Journal of Medical Microbiology Papers in Press. Published June 9, 2014 as doi:10.1099/jmm.0.074161-0

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Probiotic assessment of Enterococcus durans 6HL and Lactococcus lactis 2HL isolated from

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vaginal microflora

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Yousef Nami1, Norhafizah Abdullah*2, Babak Haghshenas1, Dayang Radiah2, Rozita Rosli1,

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Ahmad Yari Khosroushahi*3,4

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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, 4Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran

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Running title: Probiotic bacteria from vaginal microbiome

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*Corresponding authors: Ahmad Yari Khosroushahi, Email: [email protected] Address: Faculty of Pharmacy, Tabriz University of Medical Sciences, Daneshgah Street, Tabriz, Iran. P.O.Box 51664-14766; Tel. +98 411 3372250-1; Fax. +98 411 3344798; Norhafizah Abdullah, Email: nhafizah@ eng.upm.edu.my, Tel: +603 89466295, Fax: +60386567120

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1

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Abstract

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Forty-five lactic acid bacteria (LAB) were isolated from the vaginal specimens of healthy fertile

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women, and the identities of the bacteria were confirmed by sequencing of their 16S rDNA

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genes. Among these bacteria, only four isolates were able to resist and survive in low pH, bile

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salts, and simulated in vitro digestion conditions. Lactococcus lactis 2HL, Enterococcus durans

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6HL, Lactobacillus acidophilus 36YL, and Lactobacillus plantarum 5BL showed the best

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resistance to these conditions. These strains were evaluated further to assess their ability to

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adhere to human intestinal Caco-2 cells. L. lactis 2HL and E. durans 6HL were the most

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adherent strains. In vitro test under neutralized pH proved the antimicrobial activity of both

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strains. Results revealed that the growth of Escherichia coli 026, Staphylococcus aureus, and

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Shigella flexneri was suppressed by both LAB strains. The antibiotic susceptibility tests showed

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that these strains were sensitive to all nine antibiotics: vancomycin, tetracycline, ampicillin,

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penicillin, gentamycin, erythromycin, clindamycin, sulfamethoxazol, and chloramphenicol.

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These data suggest that these E. durans 6HL and L. lactis 2HL can be examined further for their

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useful properties and can be developed as new probiotics.

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Keywords: antibacterial, antibiotic, acid resistance, bile resistance, DNA sequencing

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Introduction

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The concept of eating or using live bacteria for health benefits goes back a hundred years

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(Metchnikoff, 1907). Lactic acid bacteria (LAB) are generally regarded as safe and constitute

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part of the probiotic concept, which has received significant attention over the past years

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(Metchnikoff, 1907; Salminen et al., 1998). Probiotics are live microorganisms in the form of

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single or mixed cultures; when consumed at sufficient amounts, these probiotics provide

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beneficial health effects for hosts (Guarner et al., 2008; Lilly and Stillwell, 1965; Surawicz,

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2003). Probiotics significantly affect the bioavailability of nutrients in the human body (Young

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and Huffman, 2003). These microorganisms stimulate immune mechanisms and maintain normal

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human microbiota compositions; as a result, these microorganisms reduce the risk of diarrhea

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(Binns and Lee, 2010), antibiotic-related diarrhea (Friedman, 2012), irritable bowel syndrome

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(Barouei et al., 2009; Parkes, 2010), inflammatory bowel disease (Geier et al., 2007; Gionchetti

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et al., 2002), vaginal infections (Reid and Bocking, 2003), atopic eczema (Bunselmeyer and

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Buddendick, 2010; Wickens et al., 2008), rheumatoid arthritis (Guarner et al., 2008), and cancers

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(Serban, 2013; Zhu et al., 2011). They are believed to be the dominant members of normal post-

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pubertal and premenopausal vaginal microbiota (Nam et al., 2007). Clinical proof shows that the

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vaginal and urogenital floras play a dominant role in maintaining both protection from illnesses

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and well-being of women (Burton et al., 2003).The vaginal microbiota generate a balanced

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ecosystem because of the interaction of factors of the native bacterial biota, in which the bacteria

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generally play a central role in inhibiting colonization by pathogenic organisms, including those

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responsible for yeast infections, bacterial vaginosis, urinary tract infections, and other sexually

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transmitted diseases (Shi et al., 2009).

