Inactivating conditions of therapeutic mycobacteriophages

Abstract

There is a need for new therapies to treat drug resistant nontuberculous mycobacteria (NTM) disease. Bacteriophages (phages), which are viruses that infect and kill bacteria, are actively being explored as an alternative approach for treating mycobacterial diseases. Several compassionate-use cases of phage therapy for drug resistant NTM infections exhibit favorable outcomes. To further the development of phage therapy it is important to recognize and avoid conditions that negatively impact phage activity during phage production, storage, formulation, or treatment. Conversely, there is a need to inactivate free phages in certain preclinical phage therapy experiments. In this study, we investigated three mycobacteriophages BPsΔ33HTH-HRM10, Muddy, and ZoeJΔ45 from compassionate-use NTM treatment cases for their sensitivity to a variety of conditions that included temperature, acid pH, detergents, mucus, and phage inactivating buffers. Several conditions resulted in dramatic and rapid reductions in the level of active phage while others had no effect. We also observed different sensitivities between the phages. The results provide valuable information to support further investigation and development of these phages as therapeutics.

Keywords: Bacteriophage; Nontuberculous Mycobacteria; mycobacteriophage.

Figure 1.

A. Sensitivity of phages to various temperatures. BPsΔ, Muddy, and ZoeJΔ were treated with a range of temperatures from to 22° C – 70° C. At specific time points, samples were removed and phages quantified. The mean of two independent experiments, each with three technical replicates, is plotted. Asterisks indicate significant reduction in log (PFU/ml) between a given treatment compared to the 22° C control at the same time point determined by one-way ANOVA (p<0.05) with Tukey’s post-test. B. Effectiveness of Phage Inhibition Buffer (PIB) at various pH. BPsΔ, Muddy, and ZoeJΔ were treated at a 1:10 ratio with PIB at pH 3, 4, and 5 and compared to MP Buffer pH 7.5. At specific time points, phages were quantified. The mean of two to three independent experiments, each with three technical replicates, is plotted. Asterisks indicate significant reduction in log (PFU/ml) between a given PIB pH time point compared to the MP buffer pH 7.5 control at the same time point determined by one-way ANOVA (p<0.05) with Tukey’s post-test. C. Sensitivity of phages to various pH in MP buffer. BPsΔ, Muddy, and ZoeJΔ were incubated in MP buffer at pH 2, 3, 4, and 5 and compared to MP Buffer pH 7.5. At specific time points, samples were removed and phage quantified. The mean of two to three independent experiments, each with three technical replicates, was plotted. Asterisks indicate a significant reduction in log (PFU/ml) between a given pH time point compared to the MP buffer pH 7.5 control at the same time point determined by one-way ANOVA (p < 0.05) with Tukey’s post-test. The red dashed line indicates the limit of detection. Error bars indicate standard deviation. Samples with zero PFU recovered were plotted at the level of detection.

Figure 2.. Sensitivity of phages to inclusion of detergent in the M. smegmatis culture used in agar overlays.

BPsΔ, Muddy, and ZoeJΔ 10-fold serial dilutions spotted onto agar overlays prepared with M. smegmatis resuspended in Middlebrook 7H9 media supplemented with 0.5% glycerol, 0.2% glucose (no detergent) or the same Middlebrook 7H9 media with freshly added 0.1% Tween-80 (Tw-80) or 0.1% Tyloxapol (Tylox). Plates were incubated at 37° C for 22 and 46 hours. A representative image from two experiments is shown. All images were taken with the same exposure time of 0.09 seconds using a Bio-RAD GelDOC Go.