Brain heart infusion

In terms of streptococcal species, S. sanguinis ATCC 10556 (ATCC, American Type Culture Collection), S. gordonii ATCC 10558, S. oralis ATCC 35037, and S. mutans ATCC 25175 were used. The bacteria were grown on plates containing Brain heart infusion (BHI; Sigma-Aldrich) and 1.5% agar (Wako) for plate cultures. Pre-culture was performed in an anaerobic chamber (N2: 80%; H2: 10%; CO2: 10%) at 37°C for 1 day. A single colony on the plate was cultured in BHI broth for further 24 h. A fresh culture, following liquid culture for another 4 hours, was used for the adherence experiment. The optical density of every bacterial suspension was regulated with BHI broth to 0.2 at 660 nm by a spectrophotometer (Ultrospec 2100pro; Amersham Biosciences). The bacteria for subsequent experiments were grown on disks in 24-well plates.

A suspension of two different strains was used as inoculum, composed by the reference strain ATCC 7644 and the EURL strain 12M0B098LM (Guillier, Lardeux, Michelon, & Ng, 2013). The preparation of the inoculum was done following the European Reference Laboratory for L. monocytogenes guideline (EURL, 2014) with some modifications (concentration of the inoculum and use of a mix of two L. monocytogenes strains). The challenge test was initially carried out to assess the growth potential, and then, it seemed more interesting to calculate the maximum growth rate, knowing that the use of two L. monocytogenes strains, instead of one, would not have influenced the result of the study. The strains, stored in Microbank™ (Pro‐Lab Diagnostics) at −80°C, were individually revitalized at 37 ± 1°C for 18h in BHI broth (Brain Heart Infusion, Sigma‐Aldrich Co. LLC.). In order to get adapted to the storage temperature of the product, new subcultures were set up in BHI broth and incubated at 8 ± 1°C and 12 ± 1°C until reaching the stationary phase of each bacterial strain (EURL, 2014; NACMCF, 2010). The inoculum suspension was then prepared, consisting of equal parts of liquid cultures of the two selected strains. The mixture was enumerated according to ISO 11290‐2:1988/Amd. 1:2004. The suspension was subsequently diluted (saline water) to obtain a concentration of 10³ CFU/g in the product.

B. thetaiotaomicron VPI-5482 and B. ovatus ATCC 8483 were cultured anaerobically in TYG medium, brain-heart infusion (Sigma) plus 2% (w/v) agar or minimal medium containing 0.5% (w/v) 1,6-β-glucan (Elicityl Oligotech) or glucose as sole carbon source, as described previously (2 (link)).

S. mutans UA159 (ATCC 700610) biofilms were grown for 72 h on glass microscope slides at 37°C and 5% CO₂ in brain heart infusion (Sigma-Aldrich, St. Louis, MO) supplemented with 2% (wt/vol) sucrose (Sigma-Aldrich) and 1% (wt/vol) porcine gastric mucin (type II; Sigma-Aldrich). After the growth period, the biofilm-covered slides were gently rinsed in 1% (wt/vol) phosphate-buffered saline solution (Sigma-Aldrich) and placed in petri dishes (49 ). Biofilm thickness was determined by fixing untreated samples with 4% (wt/vol) paraformaldehyde and staining with Syto 63 (Thermo Fisher Scientific, UK). Subsequently, three random confocal images were taken on three independent replicate biofilm slides, with a thickness of 51.8 ± 4.9 μm measured using COMSTAT software; thus, we used a biofilm thickness (L_b) of 55 μm in the simulations.

The antibacterial effect of honey samples was determined on Pseudomonas aeruginosa ATCC 27853, Staphylococcus epidermidis ATCC 12228 and methicillin-resistant Staphylococcus aureus ATCC 700698. Test bacteria were grown in 100 mL sterile BHI (Brain Heart Infusion, Sigma Aldrich Ltd., Budapest, Hungary), except for the MIC determination, in which case Mueller-Hinton Broth (MHB, Oxoid Ltd., London, UK) culture medium was used. Each bacterium was incubated in a shaker incubator (C25 Incubator Shaker, New Brunswick Scientific, Edison, NJ, USA) at 37 °C and at a speed of 60 rpm for 12 h [40 ]. The bacterial suspensions were diluted with clear BHI to the appropriate concentrations for each assay. For the anti-QS tests, the Chromobacterium violaceum 85WT (SZMC 6269) bacterial strain was used. The bacterium was cultivated on LB agar (Luria Bertani Broth, 10 g tryptone, 10 g NaCl, 6.6 g yeast extract, 15 g agar in 1000 mL distilled water, Sigma Aldrich Ltd., Budapest, Hungary) at 30 °C for 48 h.

Pg was cultured in broth (Brain Heart Infusion, l-cysteine hydrochloride monohydrate, yeast extract, and chloroproto-ferriheme, Sigma-Aldrich, St. Louis, MO, USA). After that, Pg was placed in an anaerobic container for 48 h at 37 °C. A total of 10⁹ colony-forming units of live Pg was suspended in 0.1 ml phosphate-buffered saline (PBS) with 2% carboxymethyl cellulose (CMC) (Sigma-Aldrich) and given to each mouse by gavage three times a week for about a month, as described previously [14 , 15 (link)]. The control group was administered 0.1 ml PBS with 2% CMC without Pg. After administration, all mice were allowed to eat and drink ad libitum.