Phage-antibiotic Synergy Research Articles

Characterization of bacteriophage vB_AbaS_SA1 and its synergistic effects with antibiotics against clinical multidrug-resistant Acinetobacter baumannii isolates.

Acinetobacter baumannii is a major cause of nosocomial infections globally. The increasing prevalence of multidrug-resistant (MDR) A. baumannii has become an important public health concern. To combat drug resistance, alternative methods such as phage therapy have been suggested. In total, 30 MDR A. baumannii strains were isolated from clinical specimens, and their antibiotic susceptibilities were determined. The Acinetobacter phage vB_AbaS_SA1, isolated from hospital sewage, was characterized. In addition to its plaque size, particle morphology, and host range, its genome sequence was determined and annotated. Finally, the antibacterial effects of phage alone, antibiotics alone, and phage/antibiotic combinations were assessed against the A. baumannii strains. Phage vB_AbaS_SA1 had siphovirus morphology, showed a latent period of 20 minutes, and a 250 PFU/cell (plaque forming unit/cell) burst size. When combined with antibiotics, vB_AbaS_SA1 (SA1) showed a significant phage-antibiotic synergy (PAS) effect and reduced the overall effective concentration of antibiotics in time-kill assessments. The genome of SA1 is a linear double-stranded DNA of 50,108bp in size with a GC content of 39.15%. Despite the potent antibacterial effect of SA1, it is necessary to perform additional research to completely elucidate the mechanisms of action and potential constraints associated with utilizing this bacteriophage.

Isolation and preliminary characterization of a novel bacteriophage vB_KquU_φKuK6 that infects the multidrug-resistant pathogen Klebsiella quasipneumoniae.

Klebsiella quasipneumoniae (previously known as K. pneumoniae K6) strains are among the multidrug-resistant hypervirulent bacterial pathogens. Phage therapy can help treat infections caused by such pathogens. Here we report some aspects of virology and therapeutic potentials of vB_KquU_φKuK6, a bacteriophage that infects Klebsiella quasipneumoniae. K. quasipneumoniae (ATCC 700603) was used to screen wastewater lytic phages. The isolate vB_KquU_φKuK6 that consistently created large clear plaques was characterized using standard virological and molecular methods. vB_KquU_φKuK6 has a complex capsid with an icosahedral head (~60 nm) and a slender tail (~140 nm × 10 nm). The phage has a 51% AT-rich linear dsDNA genome (51,251 bp) containing 121 open reading frames. The genome contains genes encoding spanin, endolysin, and holin proteins necessary for lytic infection and a recombinase gene possibly involved in lysogenic infection. vB_KquU_φKuK6 is stable at -80 to +67°C, pH 4-9, and brief exposure to one volume percent of chloroform. vB_KquU_φKuK6 has a narrow host range. Its lytic infection cycle involves a latency of 20 min and a burst size of 435 plaque-forming units. The phage can cause lysogenic infection, and the resulting lysogens are resistant to lytic infection by vB_KquU_φKuK6. vB_KquU_φKuK6 reduces the host cells' ability to form biofilm but fails to eliminate that ability. vB_KquU_φKuK6 demonstrates phage-antibiotic synergy and reduces the minimum inhibitory concentration of chloramphenicol and neomycin sulfate by about 8 folds. vB_KquU_φKuK6 cannot be directly used for phage therapy because it is a temperate bacteriophage. However, genetically modified strains of vB_KquU_φKuK6 alone or combined with antibiotics or other lytic Klebsiella phages can have therapeutic utilities in treating K. quasipneumoniae infections.

Antibacterial effect of phage cocktails and phage-antibiotic synergy against pathogenic Klebsiella pneumoniae.

The worldwide spread of antimicrobial resistance (AMR) has posed a great challenge to global public health. Phage therapy has become a promising alternative against difficult-to-treat pathogens. One important goal of this study was to optimize the therapeutic efficiency of phage-antibiotic combinations, known as phage-antibiotic synergy (PAS). Through comprehensive analysis of the phenotypic and genotypic characteristics of a large number of CRKp-specific phages, we developed a systematic model for phage cocktail combinations. Crucially, our finding demonstrated that PAS treatments not only enhance the bactericidal effects of colistin and tigecycline against multidrug-resistant (MDR) K. pneumoniae strains in in vitro and in vivo context but also provide a robust response when antibiotics fail. Overall, the optimized PAS therapy demonstrates considerable potential in combating diverse K. pneumoniae pathogens, highlighting its relevance as a strategy to mitigate antibiotic resistance threats effectively.

Phage-antibiotic synergy suppresses resistance emergence of Klebsiella pneumoniae by altering the evolutionary fitness.

