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Inhibition of Staphylococcus aureus by crude and fractionated extract from

lactic acid bacteria

Article  in  Beneficial Microbes · September 2014

DOI: 10.3920/BM2014.0021 · Source: PubMed

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Beneficial Microbes, September 2014; 5(3): 1-12 Wageningen Academic P u b l i s h e r s

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0021 1

1. Introduction

Staphylococcal infections represent a grave threat to humans. Over the past few decades, Staphylococcus aureus has gained much attention as one of the most common etiological agents of skin and soft tissue infections (Anderson et al., 2008; Charlier et al., 2009). In recent years, S. aureus has evolved into drug-resistant virulent variants (MRSA) leading to increased complications in treatment (Chambers, 2005). Thus, the use of new and novel antimicrobial substances is needed to treat S. aureus infections via non-antibiotic measures.

Lactic acid bacteria (LAB) are a group of non-spore forming Gram-positive bacteria with documented gut health potential. Numerous strains of LAB have great potential beyond gut well-being, including use as food biopreservatives and improving hypercholesterolaemia (Liong et al . , 2007; Rattanachaikunsopon and Phumkhachorn, 2010). Recently, it has been promoted that LAB have a great potential to promote skin health (Oh et al., 2006). LAB are currently seen as a feasible alternative to decolonise MRSA and treat staphylococcal skin infections, as LAB have a long history of safe use without increasing the risk of multidrug resistance of the pathogen. LAB have been reported to inhibit S. aureus isolated from foods and human vaginal tract (Charlier

Inhibition of Staphylococcus aureus by crude and fractionated extract from lactic acid bacteria

C.-B. Wong1, B.-Y. Khoo2, S. Sasidharan2, W. Piyawattanametha3,4, S.H. Kim5, N. Khemthongcharoen3,4, M.-Y. Ang6, L.-O. Chuah1 and M.-T. Liong1*

1School of Industrial Technology, Universiti Sains Malaysia, 11800, Penang, Malaysia; 2Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800, Penang, Malaysia; 3Integrated Biosensor Laboratory, National Electronics and Computer Technology Center, Pathumthani 12120, Thailand; 4Advanced Imaging Research Center, Faculty of Medicine, Chulalongkorn University, Pathumwan 10330, Bangkok, Thailand; 5Division of Food Bioscience and Technology, Korea University, Seoul 136-701, South Korea; 6Analytical Department, Fisher Scientific Sdn Bhd, Shah Alam, 40400, Selangor, Malaysia; mintze.liong@usm.my

Received: 15 February 2014 / Accepted: 4 April 2014 © 2014 Wageningen Academic Publishers

ReseaRch aRtIcleabstract

Increasing levels of antibiotic resistance by Staphyloccocus aureus have posed a need to search for non-antibiotic alternatives. This study aimed to assess the inhibitory effects of crude and fractionated cell-free supernatants (CFS) of locally isolated lactic acid bacteria (LAB) against a clinical strain of S. aureus. A total of 42 LAB strains were isolated and identified from fresh vegetables, fresh fruits and fermented products prior to evaluation of inhibitory activities. CFS of LAB strains exhibiting a stronger inhibitive effect against S. aureus were fractionated into crude protein, polysaccharide and lipid fractions. Crude protein fractions showed greater inhibition against S. aureus compared to polysaccharide and lipid fractions, with a more prevalent effect from Lactobacillus plantarum 8513 and L. plantarum BT8513. Crude protein, polysaccharide and lipid fractions were also characterised with glycine, mannose and oleic acid being detected as the major component of each fraction, respectively. Scanning electron microscopy revealed roughed and wrinkled membrane morphology of S. aureus upon treatment with crude protein fractions of LAB, suggesting an inhibitory effect via the destruction of cellular membrane. This research illustrated the potential application of fractionated extracts from LAB to inhibit S. aureus for use in the food and health industry.

Keywords: lactobacilli, antimicrobials, anti-staphylococcal

Beneficial Microbes, 2014 online ARTICLE IN PRESS

C.-B. Wong et al.

