Bacillus megaterium isolates from honey: cytotoxic effects on the human intestinal epithelial cell line Caco-2.

LÓPEZ, A. C.; MINNAARD , J.; PÉREZ, P. F. AND ALIPPI, A. M.

Congreso; Apimondia; 2011

Honey quality is influenced by microorganisms, particularly yeasts and spore-forming bacteria. Analysis of Argentinean honeys revealed that Bacillus cereus and Bacillus megaterium were commonly found. DNA sequences related to biologically active secreted factors have been detected in B megaterium (enterotoxin HBL, enterotoxin-T and cytotoxin K). Even though direct bacteria-epithelial cells interactions have been studied for B. cereus, there are no reports for B. megaterium. Previous studies with culture filtrate supernatants of B megaterium showed complete detachment of differentiated Caco-2 cells. The aim of this study was to evaluate the interaction of B. megaterium with Caco-2 cells monolayers. Epithelial cells were grown in Dulbeccos modified Eagle´s minimum essential medium (DMEM) supplemented with 10 % fetal calf serum and (37 °C, 15 days, 5 % CO2 – 95 % air atmosphere). B. megaterium strains were incubated in Luria Bertani broth (32 °C for 3 h) under agitation. After centrifugation, pellets were suspended in DMEM containing 100 µg/ml cloramphenicol and cell density was standardized. Cell monolayers were washed twice with PBS and incubated with each bacterial suspension (2 h at 37 °C in a 5% CO2 – 95 % air atmosphere). For cell association assays, the monolayers were washed three times with PBS and incubated at 37 °C with 1 ml of distilled water per well. The invasion was assessed by the aminoglycosid protection assay. Serial dilutions of samples were plated onto nutrient agar and incubated at 32°C for 16 h. After that, quantitative association and invasion were evaluated. Results showed that all the strains tested adhered to and invaded monolayers. Adhesion values were similar for all the strains under study but we found a strain dependent invading capability with strains … and … showing the highest invasion values. Epithelial cells were detached after infection when no cloramphenicol was used thus suggesting that protein synthesis is probably involved in biological activity. Presented results constitute the first insight on the direct interaction between B megaterium cells and enterocyte-like cells.