## 4 Discussion  
The central questions posed in the current study were whether refinement of plant medicines of _S. glaucescens_ with antimicrobial activity by purification from crude extract to fractions, sub-fractions and purified compounds would increase the risk of _S. aureus_ developing resistance to the antimicrobial substance, and which genes in _S. aureus_ are important for intrinsic resistance of this bacterium against crude extracts from this plant. The Luria and Delbruck fluctuation test has been used widely as a reference method to calculate and compare de novo mutation rates raised by microbes [20]. The analyses showed that mutation rates were significantly lower (\(p<0.05\)) for strains selected on plates with crude extract than on plates with fractions, sub-fractions and epifriedelanol, except for two strains against a subfraction with ethyl acetate extraction. The reason why this subfraction was different from other subfractions and why the difference was only seen in \begin{table}
\begin{tabular}{c c c c} \hline \hline
**S/No** & **Strain** & **Inactivated Gene** & **MIC (\(\upmu\)g/mL)** \\ \hline \hline
1 & Wild type of _S. aureus_ JE2 & & \\  & SAUSA300 & & \\
2 & SAUSA300\_1615 & _hemB_ & 0.094 \\
3 & SAUSA300\_1473 & _musB_ & 3 \\
4 & SAUSA300\_2406 & _cap5A_ & 3 \\
5 & SAUSA300\_0228 & _Fade_ & 3 \\
6 & SAUSA300\_1995 & _scrR_ & 3 \\
7 & SAUSA300\_1343 & _Nth_ & 3 \\
8 & SAUSA300\_0605 & _sarA_ & 3 \\
9 & SAUSA300\_2550 & _nrdG_ & 3 \\
10 & SAUSA300\_1222 & _Nuc_ & 3 \\
11 & SAUSA300\_0889 & _oppD_ & 3 \\
12 & SAUSA300\_1139 & _sucD_ & 3 \\
13 & SAUSA300\_1359 & unknown & 3 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: MIC (\(\upmu\)g/mL) of methanol extract of _S. glaucescens_ against the wild type and selected mutants of _S. aureus_ JE2 (CA- MRSA). two of our three _S. aureus_ strains tested are not known but may mean that this subfraction is more like the crude extract than the other subfractions. Further characterization is needed to determine this. Previous studies have shown that the likelihood of selecting resistant colonies was lower when bacteria were plated on a synergistic combination of antimicrobial substances than on individual substances [24; 25]. In line with this, the low rate of mutations towards crude extract from _S. glaucescens_ may be because the crude extract contains a mixture of antimicrobial substances. This explanation is challenged by the fact that the purified compound epifriedelanol has the same antimicrobial activity as the full extracts of this plant [15]. This activity has been determined by two-fold MIC assays, which are not very precise. A possible explanation is that mutation to growth in the presence of epifriedelanol happens in genes that determine the natural resistance towards other substances present in the crude extract, thus making it more difficult to mutate towards resistance to the crude extract. In terms of mechanistic understanding, this study is thus incomplete, and further studies are indicated to understand this apparent contradiction. The mutation rates determined by fluctuation assay in the current study were similar to natural rate of mutations in most microbial species, which ranges from \(10^{-9}\) to \(10^{-10}\)[24]. Strains with higher mutation rates have been shown to have an increased likelihood of acquisition of drug resistances, which may led to rapid selection of resistance and increased risk of multi drug resistance [26]; however, we saw no indications of high mutator strains in this study. In confirmation of the results from the fluctuation study, gradient concentration assay demonstrated that it took a longer time for the strains to adapt to growth with crude extract, compared to when they were sub-cultured on gradient plates with fractions, subfractions, and epifriedelanol.