2 Beneficial Microbes 5(3)

et al., 2009), mainly via the production of antimicrobial compounds, such as organic acids, diacetyl, hydrogen peroxide and antimicrobial peptides (Karska-Wysocki et al., 2009). For instance, the LAB bacteriocin nisin was found to be active against numerous food pathogens, including S. aureus, and has been employed as a food biopreservative to enhance food safety (Karam et al., 2013). Recently, LAB bacteriocins have been reported to promote wound healing via reduction of bacteria-induced inflamed acne lesions (Oh et al., 2006), thus leading to increasing interest in the utilisation of LAB in dermatology. For instance, bacteriocin from Lactococcus sp. HY 449 was able to inhibit the growth of skin inflammatory bacteria, such as S. aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and Propionibacterium acnes (Oh et al., 2006).

Many studies on the antimicrobial properties of LAB were attributed to organic acids and protein-based extracts. However, to our knowledge, little information is available on polysaccharide- and/or lipid-based antimicrobial substances produced by LAB. The aim of this study was to isolate and identify potential LAB inhibitory against a model food and dermal pathogen, S. aureus. In addition, the protein-, polysaccharide- and lipid-based cell-free supernatants (CFS) of LAB were fractionated, evaluated and characterised.

2. Materials and methods

Isolation of lactic acid bacteria

LAB strains were isolated from locally fermented products, fresh fruits and vegetables (Penang, Malaysia). Food samples were homogenised with sterilised distilled water in a blender (Waring, East Windsor, NJ, USA), serially diluted with peptone water (Merck, Darmstadt, Germany). The bacterial isolates were enumerated using the pour plate method with De Man, Rogosa and Sharpe agar (MRS; Biomark, Maharashtra, India) supplemented with 3% (v/v) L-cysteine hydrochloride (HiMedia, Mumbai, India). Plates were incubated at 37 °C and colonies were examined via Gram staining. Gram-positive rods or cocci were selected, cultured and stored at -20 °C in sterile glycerol (40% v/v) (Yeo and Liong, 2009). Cultures were activated successively three times in MRS broth supplemented with 3% (v/v) L-cysteine hydrochloride at 37 °C for 24 h prior to further analysis.

Identification of lactic acid bacteria

Total genomic DNA of each isolate was extracted using a commercial DNA extraction kit (DongSheng Biotech, Guangzhou, China P.R.) and used as a template for polymerase chain reaction (PCR) amplification. PCR primers used were 16Sf (5'-GCTGGCGGCATGCTTAACACAT-3') and 16Sr (5'-GGAGGTGATCCAGCCGCAGGT-3'). Amplification was performed in a MyCycler thermal cycler (Bio-Rad, Hercules, CA, USA) with the programme as

previously described by Ruiz et al. (2000). The sequencing of purified PCR products was performed by the Centre for Chemical Biology (CCB, Universiti Sains Malaysia, Malaysia), and nucleotide sequences of the 16S rRNA gene were analysed using the BLAST program from NCBI (http://www.ncbi.nlm.nih.gov/).

antimicrobial activity of cell-free supernatant

CFS from identified LAB strains were prepared by centrifugation and evaluated for inhibition against a clinical isolate of S. aureus (General Hospital, Penang, Malaysia) as a target pathogen using the microtiter plate assay as described by Holo et al. (1991). Unfermented MRS broth was used as a control. Microplates were incubated for 20 h at 37 °C. Growth inhibition of S. aureus was measured spectrophotometrically at 600 nm by using a HALO MPR-96 Microplate Reader (Dynamica, Zug, Switzerland) at time intervals of 2 h (Turcotte et al., 2004). LAB strains exhibiting a statistically stronger inhibition against S. aureus were selected for further analysis. To exlude antimicrobial effects attributed to organic acids, CFS of the selected LAB strains was adjusted to pH 6.5 with 1 M NaOH. The antimicrobial activity of neutralised CFS was then determined and the growth inhibition of S. aureus was calculated as: growth OD600 nm of samples% of growth OD600 nm = × 100% growth OD600 nm of control

Neutralised MRS broth was used as a control.

Determination of acetic and lactic acid

The concentration of organic acids in CFS produced by selected LAB strains was determined according to the method described by Dubey and Mistry (1996) using a high performance liquid chromatography (HPLC) system equipped with an ultraviolet-visible detector (Shidmadzu, Kyoto, Japan) set at 220 nm and a Luna C18(2) column (150×4.6 mm, 5 µm; Phenomenex, Torrance, CA, USA). HPLC grade acetic and lactic acid (Sigma-Aldrich, Steinheim, Germany) were used as standards (Yeo and Liong, 2009).

