Staphylococcus aureus stands out as one of the most virulent pathogens in the genus Staphylococcus. This characteristic is due to its ability to produce a wide variety of staphylococcal enterotoxins (SEs) and exotoxins, which in turn can cause staphylococcal food poisoning (SFP), clinical syndromes such as skin infections, inflammation, pneumonia, and sepsis, in addition to being associated with the development of inflammation in the mammary glands of dairy cattle, which results in chronic mastitis and cell necrosis. SEs are small globular proteins that combine superantigenic and emetic activities; they are resistant to heat, low temperatures, and proteolytic enzymes and are tolerant to a wide pH range. More than 24 SE genes have been well described (SEA-SEE, SEG, SEH, SEI, SEJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElR, SElS, SElT, SElU, SElV, SElW, SElX, SElY, and SElZ), being a part of different SFP outbreaks, clinical cases, and isolated animal strains. In recent years, new genes (sel26, sel27, sel28, sel31, sel32, and sel33) from SEs have been described, as well as two variants (seh-2p and ses-3p) resulting in a total of thirty-three genes from Ses, including the nine variants that are still in the process of genetic and molecular structure evaluation. SEs are encoded by genes that are located in mobile genetic elements, such as plasmids, prophages, pathogenicity islands, and the enterotoxin gene cluster (egc), and housed in the genomic island of S. aureus. Both classical SEs and SE-like toxins (SEls) share phylogenetic relationships, structure, function, and sequence homology, which are characteristics for the production of new SEs through recombination processes. Due to the epidemiological importance of SEs, their rapid assessment and detection have been crucial for food security and public health; for this reason, different methods of identification of SEs have been developed, such as liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), molecular methods, and whole-genome sequencing; providing the diagnosis of SEs and a better understanding of the occurrence, spread, and eradication of SEs. This review provides scientific information on the enterotoxins produced by S. aureus, such as structural characteristics, genetic organization, regulatory mechanisms, superantigen activity, mechanisms of action used by SEs at the time of interaction with the immune system, methods of detection of SEs, and recent biocontrol techniques used in food.

Staphylococcal enterotoxin A (SEA) is the most frequently reported in staphylococcal food poisoning (SFP) outbreaks. Aptamers are single-stranded nucleic acids that are seen as promising alternatives to antibodies in several areas, including diagnostics. In this work, systematic evolution of ligands by exponential enrichment (SELEX) was used to select DNA aptamers against SEA. The SELEX protocol employed magnetic beads as an immobilization matrix for the target molecule and real-time quantitative PCR (qPCR) for monitoring and optimizing sequence enrichment. After 10 selection cycles, the ssDNA pool with the highest affinity was sequenced by next generation sequencing (NGS). Approximately 3 million aptamer candidates were identified, and the most representative cluster sequences were selected for further characterization. The aptamer with the highest affinity showed an experimental dissociation constant (KD) of 13.36 ± 18.62 nM. Increased temperature negatively affected the affinity of the aptamer for the target. Application of the selected aptamers in a lateral flow assay demonstrated their functionality in detecting samples containing 100 ng SEA, the minimum amount capable of causing food poisoning. Overall, the applicability of DNA aptamers in SEA recognition was demonstrated and characterized under different conditions, paving the way for the development of diagnostic tools.

