Incidence and characterization of Staphylococcus aureus strains isolated from food markets

Abstract

Staphylococcus aureus is a major foodborne pathogen. The primary purpose of this research was to evaluate S. aureus contamination levels and distribution characteristics in various kinds of food. A total of 121 samples of 11 kinds of food were collected from different food markets in the summer season according to their availability and popularity, and 103 (85.1 %) samples were found to be contaminated with S. aureus. Raw meat, meat products, soybean products, and pickled vegetables are highly contaminated food. For enterotoxin genes analysis, among all the isolates, 20 (19.4 %) possessed sea gene, 2 (1.9 %) with sed gene, 8 (7.8 %) with seg gene, 4 (3.8 %) with seh gene, 24 (23.3 %) with sei gene, 5 (4.8 %) with selj gene, 24 (23.3 %) with selk gene, 44 (42.7 %) with seln gene, and 8 (7.8 %) with ser gene. The seb, sec, see, selm, and selu genes were not found in any of the isolates. For the antibiotic resistance genes tests, most isolates from the food samples were positive for at least 1 of 6 antibiotic resistance genes. The most prevalent resistance genes in samples were ermA (85/103, 82.5 %), followed by tetA (61/103, 59.2 %), chlA (60/103, 58.2 %), norA (56/103, 54.3 %), mecA (10/103, 9.7 %), and blaZ (7/103, 6.8 %). Our results showed that most isolates harbored enterotoxins-producing genes and antibiotic resistance genes, indicating the risk of foodborne S. aureus outbreaks, which drew urgent attention to the great need to improve hygienic conditions in food processing, in order to enhance the safety of food products.

Introduction

Food safety remains one of the most important global health issues, and foodborne diseases caused by microbes are a widespread public health problem (WHO 2002). Staphylococcus aureus is considered the third most economically important cause of disease in the world among the reported foodborne illnesses (Peles et al. 2007). S. aureus is a group of Gram-positive, facultative aerobic and usually unencapsulated microorganisms, which grows in a wide range of temperatures (7–48.5 °C), pH (4.2–9.3), and sodium chloride concentrations (up to 15 % NaCl) (Le Loir et al. 2003). With these characteristics, it facilitates the contamination and transmission of the organism to various kinds of foods (Zhao et al. 2013). Food stuff contamination may occur directly from infected food-producing animals or may result from poor hygiene during the production process, or the retail and storage of foods (Aydin et al. 2011; Kotzekidou 2013). Meat or poultry dishes were commonly implicated in S. aureus (55 %) outbreaks. Errors in food processing and preparation have been commonly reported, regardless of etiology; contamination by a food worker is common in S. aureus outbreaks (55 %); and vomiting is commonly (87 %) (Bennett et al. 2013). Thus, it can be seen that food poisoning caused by S. aureus is a major issue for public health programs worldwide. The literature shows that there 33 % of all bacterial food poisoning is caused by S. aureus in the United States, and estimated costs average about US$539 per illness (Bennett et al. 2013). In China, official data have shown that an annual average of about 300 million people contract foodborne diseases. The percentages of food contaminated by S. aureus have averaged 32.5 % (Hu et al. 2013). Food poisoning caused by S. aureus enterotoxin has taken place frequently in China. Actually, only a minority of patients with food poisoning seek formal medical care in China, so informative tests are reported for only a fraction. A survey in late 2010 found that Chinese considered food safety the second greatest risk they faced in daily life (after earthquakes) (Alcorn and Ouyang 2012). Contamination of foods can occur at the source of food production and during food processing and preparation, transport, and storage. S. aureus has been repeatedly detected in a diverse variety of foods. One recent study reported a high incidence of S. aureus (∼25 %) in seafood marketed in Spain, which is the largest seafood producer and the second largest consumer in the European Union (Vázquez-Sánchez et al. 2013). Another study estimated the prevalence of methicillin-resistant S. aureus (MRSA) in bulk tank milk from German dairy herds, and characterized the isolates (Kreausukon et al. 2012). The main point highlighted by these reports is that any food that provides a convenient medium for S. aureus growth may be involved in a staphylococcal food poisoning outbreak. The foods most frequently involved differ widely from one country to another, probably due to differing food habits (Hennekinne et al. 2012).

