Identification of the classical enterotoxin genes of Staphylococcus aureus in various foods by multiplex PCR assay

Abstract

Background:

An annual update of information about the prevalence of Staphylococcus aureus and staphylococcal enterotoxins (SEs) genes is required in every geographic area.

Aims:

This study was conducted to investigate the prevalence of the bacterium and type of associated enterotoxin genes in different food samples, using multiplex polymerase chain reaction (PCR) assay.

Methods:

In order to achieve these goals, 310 samples, divided into three groups (dairy products, meat, and traditional sweets groups), were collected. After determination of the prevalence of S. aureus, the existence of 16s rRNA, sea, seb, sec, sed, and see genes were evaluated using multiplex PCR assay.

Results:

Staphylococcus aureus was isolated from 103 (33%) samples. Furthermore, the meat category had the most contamination rate of S. aureus. Additionally, the kebab samples (61.5%) were the most contaminated products, followed by hamburger (47.3%), and ice cream (33.8%). Of these 103 S. aureus isolates, 72 isolates (69.9%) harbored at least one type of the classical SEs genes. The prevalence of the type A enterotoxin gene was detected higher than the other SEs genes.

Conclusion:

The results indicated that inappropriate handling of the samples in the preparation and processing steps, especially for the meat products, can lead to the spread of more bacteria. The relatively high prevalence of some classical enterotoxin genes in the isolates revealed the potential power of this bacterium to produce enterotoxins, which can lead to food-poisoning.

Introduction

Staphylococcus aureus is considered as a major public health concern, according to the definition of food-borne bacteria, published by World Health Organization (WHO) report (Le Loir et al., 2003 ▶; Angulo et al., 2008 ▶). In this regard, some, but not all strains of S. aureus, produce a wide variety of extracellular proteins and pyrogenic exotoxins, including staphylococcal enterotoxins (SEs), staphylococcal-like enterotoxins (SEls), toxic shock syndrome toxin 1 (TSST-1), and different types of hemolysins (Novick et al., 2001 ▶; Moraveji et al., 2014 ▶). These toxins cause various types of disease symptoms, including scalded skin syndrome, impetigo, toxic shock syndrome, and staphylococcal food poisoning (Novick et al., 2001 ▶; Le Loir et al., 2003 ▶).

Staphylococcal enterotoxins are resistant to heat, digestive enzymes, denaturing agents, and a wide range of pH. Hence, it does not degrade in the digestive tract, can pass through the stomach and attack the intestinal cells (Argudín et al., 2010 ▶). Among these enterotoxins, the five classical types (A, B, C, D, and E) are the most important and are responsible for 95% of staphylococcal food poisoning (Jay et al., 2005 ▶). Due to the growing of S. aureus in different extreme conditions, it can contaminate animal products during processing and preparation steps, and act as a source for SEs production and food poisoning. Therefore, there is always a potential risk of food poisoning associated with this bacterium, which needs annual checking (Chaibenjawong and Foster, 2011 ▶; Wang et al., 2017 ▶). For these reasons, rapid detection of SEs and efficient screening programs for the main source of food poisoning is critical. Therefore, the prevalence of S. aureus and the presence of classical SEs in different foods were investigated in this study, using a polymerase chain reaction (PCR) technique (Løvseth et al., 2004 ▶).

Materials and Methods

Sample collection

To assess the contamination level of S. aureus in foods, samples were aseptically collected between July 2014 and June 2015 in Gorgan, Iran. Foods were divided into three groups (dairy products, meat, and traditional sweets groups) and included raw milk, ice cream, cream, hamburger, kebab, sausage, and traditional creamy sweets. Sample sizes were calculated based on the previous study and around 100 isolates were obtained from each food group (Peyravii et al., 2013 ▶). The samples were transported to the laboratory within 6 h in a cool box.

Isolation of S. aureus

The isolation of S. aureus was done according to the ISO 6888-1:1999/reaffirmed 2005 standard methodology (ISO 6888-1, 1999), using 25 g of each sample. The colonies were checked phenotypically and confirmed genotypically for identification of S. aureus (Løvseth et al., 2004 ▶; Pelisser et al., 2009 ▶).

DNA extraction and PCR analysis

DNA of each colony was extracted, using a DNA extraction kit (CinnaGen, Iran), according to the manufacturer’s instruction. The purity and concentration of each DNA were evaluated by Spectrophotometry method (Nanodrop 1000; Thermo Scientific, Wilmington, DE, USA). Staphylococcus aureus and its five important toxins were identified, using two separate multiplex PCR reactions, according to Løvseth method (Løvseth et al., 2004 ▶). The PCR products were separated on agarose gels (1.5%) and electrophoresed at 100 V for 45 min. Each gel was stained with DNA safe-stain dye (CinnaGen, Iran), and visualized under a UV transilluminator (BTS-20, Japan). DNase free water, DNA of reference strains of S. aureus ATCC 29213 (SEA), and S. aureus NCTC 10655 (SEC) were used as negative and positive controls, respectively. A 1-Kb molecular weight marker (CinnaGen, Iran) was used to determine the size of the DNA bands.

Statistical analysis

The Pearson Chi-square and Fisher’s exact two-tailed tests were used to analyze the differences between variables, using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA). The examined items included the association of different isolation rates of S. aureus and the type of foods, and the prevalence of the toxigenic genes in isolates with various origins. The statistical significance was determined as P<0.05.

