Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells.

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Anaerobe xxx (2014) 1e8

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Clinical microbiology

Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells Q8

Q1

Yousef Nami a, Norhafizah Abdullah b, **, Babak Haghshenas a, Dayang Radiah b, Rozita Rosli a, Ahmad Yari Khosroushahi c, d, * a

Institute of Biosciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Chemical and Environmental Engineering Department, Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Biotechnology Research Center, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 February 2014 Received in revised form 29 March 2014 Accepted 28 April 2014 Available online xxx

Lactobacillus acidophilus is categorized as a probiotic strain because of its beneficial effects in human health and prevention of disease transmission. This study is aimed to characterize the probiotic potential of L. acidophilus 36YL originally isolated from the vagina of healthy and fertile Iranian women. The L. acidophilus 36YL strain was identified using 16S rDNA gene sequencing and characterized by biochemical methodologies, such as antibiotics susceptibility, antimicrobial activity, and acid and bile resistance. The bioactivity of the secretion of this strain on four human cancer cell lines (AGS, HeLa, MCF7, and HT-29) and one normal cell line (HUVEC) was evaluated by cytotoxicity assay and apoptosis analysis. This newly isolated strain was found to exhibit notable probiotic properties, such as admirable antibiotic susceptibility, good antimicrobial activity, and favorable resistance to acid and bile salt. The results of bioactivity assessment demonstrated acceptable anticancer effects on the four tested cancer cell lines and negligible side effects on the assayed normal cell line. Our findings revealed that the anticancer effect of L. acidophilus 36YL strain secretions depends on the induction of apoptosis in cancer cells. L. acidophilus 36YL strain is considered as a nutraceutical alternative or a topical medication with a potential therapeutic index because of the absence of cytotoxicity to normal cells, but effective toxicity to cancer cell lines. Ó 2014 Published by Elsevier Ltd.

Keywords: Apoptosis Anticancer Anti-microbe Antibiotic susceptibility Cytotoxicity

1. Introduction Specific lactic acid bacterial (LAB) strains, such as Lactobacillus strains, have been considered as probiotics because of their health benefits [1e4]. These strains have a long history of consumption in traditional fermented foods as natural inhabitants of healthy human gastrointestinal tracts. Probiotic bacteria are required to express high resistance to acid and bile, adhere to intestinal surfaces, and colonize in the gastrointestinal tract. Research findings have shown that several Lactobacillus bacterial strains possess the resistance to acid and bile, inhibitory activity toward the growth of

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* Corresponding author. Faculty of Pharmacy, Tabriz University of Medical Sciences, Daneshgah Street, P.O. Box 51664-14766, Tabriz, Iran. Tel.: þ98 411 3372250, þ98 411 3372251; fax: þ98 411 3344798. ** Corresponding author. Tel.: þ60 389 466295; fax: þ60 386 567120. E-mail addresses: nhafi[email protected] (N. Abdullah), [email protected] (A.Y. Khosroushahi).

pathogenic bacteria, and positive effects on the host health [5e8]. The key foundation for improving the functional properties and the biotherapeutic action of probiotic foods and pharmaceutical products is the selection of suitable probiotic candidates. Cancer is one of the main causes of human deaths. Chemotherapy and chemoprevention are applied to control metastasis and decrease human mortality [9]. Studies have discovered that some specific strains of lactobacilli can induce the production of pro-inflammatory cytokines (interleukins IL-1 and IL-6) and antiinflammatory cytokines (interleukins IL-12 and IL-10) in animal/ human body. Evaluation of the toxicity of bacterial cytotoxic agents to different cell types is performed by in vitro cytotoxicity tests and by analyzing the mechanism of apoptosis in treated cancer cells [10]. Induction of apoptosis, which can be applied to control cancer development, involves complex anticancer activity of several therapeutic substances [11]. The HeLa cervical cancer cell line is a suitable model to study cervical cancer and apoptosis [12]. Apoptosis and necrosis are major forms of cell death that both

http://dx.doi.org/10.1016/j.anaerobe.2014.04.012 1075-9964/Ó 2014 Published by Elsevier Ltd.

Please cite this article in press as: Nami Y, et al., Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells, Anaerobe (2014), http://dx.doi.org/10.1016/j.anaerobe.2014.04.012

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Y. Nami et al. / Anaerobe xxx (2014) 1e8

implicate a sequence of successive biochemical and morphological events. Apoptosis or programmed cell death occurs during embryonic growth and in the course of organ involution. Internucleosomal DNA fragmentation and phosphatidylserine membrane translocation are crucial for the differentiation of necrosis and apoptosis [13]. Although various methods are available for identification, quantification, and characterization of apoptosis, flow cytometry is the most common choice to study apoptosis because of its applicability to an extensive range of cell types, stimulants, and time. Evaluating apoptosis by flow cytometry provides information on symptoms and incidence of apoptosis that is interpreted as a sign. Besides, cellular morphological characteristic is another method to determine the cell death mode (apoptosis or necrosis). This study aims to characterize the probiotic potential of Lactobacillus acidophilus 36YL originally isolated from the vagina of healthy and fertile Iranian women. The acid and bile resistance, antibiotic susceptibility, and antimicrobial activity of this strain will be evaluated. Four different carcinoma cell lines will be used for testing the anticancer properties of L. acidophilus secretions by comparing results from paclitaxel treatment groups, as a positive control, and consequent induction of apoptotic cells through DAPI staining, DNA fragmentation, and flow cytometry which is broadly used to analyze the cytotoxicity.

