Allyson Ruffino, “Frequency of Mucoid Mutants with Phage and Antibiotic Selection”
Mentor: Alita Burmeister, Biological Sciences, Letters & Science (College of)
Poster #135
Bacteriophages (‘phages’) are viruses that infect bacteria and are a potential alternative to antibiotics. Phage U136B is obligately lytic and infects Escherichia coli using the protein TolC, an antibiotic efflux pump. However, just as bacteria evolve resistance in the presence of antibiotics, they do the same in the presence of phage. Understanding factors that drive resistance evolution is crucial to determine efficacy of potential phage therapy use. In previous work, E. coli K12 evolved resistance to phage U136B via tolC or LPS biosynthesis gene mutations. These mutants were also sensitive to clindamycin due to decreased antibiotic efflux or increased outer membrane permeability. However, a lower proportion of phage-resistant isolates had a mucoid phenotype and were more clindamycin resistant. We hypothesized that simultaneous selection by both phage U136B and clindamycin would increase the frequency of mucoid mutants. We conducted replicate selection experiments with either phage alone (N = 44) or a phage-clindamycin combination (N = 44) and quantified the frequency of mucoid mutants. We isolated sixteen representative mutants, sequenced their genomes and identified mutations by comparing to the wild-type genome using breseq. We found that E. coli selected with phage U136B had a high frequency of the nonmucoid phenotype. E. coli selected with phage U136B and clindamycin had a higher frequency of the mucoid phenotype compared to controls. Mucoid mutations mapped to Rcs Phorphoprelay genes. As expected, there was a low frequency of clindamycin and phage-resistant non-mucoid mutants. These mutants all had point mutations in tolC, resulting in predicted changes to the extracellular region of TolC. In ongoing work, we are using experimental evolution to determine which selection pressures affect the frequency of mucoidy.