Characterization of the Gain and Loss of Resistance to Antibiotics versus Tolerance to Honey as an Antimutagenic and Antimicrobial Medium in Extended‑Time Serial Transfer Experiments

Background: Honey contains several substances with antimicrobial properties that appear more recalcitrant to generating bacterial‑evolved resistance than traditional antibiotics. Objectives: This study seeks to characterize the evolution of bacteria grown for successive generations in honey as an antimutagenic medium versus ampicillin. Materials and Methods: A naive strain of Staphylococcus aureus was serially cultured for 17 days in Lysogeny broth (LB) containing sublethal concentrations of medical‑grade Manuka honey, polyfloral honey, and ampicillin. Glucose as an osmotic control and pure LB were included for comparison. A portion of each culture was removed every 24 h to (a) determine the amount of growth that occurred during the previous 24 h and (b) use as a genetic stock in serially transferred tubes containing the same inhibitory compound as used previously. Results: As indicated by an increase in growth over sequential 24‑h period, bacteria rapidly gained resistance to ampicillin in a step‑wise pattern. However, bacteria grown in Manuka and polyfloral honey never exceeded their initial growth levels. Bacteria grown in relatively high concentrations of honey for a single 24‑h period consistently lost viability after one transfer, a phenomenon that has not been reported in the literature before and which indicates the inability of bacteria to adapt to honey as an antimutagenic medium. While bacteria grown in honey did not evolve the ability to grow at higher concentrations, a single isolate grown in Manuka honey gained tolerance to Manuka by successfully surviving and proliferating beyond second transfers. Bacteria that developed antibiotic resistance were found to remain sensitive to honey. Moreover, bacteria lost their resistance to ampicillin upon a single exposure to Manuka honey.