In vitro and in vivo Activity of Lactococci Strains against Helicobacter pylori

Problem statement: Search for lactic acid bacteria that have in vitro, a significant inhibitory effect against the strains of H. pylori and to determine the inhibitory activity in vivo. Approach: The in vitro inhibitory activity of lactic acid bacteria isolat ed from milk against strains of H. pylori was determined by the agar diffusion method. Two g roups of mice were inoculated for a week with TN2GF4. After three weeks, the infected group is treated for seven days with E. faecium (B13). H pylori was detected by a count after culture of gastric b iopsy. The probiotic was determined by a count from fresh feces of mice trea ted. Results: Thirty strains of lactic acid bacteria were isolated and identified. E. faecium (B13) strain showed a highly significant inhibitio n. H. pylori was successfully detected in the gastric mucosa. E. faecium (B13) reduced the colonization in the stomach of H. pylori with a rate of 43% in a week. Conclusion: E. faecium (B13) has in vitro and in vivo an inhibitory effect against H. pylori.


INTRODUCTION
Infection with H. pylori is probably the most infection common worldwide. 20-90% of adults are infected in different countries. The infection is more common in disadvantaged, low socioeconomic status (Perez-Perez et al., 2004).
In 1994, the National Agency for Research on Cancer has determined that this is one of the pathogenic Gram-negative bacilli was recognized as a major carcinogen class (I and II) is indeed associated with gastric adenocarcinoma and gastric lymphoma of MALT type (Cavicchi and Lamarque, 2000).
Several treatment regimens have been proposed to treat H. pylori, including antibiotics and antacids, but treatment failure was observed in 20% of cases because of side effects or antibiotic resistance. Many patients after failure of initial treatment seeking other strategies in the treatment of H. pylori: alternative or addition of antibiotics (Lopez-Brea et al., 2008).
Lactic acid bacteria are considered as probiotics. Probiotics are defined as living microorganisms ingested able to exert beneficial effects on health (Ljungh and Wadstrom, 2006). Several probiotics have shown an antibacterial effect on Helicobacter pylori such as Lactobacillus acidophilus (Lin et al., 2011) and Bifidobacterium bifidum (Chenoll et al., 2011).
The aims of this study are to find lactic acid bacteria that have a significant inhibitory effect in vitro of H. pylori and to determine this antibacterial activity in vivo.

MATERIALS AND METHODS
Lactic acid bacteria: Samples of raw milk from cow, goat and sheep were taken from two regions: Miliana and Ain Defla. Other samples of camel milk were collected from Biskra and Tamanrasset.
Isolation of lactic acid bacteria was carried out on M17 medium (Pasteur Institute of Algeria) selective medium for lactococci. The Gram-positive bacteria have catalase and oxidase negative were selected for identification. They were then subjected to the pre-identification (type of fermentation and growth in hostile environments). Lactic acid bacteria were then identified at the species or subspecies by establishing their fermentation profiles using the micro-Api 20 strep identification.
These strains were transported in the middle BIO-RAD.
These strains were inoculated on Brucella agar supplemented with 10% horse blood and incubated for three days at 37°C and under a microaerophilic atmosphere.
Interaction of lactic acid bacteria with the strains of H. pylori: Lactic acid bacteria inoculated into 05 ml of M17 broth and incubated at 37°C for 24 h were tested for antibacterial activity following the agar diffusion method (Tadesse et al., 2004). The petri dishes containing Muller Hinton medium, are inoculated with one ml of H. pylori strains preculture (inoculated into 5 ml of Brucella broth and incubated at 37°C for 24 h under a microaerophilic atmosphere), after drying for 30 minutes at 37°C, filter paper discs (6mm diameter) impregnated with 50 µL of the lactic acid bacteria preculture are deposited on the surface of the agar (three replicates for each strain of lactic acid bacteria). The petri dishes were prepared and preincubated under refrigerated conditions for 2-4 h at 4°C to allow diffusion of inhibitor to be followed by incubation for 24 h at 37°C. Inhibition is considered positive if the diameter of inhibition Zone (Zi) is greater than 2 mm (Thompson et al., 1996): Zi (mm) = diameter of the inhibition zone obtained (mm) -disc diameter (6mm).