The growth of S. aureus in foods presents a potential public health hazard because many strains of S. aureus produce enterotoxins (SEs) that cause food poisoning if ingested (Peles et al. 2007). Classically, staphylococcal enterotoxins (SEs) have been divided into 5 major serological types (SEA-SEE) on the basis of their antigenic properties, but in the last few years, many other new types of SEs have been reported and their genes described (Lina et al. 2004; Peles et al. 2007; Argudín et al. 2010; Gücükoğlu et al. 2013): including staphylococcal enterotoxins (SEs; SEA to SEE, SEG to SEI, SER to SET) with demonstrated emetic activity, and staphylococcal-like (SEl) proteins, which are not emetic in a primate model (SElL and SElQ) or have yet to be tested (SElJ, SElK, SElM to SElP, SElU). Commonly, SEs and SEls have been traditionally subdivided into classical (SEA to SEE) and new (SEG to SElU) types (Argudín et al. 2010). The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity (Bania et al. 2006). However, a direct relationship of S. aureus SEs and SEls with pathogenicity has not always been established. Therefore, the detection of classical and newly identified enterotoxin genes is equally vital for the analysis of staphylococcal food poisoning (Tang et al. 2011).

Currently, the widespread use of antibiotics has provoked an exponential increase in the incidence of antibiotic resistance in several bacterial groups (Gutiérrez et al. 2012). Antibiotics are used widely in various animal foods, where they are often applied additionally for growth promotion and routine disease prevention. The use of antimicrobials in food animals creates an important source of resistant bacteria that can spread to humans (Aydin et al. 2011). Therefore, the food chain is considered a potential route of transmission of antibiotic-resistant bacteria to humans (Gutiérrez et al. 2012). Multidrug-resistant S. aureus strains are rather common, the resistance of foodborne S. aureus isolates to antimicrobial agents is an increasing problem, especially for MRSA (Sudagidan and Aydin 2013). An association between the emergence of MRSA strains in animals and the use of antibiotics in farming has been suggested. During slaughtering of MRSA-positive animals, contamination of carcasses and the environment with MRSA may occur, and, consequently, meat from these animals may become contaminated (Hennekinne et al. 2012). The antimicrobial susceptibility profiles of S. aureus isolated from clinical and subclinical samples have been reported. But knowledge about the antibiotic resistance of this pathogen isolated from food is still limited and only a few data are available (Meemken et al. 2013). The resistance genes blaZ, mecA, ermA, tetA, norA, and chlA are frequently responsible for phenotypic resistance of S. aureus to penicillin, oxacillin, erythromycin, tetracycline, quinolones, and chloramphenicol (Gao et al. 2012).

In this study, we aimed to investigate the presence of S. aureus isolates in typical food like meat, soybean products, and pickled vegetables frequently consumed by Chengdu local people in their daily lives at different food markets. And further, the enterotoxin genes and antibiotic resistance genes implicated in these isolates were characterized, in order to evaluate the risk of foodborne S. aureus outbreaks and enhance the safety of food products.

Materials and methods

Sample collection

Between August and October 2010, a total of 121 samples were collected from different food markets located in the Wuhou District of Chengdu city, Sichuan Province of China. The sampling times, sites, and numbers of samples are listed in Table 1. The samples were randomly selected according to their availability and popularity. The composition of all 121 samples was as follows: rice products (n = 3), raw meat (n = 22), cooked meat products (n = 31), dairy products (pasteurized milk) (n = 1), aquatic products (n = 3), soybean products (n = 15), dry cakes (n = 5), confection (n = 3), pickled vegetables (n = 32), vegetables and fruits (n = 4), and egg products (n = 2). From each sample at each sampling time, samples of about 100 g or 100 ml were collected. The sampled products were refrigerated rapidly and then immediately transferred to the laboratory for testing.