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Many recognized probiotics belong to the LAB genera, such as Lactobacillus, Lactococcus,

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Enterococcus, and bifidobacteria, but the most important genera commonly used as probiotics

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are Lactobacillus and bifidobacteria. Probiotics are defined as “living microorganisms which,

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upon ingestion in certain numbers, employ health advantage beyond inherent general nutrition”

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(Gorbach, 2002) and play a role in the deterrence or treatment of infectious diseases, namely,

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vaginal infections (Reid et al., 2009), allergies (Kuitunen et al., 2012), irritable bowel syndrome

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(Hoveyda et al., 2009), inflammatory bowel diseases (Damaskos and Kolios, 2008), chronically

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high cholesterol levels(Huang and Zheng, 2009), colitis (Resta-Lenert and Barrett, 2009), and

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colon cancer (Andersson et al., 2001; Liong, 2008).

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To obtain the ability to exert their probiotic effects, potential strains are expected to have

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desirable properties (Quwehand et al., 1999; Zavisic et al., 2011). Probiotics have many

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selection criteria: human origin for human usage, acid and bile tolerance, adhesion to mucosal

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surface, safe for food and clinical use, and gastric resistance (Dunne et al., 2001; Saarela et al.,

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2000).

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Another one of the most important selection criteria for probiotics is antimicrobial activity,

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which targets pathogens and the undesirable enteric elements (Klaenhammer and Kullen,

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1999). The antimicrobial effects of LAB are caused by their production of some substances,

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such as organic acids (i.e., lactic, acetic and propionic acids), hydrogen peroxide, carbon

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dioxide, bacteriocins, diacetyl, and low molecular weight antimicrobial substances (Quwehand

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and Vesterlund, 2004). The aim of this study is to isolate and characterize select LAB of the

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human vaginal tract as new potential probiotics.

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Material and Methods

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Isolation of vaginal LAB

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Vaginal samples used in this study were isolated from 40 healthy fertile women in childbearing

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age (17 to36 years). LAB were isolated by inoculating the samples onto de Man Rogosa Agar

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(MRS agar, Merck, Germany) and M17 media supplemented with 0.3% bile oxgall pH 3.0 using

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streak plate technique. The inoculated plates were incubated at 37ºC for 24 h under anaerobic

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conditions using anaerobic jars (Mitsubishi Inc. USA) equipped with anaerobic gas generating

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kits. Individual colonies were selected and subcultured in a sterile MRS medium broth. Gram

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positive and catalase negative cocci and bacilli colonies were considered as LAB and stored at -

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70ºC in MRS broth supplemented with glycerol (25% v/v).

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Identification, PCR amplification and gene sequencing

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The universal primers used for LAB were those primarily used by Mirzaei and Brazgari, (2012).

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The primers amplify an approximately 1500 bp region of the16S rDNA gene. The primer

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sequences

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TACCTTGTTAGGACTTCACC-3 .

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All amplification reactions were performed in a MWG BIOTECH (Galileo, Madrid, Spain)

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thermal cycler with the following temperature profile: an initial denaturation at 94°C for 4 min,

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followed by 32 cycles containing denaturation at 94°C for 1 min, annealing at 58°C for 1 min,

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and extension at 72°C for 1 min, and a 5 min final extension step at 72°C (Dubernet et al., 2002).

