Anaerocult a system

Pure cultures were grown under static conditions at 30°C in 10 mL liquid YEL. After 72 h cells were harvested by centrifugation (14,000 rpm for 3 min), resuspended in sterile water to final OD₆₀₀ = 0.2. For qualitative evaluation of lactose utilization, 24 well microplates containing 990 μL of modified API50CH medium were inoculated with 10 μL of cell suspension prepared as above described. Microplates were incubated under anaerobic conditions (Anaerocult A system, Merck, Darmstadt, Germany) for 7 days at 30°C. The production of acid from lactose was determined based on bromocresol purple color change (de Freitas et al., 2015 (link)). For the evaluation of the kinetics of growth on lactose, cells were inoculated to a final OD₆₅₀ = 0.01 in 200 μL YELactose within 96 well microplates. For the assessment of growth kinetics on lactose plus lactate YELactose was added with increasing concentration of Na lactate (0.0, 1.5, 3.0, and 6.0 g L⁻¹). Growth was measured automatically every hour at OD₆₅₀ using a SPECTROstar nano microplate spectrophotometer (BMG Labtech, Ortenberg, Germany). Carrying capacity (final OD₆₅₀), specific growth rate (μ), lag phase duration (λ), and area under the curve (AUC) were determined by fitting the growth curves with the Bayesian non-parametric model developed by Tonner et al. (2020) (link).

For all treatments, cells were seeded and maintained in a humidified incubator at 5% CO₂ and 37°C for 24 h before the experimental conditions were applied. For mild hypoxia (1% O₂) cells were transferred to a low-oxygen incubator prepared according to the protocol of Wright and Shay (27 (link)). In brief, cell culture dishes were placed in gas-tight containers that were flushed with a well-defined gas mixture consisting of 1% O₂, 5% CO_2, and 94% N₂ and were placed in an incubator at 5% CO₂ and 37°C for 48 h. Anoxia (<0.01% O₂) was induced by using the Anaerocult^® A-System (Merck, Darmstadt, Germany) in gas-tight containers for 24 and 48 h. Anoxic conditions were monitored through the Anaerotest^®-System (Merck, Darmstadt, Germany). Normoxic HNSCC cells were cultured under atmospheric oxygen (21% O₂) at 5% CO₂ and 37°C. Treatments with CoCl₂ (Merck, Darmstadt, Germany) at 150 µM for 48 h, staurosporine (Merck, Darmstadt, Germany) at 1 µM for 2, 4, and 6 h and erlotinib (Cell Signaling Technology, Danvers, USA) at 1 µM for 72 h was conducted at atmospheric oxygen at 5% CO₂ and 37°C.

Gardnerella vaginalis was grown overnight in sBHI medium (pH 6.5) and further sub-cultured independently in the same medium, both at pH 6.5 and pH 3.5 under anaerobic conditions using Anaerocult^® A system (Merck Millipore, Germany) in an anaerobic jar as mentioned above. An equal volume of the culture was taken to measure the absorbance (OD_{595 nm}) over time. Cells were pelleted at 4,000 × g for 10 min and stored at –20°C until preparation of whole cell lysates and determination of protein concentration and protein profile analysis by SDS-PAGE as described later. The growth curve analysis experiment including estimation of protein concentration and subsequent protein profile analysis was performed thrice. Also, cells collected at 48 h time point were plated on sheep blood agar plates (HiMedia, India) and incubated further under anaerobic conditions for 48 h at 37°C to determine the colony forming units (CFU). The CFU analysis was performed twice. The results were analyzed for statistical significance as mentioned in the section on statistical analysis.

Adhesion assays with B. infantis were performed as previously described [18 (link),19 (link)]. HT-29 cells were washed twice with PBS, and 250 µL of the bacteria and media suspensions were added to the wells, corresponding to approximately 40 bacterial cells per human cell. Bacterial cells were incubated with the HT-29 cells for 2 h at 37 °C under anaerobic conditions using an Anaerocult A system (Merck). The HT-29 cells were then washed five times with PBS to remove non-adherent bacteria. HT-29 cells were then lysed with 250 µL of 1% TritonTM X-100 (Merck) for 5 min at 37 °C. The lysates were serially diluted and enumerated by spot-plating on MRS plates to enumerate bacterial colony-forming units (CFU). The adhesion of the bacteria was determined as the percentage of original inoculum which attached, thus accounting for variations in the starting inoculum. Percentage adhesion = (CFU/mL of recovered adherent bacteria/CFU/mL of inoculum) × 100. Experiments were performed in triplicate on three separate occasions.

Four dead Lutjanus russellii (red
sea bass, size 10–20 cm) were obtained from a fishing port
located in Chonburi province, Thailand, in December 2019. Fish intestines
were collected and homogenized. The method for isolating LAB was modified
from the procedure previously described by Chen et al. (2012).^{27 (link)} 1 g portion of homogenized fish intestines was
mixed with 4 mL of normal saline (0.85% weight per volume) (w/v),
and the mixture was serially diluted. Then, 1 mL of each diluted mixture
was added to a methyl Petri dish plate. Warm, sterile MRS agar (semisolid)
was poured onto the plate and mixed by gently swirling. All plates
were incubated under anaerobic conditions (Anaerocult A system, Merck,
Darmstadt, Germany) at 30 °C for 5 days. LAB colonies were selected
from the MRS agar plates and further subcultured on the new MRS agar
plates at 30 °C for 3 days. The subcultured LAB isolates were
tested for antimicrobial activity against AV and AJ using the agar
spot assay, as described in Section 2.2.1 All isolated LAB colonies were stored
in a glycerol stock at −80 °C until use.

Bifidobacterium longum subsp. infantis (ATCC^® 15697™), Bifidobacterium longum NCIMB 8809 and Bifidobacterium breve NCFB 2258 were obtained from the American Type Culture Collection (ATCC, Middlesex, UK), the National Collection of Industrial and Marine Bacteria (Aberdeen, Scotland, UK) and the National Collection of Food Bacteria at the AFRC Institute of Food Research (Reading, UK) respectively. The strains were stored in deMan Rogosa Sharpe (MRS) (Difco, Sparks, MD, USA) broth containing 50% glycerol at − 80 °C until they were re-cultured according to the supplier’s instructions. The strains were cultured at 37 °C under anaerobic conditions generated using an Anaerocult A system (Merck). To prepare mid-exponential phase cells, overnight cultures were adjusted to an optical density of 0.3 and the cultures were then grown for a further 2 h and then adjusted to an optical density (OD_600nm) of 0.45–0.5.

Bifidobacterium longum subsp. infantis ATCC^® 15697™ (B. infantis) was obtained from the American Type Culture Collection (ATCC, Middlesex, UK). Bacterial cultures were maintained as previously described [10 (link),11 (link)] The strain was stored in deMan Rogosa Sharpe (MRS) (Difco, Sparks, MD,, USA) broth containing 50% glycerol at −80 °C. The strain was cultured twice in MRS media supplemented with L-cysteine (0.05% w/v) (Merck, Dannstadt, Germany) prior to use, and was routinely grown overnight at 37 °C under anaerobic conditions generated using an Anaerocult A system (Merck).