Staphylococcal Enterotoxins In Foods Research Articles

Microbiological Risks of Traditional Raw Cow's Milk Cheese (Koryciński Cheeses).

Traditional and regional foods have been increasing in popularity among consumers in Poland for many years. The observed trend of searching for natural and authentic taste encourages many producers to craft products from raw milk, including Koryciński cheeses. The aim of this study was to assess the microbiological hazards resulting from the presence of pathogenic bacteria in Koryciński cheeses available in retail trade. The tests were carried out using accredited methods, including the detection of the presence of Salmonella spp., the enumeration of Listeria monocytogenes, the enumeration of coagulase-positive staphylococci, and the detection of staphylococcal enterotoxins in food when the number of coagulase-positive staphylococci in the sample exceeded the limit of 105 cfu/g. The research material consisted of 45 Koryciński cheeses. The tests conducted revealed that Salmonella spp. was not detected in any of the examined cheeses. However, coagulase-positive staphylococci were present in 68.9% of the samples. In as many as 15 tested cheeses, the level of S. aureus contamination was above 105 cfu/g; therefore, these samples were tested for the presence of staphylococcal enterotoxins. The presence of staphylococcal enterotoxins was found in one Koryciński cheese. In four cheeses, the number of L. monocytogenes exceeded the level of 102 cfu/g, the limit specified in Regulation 2073/2005 on microbiological criteria for foodstuffs. The obtained research results confirm the validity of monitoring the microbiological quality of Koryciński cheeses and the need to increase awareness of ensuring proper hygienic conditions of production, including the increased risk associated with unpasteurized milk products.

Prevalence of staphylococcal toxin in food contaminated by Staphylococcus spp.: Protocol for a systematic review with meta-analysis.

Food contamination by Staphylococcus spp. enterotoxigenic strains is quite common and despite underreporting caused by the short duration of clinical symptoms and lack of medical care, staphylococcal food poisoning is one of the most common Foodborne Diseases (FBD) in the world. This study describes a systematic review protocol with meta-analysis on the prevalence and types of staphylococcal enterotoxins in food, and the profile of contaminated foods. The research will be conducted through the selection of studies reporting the analysis of staphylococcal enterotoxins in food contaminated by Staphylococcus spp. Searches will happen on the following databases: Medline (OVID), GALE, Science Direct, CAB Direct (CABI), Google Scholar, in addition to manual search in the list of references of articles, directory of theses and dissertations, and countries' health agencies. Reports will be imported into the application Rayyan. Two researchers will independently select studies and extract data, and a third reviewer will solve conflicting decisions. The primary outcome will be the identification of staphylococcal enterotoxins in food, and the secondary outcomes will include staphylococcal enterotoxin types and foods involved. To assess the risk of bias in the studies, the tool developed by the Joanna Briggs Institute (JBI) will be used. For data synthesis, a meta-analysis will be performed. However, in case that is not possible, a narrative synthesis of the most relevant results will be carried out. This protocol will serve as the basis for a systematic review that aims to relate the results of existing studies on the staphylococcal enterotoxin prevalence and types in food, and the profile of the contaminated foods. The results will broaden the perception of food safety risks, highlight existing literature gaps, contribute to the study of the epidemiological profile and may guide the allocation of health resources for the development of preventive measures related. PROSPERO registration number: CRD42021258223.

Hazard of Staphylococcal Enterotoxins in Food and Promising Strategies for Natural Products against Virulence.

Staphylococcal enterotoxins (SEs) secreted by Staphylococcus aureus frequently contaminate food and cause serious foodborne diseases but are ignored during food processing and even cold-chain storage. Notably, SEs are stable and resistant to harsh sterilization environments, which can induce more serious hazards to public health than the bacterium itself. Therefore, it is necessary to develop promising strategies to control SE contamination in food and improve food safety. Natural products not only have various pharmaceutical properties, such as antimicrobial and antitoxin activities, but they are also eco-friendly, safe, nutritive, and barely drug-resistant. Here, the hazards of SEs and the promising natural compounds with different inhibitory mechanisms are summarized and classified. The key points of future research and applications for natural products against bacterial toxin contamination in food are also prospected. Overall, this review may provide enlightening insights for screening effective natural compounds to prevent foodborne diseases caused by bacterial toxins.

