Bacillus cereus-derived α-amylase disrupts biofilm formation and quorum sensing in multidrug-resistant Klebsiella pneumoniae.

Abstract

Background and objectives: Klebsiella pneumoniae is a multidrug-resistant pathogen implicated in severe community- and hospital-acquired infections such as bacteremia, urinary tract infections, sepsis, and pneumonia. Biofilm formation, driven by extracellular polymeric substances (EPS), enhances its persistence and resistance to antibiotics. This study evaluated the anti-biofilm, antibacterial, and quorum-quenching activities of a novel α-amylase B. cereus-derived α-amylase against clinical isolates of K. pneumoniae.

Methods: The anti-biofilm activity of the enzyme was assessed via minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) assays. Biofilm architecture and viability were analyzed using confocal laser scanning microscopy (CLSM) with live/dead staining. Antibacterial efficacy was determined through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Quorum-quenching effects were evaluated using qRT-PCR to assess the expression of biofilm-associated genes (fimH and mrkD), normalized to rpoB.

Results: B. cereus-derived α-amylase exhibited MBIC and MBEC values of 64 µg/ml and 128 µg/ml, respectively; MIC and MBC ranged from 32 to 128 µg/ml. The B. cereus-derived α-amylase enzyme inhibited biofilm formation by approximately 79% ± 0.69, compared to 58% ± 2.06 by commercial α-amylase. Biofilm thickness was reduced from 179 μm to ~ 39 μm and ~ 73 μm following treatment with B. cereus-derived and commercial α-amylase, respectively. Live/dead ratios shifted significantly from 97/3% (untreated) to ~ 54/46% and 73/27% after treatment with B. cereus-derived and commercial α-amylase enzymes, respectively. Quorum-sensing gene expression was markedly downregulated following treatment with ½ MIC of B. cereus-derived α-amylase: fimH to 0.247 ± 0.045 (75.3% reduction) and mrkD to 0.187 ± 0.035 (81.3% reduction).

Conclusion: B. cereus-derived α-amylase exhibited potent anti-biofilm, antibacterial, and quorum-quenching activities against K. pneumoniae clinical isolates. These findings highlight its potential as a novel therapeutic agent for managing biofilm-associated infections, either alone or as an adjunct to conventional treatments.