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A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non eDNA networks in Pseudomonas aeruginosabiofilms

A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non eDNA networks in Pseudomonas aeruginosabiofilms
A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non eDNA networks in Pseudomonas aeruginosabiofilms
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 (CLSM) 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 P. aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but not 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.
biofilm, EPS, confocal microscopy, potassium permanganate staining
1-8
Swearingen, Matthew C.
3725ae2f-b98d-4be4-b7fe-70499e716631
Mehta, A.
d1ffa5c9-13f5-4917-ad89-df743b516d14
Nistico, Laura
7a83886a-6bf1-46a1-87dd-75a120d41603
Hill, Preston J.
4f138818-265e-46a2-bb55-4d16f9636718
Falzarano, Anthony R.
1f9953f4-40fe-4a19-9957-4c6dbbe702e7
Wozniak, Daniel J.
bfa8e8e5-5929-449b-af6a-ec2d86c47eb5
Hall-Stoodley, Luanne
94ebdc00-b549-4488-b15f-5310fb965f5b
Stoodley, Paul
08614665-92a9-4466-806e-20c6daeb483f
Swearingen, Matthew C.
3725ae2f-b98d-4be4-b7fe-70499e716631
Mehta, A.
d1ffa5c9-13f5-4917-ad89-df743b516d14
Nistico, Laura
7a83886a-6bf1-46a1-87dd-75a120d41603
Hill, Preston J.
4f138818-265e-46a2-bb55-4d16f9636718
Falzarano, Anthony R.
1f9953f4-40fe-4a19-9957-4c6dbbe702e7
Wozniak, Daniel J.
bfa8e8e5-5929-449b-af6a-ec2d86c47eb5
Hall-Stoodley, Luanne
94ebdc00-b549-4488-b15f-5310fb965f5b
Stoodley, Paul
08614665-92a9-4466-806e-20c6daeb483f

Swearingen, Matthew C., Mehta, A., Nistico, Laura, Hill, Preston J., Falzarano, Anthony R., Wozniak, Daniel J., Hall-Stoodley, Luanne and Stoodley, Paul (2016) A novel technique using potassium permanganate and reflectance confocal microscopy to image biofilm extracellular polymeric matrix reveals non eDNA networks in Pseudomonas aeruginosabiofilms. Pathogens and Disease, 74 (1), 1-8. (doi:10.1093/femspd/ftv104). (PMID:26536894)

Record type: Article

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 (CLSM) 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 P. aeruginosa biofilm. In vitro and in vivo testing indicated that KMnO4 reacted with poly-N-acetylglucosamine and Pseudomonas Pel polysaccharide, but not 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.

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More information

Accepted/In Press date: 10 October 2015
e-pub ahead of print date: 3 November 2015
Published date: February 2016
Keywords: biofilm, EPS, confocal microscopy, potassium permanganate staining
Organisations: Engineering Science Unit

Identifiers

Local EPrints ID: 384474
URI: http://eprints.soton.ac.uk/id/eprint/384474
PURE UUID: 5a042ee5-42ae-4b39-b6a6-6e1a0fde9682
ORCID for Paul Stoodley: ORCID iD orcid.org/0000-0001-6069-273X

Catalogue record

Date deposited: 04 Jan 2016 13:20
Last modified: 17 Dec 2019 01:42

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