Genomic analysis of seven mycobacteriophages identifies three novel species with differing phenotypic stabilities

Recently, case studies have been published regarding the application of mycobacteriophage (MP) therapy (MPT) in patients with multi-antibiotic-resistant infections. A major limitation in the development of MPT is the paucity of therapeutically useful MP. As there are approximately 10,000 MP that have yet to be sequenced, it is possible that characterization of this cohort would increase the repertoire of useful MP. This study aims to contribute to such a strategy, by characterizing a cohort of 7 mycobacteriophages. Sequencing analyses revealed that the MP have unique sequences, and subsequent gene annotation revealed differences in gene organization. Notably, MP LOCARD has the largest genome and operons encoding for glycosyltransferases. Taxonomic analysis executed with VIRIDIC, Gegenees and VICTOR revealed that LOCARD belongs to a different genus than the other phages and is the foundational member of one of three novel species identified in this study. LOCARD, LOCV2, and LOCV5 were selected as representative members of their species and subjected to phenotypic analyses to compare their stability under biologically and industrially relevant conditions. Again LOCARD stood out, as it was unaffected by the typical temperatures (37 °C) and salinity (0.9%) experienced in mammals, while the viability of LOCV2 and LOCV5 was significantly reduced. LOCARD was also tolerant to pH 10, low levels of antiviral detergent and was the least impacted by a single freeze-thaw cycle. When all these results are considered, it indicates that LOCARD in particular, has potential therapeutic and/or diagnostics applications, given its resilience towards physiological and storage conditions.