Lactobacillus delbrueckii susp. lactis (strain CIDCA 133) enhances uptake of pathogens by macrophages.

HUGO, A. A.; PÉREZ. P.F

Villa Carlos Paz, Córdoba. Argentina

Congreso; VI Congreso de Microbiología General; 2009

Phaghocytic activity not only contributes to the elimination of potential dangerous microorganisms but also constitute a key step in antigen presentation. Lactic acid bacteria such as lactobacilli are widely used as probiotic because of its health-promoting effects. Beneficial effects of probiotics could be ascribed, at least partially, to the activation of professional immune cells such as macrophages. In the present study, we evaluated the phagocytic activity of human macrophages, against two attaching and effacing pathogens: Enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium. We performed flow citometry studies with the human cell line U-937 in the presence of viable Lactobacillus delbrueckii subsp. lactis (strain CIDCA 133) and its extracellular products. Cells were cultured in RPMI medium suplemented with 10% (v/v) foetal bovine serum, streptomycyn and penicillin at 37ºC in a 5% CO2 atmosphere. Differentiated cells were obtained by adding 1 % (v/v) DMSO. Lactobacilli were grown in MRS broth at 37 0C and supernatans were obtained by centrifugation. They were filter sterilized, neutralized  at pH 7 (NaOH) and diluted (1/8).  EHEC strain 69160 and C. rodentium ICC 180 were grown in LB medium at 37 0C. Afterwards, they were labelled  with 0.3 mg/ml of flourescein isothiocyanate (FITC) at pH 9. Opsonization was conducted by incubation in RPMI containing 10 % (v/v) human serum. Lactobacilli were labelled with carboxifluorescein-succinimydylester in PBS at pH 7. Microorganisms and eucaryotic cells were suspended in RPMI medium with a multiplicity of infection of 20:1 (bacteria:cells) and incubated at 37 0C for 30 min. Next, cells were washed twice with cold PBS to stop phagocytosis. To determine internalized bacteria, fluorescence of externally attached microorganisms was quenched by adding 2 % (w/v) trypan blue before acquisition by flow cytometry . Uptake index was defined as the percentage of cells positive in FL1 (green) x mean fluorescence intensity. In the presence of lactobacilli, uptake of EHEC and C. rodentium by U-937 cells increased in 40 %. These results were obtained in both differentiated and undifferentiated cells. Uptake index of C. rodentium was 2 – 3 times higher than that for EHEC. When experiments were performed in the presence of culture supernatants of lactobacilli instead of whole bacteria, uptake was not modified.. These findings suggest that the enhancement of the phagocytic activity is not due to extracellular factors secreted by lactobacilli. Interestingly, low uptake indexes were obtained for strain CIDCA 133 and uptake was not modified in the presence of the gramnegative bacteria assayed. Results demonstrate that Lactobacillus delbrueckii susp. lactis (strain CIDCA 133) are capable to increase the uptake of the enteropathogens under study. This ability could be very relevant not only for the elimination of the pathogens but also to prime immune system to generate a protective response.