Fractionation of cell-free supernatant

Protein fractionation was performed by adding solid ammonium sulphate (80% (w/v) saturation) with constant stirring and left to stand for 24 h at 4 °C (Ivanova et al., 2000). The precipitates were recovered by centrifugation (8,000×g, 10 min, 4 °C), resuspended in 25 mM of ammonium acetate buffer (pH 6.5), and filtered through a 0.22 µm cellulose acetate syringe filter (Sartorious Stedium, Göttingen, Germany) prior to use. Polysaccharide fractionation was performed by adding cold ethanol (99.5%; QRec, Rawang, Malaysia) to CFS at 1:3 ratio (v/v) and left to stand for 24 h

Please cite this article as 'in press' Beneficial Microbes

Inhibition of Staphylococcus aureus

Beneficial Microbes 5(3) 3

at 4 °C (Orsod et al., 2012; Pandey et al., 2010). The crude polysaccharide fractions were recovered by centrifugation (7,000×g, 20 min, 4 °C), the pellets were resuspended in sterile deionised water at 1:1 ratio (w/v). Lipid fractionation was performed as described by Bligh and Dryer (1959) with some modifications, whereby chloroform (99.5%):methanol (99.9%) solvent (1:2; v/v) (Qrec, Selangor, Malaysia) was added to CFS at 1:3 ratio (v/v) and vortexed. Chloroform and distilled water were subsequently added to the mixture at 1:1 ratio (v/v) and vortexed. The mixture was centrifuged at 1000×g for 5 min at 4 °C and the crude lipid fraction (bottom layer) was collected and dried under nitrogen flux. The dried crude lipid fraction was resuspended in 500 µl sterile distilled water prior to use. In the same way, MRS broth was fractionated for use as a control. The microtiter plate assay described above was used to determine the antimicrobial activity of each fraction against S. aureus.

characterisation of fractionated cell-free supernatant

Fractionated CFS from LAB strains with stronger antimicrobial activities were characterised. Freeze-dried crude protein fractions were evaluated for amino acid composition according to the method as described by Bidlingmeyer et al. (1984) using a reversed phase high performance liquid chromatography (RP-HPLC) system on a Superiodex ODS column (5 µm, 4.61×50 mm, Shiseido, Tokyo) equipped with an ultraviolet-visible detector (JASCO UV detector Model Uvidec-100-VI; Japan Spectroscopic, Tokyo, Japan. A standard amino acid mixture was used (Sigma-Aldrich). The crude polysaccharide fraction was first hydrolysed in boiling water with 2 M HCl at 100 °C for 3 h, followed by neutralisation prior to determination of monosaccharide composition (Levander et al., 2001). An HPLC system equipped with a refraction index detector (Shimadzu) and a Waters Sugar Pak 1 column (300×6.5 mm, 5 µm; Waters Corporation, Milford, MA, USA) was used to analyse the monosaccharide concentrations. Column temperature was set at 85 °C with a degassed mobile phase of filtered deionised water; the flow rate was 0.2 ml/min. HPLC grade monosaccharide standards kit (Sigma-Aldrich) was used as a standard. The crude lipid fraction was converted to fatty acid methyl esters through sodium methoxide catalysis. A gas chromatography-mass spectrophotometer system (GC-MS) (GCMS-QP2010 Ultra; Shimadzu) equipped with a BPx-70 capillary column (60 m × 0.25 mm, i.d., 0.25 µm film; SGE, Ringwood, Australia) was used for the quantification of fatty acids. The initial oven temperature was 50 °C, increased to 160 °C at a rate of 4 °C/min (maintained for 10 min), followed by an increase to 200 °C at a rate of 1 °C/min (maintained for 2 min), and finally increased to 210 °C at a rate of 10 °C/min (maintained for 10 min). Helium gas was used as the carrier at a flow rate of 0.8 ml/min with split ratio 1:50. The injector temperature was 210 °C. The mass spectra were recorded at an ionisation energy of 0.97 kV with an ion source

temperature of 200 °C. The mass scan was performed at a range of 29 m/z to 550 m/z with a scan speed of 10,000. The injection volume was 10 µl. Supelco 37 Component FAME Mix (Sigma-Aldrich) was used as a standard.

scanning electron microscopy

Morphological changes of S. aureus upon treatment with the protein fraction of CFS of selected LAB strains for 20 h at 37 °C was examined using scanning electron microscope as previously described by Lye et al. (2010). Untreated cells of S. aureus were used as a control.

statistical analysis

Data analysis was carried out with SPSS software (version 19.0; IBM SPSS, Armonk, NY, USA). Repeated measures analysis of variance (ANOVA) was used for time-based analysis. One-way ANOVA was used to determine significant differences between means at a significance level of α=0.05. Tukey’s test was used to perform multiple comparisons between means. All data were presented as mean ± standard deviation from three separate runs.

