Anaerogen sachet

Fecal samples were donated by two healthy individuals (male, age 33 and 32), who did not receive antibiotic or probiotic supplementation for at least 3 months before donation. Fecal samples were collected in a sterile 50 mL Falcon tube in an airtight container together with one Anaerogen sachet (Oxoid) to obtain anaerobic conditions until transfer into an anaerobic chamber (10% CO₂, 5% H₂ and 85% N₂) within 3 h (Coy Laboratories, Ann Arbor, MI, USA). Fecal bacteria were immobilized in 1–2 mm gel beads consisting of gellan gum (2.5%, w/v), xanthan (0.25%, w/v) and sodium citrate (0.2%, w/v) under anaerobic conditions as previously described in detail^{30 (link),32 (link)}. Sixty mL of freshly produced fecal beads were transferred in the IR bioreactor containing 140 mL of nutritive medium. For bead colonization, three consecutive fed-batch fermentations were carried out by replacing 100 mL fresh nutritive medium every 8–12 h. Bacteria, growing close to the bead surface are continuously released into the growth medium due to active cell growth in the high-biomass-density peripheral layer^{29 (link),60 (link)}.

Pa^ATCC43949 was routinely grown in Lysogeny Broth (LB) with shaking (250 rpm) or on LB agar plates for 48 h at 28°C, unless otherwise stated. Where relevant, microaerobic conditions were produced by incubating agar plates in a 2.5L anaerobic jar with an AnaeroGen Sachet (Oxoid AN0025) at the indicated temperatures for 3–4 days, which lowers O₂ levels to less than 1%. Full anaerobic conditions were achieved using a Modular Atmospheric Controlled System anaerobic cabinet (DW Scientific). Haemolysis was determined on Tryptic Soy Agar base supplemented with 5% Sheep blood v/v (Oxoid). Carbon utilisation was determined using a modified version of Oxidation/Fermentation media (0.2% w/v Casein, 85 mM NaCl, 1.7 mM K₂HPO₄, 0.008% w/v Bromothymol blue, 0.8% w/v agar).

All experimental procedures and protocols were reviewed and approved by Macquarie University Human Research Ethics Committee (Reference number 5201400595) and all methods were performed in accordance with the relevant guidelines and regulations. One fecal sample each was collected from six healthy infants (4 female and 2 male) aged 5–11 months. None of the infants were given antibiotics in at least three months prior to sample submission. Infants were fed breast milk (n = 2), formula milk (n = 2) or both (n = 2). All infants were exposed to solid food prior to sample collection. Four infants were introduced to a wider range of food types compared with the other two infants (Table 1).
Fresh fecal samples were collected in a sterile container and immediately placed in an anaerobic jar (Anaero jar, Oxoid Limited, UK) with an Anaerogen sachet (Oxoid) and an anaerobic indicator (Oxoid). Samples were transported anaerobically and laboratory processing was commenced in less than two hours of collection. Fecal slurries were prepared from individual samples by homogenising in anaerobic sterile basal medium and filtering through a sterile nylon mesh cloth (985 µm) prior to using as an inoculum. Fecal slurry preparation was performed under strict anaerobic conditions as used for media preparation.

Mercury resistance levels of each microbial community were determined, as described previously for individual bacteria [26 (link)] for mercuric mercury (HgCl₂) ((Sigma-Aldrich, St. Louis, MO, USA Portugal),) and MeHg (CH₃HgCl) (Sigma-Aldrich, St. Louis, MO, USA), using nominal concentrations ranging from 0.01 to 1003 µg/mL Hg²⁺ and 0.01 to 100 µg/mL CH₃Hg⁺. Determinations of mercury resistance were carried out in duplicate at each concentration tested. After incubation at 37 °C for 24 h in the dark and under aerobic and anaerobic (anaerobic jars with AnaeroGen sachet (Oxoid, Basingstoke, UK)) conditions, bacterial growth was monitored. The mercury resistance was registered as the lowest concentration of test compounds without visible growth. All data points represent the mean ± standard deviation (STD) of 2 independent determinations (each one also performed in duplicate).

Adhesion capability was tested using overnight cell cultures grown in Man Rogosa and Sharpe (MRS, Oxoid, Milan, Italy) broth supplemented with 0.25% L-cysteine (Sigma Aldrich, Milan, Italy) (MRSc) and incubated at 37 °C under anaerobic conditions (80% N₂, 10% CO₂ and 10% H₂) using the AnaeroGen sachet (Oxoid, Milan, Italy). Then, 200 μL of a 1:100 dilution of each culture was transferred to a 96-well micro-ELISA plate (number of replicates: 32) and, after regular shaking, the absorbance at t₀ (starting time) was read at 600 nm (ELx808, BioTek-software Gen5) and the plate was incubated. After incubation at 37 °C for 120 min under aerobic conditions, the plate was washed twice using sterile PBS to remove non-adherent bacteria and air-dried for 60 min at 60 °C. Then, 200 μL of a solution of 0.25% crystal violet was added to each well and the plate was incubated at room temperature for 15 min. After incubation, the plate was rinsed twice using Milli-Q water (Millipore, Milan, Italy) to remove excess dye and 200 μL of a 98% ethanol solution was added to each well. The absorbance was read at 570 nm. The adherence index was calculated as follows: Abs570/Abs600 [38 (link)].