World Journal of Pharmaceutical Research

Israa et al. World Journal of Pharmaceutical Research

SJIF Impact Factor 5.990

Volume 4, Issue 6, 64-73. Research Article ISSN 2277 7105

EFFICIENCY OF SACCHAROMYCES BOULARDII ON BLASTOCYSTIS

HOMINIS IN LABORATORY MICE

Israa Mohammad Abd AL-Khaliq* and Dr. Sabaa Taher Mohammed.

1
Department of Microbiology, Al-Kindy College of Medicine, Baghdad University, Baghdad-
Iraq.
2
Department of Biology, College of Science, AL-Mustansyria University, Baghdad-Iraq.

ABSTRACT
Article Received on
The effectiveness of Saccharomyces boulardii yeast as vital probiotics
18 March 2015,
on this parasite was studied in the laboratory mice and compared with
Revised on 09 April 2015,
Accepted on 03 May 2015 metronidazole which used usually in the treatment of intestinal
parasites ,included Blastocystis hominis parasite, it was noticed a
gradually decreased in the numbers of parasite in stool of mice since
*Correspondence for
Author the first day of treatment parasite disappeared in the day (12 th ) from
Israa Mohammad Abd treatment with Saccharomyces yeast, and disappeared in day (13th )
AL-Khaliq from treatment with metronidazole, and when efficacy of treatment
Department of
counted for both of S. boulardii and metronidazole ,founded that this
Microbiology, Al-Kindy
yeast have a higher efficacy for treatment (87.45%) , while
College of Medicine,
Baghdad University, mitronidazole efficacy was (81.36%). The microscopic examination to
Baghdad-Iraq. histological sections of the infected mice in cecum and colon regions
showed the ability of the parasite to make histological changes
included intense inflammatory-cell infiltration, and mucosal damage. The yeast of
Saccharomyces boulardii showed higher ability in the healing and recovering damaged
tissues compared with metronidazole which showed less efficacy in healing and recovering
damaged tissue.

KEYWORDS: Blastocystis hominis ,Saccharomyces boulardii , probiotic, mitronidazole.

1- INTRODUCTION
Blastocystosis refers to a medical condition caused by infection with a protozoal parasite
called Blastocystis, which is an intestinal protozoan that inhabits the gastrointestinal tracts,
cecum and colon of humans and other animals .This genus was created by Alexieff in

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Israa et al. World Journal of Pharmaceutical Research

1911[1], it has a world wide distribution and often the most commonly reported human
intestinal protozoan in children and adults.[2] At least (20%) of patients in which the
organisms found are asymptomatic, There are many individuals that carry the parasite but are
asymptomatic. when symptomatic the usual spectrum of symptoms includes watery diarrhea
,abdominal pain ,perianal pruritis, excessive flatulence. Other symptoms include anal itching,
loss of weight, excess gas and constipation.[3] At present, the first choice of chemotherapeutic
agent is metronidazole treatment should be considered in all cases . Metronidazole is the first
line of treatment, although resistant organisms are now present in Australia and these may be
difficult to clear with treatment [4], as well as using probiotics which are defined as live
organisms, that confer benefits to the host. Their efficiency was demonstrated for the
treatment of gastrointestinal disorders, respiratory infections, and allergic symptoms.[5] There
are different routes by which probiotics may control a pathogen includes: probiotics can
modulate their physicochemical environment (nutrients, mucus, receptors availability on
epithelial cells, pH, tight junctions, and peristaltism),also it can produce biologically active
molecules such as bacteriocins, antibiotics, or oxygen peroxide that possess antimicrobial
properties and probiotics can induce immune modulation.[6] Saccharomyces boulardii is a
strain of yeast [7], it has been classified as a probiotic agent since the 1950s [8], because of its
ability to maintain and restore the natural flora in the large and small intestine and surviving
at low pH levels.[9]

This yeast has been prescribed in the past 30 years for prophylaxis and treatment of diarrheal
diseases caused by bacteria.[10] There are several clinical trials and experimental studies
strongly suggest a place for Saccharomyces boulardii as a biotherapeutic agent for the
prevention and treatment of several gastrointestinal diseases , it is also resistant to antibiotics
and proteolysis.[11]

2- MATERIALS AND METHODS

2-1 Patients
The present study included (5892) stool sample, were collected from patients of both sexes at
different ages, which were carried out during the period from September (2013) till
September (2014). All patients were obtained from those who had been admitted to / or
attended the laboratories of parasitology of the following health institutions (Central City
Teaching Hospital, AL-Imam Ali Hospital, AL-Karaama Hospital and AL-Yarmouk
Teaching Hospital ).

