# Anthraquinone Rhein Exhibits Antibacterialativity against _Staphylococcus aureus_
## 4 Discussion
Currently, exposure to antibacterial agents leads to the elimination of pathogens but not the determinants of virulence [41]. Although bacterial clearance does occur, eliminating pathogens is often insufficient to prevent tissue damage. Identifying antibacterial agents that affect the release and/or activity of toxins and enzymes is necessary to block host damage upon elimination of the etiological agent [42]. Our finding showed that Rhein alters the hemolytic capacity of _S. aureus_ up to the concentration of 3.13 \(\mu\)g/mL. Furthermore, the catalase activity was reduced by 0.96 times at the same concentration. No evidence was reported on the impact of Rhein on alpha-hemolysin and catalase activity. To date, this compound has been shown to compromise the system of iron-regulated surface determinants, responsible for the intracellular accumulation of heme molecules [33]. Mature biofilm is considered a highly organized ecosystem that contributes to the onset of chronic infections through the continuous release of planktonic bacterial forms and virulence factors. Biofilm bacteria have a 10 times higher survival rate than planktonic bacteria [43]. Antibiotic tolerance can be reversed through the breakdown of the extracellular matrix and the release of bacteria, which will then be available for antibacterial treatment [44]. Our results report Rhein's ability to disrupt mature biofilms with an average rate of 31.4 and 24% at 2 \(\times\) MIC and 1 \(\times\) MIC concentrations. No studies have reported data on the degradation of mature _S. aureus_ biofilm. Our past results has reported Rhein's ability to disrupt _S. mutans_ biofilm with minimal biofilm eradication concentrations inducing 50% values of 6.31 \(\mu\)g/mL [23]. Several evidence has demonstrated the impact of Rhein on the activity of NADH type II: quinone oxidoreductase (NDH-2) [45]. This enzyme catalyzes the oxidation of NADH and the reduction in membrane quinone, resulting in the formation of NAD+ [46]. [PERSON] et al. proved that treatment of _Cutibacterium actes_ with Rhein (6.25 \(\mu\)g/mL) completely inhibited the activity of NDH-2 [24]. This increased the (NADH)/(NAD+) ratio, with a negative impact on the glycolytic pathway and the production of ATP and pyruvate [47]. [PERSON] et al. documented the role of pyruvate in the biofilm structure of _Pseudomonas aeruginosa_. The decrease in pyruvate caused a loss of biomass with consequent alteration of biofilm integrity [48]. [PERSON] et al. showed that the hemolytic and catalase activities of _S. aureus_ require NDH-2. NDH-2 knockout strains showed drastically reduced hemolytic activity compared with the wild-type strain due to the failing production of \(\alpha\)-toxin. Moreover, NDH-2 knockout reduced the susceptibility to hydrogen peroxide compared with wild-type conditions [49]. Our findings reported the effective bacteriostatic activity of Rhein against standard and clinical isolated _S. aureus_. This strain invades and replicates within many cell types, evading the immune system through the action of \(\alpha\)-hemolysin and catalase. The first results in the death of host cells, promoting host damage, whereas catalase allows bacterial survival in macrophages, avoiding the phagocytosis process. Our studies showed that Rhein at subMIC concentrations interferes with the action of \(\alpha\)-hemolysin and catalase, thereby reducing the virulence and pathogenicity of _S. aureus_. Moreover, biofilm formation protects _S. aureus_ strains against antibiotics and hosts' defense molecules. Interestingly, Rhein disrupted the biofilm matrix, rendering the bacteria susceptible to antibiotic treatment and the action of immunity determinants. These results support Rhein as a potential antibacterial agent with antivirulence and antib Biofilm activity. Therefore, Rhein could represent a suitable therapy strategy for the treatment of _S. aureus_ infections. Additional investigation will be needed to better understand the mechanism underlying Rhein's action against _S. aureus_.