Date of Completion

2025

Document Type

Thesis

Degree Name

Bachelor of Science in Pharmacy

Keywords

antibiofilm, biofilm, eugenol, trypsin, pepsin, Pseudomonas aeruginosa, Enterococcus faecalis, extracellular polymeric substances, enzymatic biofilm disruption.

Abstract

Effective antibiofilm strategies are vital to combat increasing drug resistance due to biofilms. This study evaluated the combined effect of eugenol with the proteolytic enzymes, pepsin and trypsin, against the biofilms of Pseudomonas aeruginosa and Enterococcus faecalis. To evaluate the single and combined effect of the enzymes and eugenol, three assays were conducted; CV Assay, Coomassie Blue (CB) Assay, Resazurin Assay (RA). Broth microdilution method showed that eugenol had MIC activity against P. aeruginosa at 10 mg/mL and against E. faecalis at 5 mg/mL. Pepsin (Pep) and trypsin (Try) both showed MIC action against strains at 4 mg/mL. The minimum biofilm inhibitory concentrations showed that eugenol was effective at 20 mg/mL against both P. aeruginosa and E. faecalis, while pepsin and trypsin were at 1 mg/mL and 8 mg/mL, respectively. CV Assay showed that eugenol exhibited activity against P. aeruginosa and E. faecalis at 80.37% biofilm growth reduction (BGR) and 59.71%, respectively. Pepsin was highly effective against P. aeruginosa (91.74%) but was weak for E. faecalis (15.02%), while trypsin proved to be ineffective to both (38.53% and 0%). Combining Eugenol with either enzyme showed enhancement – specifically try+eugenol which is most effective for P. aeruginosa (97.74%). CB Assay against P. aeruginosa and E. faecalis showed the highest protein reducing activity (%PR) from the treatment of try+eugenol (85.58% and 62.46%). The Resazurin assay demonstrated that trypsin had the highest cell viability reduction against P. aeruginosa (79.07%), and try+eugenol for E. faecalis (83.14%). Statistical analysis confirmed significant differences, highlighting the potential of combination therapy in biofilm disruption.

First Advisor

James Dayrit, RPh

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