3. Results

Isolation and identification of lactic acid bacteria

A total of 42 bacterial cultures were isolated from fresh vegetables (n=22), fresh fruits (n=13) and fermented products (n=7), 39 of which were Gram-positive bacteria, and identified via 16S rRNA sequencing. The LAB isolates mainly consisted of Lactobacillus (74.4%), followed by Weissella (15.4%), Leuconostoc (5.1%) and Pediococcus (5.1%), with species such as Lactobacillus brevis, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Leuconostoc mesenteroides, Pediococcus pentosaceus, Weissella cibaria and Weissella confusa.

antimicrobial activity of isolates

CFS of LAB isolated from the three food sources exhibited inhibitory effects (P

C.-B. Wong et al.

4 Beneficial Microbes 5(3)

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B

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L. fermentum 1912a

Lactobacillus fermentum 1913AbPediococcus pentosaceus 1913Bab L. fermentum 8513a

Leuconostoc mesenteroides 8914Ac controld (untreated Staphylococcus aureus)

Lactobacillus casei 1512Af

Lactobacillus casei 1512Dcd

L. plantarum 1512Fcd

Lactobacillus brevis 1513AfLactobacillus fermentum 1512Ncd

L. plantarum 8513a

L. fermentum 8513Lgh

Leuconostoc mesenteroids 8514BdeL. fermentum 8515Ai

L. fermentum 8513Ahi

L. casei 1512BabcL. brevis 1512Mfg

Lactobacillus plantarum BT8513a

L. fermentum 8513Dab

L. fermentum 8513Mbcd

L. fermentum 8513QhiL. fermentum 8513Pecontrolj (untreated Staphylococcus aureus)

Lactobacillus paracasei 1512LbcdL. casei 1513Ccd

L. fermentum 8513Rhi

Lactobacillus fermentum 1512jcd

Weisella cibaria 1512Labcd

Lactobacillus brevis 1515ab

W. cibaria 1512Ibcd W. cibaria 8513Jabcd

L. casei 8513Ge

L. fermentum 1512Kcd

Lactobacillus casei 1513Fbcd W. cibaria 1514Hd

L. fermentum 8513Hf

W. cibaria 8513a Weisella confusa 8513Kabc

controlg (untreated Staphylococcus aureus)

Figure 1. antimicrobial activity of cell-free supernatant of lactic acid bacteria isolated from (a) fermented products, (B) fresh fruits and (c) fresh vegetables against growth of Staphylococcus aureus. each point represents the mean of triplicates from three separate runs. error bars represent standard deviation of the means. Means with different superscript letters are significantly different from one another (P

Inhibition of Staphylococcus aureus

Beneficial Microbes 5(3) 5

acetic and lactic acids

All the five strains studied produced both lactic and acetic acids, the concentration of lactic acid being higher than that of acetic acid (P

C.-B. Wong et al.

6 Beneficial Microbes 5(3)

L. plantarum 8513 and BT8513, ranging from 0.02 to 1.30%. On the other hand, the protein fraction from L. plantarum 8513 and BT8513 contained higher amounts of serine, histidine, arginine, tyrosine and methionine compared to the control, ranging from 0.20 to 1.00%. Glycine was the major component in the crude protein fractions of all samples studied and was prevalent from L. plantarum 8513 (P

Inhibition of Staphylococcus aureus

Beneficial Microbes 5(3) 7

of L. plantarum 8513 and BT8513was significantly (P

C.-B. Wong et al.

