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ISSN: 2329-8901
Journal of Probiotics & Health
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Determination and Comparasion of Antibacterial Activity of Native Bifidobacterium Strains with Similar RAPD Profile Isolated from Paired Infants Stool and Mothers Milk

Malihe Talebi1*, Morteza Eshaghi1, Meysam Hasannejad Bibalan1, Mahdi Rohani2 and Mohammad Reza Pourshafie2

1Department of Microbiology, Iran University of Medical Science, Tehran, Iran

2Pasteur Institute of Iran, Tehran, Iran

*Corresponding Author:
Malihe Talebi
Department of Microbiology
Iran University of Medical Science
Tehran, Iran
Tel: 982188884567
E-mail: [email protected]

Received Date: May 24, 2017; Accepted Date: May 29, 2017; Published Date: June 05, 2017

Citation: Talebi M, Eshaghi M, Bibalan MH, Rohani M, Pourshafie MR (2017) Determination and Comparasion of Antibacterial Activity of Native Bifidobacterium Strains with Similar RAPD Profile Isolated from Paired Infants Stool and Mothers Milk. J Prob Health 5:175. doi: 10.4172/2329-8901.1000175

Copyright: © 2017 Talebi M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Background: Nowadays using of Bifidobacteria as a probiotic candidate for microbiome engineering and developing of human gut immunity have been considered. Given that many previous studies have emphasized probiotic properties are strain specific and exposing to stress shock such as low pH can be effective on probiotic criteria, so this study was performed for assessment and comparasion of antagonist activity of native Bifidobacterium with similar genotype homolgy isolated from paired mothers milk and infants feces.

Methods: In this study, 56 mother’s milk and paired-infant feces were taken from rural area. Single colonies were picked up and confirmed by phenotypic and molecular identification. Randomly amplified polymorphic DNA (RAPD- PCR) and antibacterial activity carried out on selected strains.

Results: Amongst all 56 samples, 31 samples, including 20 stools and 11 milks were positive for Bifidobacterium including B. breve, B. longum, B. bifidum. 12 of all strains were shared between stool and milk, including 6 Bifidobacterium longum, 4 B. breve and 2 B. bifidum. Between these 12 strains, only three strains of Bifidobacterium longum presented 100% similarity in RAPD- PCR analysis and antibacterial activity were strains specific and no similar activity were observed.

Conclusion: In short, amongst all 56 paired infants stools and mothers milks, 12 different shared Bifidobacteria were isolated and identified that showed strain specific antagonist activity and some of these isolates possibly produced bacteriocin like proteins.

Keywords

Bifidobacterium; RAPD-PCR; Antibaterial activity

Introduction

Bifidobacteria are gram-positive anaerobic microorganism isolating for the first time in 1899 by Tissier from the stools of breast-fed infants [1]. Some of the Bifidobacterium species colonize in different human body sites such as human milk, gastrointestinal and genitourinary tracts [2]. Nowadays, some of the Bifidobacterium genera are used in commercial probiotic products. Probiotics are “live microorganisms when ingested in adequate amounts, confers beneficial effects to the host by improving its intestinal microbial balance” [3]. It is welldocumented that probiotics promote many aspects of human health such as innate immunity and healing of gastrointestinal disorders by different approaches, namely, competition with bacterial pathogens, production of antimicrobial compounds including organic acids [lactic, acetic, propionic acids] and bacteriocins [4,5].

The nature of gastrointestinal tract and human breast are absolutely different; for example, acidity and the presence of bile salts are the important characteristics distinguishing gut from human breast. On the other hand, previous studies have been shown probiotic traits such as antagonist activity are strains specific and isolates from different niches present variable properties and exposing to stress shock such as low pH can be effective on probiotic criteria [6-9]. So, this study was done in order to comparison of the antibacterial activity of Bifidobacterium genus isolated from mothers milk and paired infants stools presenting similar and non-similar gentype homology and RAPD profile.

Materials and Methods

Sampling

Twenty eight mother’s milk and paired-infant feces were taken from rural area from the center of Iran. Participants with following enrolled criteria selected: (a) Healthful infants and women without present or past underlying disease; (b) Normal and full-term gestation (c) Vaginally-delivered infants; and (d) lack of any usage of antibiotics and commercial probiotic products for at least 3 months before the sampling. Writing testimonial and ethic approval (IR.IUMS.REC 1395.9221133201) was obtained from the parents of the infants and Research Ethics Committee of Iran University of Medical Sciences (IUMS), respectively. The infants aged from 8 days to 22 months. The mother’s milks and infants stools were collected in a prereduced medium [10] and putted in anaerobic jar and carried to the laboratory immediately.

