Abstract
Burkholderia cepacia is an underexplored opportunistic pathogen and a food spoilage species. The bacterium may serve as an ideal model for biofilm formation and resilience. Herein, we explored the possibility of enhancing the destruction of preformed B. cepacia biofilm by combining enzymes (amylase, DNase, and protease) that potentially degrade biofilm matrices with diverse antimicrobials. Initially, the biofilm-forming ability of B. cepacia ATCC 25416 was assessed in two microbiological media. A nutrient-rich broth favored planktonic cell proliferation, whereas a nutrient-limited medium supported robust biofilm formation. The minimum inhibitory concentration (MIC) of the tested antimicrobials against planktonic cells (MIC-Plank) was determined. Ciprofloxacin and meropenem gave the smallest MIC-Plank of 4.0 and 8.0 μg/mL, respectively. The MIC of the two antimicrobials, when applied against preformed biofilm (MIC-Bio), increased to 16 μg/mL. Enzyme-antimicrobial combinations decreased the MIC-Bio of the antimicrobials to 4.0-8.0 μg/mL in a synergistic or additive manner, as measured by the fractional inhibitory concentration index (FICI). Among the tested combinations, α-amylase-ciprofloxacin exhibited a synergistic effect (FICI = 0.50), proteinase K-ciprofloxacin (FICI = 0.625), and α-amylase-meropenem (FICI = 0.750) showed an additive effect against B. cepacia biofilms. These combinations, at their MIC-Bio, were applied to preformed biofilms on stainless-steel coupons. Application of α-amylase, ciprofloxacin, and their combination significantly decreased (p < 0.0001) the biofilm populations from 8.4 ± 0.2 (untreated coupons) to 6.03 ± 0.2, 5.3 ± 0.3, and 4.5 ± 0.4 log(10) CFU/coupon, respectively. Similarly, α-amylase, meropenem, and their combination significantly decreased (p < 0.0001) the biofilm populations from 7.5 ± 0.5 (untreated coupons) to 5.8 ± 0.1, 5.6 ± 0.1, and 3.8 ± 1.0 log(10) CFU/coupon, respectively. These findings were confirmed when biofilms formed on stainless-steel coupons were examined through scanning electron microscopy. It is predicted that antimicrobial concentrations higher than MIC-Bio in the treatment combinations would eliminate residual biofilm on the coupons, but this needs to be studied. To conclude, enzyme-antimicrobial combinations offer a promising biofilm control strategy by mitigating B. cepacia preformed biofilm and minimizing risks associated with this potentially hazardous and spoilage bacterium. Such a strategy could be implemented in processing environments when food-grade antimicrobial additives are used instead of the currently tested antimicrobials.