بررسی اثر مهاری ایزوله های لاکتوباسیلوس فرمنتوم، لاکتوباسیلوس اسیدوفیلوس و لاکتوباسیلوس پاراکازئی جدا شده از ماست بر روی رشد و بیان ژنی انتروتوکسین A باکتری استافیلوکوکوس اورئوس

Introduction

Staphylococcal foodborne intoxication is the most common cause of foodborne illnesses reported in many countries and results from the consumption of foods containing sufficient amounts of one (or more) preformed enterotoxins. Symptoms of this intoxication have a rapid onset (2-8 h) and include nausea, violent vomiting and abdominal cramps with or without diarrhea. The disease is usually self-limiting and typically resolves within 24-48 h after onset. Occasionally it can be severe enough to warrant hospitalization, particularly when infants, the elderly or debilitated individuals are affected (Argudin et al., 2010; Marta et al., 2011; Schelin et al., 2011; Ventola et al., 2012;  Wallin-Carlquist et al., 2010). Among food commodities that are manipulated during processing, cheeses and dry sausages are the most frequent types of food involved in S. aureus foodborne intoxication (Cretent et al., 2011; Dal Bello et al., 2012; Marta et al., 2011; Moslehishad et al., 2013; Rode et al., 2010; Rosengren et al., 2010). The major antigen types of staphylococcal enterotoxin are Staphylococcal enterotoxin A (SEA) to SEJ; however, additional staphylococcal enterotoxins have recently been identified (Jakobsen et al., 2011). SEs are synthesized throughout logarithmic staphylococcal growth and also during the transition from exponential to stationary phase. They are active in high nanogram to low microgram quantities, and are resistant to proteolytic enzymes and conditions (heat treatment, low pH) that easily destroy the bacteria that produce them, hence they retain their activity in the digestive tract after ingestion (Argudin et al., 2010; Drezelle et al., 2009; Larkin et al., 2009). Among the Staphylococcal enterotoxins, enterotoxin A (SEA) is that most frequently responsible for staphylococcal food poisoning outbreaks. Enterotoxigenic S. aureus strains have different SE gene profiles and may simultaneously harbor several genes. The expression of these virulence factors in S. aureus is tightly controlled and regulated by a complex network of regulatory systems. Genes encoding SE exist on various genetic supports, most of which are mobile genetic elements including phages (sea, see, sep), plasmids (sed, sej, ser, ses, set) (Adams and Moss, 2000; Cha et al., 2006; Jakobsen et al., 2011; Le Marc and Valic, 2009; Moslehishad et al., 2013; Schelin et al., 2011). Among them, the sea gene, carried in the bacterial genome by a polymorphic family of temperate bacteriophages, comprises 771 base pairs and encodes an enterotoxin A precursor of 257 amino acid residues which is expressed from the mid-exponential phase of growth (Derzelle et al., 2009; Horsmon et al., 2006; Piper et al., 2012).

Currently, increasing foodstuff shelf life as well as enhancing food safety and human health using natural microflora are dominant issues in public health. Lactic acid bacteria (LAB) have considerable potential for these purposes due to their ability to produce antimicrobial agents including organic acids, bacteriocins, hydrogen peroxide and their capacity for pH reduction. LAB are widely distributed in nature and are typically involved in a large number of spontaneous food fermentations, among which LAB isolated from dairy products have recently received much attention as potential food preservatives due to their antagonistic activity against many food borne pathogens. LAB antagonism to the growth of S. aureus is well documented (Chapman et al., 2011; Charlier et al., 2009). However, the mechanism of this inhibition, and especially the inhibitory effect of LAB on S. aureus virulence factor expression including SE production, has not been comprehensively studied. Molecular approaches including transcriptomic and proteomic techniques are promising tools for increasing our understanding of the mechanisms involved in the growth inhibition of S. aureus and/or the modulation of the expression of its virulence factors by other ‘safe’ microorganisms, including LAB (Carey et al., 2008; Charlier et al., 2009; Laughton et al., 2006; Rode et al., 2010). The goal of this study is to evaluate the inhibitory activity of three Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus fermentum and Lactobacillus paracasei, all isolated from a traditional yoghurt, on growth and enterotoxin A production by S. aureus in mixed cultures.

