Probiotic Mechanism of Lactobacillus gasseri OLL2716 Strain against Helicobacter pylori

LETTER

Helicobacter pylori infection is a major risk factor for several gastroduodenal diseases, including gastric ulcer, duodenal ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and distal gastric cancer (4, 6, 9). Although it is unknown when and how the human population became infected with H. pylori in developed countries, Weyermann et al. reported that H. pylori is almost always acquired in early childhood (11). Probiotics intended for the control of H. pylori as well as for antibacterial chemotherapy against H. pylori have attracted attention (10). Current systematic reviews describe probiotics as an adjunct to first-line triple therapy and not as antibacterial chemotherapy. Whether adjunctive therapy enhances eradication rates remains controversial, but there is more evidence supporting the role of probiotics in reducing the frequency of side effects caused by the triple-therapy regimen (7). Lactobacillus gasseri OLL2716 proved effective in both suppressing H. pylori colonization of the stomach and reducing gastric mucosal inflammation (8). We reported that an L. gasseri OLL2716 strain contained in a yogurt drink can colonize the gastric mucus layer in infected patients (3). However, the mechanism by which that L. gasseri OLL2716 strain controlled H. pylori strains was unknown.

Probiotic effects of L. gasseri OLL2716 against the H. pylori ATCC 43504 strain were investigated. A total of 10 μg/ml of L. gasseri OLL2716 solution (10⁵ CFU/ml) was added to H. pylori cultured for 24 h on 5% sheep blood agar plates, and the strains were cocultured at 37°C under microaerobic conditions for 24 h and 48 h. The two cocultured strains were adhered to cover glasses by treating them with Cell-Tak (BD Biosciences, Tokyo). The cells were fixed with 2.5% glutaraldehyde and 0.1 M cacodylate buffer for 24 h, postfixed with 1% osmium tetroxide, dehydrated in increasing concentrations of ethanol (50%, 70%, and 99.5%), and subjected to critical-point drying. Each sample was examined using a VE-8800 electron microscope (Keyence, Osaka, Japan). The H. pylori strains cocultivated with L. gasseri OLL2716 for 24 h converted from rods to coccoids (Fig. 1). We cultured these H. pylori coccoids again. However, they did not grow on sheep blood agar plates.

Fig 1.

Coccoid conversion of H. pylori strains caused by L. gasseri OLL2716 or dl-lactic acid. (A) H. pylori ATCC 43504. (B) L. gasseri OLL2716. L. gasseri OLL2716 was added to H. pylori on agar plates and cocultured for 24 h (C) and 48 h (D). Coccoid conversion of H. pylori was observed after coculture for 24 h (C), and after 48 h, the number of coccoids decreased (D). Either 0.1% (0.9 g/liter) (E) or 1% (9 g/liter) (F) dl-lactic acid was added to H. pylori cultured on agar plates, and coccoid conversion of H. pylori was observed (E). However, reduction of the size and number of H. pylori organisms was observed with 9% lactic acid (F). Magnification, ×5,000. Bar, 2 μm.

The quantity of dl-lactic acid produced by Lactobacillus organisms, including L. gasseri OLL2716, was measured. Both H. pylori strains (10⁶ CFU/ml) and each Lactobacillus strain (10⁵ CFU/ml) were cocultured at 37°C under microaerobic conditions for 48 h in Brucella broth (Difco, MD) with 5% defibrinated sheep blood. The quantity of dl-lactic acid produced by each Lactobacillus strain was determined by spectrophotometry using an F kit (Roche Diagnostic, Japan, Tokyo). The seven Lactobacillus strains investigated were Lactobacillus gasseri OLL2716 and clinical isolates of Lactobacillus gasseri-1, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus fermentum-1, L. fermentum-2, and L. fermentum-3. The quantity of dl-lactic acid produced by L. gasseri OLL2716 was about 1.4 g/liter (Fig. 2). That of other Lactobacillus organisms except L. fermentum-1, -2, and -3 was almost the same. To confirm the morphological conversion of H. pylori, 10 μl each of either 0.1% (0.9 g/liter) or 1% (9 g/liter) dl-lactic acid (Wako, Osaka) was added to the H. pylori strain cultured on agar plates. After 24 h, a similar coccoid conversion of H. pylori was observed in cells treated with 0.1% dl-lactic acid (Fig. 1E). Furthermore, the number of H. pylori cells decreased when exposed to 9 g/liter of dl-lactic acid. These coccoid-shaped H. pylori cells did not grow on sheep blood agar plates. These results showed that the L. gasseri OLL2716 strain suppressed the growth of H. pylori. Generally, bacteria of a coccoid form are in a dormant state and do not multiply, although they are live bacteria. However, whether coccoid forms of Helicobacter spp. are dormant or simply dead is the subject of ongoing controversy (1).

Fig 2.

Quantities of dl-lactic acid produced by Lactobacillus gasseri OLL2716 and other Lactobacillus species.

The effect of probiotics to completely eradicate H. pylori by Lactobacillus supplementation is not yet shown. However, they can suppress H. pylori colonization (2). In this study, we confirmed coccoid conversion of H. pylori by dl-lactic acid produced by the L. gasseri OLL2716 strain and the suppression of H. pylori multiplication. Because the quantities of dl-lactic acid produced by various Lactobacillus strains, such as L. plantarum and L. salivarius, were similar, those strains may be effective against H. pylori infection. However, after screening over 200 Lactobacillus strains, Kimura found that the L. gasseri OLL2716 strain has strong anti-acid properties (5). Furthermore, this strain can compete with H. pylori in the human gastric mucus layer and suppresses H. pylori infection (3).

In conclusion, though further studies including factors other than lactate production are necessary, we showed that the mechanism of the probiotic L. gasseri OLL2716 strain against H. pylori was coccoid conversion of H. pylori due to dl-lactic acid. This is the first report that showed that the L. gasseri OLL2716 strain induces the coccoid conversion of H. pylori by using an electron microscope. The incidence of antibiotic-resistant H. pylori has increased in recent years. The combination of probiotics using L. gasseri OLL2716 and conventional eradication therapy may be effective for the control of these resistant strains. When they are ingested through yogurt, L. gasseri OLL2716 strains which are acid proof to gastric juice will be effective as probiotics against H. pylori infection.