Cultivation

The samples were transferred into selective de Man, Rogosa and Sharpe (MRS) broth medium supplemented with 0.05% (w/v) L-cysteine hydrochloride (Merck, Darmestat, Germany) and 100 mg/ liter mupirocin (Sigma-Alderich, USA) and incubated anaerobically at 37°C for 72 hours. Afterward, a tenfold serial dilutions in PBS containing 0.05% L-cysteine were prepared and plated on selective MRSc agar containing 100 mg/l mupirocin and incubated anaerobically at 37°C for 3 days. Based on the shape of the colonies, gram staining and catalase activity, single colonies were picked up.

Phenotypic and molecular identification

Phenotypic confirmation of suspicious colonies at the genus level carried out by F6PPK activity test as described previously [11]. Molecular identification was conducted based on study of Matsuki et al. [12]. Briefly; bacterial DNA was extracted by column miniprep extraction (Roche, Germany). The species and genus identification were carried out by polymerase chain reaction (PCR) as described before by specific primers (Table 1).

Isolates Primers (5'-sequence-3') Amplicon size(bp) Annealing Temp References
Biffidobacterium Species g-Bifid-F: CTC CTG-GAA-ACG-GGT-GG
g-Bifid-R: GGT-GTT-CTT-CCC-GAT-ATC-TAC-A
549-563 58 11
B.bifidum Bbif F 1: CCA CAT GAT CGC ATG TGA TTG
Bbif R 2: CCG AAG GCT TCC CAA A
278 65 11
B.breve Bbre F: CCG GAT GCT CCA TCA CAC
Bbre R: ACA AAG TGC CTT GCT CCC
288 60 11
B.longum Bilon F: TTC CAG TTG ATC GCA TGG TC
Bilon R: GGG AAG CCG TAT CTC TAC GA
831 63 11

Table 1: Species and genus specific oligonucleotides used for identification of Bifidobacterium strains.

Randomly Amplified Polymorphic DNA (RAPD-PCR)

RAPD reactions were performed according to Vincent et al. [13] by using the OPL-16 primer (5’ AGGTTGCAGG 3’). Briefly, RAPD amplification reactions consisted of 25 μL including 12.5 μL of ready to use 2X Master Mix (Amplicon, Denmark), 1 μM of primer (100 μM) and 1 μL of DNA (50 ng/μL). Amplification was performed with an initial denaturation step at 95°C for 3 min, followed by 45 cycles of denaturation at 94°C for 1 min, annealing at 35°C for 1 min and extension at 72°C for 2 min. RAPD profiles were analyzed by Bionumeric software and all of the isolates with similar pattern from one genus were considered as one strain.

Antibacterial activity

Agar spot test: Antibacterial activity of Bifidobacterium strains against 4 foodborne pathogens, including Shigella dysenteriae PTTC 1188, Salmonella typhi ATCC 19430, E. coli ATTC 4388, and Listeria monocytogenese (without distinct type number) was determined by using the agar spot test as described previously [14]. The Bifidobacteria isolates were cultured in MRSc broth for 48 h then 3 μl of bacterial suspension (108 cfu/ml) were spotted on the surface of MRSc plate and incubated anaerobically for 48 h at 37°C. The spots were covered with semi-solid BHI agar (0.5% agar) containing overnight cultured pathogenic indicator bacteria (108 cfu/ml) and cultured anaerobically for 48 h at 37°C and the inhibitory zones wider than 4 mm in diameter was considered positive.

Agar well diffusion: The inhibition zones of the cell free supernatant of Bifidobacterial growth were assessed by agar well-diffusion [14]. Summarily, 10 ml of BHI agar at 45°C were mixed with 500 μl of an overnight culture of the pathogenic strains and poured into a Petri dish. 50 μl of the cell-free supernatants filter-sterilized through a 0.22 nm pore membrane was prepared. Two types of supernatant with different PH were prepared; one with a primary PH of 4 and another neutralized to pH 6.5. These supernatants were inserted into the wells with 3 mm in diameter in the agar layer. All of the aforementioned experiments were repeated in duplicate and mean and SD of inhibition zones was calculated. The isolates producing inhibition zone in both pH rates were analyzed for the determination of peptide compounds by treating the supernatant with proteinase K.

