Accepted Manuscript Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria Abolfazl Davoodabadi, Mohammad Mehdi Soltan Dallal, Abbas Rahimi Foroushani, Masoumeh Douraghi, Mohammad kazem Sharifi Yazdi, Farzaneh Amin Harati PII:

S1075-9964(15)30015-9

DOI:

10.1016/j.anaerobe.2015.04.014

Reference:

YANAE 1439

To appear in:

Anaerobe

Received Date: 1 April 2015 Accepted Date: 25 April 2015

Please cite this article as: Davoodabadi A, Soltan Dallal MM, Foroushani AR, Douraghi M, Yazdi MkS, Harati FA, Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria, Anaerobe (2015), doi: 10.1016/j.anaerobe.2015.04.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria Abolfazl Davoodabadia, Mohammad Mehdi Soltan Dallala,b*, Abbas Rahimi Amin Haratia a

Div. of Bacteriology, Dept. of Pathobiology, School of Public Health, Tehran University of Medical

Sciences, Tehran, Iran. b

Food Microbiology Research Center, Tehran University of Medical Sciences, Tehran, Iran.

Dept. of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences,

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c

Tehran, Iran. d

Zoonosis Research Centre, Tehran University of Medical Sciences, Tehran, Iran.

Dept. of Medical Laboratory Sciences, School of Para Medicine, Tehran University of Medical Sciences.

Tehran, Iran.

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e

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Foroushani c, Masoumeh Douraghi a,b, Mohammad kazem Sharifi Yazdi d,e, Farzaneh

* Corresponding author: MM Soltan Dallal, Food Microbiology Research Center/ School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Phone: + 98-21-66462268 Fax: + 98-21-66462267

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E-mail address: [email protected]

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Abstract Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strains with Probiotic activity may have

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health Benefits for human. This study investigates the probiotic potential of Lactobacillus strains obtained from the feces of healthy infants and also explores antibacterial activity of Lactobacillus strains with probiotic potential against enteropathogenic bacteria. Fecal samples were collected from 95 healthy infants younger than 18 months. Two hundred and

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ninety Lactobacillus strains were isolated and assessed for probiotic potential properties including ability to survive in gastrointestinal conditions (pH 2.0, 0.3% oxgall), adherence to HT-29 cells and antibiotic resistance. Six strains including L. fermentum (4 strains), L.

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paracasei and L. plantarum showed good probiotic potential and inhibited the growth of enteropathogenic bacteria including ETEC H10407, S. flexneri ATCC 12022, S. sonnei ATCC 9290, S. intritidis H7 and Y. enterocolitica ATCC 23715. These Lactobacillus strains with probiotic potential may be useful for prevention or treatment of diarrhoea, but further in vitro and in vivo studies on these strains are still required.

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Key words: Lactobacillus, probiotic, infant, antibacterial, enteropathogens

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1. Introduction Lactobacillus bacteria are a heterogeneous group of lactic acid bacteria (LAB). They are gram positive, non-spore-forming, catalase negative and anaerobic or microaerophilic

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bacteria [1, 2]. Probiotics are live microorganisms, which when consumed in adequate amount confer health benefits to the host by altering indigenous microflora [3]. Probiotics bacteria, especially species of genera Lactobacillus and Bifidobacterium, are normal microflora of the complex ecosystem of the gastrointestinal (GI) tract [4, 5]. Probiotics

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bacteria must have some characteristics; they must survive under stressed conditions of the GI tract, by tolerating gastric acidity and bile toxicity [6]. Moreover, they must adhere to intestinal epithelial cells in order to persist in the gut. Probiotics bacteria should also have

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desirable antibiotic susceptibility patterns and antimicrobial effects against pathogenic microorganisms [7].

Diarrhoea is one of the major causes of morbidity and mortality among children in the developing countries [8]. Diarrhoea is the second-most common cause of death in children less than five years old, and annually causes about 760, 000 death among children [9]. Several studies have demonstrated the probiotic potential of various Lactobacillus species

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[7, 10]. Some Lactobacillus species commonly used as probiotics, with effects especially against acute diarrhoea in childhood [11].

The aim of this study was to investigate probiotic potential properties of Lactobacillus

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strains from stool of healthy infants in Iran and also determine antimicrobial activity of Lactobacillus strains with probiotic potential against some enteropathogens causing diarrhoea.

