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

McKenna J. Cruikshank; Justine M. Pitzer; Kimia Ameri; Caleb V. Rother; Kathryn Cooper; Austin S. Nuxoll; McKenna J. Cruikshank; Justine M. Pitzer; Kimia Ameri; Caleb V. Rother; Kathryn Cooper; Austin S. Nuxoll

doi:10.3934/microbiol.2024038

AIMS Microbiology

2024, Volume 10, Issue 4: 880-893. doi: 10.3934/microbiol.2024038

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Research article

Characterization of Staphylococcus lugdunensis biofilms through ethyl methanesulfonate mutagenesis

1.
Department of Biology, University of Nebraska at Kearney, Kearney, NE, United States
2.
School of Interdisciplinary Informatics, College of Information Science and Technology, University of Nebraska at Omaha, Omaha, NE, USA

Received: 27 July 2024 Revised: 10 October 2024 Accepted: 11 October 2024 Published: 17 October 2024

Staphylococcus lugdunensis is a coagulase-negative species responsible for a multitude of infections. These infections often resemble those caused by the more pathogenic staphylococcal species, Staphylococcus aureus, 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 S. lugdunensis pathogenesis. The objective of this study is to identify the essential factors for biofilm formation in S. lugdunensis. S. lugdunensis 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 S. lugdunensis surface protein A (slsA) 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, mgtE. Conversely, a mutation in the gene that codes for the von Willebrand factor binding protein, vwbl, was common among the mutants with increased biofilm formation. Following proteinase K treatment, a significant dispersal of the S. lugdunensis biofilm matrix occurred, thus confirming the presence of primarily protein-mediated biofilms; this is in agreement with previous S. lugdunensis 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 S. lugdunensis pathogenesis.
- Staphylococcus lugdunensis,
- biofilms,
- staphylococci,
- EMS mutagenesis
Citation: McKenna J. Cruikshank, Justine M. Pitzer, Kimia Ameri, Caleb V. Rother, Kathryn Cooper, Austin S. Nuxoll. Characterization of Staphylococcus lugdunensis biofilms through ethyl methanesulfonate mutagenesis[J]. AIMS Microbiology, 2024, 10(4): 880-893. doi: 10.3934/microbiol.2024038

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Abstract

Staphylococcus lugdunensis is a coagulase-negative species responsible for a multitude of infections. These infections often resemble those caused by the more pathogenic staphylococcal species, Staphylococcus aureus, 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 S. lugdunensis pathogenesis. The objective of this study is to identify the essential factors for biofilm formation in S. lugdunensis. S. lugdunensis 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 S. lugdunensis surface protein A (slsA) 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, mgtE. Conversely, a mutation in the gene that codes for the von Willebrand factor binding protein, vwbl, was common among the mutants with increased biofilm formation. Following proteinase K treatment, a significant dispersal of the S. lugdunensis biofilm matrix occurred, thus confirming the presence of primarily protein-mediated biofilms; this is in agreement with previous S. lugdunensis 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 S. lugdunensis pathogenesis.

Acknowledgments

We would like to thank Tim Foster, Trinity College, for S. lugdunensis strain N920143. Research reported in this publication was supported by the National Institute of General Medical Science of the National Institutes of Health (NIH) under award number GM103427. We would like to thank the University of Nebraska DNA Sequencing Core for their sequencing contributions on this project. The University of Nebraska DNA Sequencing Core receives partial support from the National Institute for General Medical Science (NIGMS) INBRE-P20GM103427-14 and COBRE-1P30GM110768-01 grants as well as The Fred & Pamela Buffett Cancer Center Support Grant-P30CA036727. Funding for this work was also provided by the Nebraska Research Initiative and the INSpRE core for equipment used in this project. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

MJC, JMP, ASN contributed to the conception and design of the study. ASN performed the statistical analyses. MJC wrote the first draft of the manuscript. JMP, KC, ASN contributed to writing the manuscript. MJC, JMP, CVR, KC, ASN performed experiments and generated data within the manuscript. All authors read and approved the submitted version.

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