Characterization of Bacillus cereus Toxins

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.