Results

Sampling, cultivation and identification

Amongst all 56 samples, 31 samples, including 20 stools and 11 milks were positive for Bifidobacterium. In all, 31 isolates, including B. breve, B. longum, B. bifidum were isolated and confirmed by phonotypic and molecular experiments (Table 2). 12 of strains were shared between stool and milk, including 6 Bifidobacterium longum, 4 B. breve and 2 B. bifidum which marcked and numbered with specific symbol (Figure 1).

probiotics-health-RAPD-cluster

Figure 1: RAPD cluster and simlarity of paired strains isolated from infants stool and mothers milk. Each of genus have been marked and numbered with specific symbols; B.L (B. longum), B.B (B. bifidum), BBr (B. breve), each of paired insert in one curly bracket.

BacteriaSample B.bifidum B.longum B.breve Total
Stool 7 8 5 20
Total 11 9 11 31

Table 2: Frequency of Bifidobacterium strains isolated fom milk and stool.

RAPD PCR

For the determination of genotype similarity amongst 12 paired isolates RAPD PCR was done. As shown in Figure 1, only three strains of Bifidobacterium longum present 100% similarity which BL1 and BL2 were paired but BL3 was from another paired. The other RAPD profiles indicated less than 100 similarity.

Antibacterial activity

The inhibition zones of 12 strains toward 4 indicator bacterial pathogens were determined by agar spot and agar well diffusion. In all, each isolates showed specific halo zones against indicator pathogens and the results showed no significant difference between bacterial genus and origin of isolation. Our experiments indicated neutralization of supernatants due to deletion or reduction of inhibition zone and threating of neutralized supernatant with proteinase K cause elimination of inhibition zone in reduced wells. Three isolates including B.L4, B.L6 and B.B2 due to reduction of halo inhibition toward all pathgens and threating of their supernatants with proteinase K caused deletion of halo zones. Details of the results of 12 strains shared between the stools of children and mother’s milk are shown in Table 3.

Strain Agar Spot(mm)
Mean ± SD
   
pH 4
Mean ± SD (mm)
pH 6.5
Mean ± SD (mm)
Threated supernatant
(mm)
B.L1    E.coli
Shigella dysenteriae
Salmonella typhi
Listeria monocytogenes
8 ± 0.5 10 ± 0.1 - -
- - - -
7 ± 0.1 8 ± 0.1 - -
5 ± 0.2 9 ± 0.3 - -
B.L2     E.coli
Shigella dysenteriae
Salmonella typhi
Listeria monocytogenes
- - - -
6 ± 0.4 11 ± 0.6 - -
12 ± 0.6 10 ± 0.5 - -
- - - -
B.L3     E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
12 ± 0.1 8 ± 0.6 - -
8 ±0.3 10 ± 0.7 - -
14 ± 0.6 19 ± 0.4 - -
- - - -
B.L4     E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
8 ± 0.2 8   ± 0.1 5± 0.6 -
5 ± 0.6 12 ± 0.3 6± 0.3 -
9 ± 0.6 8 ± 0.2 7 ± 0.5 -
11 ± 0.2 12 ± 0.4 8 ± 0.2 -
B.L5     E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
- - - -
- - - -
- - - -
- - - -
B.L6    E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
12 ± 0.1 14 ± 0.1 3 ± 0.3 -
8± 0.6 10 ±  0.2 4 ± 0.1 -
9±  0.4 8 ± 0.6 3 ± 0.5- -
10 ± 0.3 6 ± 0.5 6± 0.3- -
B.B1     E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
11± 0.3 11 ± 0.3   -
14 ± 0.6 16 ± 0.2 - -
10 ± 0.5 10 ± 0.3 - -
9 ± 0.7 13 ± 0.6 - -
B.B2    E.coli
Shigella dysenteriae
Salmonella typhi
          Listeria monocytogenes
7 ± 0.8 12 ± 0.1 8 ± 0.6 -
11 ± 0.6 14 ± 0.2 4 ± 0.4 -
9 ± 0.1 12 ± 0.3 5 ± 0.5 -
10 ± 0.2 15 ± 0.4 3 ± 0.7 -
B.BR1     E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
15 ± 0.3 18 ± 0.1 - -
15 ± 0.5 20 ± 0.2 - -
  - - -
- - - --
B.BR2     E.coli
Shigella dysenteriae
Salmonella typhi
          Listeria monocytogenes
13 ± 0.6 17 ± 0.6 - -
- - 3 ±  0.1 -
9 ± 0.1 20 ± 0.3 - -
8 ± 0.3 18 ± 0.3   -
B.BR3   E.coli
Shigella dysenteriae
Salmonella typhi
         Listeria monocytogenes
- - 7 ± 0.1 -
12 ± 0.3 17 ± 0.4 - -
- - - -
6 ± 0.1 16 ± 0.7 - -
B.BR4  E.coli
Shigella dysenteriae
Salmonella typhi
 Listeria monocytogenes
25 ±0.4 20 ± 0.1 - -
25 ± 0.3 24 ± 0.2 6 ± 0.3 --
20 ± 0.2 21 ± 0.3 - -
21 ± 0.3 18 ± 0.4 - -