Table 1 Sampling times, sites and numbers

Isolation and identification of S. aureus strains from food samples

Samples (25 g or 25 ml) were placed in stomacher bags containing 225 ml of brain heart infusion broth (BHI; Difco) and homogenized. The mixture was incubated at 37 °C for 24 h. The enriched sample was streaked onto Baird-Parker medium (BP; Difco) supplemented with 5 % egg yolk tellurite emulsion and incubated at 37 °C for 48 h. Presumptive gray-black to jet-black circular colonies were picked, then transferred onto trypticase soy agar (TSA; Difco) and incubated at 37 °C for 24 h. Colonies were subjected to be plated on Blood Agar and incubated aerobically at 37 °C for 24 h.

Confirmation of S. aureus isolates

The samples were examined as described above. Randomly chosen suspect colonies were picked from each plate for further phenotypic and genotypic identification. The isolates were presumed as S. aureus on the basis of their colony morphology, Gram-staining, hemolytic properties, and thermostable nuclease activity. Further, all the isolated strains were investigated and identified to the species level by PCR amplification. The presence of the target 16S rDNA (565 bp) was determined to be S. aureus species (Tang et al. 2006).

DNA extraction

Genomic DNA used for molecular analysis was isolated from each tested strain by using the extraction method described by Tang et al. (2006).

Enterotoxin genes detection

Detection of enterotoxin genes were performed by PCR. A total of 14 enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, selm, seln, ser, and selu) were examined. The each PCR primer set used in this study and the amplification condition were described previously by Tang et al. (2011). Briefly, reactions were carried out in a volume of 20 μl with a 10× PCR buffer, 2 μl; 2.0 mM MgCl₂; 200 μM of each nucleotides dATP, dTTP, dGTP, and dCTP; 1U of Taq polymerase, 1 μl of purified sample DNA, and with primers 0.5 μM. The mixes were submitted to a program with an initial denaturation step at 95 °C for 5 min, 35 amplification cycles each with 40 s at 95 °C, 50 s at 48 °C (for seh) or 52 °C (for sea, seb, sec, sed, see, selj, and selu) or 54 °C (for selk, selm, and ser) or 55 °C (for seg, seln) or 58 °C (for sei), and 50 s at 72 °C followed by an additional extension step of 10 min at 72 °C.

Antibiotic resistance genes detection

Isolated strains were analyzed for the presence of 6 target antibiotic resistance genes (Table 2). The primer pairs were designed according to the S. aureus mecA, chlA, erm, tetA, norA, and blaZ sequences by using the Primer Premier 5.0 software (PREMIER Biosoft, CA, USA). The primer pairs were also aligned by using Basic local alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST). PCR amplification was performed in a total reaction volume of 20 μl. The annealing temperature for each primer set is listed in Table 2. Primers in a concentration of 0.5 μM each and extract DNA volume of 1 μl were added to the PCR mixture (10× PCR buffer, 2 μl; 2.0 mM MgCl₂; 200 μM of each nucleotides dATP, dTTP, dGTP, and dCTP; 1U of Taq polymerase) and then subjected to amplification. Cycling conditions for the PCR included: 95 °C, 5 min; 35 repeats of the following steps: 95 °C, 40 s, annealing for 55 s, specific temperatures shown in Table 2, and 72 °C, 40 s; followed by an additional extension step of 10 min at 72 °C. Positive and negative controls were subjected to the same procedures.