Antagonism in vivo:
The results of the antagonistic effects observed in vitro have been the subject of an in vivo study performed on 36 female mice, holoxenic and have two months old (from the Pasteur Institute of Algeria).
The antagonistic effect in vivo of E. faecium (B13) is studied on TN2GF4 strain.
These mice are divided into three lots to the number of 12 mice for each lot.
Stool analysis is done for half of lot 1 (control), one mice underwent a dissection which a biopsy specimen was taken and crushed. Culture of H. pylori was performed on Brucella agar supplemented with 10% horse blood and incubated at 37 o C for 3-5 days and under a microaerophilic atmosphere.
Mice lots 2 and 3 were inoculated orally with 0.5 mL (10 9 CFU / mL ≈ 0.6DO) of H. pylori prepared for a fresh culture, incubated at 37°C for 24 h and under a microaerophilic atmosphere. The amount to be inoculated is, administered three times a week, with an interval of two days between inoculations (Sgouras et al., 2004).
After three weeks post infection, lot 3 was treated for seven days with 1 ml of milk inoculated with 10 7 cfu / ml ≈ 0.1 DO of E. faecium (B13) (Coconnier et al., 1998).
In the third and fourth weeks, six mice per group (Lot 2 and 3) and one mouse (control group) were killed and dissected aseptically. The stomach of each mouse was removed and H. pylori was detected by a count is made on the culture of gastric biopsy crushed, Gram stain, urease, catalase and oxidase.
Feces of treated mice were collected and analyzed for the presence of probiotic administration (Sgouras et al., 2004).
• Test with urea: A fragment of gastric biopsy is placed in a tube containing urea indole and incubated at 37°C for 18 h-24 h; the positive result is interpreted by the color change from orange to pink • Gram stain: A fragment of gastric biopsy crashed with two blades by setting flame and then the smear is stained with Gram's method • Culture: One biopsy specimen was crushed in 02 ml of medium BGT (glucose buffered broth). Decimal dilutions were performed; the Brucella medium supplemented with 10% horse blood was inoculated by the last dilution (10 -4 ). The petri dishes were incubated at 37°C for 3-5 days under a microaerophilic atmosphere

RESULTS
Thirty strains of lactic acid bacteria were isolated and identified. The bacterial genera found in samples of raw milk are represented by strains of Lactococcus and Enterococcus (Table  2).       (Table 3).
According to the results, it appears that the two strains E. faecium (B13) and Lc. lactis subsp. lactis (B01) have significant inhibition zones with diameters 20 and 18 mm are registered with the strain TN2GF4, 16 and 13 mm with the strain HSA3068, 15 and 11 mm with strain 26695, 15 and 13 mm with strain J99. For HPAG1 strain the best inhibition zone is found with Enterococcus faecium (B19) (diameter = 7 mm). Other inhibition zones were found with strains of lactic acid bacteria (Fig. 1). The bacterial analysis of the stool and gastric biopsies of the mouse control group revealed no presence of H. pylori and E. faecium.
During the two phases of observation, H. pylori was successfully detected in the gastric mucosa of mice infected with strain TN2GF4. The population of the TN2GF4 isolated from fragments of mouse stomach in this group ranged from 1.5 ×10 5 -1.2 ×10 6 cfu/mL.
From the 2 nd phase (30 days) there is an increase of the population of H. pylori and peaked at 2.9×10 6 cfu/mL. With a growth rate equal to 52.42% between the means of two phases of lot 2.
After 3 weeks post infection in the treated group, quantitative culture of H. pylori performed on fragments of gastric biopsies taken from 6 mice infected with TN2GF4 shows a number of bacteria ranging from 1.8 ×10 5 to 1.2 ×10 6 cfu/mL. This number starts to decrease after administration of E. faecium (B13) from 1.2 ×10 6 -1.6×10 5 cfu/mL on day 30, E. faecium (B13) reduces colonization of H. pylori in mice, in effect from 4.9×10 5 -2.8×10 5 cfu/mL or a lowering of 42.86% between the means of two phases of lot 3.
Stool analysis of treated mice revealed the presence of Enterococcus faecium (B13) with concentrations ranged from 6× 10 5 -1.03×10 6 cfu/mL. These results were confirmed by analysis of variance at the 5% applied to the evolution of H. pylori accounts at lot 2 and 3. This analysis shows that there is a highly significant difference between the infected group and the treated group. Indeed the burden of H. pylori in mice treated with E. faecium (B13) is decreased by nearly one log compared with mice receiving H pylori alone (Fig. 2).
A statistical analysis performed with a Student t test between the infected group and the group treated with E. faecium (B13) showed a highly significant difference (α = 0.05). For 3 weeks post infection, H. pylori readily colonize the stomachs of mice, with its flagella and its helical shape. It glides through the mucosa of the stomach and is anchored to the epithelial cells through adhesion. It secretes urease and in the presence of protein converts urea into ammonia and CO 2 . The production of ammonia creates a buffered microenvironment around the bacteria and can survive in acidic medium.
Ammonia is toxic to epithelial cells and will damage the surface epithelial cells (Fig. 3). However, no effect was observed in mice control group (Fig. 4).
The results clearly indicate that in vivo Enterococcus faecium (B13) was present in the microflora during the consumption period. It is then removed in a few days without lasting settlement

