D-limonene Increases Efficacy of Rifampicin as an Inhibitor of In Vitro Growth of Opportunistic Staphylococcus epidermidis RP62A

Natcha Rummaneethorn, Charlene Caoili


An increase in nosocomial infections due to Staphylococcus epidermidis has been a recent focus in the medical field. At the molecular level, the ica gene locus is responsible for biofilm formation in S. epidermidis, which contributes to its pathogenicity. Current strategies in treating biofilm-forming pathogens include antibiotics. To reduce antibiotic resistant bacteria populations, focus has shifted to naturally derived antibiotic alternatives. D-limonene is a terpenoid-based essential oil, found in citrus fruits, that has previously been shown as an effective antimicrobial agent against various related bacteria, such as S. aureus. Yet, its antimicrobial effectiveness has not been thoroughly studied in the biofilm-forming S. epidermidis strain ATCC 35894 (RP62A). Thus, the objective of this research was to study the antimicrobial effectiveness of D-limonene on S. epidermidis RP62A in comparison to the parental strain ATCC 12228. Growth studies of S. epidermidis strains ATCC 12228 and RP62A treated with various concentrations of D-limonene, rifampicin control, and a D-limonene-rifampicin drug cocktail were performed. Preliminary results indicate the inhibition of S. epidermidis ATCC 12228 when exposed to 305.6 mg/L of D-limonene. Interestingly, no effect was observed in S. epidermidis RP62A growth for studied concentrations of D-limonene. Inhibition of S. epidermidis ATCC 12228 was observed when exposed to the rifampicin control. However, S. epidermidis RP62A was observed to resume its normal growth after 16 hours of exposure to the rifampicin control. Inhibition of both strains was observed when exposed to the D-limonene-rifampicin-drug cocktail. With these results, it is hoped that the D-limonene-rifampicin drug cocktail will be further evaluated for its value in clinical settings to prevent initial nosocomial Staphylococcus infections. Future studies include obtaining the minimum inhibitory concentration (MIC) of the drug cocktail in both strains, as well as investigating the role of the ica locus in S. epidermidis RP62A’s D-limonene resistance. The significance of this research is the potential clinical use of D-limonene as a naturally derived antimicrobial agent against hospital-acquired S. epidermidis infections.


Antibiotic Susceptibility;D-Limonene;Staphylococcus epidermidis;Biofilm Formation

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