Development and validation of the Standard method EN ISO 19020 - microbiology of the food chain — Horizontal method for the immunoenzymatic detection of staphylococcal enterotoxins in foodstuffs

In the frame of the CEN Mandate M/381 from the European Commission to CEN (European Committee for Standardization), a method for the detection of staphylococcal enterotoxins in foodstuffs has been developed, validated and standardized. An extraction procedure based on dialysis concentration followed by an immuno-enzymatic detection has been defined. In addition, performance criteria (minimum values of sensitivity, specificity and level of detection) to be achieved by the commercially available immuno-enzymatic kits that could be used to detect staphylococcal enterotoxins in food matrices, were developed. A 2-stage validation study was conducted: The first stage aimed at selecting the commercial kits to be included in the second stage, which consisted in an interlaboratory study, using eight matrices covering five food categories (ready-to-eat food, meat products, milk products, dessert and fish). Results showed that two detection kits included in the study met the pre-defined performance criteria. The implementation of dialysis concentration step increased significantly the sensitivity of the method. The method developed allowed to achieve the Benchmark Dose lower limit (BMD10) estimated at 6.1 ng. In 2019, finally, the European Commission recognized this standard as the European Union reference method for the detection of staphylococcal enterotoxins in food.

Development of a Bead-Based Multiplex Assay for the Simultaneous Quantification of Three Staphylococcal Enterotoxins in Food by Flow Cytometry

Food-borne diseases are widespread; one of the reasons is contamination with staphylococcal pathogens that can produce enterotoxins of the protein, causing numerous diseases in humans. The long-term stability of the toxins requires that the methods for foodstuff quality control be refined to prevent contamination with staphylococcal enterotoxins. We developed a bead-based multiplex assay for the simultaneous detection of three staphylococcal enterotoxins (staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin H (SEH)) by flow cytometry. For detection, we used functional beads coupled with monoclonal capture antibodies, biotinylated monoclonal detection antibodies, and streptavidin conjugated to phycoerythrin. The linear ranges of detection of SEA, SEB, and SEH were 0.02–20 ng/mL, 0.2–30 ng/mL, and 0.2–20 ng/mL, respectively. Each toxin was specifically detected without cross-reactions. An efficient use of the developed method for the analysis of foodstuffs was reported.

Production of staphylococcal enterotoxins in food

Staphylococcal food poisoning results from ingestion of food contaminated with toxins produced by enterotoxigenic Staphylococcus aureus strains. Common symptoms of this intoxication include vomiting, diarrhea, and abdominal cramps. Staphylococcal enterotoxins are resistant to heat and a number of environmental factors. Certain cheeses, milk powder, and whey powder are the only foodstuffs that are being routinely examined for the presence of staphylococcal enterotoxins SEA-SEE. The newly identified enterotoxins are not included in the current examination scheme. Enterotoxin-producing staphylococci were already isolated from meat, meat products, milk, dairy products, fermented food products, vegetables, pastries and fish products. It has been demonstrated that many environmental factors associated with food processing and storage can significantly influence the level of secreted enterotoxins by S. aureus strains. Nevertheless, only a few studies on the production of staphylococcal enterotoxins were conducted in foodstuffs. Most data on their expression is based on experiments performed with a low number of S. aureus strains, and usually only SEA-SEE enterotoxins are investigated. These results inclined many authors to the conclusion that milk and dairy products are unfavorable environments for expression of staphylococcal enterotoxins. However, recent research has indicated a significant heterogeneity in the ability of enterotoxin production in milk among S. aureus strains derived from diverse sources. S. aureus strains able to secrete high levels of enterotoxins in milk and meat juice were described. This research indicates that a high number of S. aureus strains should be used for studying staphylococcal enterotoxins expression in food. It seems to be the appropriate way to assess the risk of staphylococcal food poisoning....

