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.

ABSTRACT

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.

INTRODUCTION

Infective endocarditis (IE) is a severe bacterial infection of the endocardium or heart valves, and Staphylococcus aureus remains the predominant cause of IE in the United States, with an associated higher probability of in-hospital mortality (1, 2). Bacteriophages (phages), particularly obligately lytic phages, are viruses that can locate and penetrate individual bacteria by inserting their genetic material into their host in order to take over their host’s machinery to replicate and release the progeny phage particles (3–10).

AB-SA01 (AmpliPhi Biosciences Corporation) is a bacteriophage cocktail consisting of three naturally occurring, obligately lytic myoviruses related to Staphylococcus phage K belonging to the Herelleviridae family (11, 12). Owing in part to the combination of three phages and their ability to complement each other, AB-SA01 showed broad activity against a variety of clinical S. aureus isolates (11). This makes AB-SA01 potentially favorable for clinical use as it is likely to be active against an infecting S. aureus strain and since no phage resistance or adverse reactions were observed in vivo during clinical bacteriophage-antibiotic combination (BAC) case studies in an Australian hospital (13). However, while the basic phenomenon of synergy with BACs has been documented many times, relatively little is known about the specific interactions of antistaphylococcal phages with standard-of-care (SOC) antibiotics such as vancomycin (VAN) or β-lactams, especially in microbiologically complex situations like IE. In addition to initial 24-h time-kill experiments, we focused on a multidrug-resistant clinical isolate in the context of a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) two-compartment, ex vivo model. This model is designed to mimic humanized PK/PD conditions and can therefore test novel antimicrobials while modeling standard therapeutic regimens for concomitant antibiotics (7). The model specifically recapitulates high-inoculum infections associated with clinical failure of antibiotic therapy, for which novel treatment regimens are needed.

Bacterial and phage strains, media, antibiotics, and in vitro susceptibility.

A well-characterized clinical isolate of methicillin-resistant S. aureus (MRSA) that was also daptomycin (DAP) nonsusceptible (DNS) and VAN intermediate (VISA) was used for all time-kill experiments and SEV models. Multiple mechanisms of resistance made this isolate an interesting candidate for investigating the impact of BACs. The D712 strain (DNS-VISA, agr2, USA100, and sequence type 5 [ST5]) had the following MIC susceptibilities: DAP at 4 mg/liter, VAN at 4 mg/liter, cefazolin (CFZ) at >64 mg/liter, and ceftaroline (CPT) at 0.5 mg/liter (14). AB-SA01 was provided as a stock in which each component phage was present at 1.5 × 10⁸ PFU/ml. The AB-SA01 component phages (Sa83, Sa87, and J-Sa36) lack the capacity to integrate into the bacterial genome and do not contain known bacterial virulence factors or antibiotic resistance genes (11).

Mueller-Hinton broth II (MHB) (Difco, Detroit, MI, USA) (25 mg/liter calcium, 12.5 mg/liter magnesium) was used for susceptibility tests and SEV models. Heart infusion broth (HIB) (BD, Bacto, San Jose, CA, USA) with 1.5% and 0.7% agar (Oxoid, Lenexa, KS, USA) was used for agar underlays and overlays of the phage quantification tests (modified small-drop agar overlay method [15]). VAN and CFZ were obtained commercially from Sigma Chemical Company (St. Louis, MO, USA). CPT was provided by its manufacturers (Allergan, Parsippany, NJ, and Actavis, Parsippany, NJ). Phage-mediated lysis, the release of phage progeny particles, as well as the efficiency of plating (EOP) against D712 were confirmed by the formation of individual plaques on selected S. aureus strains (Table 1).

TABLE 1.

Frequency of apparent resistance and final resistance in BAC versus AB-SA01-alone regimens^f

^aEst, estimated counts.

^bFoR, frequency of resistance [(colonies that arise on the resistance plate)/(CFU per milliliter used to create the plate)].

^cLoD, limit of detection. If the apparent frequency of resistance is calculated as 0, it is stated as <LoD.

^dS, some colonies did not grow when picked and streaked onto the plates. These are inferred to have been killed by residual phages that were picked up with the colony pick and are therefore marked “S” in the table. This was the case for all repeats for VAN+CFZ+AB-SA01 combinations.

^eEOP, efficiency of plating.

^fNumbers are PFU per milliliter for each combination of phage and bacterial isolate. Green cells indicate good clearing of the bacterial lawn by the concentrated phage sample. Yellow cells indicate moderate clearing of the bacterial lawn by concentrated phage. Pink cells indicate a very faint effect of the concentrated phage sample on the bacterial lawn. Red indicates no effect of the concentrated phage sample on the bacterial lawn. A pink or yellow cell with a titer of 0 means that no plaques were ever observed. The result is considered resistant because phages were not replicating. The partial effect may be due to “lysis-from-without” phenomena.

