 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.

Abstract Background Phage-antibiotic combinations (PAC) have been proposed for high inoculum daptomycin-nonsusceptible (DNS) MRSA infections refractory to conventional therapy. We studied PAC with synergistic activity against two DNS MRSA clinical bloods isolates (C4 and C37; DAP MIC = 4 ug/mL). Methods PAC containing DAP and/or ceftaroline (CPT) (each at ½ MIC) plus a 2-phage cocktail (Intesti13 and Sb-1, at a range of multiplicities of infection [MOI]) were tested at a high MRSA inoculum (109 CFU/mL) using: i) modified checkerboard (CB) minimum inhibitory concentration (MIC); and ii) 24h time-kill assays (TKA). Synergistic activity in CB assays was defined as either: a fractional inhibitory concentration (FIC) index ≤ 0.5 in modified CB assays; or a ≥ 2 log10 CFU/mL reduction by PACs vs the most active single-agent regimen. Significant differences between regimens were assessed by ANOVA with Tukey’s post hoc modification (P < 0.05). Results By CB assay, synergistic activity was demonstrated with Intesti13 + Sb1 (MOI of 10 to 0.01) plus either DAP or DAP + CPT (FIC ≤ 0.5 for each combination). In 24h TKA vs C4, Intest13 + Sb1 (MOI 1 and 0.1) plus either DAP or DAP + CPT demonstrated robust synergistic activity (-Δ7.21 and -Δ7.39 log10 CFU/mL, respectively) vs. the next most effective regimen of CPT + Intesti13+Sb1 (P < 0.05 each). Against C37, Intesti13 + Sb1 (MOI of 1 and 0.1) with CPT or DAP + CPT were equally potent and effective regimens (-Δ7.14 log10 CFU/mL each), but neither were significantly better than the synergistic regimen of DAP + Intesti13 + Sb1 (-Δ6.65 log10 CFU/mL).Figure 1.Bacterial quantification in 24 h high inoculum (10^9 CFU/mL) TKA of DAP and CPT combined with phages Intesti13 and Sb1 against DNS MRSA strain C4. These studies demonstrated positive phage-antibiotic synergy (PAS), with phage cocktail Intesti13 + Sb1 and either DAP or DAP + CPT. Addition of these phages to DAP or DAP + CPT combinations caused bactericidal and synergistic killing vs. CPT + phages. *, P<0.05. Abbreviations: CFU: colony forming units, GC: growth control, DAP: daptomycin, CPT: ceftaroline, MOI: multiplicity of infection, PAC: phage-antibiotic combination.Figure 2.Bacterial quantification in 24 h high inoculum (10^9 CFU/mL) TKA of DAP and CPT combined with phages Intesti13 and Sb1 against DNS MRSA strain C37. These studies demonstrate positive phage-antibiotic synergy (PAS) with phage cocktail Intesti13 + Sb1 added to DAP, CPT, and DAP + CPT at an MOI of 1 and 0.1. Abbreviations: CFU: colony forming units, GC: growth control, DAP: daptomycin, CPT: ceftaroline, MOI: multiplicity of infection, PAC: phage-antibiotic combination. Conclusion The two-phage cocktail used (Intesti13 + Sb1) demonstrated impressive synergistic activity against two DNS MRSA isolates in combination with DAP or DAP + CPT. Further experimental in vivo investigations of these candidate PACs, for treatment of high inoculum DNS MRSA infections (e.g., infective endocarditis) is warranted. Disclosures Arnold S. Bayer, MD, Akagera Medicines: Grant/Research Support|ContraFect Corporation: Grant/Research Support Michael J. Rybak, PharmD, PhD, MPH, Abbvie, Merck, Paratek, Shionogi, Entasis, La Jolla, T2 Biosystems: Advisor/Consultant

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.

