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A 96-well-plate–based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing

Nature Protocols volume 5, pages 1460–1469 (2010)Cite this article

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Abstract

A major reason for bacterial persistence during chronic infections is the survival of bacteria within biofilm structures, which protect cells from environmental stresses, host immune responses and antimicrobial therapy. Thus, there is concern that laboratory methods developed to measure the antibiotic susceptibility of planktonic bacteria may not be relevant to chronic biofilm infections, and it has been suggested that alternative methods should test antibiotic susceptibility within a biofilm. In this paper, we describe a fast and reliable protocol for using 96-well microtiter plates for the formation of Pseudomonas aeruginosa biofilms; the method is easily adaptable for antimicrobial susceptibility testing. This method is based on bacterial viability staining in combination with automated confocal laser scanning microscopy. The procedure simplifies qualitative and quantitative evaluation of biofilms and has proven to be effective for standardized determination of antibiotic efficiency on P. aeruginosa biofilms. The protocol can be performed within ∼60 h.

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Figure 1
Figure 2: Robustness of the PHLIP-calculated biofilm parameters.
Figure 3: Isopropanol treatment of biofilms as killing control.
Figure 4: Responsiveness of tobramycin-treated biofilms.
Figure 5: Application of biofilm susceptibility testing to two sputum samples.

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Acknowledgements

We thank S. Suerbaum for providing the clinical P. aeruginosa strains, M. Pletz for sputum samples and L. Szekely and his group for help and assistance in the microscopic facility at the Karolinska Institute. M.M. was supported by the International Research Training Group 1273 funded by the German Research Foundation (DFG). Financial support from Mukoviszidose e.V. and from Helmholtz-Gemeinschaft is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Chronic Pseudomonas Infection Research Group, Helmholtz Center for Infection Research, Braunschweig, Germany

    Mathias Müsken & Susanne Häussler

  2. Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany

    Stefano Di Fiore

  3. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

    Ute Römling

  4. Pathophysiology of Bacterial Biofilms, Twincore, Center for Experimental and Clinical Infection Research, a joint venture of the Helmholtz Center for Infection Research and the Medical School Hannover, Hannover, Germany

    Mathias Müsken & Susanne Häussler

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Contributions

M.M., S.D.F., U.R. and S.H. designed the study. M.M. and S.D.F. performed the experiments, M.M. and S.H. analyzed the data, S.D.F. gave technical support, and M.M. and S.H. wrote the paper.

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Correspondence to Susanne Häussler.

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Supplementary information

Supplementary Figure 1s

Visualization of the image processing procedure. (a) Original, plus (b) Background-subtracted, (c) Otsu-thresholded and (d) Outliers-removed images of (1) Syto9 fluorescence and (2) PI fluorescence as well as a (3) Colored overlay of both fluorescent images from a biofilm stack of the clinical isolate 5520 treated with 32 µg ml-1 ceftazidime (Syto9=blue, PI=yellow, overlap=white). (PDF 511 kb)

Supplementary Figure 2s

Comparison of two antibiotic treated PA14 biofilms. Individual pseudocolor merged slices of processed image stacks from PA14 biofilms treated with (a) 2 µg ml-1 and (b) 512 µg ml-1 tobramycin, starting from plane 0 (plastic surface) to plane 14 with a spacing distance of 3 µm (Syto9=blue, PI=yellow, overlap=white). (PDF 738 kb)

Supplementary Figure 3s

Responsiveness of the meropenem-treated biofilms. Distribution of the PI stained (dark), the co-localized (hatched) and the Syto9 stained (light) biovolume and CFU counts (solid white line) of PA14 and 5 clinical strains exposed to increasing concentrations of meropenem. The black dotted line marks the 80 % threshold of PI (and co-localized) fluorescence. Biovolume data are mean values of three independent replicates. The overall SD for the green, the co-localized and the red fraction are: PA14 (4.5 % / 4.9 % / 3.9 %), 5497 (6.6 % / 3.0 % / 6.7 %), 5520 (4.4 % / 3.4 % / 5.3 %), 5522 (4.0 % / 4.0 % / 3.5 %), 5524 (4.0 % / 3.7 % / 4.6 %) and 5529 (4.8 % / 2.7 % / 4.4 %). (PDF 320 kb)

Supplementary Figure 4s

Responsiveness of the ceftazidime-treated biofilms. Distribution of the PI stained (dark), the co-localized (hatched) and the Syto9 stained (light) biovolume and CFU counts (solid white line) of PA14 and 5 clinical strains exposed to increasing concentrations of ceftazidime. The black dotted line marks the 80 % threshold of PI (and co-localized) fluorescence. Biovolume data are mean values of three independent replicates. The overall SD for the green, the co-localized and the red fraction are: PA14 (3.5 % / 4.0 % / 2.2 %), 5497 (6.2 % / 3.4 % / 7.4 %), 5520 (5.5 % / 3.0 % / 7.0 %), 5522 (2.7 % / 3.4 % / 2.3 %), 5524 (6.5 % / 3.0 % / 5.3 %) and 5529 (6.1 % / 3.1 % / 5.9 %). (PDF 322 kb)

Supplementary Figure 5s

Responsiveness of the ciprofloxacin-treated biofilms. Distribution of the PI stained (dark), the co-localized (hatched) and the Syto9 stained (light) biovolume and CFU counts (solid white line) of PA14 and 5 clinical strains exposed to increasing concentrations of ciprofloxacin. The black dotted line marks the 80 % threshold of PI (and co-localized) fluorescence. Biovolume data are mean values of three independent replicates. The overall SD for the green, the co-localized and the red fraction are: PA14 (4.3 % / 3.5 % / 2.5 %), 5497 (2.4 % / 4.7 % / 5.6 %), 5520 (6.3 % / 3.4 % / 7.2 %), 5522 (4.6 % / 3.9 % / 4.0 %), 5524 (7.1 % / 4.0 % / 5.0 %) and 5529 (4.9 % / 3.2 % / 6.9 %). (PDF 315 kb)

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Müsken, M., Di Fiore, S., Römling, U. et al. A 96-well-plate–based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing. Nat Protoc 5, 1460–1469 (2010). https://doi.org/10.1038/nprot.2010.110

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