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2. Material and Methods

The study was conducted from January to December 2014. Fecal samples were collected from 95 healthy infants between 1 to 18 months of age at Farman Farmaian health care center in Tehran city, Iran. The samples were immediately cultured in MRS broth (Scharlau, Spain) and incubated under anaerobic condition at 37°C for 48-72 h. The samples were subcultured on MRS agar (Scharlau, Spain) plates and incubated under anaerobic condition at 37°C for 48. Three to four colonies of each culture were selected for further investigation. This study was approved by the Research Ethics Committee of

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Tehran University of Medical Sciences (TUMS), and informed parental consent was obtained. 2.1. Preliminary identification of Lactobacillus strains

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The fermentation of carbohydrates was determined according to Sharpe (1979) using the miniplate method described by Jayne- Williams (1976) [12, 13]. All strains were investigated for the following 14 sugars (Merck, Germany): L-arabinose, D-xylose, galactose, D-glucose, mannitol, mannose, sorbitol, cellobiose, maltose, lactose, melibiose,

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saccharose, trehalose and raffinose. Also, all strains were tested for hydrolysis of arginine, gas (Co2) production from glucose and growth at different temperatures (15°C , 45°C)

2.2. Acid and bile resistance

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[14].

One milliliter of MRS broth culture containing about 109 CFU/ml of Lactobacillus strains was transferred into 9 ml phosphate-buffered saline (PBS) (pH 2) and incubated at 37 °C for 3 h. After incubation, the number of viable bacteria was measured by plating serial dilutions on MRS agar. Acid tolerance was estimated by comparing the viable

3 h [15].

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Lactobacillus strains counts on MRS agar for surviving cells after incubation at pH 2.0 for

Lactobacillus strains that survived in the acid tolerance assay (pH 2, 3 h) were selected for bile resistance test. These strains grown in 9 ml MRS broth with and without 0.3% (w/v)

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oxgall bile (Sigma) for 8 h. Growth was recorded after 8 hour by measuring absorbance at 600 nm using spectrophotometer (Pharmacia Biotech Ultrospec 2000). Coefficient of inhibition (Cinh) was calculated using the method described by Gopal et al [16]:

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Cinh = (∆T8-T0 Control - ∆T8-T0 Treatment)/ (∆ T8-T0 Control) Where, ∆ represented the differences in absorbance between T0 (zero hours reading) and T8 (reading on the 8th hour). The experiment was done twice to ensure the reproducibility of the result. Based on calculated coefficient of inhibition (Cinh), strains were classified into non-sensitive (resistant) to 0.3% bile salt (Cinh ≈ 0), with retarded growth (0.2 0.4). L. acidophilus ATCC 4356 and L. plantarum PTCC 1058 were used as control strains in acid and bile tolerance tests. 2.3. Identification of lactobacilli by 16S rDNA gene sequencing

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The identification of the selected Lactobacillus strains with acid and bile resistance was confirmed by 16S rDNA sequence analysis. Genomic DNA was extracted according to a previously described method [17]. The PCR primer sequences were as follows: forward 5′-CTCGTTGCGGGACTTAA-3′

and

reverse

primer,

5′-

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primer,

GCAGCAGTAGGGAATCTTC-3′ (Bioneer, Korea) [18]. The reaction mixture consisted of 3 pmol primers, 1.5 mM MgCl2, 0.2 mM dNTPs, 2 µl of genomic DNA, 5 µl 10X PCR buffer and 1.5 U of Taq DNA polymerase (Sinaclon, Iran) in a final volume of 50 µl. The

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PCR program started with an initial denaturation at 94°C for 2 min, followed by 30 cycles of 94 °C for 30 s, 53 °C for 1 min and 72 °C for 1 min. PCR products were separated by agarose gel electrophoresis (1.5% w/v) and visualized by staining with ethidium bromide.

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The PCR products of strains were sent to a sequencing company (Bioneer, Korea) and the 16S rDNA sequences were compared with known sequences in GeneBank using BLAST (www.ncbi.nlm.nih.gov/blast). 2.4. Adhesion Assay

The ability of lactobacillus strains to adhere to human epithelial cells was investigated according to the method of Gopal et al., 2001 [16]. The HT-29 cell line was purchased

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from the Pasteur Institute (Tehran, Iran). HT-29 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% (v/v) fetal bovine serum and 50 unit/ml penicillin–streptomycin (Sigma). For adhesion assays, monolayers of HT-29 were

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prepared on glass cover slips that were placed in six-well tissue-culture plates (NUNC). The culture medium was replaced 2 every second day and the HT-29 cell line was used after 15 days of incubation at 37 °C in 5% CO2-95% air atmosphere.

