Mass Outbreak of Food Poisoning Disease Caused by Small Amounts of Staphylococcal Enterotoxins A and H

It was believed that food poisoning in Osaka in 2000 was due to small amounts of staphylococcal enterotoxin A (SEA) in reconstituted milk. Results of this study clearly indicate that SEH was also present in the raw material of reconstituted milk, indicating that the food poisoning was caused by multiple staphylococcal enterotoxins.     

Ecology of spontaneous fermentation in one winery during 5 consecutive years

A concentration protocol based on trichloroacetic acid precipitation was evaluated and compared with the reference method using dialysis concentration. Different quantities of purified staphylococcal enterotoxins were added to pasteurized Camembert-type cheeses. Detection of enterotoxins in these cheeses was performed using an automated detection system. Raw goat milk Camembert-type cheeses involved in a staphylococcal food poisoning were also tested. Both enterotoxin extraction methods allowed detection of the lowest enterotoxin concentration level used in this study (0.5 ng g-1). Compared with the dialysis concentration method, TCA precipitation of staphylococcal enterotoxins was 'user-friendly' and less time-consuming. These results suggest that TCA precipitation is a rapid (1 h), simple and reliable method of extracting enterotoxin from food which gives excellent recovery from dairy products.     

An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.     

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

Staphylococcus aureus in cow milk and milk products in Ambo and Bako towns,Oromia, Ethiopia: prevalence,associated risk factors,hygienic quality,and antibiogram

International Microbiology - Staphylococcus aureus (S. aureus) is a foodborne bacterial pathogens that can cause staphylococcal food poisoning and contaminate food of animal origin worldwide. The...     

Protein Standard Absolute Quantification (PSAQ) for improved investigation of staphylococcal food poisoning outbreaks

Staphylococcal enterotoxins are major causing agents of food-borne diseases. Their detection in food remnants for risk assessment or food poisoning outbreaks investigation suffers from a lack in comprehensive immunological tools. In this study, we demonstrate that the combination of immunocapture and Protein Standard Absolute Quantification (PSAQ) strategy, which uses isotope-labeled enterotoxins as internal standards for MS-based analysis, is powerful to specifically identify and quantify these contaminating agents in food matrices. This approach is believed to significantly improve the elucidation of staphylococcal food poisoning outbreaks.     

Staphylococcus aureus and food poisoning

Food-borne diseases are of major concern worldwide. To date, around 250 different food-borne diseases have been described, and bacteria are the causative agents of two thirds of food-borne disease outbreaks. Among the predominant bacteria involved in these diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food. Staphylococcal food poisoning is due to the absorption of staphylococcal enterotoxins preformed in the food. Here, we briefly review the latest data on staphylococcal enterotoxins and some papers exemplifying the interactions between S. aureus and the food matrix; environmental factors affecting staphylococcal enterotoxin production are discussed.     

A Novel ELISA Format for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.     

Modified Radioimmunoassay Determination for Staphylococcal Enterotoxin B in Foods

The sensitivity of solid-phase radioimmunoassay for the measurement of staphylococcal enterotoxin B (SEB) in foods was decreased by food constituents that react with rabbit anti-SEB with an equivalency of over 2 ng/ml. This activity was minimized by a conditioning step for anti-SEB and by removal of interfering compounds in the sample by extraction. The assay was a sequential solid-phase radioimmunoassay technique in which polystyrene test tubes were initially incubated with antisera and then with bovine serum albumin. The tubes were then conditioned with either a centrifuged aqueous cheese extract or, equally effective, reconstituted nonfat dry milk for 16 h at 4°C. Samples of milk or heat-treated and CHCl₃-extracted cheese or chicken salad slurries were incubated in the assay tubes for 6 h at 37°C. The samples were replaced by ¹²⁵I-labeled SEB and incubated for a further 2 to 4 h before the contents were removed and the tubes were washed and counted. A buffer solution containing known concentrations of toxin served as standards for assaying SEB in the food extracts. The entire assay can be accomplished within 24 h with a sensitivity of 1 ng/ml in milk and in the cheese extract or 1.3 ng/ml in the chicken salad extract.     

