Characterization of <i>Staphylococcus lugdunensis</i> biofilms through ethyl methanesulfonate mutagenesis

Abstract

<p><italic>Staphylococcus lugdunensis</italic> is a coagulase-negative species responsible for a multitude of infections. These infections often resemble those caused by the more pathogenic staphylococcal species, <italic>Staphylococcus aureus</italic>, such as skin and soft tissue infections, prosthetic joint infections, and infective endocarditis. Despite a high mortality rate and infections that differ from other coagulase-negative species, little is known regarding <italic>S. lugdunensis</italic> pathogenesis. The objective of this study is to identify the essential factors for biofilm formation in <italic>S. lugdunensis</italic>. <italic>S. lugdunensis</italic> was mutagenized through ethyl methanesulfonate (EMS) exposure, and the individual cells were separated using a cell sorter and examined for biofilm formation at 8 hr and 24 hr timepoints. Mutations that resulted in either increased or decreased biofilm formation were sequenced to identify the genes responsible for the respective phenotypes. A mutation within the <italic>S. lugdunensis</italic> surface protein A (<italic>slsA</italic>) gene was common among all of the low biofilm formers, thus suggesting that high expression of this protein is important in biofilm formation. However, other mutations common among the mutants with decreased biofilm formation were in the putative divalent cation transport gene, <italic>mgtE</italic>. Conversely, a mutation in the gene that codes for the von Willebrand factor binding protein, <italic>vwbl</italic>, was common among the mutants with increased biofilm formation. Following proteinase K treatment, a significant dispersal of the <italic>S</italic>. <italic>lugdunensis</italic> biofilm matrix occurred, thus confirming the presence of primarily protein-mediated biofilms; this is in agreement with previous <italic>S. lugdunensis</italic> studies. Additionally, all low biofilm formers exhibited decreased protein levels (1.95–2.77 fold change) within the biofilm matrix, while no difference was observed with extracellular DNA (eDNA) or polysaccharides. This study presents a unique methodology to identify genes that affect biofilm formation and sheds light on <italic>S. lugdunensis</italic> pathogenesis.</p>