Development of Elisa Test-Systems and Immune-Chromatographic Reagent Panel Designed for the Detection of Staphylococcal Enterotoxins of A and B Types

Objective of the study was the development of experimental ELISA tests and lateral flow immunoassays for detection of staphylococcal enterotoxins, A and B types.Materials and methods. Hybridomas, producing monoclonal antibodies against staphylococcal enterotoxins A and B from State Collection of the Affiliated Branch of the “48th Central Research Institute” of the Ministry of Defense of the Russian Federation, BALB/c mice and staphylococcal enterotoxins A and B were used in the research. Hybridoma cells were incubated in culture flasks and in the peritoneal cavity of BALB/c mice. Monoclonal antibodies were isolated from ascitic fluids through precipitation with saturated ammonium sulfate subsequently purified using ion-exchange chromatography. Obtained preparations of monoclonal antibodies were used for the construction of ELISA tests and immune-chromatographic reagent panels for the detection of staphylococcal enterotoxins A and B. Specific components of ELISA tests were lyophilized in protective media.Results and discussion. ELISA tests and lateral flow immunoassays which allow for detecting staphylococcal enterotoxins A and B at concentrations of 0.5 ng/ml and higher, including in food samples, have been constructed.

Pathogenic and Virulence Factor Detection on Viable but Non-culturable Methicillin-Resistant Staphylococcus aureus

Food safety and foodborne infections and diseases have been a leading hotspot in public health, and methicillin-resistant Staphylococcus aureus (MRSA) has been recently documented to be an important foodborne pathogen, in addition to its recognition to be a leading clinical pathogen for some decades. Standard identification for MRSA has been commonly performed in both clinical settings and food routine detection; however, most of such so-called “standards,” “guidelines,” or “gold standards” are incapable of detecting viable but non-culturable (VBNC) cells. In this study, two major types of staphylococcal food poisoning (SFP), staphylococcal enterotoxins A (sea) and staphylococcal enterotoxins B (seb), as well as the panton-valentine leucocidin (pvl) genes, were selected to develop a cross-priming amplification (CPA) method. Limit of detection (LOD) of CPA for sea, seb, and pvl was 75, 107.5, and 85 ng/μl, indicating that the analytical sensitivity of CPA is significantly higher than that of conventional PCR. In addition, a rapid VBNC cells detection method, designated as PMA-CPA, was developed and further applied. PMA-CPA showed significant advantages when compared with PCR assays, in terms of rapidity, sensitivity, specificity, and accuracy. Compared with conventional VBNC confirmation methods, the PMA-CPA showed 100% accordance, which had demonstrated that the PMA-CPA assays were capable of detecting different toxins in MRSA in VBNC state. In conclusion, three CPA assays were developed on three important toxins for MRSA, and in combination with PMA, the PMA-CPA assay was capable of detecting virulent gene expression in MRSA in the VBNC state. Also, the above assays were further applied to real samples. As concluded, the PMA-CPA assay developed in this study was capable of detecting MRSA toxins in the VBNC state, representing first time the detection of toxins in the VBNC state.

Highly Sensitive and Specific Detection of Staphylococcal Enterotoxins SEA, SEG, SEH, and SEI by Immunoassay

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases worldwide, resulting from the ingestion of staphylococcal enterotoxins (SEs), primarily SE type A (SEA), which is produced in food by enterotoxigenic strains of staphylococci, mainly S. aureus. Since newly identified SEs have been shown to have emetic properties and the genes encoding them have been found in food involved in poisoning outbreaks, it is necessary to have reliable tools to prove the presence of the toxins themselves, to clarify the role played by these non-classical SEs, and to precisely document SFP outbreaks. We have produced and characterized monoclonal antibodies directed specifically against SE type G, H or I (SEG, SEH or SEI respectively) or SEA. With these antibodies, we have developed, for each of these four targets, highly sensitive, specific, and reliable 3-h sandwich enzyme immunoassays that we evaluated for their suitability for SE detection in different matrices (bacterial cultures of S. aureus, contaminated food, human samples) for different purposes (strain characterization, food safety, biological threat detection, diagnosis). We also initiated and described for the first time the development of monoplex and quintuplex (SEA, SE type B (SEB), SEG, SEH, and SEI) lateral flow immunoassays for these new staphylococcal enterotoxins. The detection limits in buffer were under 10 pg/mL (0.4 pM) by enzyme immunoassays and at least 300 pg/mL (11 pM) by immunochromatography for all target toxins with no cross-reactivity observed. Spiking studies and/or bacterial supernatant analysis demonstrated the applicability of the developed methods, which could become reliable detection tools for the routine investigation of SEG, SEH, and SEI.

Staphylococcal Enterotoxins and Toxic Shock Syndrome Toxin-1 and Their Association among Bacteremic and Infective Endocarditis Patients in Egypt

Purpose. Infective endocarditis (IE) is a major complication in patients with bacteremia of Staphylococcus (S.) aureus infection. Our aim was to determine the association of the major Staphylococcal superantigens (SAgs), including Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1), among hospitalized patients diagnosed with bacteremia and those with IE. Methods. This study was conducted on 88 patients; of these, 84 (95.5%) had two positive blood cultures. Eighteen out of the 84 patients (21.4%) were diagnosed based on the modified Duke criteria by a cardiologist to have IE. The recovered isolates were screened phenotypically using ELISA followed by molecular analysis of sea, seb, sec, sed, see, and tsst-1, the major SAg coding genes, and the obtained findings were statistically analyzed. Results. Phenotypic screening for SE production of 26 selected Staphylococci (15 isolated from the IE patients (10 S. aureus and 5 coagulase negative staphylococci (CoNS)) and 11 from bacteremic patients (10 S. aureus and 1 CoNS)) using ELISA revealed that 12/26 (46%) isolates were SE producers. PCR analysis showed that 19 (73%) isolates were PCR positive for SAg genes with the highest prevalence of the sea gene (79%), followed by seb (63%) and tsst-1 (21%). The least frequent gene was sed (5.3%). Statistical correlations between bacteremic and IE isolates with respect to prevalence of SAgs showed no significant difference ( P value = 0.139, effect size = 0.572 ) indicating no specific association between any of the detected SAgs and IE. Conclusion. There is high prevalence of SEs among clinical isolates of Staphylococci recovered from patients suffering bacteremia and those with IE. No significant difference was found among Staphylococcal isolates recovered from patients with bacteremia or IE regarding both phenotypic and genotypic detection of the tested SAgs.