Enterotoxigenicity of Staphylococcus aureus Cultures Isolated from Acute Cases of Bovine Mastitis

To determine whether staphylococci causing bovine mastitis are potential causes of human intoxications, 142 cultures identified as etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were obtained from investigators in the United States and Canada, examined microscopically, and tested for carbohydrate utilization, terminal pH, catalase, coagulase, egg yolk hydrolysis, gelatin hydrolysis, cytochrome oxidase, urease production, nitrate reduction, micrococcal nuclease, phage type, and enterotoxin production. Three cultures were not confirmed as Staphylococcus aureus. Of the 157 S. aureus cultures, 23 produced staphylococcal enterotoxins. Although a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning was not proved, results indicated that staphylococcal infections of the bovine mammary gland represent a significant reservoir of enterotoxigenic strains of S. aureus.     

金黄色葡萄球菌肠毒素检测的研究进展

金黄色葡萄球菌(Staphylococcus aureus)是一种重要的人畜共患致病菌,广泛存在于自然界中,其产生的肠毒素(staphylococcal enterotoxins,SE)可通过污染食物而导致食物中毒。目前,随着人们对食品安全重视度的加深,国际上已将肠毒素的检测列入食品检验法规,因此建立灵敏、快速的肠毒素检测方法,是食品安全检测的一项重要研究内容。     

Enzyme-linked immunosorbent assay for detection of staphylococcal enterotoxins in foods.

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of staphylococcal enterotoxins in foods. The "double-antibody sandwich" protocol combines parts of several procedures reported previously. Horseradish peroxidase was conjugated to antibody specific for an enterotoxin, and the antibody-enzyme conjugate was assayed with a 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid)-H2O2 substrate solution. Enterotoxins were added to a variety of foods that were representative of those implicated in staphylococcal food poisoning outbreaks. Extracts of the foods were assayed by the ELISA and radioimmunoassay. Enterotoxin levels below 1 ng/g of food were consistently detectable by the ELISA. These results compared favorably with those of the radioimmunoassay. Experiments confirmed the interference of protein A in double-antibody sandwich ELISAs. Although protein A interference has not been demonstrated to be a problem in food extracts, we suggest a screen for protein A interference in which immunoglobulin G from nonimmunized rabbits is used. All of the known staphylococcal enterotoxins could be detected by this method. Analysis of a food product for entertoxin by the ELISA can be completed in an 8-h working day.     

Identification and Characterization of a Novel Staphylococcal Emetic Toxin

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have superantigenic and emetic activities, which cause toxic shock syndrome and staphylococcal food poisoning, respectively. Our previous study demonstrated that the sequence of SET has a low level of similarity to the sequences of other SEs and exhibits atypical bioactivities. Hence, we further explored whether there is an additional SET-related gene in S. aureus strains. One SET-like gene was found in the genome of S. aureus isolates that originated from a case of food poisoning, a human nasal swab, and a case of bovine mastitis. The deduced amino acid sequence of the SET-like gene showed 32% identity with the amino acid sequence of SET. The SET-like gene product was designated SElY. In the food poisoning and nasal swab isolates, mRNA encoding SElY was highly expressed in the early log phase of cultivation, whereas a high level of expression of this mRNA was found in the bovine mastitis isolate at the early stationary phase. To estimate whether SElY has both superantigenic and emetic activities, recombinant SElY was prepared. Cell proliferation and cytokine production were examined to assess the superantigenic activity of SElY. SElY exhibited superantigenic activity in human peripheral blood mononuclear cells but not in mouse splenocytes. In addition, SElY exhibited emetic activity in house musk shrews after intraperitoneal and oral administration. However, the stability of SElY against heating and pepsin and trypsin digestion was different from that of SET and SEA. From these results, we identified SElY to be a novel staphylococcal emetic toxin.     

