Reinforced clostridial agar

Laboratory blend of the multi-strain bacteria referred to as multi-strain or probiotic combination, PC, was prepared using industrially prepared lyophilized bacterial powders (Lallemand Health Solutions Inc., Montreal, QC, Canada) of B. longum subsp. infantis R0033, B. bifidum R0071 and L. helveticus R0052 in a ratio of 20:20:60; respectively, and as described in MacPherson et al. [22 (link)]. To rehydrate the lyophilized bacteria for both single and multi-strain blend, 1 g was mixed for 15 min at room temperature (RT) in 99 mL of phosphate buffer [0.1% soy peptone (w/v), 0.121% K₂HPO₄ (w/v), 0.034% KH₂PO₄ (w/v)] as described in MacPherson et al. and Audy et al. [22 (link),27 (link)]. Briefly, bacterial pellet from 1 ml of this bacterial suspension was washed in PBS after centrifugation at 12800 x g for 10 min at room temperature (RT) and then re-suspended in serum-free RPMI-1640 media. Individual bacteria and the multi-strain probiotic combination (PC) suspension was added to the culture flask (T25-cm² flasks containing HT-29 cells) to have a multiplicity of infection (MOI) of 100:1 for bacteria to HT-29 cell ratio. Viable counts using reinforced clostridial agar (Oxoid) were performed on the bacterial suspension and incubated 48 h anaerobically at 37°C to confirm the calculated ratio.

The three clinical P. avidum isolates T13, T14 and T15 obtained in this study were cultivated on Reinforced Clostridial Agar (Oxoid) plates for 3 days at 37°C under anaerobic conditions using the Gas-Pak™ system (Oxoid). For liquid cultures, brain heart infusion (BHI) broth (Sigma-Aldrich) was used and cultures were grown for 24 h at 37°C under anaerobic conditions.

Yeast and barley β-glucan tested in this study were purchased from Megazyme (Dublin, Ireland). D-Galactose and D-glucose were purchased from Sigma (United Kingdom). Luria-Bertani (LB) growth medium was purchased from Formedium (Norfolk, U.K.), reinforced clostridial agar from Oxoid Ltd. (Basingstoke, England) and Brain Heart Infusion from Sigma (United Kingdom). All reagents were of analytical grade.

All disaccharides tested in this study were purchased from Dextra UK (Reading, UK) or Carbosynth (Reading, UK) (Fig. S1C). l‐Rhamnose, l‐arabinose, d‐Galactose, d‐glucose and Larch Wood Arabinogalactan (LW‐AGP) were purchased from Sigma (Haverhill, UK). Luria‐Bertani (LB) growth medium was purchased from Formedium (Norfolk, UK), reinforced clostridial agar from Oxoid Ltd. and brain heart infusion from Sigma. All reagents were of analytical grade.

The cecal contents were collected aseptically into the sterilised plastic bags from the ceca of slaughtered birds. The cecal contents were immediately stored at −20 °C and processed within 24 h for the cecal microbiota population count. The collected cecal digesta homogenates were serially diluted from 10⁻¹ to 10⁻⁷. Selected agar media were used for the enumeration of targeted bacterial groups. The Escherichia. coli was enumerated using Brilliance E.coli/coliform media (CM 0956; Oxoid, UK), total anaerobic was cultured using Wilkins Chalgren Agar (CM 0619; Oxoid, UK), Pseudomonas aeruginosa was cultured using Pseudomonas Cetrimide agar (CM 0579; Oxoid, UK) Salmonella was cultured using XLD agar (CM 0469; Oxoid, UK), Clostridium was cultured using reinforced clostridial agar (CM0149; Oxoid, UK), Staphylococcus aureus was cultured using Baird parker agar (CM 0275; Oxoid, UK) and Lactobacillus was cultured using MRS agar (CM0361; Oxoid, UK). The culture plates were then incubated at 37 °C for a period of 48 h. In the case of clostridia plates, incubation was performed anaerobically at 37 °C for a period of 48 h. A colony counter was used for the enumeration of visible colonies, and the results were expressed as log₁₀ CFU/g of cecal digesta. The cecum pH was measured using a digital pH meter (AG8603, Benchtop pH Meter; Mettler Toledo, Switzerland).