DOI: 10.5061/dryad.zs7h44jq1
title: Inactivating conditions of therapeutic mycobacteriophages

description: 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
 ZoeJΔ4,5 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.
description: # Inactivating conditions of therapeutic mycobacteriophages
 [https://doi.org/10.5061/dryad.t1g1jwt8h](https://doi.org/10.5061/dryad.t1g1jwt8h) To measure phage sensitivities, phages at titers of 10^9 – 10^10 PFU/ml in mycobacteriophage (MP) buffer were exposed to different conditions at room temperature (22° C), unless otherwise indicated. At specific time points, phages were quantified as PFU/ml using an agar overlay assay with Mycobacterium smegmatis. Conditions resulting in a significant reduction in titer compared to an untreated phage control at the same time point were determined by calculating the log change (log PFUchallenge – log PFUcontrol. Log change data from replicate experiments are presented in Table 1 in the manuscript. Fig1ABCv2.csv: CSV file reporting raw data for plaque-forming units per ml (pfu/ml) and the log10 transformation of the plaque-forming units per ml [log(pfu/ml)] for phages BPs, Muddy, and ZoeJ challenged to various temperatures, PIB at various pH, and MP Buffer at various pH. Fig1A: Temperature sensitivity Fig1B: PIB + pH Sensitivity Fig1C: MP Buffer + pH Sensitivity Table1_Dryad_Raw_Data_v2.csv: CSV file reporting raw data for plaque-forming units per ml (pfu/ml) and the log10 transformation of the plaque-forming units per ml [log(pfu/ml)] for phages BPs, Muddy, and ZoeJ challenged to various therapeutic and storage conditions. Data for the conditions of Temperature, PIB, and MP Buffer are located in the file for Fig1ABC. Supplemental_Fig_1.csv: CSV file reporting raw data colony-forming units per ml (cfu/ml) and the log10 transformation of colony-forming units per ml [log(cfu/ml)] for Mycobacterium smegmatis sensitivity to phage inhibition buffer (PIB) at pH 3. Supplemental Fig 1. Mycobacterium smegmatis is not sensitive to phage inhibition buffer (PIB) at pH 3.