Neomycin Sulfate Improves the Antimicrobial Activity of Mupirocin-Based Antibacterial Ointments

Abstract

In the midst of the current antimicrobial pipeline void, alternative approaches are needed to reduce the incidence of infection and decrease reliance on last-resort antibiotics for the therapeutic intervention of bacterial pathogens. In that regard, mupirocin ointment-based decolonization and wound maintenance practices have proven effective in reducing Staphylococcus aureus transmission and mitigating invasive disease. However, the emergence of mupirocin-resistant strains has compromised the agent's efficacy, necessitating new strategies for the prevention of staphylococcal infections. Herein, we set out to improve the performance of mupirocin-based ointments. A screen of a Food and Drug Administration (FDA)-approved drug library revealed that the antibiotic neomycin sulfate potentiates the antimicrobial activity of mupirocin, whereas other library antibiotics did not. Preliminary mechanism of action studies indicate that neomycin's potentiating activity may be mediated by inhibition of the organism's RNase P function, an enzyme that is believed to participate in the tRNA processing pathway immediately upstream of the primary target of mupirocin. The improved antimicrobial activity of neomycin and mupirocin was maintained in ointment formulations and reduced S. aureus bacterial burden in murine models of nasal colonization and wound site infections. Combination therapy improved upon the effects of either agent alone and was effective in the treatment of contemporary methicillin-susceptible, methicillin-resistant, and high-level mupirocin-resistant S. aureus strains. From these perspectives, combination mupirocin-and-neomycin ointments appear to be superior to that of mupirocin alone and warrant further development.

FIG 1

Effects of neomycin on S. aureus RNase P-mediated ptRNA^Tyr processing. The mobility of precursor tRNA^Tyr in the presence (+) and absence (−) of S. aureus RNase P enzyme and the indicated concentration of neomycin (in micromolar) is shown. Densitometry measured percent activity shown (tRNA product formed) normalized to the value for DMSO-treated enzyme alone.

FIG 2

Antimicrobial zone of growth inhibition measures. (A to E) The average zones of inhibition (y axis; in square centimeters) of PEG-based ointments containing the indicated antibiotic or antibiotic mixture (x axis) toward S. aureus strain UAMS-1 (A, D, and E), USA300 (B) or BAA-1708 (C) are plotted. Significant increases in growth inhibition zones by Student's t test (n = 4), compared to the growth inhibition zones observed with 2% mupirocin, are indicated by bars and asterisks as follows: *, P ≤ 0.1; **, P ≤ 0.05.

FIG 3

Murine nasal decolonization measures. (A to C) The numbers of CFU per mouse nasal passage (y axis) after 3 days of dosing with PEG-based ointment containing the indicated antibiotic or antibiotic mixture (x axis) are plotted. Results for S. aureus strain UAMS-1 (A), USA300 (B), and BAA-1708 (C) are shown; red data points indicate low-level mupirocin-resistant isolates. Each symbol represents the value for an individual mouse. Each horizontal bar is the mean for the group of mice. Significant reductions in bacterial burden compared to the bacterial burden for mice treated with vehicle by one-way ANOVA are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 4

Murine wound decolonization measures. Shown are the numbers of CFU per lesion (y axis) following 3 days of dosing with PEG-based ointment containing the indicated antibiotic or antibiotic mixture (x axis). Results for S. aureus strains UAMS-1 (A), USA300 (B), and BAA-1708 (C) are shown. Significant reductions in bacterial burden between treatment groups by one-way ANOVA are indicated by bars and asterisks as follows: *, P ≤ 0.05; ****, P ≤ 0.0001.

FIG 5

Antimicrobial effects of PEG-based ointments toward A. baumannii and P. aeruginosa. The antimicrobial effects of PEG ointments containing vehicle, 2% mupirocin, 1% neomycin, and the combination of 2% mupirocin plus 1% neomycin toward A. baumannii strain 98-37-09 or P. aeruginosa strain PAO1 are shown.

FIG 6

Effects of PEG-based ointments on wound healing and animal health. (A) Representative wound healing images following 0, 3, 7, and 14 days of treatment with PEG-based ointments containing vehicle, 2% mupirocin, 1% neomycin, or the combination of 2% mupirocin plus 1% neomycin. (B) Average measures (n = 3) of wound contraction under the same conditions as in panel A. (C) Average body weight of animals (y axis) at the indicated day (x axis) after lesion formation and treatment with PEG-based ointment supplemented with the indicated agent.