8 Beneficial Microbes 5(3)

Gram-negative bacteria, such as Listeria monocytogenes, S. aureus, Escherichia coli and Salmonella (Atanassova et al., 2003; De Vuyst and Leroy, 2007). Glycine was detected as the major component in the crude protein fractions of CFS from L. plantarum 8513 and BT8513. Rogne et al. (2009) described the importance of glycine residues for the antimicrobial activity of plantaricin J and K. Replacing the

individual glycine residues with other residues generally reduced antimicrobial activities. Plantaricin 1.25β produced by L. plantarum TMW1.25 and the anti-Listeria bacteriocin, plantaricin C19 produced by L. plantarum C19 were reported as rich in glycine (Atrih et al., 2001), while glycine-rich features were also associated with the characteristics for bacteriocins (Remiger et al., 1999).

table 3. Fatty acids composition of crude lipid fractions extracted from Lactobacillus plantarum strains.1

Fatty acids strains of laB

L. plantarum 8513 L. plantarum Bt8513 control2

C6:0 (hexanoic acid) nd3 0.002±0.003fA 0.003±0.006eA

C8:0 (octanoic acid) 0.092±0.080fA nd 0.08±0.018eA

C10:0 (oecanoic acid) nd nd ndC11:0 (undecanoic acid) nd nd ndC12:0 (dodecanoic acid) 7.518±0.215cA 8.099±0.308cA 7.79±0.732cA

C13:0 (tridecanoic acid) nd 0.002±0.003aF ndC14:0 (myristic acid) 1.239±0.043eA 0.808±0.036dC 0.983±0.087eB

C14:1 (myristoleic acid) nd nd ndC15:0 (pentadecanoic acid) nd 0.006±0.006fA 0.026±0.045eA

C15:1(cis-10-pentadecenoic acid) nd nd ndC16:0 (palmitic acid) 17.415±0.334bA 13.897±0.356bB 18.571±1.085bA

C16:1 (palmitoleic acid) nd 0.045±0.077fA 0.005±0.008eA

C17:0 (heptadecanoic acid) nd nd 0.088±0.077eA

C17:1 (cis-10-heptadecenoic acid) 0.006±0.006fA nd ndC18:0 (stearic acid) 6.05±0.116dA 5.591±0.138dA 6.062±0.355dA

C18:1N9T (elaidic acid) 0.018±0.031fA nd 0.026±0.045eA

C18:1n9c (oleic acid) 66.635±1.011aB 71.548±0.784aA 65.534±2.274aB

C18:2N6T (linolelaidic acid) nd nd ndC18:2N6C (linoleic acid) 0.146±0.253fA nd 0.009±0.015eA

C18:3N6 (γ-linolenic acid) nd nd ndC18:3N3 (linolenic acid) nd nd ndC20:0 (arachidic acid) 0.507±0.036fA 0.129±0.223efA 0.475±0.041eA

C20:1 (cis-11-eicosenoic acid) 0.368±0.035fA 0.02±0.004efA 0.345±0.040eA

C20:2 (cis-11,14-eicosadienoic acid) nd nd ndC21:0 (heneicosanoic acid) nd nd ndC20:3N6 (cis-8,11,14-eicosatrienoic acid) nd nd ndC20:4N6 (arachidonic acid) nd 0.002±0.003fA ndC20:3N3 (cis-11,14,17-eicosatrienoic acid) nd nd 0.001±0.002eA

C22:0 (behenic acid) 0.002±0.004fA nd ndC22:1N9 (erucic acid) nd nd 0.002±0.002eA

C20:5N3 (cis-5,8,11,14,17-eicosapentaenoic acid) nd 0.001±0.003fA ndC23:0 (tricosanoic acid) 0.003±0.005fA nd ndC22:2 (cis-docosadienoic acid) nd nd ndC24:0 (tetracosanoic acid) 0.002±0.004fA nd ndC24:1 (cis-tetracosenoate acid) nd nd ndC22:6N3 (docosahexaenoic acid) nd nd nd

1 Each value is expressed as mean ± standard deviation of the mean from three separate runs. Means in the same column with different lowercase letters and means in the same row with different uppercase letters are significantly different (P

Inhibition of Staphylococcus aureus

Beneficial Microbes 5(3) 9

Hence, we postulate that glycine as the major amino acid residue attributed to the significant antimicrobial activity of L. plantarum 8513 and BT8513. Furthermore, the amino acid composition of the protein fractions from both strains confirmed the hydrophobic character of the active molecule, as amino acids, such as leucine, phenylalanine, glycine, alanine, proline, valine and isoleucine, were prevailing. Hydrophobicity is a common feature of several potent bacteriocins isolated from L. plantarum (Muriana and Klaenhammer, 1991).