product was conducted by Macrogen DNA Sequencing Service (Korea). Sequence similarity was determined via GenBank BLAST analysis [18]. 2.4. Resistance to low pH and tolerance to bile-salt condition To determine the isolate resistance for gastric acid and high bile salt conditions, a previous method was modified and employed [19]. Bacterial cells from overnight culture were harvested after centrifugation at 10,000 g at 4 C for 7 min and washed twice with PBS buffer (pH 7.2). The washed cells were respectively placed in sterile tubes at pH 3.0 for different time points (0, 1, 2, 3 h) and 0.3% (w/v) bile salts (oxgall) for 0, 1, 2, 3, and 4 h before reculturing on MRS agar. Resistance was measured in triplicate in terms of viable colony counts and enumerated on MRS agar after incubation at 37 C overnight. 2.5. Antimicrobial activity assay

The vaginal specimens obtained from 40 healthy and fertile Iranian women aged 17e36 years were evaluated for LAB isolation. The lateral vaginal wall was swabbed with sterile cotton-tipped applicators, which were stored in sterile carriers. The strain was isolated by inoculating the samples on de ManeRogosa agar (MRS agar, Merck, Germany) supplemented with 0.2% bromocresol purple (Merck, Darmstadt, Germany) with streak plate technique and was incubated anaerobically at 37 C for 48 h using an anaerobic jar. Identification was performed by phenotypic (colony and cell morphology) and molecular criteria, such as testing for Gram reaction, catalase activity [14], and 16S rDNA sequencing.

Several pathogenic organisms were selected from the Persian Type Culture Collection to detect the antagonistic substances (Table 2). Well diffusion technique was used to detect the production of inhibitory substances in the supernatant fluids of the isolates. An overnight culture of the indicator strains was applied to inoculate the appropriate agar growth media [20] at 37 C. The wells (diameter, 5 mm) were cut into agar plates, and 50 ml of filtered cell-free supernatant fluid obtained from the third subculture of the microorganisms grown in MRS broth were added to each well. The supernatant was obtained by growing the inhibitory producer strains overnight in MRS broth at 37 C. The cells were removed by centrifugation, and the supernatant were placed in the wells and allowed to diffuse into the agar at room temperature for 2 h. Consequently, the plates were incubated at optimum growth temperature of the indicator strains and were examined after 24 h to measure the diameter of the inhibition zone areola [21,22]. The inhibition zones around the wells were measured and results were expressed in terms of resistance (0 mm), moderate resistance (0e 4 mm), moderate susceptibility (4e8 mm), susceptibility (8e 12 mm) and extra susceptibility (>12 mm) by comparing with performance standards for antimicrobial susceptibility testing.

2.2. Genomic DNA extraction and amplification of 16S rDNA region

2.6. Determination of antibiotic resistance

The total DNA of isolate from the inoculated culture with a single colony was extracted using the procedure described previously by Cardinal [15]. A single colony was recultured in MRS broth at 37 C for 24 h, and 1.5 ml of the bacterial culture was then centrifuged at 10,000 g for 5 .The supernatant was discarded, and the pellet was used for DNA isolation. The extracted DNA was then suspended in 50 ml distilled water. The solved DNA sample was checked qualitatively and quantitatively by 0.8% agarose gel electrophoresis and spectrophotometry. The extracted genomic DNA of the isolate was subjected to PCR analysis. Amplification of 16S rDNA region was performed in a thermal cycler MWG BIOTECH (Galileo, Madrid, Spain) using a pair of LAB-specific universal primers (Hal6F/Hal6R) [16]. PCR amplification was performed with an initial denaturation at 94 C for 4 min, followed by 32 cycles; denaturation at 94 C for 1 min, annealing at 58 C for 1 min, extension at 72 C for 1 min, and a final extension step at 72 C for 5 min [17].

The antibiotic susceptibility of this strain was studied against the commonly prescribed antibiotics to common diseases (e.g., gentamycin, penicillin, tetracycline, ampicillin, erythromycin, vancomycin, clindamycin, chloramphenicol, and sulfamethoxazol) using disc diffusion method [23]. The isolate diluted culture (100 ml; approximately 106e107 viable cells) was diffused onto the Mueller Hinton Agar, and antibiotic discs were applied onto the surface using an antibiotic disc dispenser. The plates were incubated at 37 C under anaerobic conditions and were assessed after 24 h of

2. Materials and methods 2.1. Sampling and isolation

2.3. 16S rDNA gene sequencing PCR products from the 16S rDNA gene (1500 bp) were amplified using the Hal primer set. The sequencing of purified PCR

Table 1 Survival rate of vaginal Lactobacillus acidophilus after incubation at pH value 3.0 and bile salts o.3%. Strain

SR%

Final counts (log cfu/ml) after incubation at: 0h

1h

2h

3h

4h

Lactobacillus acidophilusa Lactobacillus acidophilusb

81

9.91

8.83

8.45

8.02

e

89

8.38

7.81

7.68

7.54

7.46

a b

Survival rate after 3 h in low pH value 3.0. Survival rate after 4 h in bile-salt condition (0.3%).