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The total reaction mixture (25 µl) contained 13 pmol of each primer, 0.2 mM of each

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deoxyribonucleotide triphosphate (Invitrogen), 1 X PCR buffer without MgCl2, 2.0 mM MgCl2,

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100ng of bacterial DNA, and 2.0 U of Taq DNA Polymerase (MP Biomedicals). The PCR

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products were resolved through electrophoresis in 1.0% (w/v) agarose gel and were visualized by

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ethidium bromide staining. PCR products were purified and sequenced by Macrogen DNA

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Sequencing Service (Korea). Sequence similarity was determined using the BLAST program of

were:

Hal6F

5 -AGATTTTGATCMTGGCTCAG-3 ′

′

and

Hal6R

5′

′

5

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the National Center for Biotechnology Information

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(http://www.ncbi.nlm.nih.gov/BLAST)(Altschul et al., 1997).

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Resistance to low pH

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To determine the acid permanency of isolated LAB, the method described previously by Claire

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et al. was performed with some modifications (Claire et al., 2006). The pH of MRS broth was

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adjusted to 3.0 with 1 M HCl before autoclaving, and pH 7.0 served as a control. Survival was

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evaluated using the log phase cultures (8 log10 cfu ml-1) by plating on MRS agar after 0, 1, 2,

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and 3 h incubation of bacterial cells in acidic MRS broth (pH 3.0) at 37°C by pour plate

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technique.

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Survival in bile salts

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Tolerance to bile salts was determined using the method of Pereira and Gibson with some

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modifications(Pereira and Gibson, 2002).MRS broth supplemented with 0.3% (w/v) oxgall and

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MRS broth without supplement as a control were inoculated with actively growing bacteria.

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Samples were incubated for 4 h at 37°C, and the bacteria maintenance was evaluated using log

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phase cultures (8 log10 cfu ml-1) by plate count on MRS agar at time points 0, 1, 3, and 4 h of

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incubation in MRS broth containing bile salts at 37°C. During the incubation for 4 h, viable

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colonies were enumerated for every hour with pour plate technique in MRS agar media.

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Survival in simulated in vitro digestion

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To determine in vitro digestion, the method described previously by Seiquer et al. was performed

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with some modifications (Seiquer et al., 2001). To recreate the gastric digestion, pepsin to a final

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concentration of 5% (w/v) was added to the samples, and the samples were adjusted to pH 3.0.

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The samples were incubated for 120 min at 37 °C with gentle agitation (110 rpm). To simulate

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intestinal digestion, the samples were adjusted to pH 6.0 and solutions of bile salts and

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pancreatin were added at a final concentration of 0.3% and 0.1% (w/v), respectively. The

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samples were incubated at 37°C for 180 min with gentle agitation at 110 rpm. To determine cell

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count, the samples were removed before and after gastric and intestinal digestion, and the

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aliquots were serially diluted and plated in triplicate on MRS agar. The plates were incubated for

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48 h under anaerobic condition.

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Adhesion of LAB to Caco-2 cells

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Bacteria were evaluated for their adhesion ability to the human colon carcinoma cell line Caco-2.

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The cells were cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine

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serum and 1% penicillin–streptomycin mixture. Cells were cultured on 24-well tissue culture

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plates and incubated at 37 °C in 5% CO2 and in a relatively humidified atmosphere until a

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confluent monolayer was achieved.

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Before the adhesion assay, the media in the wells containing a Caco-2 cell monolayer were

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removed and replaced once with fresh antibiotic-free RPMI. Then, 1×107 cfu/ml of bacteria were

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added to each well with a total volume of 1 ml and were incubated for 3 h at 37°C in an

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atmosphere of 5% (v/v) CO2. To remove non-attached bacterial cells, the wells were washed

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three times with a sterile pre-warmed PBS solution. To detach the cells from the wells, 1 ml of

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trypsin-EDTA solution (0.5% porcine trypsin and 0.2% EDTA in PBS, Sigma, USA) was added

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to each well, and the mixture was gently stirred for 5 min. To measure the viable Caco-2 cell

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count, the cells were counted in Bürker hematocytometer chamber (Merck, USA). After that, this

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cell suspension (Caco-2 and bacteria cells) was subjected to cfu determination by pure plate

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method onto MRS agar and incubated at 37°C under anaerobic conditions. Bacterial adhesion

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was expressed as total of the number of bacteria attached to viable Caco-2 cells.