Time-kill analyses for assessing BAC activity.

Time-kill experiments were performed at 1/2× MIC values or peak concentrations of the antibiotics (whichever were lower) and 7.5 × 10⁶ PFU/ml of AB-SA01 (input phage-to-bacterium ratio of ∼7.5) (Fig. 1A). While all combinations of VAN plus CPT (VAN+CPT), VAN+CFZ, and phage+VAN+CFZ were synergistic (defined here as a >2-log₁₀ CFU/ml reduction compared to the most potent agent), the combination of phage+VAN+CPT was bactericidal (bactericidal activity was defined as a >3-log₁₀ CFU/ml reduction from the baseline) (16). Phage-alone treatment revealed static activity, with colony counts in the same range as those of the initial inoculum, which was more effective than any of the other individual agents.

FIG 1.

(A) Bacterial quantification in 24-h time-kill experiments. (B) Efficacy of bacteriophage-antibiotic combinations in a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model (bacterial quantification). (C) Bacteriophage quantification in SEV models (inside SEVs) over a period of 96 h. Abbreviations: GC, growth control; VAN, vancomycin; CPT, ceftaroline; CFZ, cefazolin; AB-SA01, bacteriophage cocktail.

Ex vivo PK/PD model.

All BAC regimens were conducted using a well-characterized, simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model (Fig. 1B and C). Doses were administered as boluses (VAN at 2 g every 12 h [Q12h] [maximum concentration of drug in serum {C_max} = 72 mg/liter; half-life {T_1/2} = 6 h] and AB-SA01 and CFZ at 2 g Q8h [C_max = 404 mg/liter] [17–20] with 1 ml of 1.5 × 10⁸ PFU/ml Q12h [6 × 10⁵ PFU/ml in the total volume] [phage-to-bacterium ratio of 0.0006]) over a time period of 96 h. SEV clots were prepared by mixing the cryoprecipitate as a fibrin source with a platelet suspension (American Red Cross) and a suspension of the organism in a final inoculum of 10⁹ CFU/0.5 g, as previously described (21–23). Dynamic inflow and outflow of the fresh medium were adjusted according to the half-life of the antibiotics. SEV clots were aseptically removed from each model in duplicate, weighed, and homogenized, and 0.5 ml of the sample was then filtered (0.22-μm syringe filter) and conserved for phage quantification, while the remainder of the sample was centrifuged and resuspended in cold normal saline and plated (limit of detection of 2 log₁₀ CFU/g) using a spot plating technique (18, 21, 22). The combinations of VAN+CFZ+AB-SA01, VAN+AB-SA01, and VAN+CFZ demonstrated 6.17-log₁₀ CFU/g, 3.78-log₁₀ CFU/g, and 3.04-log₁₀ CFU/g reductions from the initial inoculum, respectively. Of remarkable interest, even the VAN+AB-SA01 combination caused a 3.78-log₁₀ CFU/g reduction in the DNS-VISA strain. Samples for antibiotic and phage quantification were taken 5 min after bolus injection. Phage quantification inside SEVs followed a trend similar to that of the main compartment of the model (broth), with about 1- to 2-log₁₀ PFU/ml-lower counts, potentially due to the lower diffusion rate inside the SEVs (Fig. 1). Interestingly, the AB-SA01-alone model had higher phage counts than either of the BAC models. This observation may be due to the absence of antibiotics, as the killing of bacteria by AB-SA01 may require longer exposures and there are also more bacterial cells available in the vegetation for phages to propagate.

Resistance tests.

Elevated MIC values were not identified in any of the regimens at the end of the 96-h model. Prior to phage exposure, the spontaneous frequency of phage resistance (FoR) within D712 populations was estimated to be <10⁻⁸ (measured according to reference 11). The development of resistance to phage treatment in 96-h samples from the PK/PD model (Table 1) was evaluated in two steps: apparent FoR and confirmation testing of restreaked single colonies using high-titer phage (10¹¹ PFU/ml), as described previously (11, 24). Not all the apparent bacteriophage-insensitive mutants (BIMs) were resistant to AB-SA01 after restreaking, and those that were had come from AB-SA01-alone models. Phage resistance at 96 h was not noted in any of the BAC treatments (VAN+AB-SA01 or VAN+CFZ+AB-SA01).

The limitations of this study include the limited number of S. aureus strains and the lack of dose escalation/de-escalation experiments. While more investigation is required, our results highlight the potential for BAC regimens to prevent bacterial resistance in difficult-to-treat, high-inoculum infections such as IE.