Materials and Methods

Bacterial strains: Staphylococcus aureus (ATCC 29213, enterotoxin A producer) obtained from the culture collection of the Pasteur Institute, Tehran, Iran was used in this study. Three lactobacillus, lactobacillus acidophilus, lactobacillus fermentum and lactobacillus paracasei isolated from traditional yoghurt, were selected for antibacterial evaluation. These isolates were identified by 16s ribosomal RNA gene sequencing according with Kandler et al. (1986) and Axelsson (2004).

Preparation of inocula: S. aureus was sub-cultured in Trypticase Soy Broth (TSB) and incubated for 18 h at 35°C. The bacterial suspension was adjusted to an optical density (OD) of 0.1 at 600 nm using a Spectronic 20 spectrophotometer (Milton Roy Company, Houston, USA). This adjustment gave a cell concentration of 107cfu ml^-1 as determined from previously prepared standard curve data. The number of cells in suspension was established by duplicate platings of 10-fold serial dilutions on BHI agar and counting the colonies after 24 h incubation at 35°C. Lactobacillus cultures were prepared by sub-culturing in MRS (de Man, Rogosa and Sharpe) broth incubated for 24 h at 35 °C and similarly titred on MRS agar.

Co-culture of S. aureus and lactobacillus isolates: Preparation of co-cultures of Lactobacillus strains and S. aureus was performed as described by Laughton et al. (2006) with some modifications. Each lactobacillus isolate (107cfu ml^-1) was grown in 10 ml TSB followed by inoculating S. aureus (105cfu ml^-1) into the medium. TSB medium inoculated individually with S. aureus or lactobacillus was used as controls. Cultures were incubated at 25 and 35°C for 0, 24, 48 and 72 h. Bacterial titres were determined by surface plate counting of aliquots of tenfold serial dilutions spread on MRS agar and Baird Parker agar plates for Lactobacillus strains and S. aureus, respectively.

Detection of staphylococcal enterotoxins: Detection and identification of enterotoxin types was performed using the RIDASCREEN SET (R-Biopharm GmbH, Darmstadt, Germany) visual immunoassay kit, following the instructions of the manufacturer. The kit utilizes monovalent capture antibodies against SEs types A to E.

RNA extraction and cDNA synthesis: Lactobacillus isolates were co-cultured in TSB with S. aureus ATCC 29213 at both 35°C and 25°C for 24 h. The effects of the lactobacillus strains on the expression of the sea gene were evaluated by real-time PCR. Briefly, bacterial cultures were centrifuged in polypropylene tubes at 12.000 ×g for 5 min at 4°C. The supernatant was removed and total RNA from bacterial cells was extracted using the Tripure Isolation Reagent (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. RNA quality was monitored by measuring the absorbance at 260 and 280 nm (A260/280 ratio) using a NanoDrop spectrophotometer (Thermo Scientific, Nanodrop, Wilmington, USA). Synthesis of cDNA from 1-1.5 ng of RNA was conducted using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, St. Leon-Roth, Germany) with random hexamer primers according to the manufacturer’s instructions. For each RNA sample a non-reverse transcribed control (NRTC) was included to detect contaminating genomic DNA. cDNA synthesis was performed in a DNA Engine ABI thermocycler 2720 (Applied Biosystems, Foster City, CA, USA.) using the following cycling conditions: 65ºC for 5 min, 42 ºC for 60 min and followed by 70 ºC for 5 min. Briefly, 1-1.5 ng of RNA was reverse transcribed with 1 µl random hexamer primers, 10 µM of each dNTP (2 µl), 4µl 5x first strand buffer, 1 µl Ribolock (RNase inhibitor 20 U/µl) and 1 µl RevertAidTM M-MuLV reverse transcriptase (200 U/µl).