Selective transport of staphylococcal enterotoxin A through in vitro generated human M cells

Staphylococcal enterotoxins are responsible for food poisoning and toxic shock syndrome due to their superantigen activity on T cells. Although their activity necessarily involves passage through the intestinal epithelium, little is known about this critical step. In the present study, we compared the in vitro transport of staphylococcal enterotoxin A through human intestinal absorptive and M cells. We found that the transport of the toxin through M cells was polarized and temperature-sensitive, in contrast with the less efficient transport of the toxin by absorptive cells. These data suggest the involvement of M cells in the intestinal absorption of staphylococcal enterotoxins.     

Detection of Staphylococcal Enterotoxin in Food

Methods are described for the extraction and serological detection of trace amounts of enterotoxins A and B in foods incriminated in outbreaks of staphylococcal food poisoning. Evidence is presented for the probable applicability of the methods for the detection of unidentified enterotoxins.     

Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk

Staphylococcus aureus RN6390 presents a diauxic growth in milk, due to amino acid limitation. Inactivation of the oligopeptide permease Opp3 (dedicated to the nitrogen nutrition of the strain) not only affects the growth of the strain but also results in reduced expression levels of three major extracellular proteases.Staphylococcus aureus is a ubiquitous gram-positive pathogen commonly found in the environment. Some S. aureus strains are able to produce enterotoxins that cause food poisoning (6). In France, contaminated dairy products are the main source of staphylococcal food-borne disease outbreaks (3). Thus, the development of this bacterium in milk is a major concern for the safety of dairy products. Our objective was to evaluate the growth of S. aureus in milk, with special attention focused on the role of the peptide transport systems.     

Establishment of highly specific and quantitative immunoassay systems for staphylococcal enterotoxin A, B, and C using newly-developed monoclonal antibodies

Staphylococcal enterotoxin (SE) activities remain after boiling or treating with proteases. The main symptoms such as vomiting and diarrhea, are caused by the ingestion of SEs. Among SEs, SEA has been reported to be the major and most toxic protein. A highly specific and simple assay system is required to diagnose staphylococcal food poisoning. Therefore, the development of a suitable assay system is strongly anticipated. In this study, we have established a highly specific and sensitive avidin-biotin sandwich ELISA (ABS-ELISA) system for SEA, SEB, and SEC1 using newly-developed monoclonal antibodies. The linearity of these systems obtained was in the range of 0.78-25 ng/ml for each SE, and furthermore, the lower concentrations of SEs could also be detected. The recoveries of SEs from murine serum, skim milk solution, and raw milk were found to be over 90%, suggesting that our systems could detect SEs without any interventions, such as these from milk or serum proteins. We were also able to quantify SEs in 22 specimens of culture supernatants of S. aureus isolated in past occurrences. Our established system should be very useful not only in the clinical field but also in various fields of investigation because of its quantifi-cation and simplicity in detecting SEs.     

Prevalence of Enterotoxigenic Staphylococci in Nose,Throat and Skin Lesions in Meat-Workers

The frequency of enterotoxigenic coagulase-positive and coagulase-negative staphylococci in nose and throat and in hand lesions was investigated in 86 meat cutters and dressers. Enterotoxin-producing staphylococci were demonstrated in nasal swabs from 22% of clinically well workers and from 42% of a group with mild coryza. The corresponding rates in throat swabs were 6 and 12%. Four of 16 superficial lesions of the hand harbored enterotoxigenic staphylococci. The implications for contamination of food and outbreaks of staphylococcal food poisoning are discussed.     

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.     

Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobadllus casei

The lysostaphin structural gene was cloned in Bacillus subtilis DSM402 and in Lactobacillus casei 102S. The gene was expressed in both organisms and active lysostaphin was released into the medium. Lysostaphin produced by these organisms induced lysis of growing and heat inactivated staphylococci. Expression in a protective starter organism is a prerequisite to produce lysostaphin in situ in fermenting foods and hence, to reduce the hygienical risk of staphylococcal food poisoning.