AN ANTIBODY MODIFIED AUTOMATED ENZYME‐LINKED IMMUNOSORBENT ASSAY‐BASED METHOD FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN*

ABSTRACT Studies were conducted with an automated enzyme‐linked immunosorbent assay (ELISA)‐based method (Vidas, Staph enterotoxin‐II [SET‐II]), exhibiting an antibody capture that had undergone modification by removal of the Fc fragment on the antibody. Raw liquid or shell eggs containing a nontoxin component with an attraction to the staphylococcal antienterotoxins were studied. Prior to ELISA testing, the eggs were homogenized and extracts collected by centrifugation. Studies showed that regardless of the ELISA‐based method used that utilized the unmodified antibodies with both the Fab1 + Fc fragments intact, positive (false positive) ELISA responses occurred with fertilized egg yolks and fertilized whole liquid or whole shell eggs. Conversely, when modified (Fab1) antibodies were used, the automated SET‐II enzyme‐linked fluorescent immunoassay was negative.PRACTICAL APPLICATIONSMost enzyme‐linked immunosorbent assays as well as other serological systems use the whole antibody that has not undergone modification for the detection of the staphylococcal enterotoxins. The use of whole antibody (Fab1 + Fc fragments) has on occasion produced false positive results. However, the antibody in which the Fc fragment has been removed leaving the Fab1 fragment provides a more specific antibody for the identification of this microbial toxin. The use of modified antibody (Fab1 fragment) represents significant improvement in antibody quality thus ensuring a greater degree of specificity without sacrificing the sensitivity of serological methods for the detection of staphylococcal enterotoxin in foods.

Significance of coagulase-positive staphylococci for microbiological food safety

Staphylococcal foodborne intoxication, in which major symptoms are vomiting and diarrhoea, occurs after ingestion of thermostable staphylococcal enterotoxins produced in food by enterotoxigenic strains of coagulase-positive staphylococci. Staphylococcal enterotoxins are normally not or only slightly inactivated during food processing, storage, distribution or during the preparation of the food in the kitchen. Therefore, if enterotoxigenic staphylococci are able to grow in food to more then 105 - 106 cfu/g/ml before they are killed there is still a risk of intoxication with consumption. The legislation of the Republic of Serbia lays down criteria for coagulase- positive staphylococci in food. However, the number of coagulase-positive staphylococci may not always be a good indicator of the presence of staphylococal enterotoxins, and the number of cells may have already decreased although the product still contains enterotoxins. The microbiological criteria for coagulase-positive staphylococci and/or staphylococcal enterotoxins in food are essential and useful to protect public health.

Atypical Toxigenic Staphylococcus and Non-Staphylococcus aureus Species on the Horizon? An Update

Staphylococcal food poisoning is a commonly reported illness caused by the ingestion of preformed staphylococcal enterotoxin in foods, With some exceptions, enterotoxin production is associated with coagulase-positive rather than coagulase-negative staphylococci. Of the coagulase-positive staphylococcal species, S. aureus was historically thought to be exclusively implicated in human foodborne illness. More recently, however, other coagulase-positive and some coagulase-negative staphylococcal species have been associated with foodborne intoxication, Coagulase activity has been used to indicate pathogenicity of a foodborne isolate, and thermostable nuclease is being suggested as a more reliable indictor of enterotoxigenicity. Evidence suggests that the metabolic expressions that are the bases of the tests may not be reliable indicators of pathogenicity. A more useful approach to determine the pathogenicity of a Staphylococcus species is to test directly for enterotoxigenicity with one of the new rapid methods. None of the conventional ancillary identification tests has been conclusively associated with enterotoxin synthesis. Furthermore, evidence exists that enterotoxin production is a characteristic of several species in the genus Staphylococcus.

