title: A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non-eDNA networks in Pseudomonas aeruginosa biofilms.
abstract: Biofilms are etiologically important in the development of chronic medical and dental infections. The biofilm extracellular polymeric substance (EPS) determines biofilm structure and allows bacteria in biofilms to adapt to changes in mechanical loads such as fluid shear. However, EPS components are difficult to visualize microscopically because of their low density and molecular complexity. Here, we tested potassium permanganate, KMnO4, for use as a non-specific EPS contrast-enhancing stain using confocal laser scanning microscopy in reflectance mode. We demonstrate that KMnO4 reacted with EPS components of various strains of Pseudomonas, Staphylococcus and Streptococcus, yielding brown MnO2 precipitate deposition on the EPS, which was quantifiable using data from the laser reflection detector. Furthermore, the MnO2 signal could be quantified in combination with fluorescent nucleic acid staining. COMSTAT image analysis indicated that KMnO4 staining increased the estimated biovolume over that determined by nucleic acid staining alone for all strains tested, and revealed non-eDNA EPS networks in Pseudomonas aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but did not react strongly with DNA or alginate. KMnO4 staining may have application as a research tool and for diagnostic potential for biofilms in clinical samples.
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Swearingen
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA 15212, USA.
authors.forename: Ajeet
authors.initials: A
authors.lastname: Mehta
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA 15212, USA.
authors.forename: Amar
authors.initials: A
authors.lastname: Mehta
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA 15212, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Nistico
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Preston J
authors.initials: PJ
authors.lastname: Hill
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Falzarano
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wozniak
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Luanne
authors.initials: L
authors.lastname: Hall-Stoodley
authors.affiliation: Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210, USA Center for Genomic Sciences, Allegheny-Singer Research Institute, Allegheny General Hospital, Pittsburgh, PA 15212, USA Department of Orthopedics, The Ohio State University, Columbus, OH 43210, USA paul.stoodley@osumc.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Stoodley
mesh_terms: D000818:Animals; D018441:Biofilms / Q000254:growth & development*; D001704:Biopolymers / Q000032:analysis*; D004396:Coloring Agents / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods*; D011196:Potassium Permanganate / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D011817:Rabbits; D013194:Staining and Labeling / Q000379:methods*; D013210:Staphylococcus / Q000502:physiology; D013291:Streptococcus / Q000502:physiology
chemical_list: D001704:Biopolymers; D004396:Coloring Agents; D011196:Potassium Permanganate
keywords: EPS; biofilm; confocal microscopy; potassium permanganate staining