Experiences with evaluation of results from antimicrobial testing on microbial biofilms formed in vitro

Barbora, Gaálová; Černáková, Lucia; Jordão, Luisa; Dohál, Matúš; Bujdáková, Helena

http://hdl.handle.net/10400.18/5718

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This presentation summarizes some experiences and troubleshooting leading to the data validation extracted from experiments focused on antimicrobial testing using various antimicrobial agents, surfaces and microorganisms (the yeast Candida albicans, bacteria Streptococcus mutans, Staphylococcus aureus, and Escherichia coli). In screening of anti-biofilm activity when many samples are tested, 3 approaches are fundamental: determination of number of survived cells (CFU), measurement of metabolic activity, and microscopy. These techniques can be combined by different ways, using various substrates or dyes. An evaluation through metabolic activity, the most easy approach, uses spectral measurements at A560 (MTT assay) or A490 (XTT assay). Substrates used in both techniques are associated with activity of dehydrogenases and are reduced in final detected product. Methods are suitable for testing biofilms formed on stable materials (for example, single or dual biofilms formed by C. albicans and S. mutans on hydroxyapatite blocks). However, compounds participating in redox reactions can significantly diminish activity of both substrates. The example of such compound is the photoactive dye methylene blue that is promising compound used in nanotechnology. Similarly, colloidal dispersions containing clay minerals interact with substrates in respect to the charge. In testing of such materials, determination of CFU is the most common choice. Taking into account those limitations, challenging approach is the molecular quantification of live/dead cells in microbial biofilms by real-time PCR. The intercalating dyes ethidium monoazide and propidium monoazide can be selected in respect to microorganisms (for example, S. mutans vs. E. coli). Mentioned methods do not allow describe many details in biofilms. Microscopic techniques in combination with appropriate dyes (CLSM) or nucleotide probes (FISH), but also SEM can help to prove a real fitness and architecture of microbial consortia.