Immunoenzymatic Detection of Staphylococcal Enterotoxins: International Interlaboratory Study

An international interlaboratory study was performed by 13 laboratories to validate a commercially available, rapid enzyme immunoassay for detection of staphylococcal enterotoxins A, B, C, D, and E in foods. The 5 enterotoxin serotypes were detected at a level of 0.5 ng/g in mushrooms and ravioli, 0.8 ng/g in meat, 1 ng/mL in milk, and 1.5 ng/g in raw milk cheese when these foods were artificially contaminated with enterotoxin A. Enterotoxins A, B, C, D, or E were also detected in culture supernatants with no protein A interference when normal rabbit serum was used. This method was validated by the French Normalization Agency for the identification of staphylococcal enterotoxins in foods and culture fluids.

A Collaborative Study on the Detection of Staphylococcal Enterotoxins in Foods with an Enzyme Immunoassay Kit (TECRA)

One of the commercially available enzyme immunoassay kits for the detection of staphylococcal enterotoxins (SEs) in foods, the TECRA screening kit (Bioenterprises Pty. Ltd., Roseville, New South Wales, Australia), has microtiter plates coated with a mixture of antibodies to all of the SEs. A collaborative study was conducted to ascertain whether specificity, sensitivity, repeatability, and reproducibility of the results obtained using this kit would meet food-safety criteria. Thirteen Canadian collaborators participated in this study to analyze both various foods to which 1.0 to 3.0 ng of SE/g of food had been added and negative control samples. In addition, the effect of animal serum in these analyses was examined. The results indicate that all collaborators (100%) were able to detect the minimum toxin levels of 1.0 ng of SEA/g of ham and 1.0 ng of SEB/g of salami and SE or SEs in other samples (chicken, turkey, and cheese) containing 2.0 to 3.0 ng/g, without any false-negative results. With regard to negative control samples, all collaborators obtained correct results except when analyzing two types of food: two collaborators (15%) showed weak false-positive results with salami and all analysts found strong false-positive results with mussels. The problem regarding specificity could be largely corrected by treating the sample with rabbit serum (0.1 volume in 1.0 volume food extract). The repeatability and reproducibility of results from the kit were acceptable.

A collaborative study on the detection of staphylococcal enterotoxins in foods by an enzyme immunoassay kit (RIDASCREEN ®)

The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) immunoassay kit, utilizes monovalent capture antibodies against SE types A to E (SEA to SEE), therefore, it simultaneously detects and identifies the enterotoxin types. A collaborative study was conducted to ascertain whether the specificity, sensitivity, repeatability and reproducibility of the kits would meet food safety criteria. Twelve Canadian laboratories participated in this study to analyze various foods to which 1.0 to 2.0 ng of SE g had been added and negative control samples. The results indicate that the sensitivity and specificity of the kit were excellent, all collaborators were able to detect the minimum toxin levels of 1.0 ng SEA g in ham and cheese, 1.0 ng SEB g in salami and turkey, and 2.0 ng SED g in other samples without any false-negative results. With regard to negative control samples, all analysts obtained correct results except for one analyst who recorded weak false-positive results with several foods detecting SEC or SEA. The overall rate of false-positive results was 0.7% for 600 triplicate assays. In addition, it was confirmed that the RIDASCREEN kit did not yield false-positive results with mussels in contrast to some other EIA kits. Overall repeatability and reproducibility of the kit were in the range of 11.69–42.57% and 17.25–68.05%, respectively.

Detection and occurrence of waterborne bacterial and viral pathogens

Detection and occurrence of waterborne bacterial and viral pathogens

Visual Screening with Enzyme Immunoassay for Staphylococcal Enterotoxins in Foods: Collaborative Study

