Gtbp filters

For epifluorescence microscopy counts, samples were fixed with formaldehyde (0.4% final concentration) (Sigma, USA) and filtered through 0.2 μm pore size polycarbonate GTBP filters (Millipore, USA). Bacteria were stained with 4′-6′-diamidino-2-phenylindole (DAPI) (Merk, Germany) for 5 min and then, rinsed in phosphate buffered saline (PBS) immediately prior to imaging. The filters were mounted on glass slides using immersion oil and were visualized in a Zeiss AXIO Imager A1 fluorescence microscope (Zeiss, Germany).

Survival in presence of capsaicin was tested by the live/dead assay using SYBR Green I (Invitrogen, Eugene, OR, USA) and propidium iodide (Sigma–Aldrich), two nucleic acid dyes differing in their ability to penetrate bacterial cells (Magi et al., 2015 (link)). Briefly, overnight-grown streptococci were suspended in 1 mL capsaicin-supplemented BHI broth [∼1 × 10⁸ colony forming units (CFU)/mL] and incubated for 15, 30, or 60 min at 37°C in 5% CO₂. After staining with 1 × SYBR Green I and 40 μg/mL propidium iodide, cells were incubated at room temperature for 25 min in the dark, harvested on GTBP filters (Ø = 0.2 μm, Millipore, Billerica, MA, USA), and examined under an epifluorescence microscope (Axioskop2, Zeiss, Milano, Italy).

Group A streptococci survival in presence of carvacrol was studied by the live/dead assay as described previously (Zandri et al., 2012 (link)) using SYBR Green I (Invitrogen, Eugene, OR, USA) and propidium iodide (Sigma–Aldrich), two nucleic acid dyes differing in their ability to penetrate bacterial cells: bacteria with intact cell membranes stain fluorescent green, whereas those with damaged membranes stain fluorescent red. Briefly, overnight grown streptococci were suspended in 1 mL carvacrol-supplemented BHIB (∼1 × 10⁸ CFU/mL) and incubated for 1, 3, or 24 h at 37^∘C in 5% CO₂. After staining with 1 × SYBR Green I and 40 μg/mL propidium iodide, samples were incubated at room temperature for 25 min in the dark, harvested on GTBP filters (Ø = 0.2 μm, Millipore, Billerica, MA, USA), and examined under an epifluorescence microscope (Axioskop 2, Zeiss, Milano, Italy).