DISCUSSION
The results from the inhibition of H. Pylori strains (TN2GF4, HPAG1, J99, 26695 and HSA3068) show that lactic acid bacteria do not have the same spectrum of action, they express more or less important variations depending firstly on the strain of H. pylori target and secondly to the indicator strain of lactic acid bacteria. The best inhibition zones were recorded by E. faecium (B13) isolated from goat's milk and Lactococcus lactis subsp. lactis (B01) isolated from cow's milk.
Strains of Enterococcus faecium isolated from Chungkukjang have proven resistance against gastrointestinal conditions such as the acidic environment and bile salts. These strains also showed a bile salt hydrolase activity, but neither hemolytic activity nor the virulence factor has been detected. For this reason, the strains could be used as input or selected cover crops in food production from fermented soybeans. This bacterium showed inhibitory activity in vitro of Listeria monocytogenes (Yoon et al., 2008) and H. pylori (Lopez-Brea et al., 2008).
Lactococcus lactis subsp. lactis (B01) have antibacterial activity against Gram-negative strains of H. pylori (J99, HSA3068, 26695, HPAG1 and TN2GF4). These results are in the same direction as those found by Joshi et al. (2006) who isolated to radish, a strain of Lactococcus lactis, which has antibacterial activity against many Gram-negative species.
The outcome after ingestion of a strain of Enterococcus faecium used in a probiotic product has been studied by Lund et al. (2002). These authors showed that in 8 out of 10 volunteers who consumed approximately 10 9 cfu daily for 10 days, the bacteria were recovered in rates between 10 3.1 and 10 6.6 cfu/ g. For three volunteers, exogenous strain was dominant among the population of E. faecium. The authors also showed that the presence of the strain was transient and did not persist after cessation of consumption.
This bacterium passes through the digestive system and is resistant to stomach acid and Biliopancreatic secretions by the rapid passage through the stomach. Tsai et al. (2004) found that the strain Enterococcus faecium TM 39 with the ability to tolerate acid and bile salts inhibit the growth of H. pylori in vitro.
Thus, certain strains of Enterococcus faecium are shown as probiotics that induce beneficial effects on consumer health. Market, these microorganisms are available as dietary supplements, such as Enterococcus faecium strain Cernelle 68 (SF 68) in a dehydrated form, marketed by Bioflorin (Sweden).
H. pylori urease activity and has a can produce ammonia by hydrolysis of urea. It can therefore contribute to hyperammonemia (Gubbins et al., 1993) but the introduction of Enterococcus faecium slowed the synthesis of NH3. The effective administration of Enterococcus faecium (SF68) was demonstrated by the work of Loguercio et al. (1995) confirms that this bacterium without urease decreases colonic ammonia synthesis. Tsai et al. (2004) have described that treatment of H. pylori with cells of strain Enterococcus faeciumTM39 significantly reduces it's binding to gastric carcinoma cells (TSGH 9201).

CONCLUSION
Lactic acid bacteria have been known for their inhibitory potency of pathogenic bacteria. Enterococcus faecium (B13) isolated from goat's milk of Miliana had inhibited in vitro the strains of H. pylori: TN2GF4, J99, 26695 and HSA3068.
Enterococcus faecium strain B13 reducing the colonization of H pylori in the stomach with a rate of 43% for a week. Enterococcus faecium B13 passes through the digestive system and is resistant to stomach acid and Biliopancreatic secretions by the rapid passage through the stomach.