Enzymatic breakdown of biofilm matrix to allow flow cytometry viability analysis of Clostridium beijerinckii cells.

Flow cytometry (FC) is a good way to enumerate the number of viable cells in suspension but is not adapted to mature biofilm analysis. The aim of this study is to investigate the effect of mechanical treatment coupled with enzymatic hydrolysis of biofilm matrix on FC viability analysis of biofilm cells. Biofilm was grown for 300 h of continuous fermentation on polyurethane foams. Fermentation was stopped, and the biofilm was detached by agitating the foams in PBS buffer with vortex agitation for 2 min. The best enzymatic hydrolysis consisted of sequential use of DNase I and proteinase K incubated for 1 h at 34°C. Biofilm cells detached from polyurethane foams were stained with both propidium iodide (PI) and carboxyfluoresceine diacetate and analyzed by FC. FC analysis performed after vortex agitation revealed the presence of high non-fluorescent events (78.9% ± 3.3%). After enzymatic treatment, a cell population was extracted from background noise and could be observed on FSC-SSC profile. The non-fluorescent events of this cell population decreased drastically to 41.9% ± 6.6%, and the percentage of viable cells was enhanced from 2.6% ± 0.9% to 38.2% ± 4.0% compared to analysis performed after mechanical treatment alone. Consequently, protease and nuclease activity are essential to hydrolyze extra polymeric substances prior to FC viability analysis in mature biofilm formed by Clostridium beijerinckii. © The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.

Citation

Maxime Carrié, Jean-Christophe Gabelle, Nicolas Lopes-Ferreira, Hélène Velly. Enzymatic breakdown of biofilm matrix to allow flow cytometry viability analysis of Clostridium beijerinckii cells. Journal of applied microbiology. 2023 Apr 03;134(4)

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PMID: 37024280

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