Antimicrobial Efficacy of Rhamnolipid Biosurfactant Against Multi-Species Endodontic Biofilm: A Comparative In Vitro Study.

OBJECTIVE

Persistent root canal infections are driven by resilient multi-species biofilms that withstand conventional irrigants. This in vitro study aimed to compare the antimicrobial efficacy of rhamnolipid (RHL) with NaOCl against a multi-species endodontic biofilm.

METHODS

A 4-species biofilm (Enterococcus faecalis, Streptococcus mutans, Fusobacterium nucleatum, Staphylococcus aureus) was developed on human dentine discs over 21 days under anaerobic conditions to simulate infected root canals. Biofilm-laden specimens were treated for 10 minutes with RHL at 1×, 2× and 4× the minimum inhibitory concentration (MIC, 50 mg/mL for E. faecalis). Standard 5.25% NaOCl and phosphate-buffered saline (PBS) served as positive and negative controls. Antimicrobial efficacy was evaluated by viable colony-forming unit (CFU) counts, quantitative real-time polymerase chain reaction (qPCR) for bacterial load (cycle threshold, CT) and confocal laser scanning microscopy.

RESULTS

Rhamnolipid demonstrated a concentration-dependent antimicrobial effect. Colony-forming unit analysis showed the greatest bacterial load reduction in the 4× MIC group (40.80 ± 12.48 CFU), followed by NaOCl (1104.0 ± 35.79 CFU), 2× MIC (1372.20 ± 29.70 CFU), MIC (1556.60 ± 25.19 CFU) and PBS (21080 ± 30.00 CFU), with all intergroup differences statistically significant. Confocal laser scanning microscopy revealed intense red fluorescence and minimal green fluorescence in the 4× MIC group, indicating predominant bacterial death. Real Time PCR results corroborated this trend, with highest CT values for S. aureus, followed by S. mutans, E. faecalis and F. nucleatum. Interestingly, F. nucleatum was more susceptible than E. faecalis in the NaOCl and PBS groups, while the reverse was observed in the RHL-treated groups.

CONCLUSION

Rhamnolipid biosurfactant, particularly at 4× MIC, demonstrated superior antimicrobial efficacy over NaOCl against multi-species endodontic biofilms, notably in reducing E. faecalis. Based on previously reported properties of RHLs, including biodegradability and potentially lower cytotoxicity, RHL may represent a promising adjunct or alternative to conventional endodontic irrigants. However, its biocompatibility, tissue interaction and clinical efficacy require further investigation.