Combinatorial Biofilm-Releasing Agents with Colistin Reduces Staphylococcus aureus Biofilm Burden Below Clinical Threshold

Biofilms are bacterial communities encased in an extracellular polymeric substance (EPS) that limits antibiotic penetration and promotes persistent infection. Biofilm formation contributes to antibiotic tolerance in pathogens such as Staphylococcus aureus, representing a major clinical challenge. We hypothesized that inducing biofilm dispersion would increase antibiotic susceptibility and improve biofilm clearance. Growth curve analysis demonstrated that glutamate, cis-2-decenoic acid (cis-DA), and amylase did not significantly affect S. aureus growth over 12 hours, whereas prolonged exposure to sodium nitroprusside (SNP) significantly reduced bacterial growth after 9 hours. Consequently, biofilm treatments were limited to 2 hours to minimize direct antimicrobial effects of SNP. Biofilm growth conditions were optimized, with mature biofilms generated after 4 days in 1:2 Brain Heart Infusion (BHI), shaking at 220 rpm. Biofilms were treated with releasing agents alone or in combination with 2x the minimum inhibitory concentration (MIC) of colistin (500 µg/mL). Although no statistically significant differences were observed between combination treatments and colistin alone, all combination treatments reduced biofilm cell counts. SNP and amylase reduced biofilm burden to near the clinical threshold (105 CFU/mL), indicating a trend toward enhanced antibiotic efficacy and suggesting that dispersion-based adjunctive therapies may help reduce bacterial loads below levels associated with persistent biofilm infections.