Apart from proteins, crude polysaccharide fractions of CFS from L. plantarum 8513 and BT8513 also inhibited the growth of S. aureus. This might be attributed to the production of exopolysaccharides (EPS). EPS from LAB was reported to exert anti-tumour and immunostimulatory activities and also antimicrobial activity against food pathogenic bacteria (Chabot et al., 2001; Fanning et al., 2012). The sugar monomers, modes of linkage, branching and substitution, which are unique in exerting functionality, might attribute to the actions of EPS. For instance, EPS from Lactobacillus kefiranofaciens ATCC 43761 consisting of equal amounts of glucose and galactose could increase mucosal immune response in mice (Chabot et al., 2001). However, studies on the antimicrobial properties of EPS from L. plantarum are limited. In the present study, the crude polysaccharide fractions of CFS from L. plantarum 8513 and BT8513, which consisted mainly of mannose and glucose at concentrations lower than the control, exhibited an inhibitory effect against S. aureus. It is postulated that the arrangement of the sugar monomers outweighed the importance of concentration in exerting antimicrobial effects. Further investigations are needed to elucidate this issue.

Lipid fractions of CFS from L. plantarum 8513 and BT 8513 also exhibited inhibition against S. aureus, suggesting the presence of antimicrobial fatty acids. Generally, long- and medium-chain fatty acids and monoglycerides are more prevalent to exhibit antimicrobial effects compared to short-chain fatty acids, mainly via growth inhibition or direct bactericidal effects (McGaw et al., 2002). In the present study, oleic acid (C18:1n9c) was the main fatty acid detected. Oleic acid has been reported to be bactericidal against antibiotic-resistant S. aureus (Shina et al., 2007). Both L. plantarum 8513 and BT8513 were grown under the same conditions but the lipid fraction of CFS from L. plantarum BT8513 contained a higher oleic acid concentration, which subsequently inhibited S. aureus better than L. plantarum 8513. To date, this study is the first to demonstrate the inhibitory effect against S. aureus of a lipid fraction from LAB strains dominated by oleic acid.

Scanning electron microscopy revealed morphological changes of S. aureus upon exposure to the protein fraction from L. plantarum 8513 and BT8513 compared

to the control. Bacteriocins and/or antimicrobial peptides could increase membrane permeability of susceptible bacteria leading to cellular deformation and cell leakage, accompanied by the release of intracellular components to the environment, thus resulting in cell lysis and eventual death (Dalie et al., 2010; Klayraung and Okonogi, 2009). We postulate that this effect is illustrated by the extracellular debris present in treated cells but not in control cells. In accordance with many other bacteriocins of L. plantarum, such bacteriocidal effects were also revealed by plantaricin MG, a bacteriocin active against Salmonella typhimurium, and plantaricin 149 (Gong et al., 2010; Lopes et al., 2009). This microscopic evaluation justified that the CFS protein fraction from L. plantarum 8513 and BT8513 exerted antimicrobial effects on S. aureus via membrane disruption and permeability.

5. conclusions

LAB strains were successfully isolated from local fermented products, fresh vegetables and fruits, and identified via 16S rRNA gene sequencing analysis. CFS of L. plantarum 8513 and BT8513 exerted a strong inhibitory effect against S. aureus, with a more prevalent effect of the CFS protein fraction. The results of the present study suggested that these LAB strains produced proteinaceous antimicrobial compounds inhibiting S. aureus via membrane disruption, as confirmed by scanning electron microscopy. These findings offer a possibility for the protective use of LAB against infections associated with S. aureus. Nevertheless, further studies are crucial to identify the proteinaceous compounds and envisage the mechanisms involved.

acknowledgements

This work was financially supported by the Science Fund Grant (305/PTEKIND/613222) provided by the Malaysian Ministry of Science, Technology and Innovation (MOSTI), the FRGS grant (203/PTEKIND/6711239) provided by the Malaysian Ministry of Higher Education (MOHE), USM RU grants (1001/PTEKIND/815085), and USM Fellowship provided by Universiti Sains Malaysia.

References

Ammor, S., Tauveron, G., Dufour, E. and Chevallier, I., 2006. Antibacterial activity of lactic acid bacteria against spoilage and pathogenic bacteria isolated from the same meat small-scale facility, screening and characterization of the antibacterial compounds. Food Control 17: 454-461.

Anderson, M.E.C., Lefebvere, S.L. and Weese, S.J., 2008. Evaluation of prevalence and factors for methicillin-resistant Staphylococcus aureus colonization in veterinary personnel attending an international equine veterinary conference. Veterinary Microbiology 129: 410-417.