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Antimicrobial activity

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The well diffusion technique was used to detect the production of inhibitory substances in the

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supernatant fluid of the isolates (Schillinger and Lucke, 1987). In the agar well diffusion assay,

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overnight cultures of the indicator strains were used to inoculate appropriate agar growth media

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at 37ºC (Dimitonova et al., 2007). Wells of 5 mm diameter were cut into agar plates, and 50 l of

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filtered cell-free supernatant fluid obtained from the third subculture of the microorganisms

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grown in MRS broth was added to each well. Supernatant fluid was obtained by growing the

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inhibitory producer strains overnight in MRS broth at 37ºC. Then, cells were removed by

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centrifugation, the supernatant fluid was placed in the wells, and the fluid was allowed to diffuse

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into the agar for 2 h at room temperature. Consequently, the plates were incubated at the

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optimum growth temperature of the indicator strains and examined after 24 h for the inhibition

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zone. At the end of the incubation, inhibition zone diameters (surrounding the wells) were

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measured (Maldonado et al., 2012; Nowroozi et al., 2004).

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Antibiotic susceptibility

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The patterns of resistance/sensitivity to antibiotics of the LAB isolates were determined using the

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disc diffusion method (Maldonado et al., 2012; Rahmati et al., 2011). LAB strains were

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incubated overnight under anaerobic conditions at 37ºC and 5% CO2. Exactly, 100 l of the

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diluted cultures (approximate 106 to 107 viable cells) were diffused onto Mueller-Hinton agar,

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and antibiotic discs were applied on the surface using an antibiotic disc dispenser. Plates were

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incubated at 37°C under anaerobic conditions and assessed after 24 h of inoculation. Inhibition

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zones around the discs were calculated using a digital caliper. The results were expressed in

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terms of resistance, moderate susceptibility, or susceptibility by comparing with the

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interpretative zone diameters given by the performance standards for antimicrobial disk

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susceptibility tests (Hoesl and Altwein, 2005).

μ

μ

8

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Results

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Isolation and identification of human vaginal LAB

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Forty-five strains were isolated from MRS and M17 agars. Among these strains were four

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Enterococcus faecium, five Enterococcus malodoratus, eight Enterococcus faecalis, three

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Enterococcus durans, four Enterococcus avium, two Enterococcus gilvus, four Enterococcus

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hirae, two Enterococcus pseudoavium, four Enterococcus lactis, two Lactobacillus casei, one

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Lactobacillus plantarum, three Lactobacillus acidophillus, and three Lactococcus lactis (Table

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1). The fact that the primers used were capable to amplify enterococci, lactobacilli, and

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lactococci genes shows that these primers are as specific as expected to amplify LAB.

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Resistance to acidic condition

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All 45 LAB strains were incubated at pH 3.0 for 3 h. After 3 h incubation, 30 strains were found

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to be resistant to low pH (pH 3.0) with survivability higher than 50%. Only five strains

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demonstrated low pH resistance with a survival rate greater than 80% (Table 1). Strains such as

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L. plantarum 5BL, E. durans 6HL, and L. lactis 2HL were the species most resistant to low pH.

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Survival in bile salts

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All bacteria were incubated for 4 h in the presence of 0.3% bile salts. Among the bacteria, only

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39 strains were resistant to 0.3% bile salts after 3 h incubation with a survival rate higher than

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50%. Only 14 strains showed bile salt resistance with survivability greater than 80% (Table 1).

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The most resistant species to bile salt conditions were E. durans 6HL and L. lactis 2HL, with

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93% survivability.

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Survival in simulated in vitro digestion

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One of the most desirable features required for probiotics is their capability to stay alive in the

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gastrointestinal tract. To further examine the strains through simulated digestion test, nine of the

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best strains that were resistant to low pH and bile salts were tested. Four strains showed

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considerable digestion survivability. The resistant strains were E. durans 6HL, L. plantarum

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5BL, L. acidophillus 36YL, and L. lactis 2HL. The highest percentage of survivability was

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observed for E. durans 6HL and L. lactis 2HL, with survivability values of 46% and 49%,

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respectively (Table 2). Based on our findings, the resistance to acid/bile condition is strain

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specific because of wide range of diversity in survivability even among same species.

