Prenylated phenolics as promising candidates for combination antibacterial therapy: Morusin and kuwanon G

Abstract

Combination of antibiotics with natural products is a promising strategy for potentiating antibiotic activity and overcoming antibiotic resistance. The purpose of the present study was to investigate whether morusin and kuwanon G, prenylated phenolics in Morus species, have the ability to enhance antibiotic activity and reverse antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. Commonly used antibiotics (oxacillin, erythromycin, gentamicin, ciprofloxacin, tetracycline, clindamycin) were selected for the combination studies. Checkerboard and time-kill assays were used to investigate potential bacteriostatic and bactericidal synergistic interactions, respectively between morusin or kuwanon G and antibiotics. According to both fractional inhibitory concentration index and response surface models, twenty combinations (14 morusin-antibiotic combinations, six kuwanon G-antibiotic combinations) displaying bacteriostatic synergy were identified, with 4-512-fold reduction in the minimum inhibitory concentration values of antibiotics in combination. Both morusin and kuwanon G reversed oxacillin resistance of methicillin-resistant Staphylococcus aureus. In addition, morusin reversed tetracycline resistance of Staphylococcus epidermidis. At half of the minimum inhibitory concentrations, combinations of morusin with oxacillin or gentamicin showed bactericidal synergy against methicillin-resistant Staphylococcus aureus. Fluorescence and differential interference contrast microscopy and scanning electron microscopy showed an increase in the membrane permeability and massive leakage of cellular content in methicillin-resistant Staphylococcus aureus exposed to morusin or kuwanon G. Overall, our findings strongly indicate that both prenylated compounds are good candidates for the development of novel antibacterial combination therapies.

Keywords: Antibacterial synergy; Antibiotics; Kuwanon G; MRSA; Membrane permeabilization; Morusin.

Fig. 1

Structures of morusin (A) and kuwanon G (B).

Fig. 2

Effects of exposure to morusin and kuwanon G on the integrity (A, B, C) and morphology (D, E, F) of S. aureus ATCC 43300 (MRSA) membrane. Fluorescence images of S. aureus ATCC 43300 (MRSA) after 60 min exposure to DMSO (control) (A), morusin (2 × MIC) (B) and kuwanon G (2 × MIC) (C). Scanning electron microscopy (SEM) of S. aureus ATCC 43300 (MRSA) after 24 h incubation: no treatment (D), treated with morusin (½ × MIC) (E) and kuwanon G (½ × MIC) (F).

Fig. 3

Effects of exposure to morusin (2 × MIC) and kuwanon G (2 × MIC) on membrane permeability of exponential-phase cultures of S. aureus ATCC 43300 (MRSA) expressed as the percentage of red fluorescent cells in the population. Each bar represents the mean acquired by counting the captured cells and the error bars represent the standard deviations. *p < 0.05 vs. control (ANOVA).

Fig. 4

Isobolograms depicting the interactions between morusin (MO) or kuwanon G (KG) and antibiotics (OX – oxacillin, CIP – ciprofloxacin, CLI – clindamycin, ERY – erythromycin, GEN – gentamicin, TE – tetracycline) against S. aureus ATCC 43300 (MRSA) (A, B), S. aureus ATCC 6538 (MSSA) (C, D) and S. epidermidis ATCC 12228 (E, F).

Fig. 5

Three-dimensional plot of the experimental percentage of growth between morusin and gentamicin against S. aureus ATCC 43300 (MRSA) (A). Three-dimensional plot of the difference between the predicted and experimental percentage of growth between morusin and gentamicin against S. aureus ATCC 43300 (MRSA) (△E model) (B).

Fig. 6

Time-kill curves of morusin, gentamicin and oxacillin (each at ½ × MIC) alone and in combination against S. aureus ATCC 43300 (MRSA).