Beneficial Microbes Please cite this article as 'in press'

C.-B. Wong et al.

10 Beneficial Microbes 5(3)

Atanassova, M., Choiset, Y., Dalgalarrondo, M., Chobert, J.M., Dousset, X., Ivanova, I. and Hacrtle, T., 2003. Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3. International Journal of Food Microbiology 87: 63-73.

Atrih, A., Rekhif, N., Moir, A.J.G., Lebrihi, A. and Lefebvre, G., 2001. Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19. International Journal of Food Microbiology 68: 93-104.

Bidlingmeyer, B.A., Cohen, S.A. and Tarvin, T.L., 1984. Rapid analysis of amino acids using pre-column derivatization. Journal of Chromatography 336: 93-104.

Bligh, E.G. and Dryer, W.J., 1959. A rapid method for total lipid extraction and purification. Canadian Journal of Physiology and Pharmacology 3: 911-917.

Chabot, S., Yu, H.L., De Léséleuc, L., Cloutier, D., Van Calsteren, M.R., Lessard, M., Roy, D., Lacroix, M. and Oth, D., 2001. Exopolysaccharides from Lactobacillus rhamnosus RW-9595M stimulate TNF IL-6 and IL-12 in human and mouse cultured immunocompetent cells, and IFN-gamma in mouse splenocytes. Lait 81: 683-697.

Chambers, H.F., 2005. Community-associated MRSA-resistance and virulence converge. The New England Journal of Medicine 352: 1485-1487.

Charlier, C., Cretenet, M., Even, S. and Le Loir, Y., 2009. Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives. International Journal of Food Microbiology 129: 30-39.

Dalie, D.K.D., Deschamps, A.M. and Richard-Forget, F., 2010. Lactic acid bacteria – potential for control of mould growth and mycotoxins. Food Control 21: 370-380.

De Vuyst, L. and Leroy, F., 2007. Bacteriocins from lactic acid bacteria: production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology 13: 194-199.

Dubey, U.K. and Mistry, V.V., 1996. Growth characteristic of bifidobacteria in infant formulas. Journal of Dairy Science 79: 1146-1155.

Fanning, H., Hall, L.J., Cronin, M., Zomer, A., MacShary, J., Goulding, P., Motherway, M.O., Shanahan, F., Nully, K., Dougan, G. and Van Sinderen, D., 2012. Bifidobacterial surface-exopolysaccharide facilitates commensal-host interaction through immune modulation and pathogen protection. Proceedings of the National Academy of Sciences of the USA 109: 2108-2113.

Gong, H.S., Meng, X.C. and Wang, H., 2010. Mode of action of plantaricin MG, a bacteriocin active against Salmonella typhimurium. Journal of Basic Microbiology 50: 37-45.

Holo, H., Nilssen, O. and Nes, I.F., 1991. Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. Journal of Bacteriology 173: 3879-3887.

Ivanova, I., Kabadjova, P., Pantev, A., Danova, S. and Dousset, X., 2000. Detection, purification and partial characterization of a novel bacteriocin substance produced by Lactococcus lactis subsp. lactis B14 isolated from boza – Bulgarian traditional cereal beverage. Biocatalysis: Fundamentals and Applications 41: 47-53.

Karam, L., Jama, C., Mamede, A.S., Boukla, S., Dhulster, P. and Chihib, N.E., 2013. Nisin-activated hydrophobic and hydrophilic surfaces: assessment of peptide adsorption and antibacterial activity against some food pathogens. Applied Microbiology and Biotechnology 97: 10321-10328.

Karska-Wysocki, B., Bazo, M. and Smoragiewicz, W., 2009. Antibacterial activity of Lactobacillus acidophilus and Lactobacillus casei against methicillin-resistant Staphylococcus aureus (MRSA). Microbiological Research 165: 674-686.

Klayraung, S. and Okonogi, S., 2009. Antibacterial and antioxidant activities of acid and bile resistant strains of Lactobacillus fermentum isolated from miang. Brazilian Journal of Microbiology 40: 757-766.

Levander, F., Svensson, M. and Radstrom, P., 2001. Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium. BMC Microbiology 1: 1-5.

