Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of <i>Bacillus licheniformis</i>

<div><p>Background</p><p>Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of <i>Bacillus licheniformis</i> D1.</p><p>Methodology/Principal Findings</p><p><i>B. licheniformis</i> strain D1 isolated from the surface of green mussel, <i>Perna viridis</i> showed antimicrobial activity against pathogenic <i>Candida albicans</i> BH, <i>Pseudomonas aeruginosa</i> PAO1 and biofouling <i>Bacillus pumilus</i> TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from <i>B. licheniformis</i> ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against <i>C. albicans</i>. Against both <i>P. aeruginosa</i> and <i>B. pumilus</i> the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces.</p><p>Conclusion/Significance</p><p>We isolated a protein from a tropical marine strain of <i>B. licheniformis</i>, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.</p></div