Phage Diversity of Six Mycobacteriophages and Host Range of 105 Mycobacteriophages in Four Mycobacteria Including Mycobacterium Tuberculosis H37Ra

Abstract

Bacteriophages (phages) are viruses that infect and generally kill bacteria. Phages are the most abundant biological entity on Earth; they are ubiquitous and as diverse as they are abundant. The focus of research at Truckee Meadows Community College since the Fall of 2014, has been on mycobacteriophages; phages that infect the host bacterium Mycobacterium smegmatis mc2155. The genometrics of six mycobacteriophages presented here: Appletree2, Cookies, Cueylyss, Guillsminger, NicoleTera, and Zulu, provide a small snapshot of the huge diversity apparent in just one subset of bacteriophages. The process by which bacteriophages adhere to a bacterial cell for infection is highly specific and not fully understood. Further elucidation into bacteriophage interactions with host bacteria is needed. In an effort to understand phage biology and phage bacterial host interactions more clearly, research into characterizing phage specificity and host range of 105 novel mycobacteriophages collected in our lab over the last four years has been underway and progress is reported here. In this study, four species of Mycobacterium were tested for infectivity, including Mycobacterium tuberculosis H37Ra. Using a spot test assay, it was found that 30.5% of the mycobacteriophage lysates tested demonstrated some degree of cross infectivity and were able to infect at least one other mycobacterial host strains. Of the 32 mycobacteriophages capable of cross infection, 50.0% were able to infect one other mycobacterial host, 46.9% were able to infect two other species of mycobacterium, and 3.1% were able to infect three other mycobacterial hosts.Of the mycobacteriophage lysates screened for host specificity, 19% infected M. tuberculosis, 17% infected M. phlei, 22% infected M. thermoresistible, and 2% infected M. cookii. To confirm infection capability, mycobacteriophages capable of infecting M. tuberculosis were further analyzed using the plaque testing assay. Of the 20 mycobacteriophages capable of infecting M. tuberculosis, it was determined that 50% were unable to infect M. tuberculosis using the plaque testing method, 20% were able to infect M. tuberculosis at a reduced rate, and 30% were able to do so at an efficiency threshold similar to that of M. smegmatis. Out of 105 mycobacteriophages tested, only six were capable of infecting M. tuberculosis efficiently, emphasizing the high specificity of these phages.Genomic DNA has been sequenced from six mycobacteriophages capable of infecting M. tuberculosis H37Ra. Cluster designation for these six phage genomes indicates that all are members of Cluster A or K. This data supports previous research on host range analysis of mycobacteriophages.Future work may include DNA sequencing of the six phages capable of infecting M. tuberculosis efficiently. Annotation of these phage genomes and comparative genomic analysis of tail fiber genes would provide further knowledge of bacteriophage host specificity.