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The cells were inoculated with approximately 1×107 CFU/ml of the strains and incubated for 2 h at 37 °C in 5% CO2-95% air atmosphere. After incubation, the monolayers were washed four times with PBS (pH 7.4), fixed with methanol and then stained with diluted giemsa (1:10, vol/vol) for 20 min. cover slips were examined microscopically under an oil immersion lens.

The numbers of lactobacilli adhered to the cultured cell lines were counted in 20 random microscopic fields and the mean ± standard deviation of adhering bacteria per 100 cells of epithelial cell-line was determined. L. rhamnosus GG (ATCC 53103) used as control.

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2.5. Antibiotic Susceptibility Lactobacillus strains were cultured in 10-ml of a mixed formulation of 90 % (w/v) Mueller–Hinton broth (Merck, Germany) and 10 % (w/v) MRS broth (Merck, Germany).

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When broth culture reached the 0.5 McFarland standard turbidity at 37 °C, cultures were streaked with a cotton swab over agar plates containing a mixed formulation of Mueller– Hinton agar added with 10 % (w/v) MRS dehydrated broth. Antibiotic disks (Mast,UK) were placed onto the agar, and plates were incubated under anaerobic condition at 37°C for

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48. Inhibition zone diameters in millimeter (mm) were measured, and results were expressed in terms of resistance (≤ 15 mm), moderate susceptibility (16–20 mm), or

2.6. Antimicrobial activity

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susceptibility (≥ 21 mm) [19].

Antimicrobial activity was carried out according to agar well diffusion assay as described previously [20]. Enteropathogenic bacteria, including ETEC H10407, S. flexneri ATCC 12022, S. sonnei ATCC 9290, Y. enterocolitica ATCC 23715 and S. enteritidis H7 (this strain, previously isolated from a patient with diarrhea in our division) were cultured in BHI broth (Merck, Germany) for 24 h, then microbial density was adjusted to 107 CFU/ml

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and cultured on nutrition agar. Lactobacillus strains were grown in MRS broth under anaerobic condition at 37°C for 20 h. Cell free culture supernatants (CFCS) were obtained by centrifuging the MRS broth (10,000g, 10 min) 100 µl of the CFCS was placed into the

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wells of the nutrition agar and the nutrition agar plates were incubated at 37°C for 14–15 h. The diameter of the clear zones around each well was measured. Lactobacillus strains with clear zones less than 11 mm, 11 to 16 mm, 17 to 22 mm and more than 23 mm,

were

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grouped as negative (-), mild (+), strong (++), and very strong (+++) inhibitor, respectively. The L. rhamnosus GG was used as positive control and sterile MRS broth was used as negative control.

Two major mechanisms of antimicrobial activity are production of organic acids, which reduce the pH and the production of hydrogen peroxide. The production of bacteriocins may be another mechanism of antimicrobial activity [21]. For these reasons, the pH of CFCS was measured and changed to 6.5 with NaOH (2.5M) and then catalase (1mg/ ml, Sigma-Aldrich, Germany) was added to CFCS and incubated at 25 °C for 1 h.

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2.7. Statistical analysis Statistical

analyses

were

performed

with

SPSS

software

(version

16.0, SPSS). One-way ANOVA test was used for statistical analysis. Results were regarded

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as statistically significant at p < 0.05. 3. Results 3.1. Acid and bile resistance

Totally 290 strains were identified as Lactobacillus by phenotypical and biochemical tests.

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strains survived in the bile resistance test (Table 1).