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inoculation. The inhibition zones around the discs were measured using a digital caliper, and results were expressed in terms of resistant, intermediate, or sensible by comparing with the interpretative zone diameters given by the Performance Standards for Antimicrobial Disk Susceptibility (NCCLS, 2007) tests for disc diffusion antibiotic susceptibility test [24,25]. 2.7. Cell lines and growth conditions Four human cancer lines, namely, AGS (human stomach carcinoma cell), HeLa (human cervical carcinoma cell), and MCF-7 (human breast carcinoma cell), HT-29 (human colorectal carcinoma cell), and one normal cell line [human umbilical vein endothelial cells (HUVEC)] were utilized to determine the anticancer effects of isolate secretion metabolites on tumor cells. The cells were routinely grown in RPMI medium supplemented with 10% heat-inactivated (30 min, 56 C) fetal bovine serum and 1% penicillinestreptomycin mixture. All experiments were performed at 37 C under 5% CO2 atmosphere and 95% humidity [26].

3

pH 7.2 thrice and utilized for apoptosis assessments by fluorescent microscopy (BX63, Olympus, Japan) equipped with U-MWU2 fluorescence filter (excitation filter BP 330e385, dichromatic mirror DM 400, emission filter LP 420) [28]. 2.11. Flow cytometry assay Apoptosis was measured by Annexin V-FITC binding using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences) according to the manufacturer’s instructions. Annexin V binds with high attraction to phosphatidylserine and could be applied to identify the cells in all stages of apoptosis [29]. Propidium iodide (PI) exclusively stained the cells with interrupted cell membranes, which could be utilized to identify late apoptotic and dead cells [30]. Cells cultured in a supernatant-free medium were applied as controls. The experiment was repeated twice with triplicate samples for each experiment. Analyses were performed on 150,000 cells obtained at a rate of 300 cells/s. FL-1 and FL-3 were represented in dot plots; viable, apoptotic, and necrotic cells were recorded.

2.8. Cell free culture supernatant (CFCS) preparation 2.12. Interpretation of results The liquid culture of L. acidophilus at the end of the exponential growth phase was centrifuged at 4000 g for 10 min to precipitate the cells. The supernatant was collected, and the pH was adjusted to 7.2 by adding 1 N NaOH and was subjected for lyophilization. The lyophilized culture supernatant, in desirable concentrations, was solved in RPMI media and sterilized by filtration through a 0.22 mm bacterial filter (Millipore) before use to treat the cancer cells.

Data were analyzed using SPSS 19.0 software (SPSS Inc., an IBM Company, Chicago, Illinois). Statistical differences in multiple groups were determined by one-way ANOVA followed by multiple mean comparisons Duncan’s test. All numerical data are displayed as mean standard deviation and p 0.05 is considered statistically significant.

2.9. Cytotoxicity assay

3. Results

Cytotoxicity effects of isolate secretion metabolites on various tumor cells were evaluated by microculture tetrazolium (MTT) assay [27]. Cells were seeded into each component of a 96-well microplate with 200 ml of the RPMI growth medium containing 1.2 104 cells in the suspension. The cells were then treated with the filtered supernatant of isolated L. acidophilus strain (5 mg/ml to 50 mg/ml) and reached 50% or 60% confluence over different incubation periods (12, 24, and 48 h). The medium was subsequently replaced with 200 ml fresh media containing 50 ml MTT solutions (2 mg/ml in PBS) and was incubated for an additional 4 h at 37 C. After incubation, the medium containing the MTT was carefully removed. Then, 200 ml of DMSO plus 25 ml Sorenson’s glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) were added to each well and were incubated for 30 min. The absorbance of each well was measured at 570 nm using a microplate reader (Biotek, ELx800, USA) after shaking for 10 s.

3.1. Isolation and identification

2.10. DAPI staining method All treated and untreated cultured cells groups were scrutinized using DAPI staining method to detect the apoptotic cells. Sterile cover slips were placed into each well of a 6-well culture plate, were seeded by cancer cells with cell density of 120 104/well, and maintained under standard culture condition. At 24 h post-seeding, treatments with L. acidophilus secretion, neat MRS medium, and paclitaxel (IC50 concentration) were performed on all cultured cells. The treated and untreated control groups were incubated for another 24 h and were prepared for the apoptosis assay. For cell staining by DAPI dye, 4% paraformaldehyde was added to each well to fix cells for 5 min. The fixed cells were permeabilized by 0.1% Triton-X100 for 5 min and stained with 50 ml DAPI dye (1:2000 dilution) per well for 3 min incubation at room temperature. The processed cells attached to the cover slips were washed with PBS at

This strain was isolated from the vaginal ecosystem of healthy and fertile Iranian women. The identification techniques illustrated Gram-positive and catalase-negative properties for isolated bacilli form bacterial strain. Results of genotypic identifications as well as 16S rDNA sequencing revealed that this isolate belongs to the L. acidophilus species. The sequence of 16S rDNA fragment represented as following: >AGCAAGTCGAGCGAGCAGAACCAGCAGATTTACTTCGGTAATGACGCTGGGGACGCGAGCGGCGGATGGGTGAGTAACACGTGGGGAACCT GCCCCATAGTCTGGGATACCACTTGGAAACAGGTGCTAATACCGGATA AGAAAGCAGATCGCATGATCAGCTTATAAAAGGCGGCGTAAGCTGTCG CTATGGGATGGCCCCGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAT TGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGA ATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGA AGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGA GGTAGTAACTGGCCTTTATTTGACGGTAATCAACCAGAAAGTCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTC CGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAAGAATAAGTCTGA TGTGAAAGCCCTCGGCTTAACCGAGGAACTGCATCGGAAACTGTTTTT CTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGGAATG CGTAGATATATGGAAGAACACCAGTGGCGAAGGCGACTCTCTGGTCTG CAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGGAGG TTTCCGCCTCTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCT TACCAGGTCTTGACATCTAGTGCCATCCTAAGAGATTAGGAGTTCCCTT CGGGGACGCTAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGT GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTATTAGTT GCCAGCATTAAGTTGGGCACTCTAATGAGACTGCCGGTGATAAACCG


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GAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGG CTACACACGTGCTACAATGGACAGTACAACGAGAAGCGAGCCTGCGAA GGCAAGCGAATCTCTGAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCA ACTCGACTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGAACGC CGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATG GAAGTCTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGGAAGGAGC.

