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IN VITRO EFFICACY OF OMX CAPSULES AGAINST HELICOBACTER PYLORI REPORT ON PRELIMINARY WORK ON OMX CAPSULES BY INVESTIGATORS: ASSOCIATE PROFESSOR JOHN LAMBERT MONASH UNIVRESITY DEPARTMENT OF MEDICINE GASTROINTESTINAL RESEARCH GROUP CLAYTON VIC. 3168 TELEPHONE: (03) 9748 7300 FAX: (03) 9748 7272 MRS. C.MISHRA RESEARCH ASSISTANT MONASH UNIVERSITY DEPARTMENT OF MEDICINE GASTROINTESTINAL RESEARCH GROUP LAYTON VIC. 3168 TELEPHONE: (03) 9550 5536 FAX: (03) 9550 5524 Introduction: Helicobacter pylori play an important role in peptic ulcer disease and gastric carcinoma. Infection occur worldwide (50-80% in some area). About 35% of the adult Australian population is infected and 15% having clinically significant| symptoms. Current therapy for the treatment of H. pylori infection is about 80% effective. Extensive and associated with side effects. Newer treatments which are safe, cost effective, reduce and/ or to replace the use of allopathic drug(s), avoid the development of bacterial resistance and simple to Administer are thus urgently required for the treatment of H. pylori infection. Our Gastrointestinal research Group at Monash University (Monash medical Centre and frankston Mornington peninsla a hospital) had shown that probiotic micro-organisms (tactic Acid Bacteria or LAB) posses antimicrobial 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 OMX capsules. This preliminary work was to investigate first objective of our project proposal sub- mitted to OMX Marketing Australia Pty Ltd (Dated 19/7/96). OBJECTIVES: The broad objective is to establish the feasibility of OMX probiotic capsules use as adjunct therapy for treatment of H. pylori infection. The first phase of the Specific objective was to : 1. Determine the spectrum of antimicrobial activity of OMX capsule against H.pylori in vitro. MATERIALS AND METHODS: (OBJECTIVE 1) OMX Capsules (supplied by OMX Marketing Australia Pty. Ltd.) CM-2B media (as recommended by OMX Co.) 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) MRS Agar and MRS broth with 0.3% Biotin (Sigma) Rogosa Agar (Oxoid) Selective media for Helicobacter - Colombia agar plate supplemented with 5% defribinated horse blood containing following antibiotics : Cefsulodin 5 mg⁻1, Trimethoprim 5 mg⁻1, Vancomycin 10mg⁻1, and Amphotericin B 5mg⁻1 (H.pylori Selective supplement, Oxoid) - (HPA Plate) Wilkins-Chalgren agar (Oxoid) suppplement with 5% horse blood (WCA) Wilkins-Chalgren agar (Oxoid) broth supplemented 5% horse blood and 0.5%cyclodextrin (WCACB) Brian heart infusion broth (Oxoid) (BHI broth) Camp Gas Pak (Oxoid) Two type strains (NCTC 1 1637 and 1 1638) and two clinical isolates of H. pylori A. ESTIMATION OF VAIBLE COUNT OF OMX CAPSULE: One OMX capsule was cur with strile blade. The content of one capsule was dissolved in 9ml of the following media: APT broth, APT brot with biotin, MRS broth, MRS broth with biotin and in a Strile distilled water for 6 and 18 hrs and incubated microaerophilically (Camp Gas Pak) on a shaker at 35°C The viability count was made on the respective plates (for example, culture from Mrs broth was spotted on Agar plate) by serial dilution in duplicate. The plates were incubated for 2 days at 35°C under microaerophilic condition. Total number of CFU/ML of sample was calculated by standard technique. B. INHIBITORY ACTIVITY OF OMX CAPSULE AGAINST H.PYLORI : Bactericidal activity of OMX 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 OMX 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 100µl dissolved OMX capsule product from different broth. APT broth, APT broth with biotin, MRS broth, MRS broth with biotin and 3% lactic add used as a control. Plates were incubated at 35oC for 4 days under microaerophilic conditions. The size of inhibitory zone was measured with Vernier callipers. The Spot on lawn assay: This assay was performed in a similar way as described above except that the 20µl of dissolved product from the capsule was spotted on a lawn of H. Pylori culture onto WCA 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 WCACB and Bill broth. The product of one OMX capsule was added into respective broth mediated incubated microaerophilically on shaker at 35 °C for 24 hrs. The viability count was made by serial dilution in sterile distilled water in duplicate and plating on. HPA plate to determine the number of vaible cells. Plates were incubated as described above. Other biochemical tests including creases oxidase and gram staining were also performed. (B) The OMX capsule was dissolved fast in a 9ml of WCACB or BHI broth for 18 hrs and then spiral shape and motile H. pylori strain (@ 10⁷ cfu/ml) was added. After incubation estimation of Viability County 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 3 times in duplicate. PH and L-Lactic acid testing: PH was measured using an insertion pH meter and L Lactic acid was measured on a DuPont Dimension