Phage-antibiotic synergy (PAS) has been recently proposed as a superior strategy for the treatment of multidrug-resistant pathogens to effectively reduce bacterial load and slow down both phage and antibiotic resistance. However, the underlying mechanisms of resistance suppression by PAS have been poorly and rarely been studied. In this study, we tried to understand how PAS suppresses the emergence of resistance using a hypervirulent Klebsiella pneumoniae (KP) strain and a novel phage H5 in combination with ceftazidime (CAZ) as a model. Our study reveals a novel mechanism by which PAS drives altered evolutionary trajectory of bacterial populations, leading to suppressed emergence of resistance. The findings advance our understanding of how PAS suppresses the emergence of resistance, and are imperative for optimizing the efficacy of phage-antibiotic therapy to further improve clinical outcomes.

Synergistic Antimicrobial Effects of Phage vB_AbaSi_W9 and Antibiotics against Acinetobacter baumannii Infection.

Acinetobacter baumannii is a challenging multidrug-resistant pathogen in healthcare. Phage vB_AbaSi_W9 (GenBank: PP146379.1), identified in our previous study, shows lytic activity against 26 (89.66%) of 29 carbapenem-resistant Acinetobacter baumannii (CRAB) strains with various sequence types (STs). It is a promising candidate for CRAB treatment; however, its lytic efficiency is insufficient for complete bacterial lysis. Therefore, this study aimed to investigate the clinical utility of the phage vB_AbaSi_W9 by identifying antimicrobial agents that show synergistic effects when combined with it. The A. baumannii ATCC17978 strain was used as the host for the phage vB_AbaSi_W9. Adsorption and one-step growth assays of the phage vB_AbaSi_W9 were performed at MOIs of 0.001 and 0.01, respectively. Four clinical strains of CRAB belonging to different sequence types, KBN10P04948 (ST191), LIS2013230 (ST208), KBN10P05982 (ST369), and KBN10P05231 (ST451), were used to investigate phage-antibiotic synergy. Five antibiotics were tested at the following concentration: meropenem (0.25-512 µg/mL); colistin, tigecycline, and rifampicin (0.25-256 µg/mL); and ampicillin/sulbactam (0.25/0.125-512/256 µg/mL). The in vitro synergistic effect of the phage and rifampicin was verified through an in vivo mouse infection model. Phage vB_AbaSi_W9 demonstrated 90% adsorption to host cells in 1 min, a 20 min latent period, and a burst size of 114 PFU/cell. Experiments combining phage vB_AbaSi_W9 with antibiotics demonstrated a pronounced synergistic effect against clinical strains when used with tigecycline and rifampicin. In a mouse model infected with CRAB KBN10P04948 (ST191), the group treated with rifampicin (100 μg/mL) and phage vB_AbaSi_W9 (MOI 1) achieved a 100% survival rate-a significant improvement over the phage-only treatment (8.3% survival rate) or antibiotic-only treatment (25% survival rate) groups. The bacteriophage vB_AbaSi_W9 demonstrated excellent synergy against CRAB strains when combined with tigecycline and rifampicin, suggesting potential candidates for phage-antibiotic combination therapy in treating CRAB infections.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some β-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

Bicyclomycin generates ROS and blocks cell division in Escherichia coli.

The role of reactive oxygen species (ROS) in the killing exerted by antibiotics on bacteria is debated. Evidence attributes part of toxicity of many antibiotics to their ability to generate ROS by interfering with cellular metabolism, but some studies dismiss the role of ROS. Bicyclomycin (BCM) is a broad-spectrum antibiotic that is the only known compound to inhibit E. coli transcription terminator factor Rho with no known other cellular targets. In the present study, we addressed this question by checking whether the induction of oxidative stress could explain the increased sensitivity to Bicyclomycin in the hns deleted strain even in Δkil background in E. coli. BCM evoked the generation of ROS in E. coli cells. BCM is known to cause the cell filamentation phenotype in E. coli. Performing fluorescence microscopic analysis, we show that bicyclomycin-dependent cell filamentation is associated with SOS response. RecA-GFP filaments were found to colocalize with the damaged DNA sites in the cell. Further analysis revealed that the genomic DNA was partitioned but the cell septum formation was severely affected under BCM treatment. Furthermore, we observed biofilm formation by E. coli after BCM treatment. We hypothesize that ROS production after BCM treatment could lead to cell filamentation in bacteria. A better understanding of the mode of toxicity of BCM will help us design better antibiotic treatment regimes for clinical practices, including combinatorial drug therapies. The cell filamentation phenotype observed after BCM treatment makes this antibiotic a promising drug for phage-antibiotic synergy (PAS) therapy.

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

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.

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.

