Treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) Infection with Rubus multibreatus Le’Vl Leaf Extract

Abstract

Background: Rubus multibreatus Le’vl is a wild-type shrub of the Rosacea family, commonly used in traditional Chinese medicine. It is claimed to invigorate the kidney and liver and reduce inflammation. From other literature, the Rubus genus has been defined by the presence of diterpene glycosides, with or without the presence of triterpene glycosides, and classifying its phyto-chemical activity owing to a number of oleanane ursane glucosides.
 Research objectives: To determine whether or not the plant had medicinal properties; To determine the structure of the metabolite(s) responsible for its medicinal properties; To determine the in-vivo activity of the active plant extract and medicinal significance.
 Study design: Solvent extraction using different solvents with different polarities was used to test for antibacterial activity. The choice solvent extract was tested for metabolites via chemical tests and HPLC. The pure compound(s) identified from HPLC results were tested for antibacterial activity to determine the compound(s) responsible for antibacterial activity. The extent of bacterial inhibition was determined by MBC (minimum bacterial concentration), bacterial growth curve, SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis), and qPCR (quantitative polymer chain reaction) amplification of viral RNA. The histopathological changes of the liver and kidney, inflammatory cytokines, and IgG were used to explain relief from, or lack thereof, upon treatment with choice plant extract(s).
 Methodology: Antibacterial activity was determined using both gram-positive and gram-negative bacteria; MSSA S. aureus ATCC25923, E. coli serotype 078, and S. typhi ATCC14028. Different solvent extracts were used to test for its antibacterial activity in vitro. The powdered leaf sample was first macerated in ethanol (90%) and concentrated using different organic solvents with different polarities; ethyl acetate, dichloromethane, and n-butanol. The ethyl acetate leaf extract of the plant, which was rich in flavonoids, phenolic acids, tannins, resins, and saponins, was preferred as the crude extraction method, to the other organic solvents; dichloromethane and n-butanol, whose extracts showed no antibacterial activity towards the gram-positive MSSA S. aureus ATCC25923, E. coli serotype 078 and S. typhi ATCC14028. Only the ethyl acetate extract showed antibacterial activity towards methicillin-susceptible S. aureus (MSSA). Further experiments were done using the methicillin-resistant S. aureus TCH1516 (ATCCBAA-1717) (USA300), which poses a greater threat to the modern-day treatment of bacterial infections. A murine model approach using specific pathogen-free (SPF) mice was used to investigate whether the extract could inhibit S. aureus in-vivo, as it did in-vitro, to determine the bacterial concentration that could cause infection and mortality, and the significance of the plant extract as treatment, during the infection period, both at the beginning of infection and post-infection.
 Results: The histopathological analyses of the liver and kidney, expression of inflammatory cytokines; IFN-?, TNF-R1, IL-1?, 1L 6, and IgG, showed the significance of the leaf extract in the treatment of methicillin-resistant S. aureus infection. HPLC analyses of the ethyl acetate extract confirmed the presence of 6 compounds; kaempferol, quercetin, gallic acid, ellagic acid, caffeic acid, and ferulic acid, and the absence of rutin. The most active compound of the 6, against S. aureus, was identified to be gallic acid, at a minimum bacterial concentration value (MBC) of 0.1111µg/µL while ellagic acid and ferulic acid followed next at 0.6666µg/µL and 1.7776µg/µL respectively. This, however, is not to say that another compound was not present, that could have inhibited the bacteria. Mass spectroscopic analysis may identify other unknown compounds that could significantly have useful medicinal properties.
 Conclusion: Rubus multibreatus Le’vl certainly has antibiotic properties against Staphylococcus aureus and gallic, ellagic, and ferulic acids can be attributed to the anti-staphylococcal activity, however, only three bacterial species were used; S. aureus, E. coli and S. typhi, and it was not determined whether the plant or the pure compound metabolites; kaempferol, quercetin, gallic acid, ellagic acid, caffeic acid, and ferulic acid, had other antimicrobial activities; anti-anthelmintic, anti-fungal or anti-viral properties. The histopathological analyses of the liver and kidney, expression of inflammatory cytokines; IFN-?, TNF-R1, IL-1?, 1L 6, and IgG, showed the significance of the leaf extract in the treatment of methicillin-resistant S. aureus infection. Rubus multibreatus Le’vl plant extract improved survival, reduced inflammation in the anal glands, and cleared kidney abscesses. Both IL-6 and IFN-? expression was reduced upon administration of the plant extract, however, IL-6 expression was not reduced when treatment was administered post-infection. IFN-? was significantly reduced in the post-infection group, compared to the positive control group, which expressed 3-4 times as much. R. multibreatus Le’vl was also effective in reducing neutrophilia and necrosis, and overall preventing the formation of bacterial-like cells as seen in the liver of the positive control group.