Untersuchungen zu Bacillus cereus Enterotoxin-Komplexen auf zellulärer Ebene

This paper deals with the in vitro activity of the B. cereus
enterotoxin-complexes Hemolysin BL (HBL) and non-hemolytic
enterotoxin (Nhe) on cells. For that purpose the cytotoxic and
hemolytic activity of crude B. cereus culture supernatants was
analyzed in cell culture assays and on blood agar plates. Specific
monoclonal antibodies were used for the detection of single
components. By using neutralisation assays and subtractive
immoaffinity chromatography the inactivation of the specific toxin
complexes could be achieved. When testing different cell lines
(Vero-, Neuro2A-, Jurkat-, hybridoma cells) for their sensibility
against culture supernatants of toxic B. cereus isolates, Vero
cells proved to be the most sensitive. The sensitivity pattern of
hybridoma cells differed from the other cell lines, as HBL
containing supernatants showed higher cytotoxicity than
supernatants of single Nhe-producers. Analyses of 18 B. cereus
isolates revealed that the 5 - 40 % of the cytotoxic activity
measurable in culture supernatants could be attributed to HBL. A
strong correlation (r2 = 0,938) could be found between remaining
cytotoxic activity and the detectable L2 concentrations. The
results of the neutralisation assays could be verified by
subtractive immunoaffinity chromatography of Nhe B and testing the
Nhe B negative, HBL positive B. cereus culture supernatants (Nhe
B-/HBL+). Furthermore it could be demonstrated for the first time
that Nhe B plays a major part in the appearance of the
discontinuous hemolytic pattern typical for HBL producers. After
sensibilisation with tunicamycin Vero cells showed a substantial
increased cytotoxic activity (63 – 132 %). The same effect could be
observed in Fumonisin B pretreated cells for crude supernatants,
but not for the Nhe B-/HBL+ preparation. Neither treatment with
swainsonine, castanospermine nor enzymatic treatment with sialidase
and N-glycosidase F resulted in an alteration of toxicity.