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2-2 Collection of stool sample

Stool specimens were collected in sterile, clean, and dry plastic containers with tight lids
specially made for this purpose. Each container was given number representing the patient.
Each stool sample after making general stool examination and detection Blastocystis hominis
in the slide, this positive sample used for culturing this parasite.

2-3 Culture
A typical sample which contain[4] parasites of B.hominis or more in the field of the slide was
taken , and cultured in modified Jonesmedium. Approximately (50 mg) of each fecal
specimen was inoculated into a screw - cap autoclaved tube containing modified Jones
medium . The tubes were incubated at (37C) for (24, 48 hr) , then after (24 and 48 hr) One
drop of sample was examined on direct microscopic examination of the culture and
calculated the numbers of cysts (C) or Trophozoite (T). Determination can be done by the
numbers of C or T in one ml of sample by using the equation of Al-Idrrise et al. 2008.[12]

2-4 Determination of the infective dose

Parasitic dose for infection was prepared according to Robert -Thompson et al.1976.[13]
parasites were counted and the number were adjusted at ( 1x103) cell /0.1 ml and then
inoculated orally to cause infection into mice.

2-5Preparation of yeast Saccharomyces boulardii cells

Lyophilized Saccharomyces boulardii (bioflor - laboratoss- biocodex- France) were obtained
from the scientific offices, then reactivated by cultured on sabouraud dextrose broth at pH [6],
and incubation at mixing incubator for ( 48 hr ) at (30 C).

At the end of incubation ,the yeast were recovered by centrifugation for(10min). at (1000
rpm). The deposit was diluted in (0.05) NaCl and counted under the microscope to obtained (
1107 cells/0.1ml).[14]

2-6 Animals
(24) albino mice males with ages range between (8-10 weeks), were obtained from National
Control Center For Drugs and Researches (NCCDR) . Mice were put in plastic clean cages ,
and stool of them was examined before the beginning of the experiment to make sure of
clearance mice from any intestinal parasites.

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2-6 The experimental design

Immunosuppressed of[24] mice by dexamethazone according to Zhongguo et al. 2006.[15]
After (5 days), only[18] mice were inoculated orally by micropipette with (0.1 ml) of prepared
inoculums of Blastocystis hominis.

In the next day all mice feces was examined to confirm the presence of the parasite in the
stool and occurance of the infection ,then the infected mice were divided into[3] groups
with[6] mice,. the remaining six mice which has been not infected , kept as negative control
group.

Then each group was inoculated as follow

- Group one: mice given orally (0.1 ml) contain ( 1x103 cell) from Saccharomyces.
- Group two: mice given (0.1ml) of metronidazole (30 mg / kg / day) orally as asingle dose
per day.
- Group three: mice were not infective , given orally (0.1 ml ) of normal saline and
concidared as a positive group.
-Group four: mice were given orally (0.1ml) of (pbs) .This group concidared as a
negative control.

2-7 Enumeration of Blastocystis hominis

Parasite in feces were enumerated as Shulka et al. 2008.[16] Briefly, mice feces were
collected of the first three groups daily from each mouse.
One gram of fecal sample was dissolved in (10 ml) of normal saline, homogenized then
counted every day.

2-9 Sufficient treatment calculation

Sufficient treatment for Saccharomyces boulardii and metronidazole were measured
according to the method of Xioa et al.1996.[17]

2-10 Histological study

Mice were sacrificed (2weeks) post infection. Histological sections prepared from their
intestine and were examined with light microscope.

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3- RESULTS AND DISCUSSION

The present study evaluated the effect of Saccharomyces boulardii as probiotic
supplementation on Blastocystis hominis in albino mice and compare it with metronidazole.
In this study it was found that incubation period of this parasite appeared within (24-48 hr).

