A single-stranded DNA aptamer that selectively binds to Staphylococcus aureus enterotoxin B

The bacterium Staphylococcus aureus is a common foodborne pathogen capable of secreting a cocktail of small, stable, and strain-specific, staphylococcal enterotoxins (SEs). Staphylococcal food poisoning (SFP) results when improperly handled food contaminated with SEs is consumed. Gastrointestinal symptoms of SFP include emesis, diarrhea and severe abdominal pain, which manifest within hours of ingesting contaminated food. Immuno-affinity based methods directly detect, identify, and quantify several SEs within a food or clinical sample. However, the success of these assays depends upon the availability of a monoclonal antibody, the development of which is non-trivial and costly. The current scope of the available immuno-affinity based methods is limited to the classical SEs and does not encompass all of the known or emergent SEs. In contrast to antibodies, aptamers are short nucleic acids that exhibit high affinity and specificity for their targets without the high-costs and ethical concerns of animal husbandry. Further, researchers may choose to freely distribute aptamers and develop assays without the proprietary issues that increase the per-sample cost of immuno-affinity assays. This study describes a novel aptamer, selected in vitro, with affinity to staphylococcal enterotoxin B (SEB) that may be used in lieu of antibodies in SE detection assays. The aptamer, designated APT(SEB1), successfully isolates SEB from a complex mixture of SEs with extremely high discrimination. This work sets the foundation for future aptamer and assay development towards the entire family of SEs. The rapid, robust, and low-cost identification and quantification of all of the SEs in S. aureus contaminated food is essential for food safety and epidemiological efforts. An in vitro generated library of SE aptamers could potentially allow for the comprehensive and cost-effective analysis of food samples that immuno-affinity assays currently cannot provide.     

Molecular epidemiological characterization of Staphylococcus aureus isolates originating from food poisoning outbreaks that occurred in Tokyo,Japan

Staphylococcal food poisoning (SFP), one of the commonest food‐borne diseases, results from the ingestion of one or more staphylococcal enterotoxins (SEs) produced in foods by Staphylococcus aureus. In the present study, 203 S. aureus strains originating from 83 outbreaks that had occurred in Tokyo were examined for their coagulase type and genotype of SEs to analyze their molecular epidemiological characteristics. The representative subsets of the 83 S. aureus isolates were analyzed by multilocus sequence typing (MLST) and S. aureus pathogenicity island (SaPI) scanning. The isolates were integrated into eight specific clonal complexes (CC) s; CC81, CC8, CC6, CC5, CC508, CC59, CC20 and CC30. The profiles of the coagulase type, SE/SEl genotype and the suspected type of enterotoxin‐encoding mobile genetic element (MGE) indicated a correlation with each CC. SaPI scanning showed fixed regularity between the distributions of genomic islands, including SaPIs, and the phylogenetic lineage based on MLST. These results indicate that the S. aureus isolates, which classified into eight CCs, have distinguishable properties concerning specific coagulase type, enterotoxin genotype and MGE type. Strains of S. aureus harboring these particular elements possess the potential to cause SFP.     

Staphylococcal Enterotoxins B and C

Abstract

The staphylococcal enterotoxins (SEs) are a subgroup of related protein exotoxins in the pyrogenic toxin (PT) family produced by Staphylococcus aureus and Streptococcus pyogenes (1). Like other members of the PT family, the SEs are superantigens and elaborate a set of biological activities linked to their ability to stimulate cells of the immune system (2). These activities contribute to their ability to induce toxic shock syndrome, immunosuppression, and probably other diseases (3). However, as is evident from the fact that they are designated as enterotoxins, the SEs are distinguishable from other members of the PT family by their ability to induce gastroenteritis when ingested. Hence, they are the causative agents in staphylococcal food poisoning (SFP), a very common form of food-associated gastroenteritis in the United States and worldwide (4).     

Murine monoclonal antibodies reactive with staphylococcal enterotoxins A, B, C2, D, and E

By fusion of mouse spleen cells immunized with five different staphylococcal enterotoxins (SEA, SEB, SEC2, SED, and SEE) with myeloma cells, we obtained 15 hybridomas producing monoclonal antibodies (mAbs). Four mAbs were reactive with both SEA and SEE, whereas 8 mAbs were reactive with SEB and SEC2. One mAb reacted with SEA, SED, and SEE. The other two mAbs were found to be reactive with all five serotypes of SEs. The mAbs specific for five serotypes of SEs were found to be most reactive with SED, reactive with SEA, and slightly less reactive with SEB, SEC2, and SEE. Those mAbs with specificities for all serotypes of SEs may be valuable to prepare immunoadsorbent(s) for isolation of SEs and to detect SEs in foods and clinical specimens involved in outbreaks of staphylococcal food poisoning.     