Liong, M.T. and Shah, N.P., 2005. Production of organic acids from fermentation of mannitol, fructooligosaccharide and inulin by a cholesterol removing Lactobacillus acidophilus strain. Journal of Applied Microbiology 99: 783-793.

Liong, M.T., Dunshea, F.R. and Shah, N.P., 2007. Effects of a symbiotic containing Lactobacillus acidophilus ATCC 4962 on plasma lipid profiles and morphology of erythrocytes in hypercholesterolaemic pigs on high- and low- fat diets. British Journal of Nutrition 98: 736-744.

Lopes, J.L.S., Nobre, T.M., Siano, A., Humpola, V., Bossolan, N.R.S., Zaniquelli, M.E.D., Tonarelli, G. and Beltramini, L.M., 2009. Disruption of Saccharomyces cerevisiae by plantaricin 149 and investigation of its mechanism of action with biomembrane model systems. Biochimica et Biophysica Acta 1788: 2252-2258.

Lye, H.S., Rahmat-Ali, G.R. and Liong, M.T., 2010. Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract. International Dairy Journal 20: 169-175.

McGaw, L.J., Jager, A.K. and Van Staden, J., 2002. Isolation of antibacterial fatty acids from Schotia brachypetala. Fitoterapia 73: 431-433.

Muriana, P.M. and Klaenhammer, T.R., 1991. Purification and partial characterization of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088. Applied and Environmental Microbiology 57: 114-121.

Oh, S., Kim, S.H., Ko, Y., Sim, J.H., Kim, K.S., Lee, S.H., Park, S. and Kim, Y.J. 2006. Effect of bacteriocin produced by Lactococcus sp. HY449 on skin-inflammatory bacteria. Food and Chemical Toxicology 44: 552-559.

Orsod, M., Joseph, M. and Huyop, F., 2012. Characterization of exopolysaccharides produced by Bacillus cereus and Brachybacterium sp. isolated from Asian sea bass (Lates calcarifer). Malaysian Journal of Microbiology 8: 170-174.

Pandey, A., Naik, M. and Dubey, S.K., 2010. Hemolysin, protease, and EPS producing pathogenic Aeromonas hydrophila strain An4 shows antibacterial activity against marine bacterial fish pathogens. Journal of Marine Biology 1: 1-9.

Rattanachaikunsopon, P. and Phumkhachorn, P., 2010. Lactic acid bacteria: their antimicrobial compounds and their uses in food production. Annals of Biological Research 1: 218-228.

Please cite this article as 'in press' Beneficial Microbes

Inhibition of Staphylococcus aureus

Beneficial Microbes 5(3) 11

Remiger, A., Eijsink, V.G.H., Ehrmann, M.A., Sletten, K., Nes, I.F. and Vogel, R.F., 1999. Purification and partial amino acid sequence of plantaricin 1.25α and 1.25β, two bacteriocins produced by Lactobacillus plantarum TMW1.25. Journal of Applied Microbiology 86: 1053-1058.

Rogne, P., Haugen, C., Fimland, G., Nissen-Meyer, J. and Kristiansen, P.E., 2009. Three-dimensional structure of the two-peptide bacteriocin plantaricin JK. Peptides 30: 1613-1621.

Ross, R.P., Morgan, S. and Hill, C., 2002. Preservation and fermentation: past, present and future. International Journal of Food Microbiology 79: 3-16.

Ruiz, A., Poblet, M., Mas, A. and Guillamon, J.M., 2000. Identification of acetic acid bacteria by RFLP of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer. International Journal of Systematic and Evolutionary Microbiology 50: 1981-1987.

Shina, S.Y., Bajpaia, K.V.K. and Kang, H.R., 2007. Antibacterial activity of eicosapentaenoic acid (EPA) against foodborne and food spoilage microorganisms. LWT – Food Science and Technology 9: 1515-1519.

Trias, R., Baneras, L., Montesinos, E. and Badosa, E., 2008. Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi. International Microbiology 11: 231-236.

Turcotte, C., Lacroix, C., Kheadr, E., Grignon, L. and Fliss, I., 2004. A rapid turbidometric microplate bioassay for accurate quantification of lactic acid bacteria bacteriocins. International Journal of Food Microbiology 90: 283-293.

Yeo, S.K. and Liong, M.T., 2009. Effect of probiotics on viability and growth characteristics of probiotics in soymilk. Journal of the Science of Food and Agriculture 90: 267-275.

Beneficial Microbes Please cite this article as 'in press'

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