Staphylococcus species in food produce Staphylococcal enterotoxins (SEs) that cause Staphylococcal food poisoning (SFP). More than 20 SE types have been reported, among which Staphylococcal enterotoxin A (SEA) has been recognized as one of the most important SEs associated with SFP. However, the regulatory mechanisms underlying its production remain unclear. Previously, we identified a major SFP clone in Japan, CC81 subtype-1, which exhibits high SEA production. In this study, we attempted to identify the factors contributing to this phenomenon. Thus, we demonstrated that the attenuation of the activity of endogenous regulator, Staphylococcal accessory regulator S (SarS), and the lysogenization of a high SEA-producing phage contributed to this phenomenon in CC81 subtype-1. Furthermore, our results indicated that SarS could directly bind to the promoter upstream of the sea gene and suppress SEA expression; this low SarS repression activity was identified as one of the reasons for the high SEA production observed. Therefore, we revealed that both exogenous and endogenous factors may probably contribute to the high SEA production. Our results confirmed that SE production is a fundamental and critical factor in SFP and clarified the associated production mechanism while enhancing our understanding as to why a specific clone frequently causes SFP.
OBJECTIVE: The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Staphylococcal food poisoning (SFP) is a common food-borne illness often associated with contamination during food handling. The genes for Staphylococcal enterotoxin (SE) isoforms SEA and SEB are frequently detected in human nasal Staphylococcus aureus isolates and these toxins are commonly associated with SFP. Past studies described the resistance of preformed SE proteins to heat inactivation and their reactivation upon cooling in foods. Full thermodynamic analyses for these processes have not been reported, however. The thermal stabilities of SEA, SEB, and SEH and reversibility of unfolding in simple buffers were investigated at pH 4.5 and pH 6.8 using differential scanning calorimetry (DSC). SEA and SEB unfolding was irreversible at pH 6.8 and at least partially reversible at pH 4.5 while SEH unfolding was irreversible at pH 4.5 and reversible at pH 6.8. Additional studies showed maximum refolding for SEB at pH 3.5-4.0 and diminished refolding at pH 4.5 with increasing ionic strength. SE-stimulated secretion of interferon-gamma by human peripheral blood mononuclear cells was used to assess residual SE biological activity following heat treatments using conditions matching those used for DSC studies. The biological activities of SEB and SEH exhibited greater resistance to heat inactivation than that of SEA. The residual activities of heat-treated SEB and SEH were measurable but diminished further in the presence of reconstituted nonfat dry milk adjusted to pH 4.5 or pH 6.8. To different extents, the pH and ionic strengths typical for foods influenced the thermal stabilities of SEA, SEB, and SEH and their potentials to renature spontaneously after heat treatments.

Staphylococcal enterotoxins (SEs) produced by enterotoxigenic Staphylococcus aureus in food cause staphylococcal food poisoning. We recently reported a foodborne outbreak due to S. aureus harboring new SE/SE-like (SEl) genes (seg, sei, sem, sen, seo, and selu) related to enterotoxin gene cluster (egc) 2 as with other research groups. However, the pathogenicity of SEs production remains unclear. Therefore, we herein investigated egc2-related SEs production from S. aureus isolates and leftover food items during a foodborne outbreak using a sandwich enzyme-linked immunosorbent assay suitable for the quantification of SEs. S. aureus isolates produced markedly high levels of egc2-related SEs, and the leftover food item \"Sushi\" contained SEs over the toxin dose that causes food poisoning symptoms. A representative isolate was subjected to whole-genome sequencing. The isolate was homologous with previously reported ST45 strains, particularly the unique genomic island νSaβ structure mostly consisting of egc2. The present study indicates that egc2-related SEs are food poisoning causative agents based on high SE production levels within an actual foodborne outbreak.

Staphylococcal food poisoning (SFP), caused by the contamination of staphylococcal enterotoxins, is a common foodborne disease worldwide. The aims of this study were: (1) to investigate classical staphylococcal enterotoxin genes, sea, seb, sec, sed, and see, among Staphylococcus aureus and coagulase-negative staphylococci (CNS) associated with bovine mastitis; (2) to determine the effect of temperature on the expression of classical staphylococcal enterotoxin genes in staphylococci in milk. The detection of classical staphylococcal enterotoxin genes was performed using S. aureus (n = 51) and CNS (n = 47). The expression of classical enterotoxin genes, including sea, seb, sec, and see, was determined during the growth of staphylococci in milk subjected to ultra-high-temperature processing at two different temperatures: 8 °C and room temperature. Classical staphylococcal enterotoxin genes were expressed more frequently in S. aureus (35.30%) than in CNS (12.77%). The sec gene was most frequently detected in S. aureus (29.41%) and CNS (6.38%). Moreover, the expression of sea and sec was significantly higher at room temperature than at 8 °C after 16 h of incubation (p < 0.05). These results emphasize the importance of maintaining the storage temperature of milk below 8 °C to reduce the risk of SFP.

