Decay and damage of therapeutic phage OMKO1 by environmental stressors

Abstract

Antibiotic resistant bacterial pathogens are increasingly prevalent, driving the need for alternative approaches to chemical antibiotics when treating infections. One such approach is bacteriophage therapy: the use of bacteria-specific viruses that lyse (kill) their host cells. Just as the effect of environmental conditions (e.g. elevated temperature) on antibiotic efficacy is well-studied, the effect of environmental stressors on the potency of phage therapy candidates demands examination. Therapeutic phage OMKO1 infects and kills the opportunistic human pathogen Pseudomonas aeruginosa. Here, we used phage OMKO1 as a model to test how environmental stressors can lead to damage and decay of virus particles. We assessed the effects of elevated temperatures, saline concentrations, and urea concentrations. We observed that OMKO1 particles were highly tolerant to different saline concentrations, but decayed more rapidly at elevated temperatures and under high concentrations of urea. Additionally, we found that exposure to elevated temperature reduced the ability of surviving phage particles to suppress the growth of P. aeruginosa, suggesting a temperature-induced damage. Our findings demonstrate that OMKO1 is highly tolerant to a range of conditions that could be experienced inside and outside the human body, while also showing the need for careful characterization of therapeutic phages to ensure that environmental exposure does not compromise their expected potency, dosing, and pharmacokinetics.

Fig 1. Inactivation & fitness suppression of OMKO1 by thermal stress.

A. To measure phage particle survival of heat stress at different temperatures, OMKO1 was exposed to one of a range of temperatures for 5, 30, 60, or 90 minutes, then titered. Percent survival is plotted relative to the titer of the 55°C treatment after 0 minutes. The dotted line denotes the limit of detection, with the timepoint where survival fell below the limit of detection plotted as an asterisk. 80°C was also tested but caused such rapid particle decay that all measures fell below the limit of detection. B. To measure phage particle survival of heat stress over longer periods of time, five biological replicates of phage OMKO1 were exposed to 70°C for 5, 90, 180, 270, or 360 minutes, then titered. Percent survival is plotted relative to the source stock titer. The dotted line denotes the mean limit of detection across all five batches. C. To determine whether phage fitness is affected by a history of heat stress exposure, 70°C heat shocked phages or unshocked control phages (0 min) were inoculated with bacteria and grown overnight while measuring bacterial density. As a metric of phage fitness, the peak bacterial density was computationally determined. Thus, higher peak bacterial densities indicated phages with lower fitness. The dotted line denotes the absence of bacterial growth. Bacteria were also grown in the absence of phage in LB media (“+ Ctrl”) or LB media that had been heat shocked for 360 mins at 70°C (“+ Ctrl Shock”). Heat shock treatments that are not significantly different from each other via Tukey Honest Significant Differences are indicated by the same shared letter (a or b).

Fig 2. Phage OMKO1 decay is not accelerated by saline concentration.

Fig 3. Degradation of phage OMKO1 under different urea concentrations.