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Adhesion assay to Caco-2 cells

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The four digestion-resistant LAB strains were examined for their capability to adhere to Caco-2

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cells. The results showed that E. durans 6HL and L. lactis 2HL were the most adherent strains,

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with adhesion values of 4.8×104 cfu/ml and 2.8×105 cfu/ml, respectively.

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Activity against pathogenic microorganisms

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The results of inhibitory action of two selected vaginal strains against pathogenic

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microorganisms are shown in Table 3. In vitro tests under neutralized pH were performed. The

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results demonstrated antimicrobial activity for both examined strains. These strains were

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recognized as active because the capability to inhibit the growth of one or more target pathogenic

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strains was observed. The growth of Escherichia coli 026, Staphylococcus aureus, and Shigella

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flexneri were suppressed by both LAB strains. Results showed that the inhibitory action of L.

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lactis 2HL was better than that of E. durans 6HL.

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Antibiotic susceptibility

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E. durans 6HL and L. lactis 2HL indicated good sensitivity to all nine antibiotics tested in this

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study, such as vancomycin, tetracycline, ampicillin, penicillin, erythromycin, clindamycin,

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gentamycin, chloramphenicol, and sulfamethoxazol. The areolas around the antibiotic disks in

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figure 1 illustrate E. durans 6HL good susceptibility for all nine examined antibiotics.

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Discussion

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The majority of bacteria commonly recognized as probiotic are LAB, which are commensal

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microorganisms in the human genital and gastrointestinal tract. These bacteria have

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demonstrable useful properties and have a long history of being safe (Nueno-Palop and Narbad,

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2011; Petricevic et al., 2012). Among the genital microflora, vaginal microbiota from healthy

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women can be considered as a good reservoir of probiotics isolation. Presence of these probiotics

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in vaginal microbiota plays a significant role in the prevention of vaginal infections and health

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maintenance (Strus et al., 2012). This study was undertaken to assess the potential probiotic

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properties of LAB isolated from Iranian vaginas.

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Increasing interest has been given to the use of LAB isolated from human genitalia and

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gastrointestinal tract as probiotics to treat urogenital tract infections (Okkers, 1999). The

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potential application of vaginal LAB isolated from healthy women as probiotics has not been

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investigated in detail. In this study, vaginal swabs were collected from the lateral vaginal wall of

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40 healthy Iranian women. As a result of the identification tests, 45 LAB isolates were identified

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as 4 E. faecium, 5 E. malodoratus, 8 E. faecalis, 3 E. durans, 4 E. avium, 2 E. gilvus, 4 E. hirae,

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2 E. pseudoavium, 4 E. lactis, 2 L. casei, 1 L. plantarum, 3 L. acidophillus, and 3 L. lactis (Table

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1). Enterococci were predominant among the LAB genera; this genus represented 80% of the

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isolates, followed by lactobacilli (13%), and lactococci (7%). Similar results have been reported

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by other researchers. Redondo-Lopez et al. (Redondo-Lopez et al., 1990) reported that L.

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acidophillus was the dominant species among lactobacilli of vaginal microflora.

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A requirement for bacteria to be recognized as a probiotic is their capability to stay alive while

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passing through the upper digestive tract to reach the large intestine, where their useful actions

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are expected (Bezkorovainy, 2001; Tuomola et al., 2001). To be colonized in the intestine,

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probiotic bacteria have to adhere to the intestinal mucosa to avoid being removed from the colon

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by peristalsis. Nine vaginal isolates were examined for this test, and among them, only four

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strains were able to survive exposure to the simulated digestion conditions of the stomach. Their

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adhesion ability to Caco-2 cells was evaluated for further analysis. In conclusion, we found that

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the strains L .lactis 2HL and E. durans 6HL were the strains most resistant to digestion

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conditions and were the best strains to adhere to Caco-2 cells. This screening showed that these

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strains could be candidates as new probiotics.

