Columbia agar

All strains are listed in supplementary S1 Table in S1 Text. Streptococcus pneumoniae D39 and derivates were routinely grown in C+Y medium or on Columbia agar (Merck) supplemented with 2% (v/v) defibrillated horse-blood (SSI-Denmark) at 37°C with 5% CO₂. Spectinomycin, kanamycin, or chloramphenicol were added at 100, 250 or 0.4 μg/mL, respectively, when appropriate. For growth and induction experiments, strains were grown in chemically defined medium (CDM) prepared as described previously with addition of 10 U/mL catalase (Merck) [76 (link)].

Urine samples were cultured using a calibrated pipette to deliver 10 μl and 100 μl of samples onto Columbia agar (Merck, Germany) supplemented with 5% sheep blood and onto MacConkey agar (Merck, Germany). The blood agar plates were incubated aerobically, and the MacConkey agar plates were incubated aerobically. All samples were incubated at 37°C for 24 h until adequate growth was present. Primary plates were carefully inspected for colonies of E. coli, which were plated onto sheep blood agar plates; these plates were incubated at 37°C for 24 h. Suspected colonies were transferred to the Eosin Methylene Blue agar (EMB agar, Merck, Germany) plates and incubated at 37°C for 24 h. Metallic green colonies with typical E. coli morphologies of the EMB agar plates were identified as E. coli using standard techniques, including indole, Methyl Red–Voges-Proskauer (MR-VP), Triple Sugar Iron agar (TSI), and citrate biochemical testing and analysis with an API-20E system (BioMérieux, Marcy l'Etoile, France) [16 ].

Trypticase soy broth (TSB) (Merck, Germany), mannitol salt agar (MSA) (Merck, Germany), and cetrimide agar (CA) (Merck, Germany), Columbia agar (Merck, Germany) containing 5% sheep blood, and BHI (Merck, Germany) containing 6% NaCl were used as culture media to cultivate and recover the slow-growing phenotypes, S. aureus, and P. aeruginosa strains. The plates were incubated in both ambient air and 5% CO₂, at 37 °C and 25 °C.

The extracellular protease production was determined by the plate assay in Luria Bertani (Merck, Darmstadt, Germany) agar medium supplemented with 1% of skimmed milk (w/v). A bacterial suspension of 0.5 McFarland was prepared and subsequently inoculated (5 μL), then all plates were incubated at 37 °C for 24 h in a normal atmosphere. The presence of extracellular proteases was revealed by the formation of clear halos around the colonies which were measured. The halos were classified as negative (−) in the absence of a halo, as weak positive (+/−) in the presence of a halo less than 11 mm, as positive (+) in the presence of a halo less than 13 mm, and as strongly positive (++) in the presence of a halo less than 15 mm [38 (link)].
The hemolytic activity was determined by the plate assay using a Columbia Agar with 5% sheep blood (Merck, Darmstadt, Germany). A bacterial suspension of 0.5 McFarland was prepared, and subsequently, 5 μL was inoculated and plates incubated at 37 °C for 24 h in a normal atmosphere. The production of hemolysins was identified by the presence of clear (β-hemolysis) or diffuse (α-hemolysis) halos around the colonies. The absence of a halo shows that there was no production of hemolysins [39 (link)].

6×10⁶ WT or Pik3r1^−/− fetal liver-derived neutrophils were incubated with 1.5×10⁶ serum-opsonized MSSA (strain Wood 46) in 300 μl PBS for 60 min. Samples were added to ice-cold Difco nutrient broth (BD, Franklin Lakes, NJ, USA) with 10% saponin and sonicated to liberate ingested bacteria [32 (link)]. Surviving bacteria were enumerated by plating on Columbia agar (Sigma, St. Louis, MO, USA).

All samples collected for bacteriological analysis were analyzed using routine bacteriological procedures in the local veterinary diagnostic laboratories, in accordance with the safety regulation in force. In brief, the urine samples collected from the 13-year-old Border Collie (first reported case) were directly cultivated using Chocolate PolyViteX (BioMerieux, Craponne, France) and chromogenic CPS media (BioMerieux, Craponne, France). In contrast, tissue biopsies of the scrotal region collected from the 5-year-old German Shepherd (second reported case) were cut with a surgical single-use scalpel, and 10 g was homogenised and diluted in 1/2 to 1/5 ratios in phosphate-buffered saline (PBS) solution (0.9%). The homogenate was then plated onto Chocolate PolyViteX and Columbia agar (Sigma-Aldrich, Saint-Quentin-Fallavier, France) with sheep’s blood (5%) media. The resulting colonies of the two cases were purified via replating and analyzed via MALDI-TOF for first-line identification purposes. The two isolates (20-02069-2828 and 22-03912-5948) suspected to be Brucella were transferred to the French National Reference Laboratory for Animal Brucellosis (ANSES, Maisons-Alfort, France) to confirm the Brucella genus and determine the species, in accordance with the safety regulations in force.