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Background and objectives <p> <i>Klebsiella pneumoniae</i> 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 <i>B. cereus</i>-derived α-amylase against clinical isolates of <i>K. pneumoniae</i>. </p> Methods <p>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 (<i>fimH</i> and <i>mrkD</i>), normalized to <i>rpoB</i>.</p> Results <p><i>B. cereus</i>-derived α-amylase exhibited MBIC and MBEC values of 64&#xa0;µg/ml and 128&#xa0;µg/ml, respectively; MIC and MBC ranged from 32 to 128&#xa0;µg/ml. The <i>B. cereus</i>-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&#xa0;μm to ~ 39&#xa0;μm and ~ 73&#xa0;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 <i>B. cereus</i>-derived and commercial α-amylase enzymes, respectively. Quorum-sensing gene expression was markedly downregulated following treatment with ½ MIC of <i>B. cereus</i>-derived α-amylase: <i>fimH</i> to 0.247 ± 0.045 (75.3% reduction) and <i>mrkD</i> to 0.187 ± 0.035 (81.3% reduction).</p> Conclusion <p><i>B. cereus</i>-derived α-amylase exhibited potent anti-biofilm, antibacterial, and quorum-quenching activities against <i>K. pneumoniae</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月29日

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  • Abd-El-Rahman A. Mustafa,
  • Amal M. Abo-Kamer,
  • Lamiaa A. Al-Madboly

摘要

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.