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The growth of pathogens is inhibited by producing antagonistic substances produced during the

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growth of LAB which decrease the introitus by E. coli via lessening the vaginal pH to 4.5.

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(Tomas et al., 2003).

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In vitro growth of many enteric pathogens has been demonstrated to be inhibited by LAB. LAB

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can treat a wide range of gastrointestinal disorders and can be used in both humans and animals

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(Aslim and Kilic, 2006). L. lactis 2HL has been demonstrated to strongly inhibit the in vitro

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growth of Streptococcus mutans, S. flexneri, Gardnerella vaginalis, Bacillus cereus, E. coli 026,

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and Salmonella typhimurium. The growth of other enteropathogenic bacteria, such as E. coli

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0157, S. aureus, Listeria monocytogenes, Klebsiella pneumonia, Pseudomonas aeroginosa,

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Candida albicans, Serratia marcesens, and Staphylococcus saprophyticus, was inhibited by L.

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lactis 2HL. The growth of five other strains, namely, S. flexneri, L. monocytogenes, S. aureus, E.

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coli 026, and S. typhimurium, was inhibited by E. durans 6HL.

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Annually, 1 million women are affected by urogenital tract infections (UGTI) worldwide

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(MacPhee et al., 2010). UGTI are responsible for high health care costs as well as high morbidity

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and mortality rates in females (Donders et al., 2009). Although Iranian women are not an

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exception, not much official epidemiological data are available on the subject. The most

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common pathogen associated with desquamative inflammatory vaginitis is E. coli (Donders et

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al., 2009). According to Barzegari and Saei (2012), probiotics must be isolated with respect to

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native microbiomes and be used in the same population in the interest of efficacy. The efficiency

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of a probiotic can be found in the target population before usage to guarantee the health and

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therapeutic profits. Therefore, two native (E. coli 026) and non-native (E. coli 0157) pathogenic

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organisms were assessed in this study. The growth of the native strain was inhibited by both

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LAB strains, but the growth of the non-native strain was only inhibited by L. lactis 2HL.

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Vancomycin resistance is one of the most important concerns in antibiotic resistance because

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vancomycin is one of the last antibiotics largely effective against clinical infections caused by

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multidrug-resistant pathogens (Johnson et al., 1990; Woodford et al., 1995). We determined the

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susceptibility of strains 2HL and 6HL to several antibiotics. Both strains were sensitive to

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tetracycline,

285

sulfamethoxazol, gentamycin, and, more importantly, vancomycin, which is the antibiotic of last

286

resort. In many cases, our results are similar to previously obtained results (Lim et al., 1993;

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Tynkkynen et al., 1998).

288

In conclusion, we identified two strains (E. durans and L. lactis) from the human vaginal tract

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that can survive the digestive system and potentially colonize the intestinal epithelial cells. We

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also show that these strains have good ability to inhibit the growth of many pathogenic

291

microorganisms and are sensitive to a broad range of antibiotics. These strains have good

292

potential to be used as probiotics.

ampicillin,

penicillin,

clindamycin,

293 294 295 296 297 298 13

chloramphenicol,

erythromycin,

299 300

Ethical issues No ethical issues to be promulgated.

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Conflict of interest statement The authors declare that there are no conflicts of interests. No writing assistance was utilized in the production of this manuscript.

304 305 306

Acknowledgment The financial support of the University Putra Malaysia and Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran are gratefully acknowledged.