Phage Therapy as an Emerging Antibiotic Alternative

In 2019, nearly 5 million deaths worldwide were attributed to antimicrobial resistance (AMR), underscoring its profound threat to global public health. AMR causes traditional antibiotics to lose effectiveness against bacterial infections due to the emergence of resistant bacteria. Consequently, phage therapy, which employs bacteria viruses to combat bacterial infections, has become a potential solution. Phages can enhance antibiotic sensitivity by targeting bacterial mutants, influencing the evolution of populations and impacting receptors responsible for antibiotic efflux from cells. Despite the advantages, several limitations to the use of bacteriophages exist. Phage-Antibiotic Synergy (PAS) may address these limitations. PAS describes the phenomenon of improved antimicrobial effect caused by stimulated phage replication in the presence of sublethal concentrations of antibiotics. Phage-antibiotic therapy has effectively reduced the emergence of phage-resistant and antibiotic-resistant strains simultaneously.

Phage-antibiotic synergy: Cell filamentation is a key driver of successful phage predation.

Phages are promising tools to fight antibiotic-resistant bacteria, and as for now, phage therapy is essentially performed in combination with antibiotics. Interestingly, combined treatments including phages and a wide range of antibiotics lead to an increased bacterial killing, a phenomenon called phage-antibiotic synergy (PAS), suggesting that antibiotic-induced changes in bacterial physiology alter the dynamics of phage propagation. Using single-phage and single-cell techniques, each step of the lytic cycle of phage HK620 was studied in E. coli cultures treated with either ceftazidime, cephalexin or ciprofloxacin, three filamentation-inducing antibiotics. In the presence of sublethal doses of antibiotics, multiple stress tolerance and DNA repair pathways are triggered following activation of the SOS response. One of the most notable effects is the inhibition of bacterial division. As a result, a significant fraction of cells forms filaments that stop dividing but have higher rates of mutagenesis. Antibiotic-induced filaments become easy targets for phages due to their enlarged surface areas, as demonstrated by fluorescence microscopy and flow cytometry techniques. Adsorption, infection and lysis occur more often in filamentous cells compared to regular-sized bacteria. In addition, the reduction in bacterial numbers caused by impaired cell division may account for the faster elimination of bacteria during PAS. We developed a mathematical model to capture the interaction between sublethal doses of antibiotics and exposition to phages. This model shows that the induction of filamentation by sublethal doses of antibiotics can amplify the replication of phages and therefore yield PAS. We also use this model to study the consequences of PAS on the emergence of antibiotic resistance. A significant percentage of hyper-mutagenic filamentous bacteria are effectively killed by phages due to their increased susceptibility to infection. As a result, the addition of even a very low number of bacteriophages produced a strong reduction of the mutagenesis rate of the entire bacterial population. We confirm this prediction experimentally using reporters for bacterial DNA repair. Our work highlights the multiple benefits associated with the combination of sublethal doses of antibiotics with bacteriophages.

Sequential treatment effects on phage–antibiotic synergistic application against multi-drug-resistant Acinetobacter baumannii

Bacteriophage (phage) therapy, exploiting phages which are the natural enemies of bacteria, has been re-introduced to treat multidrug-resistant (MDR) bacterial infections. However, some intrinsic drawbacks of phages are overshadowing their clinical use, particularly the narrow host spectrum and rapid emergence of resistance upon treatment. The use of phage-antibiotic combinations exhibiting synergistic bacterial killing [termed 'phage-antibiotic synergy' (PAS)] has therefore been proposed. It is well reported that the types and doses of phages and antibiotics are critical in achieving PAS. However, the impact of treatment order has received less research attention. As such, this study used an Acinetobacter baumannii phage vB_AbaM-IME-AB2 and colistin as a model PAS combination to elucidate the order effects in-vitro. While application of the phage 8 h before colistin treatment demonstrated the greatest antibacterial synergy, it failed to prevent the development of phage resistance. On the other hand, simultaneous application and antibiotic followed by phage application were able to suppress/delay the development of resistance effectively, and simultaneous application demonstrated superior antibacterial and antibiofilm activities. Further in-vivo investigation is required to confirm the impact of treatment order on PAS.

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 β-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 β-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.

"Two Is Better Than One": The Multifactorial Nature of Phage-Antibiotic Combinatorial Treatments Against ESKAPE-Induced Infections.

Phage-antibiotic synergy (PAS) has been extensively explored over the past decade, with the aim of developing more effective treatments against multidrug-resistant organisms. However, it remains unclear how to effectively combine these two approaches. To address this uncertainty, we assessed four main aspects of PAS interactions in this review, seeking to identify commonalities of combining treatments within and between bacterial species. We examined all literature on PAS efficacy toward ESKAPE pathogens and present an analysis of the data in papers focusing on: (1) order of treatment, (2) dose of both phage and antibiotics, (3) mechanism of action, and (4) viability of transfer from in vivo or animal model trials to clinical applications. Our analysis indicates that there is little consistency within phage-antibiotic therapy regimens, suggesting that highly individualized treatment regimens should be used. We propose a set of experimental studies to address these research gaps. We end our review with suggestions on how to improve studies on phage-antibiotic combination therapy to advance this field.