3.2. Resistance to low pH and tolerance to bile-salt condition Acid and bile assay revealed that L. acidophilus 36YL strain can grow in MRS broth following the decrease in pH to 3.0, and the resistance to this pH with 81% survivability. This strain also showed superior resistance to bile salt (oxgall) with about 89% survivability. This strain showed high resistance to low pH, and the viable count was found to be >8 log cfu/ml after 3 h exposure to pH 3. Table 1 shows the percentages of growth of the strain and final counts (in log cfu/ml) after 1, 2, and 3 h incubation at pH 3. This strain is resistant to bile salts even after 4 h exposure and retains its viability with small reduction in viable counts (12 mm; R: resistant; SR: semi resistant; SS: semi sensitive; S: sensitive; ES: extra sensitive; CIP: Collection of Bacteries de l’Institut Pasteur, Paris, France. ATCC: American Type Culture Collection, Virginia, USA. NCTC: National Collection of Type Cultures, London, UK. PTCC: Persian Type Culture Collection, Tehran, Iran.


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Antibiotics

Disk content

Mean zone diameter (mm)

Susceptibilitya

Vancomycin Tetracyclin Penicillin Ampicillin Chloramphenicol Clindamycin Erythromycin Gentamycin Sulfamethoxazol

30 mg 30 mg 10 mg 10 mg 30 mg 2 mg 15 mg 10 mg 25 mg

21 23 17 19 26 18 20 7 6

S S S S S I S R R

a

3.7. Apoptosis detection by flow cytometry

S: Sensible; R: Resistant; I: Intermediate.

shrinking cells with condensed (early apoptosis) or fragmented (late apoptosis) nuclei (Fig. 2b). The results revealed that the numbers of apoptotic cells with the condensed and fragmented nuclei were significantly higher than the blue (in the web version) intact normal cells (Fig. 2b). The treated HeLa cells displayed the very distinctive signs of apoptosis, including formation of micronuclei, cell shrinkage, membrane blebbing, nucleus fragmentation, and apoptotic bodies (Fig. 2b). These characteristics proved that apoptosis could be considered as a main cytotoxic mechanism. None of these signals were observed in untreated HeLa cells

Annexin V-FITC/PI flow cytometry analysis was performed to confirm that the metabolite secreted from selected vaginal L. acidophilus strain induced apoptosis on HeLa cancer cell. These cells were treated with 50 mg/ml L. acidophilus supernatant for 24 and 48 h. The dual parameter fluorescent dot blots revealed the viable cell population in the lower left quadrant (Annexin V/PI), the cells at the early apoptosis are in the lower right quadrant (Annexin Vþ/PI), and the cells at the late apoptosis are in the upper right quadrant (Annexin Vþ/PIþ). Fig. 3aed shows that 0.29% and 0.27% of the cells were Annexin Vþ/PI in untreated cells, whereas 0.36% and 4.33% of cells were Annexin Vþ/PIþ after 24 and 48 h. Our findings indicated that the cytotoxicity of L. acidophilus supernatant on HeLa cells occurred during apoptosis. A total of 31.69% and 63.34% of the treated cells were induced at early and late stages of apoptosis after 24 and 48 h of incubation. These results revealed that significant differences existed in apoptotic induction by L. acidophilus supernatant between 24 h and 48 h on human

120

120

(a)

(b)

100

AGS Cell Viability (%)

MCF7 Cell Viability (%)

5

(Fig. 2a). Membrane blebbing, cell shrinkage, and the appearance of micronuclei were prominent apoptotic features of HeLa cells treated with L. acidophilus, but the untreated HeLa cells failed to induce any apoptotic morphologic changes.

Table 3 Antibiotics susceptibility.

80 60 40

* *

20 0

100 80 60

*

40

*

20 0

CON

Dla

TAXOL

L. acidophillus

CON

Concentration of L. acidophillus metabolite (50 μg/ml)

Dla

TAXOL

L. acidophillus


120

120

(c)

(d)

100

HT-29 Cell Viability (%)

HeLa Cell Viability (%)

80 60 40

*

*

TAXOL

L. acidophillus

20 0

100 80 60 40

20

*

*

TAXOL

L. acidophillus

0

CON

Dla

CON

Concentration of L. acidophillus metabolite (50 μg/ml) 120

HUVEC Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

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Dla


(e)

100 80 60

40

* 20

0 CON

Dla

TAXOL

L. acidophillus

Fig. 1. Effect of L. acidophilus secretion metabolites on the viability of MCF-7 (a), AGS (b), HeLa (c), HT-29 (d), cancer cells and HUVEC (e) normal cell by 50 mg/ml concentration for 24 h. Data are expressed as mean of viability percentage S.D (error bars). Asterisks denote statistically significant differences (*p 0.05). CON: RPMI and MRS were used as control. DLa: L. acidophilus isolated from dairy products was used as a reference strain for comparison. Taxol: used as a positive control.


