Enhancing the destruction of Burkholderia cepacia biofilm on stainless steel coupons by combining matrix-degrading enzymes with antimicrobials

<i>Burkholderia cepacia</i> 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 <i>B. cepacia</i> biofilm by combining enzymes (amylase, DNase, and protease) that potentially degrade biofilm matrices with diverse antimicrobials. Initially, the biofilm-forming ability of <i>B. cepacia</i> 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 <i>B. cepacia</i> 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 (<i>p</i> < 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 log₁₀ CFU/coupon, respectively. Similarly, α-amylase, meropenem, and their combination significantly decreased (<i>p</i> < 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 log₁₀ 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 <i>B. cepacia</i> 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.