## 3 Biological Activities
### Antibacterial Activity
The biofilm formation was inhibited by 67.1%, 69.5%, and 70.1% with 225 \(\upmu\)M citral after treatment at 25 \({}^{\circ}\)C for 24, 48, and 72 h, respectively. Compared with the control, cells treated with citral showed significant and dose-dependent inhibition of biofilm formation of _C. sakazakii_[75]. The authors mention that the enhanced antibiofilm effect of citral at 12 \({}^{\circ}\)C could be due to the lower metabolic and growth rates of C. _sakazakii_ at this temperature, potentially resulting in a weaker biofilm that is more sensitive to citral. In this sense, studies evaluating the antibiofilm potential of citral must consider the biological variability and factors that might influence biofilm inhibition, such as incubation temperatures, pH, ionic strength, surface adhesion materials, and O\({}_{2}\) and nutrient concentration. In a recent study, the antibiofilm potential of citral against MRSA was evaluated, and the possible mode of action was deciphered, finding that citral inhibited biofilm formation by MRSA without affecting growth at 100 \(\upmu\)g/mL. Microscopic analysis showed that citral greatly hindered the surface adherence of MRSA. Genetic ontology and String analysis revealed that citral differentially regulates proteins involved in pleiotropic transcriptional repression (_CodY_), cell wall homeostasis (_IsaA_), regulation of exotoxin secretion (_SaeS_), adhesion cell, hemolysis, capsular polysaccharide biosynthesis, and pathogenesis. Gene expression analysis and in vitro assays further validated the alteration in the synthesis of slime, hemolysis, lipase, staphyloxanthin, and oxidative susceptibility [7]. MRSA is the main pathogen in biomaterial-associated infections and is well-known to form biofilms in foreign materials. For this reason, [PERSON], [PERSON] [74] evaluated the synergistic antibiofilm activity of citral in combination with thymol to inhibit the adhesion of MRSA to titanium surfaces. The antibiofilm coating effectively inhibited MRSA adhesion in vitro, and the antibiofilm activity of the coating was not affected by plasma conditioning. Furthermore, the antibiofilm coating was neither hemolytic nor toxic to peripheral blood mononuclear cells. Citral was evaluated against mature biofilms of _S. aureus_ SC-01, _L. monocytogenes_ EGD-e, or _E. coli_ MG1655 in a range of 500-2000 \(\upmu\)L/L. The MICs were 200 mL/L for _L. monocytogenes_ and 500 mL/L of citral for _S. aureus_ SC-01 and _E. coli_, and the concentration of 500 mL/L of citral was selected to be tested against mature biofilms of 72 h of incubation. Citral at 45 \({}^{\circ}\)C for 60 min reduced more than five logarithmic cycles of sessile cells that are part of mature biofilms of the three species. These results demonstrate the great potential of citral present in EOs for eradicating biofilms formed by foodborne pathogenic microorganisms [50]. In another study, citral inhibited the biofilm formation of _S. aureus_ strains. At an undergrowth inhibitory concentration of 0.25 \(\upmu\)L/mL, a 46.5% inhibition on biofilm formation of the clinical _S. aureus_ isolate was observed, while the _S. aureus_ strain DMST 4745 showed 74.6% of the biofilm formations. This inhibitory effect increased with the increasing concentration of the tested agents [48]. Several studies evaluating the antibiofilm potential of natural compounds such as citral measure the biofilm only as biomass and do not consider the effect of these compounds on the cell viability and EPS components. In this sense, a deeper analysis of the impact of this compound on biofilm as a multicomponent system should be considered, as well as their effect on other virulence factors, to have a broad overview of their antipathogenic effect. Additionally, despite the relevant information of studying citral against single-species biofilms to have an approach to the possible mode of action, several investigations have not evaluated its effect against multispecies- biofilms, as commonly found in the natural environments.