Novel Platform for the Detection of Staphylococcus aureus Enterotoxin B in Foods

Abstract

bStaphylococcal contamination of food products and staphylococcal food-borne illnesses continue to be a problem worldwide. Screening of food for the presence of Staphylococcus aureus and/or enterotoxins using traditional methods is laborious. Reliable and rapid multiplex detection methods from a single food extract or culture supernatant would simplify testing. A fluorescence-based cytometric bead array was developed for the detection of staphylococcal enterotoxin B (SEB), using magnetic microspheres coupled with either an engineered, enterotoxin-specific V␤ domain of the T-cell receptor (V␤-TCR) or polyclonal antibodies. The binding affinity of the V␤-TCR for SEB has been shown to be in the picomolar range, comparable to the best monoclonal antibodies. The coupled beads were validated with purified enterotoxins and tested in a variety of food matrices spiked with enterotoxins. The V␤-TCR or antibody was shown to specifically bind SEB in four different food matrices, including milk, mashed potatoes, vanilla pudding, and cooked chicken. The use of traditional polyclonal antibodies and V␤-TCR provides a redundant system that ensures accurate identification of the enterotoxin, and the use of labeled microspheres permits simultaneous testing of multiple enterotoxins from a single sample. Staphylococcus aureus is among the top five common pathogens associated with food-borne illness nationwide. The Centers for Disease Control and Prevention (CDC) estimates that there are about 240,000 illnesses with 1,000 hospitalizations and 6 deaths associated with staphylococcal food poisoning (SFP) annually (1). SFP is directly linked to small (25-to 30-kDa) exotoxins, known as staphylococcal enterotoxins (SEs), that are heat resistant, tolerate low pH, and often persist long after the microbe has been rendered nonviable. SEs are superantigens that induce nonspecific T-cell activation, which can rapidly cascade to a massive release of inflammatory mediators and may lead to toxic shock (2, 3). SFP occurs when improperly handled food contaminated with as little as 100 ng of SE is consumed. SFP is marked by severe gastrointestinal symptoms such as emesis, diarrhea, and/or abdominal pain after a 4-h incubation period (4).Suspect food products are typically analyzed by using traditional culture techniques followed by identification of enterotoxigenic staphylococcal strains. Food extracts positive for bacteria may then be evaluated for the presence of preformed SEs (5). A recent outbreak in Japan from milk contaminated with staphylococcal enterotoxin A (SEA) illustrated the challenges of conventional methods, since viable bacteria were not detected (6, 7). PCR methods would be a useful screening tool for samples that are culture negative and enterotoxin positive (8). PCR methods are sensitive and can detect low levels of gene targets; however, this does not verify the presence of expressed proteins, and it is unacceptable as a definitive method for SE detection in the regulatory arena. Therefore, immunological techniques using either m...

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

“…By way of comparison, the assay used in the present study with biotinylated Vβ-SEB coated streptavidin beads enabled detection of SEB at concentrations as low as 3 pg/ml in singleplex assay. This was 40 fold better than our previous effort of detecting SEB where Vβ-SEB was coated on paramagnetic beads by means of amine coupling [ 31 ]. We believe that the site-specific biotinylation of Vβ protein allowed its immobilization on streptavidin coated in an optimal orientation hence leading to improved sensitivity of toxin detection, than immobilizing Vβ via amine coupling methods which may have yielded heterogeneous orientations.…”

“…Commercial kits based on ELISA and ELFA using anti-enterotoxin antibody or antibody fragments (Fab) are available with reported sensitivities for staphylococcal enterotoxins A-E of 0.25 to 1 ng/ml. To date, such assays are limited to analysis of individual toxins [ 11 , 31 ].…”

“…For comparison, we had recently used direct coupling of the Vβ-SEB to detect 125 pg/ml SEB in different food matrices [ 31 ]. We hypothesized that site-specific biotinylation of high-affinity Vβ proteins would allow their immobilization on streptavidin pre-coated beads in an optimal orientation and indeed the biotin approach yielded a 40-fold improvement to 3 pg/ml.…”

Stress-caused anergy of leukocytes towards Staphylococcal enterotoxin B and exposure transcriptome signatures

“…The identified host origin pathogen-specific or stress fingerprints have the potential to be used as specific markers if they are used in groups (similar to DNA fingerprinting patterns) on a chip, for example. 29 This study was restricted to male subjects owing to the all male composition of the RASP (Army Rangers) population. Some of our findings may not be applicable to female subjects (as female subjects may react differently towards RASP or SEB or both).…”

Isolation and Epitope Mapping of Staphylococcal Enterotoxin B Single-Domain Antibodies

“…To that end, there have been recent efforts to develop sensing platforms for SEB including photonic crystal-based lab-on-a-chip assays [8], fluorescence-based flow cytometry assays [9], and aptamer-based fluorescence resonance energy transfer (FRET) assays [10]. Each of these approaches requires sensitive and selective recognition elements such as monoclonal antibodies (mAbs), aptamers, or receptors specific to SEB able to bind the toxin in complex sample matrices and elicit a measurable response.…”