Biofilms are a frequent cause of food contamination of potentially pathogenic bacteria, such as Staphylococcus aureus. Given its vast role in human disease, the possible impact of biofilm-producing S. aureus isolates in a food processing environment is evident. Sixty-nine S. aureus isolates collected from one firm following multiple staphylococcal food poisoning outbreak investigations were utilized for this analysis. Strain evaluations were performed to establish virulence determinants and the evolutionary relationships using data generated by shotgun whole-genome sequencing (WGS), along with end point polymerase chain reaction (PCR) and in vitro phenotypic assessments. S. aureus isolates were grouped into six well-supported clades in the phylogenetic tree, with the relationships within the clades indicating a strong degree of clonal structure. Our analysis identified four major sequence types 47.8% ST1, 31.9% ST45, 7.2% ST5, and 7.2% ST30 and two major spa types 47.8% t127 and 29.0% t3783. Extrapolated staphylococcal enterotoxin (SE) analysis found that all isolates were positive for at least 1 of the 23 SEs and/or SE-like toxin genes. Enterotoxigenic assessments found that 93% of the isolates expressed a classical SE(A-E). SE gene concurrence was observed at 96.2%, based on PCR and WGS results. In total, 46 gene targets were distinguished. This included genes that encode for adhesion and biofilm synthesis such as clfA, clfB, bbp, ebpS, ica, bap and agr. Our evaluation found agr group III to be the most prevalent at 55%, followed by 35% for agr group I. All isolates harbored the complete intercellular adhesion operon that is recognized to contain genes responsible for the adhesion step of biofilm formation by encoding proteins involved in the syntheses of the biofilm matrix. Phenotypic characterization of biofilm formation was evaluated three times, with each test completed in triplicate and accomplished utilizing the microtiter plate method and Congo red agar (CRA). The microtiter plate results indicated moderate to high biofilm formation for 96% of the isolates, with 4% exhibiting weak to no biofilm development. CRA results yielded all positive to intermediate results. The potential to inadvertently transfer pathogenic bacteria from the environment into food products creates challenges to any firm and may result in adulterated food.

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.

Staphylococcal enterotoxins (SEs) are among the leading causes of food intoxications, affecting consumer health even in nanogram (ng) amounts. In the European Union, certain food safety criteria are specified, including the absence of SEs in cheeses, milk powder and whey powder. Until 2019, the analytical reference method used was the European Screening Method, which was replaced by EN ISO 19020. For the official laboratories involved in food control, the German Reference Laboratory for coagulase-positive staphylococci including Staphylococcus aureus organized three interlaboratory proficiency tests (ILPTs) to detect SE type A in food during the years 2013-2018. The selected food products (cream cheese and vanilla pudding) were successfully tested beforehand with regard to easy handling, homogeneity and stability of the added toxin. In 2013, ILPT participants overall were not competent in detecting SE type A in food. The following factors were identified to improve the performance: (i) concentration of sample extract using dialysis; (ii) selection of a sensitive detection kit; and (iii) proper sample handling. By taking these factors into account and instructing and training the laboratories, their competence greatly improved. In 2018, all performance criteria (specificity, sensitivity and accuracy) were >90%, even at very low concentrations of SE type A of approximately 0·01 ng g-1 food.

Staphylococcal enterotoxins are one of the most important causative agents of food poisoning. These molecules function as both gastrointestinal toxins and superantigens (SAgs) which can simultaneously bind MHC-II and T cell receptor leading to a non-specific polyclonal T cell activation and massive proinflammatory cytokine release. Common symptoms include vomiting and diarrhea; however, in more severe cases, systemic dissemination may result in toxic shock syndrome and can be lethal in a few hours. Only small amounts of these heat-stable toxins are needed to cause the disease. Therefore, it is highly important to detect quickly low concentrations of SAgs in biological samples. In this work, we report a surface plasmon resonance (SPR)-based capture immunoassay for the detection of the SAg SEG. We analyzed the use of different amplification strategies. The SPR-based double-antibody sandwich approach could detect picomolar levels of SEG. The use of antibody-coated silica nanoparticles (AbSiNPs) as an alternative enhancing reagent also detected SEG in the picomolar range. Although AbSiNPs did not improve the limit of detection, for the same amount of SAg tested, AbSiNPs gave a higher response level than free antibodies. This work highlights the suitability of silica nanoparticles for signal amplification in SPR-based biosensors. Overall, SPR biosensors offer the capability for continuous real-time monitoring and high sensitivity that can be befitting for the detection of enterotoxins in food industries, laboratories and regulatory agencies.