Synthetic exfoliative toxin A and B DNA probes for detection of toxigenic Staphylococcus aureus strains

Abstract

Two methods for the detection of exfoliative toxin (ET) from Staphylococcus aureus were compared: (i) a phenotypic assay, electrosyneresis, and (ii) a genotypic assay, staphylococcal DNA hybridization with oligodeoxynucleotide probes. The probes were chosen from the previously determined sequences of serotype A and B of ET, one probe for serotype A and another for serotype B. Strains exhibiting ET production in electrosyneresis always possessed the ET gene(s). Conversely, some strains not exhibiting ET production in electrosyneresis harbored the ET gene(s). The latter strains produced low levels of ET. ET-negative phage group 2 strains of S. aureus as well as tested coagulase-negative staphylococci did not possess the ET gene(s). The sensitivity of the DNA hybridization technique was 106 bacteria or 100 ng of genomic DNA.

Clinical, Microbial, and Biochemical Aspects of the Exfoliative Toxins Causing Staphylococcal Scalded-Skin Syndrome

“…Simple enzymelinked immunosorbent assays (204) are not as sensitive as RIA, but a double-antibody procedure, in which a secondary rat polyclonal antibody is used, has attained the sensitivity of RIA (156). Other methods developed include slide latex agglutination (187), electrosyneresis and DNA hybridization with oligodeoxynucleotide probes (204,216), and genomic DNA fingerprinting after SmaI digestion of DNA extracted from S. aureus strains (95). Unfortunately, most of these tests have been developed for research purposes and are not suitable as routine tests in hospital laboratories because they tend to be time-consuming, laborious, and expensive.…”

Exfoliative toxin detection using reversed passive latex agglutination: clinical and epidemiologic applications

“…Among immunologic methods, electrosyneresis is well adapted to the epidemiologic study of ET-producing S. aureus isolates, since this method is rapid and can be used to test many isolates simultaneously. However, its sensitivity is relatively low (detection limit for ET protein, 5 g/ml) (19). Another immunologic method, Ouchterlony immunodiffusion, is even less sensitive (50 to 60 g/ml) (2, 13).…”

“…The definitive diagnosis of SSSS requires detection of ET from clinical isolates of S. aureus. Several methods for detecting ET have been developed: the newborn mouse bioassay (2,12), Ouchterlony immunodiffusion (2), radial immunodiffusion (2,26), electrosyneresis (19), slide latex agglutination (13), enzyme-linked immunosorbent assay (ELISA) (16), radioimmunologic assay (26), and molecular techniques such as a probe assay (19) and PCR (6,21). These methods require expensive instruments, complicated procedures, and/or radioactive probes.…”

Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

“…Thus, ET-producing strains of S. aureus which excreted small amounts of toxin could be misidentified by immunological methods. Rifai et al (15) applied DNA hybridization to the analysis of S. aureus for the presence of eta and etb genes using oligonucleotide probes and showed that the lowest detection limit is 106 bacteria or 100 ng of purified genomic DNA. The hybridization procedure used to identify eta and etb genes, however, requires a great deal of time.…”

“…The agar plate technique using anti-ET serum (6), gel immunoprecipitation (7,8), enzyme-linked immunosorbent assay (ELISA) (14), radioimmunological assay (1,23), biological assay (11) and Southern blot hybridization (15) have been used to screen for exfoliative toxin (ET) production by S. aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS). It takes a great deal of time to identify ETs and ET genes by these methods, and in some cases, a strain with low ET excretion could be misidentified.…”