Assessment of the potential of phage-antibiotic synergy to induce collateral sensitivity in Salmonella Typhimurium

This study was designed to evaluate the synergistic effect of phage and antibiotic on the induction of collateral sensitivity in Salmonella Typhimurium. The synergistic effects of Salmonella phage PBST32 combined with ciprofloxacin (CIP) against S. Typhimurium KCCM 40253 (STKCCM) were evaluated using a fractional inhibitory concentration (FIC) assay. The CIP susceptibility of STKCCM was increased when combined with PBST32, showing 16-fold decrease at 7 log PFU/mL. The combination of 1/2 × MIC of CIP and PBST32 (CIP[1/2]+PBST32) effectively inhibited the growth of STKCCM up to below the detection limit (1.3 log CFU/mL) after 12 h of incubation at 37 °C. The significant reduction in bacterial swimming motility was observed for PBST32 and CIP[1/4]+PBST32. The CIP[1/4]+PBST32 increased the fitness cost (relative fitness = 0.57) and decreased the cross-resistance to different classes of antibiotics. STKCCM treated with PBST32 alone treatment exhibited the highest coefficient of variation (90%), followed by CIP[1/4]+PBST32 (75%). These results suggest that the combination of PBST32 and CIP can be used to control bacterial pathogens.

1691. Bacteriophage and Protein Synthesis Pathway Inhibitor Combinations: Antibiotic mechanism of action drives antagonistic interactions

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 &amp;gt;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&amp;lt; 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 &amp;gt;4) while phage-DAP was synergistic (FIC 0.5) (ANOVA range of mean differences 0.52 to 2.59 log10 CFU/mL; P&amp;lt; 0.001). For P. aeruginosa, phage-AZM and phage-GEN were antagonistic (FIC &amp;gt;4) and additive (FIC=1), respectively (ANOVA range of mean differences 1.04 to 1.95 log10 CFU/mL; P&amp;lt; 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.

Synergy of a virulent phage (φAB182) with antibiotics leading to successful elimination of biofilms formed by MDR Acinetobacter baumannii

Emergence of multiple drug resistant (MDR) strains of Acinetobacter baumannii and a withering drug discovery pipeline necessitates the search for effective alternatives to replace or synergize with currently used antibiotics. In this report, we have described the synergy assessment of a virulent Acinetobacter baumannii phage φAB182 with a wide range of antibiotics. Myophage φAB182 was isolated from sewage against MDR A. baumannii and exhibited maximum stability at 25 °C and pH 7. It also had a short latent period of 9min with a large burst size of 287. The phylogenetic analysis of its major capsid protein gene indicated an 84.15% similarity to the lyticA. baumannii phage Acj9. In the presence of antibiotics, phage φAB182 showed the highest synergy (p<0.0001) with colistin, followed by polymixin B, ceftazidime and cefotaxime and this synergistic effect was further validated by time kill kinetics. The combined action of phage φAB182 with colistin, polymixin B, ceftazidime and cefotaxime was also synergistic for the eradication of biofilms formed by A. baumannii as measured by MBECcombination/MBECantibiotic values (<0.25). We thus propose bacteriophage φAB182 as a potential antibacterial candidate in combination therapy. The findings from this study strongly support the use of phage antibiotic synergy for the successful treatment of biofilm forming MDR A. baumannii infections.

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

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. 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. 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. 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.

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.

An Optimized Checkerboard Method for Phage-Antibiotic Synergy Detection.

Phage-antibiotic synergy is a promising therapeutic strategy, but there is no reliable method for synergism estimation. Although the time-kill curve assay is a gold standard, the method is not appropriate for fast and extensive screening of the synergy. The aim is to optimize the checkerboard method to determine phage-chemical agent interactions, to check its applicability by the time-kill curve method, and to examine whether the synergy can be obtained with both simultaneous and successive applications of these agents. In addition, the aim is to determine interactions of the Pseudomonas phage JG024 with ciprofloxacin, gentamicin, or ceftriaxone, as well as the Staphylococcus phage MSA6 and SES43300 with ciprofloxacin, gentamicin, and oxacillin. The results show that the optimized checkerboard method is reliable and that results correspond to those obtained by the time-kill curve. The synergy is detected with the phage JG024 and ciprofloxacin or ceftriaxone against Pseudomonas aeruginosa, and the phage SES43300 with ciprofloxacin against MRSA. The synergy was obtained after simultaneous applications, and in the case of P. aeruginosa, after application of the second agent with delay of one hour, indicating that simultaneous application is the best mode of synergy exploitation for therapy. The checkerboard method can be used for thorough clinical studies on synergy and in the future for personalized therapy when infections are caused by multiple resistant bacteria.