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Table 1 Screening of LAB strains for their resistance to low pH (pH 3.0) and bile salts (0.3% v/v) Strain Species Low pH Bile salt code survival survival (%) (%) 20BL Enterococcus faecium 48 68 15BL Enterococcus faecium 56 76 36Y Enterococcus faecium 78 88 15BS Enterococcus faecium 53 73 20BS Enterococcus malodoratus 58 78 19B Enterococcus malodoratus 41 61 14H Enterococcus malodoratus 37 57 10HS Enterococcus malodoratus 24 44 6BL Enterococcus malodoratus 47 57 13B Enterococcus faecalis 52 72 1H Enterococcus faecalis 21 31 9B Enterococcus faecalis 49 61 17H Enterococcus faecalis 61 74 19HS Enterococcus faecalis 18 34 16H Enterococcus faecalis 73 89 7BL Enterococcus faecalis 51 70 1HL Enterococcus faecalis 33 53 12HL Enterococcus durans 54 74 6HL Enterococcus durans 87 93 10HL Enterococcus durans 58 68 17BL Enterococcus avium 71 91 19YC Enterococcus avium 46 66 18HL Enterococcus avium 59 79 19Y Enterococcus avium 22 32 7BS Lactobacillus acidophillus 64 72 15HL Lactobacillus acidophillus 72 81 36YL Lactobacillus acidophillus 81 89 8BS Enterococcus gilvus 74 87 15HS Enterococcus gilvus 62 73 20HL Enterococcus hirae 77 90 2BS Enterococcus hirae 41 57 14BS Enterococcus hirae 61 78 20HS Enterococcus hirae 30 43 5HL Enterococcus pseudoavium 75 87 12HS Enterococcus pseudoavium 59 67 5BL Lactobacillus plantarum 88 91 18HS Lactobacillus casei 79 88 17Y Lactobacillus casei 53 72 1BL Enterococcus lactis 33 42 7HL Enterococcus lactis 47 59 16B Enterococcus lactis 58 68 2BL Enterococcus lactis 67 89 2HL Lactococcus lactis 88 93 17YL Lactococcus lactis 84 88 13HL Lactococcus lactis 62 76 The low pH survivability was measured after 3 h and bile salt survivability was determined after 4 h.

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Table 2 Survival of low pH and bile salt resistant strains in simulated digestion condition and their capacityto adhere to Caco-2 cell line. Strain Species Low pH Bile salt Digestion Adhesion to Caco-2 code survival survival survival no. of cells (cfu/mL) (%) (%) (%) 5HL Enterococcus pseudoavium 75 87 0 36YL Lactobacillus acidophillus 81 89 34 2.7 × 104 3.2 × 103 5BL Lactobacillus plantarum 88 91 29 4.8 × 104 6HL Enterococcus durans 87 93 46 2.8 × 105 2HL Lactococcus lactis 88 93 49 17YL Lactococcus lactis 84 88 0 36Y Enterococcus faecium 78 88 0 20HL Enterococcus hirae 77 90 0 18HS Lactobacillus casei 79 88 0

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Table 3 The inhibitory effect of strains against pathogenic bacteria Indicator organisms LAB strains 2HL 6HL Salmonella typhimurium 13.2±0.6 3±0.5 Escherichia coli 026 15±0.3 5±0.4 Escherichia. coli 0157 11.9±0.4 0±0.0 Staphylococcus aureus 10.7±0.6 6±0.6 Bacillus cereus 13±1.3 0±0.0 Gardnerella vaginalis 21.3±0.8 0±0.0 Listeria monocytogenes 9±1.0 2.9±0.3 Klebsiella pneumoniae 10±0.3 0±0.0 Enterococcus faecalis 12±0.8 0±0.0 Shigella flexneri 13.1±0.4 7.7±0.8 Pseudomonas aeroginosa 11±0.3 0±0.0 Candida albicans 8±1.3 0±0.0 Serratia marcesens 9±0.3 0±0.0 Streptococcus mutans 14.4±0.4 0±0.0 Staphylococcus saprophyticus 11±1.0 0±0.0

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Notes: values are mean ± standard deviations of triplicate measurements. Data represents the mean diameter (mm) of areola around replicated wells.

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Figure 1The susceptibility spectrum of strain 6HL (Enterococcus durans) against antibiotics including vancomycin, tetracycline, ampicillin, penicillin, erythromycin, clindamycin, gentamycin, chloramphenicol and sulfamethoxazol

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