Antibiotic Disk Susceptibility Tests as a Way to Economically Screen Aminoglycoside Acetyltransferase Mutant Enzymes for Activity
Damon Holt, Alejandra Portillo, Leyla Riyhani, Gabriela Tozlovan, Ron Painter
Department of Chemistry & Biochemistry
Faculty Supervisor: Misty L. Kuhn
Antibiotic resistance is a significant threat to human health. A lack of new antibiotics in the pipeline requires a critical stewardship attitude toward the antibiotics currently available as therapeutics. To achieve this, a broader understanding of the mutational adaptability of currently known antibiotic resistance proteins is required. One class of proteins that are pivotal in bacterial resistance to aminoglycoside antibiotics includes the aminoglycoside N-acetyltransferases (AACs). Our laboratory is currently interested in the AAC(6')-Ig enzyme from the bacterium Acinetobacter haemolyticus. Therefore, this study investigates the effects of a series of 64 AAC(6')-Ig active site residue mutations on enzyme-mediated modification of four aminoglycoside antibiotics via antibiotic disk susceptibility tests. Since one of the substrates (AcCoA) that is required for enzymatic reactions is costly (~$1K per 100 mg), we took advantage of the AcCoA produced inside of the E. coli bacterium during its growth on rich media as an economical strategy for screening these mutant enzymes for activity. Our specific objectives included testing and analyzing the impact of individual amino acid substitutions on bacterial resistance to tobramycin, neomycin, kanamycin, and amikacin. We found several examples of mutants that successfully acetylated these antibiotics, which provides preliminary insight into their activity and resistance profiles.