The Virulence Index: A Metric for Quantitative Analysis of Phage Virulence

Background: One of the main challenges in developing phage therapy and manufacturing phage products is the reliable evaluation of their efficacy, performance, and quality. Since phage virulence is intrinsically difficult to fully capture, researchers have turned to rapid but partially inadequate methods for its evaluation. Materials and Methods: This study demonstrates a standardized quantitative method to assess phage virulence based on three parameters: the virulence index ( V P )-quantifying the virulence of a phage against a host, the local virulence ( v i )-assessing killing potential at given multiplicities of infection (MOIs), and MV 50 -the MOI at which the phage achieves 50% of its maximum theoretical virulence. This was shown through comparative analysis of the virulence of phages T4, T5, and T7. Results: Under the conditions tested, phage T7 displayed the highest virulence, followed by phage T4 and, finally, by phage T5. The impact of parameters such as temperature and medium composition on virulence was shown for each phage. The use of the method to evaluate the virulence of combinations of phages-for example, for cocktail formulation-is also shown with phages T5 and T7. Conclusions: The method presented provides a platform for high-throughput quantitative assessment of phage virulence and quality control of phage products. It can also be applied to phage screening, evaluation of phage strains, phage mutants, infection conditions and/or the susceptibility of host strains, and the formulation of phage cocktails.