Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes

Abstract

To investigate the distribution of staphylococcal enterotoxin (SE) A to I (SEA to SEI) genes (sea to sei) in Staphylococcus aureus, 146 isolates obtained in Japan from humans involved in and samples from food poisoning outbreaks, healthy humans, cows with mastitis, and bovine raw milk were analyzed by multiplex PCR. One hundred thirteen (77.4%) S. aureus isolates were found to be positive for one or more se genes. The se genotype was classified into 14 genotypes. seg and sei coexisted in the same S. aureus strain. The newly developed sandwich enzyme-linked immunosorbent assay showed that most seh-harboring S. aureus isolates were able to produce a significant amount of SEH. However, most of the S. aureus isolates harboring seg and about 60% of the isolates harboring sei did not produce a detectable level of SEG or SEI, while reverse transcription-PCR analysis proved that the mRNAs of SEG and SEI were transcribed in S. aureus strains harboring seg and sei genes. These results suggest the importance of quantitative assessment of SEG and SEI production in foods in order to clarify the relationship between these new SEs and food poisoning.

FIG. 1.

Specificities of sandwich ELISA systems for detection of SEG, SEH, and SEI. SEA, SEB, SEC2, SED, and SEE (100 ng/ml each) were purified from the culture supernatant of each strain by the method of Shinagawa et al. (21). rSEG, rSEH, and rSEI were each subjected to sandwich ELISA.

FIG. 2.

Frequency distribution of the productivity of SEG, SEH, and SEI by S. aureus isolates. All S. aureus isolates were grown in brain heart infusion broth-1% yeast extract at 37°C for 48 h. The culture supernatants were preincubated with 20% (vol/vol) normal rabbit serum overnight and were diluted with phosphate-buffered saline-Tween 20 to reduce the background caused by protein A. These pretreated culture supernatants were each subjected to sandwich ELISA, and the concentrations of SEG, SEH, and SEI were determined by use of standard curves.

FIG. 3.

Detection by RT-PCR of SEG and SEI mRNAs in S. aureus isolates harboring seg and sei genes. Total RNAs purified at the mid-log phase from cultures of S. aureus isolates harboring seg and sei were subjected to RT-PCR analysis. Lanes: F1, Fukuoka 1; S1, Saga 1; A1, Aomori 1; M, HaeIII-digested φX174 as a size marker; PC, genomic DNA of Fukuoka 1 as a positive control; NC, Milli-Q water as a negative control; +, with RT; −, without RT.