Real-time PCR: Real-time PCR was performed on cDNA (1 µl) template in 20 µl final volume, containing 10 µl of Power SYBR Green^© II PCR master mix (Thermo Scientific, Southampton, UK), 0.5 µl of each primer and 8 µl nuclease-free water. Forward and reverse sea primers were 5′-TTGGAAACGGTTAAAACGAA-3′ and 5’-GAACCTTCCCATCAAAAACA-3’, respectively; forward and reverse primers for the 16s rRNA reference gene (Lee et al. 2007) were 5′-CCGCCTGGGGAGTACG-3′ and 5′-AAGGGTTGCGCTCGTTGC-3′, respectively. Real-time PCR conditions were as follows: initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 10 sec, primer annealing at 55 °C for 30 sec and extension at 72 °C for 30 sec, followed by melting curve analysis at 65-95 °C (temperature transition rate of 0.1 °C/sec) based on continuous fluorescence reading. Fluorescence data were collected at the end of each cycle on a Quantitative PCR system (Rotorgene-6000, Corbett, Sydney, Australia). The relative expression of the sea gene was calculated versus the calibration sample and the endogenous control (16s rRNA) to normalize the sample input amount, and the levels of sea expression of treated and untreated samples were compared. All determinations were done in triplicate.

Statistical analysis: Experimental data were analyzed using the SPSS 17.0 statistical software. Data were expressed as the mean ± SD. Statistical differences were examined using independent student’s t-Test. A p value less than 0.05 was considered to be statistically significant.

Results

 Effect of lactobacillus strain on S. aureus growth: The effect of LAB strains on S. aureus counts at 25 °C is shown in Fig. 1. In the absence of LAB inoculation S. aureus grew during the 24 h incubation period to 9.4 log10 CFUml^-1 and bacterial counts remained unchanged over the following 48 h. Co-culture with LAB strains however had a significantly inhibitory effect on S. aureus growth since the cell densities of S. aureus co-cultured with L. fermentum, L. paracasei and L. acidophilus rose to only 6.3, 6.22 and 6.36 log after 24 h, respectively. No significant changes were found in S. aureus counts during the following 48 h incubation period. The results of S. aureus growth at 35 °C in the presence of LAB strains are shown in Fig. 2. LAB strains reduced the cell densities of S. aureus by 2-3 log compared to the control at all evaluation times. There were statistically significant differences between the results at 25 and 35 °C; the inhibition being greater at 25 °C compared to 35 °C.

 Enterotoxins assay: The pattern of toxin production by S. aureus in the presence of LAB strains is shown in Table 1. The production of enterotoxins (A, E, C) in the co-culture and control media was confirmed using a RIDASCREEN kit. All the LAB strains inhibited the production of SEA, SEC and SEE at 25 °C as well as at 35 °C.

 Gene expression assay: The primers specific for the sea and 16s rRNA genes yield PCR products of 120 and 240 bp, respectively (Fig. 3). Real-time PCR was performed to assess sea gene expression in bacterial cultures relative to the expression of the 16s rRNA gene (endogenous control). No background signal was seen for controls lacking template or reverse transcriptase. The results of sea gene expression of S. aureus cultured in the presence of LAB strains at two different temperatures are shown in Fig. 4. Co-culture of S. aureus in the presence of each of the Lactobacillus strains showed reduced levels of the sea gene transcript compared to the control. However, the level of this reduction was strain and temperature dependent. At 25 °C the transcript level of sea in S. aureus co-cultured with L. fermentum, L. paracasei and L. aciduphilus was decreased 8.79, 7.08 and 4.78 fold, respectively, compared to the control, whereas at 35°C, these reductions were, 3.26, 5.21 and 9.86 fold respectively. Moreover, the greatest sea down-regulation was observed following co-incubation of S. aureus with L. fermentum at 25 °C while L. acidophillus showed more inhibition at 35 °C.

Table 1. Results of growth indices in different groups at 30 and 60 days of experiment.

Significant differences with control in level of 0.05 are marked by * sign.

Table 2. Effect of different concentration of Aloe vera on hematological parameters.

Significant differences with control in level of 0.05 are marked by * sign.

Table 3. The serum Lysozyme and bactericidal activity as well as serum total protein and globulin level in experimental groups.

Significant differences with control in level of 0.05 are marked by * sign.

Discussion

From the food safety and human health points of view, inhibition of the growth and/or toxin production of foodborne pathogens including S. aureus by LAB are promising methods. LAB isolated from traditional foods are probably the most suitable candidates for improving the microbiological safety of foods since they are well adapted to the conditions of fermented products and should therefore be more competitive than LAB isolated from non-fermented sources. Possible mechanisms responsible for the antagonistic effects of LAB on S. aureus include competition for nutrients and the production of bacteriocins, hydrogen peroxide and fatty acids.

