Abstract
Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD50 values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.
Highlights
Staphylococcus aureus and group A Streptococcus pyogenes secrete a family of pyrogenic toxins including staphylococcal enterotoxins A, B, C (SEA, SEB, SEC), toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxins (SpeA, SpeC)
We report the development of a method for multiplex detection of three staphylococcal (SEA, SEB, TSST-1) and two streptococcal toxins (SpeA and SpeC), which could be used for detecting toxins in food, in supernatants derived from blood cultures of Staphylococcus aureus and/or Streptococcus pyogenes, or potentially directly in clinical samples
Pus and debrided tissue at surgical incision in sternum of 4 month-old infant, Japan Not reported Bloodstream of an adult female ICU patient, US Pharyngeal smear of a patient, Japan Archaic methicillin resistant S.aureus (MRSA) strain, UK Food associated with a staphylococcal food poisoning outbreak, US Patient with menstrual TSS, US Not reported Not reported Bloodstream sample, US Isolated from wound, US Isolated from wound, US Isolated from peritoneal fluid, US Not reported Not reported Not reported Not reported Not reported
Summary
Staphylococcus aureus and group A Streptococcus pyogenes secrete a family of pyrogenic toxins including staphylococcal enterotoxins A, B, C (SEA, SEB, SEC), toxic shock syndrome toxin-1 (TSST-1) and streptococcal pyrogenic exotoxins (SpeA, SpeC). A common property of these toxins is that they possess superantigen (SAg) activity that results in overstimulation of the immune system. These T-cell mediated reactions are associated with hyperinflammation and in some cases, organ failure or death. These toxins have been implicated in sepsis, PLOS ONE | DOI:10.1371/journal.pone.0135986. These toxins have been implicated in sepsis, PLOS ONE | DOI:10.1371/journal.pone.0135986 August 25, 2015
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