Selected foods containing 4-10 ng each of a mixture of Staphylococcus aureus enterotoxin serotypes A-E were tested by 15 collaborators. An enzyme-linked immunosorbent assay (EIA) was used with polyvalent antisera to these serotypes in a polyclonal antibody double "sandwich" configuration. Controls were free of toxin. Foods (25 g test samples) were homogenized with Tris (0.25 M, pH 8.0) and centrifuged. The food extract was filtered through cotton and mixed with sample additive. For the EIA, 200 microL aliquots of the treated extracts were added to previously washed microtiter wells coated with antibody to staphylococcal enterotoxin serotypes A-E. Wells were washed and treated with the polyvalent antisera (A-E)-enzyme conjugate, and then washed again. Substrate was added, and wells were incubated. After incubation, stop solution was added. Results were determined visually and by measuring absorbance using a microtiter plate reader. In foods containing enterotoxin, bluish-green color was developed (positive result). Test solutions with absorbances > 0.200 were considered positive; those with absorbances < or = 0.200 were negative. The method is sensitive and specific, and allows the rapid assay of staphylococcal enterotoxins in foods without differentiating their serotypes. The method has been adopted first action by AOAC INTERNATIONAL.

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.

Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning

Four commercial kits, three based on sandwich ELISA techniques and one on latex agglutination were compared for the detection of staphylococcal enterotoxins in foods from outbreaks of food poisoning. Enterotoxin was detected in 14 of 18 foods with the Swiss SET-EIA and in 9 or 10 with the Unipath SET-RPLA and two ELISAs from Transia. The advantages and disadvantages of the four methods are discussed.

Simple and Rapid Inhibition Enzyme Immunoassay for the Detection of Staphylococcal Enterotoxin B in Foods

An absorption-inhibition ELISA (AI-ELISA) was developed to detect, within an hour, the staphylococcal enterotoxin B (SEB) in solutions and food homogenates. The SEB-containing sample is first incubated with a standardized concentration of antibody. The quantity of nonabsorbed anti-SEB Ab is evaluated by incubating a SEB-coated dipstick in the reaction mixture. The protein A-peroxidase conjugate is then used to detect the anti-SEB Ab bound to SEB-coated dipsticks. The enzymatic activity developed in a solution containing the substrate and a chromogen is inversely related to the SEB concentration. Concentrations as low as 0.01 to 1 μg SEB/ml can be consistently detected within 20 min. In unconcentrated food homogenates, 100 ng of SEB per ml could be detected by this AI-ELISA. The minimal level of sensitivity could be lowered to at least 1 ng SEB/g of food. The rapidity of the AI-ELISA, its simplicity, and repeatability make this immunoassay a promising tool for routine detections of staphylococcal enterotoxins in foods.

Rapid detection of staphylococcal enterotoxins in foods with a modification of the reversed passive latex agglutination assay.

A modification of the reversed passive latex agglutination kit assay for detection of staphylococcal enterotoxins by centrifugation of microtitration plates reduces the incubation time of the assay from 20-24 h to 4 h. Enterotoxins can therefore be detected in foods within the working day.

ELISA with enzyme amplification for sensitive detection of staphylococcal enterotoxins in food

A highly sensitive amplified ELISA in microtitre plates for the detection of staphylococcal enterotoxins compared favourably with a commercially available kit. The amplified ELISA demonstrated a reduced effect of the food sample matrix and an ease of handling. Using the amplified ELISA, the influence of different parameters (pH, time, temperature, re-extraction, concentration procedure) on the recovery efficiency and assay reliability was tested on foods implicated in food poisoning outbreaks as well as on spiked foods containing low levels of enterotoxins.

Detection of staphylococcal enterotoxins in dairy products by the reversed passive latex agglutination (SET-RPLA) kit

The SET-RPLA is a commercially available kit for the detection of staphylococcal enterotoxins in foods. Previous reports have shown that non-specific reactions occur on use of the kit with cheeses and thereby restrict its use. In this study a variety of dairy products were tested and non-specific reactions were found to be associated only with those products rennetted during manufacture. These reactions can be obviated by addition of 10 mM sodium hexametaphosphate to the diluent provided in the kit, without affecting the ability to detect staphylococcal enterotoxins in dairy products. The sensitivity of the SET-RPLA was demonstrated to be 0.25 ng/ml.