It was noticed that orally inoculation of this yeast in infected mice led to reduce the shedding
2
of this parasite in stool of mice since the first day of treatment (2.50 x10 cell /gm ), and
continue to decrease gradually with days till stopped shedding of parasite and became (zero)
in day (12th) post inoculation, compare with mitronidazole showed a slight decrease occurred
after first five days post inoculation ( 2.83 2.67 , 2.00 , 2.00 , 2.00 x10 2cell /gm) respectively
, then shedding of parasite start decrease gradually till reach to zero at day (13 th ) ,when the
parasite completely disappear in the mice stool ,as seen in table (1). While the infected mice
in positive control group maintained on shedding the parasite in stool with continuously
increased in number of parasite till day (13th ) post inoculation was reached to (11.50
x102cell /gm). There was no significant differences between the mean of the two treatment
groups.group treated with.

Table (1): Mean number of Blastocystis hominis in treatment and control groups SD x
102
Means SD LSD
Days
S.boulardii Mitronidazole Control value
1 2.50 0.54 2.83 0.75 3.33 0.51 0.756
2 2.17 0.41 2.67 0.81 4.33 0.51 0.745
3 1.50 0.54 2.00 0.63 5.00 0.63 0.745
4 1.33 0.51 2.00 0.63 5.33 0.51 0.686
5 1.00 0.00 2.00 0.63 6.00 0.63 0.778
6 0.667 0.21 1.50 0.54 6.50 0.54 0.661
7 0.667 0.21 1.33 0.05 6.50 0.83 0.789
8 0.667 0.21 0.833 0.41 7.67 0.81 0.745
9 0.667 0.21 0.667 0.21 7.83 0.75 0.745
10 0.333 0.05 0.667 0.21 8.67 0.82 0.778
11 0.166 0.04 0.500 0.05 10.16 1.16 0.962
12 0.00 0.00 0.333 0.05 10.16 1.16 0.908
13 0.00 0.00 0.00 0.00 11.50 1.04 0.745
when applied the equation of treatment efficacy, found that Saccharomyces boulardii
efficacy was (87.45%) while mitronidazole efficacy was (81.36%) .Table (2).

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Table (2): Efficacy treatment for treatment groups.

Type of treatment dose / ml Efficiency of treatment(%)
Saccharomyces boulardii 0.1 87.45
Mitronidazole 0.1 81.36

To study the pathogensis of Blastocystis hominis parasite, the positive control group regarded
as the best example to study the pathogensis of parasite in the mice, mice were sacrificed
after (2weeks) post infection.

Slides of histological examination of the cecum and colon of positive control group showed
intense inflammatory-cell infiltration, mucosal damage, inflammation or increased numbers
of infiltration of lymphocytes in the mucosa compared with negative control group, as shown
in figure (1), figure (2) and figure (3).

Figure (1): Section of large intestine (colon) in mice of negative control group showing
normal structure appearance of colonic mucosa with normal mucus secretion.(H&E),
100 x.

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Figure (2): Section of large intestine (colon) in mice of positive control group, showing
infiltration of lymphocytes and damage in mucosa tissue .(H&E), 200 x.

Figure (3): Section of large intestine (colon) in mice of positive control group, showing
damage in mucosa tissue and hydropic degeneration.(H&E), 400 x.

The results of histopathological study confirm the results of experimental study, the results
showed that , Saccharomyces boulardii led to heal large intestinal tissues which damaged
by Blastocystis , figure (4) . While mitronidazole caused damage in the tissues of large
intestine which noticed through the infiltration of lymphocytes occurred, figure (5).

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Figure (4): Section of large intestine (colon) in mice treated with Saccharomyces
boulardii, showing near normal appearance of colonic mucosa.(H&E),10x.

Figure (5): Section of large intestine (colon) in mice treated with mitronidazole ,showing
infiltration of lymphocytes and short length of villi . (H&E) , 20x.

S. boulardii has several different types of mechanisms of action, which may be classified
into three main areas: luminal action, trophic action[18], and the third action in regulation of
immune response.[19]

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The luminal action of this yeast include antimicrobial activity, The another luminal action
achieved by antitoxin effects .The second type of mechanism of action is the trophic action
on the intestinal mucosa ,and the third type of action of S. boulardii , is in regulation of
immune response.

There is increasing evidence that the gastrointestinal microflora is a major regulator of the
immune system, not only in the gut, but also in other organs. The efficacy and safety yeast
Saccharomyces boulardii has been prescribed in the past (30 years) for prophylaxis and
treatment of diarrheal diseases caused by bacteria . Importantly, S. boulardii has
demonstrated clinical and experimental effectiveness in gastrointestinal diseases with a
predominant inflammatory component, indicating that this probiotic might interfere with
cellular signaling pathways common in many inflammatory conditions.[10]

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