Table 2 Primers for detection of antibiotic resistance genes

Results

Identification of S. aureus isolates

The S. aureus could be identified by colony morphology on Baird-Parker agar, Gram-staining, hemolytic properties, thermostable nuclease activity (TNase tests appeared pink halo) on toluidine blue-DNA agar and 16S RNA PCR detection. Randomly chosen suspect colonies were picked from each plate. Presumptive gray-black to jet-black circular colonies were re-picked on Baird-Parker agar, incubated at 37 °C for 24 h. Colonies were subjected to plating on Blood Agar and incubated aerobically at 37 °C for 48 h. The hemolytic activities were investigated with the presence of clear zones around the colonies due to the erythrocyte-lysing. Among the isolated strains, 15 isolates exhibited strong hemolytic activities because of a large zonal radius of complete hemolysis on sheep blood agar; the other 88 isolates did not present any hemolytic activities. For the thermostable nuclease activity analysis, 34 isolates could produce thermostable nuclease activities due to the presence of a pink halo on toluidine blue-DNA agar. The presence of the targets of S. aureus-specific part of 16S rDNA gene (565 bp) in 1 % agarose gel was determined to be S. aureus species.

Detection ratio and distribution of S. aureus in food samples

A total of 103 (85.1 %) of the 121 food samples examined were found to be contaminated with S. aureus. The detection ratio was very high (shown in Table 3). The positive rates in aquatic products and egg products were highest (3/3 and 2/2, both 100 %, respectively), followed by raw meat and meat products (21/22, 95.5 %, and 29/31, 93.5 %, respectively), soybean products (13/15, 86.7 %), dry cakes and pickled vegetables (4/5, 80 %, and 25/32, 78.1 %, respectively), vegetables and fruits (3/4, 75 %), and rice products (2/3, 66.7 %). The dairy product (pasteurized milk) was not found to be contaminated with S. aureus.

Table 3 The distribution of S. aureus in samples

Distributions of sea to selu genes in S. aureus isolates

The results of the PCR analysis of 14 SEs and SEls genes are shown in Table 4. Among all the isolates (n = 103), 20 (19.4 %) possessed sea gene, 2 (1.9 %) with sed gene, 8 (7.8 %) with seg gene, 4 (3.8 %) with seh gene, 24 (23.3 %) with sei gene, 5 (4.8 %) with selj gene, 24 (23.3 %) with selk gene, 44 (42.7 %) with seln gene, and 8 (7.8 %) with ser gene. The seb, sec, see, selm, and selu genes were not found in any of the isolates. The detection rate of the newly recognized SEs and SEls genes (seg through selu) was higher than that of the classical SEs genes (sea through see). Comparatively, the frequency of individual gene of SEs and SEls was variable, and was as follows: seln > sei = selk > sea > seg = ser > selj > seh > sed.

Table 4 The distributions of sea to selu genes in S. aureus isolates of food samples

Distributions of antibiotic resistance genes

The detection results of six antibiotic resistance genes reflected the prevalence of these genes. Most isolates from food samples in this study were positive for at least one of the six antibiotic resistance genes. The prevalence of the six resistance genes in samples were ermA (85/103, 82.5 %), tetA (61/103, 59.2 %), chlA (60/103, 58.2 %), norA (56/103, 54.3 %), mecA (10/103, 9.7 %), and blaZ (7/103, 6.8 %), respectively (Table 5).

Table 5 Detection rate of 6 antibiotic resistance genes in S. aureus isolates of food samples

Discussion

Staphylococcus aureus is one of the most commonly identified bacteria that cause food pollution. Food poisoning caused by S. aureus accounted for a large proportion, whether in developed countries or in developing countries (Argudín et al. 2010). The changing epidemiology of S. aureus over recent decades linked to food production highlights the necessity of monitoring the S. aureus that are circulating through the food chain (Argudín et al. 2012). Moreover, investigation and determination of S. aureus contamination in food samples is significant with regard to food safety. In this survey, we analyzed 103 isolates from 10 types of food categories which were consumed frequently by local Chengdu people. We evaluated S. aureus contamination levels and described characterizations of food-related isolates by using microbiology methods and molecular biology methods, respectively. Although our study is a limited survey on the prevalence of S. aureus in foods from different local markets, these tested foods are consumed frequently by Chengdu local people. Due to the fact that our sampling time was in summer, the results reflected the potential high risk of S. aureus to consumers in this season.