The inhibitory potential of LAB on S. aureus growth has been described in a number of studies (Alomar et al., 2008; Charlier et al., 2009; Cretent et al., 2011; Delbes et al., 2006; Kazemi Darsanaki et al., 2012; Otero et al., 2006; Rode et al., 2010; Rosengren et al., 2010). In the current work the effects of three lactobacillus strains isolated from yoghurt (L. acidophilus, L. paracasei and L. fermentum) on the growth of S. aureus were studied under co-culture conditions. The bacterial counts of S. aureus in co-cultures were found to be significantly lower than that in control medium, and growth inhibition at 25 °C was seen to be greater than that at 35 °C. It is well documented that the degree of growth inhibition varies depending on growth medium, the temperature and the strain tested (Chapman et al., 2011; Cotter et al., 2013; Lee and Salminen, 2009; Varalakshmi et al., 2014). Our experimental growth results are in accordance  with Troller and Frazier (1963) who reported that the maximum inhibition of growth of S. aureus in association with other organisms occurred at temperatures of 20 to 25 °C. Pereira et al. (2009) reported similar findings regarding the inhibition of S. aureus by psychrophilic saprophytes. Previous reports also suggest that the growth of S. aureus is inhibited to a greater degree at temperatures lower than 30 °C when cultured in the presence of other microorganisms (Alomar et al., 2008; Dos Santos et al., 2016; Haines and Harmon, 1973; Troller and Frazier, 1963).

Among S. aureus virulence factors, enterotoxins play a particularly important role and are the principal threat in foodstuffs. In our study, the results of enterotoxin detection show that the production of SEA, SEC and SEE is suppressed in the presence of the lactobacillus isolates studied. This inhibitory effect has also been reported by other researchers (Haines and Harmon, 1973; Troller and Frazier, 1963). Despite the importance of enterotoxins, regulation of their expression and that of enterotoxin-like proteins is still poorly documented. Moreover, the effect of bacteria-bacteria interactions on enterotoxin gene expression in S. aureus has not been well studied.

Molecular approaches including transcriptomics are promising methods for increasing our knowledge of the mechanisms involved in the inhibition of the production of bacterial virulence factors via microbial interactions (Garzoni et al., 2007; Rode et al., 2010). Indeed, reverse transcriptase PCR has been used previously to study S. aureus SEs expression (Hor et al., 2014; Wallin-Carlquist et al., 2010; Young-Duck et al., 2007). In the present work the effects of lactobacillus isolates on sea expression levels in S. aureus was investigated employing real-time PCR because of its increased sensitivity and specificity compared to conventional methods (Garzoni et al., 2007; Horsmon et al., 2006; Ruijter, 2009). We have shown that lactobacillus isolates reduce sea expression in S. aureus. Specifically, our data reveal that co-culture with L. fermentum results in the greatest down regulation of sea gene expression at 25 °C, whilst at 35 °C this is achieved by L. acidophilus. To date, very few gene expression studies have been carried out to evaluate the impact of probiotic bacteria on the gene expression of SEs or other exotoxins (Cotter et al., 2013; Even et al. 2009). Recently, Laughton et al. (2006) reported that L. ruteri produces a low molecular weight soluble compound which is able to interfere with the expression of an exotoxin gene in S. aureus. It was indicated that the impact of L. lactis on enterotoxin expression was enterotoxin type dependent; L. lactis strongly decreased the expression of sec and sel, while slightly favoring the expression of sea (Even et al., 2009).

In conclusion, the results of this study reveal that the lactobacillus isolates studied reduced both S. aureus growth and its production of enterotoxin. Real time PCR analysis showed down-regulation of sea which was both temperature and strain dependent. These results indicate the potential for application of these isolates as natural agents for the prevention of bacterial growth/toxin production with a view to improving the quality and safety of susceptible foods.

Acknowledgements

We are grateful to the Research Council of Faculty of Veterinary Medicine, University of Tehran and the Department of Medical Genetics, Tehran University of Medical Sciences for supporting this research, and also to Dr. Andrew MacCabe of the Instituto de Agroquímica y Tecnología de Alimentos (IATA), Valencia, Spain, for critical reading of the manuscript.