Enterotoxigenic potential of Staphylococcus spp. isolates recovered from raw milk and artisanal cheese

In this work, we investigated the phenotypic profile of Staphylococcus spp. isolates recovered from raw milk and artisanal cheese, and their enterotoxigenic potential through the detection of classical enterotoxin genes (sea, seb, sec, sed and see). A total of 104 isolates (58 coagulase-positive Staphylococcus – CoPS; and 46 coagulasenegative StaphylococcusCoNS) were used, of which 33 were retrieved from raw milk and 71 from artisanal cheese produced in the Serrana region of Santa Catarina. Identification of CoPS was conducted via biochemical tests. Detection of the genes sea, seb, sec, sed, and see was carried out by multiplex PCR technique. Among the 58 CoPS analyzed, 64% were identified as S. aureus, 22% as S. scheiferi coagulans, 12% as S. hyicus and as 2% S. intermedius. In the present study was noted that 40% of CoPS isolates retrieved from milk harbored seb gene, while only one from artisanal cheese was positive for gene sea. In this study all CoNS samples investigated were negative for enterotoxins genes. The enterotoxigenic potential of CoPS, is an issue of great importance for public health. For that reason, it is necessary that cheese factories strictly follow the safety processes involved in manufacturing.


INTRODUCTION
S. aureus is considered the most important species among Staphylococcus spp. due to its versatile feature to produce several endotoxins, especially the staphylococcal enterotoxins (SE). However, Valle et al. (1990) described that other coagulase-producing staphylococci exhibited the same characteristic. Later, Pereira et al. (2001) demonstrated that some coagulase-negative staphylococci (CoNS) were also able to produce toxins under laboratory conditions.
Enterotoxins are proteins of low molecular weight, which are resistant to inactivation by stomach proteases and withstand temperatures up to 126.7°C/6.2 min. For that reason, in some cases they are able to survive in food even after thermal treatment (thermoresistant toxins) (Santana et al. 2010).
The genes encoding staphylococcal enterotoxins can be present in bacteriophages (sea and sec), plasmids (seb, sed and selj), staphylococcal chromosome (seb, sec, seh, sei, selk, sell, selm, seln, selo, selp and selq) and pathogenicity islands (sec, seb, selk and selq) (Jarraud et al. 2001, Zhang et al. 1998. According to Sanitary Surveillance Agency (Serviço de Vigilância Sanitária) from Ministry of Health, S. aureus was the third most frequent pathogen associated to foodborne disease outbreaks of known causative agent between the years of 2007 and 2017 in Brazil (Brasil 2017). This is an information that raises concern, as it represents a potential risk for public health, especially due to the production of enterotoxins, which are capable of inducing intoxication within few hours after their ingestion.
Raw milk and dairy products are among the most commonly contaminated food by Staphylococcus spp. (Santana et al. 2010), especially those that are handcrafted such as artisanal cheese (Argudin et al. 2010, Loir et al. 2003. In this context, the objective of this work was to characterize phenotypically the isolates of CoPS and to verify the enterotoxigenic potential of Staphylococcus spp. isolates recovered from milk and serrano artisanal cheese samples, produced in the southern region of Brazil, through the detection of staphylococcal enterotoxin genes (sea, seb, sec, sed and see).

Bacterial sample collection
For this study we tested 58 coagulase-positive (CoPS) and 46 coagulase-negative (CoNS) staphylococci bacterial samples recovered from raw milk (n=33) and serrano cheese (n=71), which is produced in the Serrana region of Santa Catarina region.
These isolates were previously collected during the study conducted by (Pontarolo et al. 2017)  Samples were stored at -20°C in brain heart infusion broth (BHI) with 20% glycerol. Samples were recovered and purified in BHI agar, and consequently, incubated in a bacteriological incubator for 24h at 37°C.

DNA extraction and detection of enterotoxin genes
Genomic DNA extraction of the samples followed the protocol described by (Doyle & Doyle 1987) with some modifications. A volume of 200 µL of each bacterial inoculum (colonies were previously incubated in 200 µL of BHI broth for 24h at 37°C) was mixed with 500µL of chloroform: isoamyl alcohol (24:1) and preheated in water-bath for 30 min at 56°C. After that, samples were centrifuged for 10 min at 12,000 rpm. We transferred supernatants to new sterile microtubes and supplemented them with 600 µL of cold 70% alcohol. Then, the microtubes, containing the mixture, were further centrifuged for 20 min at 13,500 rpm. Subsequently, supernatant of each sample was discarded, and microtubes were let to dry completely at 56°C. DNA samples were resuspended in 200 µL of ultra-pure water and stored at -20°C until use.
To detect enterotoxin (sea, seb, sec, see and sed) and femA genes, we used multiplex PCR technique based on the standardized methodology proposed by (Freitas 2005) with modifications. PCR mixture of each sample consisted of 2 µL of genomic DNA, 0.4 pmol of each primer (Table I), 200mM of each dNTP, 1x PCR buffer, 2 mM of MgCl 2 , 1.25 U of Platinum Taq DNA polymerase, resulting in a final volume of 25 µL. Amplification reaction was carried out as follows: initial denaturation step at 94 o C for 5 min, 35 cycles of denaturation at 94 o C for 2 min, annealing at 52 o C for 2 min, extension at 72 o C for 3 min, and final extension at 72 o C for 7 min. To avoid production of unspecific amplicons, we established a set of two Multiplex-PCR assays with different primers. One of them contained the primers sea, sec and see, while the other had the primers seb, sed and femA. S. aureus ATCC 13565 (sea), ATCC 14458 (seb), ATCC 19095 (sec), ATCC 23235 (sed) and ATCC 27664 (see) were used as positive quality control. Gene 16S and ultrapure water were used, respectively, as internal and negative quality controls for the amplification reactions.