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Seventy six strains survived in acidic condition. Among these 76 strains, 27 Lactobacillus

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Table 1. Acid and bile resistance of 27 Lactobacillus isolates

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h3 8.23±0.11 7.05±0.13 8.17±0.09 9.34±0.17 8.07±0.17 7.54±0.12 9.25±0.09 8.34±0.13 8.62±0.20 8.25±0.13 8.38±0.21 9.15±0.14 8.95±0.10 9.11±0.09 8.70±0.21 9.72±0.15 7.49±0.19 9.14±0.11 9.04±0.16 9.19±0.21 8.01±0.14 7.59±0.15 5.30±0.19 8.22±0.16 7.38±0.17 9.16±0.16 8.48±0.21 8.54±0.23 8.25±0.15

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h0 9.01±0.09a 8.82±0.12 9.34±0.05 9.63±0.19 8.17±0.14 8.79±0.06 9.32±0.05 9.49±0.15 9.43±0.23 9.54±0.07 9.61±0.14 9.47±0.10 9.75±0.04 9.77±0.12 9.85±0.17 9.81±0.17 9.65±0.13 9.74±0.15 9.73±0.23 9.62±0.12 9.32±0.09 8.61±0.11 7.17±0.22 9.61±0.18 8.49±0.13 9.61±0.24 9.04±0.17 9.41±0.21 9.27±0.09

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a

Strain identity ATCC 4356 PTCC 1058 84-3 4-1 11-2 19-5 26-1 26-3 40-3 40-2 40-5 49-1 61-3 63-2 64-1 89-4 89-1 89-2 89-3 90-2 19-2 19-1 6-4b 36-10 34-5 83-2 2-2 24-6 84-2

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Strains L. acidophilus L. plantarum L. brevis L. brevis L. casei L. fermentum L. fermentum L. fermentum L. fermentum L. fermentum L. fermentum L. fermentum L .fermentum L. fermentum L. fermentum L. fermentum L. fermentum L. fermentum L. fermentum L. fermentum L. paracasei L. paracasei L. paracasei L. plantarum L. plantarum L. plantarum L. reuteri L. rhamnosus L. rhamnosus

Bile resistance (0.3%, h8) Cinh 0.33 0.31 0.33 0.19 0.37 0.19 0.24 0.35 0.28 0.37 0.38 0.35 0.29 0.22 0.08 0.33 0.34 0.38 0.37 0.31 0.01 0.01 0.05 0.21 0.30 0.23 0.25 0.38 0.26

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Acid resistance (pH:2)

Each value represents the mean value± stand deviation (SD) from two trials (log CFU/ml).

3.2. Adhesion assay

The adherence potential of 27 Lactobacillus strains with acid and bile tolerance was examined with HT-29 epithelial cells (Fig. 1). Fourteen strains exhibited adhesive properties. Among these, 6 strains have higher adherence potential than to the positive control, L. rhamnosus GG (Fig. 2). These 6 strains were L. fermentum 40-2, L. fermentum 40-3, L. fermentum 89-1, L. fermentum 26-3, L. paracasei 6-4b and L. plantarum 34-5. The other 13 strains did not show any attachment. 8

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Fig. 1. Adherence to the epithelial cells (HT-29). Each adhesion index value represents the mean numbers ± stand deviation (SD) of bacteria adhering per 100 epithelial cells. Each adhesion assay was performed in duplicate.

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* Significant differences (p < 0.05) between the adhesion index values of positive control

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strain L. rhamnosus GG and the tested strains. The error bars show standard deviations.

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Fig. 2. Adherence to the HT-29 cells. A: L. fermentum 40-2; B: L. rhamnosus GG.

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3.3. Antibiotic Susceptibility In order to investigate probiotic characteristic, the antibiotic susceptibility of 27 Lactobacillus strains with acid and bile tolerance was performed. Table 2 shows the

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antibiotic susceptibility patterns of the strains. Most Lactobacillus strains were susceptible to most tested antibiotics. However, 25 strains were resistant to vancomycin and streptomycin.

Table 2. Susceptibility of 27 Lactobacillus strains to antibiotics using the disc diffusion

Antibiotics DA2

E15

GM10

PG10

S S S S S S S S S S S S S S S S S S S S S S S S S S S

S S S S S S S R S R S S S S S S S S S S MS S S S S S S

S S S S S S S S S S S S S S S S S S S S S S S S S S S

MS R S MS R MS MS S MS S MS MS R MS S R R MS MS R R S MS S MS MS MS

R S R S S S S S S S MS S S S S S S S S S S S S S S S S

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RP5

S10

T30

V30

S S S S MS S S S S S MS MS S MS MS S S S S R S MS S S S S S

R R R R R R R R R R R R R R R R R R R R R R R R MS MS R

MS S S S MS S S R S S S S S S R S MS S S R S R S MS S MS MS

R R R R R R R R R R R R R R R R R R R R R R S S R R R

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Ch30

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Strain identity 2-2 19-2 19-1 11-2 19-5 26-1 26-3 34-5 24-6 36-10 40-3 40-2 40-5 49-1 4-1 6-4b 61-3 63-2 64-1 83-2 84-2 84-3 89-4 89-1 89-2 89-3 90-2

