Live dead baclight fluorescent stain

Plaque acid tolerance was evaluated using a previously validated method (Neilands et al., 2012 (link); Senneby et al., 2017 (link)). Briefly, 25 µl plaque sample was mixed with 75 µl TYE medium (1.7% tryptone, 0.3% yeast) containing 20 mM glucose and 40 mM phosphate/citrate buffer adjusted to pH 3.5 and incubated aerobically at 37°C for 2 hours. Following incubation, the cells were stained with LIVE/DEAD^® BacLight™ Fluorescent Stain (Molecular Probes) and transferred into an Ibidi mini flow cells (Ibidi GmbH). Flow-cells were then viewed with confocal laser scanning microscopy (CLSM) using a Nikon Eclipse TE2000 microscope (Nikon Corp.) with an Ar laser (488 nm laser excitation). Images were acquired with a Photometrics Prime 95B camera using Nikon NIS-Elements software. Ten randomly selected images from each sample were saved for further analysis. All confocal images were examined by an experienced oral microbiologist and given a score 1-5 as described by Senneby et al. (2017) (link). This method has been shown to have high intra-rater agreement and the scoring corresponding well to the percentage obtained by manually counting the cells (Senneby et al., 2017 (link)).

The assay described by Fernández de Palencia, et al.^{34 (link)}, with the modifications of Perez, et al.^{16 (link)}, was followed for the simulation of bacterial transit through the GIT. Briefly, approximately 10¹⁰ cfu mL⁻¹ of the wt and ΔagdiΔtdc strains from late exponential phase cultures (in GM17 supplemented with 20 mM agmatine and 10 mM tyrosine) were collected and mixed with the electrolyte solution (supplemented with the same concentrations of substrates). Cells were exposed first to lysozyme and then pepsin plus a successive reduction in pH to simulate gastric stress conditions. Gastrointestinal stress was mimicked by exposure of samples incubated at pH 5, 4.1, 3.0, 2.1 and 1.8 (gastric conditions), followed by their incubation in the presence of bile salts and pancreatin at pH 8 (small intestine conditions, GI). Colonic stress was simulated with the sample originally at pH 3 adjusted to pH 7 and incubated overnight. For each condition, cell viability was measured using the LIVE/DEAD^® BacLight fluorescent stain (Molecular Probes, Netherlands) as previously described^{34 (link)}. The values provided are the mean of three independent replicates, expressed as a percentage of the untreated control. BA accumulation at the end of the assay was quantified as described below.