RESULTS
A total of 104 Staphylococcus spp. ( 58 CoPS and 46 CoNS) isolates retrieved from milk and serrano artisanal cheese samples were used in this study. Through phenotypic analyses of CoPS, we observed a predominance of S. aureus (64%) in relation to the other species (Table II). In addition, all samples identified as S. aureus revealed the presence of femA gene.
Out of the 58 CoPS isolates, nine of them (15.51%) carried at least one of the enterotoxin genes investigated. Of the 20 isolates from raw milk, 8 of them (40%) harbored seb gene, and were characterized as: S. aureus (n=5), Staphylococcus scheiferi coagulans (n=2) and Staphylococcus hyicus (n=1). In relation to the 38 CoPS isolates obtained from raw milk, only one (S. aureus) harbored gene sea. It is important to mention that there was no relation between isolate origin (raw milk and cheese) and presence of enterotoxin genes. All CoNS samples investigated in this study were negative for enterotoxins genes. Table I. Oligonucleotides used to detect enterotoxin-producing genes and femA gene in Staphylococcus spp.

Primer
Sequence 5 One of the main concerns of food microbiology is staphylococcal food poisoning, a common widespread foodborne disease caused by the ingestion of staphylococcal enterotoxins, which are usually produced by CoPS enterotoxigenic strains, mainly S. aureus (Borges et al. 2008), but also from other CoNS species (Pereira et al. 2001, Valle et al. 1990. In this study, a total of 104 Staphylococcus spp. (58 CoPS and 46 CoNS) recovered from raw milk and artisanal cheese were investigated. In relation to CoPS identification, we observed that the majority of the samples were identified as S. aureus (63.79%). In addition, all these S. aureus isolates exhibited the presence of gene femA. As described above and in other studies (Senger & Bizani 2011) also found similar results to ours. The authors investigated a total of 60 samples of Minas Frescal cheese (half of them handcrafted and the other half industrially manufactured) commercialized in the city of Canoas, Rio Grande do Sul, Brazil. They also observed that 40% of bacterial isolates retrieved from artisanal cheese, and 23.3% from industrial cheese, were contaminated with S. aureus. Interestingly, we also identified the species S. scheiferi coagulans (22%), S. hyicus (12%) and S. intermedius (2%) among the isolates. Interestingly, a study conducted in Sweden revealed that 69% of milk samples and 6% of dairy products were contaminated by CoPS (Rosengren et al. 2010).
Ingestion of food containing preformed staphylococcal enterotoxins causes sudden and rapid onset illness with symptoms like nausea, vomiting, abdominal cramps, and diarrhea. For that reason, the presence of enterotoxic staphylococci in foods is an important health problem, especially for consumers of raw dairy products (Santana et al. 2010), In this study, nine (15%) of the 58 CoPS isolates were positive for at least one of the five enterotoxin genes investigated.
In relation to the origin of the samples, 40% of them recovered from milk showed the presence of gene seb, while 3% of them retrieved from artisanal cheese harbored sea gene. The sea gene is carried by a prophage and can be easily disseminated among Staphylococcus spp. strains. Its product, enterotoxin A, is frequently associated with food poisoning since it is toxic at low concentrations. However, the seb gene can be carried by a plasmid, chromosome and islands of chromosomal pathogenicity (Jarraud et al. 2001). In their study (Ercoli et al. 2017), on the Investigation of a Staphylococcal Food Poisoning Outbreak from a Chantilly Cream Staphylococcus spp. in sheep cheese isolates, especially by the presence of the genes seb (36.8%), sea (5.3%), and sea + seb (5.3%) (Holecková et al. 2002). In their study (Mello et al. 2016) performed in different Brazilian States, including the South, Southeast and Northeast, observed the prevalence of the sea gene in 18.2% followed by the seb gene in 7.7% of cow's milk isolates with subclinical mastitis.
On the other hand, none of the five enterotoxin genes investigated were found in the CoNS isolates from this work. As reported to (Rosec & Gigaud 2002) reported in their study a low incidence of classic enterotoxin genes in 332 Staphylococcus spp. isolates recovered from different food samples in France. Similar to our results (Borelli et al. 2011) also did not detect the presence of the genes sea, seb, sec and, sed in staphylococcal isolates recovered from Minas cheese produced with raw milk. In addition (Hunt et al. 2012) demonstrated that 83.2% of isolates recovered from raw milk and cheese did not harbor the enterotoxin-coding genes studied. It is important to note that growth and enterotoxin production by enterotoxic strains of Staphylococcus sp. in foods are influenced by varied factors, such as temperature, pH, water activity (aw), inoculum size, atmospheric composition, carbon and nitrogen sources, salt levels, and competing microflora. In addition, lactic acid bacteria, present in the natural microflora of raw milk, are able to influence not only the synthesis of staphylococcal enterotoxins (they can reduce pH of milk, produce bacteriocins and hydrogen peroxide), but they are also considered nutritional competitors (Novick 2003). This could explain the low incidence of staphylococci carrying enterotoxin genes, especially in cheese, since the acidification of the substrate as consequence of lactic bacteria proliferation.
Although the presence of genes encoding enterotoxins does not necessarily means that they will be produced, it does emphasize the potential of the bacteria to trigger enterotoxin production under optimal conditions in food (Santana et al. 2010).
The presence of enterotoxin genes, even at low quantity, emphasizes the enterotoxigenic potential of CoPS, which is an important public health issue. Therefore, it is necessary that cheese and dairy factories strictly follow all processes and flows influencing food safety, in order to assure a bacteriological safe and stable product. Although the enterotoxin genes were not detected in the CoNS isolates, we expect that these data will assist the current legislation to establish a minimum detection limit of CoNS in foods.