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Fig. 2. Detection of normal and apoptotic cells of HeLa cancer cells without treating by L. acidophilus supernatant (a) and with treating by L. acidophilus supernatant (b) after 24 h incubation.

cervical carcinoma cells. The cells stained with PI alone (V/PIþ) underwent necrosis. Both treated and untreated cell lines demonstrated a slight time-dependent increase in the necrotic population shown in the upper left quadrant (V/PIþ, Fig. 3). The treated cells showed a slight time-dependent decrease in early apoptotic cells, but a considerable time-dependent increase in the late apoptotic cells. The percentage of viable cells among treated HeLa cells were markedly reduced from 68% to 9%, and the percentage of late apoptotic cells markedly increased from 7% to 43% by increasing the incubation time from 24 h to 48 h. The percentage of early apoptotic cells slightly decreased with increasing incubation time, which

reached 25% to 21% after 24 and 48 h incubation. The predominance of early apoptotic cells at 24 h and late apoptotic cells at 48 h implied a significant (p < 0.05) apoptotic induction on HeLa cells in a time-dependent manner by L. acidophilus secretions compared with that in the untreated control. 4. Discussion The prominence of vaginal lactobacilli as an obstruction to infections is of substantial interest. The previous experiments have validated that the vaginal LAB protective role in health among

Fig. 3. Flow cytometric analysis of cervical cancer cells (HeLa cell) after incubation without treating with L. acidophilus supernatant for 24 h (a) and 48 h (b) and with treating by L. acidophilus supernatant for 24 h (c) and 48 h (d). Dots with Annexin V/PI (lower left), Annexin Vþ/PI (lower right), and Annexin Vþ/PIþ (upper right) Annexin V/PIþ (upper left) features represent viable intact, early apoptotic, dead late apoptotic cells, and necrotic cells respectively.


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women would benefit from the improvement of simple techniques, which allowed identification and detection. In this study, different in vitro procedures were used to characterize the antagonistic and anticancer properties of vaginal L. acidophilus 36YL strain. This strain revealed the inhibition of the growth of potential human pathogens and other undesirable bacteria. The results showed that this strain can be considered as a probiotic that possesses the best antagonistic and anticancer properties and good susceptibility spectra against most antibiotics. Probiotics are recently defined as live microorganisms which confer a health advantage on the host upon consumption in sufficient and suitable amounts. Requirements should be fulfilled to sustain a certain strain as a probiotic [33,34], which were confirmed in previous, well-designed human studies. To show optimistic health effects, the lactobacilli have to resist the stressful conditions of the gastrointestinal tract that contains bile [35,36]. Testing the survival of probiotic strains, particularly lactobacilli, at in vitro simulated gastrointestinal tract conditions could forecast the real survival of these strains in vivo when used in an unprotected method. Several Lactobacillus strains lose their viability upon exposure to low pH for 3 h. Our outcomes on the viability of the lactobacilli at pH 3 agree with the previous data [25,36e39]. Probiotic strains should be sensitive to antibiotics and should not carry transmissible antibiotic resistance genes if they are resistant to antibiotics [40e43]. In this study, the vaginal and probiotic L. acidophilus 36YL strain was tested for antibiotic susceptibilities. The resistance to vancomycin is of foremost concern, because vancomycin is one of the remaining antibiotics broadly effective against clinical infections produced by multidrugresistant pathogens [44,45]. LAB (e.g., Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus plantarum, and Leuconostoc spp.) and pediococci species are resistant to vancomycin, which is commonly intrinsic and non-transmissible [46e48]. In this study, we found that this strain has good sensitivity to numerous tested antibiotics, particularly vancomycin (Table 3). The results are similar to the previously reported findings [49e51]. Our findings are consistent with previously reported results [42,52]. While several health effects have been ascribed to LAB, their anticancer activity remains controversial because of the absence of a direct experimental sign showing cancer suppression in man as a result of LAB consumption. However, indirect proofs based largely on laboratory studies exist. The majority of studies on anticancer effects of LAB have dealt with colorectal cancer [53e56]. Hence, we decided to evaluate the anticancer activity of this strain on breast, stomach, cervical and colorectal cancer cell lines. The metabolites secreted from L. acidophilus 36YL strain exhibited the most potent cytotoxic effect against human colorectal cancer cells (HT-29) and human cervical cancer cells (HeLa). Probiotic organisms inhibit the proliferation of mammalian cells in both cell lines [57,58]. The induction of apoptotic cells by conjugated linoleic acid produced by various probiotic strains was established in Caco-2 and HT-29 mammalian cancer cell lines [59]. In this study, four tumor cell lines, such as HeLa, HT-29, AGS, and MCF-7 to avoid line-specific effect assessments, and one normal cell line (HUVEC) were chosen. The results demonstrated a significant inhibition on the growth of four evaluated cancer cell lines by the secreted metabolites of L. acidophilus. However, the vaginal LAB strain secretions decreased the proliferation and viability in all cancer cell lines, but did not show significant inhibitory or toxic effects on HUVEC normal cells. Apoptosis denotes an organized and energy-dependent procedure leading to cell death. This mode of cell death is physiological, genetically controlled, and of central importance to tissue progress and homeostasis [60,61]. Apoptotic cells possess morphological features, including membrane blebbing, nuclear condensation, and

