According to CDC, every year at least 2 million people are affected and 23,000 dies as a result of antibiotic resistance in U.S. It is considered one of the biggest threats to global health. More and more bacterial infections are becoming harder to treat. One such infection is fire blight, one of the most destructive disease of apple and pear trees. It is caused by bacteria Erwinia amylovora and its outbreaks have been known to destroy entire orchards in a single season. The conventional method of treatments includes use of antibiotics like streptomycin and oxytetracycline but the incidences like presence of multi-drug resistant bacteria in the mammals grazing in the fields have raised concerns. Phage therapy is considered one of the few ways available to combat bacterial resistance and prevent fire blight. In this method, a cocktail of highly lytic bacteriophages is prepared and sprayed on the trees at different time intervals. Bacteriophages are an “intelligent” drug. They multiply at the site of the infection until there are no more bacteria and then they are excreted back into the nature. These phenomena make them more efficient than an antibiotic, which kills all kind of bacteria including good bacteria and can be maintained in the environment for long periods of time. These qualities of bacteriophage have resulted in many commercially available phage therapies. The initial part of this research focuses on isolation, characterization and genomic comparison of bacteriophages that infect a plant pathogen E.amylovora of Erwiniaceae family of Enterobacteriales order. In this study, 28 novel bacteriophages were isolated, fully sequenced, characterized and grouped into seven families based on phage homology. To take this further, we characterized a novel jumbo family of bacteriophages that has a small burst size of 4.6-4.9 and are most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity. These bacteriophages are shown to infect Erwinia and Pantoea bacterial strains, but no infection of 9 other bacterial strains tested, was seen, under laboratory conditions. The results of this work provide an insight on special characteristics that makes bacteriophage so unique and adaptable. The final part of this research explores the enormous diversity of bacteriophages. In 2014 Grose and Casjens grouped 337 fully sequenced tailed phages into 56 diverse clusters (32 lytic and 24 temperate). We further expanded our current understanding of these clusters by performing the comprehensive analysis of genomes and proteomes of 1037 tailed bacteriophages, posted on GenBank. The results of this work provide insights into diversity and relatedness of bacteriophages and the data is posted on GenBank.

Bacteriophages (phages) are the smallest biological entity on the planet. They provide vast amounts of valuable knowledge to biologists. Phage genomes are relatively simple compared to the organisms they infect (prokaryotes) and yet continually point to the complexity surrounding molecular- and microbiological mechanisms of life. By studying phages we can learn of the systems of gene expression, protein interaction and DNA organization. Phages are useful not only from an academic perspective, but may also have useful clinical applications. In the face of the rise of antibiotic-resistant bacterial “super pathogens”, scientists and researchers turn to phages as alternative treatments to these types of infections. Phages are capable of infecting and killing even the deadliest of bacterial pathogens, such as carbapenem-resistant Enterobacteriaceae (CRE) or Bacillus anthracis, and may prove increasingly useful in the future for combatting harmful pathogens. This thesis looks at several aspects of phage biology—from the underlying genetics contributing to phage virulence, to the clinical application of phage therapy to treat infections. First, a look at CRE-Klebsiella pneumoniae isolates and phages capable of infecting some strains may reveal a potential therapeutic approach in the future. Additionally, genomic analysis reveals interesting features that may explain aspects of phage virulence and evolutionary history. Then, a collection of genetically diverse phages is used in infection assays on pathogenic strains of Bacillus anthracis to establish the first-reported phages capable of infecting these strains. Finally, the process of preparing phage samples for therapeutic application is explored in-depth to conclude with discussion of clinical application. During the course of these projects over 25 phages were isolated, as many phage genomes were assembled and annotated, resulting in the preparation of two genome announcements and near-completion of two publishable first-author papers (chapters II and III). In addition, participation in a variety of collaborative efforts may lead to a handful of co-author papers and on various topics, including phage biology and application.

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Antibiotic resistant strains of bacteria continue to increase in prevalence, hindering the ability of clinicians to treat infection. One disease exacerbated by this trend is osteomyelitis, or bone infection. When osteomyelitis is induced by these antibiotic resistant strains, patients can experience prolonged hospital visits, greater economic burdens, amputation, and even death. Due to the limitations of antibiotics to clear these infections, we sought to identify new therapeutic options for osteomyelitis. Our aim was to first develop an in vivo implant-related model of osteomyelitis. We then wanted to explore the potential of novel CRISPR-Cas9 modified bacteriophage to treat infection. In vitro and in vivo investigations demonstrated that bacteriophage therapeutic may be a viable option for infection mitigation. Furthermore, our in vivo model of osteomyelitis proved to be reliable, consistent, and challenging. Future research will utilize this model as a platform for optimizing therapeutic regimen and delivery vehicle(s) for antimicrobial therapeutics.

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