Anaerobe basal agar

The bacterium used in this study was P gingivalis (ATCC33277), which was grown in anaerobe basal agar (Oxoid, Hampshire, Unites Kingdom) with 5% sheep blood (BD Diagnostic Systems, Germany) at 37 °C for 72 h in an anaerobic jar (Oxoid) before preparing the inoculum in each experiment.

Intestinal content samples, from ileum, caecum, and distal colon faeces, were collected from the same mice and immediately used to inoculate 2 mL of Anaerobe Basal Broth (Oxoid Ltd., Cheshire, UK). The inoculated medium was cultured for 48 h at 37°C under strict anaerobic conditions (80% N₂, 10% CO₂, and 10% H₂) in an anaerobic chamber (model 1024; Forma Scientific, Marietta, OH). Then, aliquots of 100 µL were used to prepare tenfold serial dilutions. The diluted samples were spread on the surface of non-selective agar media (Anaerobe Basal Agar, Oxoid, UK), and counting of colony-forming units (CFUs) was conducted after 48 h. The rest of the culture was centrifuged (4000g for 5 min), and 1 mL of the supernatant was frozen in liquid nitrogen and preserved at −80°C until they were processed to determine the amine content.
The CFUs were counted, at the appropriate dilution, to determine the amount from the different intestinal segments to normalise the amines produced to the media.
Gram stain was used to classify the bacteria as either Gram-positive or Gram-negative.

Samples were cultured immediately, once presumptive positive results from the PCR were obtained, to attempt recovery of C. ureolyticus. Enriched samples were inoculated onto NAV Agar [33 (link)], consisting of (l^-1) 46 g of Anaerobe Basal Agar (Oxoid), 10 g Agar (Sigma Aldrich) and NAV supplement described previously. Each sample was inoculated in triplicate and incubated anaerobically using AnaeroGen 2.5 L gas packs (Oxoid) at 37°C for up to 14 days. Plates were checked during that period at regular intervals (3–4 days) for presumptive C. ureolyticus colonies: flat, translucent, spreading colonies. Colony PCR with C. ureolyticus - hsp60 primers was performed on putative colonies. All PCR positive isolates were sent for 16S rRNA sequencing using fD1 and rP1 primers [35 (link)] to confirm their identity.