Staphylococcus aureus are ubiquitous and are impossible to eliminate from our environment. Temperature and time abuse of foods commonly contributes to outbreaks caused by S. aureus, regardless of where the food was prepared (Bennett et al. 2013). Normally, staphylococcal food poisoning is mainly concentrated in May to October, the season of high temperatures. Our study provides information about the incidence of S. aureus in 11 kinds of food sampled from different dates in August to October. The season has a high temperature which is suitable for S. aureus growth; therefore, we recovered 103 S. aureus strains out of 121 food samples, giving an overall incidence of 85.1 % (103/121). We found there was a high incidence of S. aureus in cooked meat products, soybean products, and pickled vegetables, which was 93.5, 86.7, and 78.1 %, respectively. Based upon the fact that contamination of foods with S. aureus can occur at the source of food production and during food processing and preparation, transport, and storage (Bennett et al. 2013), the contamination of cooked meat products, soybean products, and pickled vegetables might possibly come from the food handling process. Meat products, soybean products, and pickled vegetables are widely consumed by Chengdu local people. The high percentage of positive samples for S. aureus in these foods draws our attention to an urgent need to improve proper food handling practices and good personal hygiene. The positive rates in aquatic products and egg products were 100 % (3/3, 2/2), which might possibly be partly because our sample size was insufficient to accurately estimate the prevalence rates in these two kinds of food. In the future, we will increase the sample size for further study. In this study, our results were higher than other reports due to the sampling time of the summer season. In the research of Hu et al. (2013) and Aydin et al. (2011), the positive rates of S. aureus were only 15.6 % (32/205) and 14.4 % (150/1070), respectively. Some reports have also described a slightly higher incidence of meat products [48.7 % (80/164)] (Güven et al. 2010). Even though our study is a limited survey, our results demonstrated the spread of S. aureus within food products would be a major concern. According to other reports (Doyle et al. 2011), the main reservoirs of S. aureus are the nasal cavity and the skin of humans. Food handlers are the primary source of contamination of foods. Any time a food is exposed to human handling, asymptomatic food handlers may harbor S. aureus and can contaminate food during preparation. At least 30–50 % of individuals carry these organisms in their nasal passages and throats, or on their hands. Food handlers should practice good personal hygiene and should be educated in proper food handling practices (Cliver and Riemann 2002). Only by improving sanitation and hygiene procedures, can contamination levels of S. aureus be effectively reduced.

Moreover, S. aureus produces a spectrum of enterotoxin that is recognized as the main reason for causing staphylococcal food poisoning. Numerous studies on the distribution of enterotoxigenic staphylococci isolated from food vary from one report to another in the percentage of enterotoxigenic strains and the distribution of enterotoxin genes (Le Loir et al. 2003). Morandi et al. (2007) reported frequent enterotoxin A and D findings in many cow dairy products. In this study, the only detected classical enterotoxin genes were sea and sed genes. Our finding was in agreement with that reported above. We did not isolate any S. aureus strains that harbored seb, sec, or see genes. The 103 S. aureus isolates showed a high level of diversity in their genotypes, which might be caused by the need to adapt to the different environments. The surveys of S. aureus isolated from food and other sources demonstrate that the percentage of enterotoxigenic strains increases considerably if the genes coding for the newly described enterotoxin-like superantigens (SEG to SEIU) are considered together with the so-called classical enterotoxins (SEA to SEE) (Bania et al. 2006). The prevalence of the seln, selk, sei, and sea genes was dominant in these isolates, which could reflect their potential risk in causing staphylococcal foodborne disease. All the classical gene-positive strains were accompanied by the genes of new enterotoxins, and classical gene-negative strains were still found to carry new enterotoxins genes, in which the new enterotoxin genes were more frequently detected than the classical enterotoxin genes. Although a report has shown that SEA is the predominant cause of foodborne intoxications caused by S. aureus (Le Loir et al. 2003), some cases of food poisoning (about 5 % according to estimates) in which none of the classical enterotoxins were detected can be attributed to new enterotoxins (Su and Wong 1995; Rosec and Gigaud 2002). The high prevalence of newly discovered genes in food-derived S. aureus, especially for seg, seh, and sei genes, which have been shown to have emetic activity, was thus considered as a potential causative agent in food poisoning. In this study, seg, seh, and sei genes were found in 4, 8, and 20 isolates, respectively, which showed a high potential risk to human health. According to a recent estimate, the main cause of food poisoning is microbial pathogens, accounting for about 48.3 % of the total cases of illness in China (Chen et al. 2010). Food poisoning attributed to S. aureus accounts for about 25 % of foodborne illnesses in China, whereas, in some areas, it accounts for 40 % (Tang et al. 2011). In this study, our limited sampling and microbiological analyses showed a high risk of contamination of toxigenic S. aureus in different kinds of food, which are frequently consumed by Chengdu local people, indicating a higher opportunity for this organism to spread epidemics, resulting in food poisoning.

