Bcet rpla

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.

The absence of major enterotoxins in MB40 was confirmed using kits commercially available at the time of study: 3M Tecra™ (St. Paul, MN, USA) and Oxoid™ BCET-RPLA (Hampshire, UK). The 3M Tecra™ kit is able to detect Bacillus diarrheal enterotoxin (BDE) at or above a concentration of 1 ng/mL. The Oxoid kit is able to detect B. cereus enterotoxins (BceT) at a concentration at or above 2 ng/mL. Each kit included internal positive and negative controls that were run alongside each trial.
A frozen stock vial of MB40 was used to streak a trypticase soy agar (TSA) plate for isolation. TSA plates were incubated overnight at 35 ± 2 °C and well-isolated colonies were used to inoculate 100 mL of MB40 growth medium. After 16–20 h of growth at 35 ± 2 °C with shaking, a 1 mL sample was harvested from each flask; the cells were separated from the spent medium via centrifugation. The assays were completed and data analyzed according to the manufacturer’s protocol. Three trials were run independently of each other and all data were independently interpreted by at least two researchers. All interpretations were completed in a single-blinded manner such that the researcher interpreting the data did not know which samples were controls or samples. All data reported are an average of at least three trials.