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Competition of Lactobacillus paracasei with Salmonella enterica for adhesion to Caco-2 cells.

Jankowska A, Laubitz D, Antushevich H, Zabielski R, Grzesiuk E - J. Biomed. Biotechnol. (2008)

Bottom Line: Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively.Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact.In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland.

ABSTRACT
Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs), Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously), and preincubation (L. paracasei was incubated with Caco-2 cells first, and then S. enterica was added). In coincubation experiment, the presence of L. paracasei decreased S. enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs) acted weaker as inhibitors of Salmonella adhesion in comparison to the whole L. paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

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Two-hour coincubation experiments. Adhesion of S. enterica to Caco-2 cells under different conditions: (a) S. enterica was incubated with Caco-2 cells; (b) S. enterica and L. paracasei were coincubated; (c)   L. paracasei overnight culture was washed with and resuspended in isotonic salt solution and coincubated with S.enterica; (d) L. paracasei overnight culture was centrifuged and spent supernatant (SCS) was incubated with S. enterica. S. enterica was incubated with SCS fractions: (e) I (>30 kDa); (f) II (30–10 kDa); (g)III (10–5 kDa); (h)IV (<5 kDa). Numbers represent theamount of adhered bacteria per 1 Caco-2 cell. Mean and SEM, t-test. Asterisks indicate the statistical differences: *P < .05, **P < .01, ***P < .001.
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fig2: Two-hour coincubation experiments. Adhesion of S. enterica to Caco-2 cells under different conditions: (a) S. enterica was incubated with Caco-2 cells; (b) S. enterica and L. paracasei were coincubated; (c) L. paracasei overnight culture was washed with and resuspended in isotonic salt solution and coincubated with S.enterica; (d) L. paracasei overnight culture was centrifuged and spent supernatant (SCS) was incubated with S. enterica. S. enterica was incubated with SCS fractions: (e) I (>30 kDa); (f) II (30–10 kDa); (g)III (10–5 kDa); (h)IV (<5 kDa). Numbers represent theamount of adhered bacteria per 1 Caco-2 cell. Mean and SEM, t-test. Asterisks indicate the statistical differences: *P < .05, **P < .01, ***P < .001.

Mentions: Coincubation of L. paracasei and S. enterica with Caco-2 cells for 2 and 4 hours resulted in about 4- and 2-fold decrease of S. enterica adhesion to Caco-2 cells,respectively, as compared to the experiment with incubation of S. enterica alone (Figures 2(b) versus 2(a) and Figures 3(b) versus 3(a)). A significant decrease (P < .05) in the number of S. enterica adhering to Caco-2 cells was also observed in a coincubation study when L. paracasei devoid of its growing medium and resuspended in salt solution was used (Figures 3(c) versus 3(a)). However, the decrease was statistically significant only after 4-hour incubation, and the effect was significantly weaker than that when L. paracasei was used with its incubation medium (Figures 2(c) versus 2(b) and Figures 3(c) versus 3(b)). The later indicated that not only L. paracasei by itself but also the substances secreted by L. paracasei to the medium might counteract the adhesion of S. enterica to Caco-2 cells. This was confirmed in the studies with the use of supernatant obtained after centrifugation of L. paracasei overnight culture (SCS). Four-hour incubation of S. enterica with SCS led to a significant decrease of adhesion of S. enterica to Caco-2 cells (Figures 3(d) versus 3(a)) while the 2-hour incubation did not show statically significant effect (Figures 2(d) versus 2(a)). The results of incubation of S. enterica with four separate L. paracasei SCS fractions are shown on Figures 2(e)–2(h). Besides fraction IV (<5 kDa), the presence of remaining fractions resulted in significant reduction of S. enterica adhesion to Caco-2 cells (Figures 3(e) versus 3(a), 3(f) versus 3(a) and 3(g) versus 3(a)). Among these fractions, the strongest inhibition was observed for fraction II (30–10 kDa). Fresh MRS-broth medium did not influence the adhesion of S. enterica to Caco-2 cells (data not shown).


Competition of Lactobacillus paracasei with Salmonella enterica for adhesion to Caco-2 cells.

