#17-108

Complete

Date Full Report Received

Date Abstract Report Received

Investigation

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Staphylococcus aureus is an opportunistic pathogen of major concern in both human and animal health. While this bacterium can cause life-threatening illness, it more commonly asymptomatically colonizes the nasal passages and skin of healthy people. S. aureus can also be carried by livestock animals including swine, and there is evidence that S. aureus can be transmitted back and forth between humans and swine. This makes swine a potential reservoir for S. aureus, which may pose a risk to workers in swine production facilities, and potentially to the larger community. Bacteriophages (phages) are bacterial viruses and they are major predators of bacteria in natural environments. The continued emergence of antibiotic resistant bacteria, including resistant strains of S. aureus, has led to increased interest in novel antibacterial strategies, including the use of phages. Objectives: Our preliminary data suggested the previously isolated S. aureus phage may be effective in combating S. aureus prevalence in swine production environments. To evaluate the utility of these phage the goals of this project were to i) investigate the host range that these isolated phage have against a variety of both swine and human S. aureus isolates either alone or in combination and ii) to select broad host range phage and test their ability to eliminate phage-resistant S. aureus isolates as well as to eliminate S. aureus biofilms. Methods: 14 phage were tested at two different concentrations (108 and 106 PFU/ml) for their ability to effectively infect a panel of 17 different S. aureus isolates from both human and swine sources in a liquid plate reader assay. Five of these phage were previously isolated, and the other 10 were phage isolated during the first awarded NPB grant titled “Characterization of Staphylococcus aureus Bacteriophages in Swine Production Environments”. Phage resistant S. aureus were isolated for four different phage and the ability of five phage to overcome these isolates were tested in a liquid plate reader assay. Five different phage cocktails were evaluated in their ability to infect six different S. aureus isolates in liquid culture assays and are currently being tested in their ability to control biofilm formation by the same strains. A panel of 16 S. aureus isolates were evaluated for their ability to form a biofilm in a 96-well tissue culture plates, a common model system for studying biofilms. Lastly, attempts to sequence myophages that have failed sequencing on three different platforms is ongoing. Results: Novel isolated phage display a varied host range, with podophages displaying the most limited host range and myophages displaying the most varied host range with some infecting 80% of the tested strains. These observations are the same for the previously isolated phages K and 44AHJD. However, while 44AHJD is a podophage, it has a slightly more expanded host range than the novel podophages tested. Phage-resistant mutants, if seen, usually caused regrowth of the culture at approximately 18 hours into the 20 hour assay. Therefore, phage-resistant S. aureus isolates were isolated for four different phage. These phage-resistant S. aureus isolates were tested in the liquid culture assay against a panel of five phage to determine if other phage could still infect these phage-resistant S. aureus mutants. This data was then used to generate different phage cocktails to ensure that if an S. aureus isolate became resistant to one phage, it would be sensitive to another phage in the formulations. Biofilm 96-well plate assays for 16 S. aureus isolates have shown extremely varied results between strains as well as the positive control (ATCC 25923). The strain with the strongest biofilm formation ability besides the positive control is human isolate NRS70, which is an ST5, USA100 and SccMec type II strain. The strongest biofilm former of the swine isolates tested in this assay was PD18 which is a ST9 methicillin-sensitive (MSSA) nasal swine strain isolated in North Carolina. Sequencing of previously isolated podophage was finished with a total of sixteen podophage genomes sequenced. Comparison of the podophage genomes to understand the observed difference in things such as plaque morphology and host range is ongoing. Additionally, all attempts to sequence a set of previously isolated large myophages have failed, suggesting hypermodification of phage DNA. Experiments to identify which base in these phage is modified by using thin layer chromatography (TLC) is ongoing.