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A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results.Staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST), and exfoliative toxins A and B belong to a family of related pyrogenic toxins produced by Staphylococcus aureus (4, 10). By virtue of its variety of enterotoxins S. aureus is an important food-borne pathogen. In addition to the well-characterized SEA, SEB, SEC, SED, and SEE, new serological types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, and SEU) have been identified in recent years (5,6,7,11,13,15,16,18). Five of the SE genes (seg, sei, sem, sen, and seo) belong to the same enterotoxin gene cluster (egc), and detection of one of these genes usually indicates the presence of all five enterotoxin genes (5, 11). For routine detection of SEs, commercially produced kits, such as reverse passive latex agglutination assays and enzyme-linked immunosorbent assays, are most commonly used. However, these methods are, to date, designed only to detect SEA, SEB, SEC, SED, and SEE. As an alternative to these more traditional methods, the PCR approach can provide detection of toxin genes and is presently designed to detect at least nine SE genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) (1,9,12,14).The objective of this study was initially to establish the multiplex PCR method for detection of the toxin genes in staphylococcal isolates published by Monday and Bohach (12). According to their article, any of the nine SE genes (sea, seb, sec, sed, see, seg, seh, sei, and sej), the TSST gene, and the 16S rRNA gene can be amplified in a single multiplex reaction (results were not shown). However, while establishing the described method on two different types of thermocyclers, no detection of seb, sec, and tsst could be observed, and the amplification of sec and sei gave various results. Some modifications and adaptations were therefore considered necessary.The following modifications were made to Monday and Bohach's method (12): (i) using another DNA isolation method; (ii) redesigning four primers (seb-sec forward, seb reverse, sei forward, and tsst reverse); (iii) splitting the multiplex into two PCRs; (iv) significantly decreasing the amount of 16S rRNA primers; and (v) adding four cycles to the PCR program.Bacterial isolates and DNA isolation. The S. aureus isolates used in this study are listed in Table 1. One colony of each isolate was incubated overnight in 3 ml of brain heart infusion broth medium at 37°C with agitation. Overnight culture (1.5 ml) was transferred to an Eppendorf tube and was centrifuged for 2 min at 17,500 ϫ g. The pellet was resuspended in 1ϫ Tris-EDTA buffer, and the cells were then lysed with 10 l of 10-mg/ml ...

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.

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Aim:To investigate bacteriological quality in organically grown leaf lettuce, including the presence of selected pathogenic bacteria, and to obtain information about organic lettuce production, including fertilizing regimes. Methods and Results: Altogether 179 samples of Norwegian organically grown lettuce were collected from 12 producers. Escherichia coli was isolated from 16 of the lettuce samples, but in 12 of these contamination was sufficiently low (<100 CFU g)1 ) that they would be considered to be of acceptable bacteriological quality.Escherichia coli O157 and Salmonella were not detected in any of the samples. Listeria monocytogenes serogroups 1 and 4 were isolated from two samples. Conclusions: Organic lettuce produced in Norway was generally of acceptable bacteriological quality, but the results show that contamination of organic lettuce with E. coli and L. monocytogenes do occasionally occur. Significance and Impact of the Study: These results suggest that organically grown lettuce may be contaminated with E. coli and L. monocytogenes during cultivation.