discrete autoanalyser (DuPont. Wilmington, USA). RESULTS: A. TOTAL VIABLE COUNTS FROM OMX CAPSULE: Several media were used to determine the total viable number of organisms present in OMX capsule. The results are shown in Table 1. It was found that OMX capsule dissolved for 18 hrs in APT with/or without biotin gives good recovery of the organisms from the capsule. The results are the mean of duplicate test. The colonies recovered from the capsule dissolved in water and plated on CM-2B media were very tiny compared to APT or MRS media. Table 1: Total count from OMX capsule dissolved in various media for 6 hrs and 18hrs. MEDIA Water APT APT+ Biotin MRS MRS + Biotin CFU/ML 6hrs 4 X 10₃ 5 X 10⁵ 9 X 10⁵ 3 X 10⁴ 2 X 10⁴ 18hrs 9 X 10⁵ 18 X 10⁶ 9 X 10⁶ 7 X 10⁶ 5 X 10⁶ B. INHIBITORY ACTIVITY OF OMX CAPSULE AGAINST H. PYLORI Well duffusion 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 hrs or for 18 hrs. How- every, there was no difference in the size of inhibitory zone from test sample dissolved from two different time intervals. APT with/or without biotin, mRS with/or without biotin (without OMX 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 Helicobacter pylori strains (2 type’s strains and 2 clinical isolates) by OMX product dissolved in various broth media for 18 hrs and tested by an agar well diffusion assay: MEDIA: One OMX capsule dissolved in : Water APT ATY + Biotin MRS MRS + Biotin 3% lactic acid H. pylori NCTC 11637 (Size -mm) 6mm 11mm 12mm 12mm 11mm 10mm H. pylori NCTC 11638 Clinical Isolate A Clinical isolate B 6mm 12mm 12mm 12mm 11mm 11mm 5.5mm 10mm 11mm 12mm 10mm 11mm 6mm 12mm 12mm 12mm 10mm 11mm Viability assay: A. When one McFarland of H. pylori strain and product of OMX capsule was incubated together for one day there was only one log cycle decrease in number of H. pylori cells counted from HPA plate. From the HPA plate, biochemical tests - rapid urease, oxidase and Gram staining was performed confirm the presence of H. pylori. The Rapid urease test directly from the broth media (WCACB 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 strains tested both in WCACB and BHI. B. When OMX capsule dissolved 18 hrs 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 and fig. 1). The rapid unease 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 contains H pylori strain (without OMX capsule) alone grew alter the incubation Table 3: Inhibition of H. pylori strains (NCTC 11637 & 11 639) after 24 hrs in WCACB broth with pre-dissolved OMX capsule for 18 hrs. Sample H. pylori NCTC 11637 H. pylori NCTC 11637 + OMX capsule H. pylori NCTC 11638 H. pylori NCTC l 1638 + OMX capsule 0 hrs 2 x 10⁷ cfu/ml 2 x 10⁷ cfu/ml Time 24 hrs 9 x 10⁹ cfu/ml 3 x 10⁴ cfu/ml 5 x 10⁷ cfu/ml 5 x 10⁷ cfu/ml 8 x 10⁹ cfu/ml 8 x 10₃ cfu/ml Addition of Urea (7 mM/1) did not affect the inhibitory activity of the OMX product. The pH of OMX capsule after 18 hrs of incubation in APT broth was 5.1 compared to 6.5 pH from a 6 hrs 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 larger zone of inhibition H. pylori. The L-Lactic acid concentration (mmo1/L) of one OMX capsule dissolved in APT broth for 18 hrs 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 which was not significant. However, when OMX capsule dissolved first for 18 hrs and then incubated with H. pylori, there was significant reduction in growth An APT medium is suitable for recovery of LAB from the capsule. The mechanism for the inhibition H. pylori is unclear. The results suggest the various metabolites including lactic and acetic acid, other organic acid, hydrogen peroxide carbon dioxide diacetyl, bacteriocins and combination of various components may be involved (Ref 4). It is important to identify and charaterise the substance(s) with 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 OMX capsule in gastric juice. A study to determine the ability of LAB from OMX capsule to adhere human intestinal cells in vitro is also essential. Phase l of the project proposal is finished. The next phase of the work is to carry out a small pilot clinical trial to determine the efficacy of the OMX capsule in human subjects. Fig1. Inhibition of H. pylori strains (NCTC 11637 AND 11638) after 24 hrs in WCACB broth with pre-dissolved OMX capsule of 18 hrs. 24 TIME (HRS) 11637 11637+OMX 11638 11638+OMX REFERENCES: 1. In vitro inhibition of Helicobacter pylori NCTC 11637 by organic acids and lactic acid bacteria P D Midolo, J R Lambert, R Hull, F Luo and ML Grayson. J of Appl. Bact. 1995, 79:475-479. 2. Anti-Helicobacter pylori effect of yoghurt. C Mishra, J R Lambert and P Midolo. J Gastroentrol hepatol 1996:11: suppl l A 34. 3. In vitro and in vivo efficacy of yoghurt on Helicobacter pylori. C Mishra, L R Lambert and P Midolo, G Cardaci, S Lin and L Nicholson. Microbiology Australia 1996:17:4 Pol 31. 4. Production of anti-microbial substances by probiotics. C Mishra and J Lambert. Asia Pacific J Clin Nutr 1996:5:20-24.