IN VITRO EFFICACY OF OM-X CAPSULES AGAINST HELICOBACTER PYLORI
In the United States the OM-X capsule is known as Probiotics12Plus
Report on Preliminary Work in 1998 on OM-X Capsules by:
Associate Professor John Lambert
Monash University, Department of Medicine, Gastrointestinal Research Group
Mrs. C. Mishra, Research Assistant Monash University, Department of Medicine, Gastrointestinal Research Group
Introduction: Helicobacter pylori play an important role in peptic ulcer disease and gastric carcinoma. Infection occurs worldwide (50 percent to 80 percent in some areas). About 35 percent of the adult Australian population are infected and 15 percent have clinically significant symptoms. Current therapy for the treatment of H. pylori infection is about 80 percent effective, expensive and associated with side effects. New treatments that are safe, cost effective, reduce and/or replace the use of allopathic drug(s), avoid the development of bacterial resistance and are simple to administer are urgently required for the treatment of H. pylori infection.
Our Gastrointestinal Research Group at Monash University (Monash Medical Centre and Frankston Mornington Peninsula Hospital) had shown that probiotic micro-organisms (Lactic Acid bacteria or LAB) possess anti-microbial activity against H. pylori in vitro (Ref. 1,2&3). Our initial in vitro study prompted us to determine the fate of H. pylori in the presence of ingredients of OM-X capsules.
From the literature provided on the OM-X capsule, it should be noted that this capsule is a concentrate of wild fruits, herbs, seaweed and 12 strains of Lactic Acid Bacteria. Each capsule contains 18 amino acids, 9 vitamins, 5 minerals and 59 million live lactic acid bacteria from four groups namely Lactobacillus, Streptococcus, Enterococcus and Bifidobacterium from various fermented foods. Its inventor, Dr. Iichiroh Ohhira from Japan, has shown that Enterococcus faecalis TH10 has bactericidal activity against Methicillin-Resistant Staphylococcus aureus. The active component is not a peptide or a protein.
This preliminary work was to investigate the first objective of our project proposal submitted by OMX Marketing Australia Pty. Ltd. (Dated July 19, 1996).
Objectives: The broad objective is to establish the feasibility of OM-X probiotic capsules use as adjunct therapy for the treatment of H. pylori infection.
The first phase of the Specific objective was to–determine the spectrum of antimicrobial activity of OM-X capsule against H. Pylori in vitro.
Materials and supplies used in the research:
? OM-X capsules (supplied by OMX Marketing Australia Pty. Ltd.).
? CM-2B media (as recommended by OMX company).
? All-purpose medium with Tween (Difco) (APT Agar and APT broth).
? APT Agar and APT broth with 0.3% Biotin (Sigma).
? De Man, Rogosa and Sharpe Agar and broth (Oxoid) (MRS agar and MRS broth).
? Rogosa Agar (Oxoid).
? Selective media for Helicobacter ? Colombia agar plate supplemented with 5% defribinated horse blood containing the following antibiotics: Cefsulodin 5mgl-1, Trimethoprim 5mg-1, Vancomycin 10mgl-1, and Amphotericin B 5mgl-1 (H. pylori selective supplement, Oxoid) (HPA plate).
? Wilkins-Chalren agar (Oxoid) supplemented with 5% horse blood (WCA).
? Wilkins-Chalren agar (Oxoid) broth supplemented 5% horse blood and 0.5% cyclodextrin (WCACB).
? Brain heart infusion broth (Oxoid) (BHI broth).
? Camp Gas Pak (Oxoid).
? Two type strains (NCTC 11637 and 11638) and two clinical isolates of H. pylori.
A. Estimation of Viable Count of OM-X Capsule:
One OM-X capsule was cut with a sterile blade. The content of one capsule was dissolved in 9 ml of the following media: APT broth, APT broth with biotin, MRS broth, MRS broth with biotin and in a Sterile distilled water for 6 to 18 hours and incubated microaerophilically (Camp Gas Pak) on a shaker at 35 degrees centigrade. The viability count was made on the respective plates (for example, culture from MRS broth was spotted on MRS Agar plate) by serial dilution in duplicate. The plates were incubated for two days at 35 degrees centigrade under microaerophilic condition. Total number of CFU/ML of sample was calculated by standard technique.
B. Inhibitory Activity of OM-X Capsule Against H. Pylori:
Bactericidal activity of OM-X product was determined by the loss of viability of H. pylori, the well diffusion assay and Spot on lawn assays.
The Well Diffusion Assay: One OM-X capsule was dissolved and incubated as described above. The assay was performed on a WCA media swabbed over with a four different H. pylori strains (@ 107 cfu/ml). A well was cut using a sterile metal borer and was filled with 100ul-dissolved OM-X capsule product from different broth. APT broth, APT broth with biotin, MRS broth, MRS broth with biotin and 3% lactic acid was used as a control. Plates were incubated at 35 degrees centigrade for four days under microaerophilic conditions. The size of the inhibitory zone was measured with Vernier calipers.
The Spot on Lawn Assay: This assay was performed in a similar way as described above except that the 20ul of dissolved product from the OM-X capsule was spotted on a lawn of H. pylori culture onto SCA media.
Viability assay was performed in two ways:
(a) A one McFarland (@ 107 cfu/ml) suspension of the spiral shape and motile H. pylori strain was made in 9 ml of SCACB and BHI broth. The product of one OM-X capsule was added into the respective broth media and incubated microaerophilically on a shaker at 35 degrees centigrade for 24 hours. The viability count was made by serial dilution in sterile distilled water in duplicate and plating on HPA plate to determine the number of viable cells. Plates were incubated as described above. Other biochemical tests including urease, oxidase, and gram staining were also performed.