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method

Antibiotics (Disk potency): Ch30: Chloramphenicol (30µg); DA2: Clindamycin (2µg); E15: Erythromycin (15µg); GM30: Gentamicin (10µg); PG10: Penicillin G (10µg); RP5: Rifampicin (5µg); S: Streptomycin (10µg); T30: Tetracycline (30µg); V30: Vancomycin (30µg). R: resistance; MS: moderate susceptibility; S: susceptibility

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3.4. Antimicrobial activity Six Lactobacillus strains which showed good tolerance to acid and bile conditions and have good adherence potential to the HT-29 epithelial cells, were investigated for antimicrobial

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activity against the enteropathogenic bacteria (Table 3). Only CFCS of L. plantarum 34-5 with no treatment, like the L. rhamnosus GG exhibited inhibition zone against all the enteropathogenic bacteria. The growth of S. flexneri ATCC 12022 and ETEC H10407 were inhibited by all the six Lactobacillus strains.

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L. fermentum 40-2 showed inhibition zone against ETEC H10407, S. flexneri ATCC 12022, S. sonnei ATCC 9290 and Y. enterocolitica ATCC 23715. L. fermentum 40-3 showed inhibition zone against ETEC H10407, S. flexneri ATCC 12022 and Y. enterocolitica

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ATCC 23715. L. fermentum 89-1 showed inhibition zone against ETEC H10407, S. flexneri ATCC 12022, S. sonnei ATCC 9290 and S. intritidis H7. L. fermentum 26-3 inhibited the growth of ETEC H10407, S. flexneri ATCC 12022 and Y. enterocolitica ATCC 23715. L. paracasei 6-4b inhibited the growth of ETEC H10407, S. flexneri ATCC 12022, S. intritidis H7 and Y. enterocolitica ATCC 23715.

Table 3. Antimicrobial activity of Cell free culture supernatants (CFCS) of the

ETEC

S. flexneri

S. sonnei

S. intritidis

Y. enterocolitica

H10407

ATCC 12022

ATCC 9290

H7

ATCC 23715

++

+

+

++

a

+

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L. rhamnosus GG

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Lactobacillus strains against the enteropathogenic bacteria.

+

++

+

-

+

L. fermentum 40-3

+

++

-

-

+

L. fermentum 89-1

+

++

+

+

-

L. fermentum 26-3

+

++

-

-

+

L. paracasei 6-4b

+

+++

-

+

++

L. plantarum 34-5

+

++

+

+

++

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L. fermentum 40-2

; Interpretation of zone inhibition diameter. –, less than 11 mm; +, 11–16 mm; ++, 17–22

mm and +++, more than 23 mm.

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When CFCS of the Lactobacillus strains was adjusted to pH 6.5 and treated with catalase, the antimicrobial activity of the CFCS was disappeared. These findings suggest that the production of bacteriocins or bacteriocin-like compounds did not involve in the mechanism

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of antimicrobial activity. 4. Discussion

The probiotic potential of lactobacilli strains naturally isolated from the human GI-tract should be emphasized. LAB such as lactobacilli with human origin have more probiotic

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potential than LAB from the other sources due to their adaptation to the human GI environment [22]. Several studies have demonstrated the probiotic potential of various Lactobacillus strains with human origin and most current successful probiotic strains are

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indicated to be of human origin [22-24].

In this study 95 fecal samples from healthy infants were collected and 3 to 4 isolates (n=290) from each samples selected for investigation of probiotic potential. Probiotic bacteria must survive in the stressful condition of gastrointestinal tract, such as acidic pH and bile. Our results showed that 27 lactobacillus isolates survived in acidic pH and 0.3% bile.

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In this study, we tested adherence potential of 27 Lactobacillus strains with tolerance to acidic pH and 0.3% bile to HT-29 cells. Six strains include L. fermentum 40-2, L. fermentum 40-3, L. fermentum 89-1, L. fermentum 26-3, L. paracasei 6-4b and L.

