Advances in Infectious Diseases, 2013, 3, 200-204
http://dx.doi.org/10.4236/aid.2013.33029 Published Online September 2013 (http://www.scirp.org/journal/aid)
Identification of Bifidobacterium animalis,
Bifidobacterium adolescentis and Bifidobacterium bifidum
from Stool of Children and Detection of Their
Antibacterial Properties
Mansour Amin1,2*, Ahmad Farajzadeh Sheikh1, Ham e d Goodarzi1,2, Mehdi Sormeh2
1Department of Microbiology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran;
2Infectious and Tropical Diseases Research Center, Ahvaz Jundishapur University of Medical Sciences (AJUMS), Ahvaz, Iran.
Received April 6th, 2013; revised May 6th, 2013; accepted June 6th, 2013
Copyright © 2013 Mansour Amin et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Introduction and Objective: The genus Bifidobacterium can generally be found in quantity in the habitats such as hu-
man and animal gastrointestinal tract, dental caries, vagina and oral cavity. The aim of this study was to isolate Bifido-
bacterium from stool and determine their inhibitory effect against some pathogens. Materials and Methods: 130 sam-
ples were collected by wet swabs and kept in sterile tubes containing MRS broth media. And Bifidobacterium isolated
from stool was enriched in Man-Rogosa-Sharpe medium (MRS) broth and isolated by growing on MRS agar medium
and characterized by phenotypic characteristics and PCR technique at genus and species levels. The antimicrobial sub-
stance was extracted from ethyl acetate solvent and the antimicrobial activity against some pathogenic bacteria, such as
Salmonella typhi and Shigella sonnei, were investigated. Results: Eleven Bifidobacterium bifidum and four Bifidobac-
terium adolescentis, which were isolated from fresh stool, were identified by PCR. Antimicrobial substance from MRS
broth medium was extracted. This antimicrobial compound showed a potent inhibitory activity against four tested bac-
teria. These bacteria produced acetic acid and lactic acid as inhibitory substances that were different from bacteriocins.
Conclusion: Fresh stool may be used as a source of antimicrobial lactic acids bacteria, Bifidobacterium bifidum and
adolescentis as two probiotics can establish themselves in gut and urogenital tract to prevent the human body from ad-
verse effects of pathogens.
Keywords: Bifidobacterium; PCR; Antimicrobial Substance; Stool
1. Introduction
Bifidobacteria were first discovered in infant feces by
Tissier and named it Bacillus bifidus [1]. Bifidobacteria
are a natural resident of the human and animal gastroin-
testinal tract, dental caries and vagina, oral cavity, urine
and blood [2]. Several Bifidobacterium strains are now
being used as probiotics by using established criteria
which belong to the Bifidobacterium animalis, Bifido-
bacterium bifidum, Bifidobacterium breve, Bifidobacte-
rium longum fermentum and some other species of this
genus [2,3]. For human nutrition, probiotics are defined
as live microbial food supplements or components of bac-
teria which have been shown to have beneficial effects on
human health [4]. Some of the health benefits which
have been claimed for probiotics include the following:
Diarrhea Diseases, Radiation induced diarrhea, Inflam-
matory Bowel Disease, Crohn’s disease, Ulcerative coli-
tis, Prevention of colon cancer, Lactose Intolerance, Irri-
table Bowel Syndrome (IBS), Pouchitis, Constipation,
Helicobacter pylori, Pancreatitis, Hepatic encephalopa-
thy, Nonalcoholic fatty liver disease (NASH), Allergy,
Urogenital infections and HIV, Probiotics in Pregnancy,
reduction of serum cholesterol and improvement of the
normal microflora [5]. Certain strain of bacteria has been
discovered over the years to have probiotic properties,
mainly consisting of lactic acid producing bacteria (Lac-
tobacilli, Streptococci, Enterococci, Lactococci, Bifido-
bacteria), Bacillus and fungi such as Saccharomyces and
Aspergillus [6]. The key enzyme of hexose catabolism in
*Corresponding author.

Identification of Bifidobacterium animalis, Bifidobacterium adolescentis and Bifidobacterium
bifidum from Stool of Children and Detection of Their Antibacterial Properties
201
Bifidobacterium is the fructose-6-phosphate phosphoke-
tolase which splits the hexose phosphate to erythrose-
4-phosphosphate and acetyl phosphate. Several species,
including Bifidobacterium adolescentis, are members of
the normal intestinal tract and appendix, dental caries and
vagina flora of healthy humans. Other species, such as
Bifidobacterium animalis subsp. lactis and Bifidobacte-
rium urinalis, are commonly isolated from dairy products
as well as from human urine and they play an important
role as probiotics in human and animal nutrition [2]. Bi-
fidobacterium produces acids, hydrogen peroxide, bacte-
riocins and biosurfactants, and thus confers protection of
the host. Also Bifidobacterium as a natural vaginal flora
plays an important role in the reproductive health of
woman by maintaining acidic vaginal PH, providing
colonization resistance and preventing the growth of
pathogens [7-9]. The acidic PH itself act as a natural de-
fense against sexually transmitted disease and AIDS
[10,11]. The lactic acid Bacteria (LAB) is one of the
most important groups of microorganisms to mankind,
being involved in the products (cheese, yogurt and kefir)
[12] and stool, blood, urine, dental caries [2]. The aim of
this study was to isolate Bifidobacterium animalis, Bifi-
dobacterium adolescentis and Bifidobacterium bifidum
from fresh stool by PCR and to examine their antimicro-
bial activity against some microorganisms such as Sal-
monella typhi, Shigella dysenteriae.