Abstract Background The urgency to find new alternatives has increased as multidrug-resistant organisms enhance their defense mechanisms. In recent years, there has been a renewed interest in bacteriophages as potential alternatives or adjuncts to antibiotic therapy. However, there is limited information on the impact of bacteriophages with the innate immune system. Cathelicidin LL-37, a cationic peptide with antimicrobial properties, functions in coordination with the immune system to eradicate pathogens. We aim to evaluate how the survival of daptomycin (DAP) and ampicillin (AMP) resistant or non-susceptible Enterococcus faecium isolates are impacted by the interaction between bacteriophage, LL-37, and the addition of DAP/AMP. Methods Two E. faecium clinical isolates were utilized: R497 and HOU503. Bacteriophages provided by the Naval Medical Research Command, NV- 503-01 and NV-497 were quantified and propagated to approximately 108 PFU/mL. LL-37 was prepared at a final concentration of 640μM and diluted in RPMI intentionally to a suboptimal concentration of 0.5μM to adjust for a 70-80% survival rate. This was done to detect a synergistic interaction more easily with the addition of phage. Samples were incubated at 37 °C and plated in triplicate on brain heart infusion agar at 2h. After 24h of incubation, colonies were counted for and analyzed. Data was expressed as a mean percent survival with standard deviation. ANOVA with Tukey's HSD post-hoc test was used to determine the variations between each combination used. Results The targeted bacterial survival rate against E. faecium isolates was attained by LL-37 at a concentration of 0.5μM. However, when R497 and HOU503 was combined with LL-37, bacteriophage, and DAP/AMP the percentage of bacterial survival was significantly (P< 0.05) lower than that of the growth control, LL-37, AMP/DAP, and bacteriophage alone. Conclusion By conducting bacterial survival assays, we observed a notable enhancement in the elimination of multi-drug resistant E. faecium when LL-37, bacteriophage, and DAP/AMP were added. Examining this interaction over longer periods, both with and without varying antimicrobials, will yield more understanding regarding the potential involvement of bacteriophage in the innate immune system's response to fighting infections. Disclosures Michael J. Rybak, PharmD, PhD, MPH, Abbvie, Merck, Paratek, Shionogi, Entasis, La Jolla, T2 Biosystems: Advisor/Consultant

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.

ABSTRACT 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 log 10 CFU/cm 2 ). 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 log 10 CFU/cm 2 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.

ABSTRACT 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 log 10 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 log 10 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.

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. 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. 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. 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.

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.

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.

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.

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).

Abstract Background Bacteriophage (phage) to augment antibiotic efficacy is a possible therapeutic option in the era of antimicrobial resistance. Studies to date have assessed phage-antibiotic synergy (PAS), however, its efficacy may be dependent upon antibiotic mechanism of action. Here, we report our in-vitro evaluation of phage-antibiotic antagonism (PAA) among phage and protein synthesis inhibitor combinations in multidrug-resistant clinical strains of P. aeruginosa, S. aureus, and E. faecium. Methods The following bacteria (phage) regimens were evaluated: 10266 (EM) and R9010 (14207) (P. aeruginosa) against gentamicin (GEN), azithromycin (AZM), and ciprofloxacin (CIP); N315 and 494 (Intesti) (S. aureus), and R497 and HOU503 (NV-497) (E. faecium) against linezolid (LNZ), minocycline (MIN), and daptomycin (DAP). Modified checkerboard (CB) MIC assays were used for preliminary screening followed by 24h time kill analyses (TKA). For CB, synergy, additive activity, and antagonism were defined as an FIC index of ≤0.5, 1–4, and >4, respectively. In 24h TKA, synergy and additivity were defined as a ≥2 and ≥1 log10 CFU/mL reduction from baseline, while antagonism was defined as phage-antibiotic combinations with CFU/mL higher than the most effective single treatment at 24h. Data were compared by one-way ANOVA and Tukey (HSD) test (P< 0.05). Results In CB analyses and 24h TKA of S. aureus and E. faecium isolates, phage-LZD and phage-MIN combinations were antagonistic (FIC >4) while phage-DAP was synergistic (FIC 0.5) (ANOVA range of mean differences 0.52 to 2.59 log10 CFU/mL; P< 0.001). For P. aeruginosa, phage-AZM and phage-GEN were antagonistic (FIC >4) and additive (FIC=1), respectively (ANOVA range of mean differences 1.04 to 1.95 log10 CFU/mL; P< 0.001). Conclusion Our results suggest that antibiotics that act on the protein synthesis pathway may lead to PAA, however, PAA interactions may be highly dependent upon antibiotic mechanism of bacterial inhibition (i.e., location of ribosomal protein synthesis inhibition, bactericidal vs. bacteriostatic) protein synthesis inhibit. Studies assessing PAA in a wider array of phage-antibiotic combinations are warranted. Disclosures Cesar A. Arias, MD, PhD, Entasis Phramceuticals: Grant/Research Support|MeMed Diagnostics: Grant/Research Support|Merck: Grant/Research Support.

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 log(10), CFU/mL reduction). With the VISA-DNS strain 8015 and DNS strain 684, we detected antibiofilm synergy between Sb-1 and DAP in the phage+daptomycin regimen (>2 log(10) 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.

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 Acinetobacter baumannii 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.

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.

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.