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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 Ethical issues 101 102 No ethical issues to be promulgated. 103 104 Uncited reference Q7 105 106 [31]. 107 108 Acknowledgment 109 110 The financial support of the University Putra Malaysia and the 111 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, 112 Iran is gratefully acknowledged. Q3,4 113 114 References 115 116 [1] Guarner F, Perdigon G, Corthier G, Salminen S, Koletzko B, Morelli L. Should 117 yoghurt cultures be considered probiotic? Br J Nutr 2005;93:783e6. [2] Guarner F, Malagelada JR. Gut flora in health and disease. Lancet 2003;360: 118 512e9. 119 [3] Shanahan F. Probiotics and inflammatory bowel disease: from fads and fan120 tasy to facts and future. Br J Nutr 2002;88:5e9. [4] Alvarez-Olmos MI, Oberhelman RA. Probiotic agents and infectious diseases: a 121 modern perspective on a traditional therapy. Clin Infect Dis 2001;32:1567e 122 76. 123 [5] Jacobsen CN, Nielsen VR, Hayford AE, Moller PL, Michaelsen KF, Paerregaard A. Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by 124 in vitro techniques and evaluation of the colonization ability of five selected 125 strains in humans. Appl Environ Microbiol 1999;65:4949e56. 126 [6] Gorbach SL. The discovery of Lactobacillus GG. Nutrition 1996;31:2e4. [7] Greene JD, Klaenhammer TR. Factors involved in adherence of lactobacilli to 127 human Caco-2 cells. Appl Environ Microbiol 1994;60:4487e94. 128 [8] Kirjavainen PV, Ouwehand AC, Isolauri E, Salminen SJ. The ability of probiotic 129 bacteria to bind to human intestinal mucus. FEMS Microbiol Lett 1998;167: 130 185e9. the formation of apoptotic bodies. A biochemical hallmark of apoptosis is the cleavage of chromatin into small fragments (oligonucleosome) described as “DNA ladders” when seen in electrophoresed gels [61e65]. DNA ladder assay is a simple and available technique, which is valuable for rapid screening of apoptotic changes in cell populations. This method allows working with cell lysates and does not demand any special laboratory tools. Therefore, the DNA fragmentation pattern should not be utilized as the only sign of apoptosis [66]. The mechanism of cell death has an excessive effect on the resulting response in the surrounding tissue. Death by necrosis acts as a natural adjuvant that induces oxidative stress and production of numerous pro-inflammatory cytokines [67]. By contrast, death by apoptosis is a controlled event usually with minimal loss of membrane integrity until the later stages (secondary necrosis). This mode of cell death frequently encompasses phagocytosis by resident tissue macrophages and the release of anti-inflammatory cytokines [68]. The assay consumes PI excluded as membrane integrity dye because viable cells will exclude low concentrations of this substance. Dual staining with FITC-conjugated Annexin-V allows discrimination of healthy viable cells, early apoptotic cells, and late apoptotic/necrotic cells. The findings of apoptosis detection methods, namely, DAPI staining and flow cytometry, support the conclusion that L. acidophilus induces cytotoxicity at least in part through the apoptotic pathway. The newly identified and probiotic L. acidophilus 36YL strain tested in this study possesses considerable properties against most of the tested antibiotics and pathogenic bacteria. This strain also has a good capability to survive in GIT conditions and reveals the best properties regarding acid and bile resistance. This study implied that the potential of metabolites produced by this strain could be accounted as a nutraceutical alternative or a topical medication with a good therapeutic index because of the absence of cytotoxicity in normal mammalian cells.