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.     

Staphylococcal enterotoxin genes are common in Staphylococcus aureus intestinal flora in Sudden Infant Death Syndrome (SIDS) and live comparison infants

Pathological and epidemiological findings in sudden infant death syndrome (SIDS) suggest an infectious aetiology with indications of involvement of staphylococcal enterotoxins (SEs). While SEA, SEB and SEC have been found in the sera and tissues of SIDS cases, little is known about the role of intestinal Staphylococcus aureus or the roles of later-described toxins SEE, SEG, SEH, SEI and SEJ in SIDS. We used a molecular-based approach to define whether the intestinal tract could be a source of SEs to support the staphylococcal toxic shock hypothesis for SIDS. Intestinal contents from 57 SIDS infants and faeces from 79 age- and gender-matched live comparison infants were cultured and tested for S. aureus and sea-b-c-e-g-h-j and TSST using PCR. High proportions of infants in both groups carried toxigenic and nontoxigenic S. aureus . Significantly greater proportions of SIDS compared with comparison babies were positive for S. aureus (68.4% vs. 40.5%) and for SE genes (43.8% vs. 21.5%), suggesting a possible role in SIDS. The results indicate that colonization by S. aureus with SE genes is common in infants; however, their detection is unlikely to be a strong predictive tool for SIDS. Other factors (including immune response) may reveal a specific susceptibility to SEs in SIDS infants.     

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.     

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.     

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.     

Molecular analysis of Staphylococcus aureus isolates associated with staphylococcal food poisoning in South Korea

AIMS: To investigate the molecular epidemiological study of Staphylococcus aureus from staphylococcal food poisoning (SFP) incidents in South Korea. METHODS AND RESULTS: Three hundred and thirty-two strains isolated from ten provinces between June 1999 and January 2002 were characterized by staphylococcal enterotoxin genes, toxic shock syndrome toxin 1 (tst) gene, and exfoliative toxin genes. Toxin genotypes were sea-seh (n=197), sea (n=51), sea-seg-sei (n=14), seg-sei (n=10), seb (n=10), seb-sed-seg-sei-sej (n=3), sea-seg-seh-sei (n=1), sea-seb (n=1), sea-sec (n=1), seg-sei plus eta (n=4), and sea-seg-sei plus tst (n=40). Most of the strains could be classified into three clusters of pulsed-field gel electrophoresis (PFGE) types A and B with coagulase type VII and type E with coagulase type IV. Of the ten sequence types (ST), ST1, ST59, and ST30 were frequently showed by multilocus sequence typing. CONCLUSIONS: The strain belonging to PFGE pattern A with sea-seh gene, coagulase VII, and ST1 was the most epidemic clone of SFP incidents in Korea.     

Genotypes, exotoxin gene content, and antimicrobial resistance of Staphylococcus aureus strains recovered from foods and food handlers

Staphylococcal food poisoning, one of the most common food-borne diseases, results from ingestion of one or more staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus in foods. In the present study, 64 S. aureus isolates recovered from foods and food handlers, associated or not associated with food-poisoning outbreaks in Spain, were investigated. They were assigned to 31 strains by spa typing, multilocus sequence typing (MLST), exotoxin gene content, and antimicrobial resistance. The strains belonged to 10 clonal complexes (CCs): CC5 (29.0%), CC30 (25.8%), CC45 (16.1%), CC8, CC15 (two strains each), CC1, CC22, CC25, CC59, and CC121 (one strain each). They contained hemolysin genes (90.3%); lukED (77.4%); exfoliatin genes eta, etd (6.5% each), and etb (3.2%); tst (25.8%); and the following enterotoxin or enterotoxin-like genes or clusters: sea (38.7%), seb (12.9%), sec (16.1%), sed-selj with or without ser (22.9%), selk-selq (6.5%), seh, sell, selp (9.7% each), egc1 (32.3%), and egc2 (48.4%). The number of se and sel genes ranged from zero to 12. All isolates carrying tst, and most isolates with genes encoding classical enterotoxins (SEA, SEB, SEC, and SED), expressed the corresponding toxin(s). Two CC5 isolates from hamburgers (spa type t002, sequence type 5 [ST5]; spa type t2173, ST5) were methicillin resistant and harbored staphylococcal cassette chromosome mec (SCCmec) IVd. Six (19.4%) were mupirocin resistant, and one (spa type t120, ST15) from a food handler carried mupA (MIC, 1,250 μg/ml). Resistance to ampicillin (blaZ) (61.3%), erythromycin (ermA-ermC or ermC) (25.8%), clindamycin (msrA-msrB or msrB) (16.1%), tetracycline (tetK) (3.2%), and amikacin-gentamicin-kanamycin-tobramycin (aphA with aacA plus aphD or aadD) (6.5%) was also observed. The presence of S. aureus strains with an important repertoire of virulence and resistance determinants in the food chain represents a potential health hazard for consumers and merits further observation.     

