Columbia agar

The pathogenic loads were surveyed in fecal samples at defined time points p.i., and upon necropsy in luminal samples derived from the colon, ileum, duodenum and stomach and additionally, in ex vivo biopsies taken from mesenteric lymph nodes (MLN), spleen, lungs, liver, and kidneys by culture as reported earlier [36 (link),38 (link)]. Briefly, by using sterile pestles respective samples were homogenized in sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA). Serial dilutions were then streaked onto karmali agar and Columbia agar (supplemented with 5% sheep blood; both from Oxoid, Wesel, Germany) and incubated in a jar containing CampyGen gas packs (Oxoid, Wesel, Germany) under microaerophilic conditions at 37 °C for at least 48 h. In order to survey systemic translocation, cardiac blood (0.1 mL) was directly plated onto Columbia agar (supplemented with 5% sheep blood) and incubated likewise. The detection limit of viable pathogens was 100 CFU per g.

Staphylococcaceae were first enriched in 6.5% NaCl Brain Heart Infusion (BHI) broth (HiMedia, Mumbai, India) at 37 °C overnight, and then sub-cultured onto Columbia agar (Oxoid, Hampshire, UK) supplemented with 5% sheep blood, after which the plates were incubated at 37 °C for 24–48 h. Colonies that resembled staphylococci or mammaliicocci were chosen based on their distinct morphologies, and purified on Columbia agar with 5% sheep blood. The isolates were grown at 37 °C for 24–48 h, and subsequently stored at −80 °C in 50% BHIB and 50% glycerol until further analysis.

The ability to produce and secrete hemolysins and hemolysin BL (HBL) was assessed by streaking bacterial cells on blood agar (Columbia agar containing 5% horse-blood, Oxoid, Basingstoke, UK) and sheep blood agar (Columbia agar containing 5% sheep-blood, Oxoid), respectively. Plates were incubated at 30 °C for 18 h. Hemolysin production was checked by visually evaluating the presence of a halo of incomplete hemolysis immediately around the colonies. HBL secretion was tested by observing the formation of an unusual discontinuous zone of hemolysis surrounding colonies [10 (link)]. Diarrheal toxin was detected in filtered culture supernatants by using the B. cereus enterotoxin-reversed passive latex agglutination (BCET-RPLA, Oxoid, Basingstoke, UK) kit accordingly to the manufacturers. The production of phosphatidylcholine-specific phospholipase C (PC-PLC) was evaluated by agar-diffusion assays by using 0.15% l-α-phosphatidylcholine (Sigma-Aldrich, Milan, Italy) [15 (link)]. Protease secretion was checked by seeding bacterial cells on 1.5% skim milk (Oxoid, Basingstoke, UK), followed by incubation at 37 °C for 18 h [16 (link)]. The presence of a clear degradation halo around colonies was indicative of the presence of proteolytic activities. Experiments were repeated three times in separate days.