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[9] Miranda CL, Stevens JF, Hemlmrich A, Henderson MC, Rodriguez RJ, Yang YH, et al. Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Human lupulus) in human cancer cell lines. Food Chem Toxicol 1999;37: 271e85. [10] Hsu YL, Kuo PL, Lin LT, Lin CC. Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells. J Pharmacol Exp Ther 2005;313:333e44. [11] Li Y, Ahmed F, Ali S, Philip PA, Kucuk O, Sarkar FH. Inactivation of nuclear factor kappaB by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agent in human cancer cells. Cancer Res 2005;65:6934e42. [12] Rahmati M, Bannazadeh H, Fazli D, Tahmasebzadeh F, Farrokhi A, Rasmi Y. The apoptotic and cytotoxic effects of Polygonum avicular extract on HeLa-S cervical cancer cell line. Afr J Biochem Res 2011;5:373e8. [13] Dartsch DC, Schaefer A, Boldt S, Kolch W, Marquardt H. Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis. Apoptosis 2002;7:537e48. [14] Sneath PA, Mair NS, Sharpe ME, Hoh JG. Bergey’s manual systematic bacteriology. 9th ed. Baltimore: Williams and Wilkins; 1986. pp. 1208e35. [15] Cardinal MJ, Meghrous J, Lacroix C, Simard RE. Isolation of Lactococcus lactis strain producing inhibitory activity against Listeria. Food Biotechnol 1997;11. [16] Lane DJ, Pace B, Olsen GJ, Stahlt DA, Sogint ML, Pace N. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 1986;82:6955e9. [17] Dubernet S, Desmasures N, Gueguen M. A PCR-based method for identification of lactobacilli at the genus level. FEMS Microbiol Lett 2002;214:271e5. [18] Endo A, Okada S. Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis. J Biosci Bioeng 2005;3:216e21. [19] Claire L, Glenn V, Gibson R, Robert A. Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium. J Appl Microbiol 2006;100:846e53. [20] Dimitonova SP, Danova ST, Serkedjieva JP, Bakalov BV. Antimicrobial activity and protective properties of vaginal lactobacilli from healthy Bulgarian women. Anaerobe 2007;13:178e84. [21] Maldonado NC, De Ruiz CS, Otero MC, Sesma F, Nader-Macías ME. Lactic acid bacteria isolated from young calves e characterization and potential as probiotics. Res Vet Sci 2012;92:342e9. [22] Nowroozi J, Mirzaii M, Norouzi M. Study of Lactobacillus as probiotic bacteria. Iran J Publ Health 2004;33:1e7. [23] Galvez AA, Dubois-Dauphin R, Ghalfi H, Campos D, Thonart P. Description of two Enterococcus strains isolated from traditional Peruvian artisanalproduced cheeses with a bacteriocin-like inhibitory activity. Biotechnol Agron Soc Environ 2009;13:349e56. [24] Barzegari A, Saei AA. Designing probiotics with respect to the native microbiome. Future Microbiol 2012;7:571e5. [25] Fernandez MF, Boris S, Barbes C. Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol 2003;94:449e 55. [26] Merghoub N, Benbacer L, Amzazi S, Morjani H, El Mzibri M. Cytotoxic effect of some Moroccan medicinal plant extracts on human cervical cell lines. J Med Plants Res 2009;3:1025e50. [27] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;16:55e63. [28] Paolillo R, Romano Carratelli C, Sorrentino S, Mazzola N, Rizzo A. Immunomodulatory effects of Lactobacillus plantarum on human colon cancer cells. Int Immunopharmacol 2009;9:1265e71. [29] Westermann B. Molecular machinery of mitochondrial fusion and fission. J Biol Chem 2008;283:13501e5. [30] Zavisic G, Radulovic Z, Vranic V, Begovic J, Topisirovic L, Strahinic I. Characterization and antimicrobial activity of vaginal Lactobacillus isolate. Arch Biol Sci Belgr 2011;63:29e35. [31] Todorov SD, Botes M, Danova ST, Dicks LM. Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions. J Appl Microbiol 2007;103:629e39. [32] Prinsloo S, Pieters R, Bezuidenhout CC. A cell viability assay to determine the cytotoxic effects of water contaminated by microbes. S Afr J Sci 2013;109:1e4. [33] Collins JK, Thornton G, Sullivan GO. Selection of probiotic strains for human applications. Int Dairy J 1998;8:487e90. [34] Lee J, Yun HS, Cho KW, Oh S, Kim SH, Chun T, et al. Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity. Int J Food Microbiol 2011;148:80e6. [35] Chou LS, Weimer B. Isolation and characterization of acid and bile tolerant isolates from strains of Lactobacillus acidophilus. J Dairy Sci 1999;82:23e31. [36] Pan X, Chen F, Wu T, Tang H, Zhao Z. The acid, bile tolerance and antimicrobial property of Lactobacillus acidophilus NIT. Food Control 2009;20:598e602. [37] Zhang Y, Zhang L, Ma W, Yi HX, Yang X, Du M, et al. Screening of probiotic lactobacilli for inhibition of Shigella sonnei and the macromolecules involved in inhibition. Anaerobe 2012;1e6. [38] Zhang Y, Zhang L, Dua M, Yi H, Guoa C, Tuo Y, et al. Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food. Microbiol Res 2011;167:27e31.