Jankowska A, Laubitz D, Antushevich H, Zabielski R, Grzesiuk E - J. Biomed. Biotechnol. (2008)

Two-hour coincubation experiments. Adhesion of S. enterica to Caco-2 cells under different conditions: (a) S. enterica was incubated with Caco-2 cells; (b) S. enterica and L. paracasei were coincubated; (c)   L. paracasei overnight culture was washed with and resuspended in isotonic salt solution and coincubated with S.enterica; (d) L. paracasei overnight culture was centrifuged and spent supernatant (SCS) was incubated with S. enterica. S. enterica was incubated with SCS fractions: (e) I (>30 kDa); (f) II (30–10 kDa); (g)III (10–5 kDa); (h)IV (<5 kDa). Numbers represent theamount of adhered bacteria per 1 Caco-2 cell. Mean and SEM, t-test. Asterisks indicate the statistical differences: *P < .05, **P < .01, ***P < .001.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279207&req=5

fig2: Two-hour coincubation experiments. Adhesion of S. enterica to Caco-2 cells under different conditions: (a) S. enterica was incubated with Caco-2 cells; (b) S. enterica and L. paracasei were coincubated; (c) L. paracasei overnight culture was washed with and resuspended in isotonic salt solution and coincubated with S.enterica; (d) L. paracasei overnight culture was centrifuged and spent supernatant (SCS) was incubated with S. enterica. S. enterica was incubated with SCS fractions: (e) I (>30 kDa); (f) II (30–10 kDa); (g)III (10–5 kDa); (h)IV (<5 kDa). Numbers represent theamount of adhered bacteria per 1 Caco-2 cell. Mean and SEM, t-test. Asterisks indicate the statistical differences: *P < .05, **P < .01, ***P < .001.
Mentions: Coincubation of L. paracasei and S. enterica with Caco-2 cells for 2 and 4 hours resulted in about 4- and 2-fold decrease of S. enterica adhesion to Caco-2 cells,respectively, as compared to the experiment with incubation of S. enterica alone (Figures 2(b) versus 2(a) and Figures 3(b) versus 3(a)). A significant decrease (P < .05) in the number of S. enterica adhering to Caco-2 cells was also observed in a coincubation study when L. paracasei devoid of its growing medium and resuspended in salt solution was used (Figures 3(c) versus 3(a)). However, the decrease was statistically significant only after 4-hour incubation, and the effect was significantly weaker than that when L. paracasei was used with its incubation medium (Figures 2(c) versus 2(b) and Figures 3(c) versus 3(b)). The later indicated that not only L. paracasei by itself but also the substances secreted by L. paracasei to the medium might counteract the adhesion of S. enterica to Caco-2 cells. This was confirmed in the studies with the use of supernatant obtained after centrifugation of L. paracasei overnight culture (SCS). Four-hour incubation of S. enterica with SCS led to a significant decrease of adhesion of S. enterica to Caco-2 cells (Figures 3(d) versus 3(a)) while the 2-hour incubation did not show statically significant effect (Figures 2(d) versus 2(a)). The results of incubation of S. enterica with four separate L. paracasei SCS fractions are shown on Figures 2(e)–2(h). Besides fraction IV (<5 kDa), the presence of remaining fractions resulted in significant reduction of S. enterica adhesion to Caco-2 cells (Figures 3(e) versus 3(a), 3(f) versus 3(a) and 3(g) versus 3(a)). Among these fractions, the strongest inhibition was observed for fraction II (30–10 kDa). Fresh MRS-broth medium did not influence the adhesion of S. enterica to Caco-2 cells (data not shown).

Bottom Line: Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively.Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact.In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jabłonna, Poland.

ABSTRACT
Competition of commensal and probiotic bacteria with pathogens for adhesion and colonization is one of the important protective mechanisms of gastrointestinal tract. In this study, we examined the ability of Lactobacillus paracasei to inhibit the adhesion of pathogenic Salmonella enterica to human colon adenocarcinoma Caco-2 cells. Caco-2 cells were grown for 6 or 21 days to obtain nondifferentiated or well-differentiated cells, respectively. In adhesion experiments, bacteria were added to the cells for 2 or 4 hours. The number of attached bacteria was expressed as colony-forming units (CFUs), Caco-2 cells were counted in hematocytometer. Both bacterial strains used adhered better to well-differentiated than to nondifferentiated Caco-2 cells, however, the amount of Salmonella adhered to Caco-2 after 2 hours of contact was 12-fold higher in comparison to L. paracasei and almost 27-fold higher after 4 hours of contact. Two types of experiments were done: coincubation (both bacteria were added to Caco-2 cells simultaneously), and preincubation (L. paracasei was incubated with Caco-2 cells first, and then S. enterica was added). In coincubation experiment, the presence of L. paracasei decreased S. enterica adhesion by 4-fold and in preincubation experiment even 7-fold. Generally, Lactobacillus spent culture supernatants (SCSs) acted weaker as inhibitors of Salmonella adhesion in comparison to the whole L. paracasei culture in coincubation experiment. In conclusion, the displacement of pathogens by lactic acid bacteria and its secretions showed here depends on the time of bacteria-epithelial cell contact, and also on the stage of Caco-2 differentiation.

Show MeSH
Related in: MedlinePlus