(b) The OM-X capsule was dissolved first in a 9 ml of SCACB or BHI broth for 18 hours and then spiral shape and motile H. pylori strain (@ 107 cfu/ml) was added. After incubation estimation of viability, count was done as described above.
WCACB and BHI broth with H. pylori strain alone (@ 107 cfu/ml) was used as a control. All tests were repeated three times in duplicate.
pH and L-Lactic Acid Testing: The pH was measured using an insertion pH meter and L-Lactic acid was measured on a DuPont Dimension discrete auto-analyzer (DuPont, Wilmington, USA).
A. TOTAL VIABLE COUNT FROM OM-X CAPSULE
Several media were used to determine the total viable number of organisms present in an OM-X capsule. The results are shown in Table 1. It was found that an OM-X capsule dissolved for 18 hours in APT with/or without biotin gives good recovery of the organisms from the OM-X capsule. The results are the mean of duplicate tests. The colonies recovered from the OM-X capsule dissolved in water and plated on CM-2B media were very tiny compared to APT or MRS media.
Table 1. Total count from an OM-X capsule dissolved in various media for 6 and 18 hours.
6 HOURS 18 HOURS
Water 4×103 9×105
APT 5X105 18×106
APT + Biotin 9×105 9×106
MRS 3×104 7×106
MRS + Biotin 2×104 5×106
B. Inhibitory Activity of OM-X Capsule Against H. pylori:
Well Diffusion Assay: Table 2 shows the average inhibitory zone of four different strains of H. pylori strains tested. The capsule was dissolved in various media either for 6 hours or for 18 hours. However, there was no difference in the size of the inhibitory zone from test samples dissolved for two different time intervals. APT with/or without biotin, MRS with/or without biotin (without OM-X capsule) didn?t show any inhibition against H. pylori strains.
Spot on lawn assay: Did not show any inhibition of H. pylori.
Table 2. Inhibition of H. pylori strains (two type strains and two clinical isolates) by OM-X product dissolved in various broth media for 18 hours and tested by an agar well diffusion assay.
MEDIA: One H. pylori NCTC H. pylori NCTC Clinical Clinical
OM-X capsule 11637 11638 Isolate A Isolate B
Dissolved in: (Size: mm)
Water 6mm 6mm 5.5mm 6mm
APT 11mm 12mm 10mm 12mm
APT + Biotin 12mm 12mm 11mm 12mm
MRS 12mm 12mm 12mm 12mm
MRS + Biotin 11mm 11mm 10mm 10mm
3% Lactic Acid 10mm 11mm 11mm 11mm
A. When one McFarland of H. pylori strain and product of OM-X capsule was incubated together for one day there was only one log cycle decrease in number of H. pylori cells counted from the HPA plate. From the HPA plate, biochemical tests?rapid urease, oxidase and Gram staining?were performed to confirm the presence of H. pylori. The rapid Urease test directly from the broth media (SCACB and BHI) was also positive and Gram staining showed gram negative spiral bacilli along with gram positive bacilli and cocci from the capsule. The results were similar for all the stains tested both in SCACB and BHI.
B. When an OM-X capsule dissolved 18 hours prior to addition of H. pylori cells and count was made after further incubation, there was 3-4 log cycle decreased in viable cells on HPA plate (Table 3). The rapid Urease test directly from the broth media was negative and gram staining showed very few spiral gram-negative bacilli along with gram-positive bacilli and cocci. The WCACB and BHI broth contained H. pylori strain (without OM-X capsule) alone grew well after incubation.
Table 3. Inhibition of H. pylori strains (NCTC 11637 & 11638) after 24 hours in SCACB broth with pre-dissolved OM-X capsule for 18 hours.
0 Hours 24 Hours
H. pylori NCTC 11637 2×107 cfu/ml 9×109 cfu/ml
H. pylori NCTC 11637 + OM-X 2×107 cfu/ml 3×104 cfu/ml
H. pylori NCTC 11638 5×107 cfu/ml 8×109 cfu/ml
H. pylori NCTC 11638 + OM-X 5×107 cfu/ml 8×103 cfu/ml
Note: Addition of Urea (7mm/1) did not affect the inhibitory activity of the OM-X product. The pH of the OM-X capsule after 18 hours of incubation in APT broth was 5.1 pH compared to 6.5 pH from a 6 hour sample. Individual isolated colonies (15) were picked up from the APT plate. Four different types of organisms were found. It was noticed that the lower the pH and higher the lactic acid concentration, the zone of inhibition of H. pylori was larger. The L-Lactic acid concentration (mmo1/L) of one OM-X capsule dissolved in APT broth for 18 hours was 32.3.
CONCLUSION AND FUTURE WORK:
The results of the well diffusion assay showed inhibition of all H. pylori strains tested. While growing together in a broth media there was only one log cycle decrease in H. pylori count that was not significant. An APT medium is suitable for recovery of LAB from the OM-X capsule.
The mechanism for the inhibition of H. pylori is unclear. These results suggest that various metabolites including lactic and acetic acid, other organic acid, hydrogen peroxide, carbon dioxide, diacetyl, bacteriocins and combination of various components may be involved (Reference 4). It is important to identify and characterize the substance(s) with the highest anti-H pylori activity to achieve the optimum results.
It is also essential to assess the survival of H. pylori in the presence of an OM-X capsule in gastric juice. A study to determine the ability of LAB from an OM-X capsule to adhere to the human intestinal cells in vitro also is essential.