Effects of multispecies probiotic combination on helicobacter pylori infection in vitro

Abstract

Probiotic bacteria alleviate many gastrointestinal symptoms, but the current trend of combining bacteria for additional benefit may make their effects more complex. We characterize four probiotics and their combination in terms of pathogen adhesion, barrier function, cell death, and inflammatory response in Helicobacter pylori-infected epithelial cells. H. pylori-infected Caco-2 cells were pretreated with Lactobacillus rhamnosus GG, Lactobacillus rhamnosus Lc705, Propionibacterium freudenreichii subsp. shermanii Js, Bifidobacterium breve Bb99, or all four organisms in combination. We evaluated the adhesion of H. pylori by in situ immunofluorescence; epithelial barrier function by measurement of transepithelial resistance; apoptosis by measurement of caspase 3 activation; cell membrane leakage by measurement of lactate dehydrogenase release; and inflammation by measurement of interleukin-8 (IL-8), IL-10, prostaglandin E(2) (PGE(2)), and leukotriene B(4) (LTB(4)) release. All probiotics inhibited H. pylori adhesion. L. rhamnosus GG, L. rhamnosus Lc705, P. freudenreichii subsp. shermanii Js, and the combination inhibited H. pylori-induced cell membrane leakage. L. rhamnosus GG, L. rhamnosus Lc705, and the combination initially improved epithelial barrier function but increased the H. pylori-induced barrier deterioration after incubation for 24 to 42 h. L. rhamnosus GG, L. rhamnosus Lc705, and P. freudenreichii subsp. shermanii Js inhibited H. pylori-induced IL-8 release, whereas L. rhamnosus GG, L. rhamnosus Lc705, and B. breve Bb99 suppressed PGE(2) release. None of these anti-inflammatory effects persisted when the probiotics were used in combination. The combination thus increased the levels of IL-8, PGE(2), and LTB(4) released from H. pylori-infected epithelial cells. The proinflammatory actions of the individual components dominated the anti-inflammatory effects when the probiotic bacteria were used in combination. Our results stress that the therapeutic response can be optimized if probiotic strains are characterized before they are used in combination.

FIG. 1.

Cells cultured on 96-well plates and allowed to differentiate for 15 days were incubated with H. pylori at the indicated concentrations with and without a 1-h preincubation with the indicated concentrations of the probiotic strains. The level of adhesion of H. pylori at 10⁸ CFU/ml was assigned a value of 100%, and the effects of the probiotics were compared with this control value. All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of treatment. Data are the means ± standard errors of the means (n = 5 to 7). *, P < 0.05 compared to the results obtained with H. pylori alone; **, P < 0.01 compared to the results obtained with H. pylori alone; ***, P < 0.001 compared to the results obtained with H. pylori alone. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.

FIG. 2.

TER from 0 to 42 h after preincubation with and without probiotic strains. Cells were allowed to differentiate for 21 days on semipermeable inserts in 12-well plates. The indicated concentrations of H. pylori with and without a 1-h probiotic pretreatment were added to the apical compartments of culture inserts, and TER across the epithelial monolayer was measured. All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of treatment. Data are the means ± standard errors of the means (n = 6). *, P < 0.01 compared to the results for the uninfected control. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.

FIG. 3.

Effects of probiotics and H. pylori on LDH release and caspase 3 activity at 8 h. Caco-2 cells cultured on 12-well plates, allowed to differentiate for 21 days, and pretreated for 1 h with the probiotics at the indicated concentrations were infected with H. pylori at a concentration of 10⁸ CFU/ml. The LDH activity in the conditioned medium was evaluated and compared to that for untreated cells. Caspase 3 activity was evaluated from cell lysates. All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of treatment. Data are the means ± standard errors of the means (n = 4 to 5). ***, P < 0.001 compared to the results obtained with the uninfected control; ⧫⧫⧫, P < 0.001 for H. pylori not pretreated versus the results for pretreated H. pylori. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.

FIG. 4.

Effects of probiotics and H. pylori on LDH release and caspase 3 activity at 24 h. Caco-2 cells cultured on 12-well plates, allowed to differentiate for 21 days, and pretreated for 1 h with the probiotics at the indicated concentrations were infected with H. pylori at a concentration of 10⁸ CFU/ml. The LDH activity in the conditioned medium was evaluated and compared to that for untreated cells. Caspase 3 activity was evaluated from cell lysates. All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of treatment. Data are the means ± standard errors of the means (n = 4 to 5). *, P < 0.05 compared to the results for the uninfected control; ***, P < 0.001 compared to the results for the uninfected control; ⧫, P < 0.05 for H. pylori not pretreated versus the results for pretreated H. pylori; ⧫⧫, P < 0.01 for H. pylori not pretreated versus the results for pretreated H. pylori; ⧫⧫⧫, P < 0.001 for H. pylori not pretreated versus the results for pretreated H. pylori. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.

FIG. 5.

Levels of IL-8 in the culture medium after 42 h of H. pylori infection with and without probiotic pretreatment. Cells cultured on semipermeable inserts in 12-well plates were allowed to differentiate for 21 days. After 1 h of pretreatment with and without probiotics at a concentration of 10⁸ CFU/ml, H. pylori was added at 10⁷ CFU/ml (Hp 10) or 10⁸ CFU/ml (Hp 100). All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of each treatment. Data are the means ± standard errors of the means (n = 6). *, P < 0.05 compared to the results for the uninfected control; ***, P < 0.001 compared to the results for the uninfected control; ⧫, P < 0.05 for H. pylori not pretreated versus the results for pretreated H. pylori; ⧫⧫, P < 0.01 for H. pylori not pretreated versus the results for pretreated H. pylori; open bars, apical compartment; shaded bars, basal compartment. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.

FIG. 6.

Levels of PGE₂ in the culture medium after 42 h of H. pylori infection with and without probiotic pretreatment. Cells cultured on semipermeable inserts in 12-well plates were allowed to differentiate for 21 days after they reached confluence. After 1 h of pretreatment with and without probiotics at a concentration of 10⁸ CFU/ml, H. pylori was added at 10⁷ CFU/ml (Hp 10) or 10⁸ CFU/ml (Hp 100). All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of treatment. Data are the means ± standard errors of the means (n = 6). *, P < 0.05 compared to the results for the control; **, P < 0.01 compared to the results for the control; ***, P < 0.001 compared to the results for the control; ⧫⧫, P < 0.01 for H. pylori not pretreated versus the results for pretreated H. pylori; ⧫⧫⧫, P < 0.001 for H. pylori not pretreated versus the results for pretreated H. pylori. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.

FIG. 7.

Levels of LTB₄ in the culture medium after 42 h of H. pylori infection with and without probiotic pretreatment. Cells cultured on semipermeable inserts in 12-well plates were allowed to differentiate for 21 days. After 1 h of pretreatment with and without probiotics at a concentration of 10⁸ CFU/ml, H. pylori was added at 10⁷ CFU/ml (Hp 10) or 10⁸ CFU/ml (Hp 100). All bacteria were suspended in fresh culture medium, and control cultures received fresh culture medium instead of treatment. Data are the means ± standard errors of the means (n = 6). ***, P < 0.001 compared to the results for the control; ⧫, P < 0.05 for H. pylori not pretreated versus the results for pretreated H. pylori; ⧫⧫⧫, P < 0.001 for H. pylori not pretreated versus the results for pretreated H. pylori. Hp, H. pylori; LGG, L. rhamnosus GG; Lc705, L. rhamnosus Lc705; PJS, P. freudenreichii subsp. shermanii Js; BB, B. breve Bb99; Comb., all four probiotic strains in combination.