Further, bacterial resistance has been reported for more than 30 years, and S. aureus is one of the most extensively studied pathogens in clinics both on humans and on animals (Hu et al. 2013). The detection pattern of antibiotic resistance genes largely reflected the antibiotic usage patterns and the prevalence of antibiotic resistance genes. In this study, we analyzed the prevalence of antibiotic resistance genes in these food-derived isolates. A total of 103 S. aureus food isolates were examined on the basis of the presence of ermA, tetA, chlA, norA, mecA, and blaZ genes and the rate of isolates detected was high, most of which were positive for at least 1 of 6 antibiotic resistance genes. Multi-antimicrobial resistance was observed among these food-originated isolates, which indicated that the presence of these antimicrobial-resistant S. aureus might be a potential health hazard for humans. Among the antibiotic resistance genes analyzed, the high prevalence was ermA (82.5 %), tetA (59.2 %), chlA (58.2 %), and norA (54.3 %), indicating the wide distribution of these genes. In many developing countries, some antibacterial agents have been rendered ineffective. Misuse and overuse of these antibiotics could have contributed to this trend. In China, the usage of antibiotics is estimated to be more than 25,000 t each year. Excessive use of antibiotics in human therapy and animal feeding has led to a serious problem of the antibiotic contamination in China (Jiang et al. 2013). Under the circumstances, the rapid emergence of MRSA infections is one of the most surprising events in infectious diseases in recent years (DeLeo and Chambers 2009). The prevalence of hospital-associated MRSA (based on the detection of the mecA gene) may vary from 1.5 to 20 % (Shittu et al. 2011). According to recent reports, MRSA has been successfully isolated from animal food (Hu et al. 2013). In this study, the detection rate of mecA gene in food-derived isolates was 9.7 %, which was consistent with the reported data. The rate of MRSA strains contamination suggests that improvements in food safety and personal hygiene guidelines may be advisable to reduce the risk of spread of these MRSA strains. From our results, although the detection rates of mecA and blaZ genes were lower (9.7 and 6.8 %, respectively) than the other four antibiotic resistance genes (ermA, tetA, chlA, and norA), they still could pose serious problems in the future as potential reservoirs for resistance and virulence factors, and could lead to the emergence and spread of severe infections. Government should strengthen agricultural drug supervision at the animal breeding level, so that drug resistance can be controlled effectively.

Taken together, our study provides baseline information on the nature of the antibiotic resistance genes from S. aureus food isolates in Chengdu, Sichuan. The results draw urgent attention to the great need to understand the pathways and mechanisms of the antibiotic resistance genes released into different kinds of food in the study area, and to seek effective ways to reduce their spread for the benefit of public health. This study reveals the detection ratio and characteristics of S. aureus isolates from food. The use of phenotypic and molecular methods could provide useful information on the distribution and characterization of the enterotoxin genes and antibiotic resistance genes stated above. Many of the S. aureus isolates are enterotoxigenic, which is a potential hazard for human health. The high prevalence of antibiotic resistance genes is also a potential health hazard for human health.