Antibacterial activity of rhodomyrtone on Clostridium difficile vegetative cells and spores in vitro.

The increasing incidence and severity of diarrhoea and colitis caused by Clostridium difficile, together with a high rate of relapse following treatment with currently recommended antimicrobials, calls for novel interventions for C. difficile infection (CDI). Rhodomyrtone, a bioactive compound derived from the leaves of the rose myrtle (Rhodomyrtus tomentosa) has demonstrated antibacterial activity against several Gram-positive bacteria. This study compared the in vitro antimicrobial activity of rhodomyrtone on C. difficile with that of vancomycin, a recommended agent for the treatment of CDI. Determination of the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of rhodomyrtone and vancomycin for ten C. difficile isolates showed that the MICs of rhodomyrtone for C. difficile vegetative cells (0.625-2.5 mg/L) were comparable with that of vancomycin (1.25 mg/L), but the MBCs of rhodomyrtone (1.25-5 mg/L) were significantly lower than those for vancomycin (5 mg/L to ˃40 mg/L; P < 0.001). Time-kill assays showed rapid bactericidal activity for rhodomyrtone, with ≥99% killing within 4 h. Rhodomyrtone was also four-fold more potent than vancomycin in inhibiting C. difficile spore outgrowth. Transmission electron microscopy of rhodomyrtone-treated C. difficile revealed cell lysis and evidence of defective cell division and spore formation. These studies indicate that rhodomyrtone should be further investigated as a potential treatment for CDI.

Rhodomyrtus tomentosa Leaf Extract Inhibits Methicillin-Resistant Staphylococcus aureus Adhesion, Invasion, and Intracellular Survival in Human HaCaT Keratinocytes.

Methicillin-resistant Staphylococcus aureus (MRSA) has an ability to invade nonprofessional phagocytic cells, resulting in persistent infections and most likely host cell death. Series of our studies have claimed pronounced antibacterial efficacy of Rhodomyrtus tomentosa leaf extract. This study was to further investigate potency of the extract in intracellular killing of human HaCaT keratinocytes. Pretreatment of MRSA with the extract resulted in a remarkable reduction in the bacterial adhesion to HaCaT keratinocytes, compared with untreated control (p < 0.001). In addition, at least 60% inhibition of the bacterial invasion into HaCaT cells was observed. Intracellular killing assay demonstrated that the extract exhibited strong antibacterial activity against intracellular MRSA at nontoxic concentrations (128 mg/L), which may have resulted from the increase in bactericidal activity under phagolysosomal pH. Transmission electron microscopy displayed the effects of the extract on alterations in the bacterial cell morphology with cell lysis. Fluorescence microscopy revealed that the extract decreased MRSA-induced apoptosis in HaCaT cells. In addition, cytotoxicity of HaCaT cells caused by MRSA supernatant was reduced at least 50% by the extract. The potential activities of R. tomentosa extract may be useful in an alternative treatment of MRSA infections in slight acidic compartments, particularly skin infections.

Rhodomyrtone modulates innate immune responses of THP-1 monocytes to assist in clearing methicillin-resistant Staphylococcus aureus.

BACKGROUND: The increasing resistance of Staphylococcus aureus to conventional antibiotics poses a major health problem. Moreover, S. aureus can survive within phagocytes, thus evading some antibiotics and the innate immune response. Rhodomyrtone, a bioactive compound from the leaves of Rhodomyrtus tomentosa, possesses potent antibacterial activity against methicillin-resistant S. aureus (MRSA). This study was to investigate the immunomodulatory effects of rhodomyrtone on THP-1 monocytes in response to MRSA. METHODS: THP-1 monocytes were stimulated with heat-killed MRSA, followed by treatment with rhodomyrtone. The cell pellets were prepared to detect pro-inflammatory molecules using real-time PCR. The supernatants were collected to assess nitric oxide production using Griess assay. Assays for phagocytosis and bacterial killing by THP-1 monocytes were performed to determine if they were affected by rhodomyrtone. RESULTS: Expression of pro-inflammatory molecules including IL-1ß, TNF-α, IL-6, and iNOS was enhanced in THP-1 monocytes stimulated with high doses of heat-killed MRSA (108 to 109 cfu/ml). In contrast, monocytes stimulated with MRSA at lower doses (106 to 107 cfu/ml) did not induce the expression of these cytokines. However, rhodomyrtone significantly increased the expression of pro-inflammatory mediators, IL-6 and iNOS in monocytes stimulated with heat-killed MRSA at low doses, and displayed some anti-inflammatory activity by reducing TNF-α expression in monocytes stimulated with heat-killed MRSA at high doses. Treatment with rhodomyrtone also significantly up-regulated the expression of the key pattern recognition receptors, TLR2 and CD14, in THP-1 monocytes stimulated with heat-killed MRSA at 106 to 109 cfu/ml, while heat-killed MRSA alone did not induce the expression of these molecules. The ability of rhodomyrtone to eliminate MRSA from the monocytes was observed within 24 h after treatment. CONCLUSION: Rhodomyrtone enhanced the expression of pattern recognition receptors by monocytes in response to MRSA. Increased expression of these receptors might improve MRSA clearance by modulating pro- and anti-inflammatory cytokine responses.