Anaerobiosis

B. cereus ATCC 14579 cells were grown in a batch bioreactor on MOD medium supplemented with 30 mM glucose as the carbon source (Rosenfeld et al., 2005 (link)) and buffered at pH 7.2 with 2 M KOH. The bioreactor was an autoclavable 3-liter glass BioFlo®/CelliGen®115 (New Brunswick Scientific) with a working volume of 2 liters. It was equipped with a polarographic oxygen electrode (Mettler Toledo), a pH electrode (Mettler Toledo), and a redox-combined electrode (AgCl, Mettler Toledo). Sterile gas was fed through the culture at a constant flow set to 20 mL/h. For oxic conditions, oxygen saturation was maintained at 100% by automatic adjustment of the stirring speed. For anoxic conditions, a dissolved oxygen tension value (pO₂) of 0% was obtained with a constant flow of pure nitrogen (high- ORP condition) or hydrogen gas (low-ORP condition). Each bioreactor was inoculated with a subculture grown for 8 h (exponential growth phase) in glucose-containing MOD medium under aerobiosis or anaerobiosis. Cells from the inocula were harvested by centrifugation (7000 × g for 5 min at room temperature), washed in fresh medium, and then diluted to achieve an initial optical culture density at 600 nm of 0.02. Batch cultures were carried out at 37°C under a 300 rpm agitation speed.

Measuring of gas-exchange was performed via a continuous breath-by-breath method and averaged over intervals of 15 s. We used a mobile equipment with a small, low-dead-space respiratory valve (88 ml) and a pediatric mouthpiece (Metamax^®, Cortex Biophysic GmBh Co., Leipzig, Germany), which was fitted on the children’s back. The cardiopulmonary exercise equipment weighed 600 g. The backpack for carrying the Metamax was adjusted as to fit the small proportions of preschool children.
According to the recommendations of Wasserman et al., ventilatory thresholds VT₁ (first ventilatory threshold, start of anaerobiosis and accumulation of lactate) and VT₂ (second ventilatory threshold, point of ventilatory compensation of lactic acidosis) were determined using the V-slope method (Beaver et al. 1986 (link)). Single regression analysis was used to determine OUES slope by plotting $\dot{V}{O}_2$ (ml/min) against the logarithm of $\dot{V}E$ (ml/min), reflecting the relation between oxygen uptake $\dot{V}{O}_2$ (ml/min) and minute ventilation $\dot{V}E$ (ml/min) during incremental exercise, thus showing the effectiveness of $\dot{V}{O}_2$ (Akkerman et al. 2010 (link); Baba et al. 1996 (link)).
Physiological criteria for completion of a valid peak $\dot{V}{O}_{2peak}$ test included two of the following three criteria: (1) peak HR within 5% of the age-predicted maximum, (2) RER ≥ 1.0, and (3) volitional fatigue. Criteria for $\dot{V}{O}_{2max}$ were (1) peak HR ≥ 200 beats per minute (bpm), (2) HR ≥ 85% of the age-predicted maximum, (3) RER ≥ 1,1 (Tuan et al. 2018 (link)) or (4) leveling off, respectively, monitored both indoors and outdoors. Since the latter is difficult to determine in children, special consideration was applied when observing a true plateau (Armstrong et al. 1991 (link)). The subjects were asked, not to consume food or drinks rich in carbohydrates 2 h prior to the tests. Emphasis was given on similar test conditions including time of day or temperature. Due to COVID-19 pandemic contact restrictions, this could not be guaranteed for all the outdoor tests, as some had to be postponed and then be performed at a later date. Still, extreme temperature conditions were avoided. Indoor tests were performed in an air-conditioned room (20 °C) within the hospital. Outdoor temperatures varied from around 5 °C to 25 °C. Indoor tests always took place prior to the outdoor tests as a 12-lead ECG could only be used during the indoor test and we wanted to exclude arrhythmias in a secure environment.

A total of 24 specific pathogen-free chickens (11 weeks of age) were grouped, with 6 birds per cage. Three groups were inoculated with 0.1 mL of A. butzleri culture (1.0 × 10⁹ UFC/mL) by gavage (Day 0), and one group was kept as the negative control (non-inoculated). One bird was euthanized per day (day 1 to day 6), for the observation of gross lesions and to collect tissues for histopathology. Fecal samples were collected daily for 7 days. The fecal samples were diluted 1:9 in selective enrichment broth as described by Johnson, Murano (1999) and incubated in anaerobiosis at 30 °C for 48 h. Then, 10 μL of cultured broth was placed on a sterile cellulose membrane (0.45 μm) on Johnson and Murano agar. After one hour, the filters were removed, and the broth was seeded and incubated at 30 °C for 48–72 h.

Subgingival-derived biofilms from patients P30 and P31 grown in McBain-anaerobiosis in the AAA model were further visualized using confocal-laser scanning microscopy (CLSM) to compare control and Aii20J-treated biofilms. Biofilms formed onto the glass coverslips were stained with two nucleic acid dyes; SYTO 9, which is membrane permeable and enters all bacterial cells, and propidium iodide (PI), which only enters membrane-damaged cells (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo Fisher Scientific, Waltham, US). The staining solution was prepared by mixing SYTO 9 and PI in a 1:1 proportion and then diluting it 150-fold in sterile PBS (pH 7.4). Approximately 50 μL of the staining solution were poured onto each biofilm. Samples were kept for 15 minutes at room temperature and protected from light before image acquisition with a Leica Stellaris 8 FALCON confocal microscope, with the objective HC PL APO CS2 20x/0.75 Dry (Leica Microsystems GmbH, Wetzlar, DE). Biofilms were observed using the 499 nm laser excitation and 504 – 555 nm emission band for SYTO 9, and 561 nm excitation laser and 570 – 675 nm emission band for PI. Photographs were acquired at 20x magnification. To calculate the area covered by biofilms, eight fields for each condition were randomly selected and processed using the Leica Application Suite X software (LAS X v.3.7.4, Leica Microsystems). The area emitting photons from the SYTO 9 and PI channels was divided by the total field of view. The results are expressed in percentage of the area covered by biofilm structures in each condition.