Results

The frequency of S. aureus in the food materials

Among 312 food materials, 103 (33%) analyzed samples were contaminated with S. aureus. Although the meat category had the most contamination of S. aureus, no significant correlation was observed between three-food categories and the bacterium pollution rate (P=0.1). Additionally, the kebab samples (61.5%) were the most contaminated products, followed by hamburger (47.3%), and ice cream (33.8%). According to this, a weak significant correlation was detected between food types and the prevalence of S. aureus (P=0.047). Table 1 shows the details of the prevalence of S. aureus in the foods.

Table 1.

Prevalence of Staphylococcus aureus in the various food samples and their enterotoxin genes

Category	Source/number of samples	No. of Staphylococcus aureus isolates (%)	Prevalence of enterotoxin genes (%*)
			sea	seb	sec	sed	see
Meat	Hamburger/38	18 (47.3)	6 (33.3)	0 (0)	1 (5.6)	5 (27.8)	0 (0)
	Kebab/13	8 (61.5)	3 (37.5)	0 (0)	0 (0)	4 (50)	0 (0)
	Sausage/13	2 (15.3)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
	All/64	28 (43.8)	9 (32.1)	0 (0)	1 (3.5)	9 (32.1)	0 (0)
Dairy products	Raw milk/90	28 (31.1)	6 (21.4)	0 (0)	3 (10.7)	11 (39.3)	0 (0)
	Ice cream/59	20 (33.8)	8 (40)	3 (15)	3 (15)	4 (20)	0 (0)
	Cream/13	2 (15.3)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)
	All/162	50 (30.9)	14 (28)	3 (6)	6 (12)	15 (30)	0 (0)
Traditional sweets	Traditional sweets/86	25 (29.1)	10 (40)	0 (0)	2 (8)	3 (12)	0 (0)
All samples	All samples/312	103 (33)	33 (32)	3 (2.9)	9 (8.7)	27 (26.2)	0 (0)

^* Based on the isolates number in each source

Detection of classical SEs genes

Of these 103 S. aureus isolates, 72 isolates (69.9%) harbored at least one type of the classical SEs genes. Among them, five isolates harbored two genes simultaneously, as follows; two isolates, obtained from ice cream, had sea + sec, and sea + seb genes, respectively. Furthermore, two milks’ isolates had sec and sed genes. One isolate, which was obtained from hamburger, had sea and sed genes, simultaneously. None of the isolates had see gene. Table 1 also shows the genes distribution in detail. Based on the prevalence of the enterotoxin genes, the isolates with type A enterotoxin had a higher prevalence in the ice cream and traditional sweets samples, compared to the other food samples, but this difference was not statically significant (P=0.3).

Discussion

Annual review and analysis of food contamination with S. aureus and its enterotoxins in each geographic area in every country is crucial. In this study, the most contaminated foods were kebab and hamburger samples, in the category of meat product. It has been shown that the rate of contamination of S. aureus in food samples can be very variable. It has been reported that this rate can be very low, moderate, or even very high (Peyravii et al., 2013 ▶; Santos et al., 2014 ▶; Tang et al., 2015 ▶). Several factors, including different sampling, detection methods and their sensitivity, sample size, seasonal effects, the ecological and geographical origin of the isolates can be involved in this variation (Kamarehei et al., 2013 ▶). However, in most studies with any prevalence rate, the meat and dairy products are at the top of the contaminated foods, consistent with the results obtained from this study. In this regard, Wang et al. (2017) ▶ stated that one of the main reasons for the food-poisoning in the world is related to the meat products contaminated with S. aureus. One of the reasons for this contamination in the meat products can be more manipulation of the samples in the preparation and processing steps (Kraushaar et al., 2017 ▶; Wijesurendra et al., 2017 ▶). Manipulation of the food products, especially when other food materials such as onion and animal fats are added to them and mixed with hands, can lead to the transfer of the bacterium from other materials or workers’ hands to food or at least it can increase the chance of the transfer. Both the kebab and hamburger are manipulated more than the rest of the analyzed samples, and the results of this study showed the high contamination rate in these products, which reinforces this theory. It seems that the use of automatic machines in food preparation and processing or disinfection of additive substances and hands of workers can help to reduce the chance of contamination. The prevalence of S. aureus in the dairy products obtained in this study was also consistent with Imani Fooladi et al.’s (2010) ▶ investigation published, which reported about 32% of the dairy products were contaminated. Non-standard milking, for example using dirty milking machine or hands, can also transfer the S. aureus, which is natural flora of cow’s skin, to the dairy products.

Most of the isolates analyzed in this study possessed sea, followed by sed genes. Other investigators have also suggested the dominance of the sea (Rall et al., 2008 ▶; Argudín et al., 2012 ▶; Kamarehei et al., 2013 ▶; Seyoum et al., 2016 ▶). The data from the present research showed more than one enterotoxin gene can be present in one isolate, simultaneously. Although the prevalence of S. aureus with several genes is low, it should not be neglected. In order to reduce the risk of the S. aureus contamination in foods and improve health programs, more controlling strategies should be implemented in all stages of food preparation. In 2009, Pelisser et al. stated that approximately 10⁶ CFU/g of S. aureus count is necessary for enterotoxins production. This point should be considered on issues related to food hygiene and several food preservation strategies should be implemented to reduce bacterial replication in food products.

The results of this study suggested that the contamination with S. aureus with the potential power of toxin production is still a potential health risk, especially in food with high manipulation at the time of preparation. The relatively high prevalence of the bacteria carrying enterotoxin A gene and their distribution in different food can be alarming, furthermore, the controlling programs are not still enough and more government oversight should be applied. Moreover, it was shown that more attention to food health issues should be the main concern of food factories and regulatory agencies. It also shows that molecular methods provide rapid, accurate, and economical means for S. aureus screening in food safety evaluations.

Conflict of interest

There was no conflict of interest.