rhodomyrtone and Staphylococcal-Infections

Sigma factor B (SigB) controls the expression of Staphylococcus aureus genes including virulence factors and plays a role in the bacterial secretion system through membrane vesicle production. Inhibition of SigB could attenuate SigB dependent virulence and secretion system. The objective of this study was to determine the effects of rhodomyrtone on SigB and virulence factors related to SigB. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values of rhodomyrtone against 67 clinical methicillin-resistant S. aureus isolates were 0.25-8 μg/ml, which were similar to those of vancomycin. Using luciferase gene fused to SigB dependent promoters of asp23, five time reduction in SigB activity was observed when the bacteria were treated with rhodomyrtone for 3 h. Rhodomyrtone significantly reduced SigB activity in a concentration dependent manner in exponentially growing cells (P < 0.05). In addition, sigB mutant was more sensitive towards increasing concentrations of rhodomyrtone than the wild type and yabJ-spoVG mutant. Rhodomyrtone at 0.625 μg/ml reduced the growth of sigB mutant by approximately 99%, compared with the yabJ-spoVG mutant and the wild type. Membrane vesicles were significantly reduced in the bacterial cells when treated with 0.5 × MIC rhodomyrtone (P < 0.05). Decreased haemolytic activity was detected within rhodomyrtone-treated membrane vesicles. The results indicated that rhodomyrtone inhibited S. aureus SigB activity during exponentially growing phase and inhibited haemolytic activity within membrane vesicles.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Cell Membrane; DNA-Binding Proteins; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Mutation; Sigma Factor; Staphylococcal Infections; Staphylococcus aureus; Vancomycin; Virulence; Virulence Factors; Xanthones

An ethanolic extract from Rhodomyrtus tomentosa leaves (RTL) was studied as a natural alternative to control Staphylococcus aureus, which is an important pathogen responsible for bovine mastitis. The minimal inhibitory concentrations (MICs) of the RTL extract and of rhodomyrtone, a pure compound isolated from the plant, were determined by a microdilution method. Rhodomyrtone and the RTL extract exhibited antibacterial activity against S. aureus, including its persistent phenotype (SCV: small-colony variant) and a biofilm hyperproducer strain, with MICs of 0.25-0.5 and 8-16 µg/mL, respectively. Time-kill kinetics showed a strong bactericidal activity for both the RTL extract- and rhodomyrtone-treated bacteria at 2 × MIC as early as 4 h post-exposure. An additive effect of the extract at 0.5 × MIC was observed in a combination with oxytetracycline or pirlimycin against S. aureus by showing a 64- to 128-fold reduction in antibiotic MICs. Moreover, the RTL extract significantly decreased the number of intracellular SCVs inside bovine mammary epithelial cells. However, the extract or its combination with pirlimycin only slightly improved the activity of pirlimycin against the bacterial colonization of mouse mammary glands. In vitro MICs determined in the presence of casein indicated that the limited activity of the RTL extract in the murine model of mastitis could be linked to neutralization of active components by milk proteins. While the RTL extract showed interesting antibacterial properties in vitro, to be considered as an alternative to antibiotics in dairy farms, formulation studies are needed to cope with the observed reduction of activity in vivo.

Topics: Animals; Anti-Bacterial Agents; Cattle; Disease Models, Animal; Epithelial Cells; Female; Mastitis, Bovine; Mice; Microbial Sensitivity Tests; Myrtaceae; Plant Extracts; Staphylococcal Infections; Staphylococcus aureus; Xanthones

The anti-staphylococcal activity of an ethanol extract of Rhodomyrtus tomentosa and its pure compound, rhodomyrtone, as well as their effects on staphylococcal biofilm formation and biofilm-grown cells were assessed. MIC and minimal bactericidal concentration values of the ethanol extract and rhodomyrtone against planktonic cultures and biofilms of five clinical strains each of Staphylococcus aureus and Staphylococcus epidermidis, and American Type Culture Collection (ATCC) strains of both species, were 32-512 and 0.25-2 µg ml(-1), respectively. Results from time-kill studies indicated that rhodomyrtone at a concentration of 4× MIC could reduce the number of Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis ATCC 35984 cells by 99.9% within 3 and 13 h, respectively. The ability of rhodomyrtone and the ethanol extract to prevent biofilm formation and kill mature biofilms was assessed: both demonstrated better activity than vancomycin at inhibiting staphylococcal biofilm formation. In addition, the viability of 24 h and 5-day staphylococcal biofilm-grown cells decreased after treatment with the ethanol extract and rhodomyrtone. The ability to reduce biofilm formation and kill mature biofilms occurred in a dose-dependent manner. Scanning electron microscopy clearly confirmed that treatment with rhodomyrtone at 16× MIC could reduce 24 h biofilm formation and the numbers of staphylococci, whilst at 64× MIC this compound destroyed the organisms in the 5-day established biofilm. These results suggest that rhodomyrtone has the potential for further drug development for the treatment of biofilm-forming staphylococcal infections.

Topics: Anti-Bacterial Agents; Biofilms; Microbial Sensitivity Tests; Microbial Viability; Myrtaceae; Plant Extracts; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Vancomycin; Xanthones