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plantarum 34-5 showed good adherence properties. Attachment to intestinal epithelial cells is another important property of probiotic bacteria. This property is required for colonization and persistence of probiotic bacteria in the

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gastrointestinal tract [25]. In recent years, several studies have been documented the usefulness of human intestinal cell-lines, e.g. HT-29, Caco-2 and HT29-MTX, as in vitro cellular models for assessing the adhesive properties of probiotic strains [15, 16]. Several previous studies have been reported that L. rhamnosus GG as a positive control bind to HT-29 cells [10, 26]. The adhesion index values observed for our strains like L. fermentum 40-2 and L. fermentum 40-3 were 168± 7 and 160± 9 respectively, which was significantly higher than adhesion index of 46± 5 obtained for L. rhamnosus GG in this study. Tsai et al. [15] reported an adhesion index for L. paracasei En4 with Caco-2 cells as

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57± 4.93, which was less than that adhesion index for L. paracasei 6-4b (102± 8) in this study. Most Lactobacillus strains were resistant to vancomycin and streptomycin. Several species

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of Lactobacillus are intrinsically resistant to vancomycin [23]. The vancomycin resistance in some species of Lactobacillus spp. (e.g., L. rhamnosus, L. casei, L. plantarum, L. fermentum, L. brevis, L. curvatus) has been reported to be chromosomally encoded and not inducible or transferable [24]. Previous studies have also reported high level of resistance to

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streptomycin for all investigated lactobacilli [27, 28]. Katla et al. suggested that lactobacilli may have a natural reduced susceptibility to aminoglycosides, perhaps due to low cell membrane permeability [29]. Other studies have also reported that some Lactobacillus

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strains are known to be naturally resistant to vancomycin and streptomycin, and such resistances are usually intrinsic, chromosomally encoded, not transmissible and do not usually form a safety concern [23, 30].

The inhibition of enteropathogenic bacteria appeared to be due to the production of organic acids or hydrogen peroxide produced by the Lactobacillus strains. Previously, several studies have reported that a pH-dependent mechanism was involved in the antimicrobial

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activity of Lactobacillus strains [31, 32].

Tsai et al. [15] have been previously reported that L. acidophilus RY2, L. salivarius MM1 and L. paracasei En4 isolated from healthy infant feces significantly inhibit the growth of

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ETEC. In acordance with other reports, this study showed that Lactobacillus strains have antibacterial effect against enteropathogen bacteria [24, 25, 33]. LAB such as lactobacilli with human origin have more probiotic potential than LAB from

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the other sources due to their adaptation to the human GI environment [22]. Several studies have demonstrated the probiotic potential of various Lactobacillus strains with human origin and most current successful probiotic strains are indicated to be of human origin [2224].

In conclusion, this study showed that Lactobacillus strains with good probiotic potential could be isolated from fecal of healthy infants. The present study indicates that fecal microflora of healthy infants is a good origin for isolation of different Lactobacillus species with probiotic potential. Some Lactobacillus strains like L. plantarum 34-5 have

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antibacterial effect against enteropathogenic bacteria. These results suggest that our Lactobacillus strains with probiotic potential may be useful for prevention or treatment of

Acknowledgements

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diarrhoea, but further in vitro and in vivo studies on these probiotic strains are still required.

This work was supported by the Vice-Chancellor for Research grant (no. 21491) of Tehran

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University of Medical Sciences (Tehran, Iran).

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32. Servin AL. Antagonistic activities of lactobacilli and bifidobacteria against microbial pathogens. FEMS Microbiol Rev 2004; 28 (4): 405-440. 33. Coconnier MH, Liévin V, Bernet Camard MF, Hudault S, Servin AL. Antibacterial effect of the adhering human Lactobacillus acidophilus strain LB. Antimicrob Agents Chemother 1997; 41 (5): 1046-1052.

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Core findings of the this study are;  Fecal sample of healthy Iranian infants is a good origin for isolation of

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native probiotic Lactobacillus strains.

 Latobacillus strains in this study have good probiotic potential.

 Latobacillus strains inhibit the growth of enteropathogenic bacteria

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including, ETEC H10407, S. flexneri ATCC 12022, S. sonnei ATCC 9290,

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S. intritidis H7 and Y. enterocolitica ATCC 23715.

Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria.

Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strai...
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