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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.