Bacteriophage Felix O1, also known as enterobacteria phage O1, has been used to type Salmonella Typhi and is an excellent candidate for use in bioremedial and therapeutic applications. It has extremely high intra-species specificity and is strictly virulent in nature, unable to undergo lysogeny. To facilitate the development of the bacteriophage for use in these areas, the full sequence of the genome had been elucidated previously. In this work, identification and classification of functional coding sequences via reverse transcriptase-polymerase chain reaction was performed. All of the 115 putative open reading frames (ORFs) studied were found to be functional. 53.0%, 9.6%, and 18.3% of the ORFs investigated were found to initiate expression early, middle or late in the lytic cycle, respectively. Expression of the remaining 19.1% ORFs was evident when the amount of total RNA was increased and when samples were taken at a later time point. Comparisons between bacteriophage Felix O1 and the phage with the most shared homologs, phage T4, revealed many similarities in times of gene expression. / Master of Science

Bacteriophage Felix O1 was studied for applicability as a Salmonella intervention. Felix O1's potential as a Salmonella therapeutic was explored, as was its utility as a food application. Felix O1 is specific for and infects most serovars within the genus Salmonella. The entire 86.155-kb sequence of the phage's linear, double-stranded chromosome was determined. 213 open reading frames (ORFs) were found, including 23 homologues of phage genes (e<0.008). Homology searches do not indicate genes that would be expected to increase virulence of Salmonella. Thirteen T4 homologues were found, including rIIA and rIIB, rapid lysis genes of T-even phages. Site-directed mutagenesis of the rIIB region was attempted by homologous recombination with plasmids containing luxAB of Vibrio harveyi. No DrIIB luxAB+ recombinants resulted from the methods tried. Serial in vivo passage was used to select for a longer-circulating Felix O1 mutant using the modified methods of Merril et al., (1996). No difference was found in the clearance of wild-type (WT) and Felix O1 following nine serial passages. Injection of 10⁹pfus yielded 24-hour concentrations of 6.5 and 4.9 log10 pfus/ml plasma for WT and 9th passage, respectively. Both isolates were undetectable in plasma by 72 hours, but remained in spleens at 96 hours. A large-plaque Felix O1 variant (LP) isolated during in vivo serial passage was compared with WT for Salmonella growth suppression. Spectrophotometric measurement of BHI cultures indicated greater suppression of S. typhi by LP than by WT, a difference not seen with S. typhimurium DT104. Both isolates suppressed 24-hour S. typhimurium DT104 growth on experimentally-contaminated chicken frankfurters at 22°C. Untreated frankfurters yielded 6.81 log10 Salmonella cfus/g, whereas WT and LP-treated samples yielded 5.01 and 4.70 log10 cfus/g, respectively. Both phages suppressed the Salmonella typhimurium DT104 growth (p<0.0001), but the isolates did not perform differently (p=0.5088). Presence of Salmonella caused a higher yield of WT phage than from the uninoculated group (p=0.0011), but did not affect LP yield (p=0.4416). With Salmonella present, the 24-hour LP concentration was lower than WT concentration. This supports the surmised LP rapid-lysis phenotype since T4 rapid-lysis mutants typically exhibit lower burst sizes than wild-type phage. / Ph. D.

There is an increasing incidence of antimicrobial-resistant organisms isolated from food and food products. Coupled with that rising incidence is increased media scrutiny and coverage of outbreaks of foodborne illnesses. Consequently, consumers increasingly demand safer food, and that the antimicrobial measures used be other than antimicrobial drugs. A possible solution is to use bacteriophages, or the purified holin and endolysin proteins that make them lethal and lytic, as antimicrobial food treatments or additives. The bacteriophage Felix O1 is a promising candidate for development as an anti-Salmonella food treatment. This dissertation describes the work done to determine if these proteins could be of value as bioremedial agents. Endolysin treatments of Gram negative bacteria require two agents: the lytic endolysin, and a second agent to permeabilize the outer membrane of the bacterium. The holin protein was proposed as an outer membrane permeabilization agent. Methods used to locate the holin gene included BLAST analysis, analysis of putative Felix O1 proteins for transmembrane domains, and examination of the lysin sequence for an N-terminal signal sequence. Analyses did not reveal a promising candidate. Cloning of rIIA as a potential holin was attempted without success. Results of various analyses are discussed, as are chemical alternatives to the use of purified holin as a permeabilization agent. The endolysin, LysO1, was successfully cloned and characterized. PHYRE analysis predicted that the enzyme structure is composed of α helices arranged into two lobes, with the active site in a cleft between them. The enzyme lysed all tested strains of Salmonella and a tested strain of the foodborne pathogen Escherichia coli. Campylobacter jejuni susceptibility remains ambiguous, and the enzyme had no effect on Listeria monocytogenes or Micrococcus luteus. LysO1 was most active at alkaline pH and low ionic strength; optimal activity was observed in 25 mM buffer at pH 10. If removed from frozen storage, the enzyme was most thermostable at 30 °C. Lytic activity was adversely affected by the presence of the divalent cations calcium, magnesium, and zinc, and by high ionic strength. Considerable time was devoted to development of the activity assay used to further characterize the enzyme, and details of those experiments are provided. Logical extensions of the research project, such as further characterization and testing needed to obtain government approval for widespread use of the treatment, and possible pursuit of treatment based on an enzyme derivative such as an antimicrobial peptide, are discussed. / Ph. D.