2. Material and Method
In total, 130 samples were collected by wet swabs and
kept in sterile tubes containing MRS broth media. The
sources of samples were fresh stool. The entire sample
tubes where Man-Rogosa-Sharpe medium incubated at
37˚C and 5% CO2 conditions for 1days, then subcultured
on (MRS) agar (Hi-media, India) for 24 h. The colonies
were characterized by phenotypical properties including
morphology, gram positive staining and absence of cata-
lase, oxidase and motility [13]. The DNA of the bacteria
was extracted from single colonies after growing the Bi-
fidobacterium on MRS agar under anaerobes conditions
overnight as described previously [14]. For preliminary
detection of Bifidobacterium, the PCR assay was per-
formed using the primers cited in Table 1.
The composition of PCR mixture was 50 mM KCl, 10
mM HCL (PH: 8.5), 1.5 µl MgCl2, 12 µl dNTPs, 2 µl of
each primer, 0.5 µl of Taq polymerase and 10 µl of DNA
template in final volume of 25 µl. The PCR conditions
were initial denaturation one cycle of 94˚C for 5 min,
followed by 35 cycles of 94˚C for 20 s, annealing of
55˚C for 20 s, extension of 72˚C for 30 s and then a final
extension at 72˚C for 5 min for Bifidobacterium adoles-
centis and Bifidobacterium bifidum and for Bifidobacte-
rium animalis 9 min at 94˚C for initial denaturation and
35 cycles of 30 s at 94˚C for denaturation, 30 s at 62˚C
for annealing, 30 s at 72˚C for extension, followed by 10
min at 72˚C for a final extension using a thermocycler
(Techgene, UK). The PCRproducts were analyzed on 1%
agarose gel. The confirmed species by PCR were tested
for antimicrobial properties. Antimicrobial compound
was isolated using ethyl acetate solvent from Bifidobac-
terium adolescentis and Bifidobacterium bifidum sepa-
rately. After 4 days incubation, the MRS broth media
containing bacteria was mixed with ethyl acetate and
agitated with a magnatic stirrer for 1 day. Then the media
was allowed to settle for 30 min. following settlement,
the solution was separated into two phases, which the
supernatant was comprised of the extracted antimicrobial
compound. The color of ethyl acetate was turned yellow
after agitation. The supernatant then was dried at 45˚C.
The quantity of antimicrobial compound was determined
as 70 mg.
The Minimal Inhibitory Concentration (MIC) of this
antimicrobial substance determined using modified E.
test, by incorporating 20 µl of the each extract in paper
discs. The final concentration of the extract in disc was
estimated as 1.06 mg in total incorporated volume [17].
Table 1. The primers used in this study [15,16].
Species primer size
Pbi F1:5’-CCGGAATAGCTCC-3’
Bifidobacterium spp. Pbi R2:5’-GACCATGCACCACCTGTGAAA-3’ (914 bp)
Ban F2:5’-AACCTGCCCTGTG-3’
B. animalis Pbi R1:5’-GCACCACCTGTGAACCG-3’ (925 bp)
BIA-1:5’- GGAAAGATTCTATCGGTATGG-3’
B. adolescentis BIA-2:5’-CTCCCAGTCAAAAGCGGTT-3’ (244 bp)
BiBIF-1:5’-CCACATGATCGCATGTGATTG-3’
B. bifidum BiBIF-2:5’-CCGAAGGCTTGCTCCCAAA-3’ (278 bp)
The standard strains of B. animalis, B. adolescentis and B. bifidum as positive controls were included in each set of PCR amplification; DNA template in a final
volme of 25 µl. All the reagents were purchased from Cinnagen Company, Tehran, Iran. u

Identification of Bifidobacterium animalis, Bifidobacterium adolescentis and Bifidobacterium
bifidum from Stool of Children and Detection of Their Antibacterial Properties
202
The target bacteria were some clinically isolated patho-
gens including Shigella dysenteiae. The standard strains
which were included in the study Salmonella typhi
(PTCC 1609), staphylococcus aureus (PTCC 1112), and
Bacillus cereus (PTCC 1247) (Persian type culture col-
lection (PTCC)) obtained from collection center of fungi
and bacteria, Tehran, Iran. The experiments were re-
peated 3 times and the results were constant in all tests.
