Detection of Staphylococcus aureus Enterotoxins A to D by Real-Time Fluorescence PCR Assay

Abstract

Staphylococcus aureus is one of the most significant pathogens causing nosocomial and community-acquired infections. Among the secreted staphylococcal virulence factors, there is a growing list of enterotoxins which can induce gastroenteric syndrome and toxic shock syndrome. Here, we developed a real-time fluorescence PCR assay (TaqMan PCR) for the detection of genes encoding staphylococcal enterotoxins A, B, C1, and D (SEA, SEB, SEC1, and SED) of S. aureus as well as the mecA gene encoding methicillin resistance and the femB gene as a specific genomic marker for S. aureus. SEA to SED were selected because they are the four classically described enterotoxins of S. aureus and because they were detected by latex agglutination. In order to evaluate the reliability of TaqMan PCR, we investigated 93 isolates of S. aureus derived from patients at our hospital over 5 months and compared the results with data obtained by a commerciall available reversed passive latex agglutination assay (SET-RPLA) for these isolates. Thirteen enterotoxin genes were detected by TaqMan PCR; however, no proteins expressed by these genes were detected by SET-RPLA. As a result, more isolates of S. aureus (n ‫؍‬ 44) were found positive by TaqMan PCR for one or more enterotoxin genes than by SET-RPLA for the respective proteins expressed by these genes (n ‫؍‬ 40). We conclude that TaqMan PCR is more sensitive because it offers the possibility for determining enterotoxins on a genotypic basis. Additionally, the assay allows the parallel detection of genes for SEA to SED and methicillin resistance in S. aureus. Furthermore, real-time PCR is well suited for screening large numbers of samples at the same time, allowing rapid, reliable, efficient, and cost-saving routine laboratory diagnosis.

Immunoquantitative Real-Time PCR for Detection and Quantification of Staphylococcus aureus Enterotoxin B in Foods

“…Use of iqPCR for detection of SEB in early growth phases revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. Some other findings suggest that SEB production is related to the late exponential phase and the transition to the stationary phase (6,15,23), which can be the case for some other members of the SE family. A significant increase in SEB production was noticed when the S. aureus counts reached 5 log CFU ml Ϫ1 .…”

Comparison of the BD Max Methicillin-Resistant Staphylococcus aureus (MRSA) Assay and the BD GeneOhm MRSA Achromopeptidase Assay with Direct- and Enriched-Culture Techniques Using Clinical Specimens for Detection of MRSA

“…From the remaining ACP sample buffer, 100 l was removed and directly streaked onto cefoxitin-containing BBL CHROMagar MRSA agar plates (BD), which were inspected after 24 h and 48 h. Moreover, 100 l of the sample buffer was inoculated into 5 ml of Trypticase soy broth-6.5% sodium chloride (BD) for overnight enrichment of S. aureus, followed by plating. Mauve colonies were confirmed by latex agglutination (Pastorex StaphPlus; Bio-Rad, Marbes-la-Coquette, France), growth on DNase and Oxa-screen plates (BD), and in-house PCR for mecA and femB (13,15).…”

Validation of Virulence and Epidemiology DNA Microarray for Identification and Characterization of Staphylococcus aureus Isolates

“…Molecular assays based on PCR have been reported for the detection of MRSA (4,6,7,9,10,30), the identification of staphylococcal species (17,20,21), or the identification of specific virulence genes (5,11,14,15,18,19,22,24,26,33). DNA microarrays can identify, subtype, and detect acquired antibiotic resistance determinants simultaneously (1,23,32,35); however, their clinical value has been limited by a complicated methodology that is unsuitable for routine use in diagnostic microbiology laboratories.…”