Table 3: Antibacterial Activity of paired Bifidibacterium isolates against indicator pathogens.

Antibacterial activities of three isolates showing 100% RAPD pattern similarity including B.L1, B.L2 and B.L3 (Figure 2) indicated no difference between agar spot and Non-neutralized supernatant well diffusion method. All of three isolates formed variable halo zone against Salmonella typhi and B.L3 only inhibited L. monocyogenes. B.L1 inhibited all pathogene indicator except Shigella dysenteriae.

probiotics-health-Antibacterial-activity

Figure 2: Antibacterial activity of Bifidobacterium longum with similar RAPD pattern isolated from mothers milks and infants feces by Agar spot and Non-neutralized supernatant well diffusion.

Discussion

Human Innate immunity is one of the most important immune pathways providing by some approaches including production of antibacterial compound. Nowadays, microbiome engineering and usage of probiotic products is an important concept for developing of gut immunity [15]. It is well-documented that probiotics including Bifidobacteria (one the most wildely used probiotics) develop gasteroitestinal immunity by production of antibacterial substance [8]. The previous studiese have proven that probitic properties are strain specific and origin of isolation may be affect on these traits [16], therefore assessment of antibacterial activity of each probiotic candidates seems to be vital.

In the present investigation, sampling according to the enrolled criteria was done from rural area in the center of Iran,. Different Bifidobacterium genera were isolated from paired mothers milk and infants feces and RAPD profiles were determined and the similarity of patterns were analyzed by Bionumeric software. Afterward the antagonist activity of Bifidobacterium genera toward 4 important enteric pathogens was assayed. 58 paired samples were collected that 31 different strains from 31 samples were isolated. Amongst all isolates only 12 strains were paired. As shown in Figure 1, only three Bifidobacterium longum including B.l1, B.l2 and B.l3 showed 100% similarity. B.l1 and B.l2 were isolated from one paired mother and infants. Since the establishment of the Bifidobacterium species in the intestinal tract occur after 20 days of age [16] and B.l2 was isolated from 11 days old baby, therefore in line with Solis et al. it is may be due to direct transfer from mother to infant [17]. Bl3 was from mother milk of another paired. Two Bifidobacterium bifidum presented 90% similarity. Amongst four Bifidobacterium breve, paired 5 showed 90% homology but paired 6 presented the lowest similarity.

According to Table 2, the results of agar spot and non-neutralized supernatant were approximately similar but the neutralization of supernatant caused reduction or deletion of halo zones. Similar to results of servin et al, our outputs indicated that antibacterial traits of isolates were strain specific and origin of isolates had no effect on this issue. Also no significant difference was observed between type of Bifidobacterium genus and antibacterial activity profiles [18]. Despite high genotype homology between B.L1 and B.L2 isolating from same paired but no significant antagonist similarity were seen (Figure 2).

In order to investigate the possibility of presence of peptide compounds in neutralized supernatant, a part of neutralized cell free supernatant threated with proteinase K and agar well diffusion method was done. Three isolates including B.l4, B.l6 and B.B2 showed inhibitor activity against pathogens in neutralized and non-neutralized supernatant while treated neutralized supernatant did not inhibit pathogen indicators. Simliar to Cherif et al. it may be due to production of bacteriocins like proteins which should be assessed in next studies by molecular experiments [19]. B.l1, B.L2 and B.L3 did not display similar reaction.

In short, amongst all 56 paired infants stools and mothers milks, 12 different shared Bifidobacteria were isolated and identified that three isolates showing 100% RAPD profile similarity presented strain specific antagonist activity moreover some of the isolates possibly produced bacteriocin like proteins which for the confirmation more experiments should be done.

Fundning

This work was supported by Iran University of Medical Science (IUMS) (Grant Number 9221133201).

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