The E. test and ANOVA variance were used for data
analysis by application of SPSS software (SPSS Inc.
No.15, Chicago, IL, USA).
3. Results
All of the preliminary isolated Bifidobacterium were
subjected to PCR for confirmation of specific primers
and species—specific primers. Using species-specific, 11
of them were confirmed Bifidobacterium bifidum (Fig-
ure 1) and 6 were Bifidobacterium adolescentis (Figure
2). The confirmed strains by PCR were tested for their
antimicrobial properties.
The activity of antimicrobial substances was tested
against target pathogens after adjustment of pH at 7 using
5M NaOH. The antimicrobial compounds showed potent
inhibitory activity against two tested bacteria Salmonella
typhi and shigella dysenteiae. The MICs of two selected
antimicrobial compounds obtained from Bifidobacterium
bifidum were determined using modified E. test [17]
(Figures 3 and 4).
The important point of this study was isolation the Bi-
fidobacterium species from fresh stool. In the other hand
we know that these bacteria are probiotic and can protect
our body against the pathogenic bacteria. The obtained
results revealed that the mentioned stools have many
Bifidobacterium and Lactobacillus and these bacteria
produce antibiotic in addition the organic acids and hy-
drogen peroxide. The antimicrobial compounds isolated
from Bifidobacterium adolescentis and Bifidobacterium
bifidum showed activity against some pathogenic bacte-
ria such as Salmonella typhi and Shigella dysenteriae.
The obtained MICs were between 25 - 400 µl ML (Table
2). The considerable point in this study was extraction of
a compound with antimicrobial activity which was only
dissolved in ethyl acetate but was non-dissolvable in wa-
ter solvents.
4. Discussion
Bifidobacterium can inhibit the growth and attachment of
pathogens to epithelial cells. These organisms are pro-
duced compounds as hydrogen peroxide and bacterio-
cin-like that can kill the pathogenic microorganisms in
human body [9,18,19]. In a study that was performed by
Shuhaimi et al. [20] Bifidobacterium infantis G4 isolated
Figure 1. Agarose gel of PCR products amplified (278 bp)
by species-specific primers of B. bifidum, 1: marker 100 bp,
2: Control negative, 3: control positive, 4 - 12: positive iso-
lates.
Figure 2. Agarose gel of PCR products (244 bp) amplified
by species-specific primers of B. adolescentis, 1: marker 100
bp, 2: Control negative, 3: control positive, 4 - 9: positive
isolates.
from infant stool, and was tested for their antibacterial
activity, antimicrobial susceptibility and adherence prop-
erties to human colon carcinoma HT29 cell lines. The
isolate was observed to be effective in inhibiting the
growth of pathogens namely Salmonella enterica ssp.
enterica serovar Enteritis, vibrio cholera, Escherichia
coli, Bacillus cereus, Pseudomonas aeruginosa and Lis-
teria monocytogenes. S. Kozhakhmetov [21] reported
detection of Bifidobacterium spp. (Bifidobacterium ado-
lescentis 180, Bifidobacterium breve 204, Bifidobacte-
rium breve 584, B. breve 587) to be effective in inhibit-
ing the growth of pathogens namely E. coli, P. mirabilis,
P. mirabilis, S. aureus, Salmonella enterica subs. en-
terica serovar Typhimurium, Bacillus spp. Α.
In a study, Bifidobacterium longum (NCFB 2259) was

Identification of Bifidobacterium animalis, Bifidobacterium adolescentis and Bifidobacterium
bifidum from Stool of Children and Detection of Their Antibacterial Properties
203
Figure 3. E test representing MIC of antimicrobial com-
pound obtained from B. bifidum against Shigella dysen-
teriea.
Figure 4. E test representing MIC of antimicrobial com-
pound obtained from B. bifidum against Salmonella typhi.
effective in inhibiting the growth of pathogens E. coli
O157:H7, one of the leading causes of bacterial food
borne diseases [22]. It is expected that the antimicrobial
compound of B. adolescentis was not much different
from active compounds Bifidobacterium bifidum. The
MIC values for gram negative and gram positive indi-
cated that gram positive and negative bacillus were more
sensitive to bifidoba cterium antimicrobial compound
than gram positive cocci.
5. Conclusion
The objectives of this study showed that about 13% of
Table 2. MIC of antimicrobial compound obtained from B.
bifidum and B. adolescentis against pathogenic bacteria
using E. test.
Bacteria MIC (µg·mL−1)
B. bifidum B. adolescentis
Salmonella typhi 200 200
Shiglla dysentriae 200 200
Bacillus cereus 120 140
Staphylococcus aureus180 220
healthy peoples in Ahvaz city—Iran can be supported
from rectal pathogens by Bifidobacterium probiotics but
others are at risk of being attacked by harmful microbes.
The food containing probiotics may be colonized by the
Bifidobacterium and Lactobacillus species in the rectum
through oral—fecal track.
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