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Frederick M. Cohan




Due to a variety of misuses, including but not limited to over-prescription, use during viral infections, and overuse in agriculture, antibiotic-resistant bacteria have become a significant issue in human populations across the globe. As a result of this rise in resistant bacteria, the efficacy of antibiotics across many different classes has declined, which in and of itself can exacerbate the issue to resistance. As an alternative to a purely antibiotic therapy for bacterial infection, some research groups have sought to combine the antimicrobial power of heavy metals, particularly silver, with their antibiotic of choice in order to bolster the power of the weakened antibiotic. This is dubbed synergistic therapy. Though silver is a good choice as an adjuvant for antibiotics, it demonstrates a much more powerful effect in gram-negative bacteria. The goal of this study is to establish this same relationship with copper – which demonstrates a stronger inhibitory effect in gram-positive bacteria – and two ribosome-inhibiting antibiotics, tetracycline and kanamycin. These experiments were conducted on twelve closely related strains of Bacillus subtilis ssp. spizizenii. We also sought to quantify the magnitude of this inhibition and determine if there was a differential response to the treatments across all twelve strains.

Twelve closely related strains, all of the same putative ecotype (PE15) were selected from our stock, were isolated from the Radio Facility Wash site in Death Valley. The twelve strains were originally isolated from the North-facing slope, the South-facing slope, or the bottom of the canyon (called the Arroyo). Due to their location of origin, we hypothesized that they may be differentially tolerant to copper. According to previous research, this tolerance to copper may serve as a proxy for antibiotic resistance, as copper exposure can drive selection to antibiotic resistance genes. Strains were then exposed to different levels of copper and antibiotic to determine a dosage that would not inhibit growth, to mimic tolerance to treatment in a clinical setting. The strains were then exposed to a combinatorial treatment, and growth was scored.

I determined that there was a significant synergistic inhibition across nearly all strains in both the copper + kanamycin and copper + tetracycline treatments. Although we hypothesized that strains would perform differentially to the treatment due to the microhabitat they were isolated from, this was not this case. I also determined that we were not observing adaptive evolution to the treatments by selecting for mutants that were more tolerant, but we were observing a true, robust synergistic relationship.

There are many new directions in which to expand this project. Primarily, it would be prudent to include some non-PE15 strains in the experiment. This would allow us a greater opportunity to observe differential response to the treatments across the strains. This would then allow us to make greater conclusions on this treatment as it relates to antibiotic-resistant bacteria. Additionally, it would make great sense to include antibiotics with different mechanisms of action in the experiment. We now know that ribosome-inhibiting antibiotics have a robust effect when used with copper, but the inclusion of other antibiotics could help establish a copper-centric paradigm of synergistic treatments. Finally, it might be possible to experimentally induce antibiotic resistance in the strains that were used in this experiment or use known mutants. This would remove the need for a copper-proxy of resistance and would give this project a greater clinical relevance.



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