[39] Charteris WP, Kelly PM, Morelli L, Collins JK. Development of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J Appl Microbiol 1998;84:759e68. [40] Saarela M, Mogensen G, Fonden R, Matto J, Mattila-Sandholm T. Probiotic bacteria: safety, functional and technological properties. J Biotechnol 2000;84: 197e215. [41] Salminen S, Von Wright A, Morelli L, Marteau P, Brassart D, Vos De WM, et al. Demonstration of safety of probiotics e a review. Int J Food Microbiol 1998;44:93e106. [42] Zhou JS, Pillidge CJ, Gopal PK, Gill HS. Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. Int J Food Microbiol 2005;98:211e7. [43] Argyri AA, Zoumpopoulou G, Karatzas KA, Tsakalidou E, Nychas G-JE, Panagou EZ, et al. Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiol 2013;33:282e91. [44] Woodford N, Johnson AP, Morrison D, Speller DCE. Current perspective on glycopeptide resistance. Clin Microbiol Rev 1995;8:585e615. [45] Johnson AP, Uttley AHC, Woodford N, George RC. Resistance to vancomycin and teicoplanin: an emerging clinical problem. Clin Microbiol Rev 1990;3: 280e91. [46] Klein G, Pack A, Bonaparte C, Reuter G. Taxonomy and physiology of probiotic lactic acid bacteria. Int J Food Microbiol 1998;41:103e5. [47] Handwerger S, Pucci MJ, Volk KJ, Liu JP, Lee MS. Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate. J Bacteriol 1994;176:260e4. [48] Jamalifar H, Rahimi H, Samadi N, Shahverdi A, Sharifian Z, Hosseini F, et al. Antimicrobial activity of different Lactobacillus species against multi-drug resistant clinical isolates of Pseudomonas aeruginosa. Iran J Microbiol 2011;3:21e5. [49] Tynkkynen S, Singh KV, Varmanen P. Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes. Int J Food Microbiol 1998;41:195e204. [50] Choi SY, Chang CE, Kim SC, So J-S. Antimicrobial susceptibility and strain prevalence of Korean vaginal Lactobacillus spp. Anaerobe 2003;9:277e80. [51] Lee H, Yoon H, Ji Y, Kim H, Park H, Lee J, et al. Functional properties of Lactobacillus strains isolated from kimchi. Int J Food Microbiol 2011;145: 155e61. [52] Olukoya DK, Ebigwi SI, Adebawo OO, Osiyemi FO. Plasmid profiles and antibiotic susceptibility patterns of Lactobacillus isolated from fermented foods in Nigeria. Food Microbiol 1993;10:279e85. [53] Thirabunyanon M, Hongwittayakorn P. Potential probiotic lactic acid bacteria of human origin induce antiproliferation of colon cancer cells via synergic actions in adhesion to cancer cells and short-chain fatty acid bioproduction. Appl Biochem Biotechnol 2013;169:511e25. [54] Motevaseli E, Shirzad M, Akrami SM, Mousavi AS, Mirsalehian A, ModarressiHossein M. Normal and tumor cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate. J Med Microbiol; 2013. Q6 [55] Rafter J. the effects of probiotic on colon cancer development. Nutr Rev 2004;17:277e84. [56] Hirayama K, Rafter J. The role of probiotic bacteria in cancer prevention. Microbes Infect 2000;2:671e86. [57] Lee JW, Shin JG, Kim EH, Kang HE, Yim IB, Kim JY, et al. Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum. J Vet Sci 2004;5:41e8. [58] Pessi T, Sutas Y, Saxelin M, Kallioinen H, Isolauri E. Antiproliferative effects of homogenates derived from five strains of candidate probiotic bacteria. Appl Environ Microbiol 1999;65:4725e8. [59] Ewaschuk JB, Walker JW, Diaz H, Madsen KL. Bioproduction of conjugated linoleic acid by probiotic bacteria occurs in vitro and in vivo in mice. Nutrition 2006;136:1483e7. [60] Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implication in tissue kinetics. Br J Cancer 1972;68:239e57. [61] Wyllie A, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980;68:251e306. [62] Gonzalez-Marin C, Gosalvez J, Roy R. Types, causes, detection and repair of DNA fragmentation in animal and human sperm cells. Int J Mol Sci 2012;13: 14026e52. [63] Matalova E, Spanova A. Detection of apoptotic DNA ladder in pig leukocytes and its precision using LM-PCR (ligation mediated polymerase chain reaction). Acta Vet Brno 2002;71:163e8. [64] Saraste A. Morphologic criteria and detection of apoptosis. Herz 1999;24: 189e95. [65] Enari M, Sakahira H, Yokoyama H. A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 1998;391:43e50. [66] Collins RJ, Harmon BK, Gobe GC, Kerr JFR. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int J Radiat Biol 1992;61:451e3. [67] Anderson HA, Englert R, Gursel I, Shacter E. Oxidative stress inhibits the phagocytosis of apoptotic cells that have externalized phosphatidylserine. Cell Death Differ 2002;9:616e25. [68] Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGFbeta, PGE2, and PAF. J Clin Invest 1998;101:890e9.


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Draft Genome Sequence of the Probiotic Strain Lactobacillus acidophilus ATCC 4356.

Functional Profile Evaluation of Lactobacillus fermentum TCUESC01: A New Potential Probiotic Strain Isolated during Cocoa Fermentation.

The domestication of the probiotic bacterium Lactobacillus acidophilus.

Enteric coating of granules containing the probiotic Lactobacillus acidophilus.

Vaginal suppositories containing Lactobacillus acidophilus: development and characterization.

Effects of Lactobacillus plantarum A7 with probiotic potential on colon cancer and normal cells proliferation in comparison with a commercial strain.

Effects of probiotic supplement (Bacillus subtilis and Lactobacillus acidophilus) on feed efficiency, growth performance, and microbial population of weaning rabbits.

Converging effects of a Bifidobacterium and Lactobacillus probiotic strain on mouse intestinal physiology.

Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines.

Lectin-like activity of Lactobacillus acidophilus strain JCM 1026.

Reduction of Aflatoxin B1 Toxicity by Lactobacillus plantarum C88: A Potential Probiotic Strain Isolated from Chinese Traditional Fermented Food "Tofu".

Complete Genome Sequence of Lactobacillus acidophilus FSI4, Isolated from Yogurt.

Adhesion of human Lactobacillus acidophilus strain LB to human enterocyte-like Caco-2 cells.

Complete Genome Sequence of Probiotic Lactobacillus plantarum Strain FMNP01, Isolated from Mango Fruit.

Complete genome sequence of Lactobacillus reuteri I5007, a probiotic strain isolated from healthy piglet.

Whole Genome Sequence of the Probiotic Strain Lactobacillus paracasei N1115, Isolated from Traditional Chinese Fermented Milk.

Some probiotic and antibacterial properties of Lactobacillus acidophilus cultured from dahi a native milk product.

Technological properties and probiotic potential of Lactobacillus fermentum strains isolated from West African fermented millet dough.

Survival of Lactobacillus acidophilus and Bifidobacterium bifidum in ice cream for use as a probiotic food.

Selection, characterisation and evaluation of potential probiotic Lactobacillus spp. isolated from poultry droppings.

Plant extract enhances the viability of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus in probiotic nonfat yogurt.

Inoculated fermentation of green olives with potential probiotic Lactobacillus pentosus and Lactobacillus plantarum starter cultures isolated from industrially fermented olives.

Genome architecture of Lactobacillus plantarum PS128, a probiotic strain with potential immunomodulatory activity.

Draft Genome Sequence of Lactobacillus brevis Strain 3M004, a Probiotic with Potential Quorum-Sensing Regulation.