In Vitro Efficacy of OM-X Capsules Against Helicobacter Pylori

In Vitro Efficacy of OM-X Capsules Against Helicobacter Pylori 

Report on Preliminary Work on OM-X Capsules 

By John Lambert 1998

Introduction: Helicobacter pylori play an important role in peptic ulcer disease and gastric carcinoma. Infection occurs v~vidwkk (50 percent to 80 percent in some areas). About 35 percent of the adult Australian population are infected and 15 peTccrt havc clinically significant symptoms. Current therapy for the treatment of H. pylori infection is about 80 percent effective, e.xpcnsi?.v and associated with side effects. New treatments that are safe, cost effective, reduce and/or replace the use of allopathic drug(s). a%vid the development of bacterial resistance and are simple to administer are urgently required for the treatment of H. pylori infection.

The Gastrointestinal Research Group at Monash University (Monash Medical Centre and Frankston Mornington Perunsula Hospital) had shown that probiotic micro-organisms (Lactic Acid bacteria or LAB) possess anti-microbial activity against H. pvlori in invitro (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. scauted and 12 strains of Lactic Acid Bacteria. Each capsule contains 18 arnino 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 bactercidal activity against Methicillin-Resistant Staphylococcus aureus. The active component is not a peptide or a protein.

The 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 capsules against H. Plori in vitro.

Materials and Supplies Used in the Research

• OM-X capsules (supplied by OMX Marketing Australia Pty. Ltd.). 
• CM-2B media (as recommened 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 containg the following antibiotics" Cufsulodin 5mgl^-1, Trimethoprim 5mb^-1, Vancomycin 10mg^-1, and Amphotericin B %mgl^-1 (H. pylori selective supplement, Oxoid)(HPA plate). 
• Wilkins-Chalren agar (Oxoid) supplemented with 5% horse blood (WCA). 
• Wilkins-Chalren agar (Oxoid) broth supplement 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 streile 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 Fas Pak) on a shaker at 35 degrees centigrade. THe viability count was made on the respective plater (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 dgrees centigrade under microaerophilic condiion. Total number of CFU.ML of sample was calculated by standard technique.

B. Inhibitory Activity of OM-X Against H. Pylori Bactericidal activity of OM-X 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 (@10^t 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 centrigrade 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 (@10^7 cfu/ml) suspension of the spiral shape and motile H. oykir straub 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 microaerophillically on a shaker at 35 degrees centigrade for 24 hours. The viability count was made by serial dulution in sterile distilled waer in duplicate and plating on HPA plae to determine the number of viable cells. Plates were incubated as described above. Other biochemical test 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 (@10^7 cfu/ml) was added. After incubation estimation of viability, count was done as described above.

WCACB and BHI broth with H. pulori strain alone (@10^7 cfu/ml) was used as a control. All test were repeated three times in duplicate. 

ph and L-Lactic Acid Testing: THe ph was measured using and insertion pH meter and L-Lactic acid was measured on a DuPont Dimension discrete auto-analyzed (Dupont, Wilmington, U.S.A.). 

Results:

A. Total Viable Count from OM-X Capsule 
Several media were used to determine the total viable number of organisms present in and OM-X capsule. 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.

B. Inhibitory Activity of OM-X capsule Against H. pylori: 
Well Diffusion Assay: 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.

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 BPA 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. 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.