The Sensitivity of Staphylococcus aureus to pure flavonoids and synthetic antimicro bials

Abstract

Staphylococcus aureus is an opportunistic pathogen that forms part of the normal flora of the nose, skin, mouth and other parts of the body. S. aureus infections range from superficial skin infection to more serious diseases such as pneumonia, meningitis or septicaemia. It has an ability to rapidly develop resistance to many classes of antibiotics. Methicillin resistant S. aureus (MRSA) poses a big problem because of intrinsic resistance to virtually all ~-lactams, and its tendency to develop resistance to unrelated antibiotics. Several studies have demonstrated the synergistic activity between plant extracts, their flavonoids and antimicrobial agents. Therefore this study was initiated to determine whether the flavonoids chrysin, naringenin and 7- hydroxyflavone can affect the susceptibility of methicillin sensitive S. aureus (MSSA) and MRSA in combination with methicillin and vancomycin, and also to assess the effect these flavonoids have on the expression of efilux pump genes present in the two strains. Flavonoids are a naturally occurring group of phenolic compounds which have been recognized as important due to their health benefits. Interest in the antimicrobial activity of flavonoids has grown over the last two decades and literature has shown that flavonoids such as chrysin and naringenin possess antimicrobial properties. The antibacterial activity of three flavonoids (7- hydroxyflavone, naringenin and chrysin) was determined, as well as their possible synergistic activity with ampicillin, methicillin and vancomycin against MSSA ATCC 25923 and MRSA ATCC 33591 strains, using disk diffusion and microdilution assays. Neither chrysin, naringerin nor 7-hydroxyflavone produces inhibition zones in the disk diffusion studies. Chrysin and 7- hydroxyflavanone were insoluble in solvents for microdilution studies. In the microdilution assays, naringenin showed inhibitory activity on its own and combinations with methicillin and vancomycin produced additive antibacterial activity against both MSSA and MRSA. Increase growth inhibition was especially observed when naringenin was combined with sub-inhibitory levels of the antibiotics. Efflux pumps actively transport solutes out of cells. These pumps may be specific to one agent or may transport structurally different compounds. The pumps that transport structurally different compounds are associated with multiple drug resistance. Currently more than ten efflux pumps have been described for S. aureus. The presence of efflux pump genes was assayed by PCR and quantitative RT -PCR respectively. The results of this study showed that MSSA ATCC 25923 and MRSA ATCC 33591 share several efflux systems, which are expressed at basal levels. With conventional PCR GyrA, NorAp, NorC, MdeA, SMR and NorB were detected in both strains of S. aureus. NorA, SepA and QacAIB were not detected in MSSA ATCC 25923. MepA and SepA were not detected in the MRSA strain. NorA was only detected in the MRSA strain. In contrast, in the Real-time PCR experiments NorB and NorAp were detected in both strains. Whereas MepA was only detected in MSSA ATCC 25923 strain, SMR was detected only in the MRSA ATCC 33591 strain. Resistance mechanisms are varied and complex, but this study indicated that flavonoids can elevate the susceptibility of MSSA and MRSA strains to the antibiotics used. The exact mechanism by which flavonoids counter bacterial infection is not clearly understood. Further studies are recommended to investigate if the additive effects observed by the combination of naringenin with antibiotics are the result of a knockdown of any of the efllux pump genes.