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.     

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.     

Applicability of an immunoblot technique combined with a semiautomated electrophoresis system for detection of staphylococcal enterotoxins in food extracts.

We studied the usefulness of an immunoblot technique for the detection of staphylococcal enterotoxins (SEs) in strains and food extracts. Food samples (milk, yogurt, hot dog sausage, cheese, and mayonnaise) were artificially contaminated with SEA through SEE. Protein A did not interfere with the results; it appeared on electrophoresis gels as bands with molecular weights higher than those of the SEs. Other food proteins were not revealed by the technique. The immunoblot technique proved to be fast, specific, and sensitive for the detection of SEs in foods.     

Applicability of an immunoblot technique combined with a semiautomated electrophoresis system for detection of staphylococcal enterotoxins in food extracts.

We studied the usefulness of an immunoblot technique for the detection of staphylococcal enterotoxins (SEs) in strains and food extracts. Food samples (milk, yogurt, hot dog sausage, cheese, and mayonnaise) were artificially contaminated with SEA through SEE. Protein A did not interfere with the results; it appeared on electrophoresis gels as bands with molecular weights higher than those of the SEs. Other food proteins were not revealed by the technique. The immunoblot technique proved to be fast, specific, and sensitive for the detection of SEs in foods.     

Characterization and identification of coagulase-negative, heat-stable deoxyribonuclease-positive staphylococci

Various characteristics of 13 coagulase-negative, weakly heat-stable deoxyribonuclease-positive staphylococci from human, veterinary and food sources were determined in an effort to identify them. Nine of the isolates were identified as coagulase-negative Staphylococcus aureus (2), Staphylococcus xylosus (2), Staphylococcus simulans (3), Staphylococcus capitis (1) and Staphylococcus sciuri subsp. lentus (1); the other four isolates, from food and veterinary sources, could not be identified as currently accepted or proposed species. Teichoic acid and peptidoglycan compositions were used as key taxonomic characteristics. The determination of heat-stable deoxyribonuclease activity can be useful to detect coagulase-negative S. aureus strains. However, this activity also appears to be present in strains of other staphylococcal species.     

Characterization of a novel staphylococcal enterotoxin-like superantigen,a member of the group V subfamily of pyrogenic toxins

Staphylococcus aureus is an important human pathogen, causing a variety of diseases. Major virulence factors of this organism include staphylococcal enterotoxins (SEs) that cause food poisoning and toxic shock syndrome. Our study identified a novel enterotoxin-like protein that is a member of the new subfamily (group V) of pyrogenic toxin superantigens (PTSAgs) and examined its biochemical and immunobiological properties. The gene encoding the SE-like protein is directly 5' of another recently identified PTSAg, SEK. The SE-like protein had a molecular weight of 26000 and an experimentally determined isoelectric point between 7.5 and 8.0. We demonstrated that the PTSAg had many of the biological activities associated with SEs, including superantigenicity, pyrogenicity, and ability to enhance endotoxin shock, but lacked both lethality in rabbits when administered in subcutaneous miniosmotic pumps and emetic activity in monkeys. Recombinant protein stimulated human CD4 and CD8 T cells in a T cell receptor variable region, beta chain (TCRVbeta) specific manner. T cells bearing TCRVbeta 2, 5.1, and 21.3 were significantly stimulated.     

RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production

Staphylococcal enterotoxins (SEs) are emetic toxins that cause food poisoning. SEs also function as powerful pyrogenic toxin superantigens that stimulate non-specific T-cell proliferation. Together with the hemolysins, SEs have been largely implicated as virulence factors in multiple infection models. Recent biochemical and genetic analyses have demonstrated that production of some of these toxins is partially regulated by quorum sensing mechanisms where proteins and peptides activate the accessory gene regulator (agr). Because toxin production is central to bacterial pathogenesis, therapeutic strategies alternative to antibiotics, and based on rational interference of the quorum sensing systems involved, are currently being developed. This approach would lead to repression of toxin production and, thus, to disease prevention. Here we provide evidence to conclude that synthetic analogs of the RNAIII inhibiting peptide (RIP) and antibodies to its target molecule TRAP function in vitro as efficient suppressors of agr-regulated exotoxin production by Staphylococcus aureus.     

Enterotoxigenic potential of Staphylococcus intermedius.

Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast to Staphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive species S. intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine, n = 247; equine, n = 23; feline, n = 9; other, n = 2) and 11 human isolates of S. intermedius was tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genes sea, seb, sec, sed, and see. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%) S. intermedius isolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for the sec gene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number of S. intermedius isolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission of S. intermedius from animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.