Staphylococcal Enterotoxin Type R Pseudogene Presence in Staphylococcus aureus Reference and Outbreak Strains.

Novel staphylococcal enterotoxins (SEs) expressed by Staphylococcus aureus strains have been described throughout the years, among these being the SE protein SER. To further characterize this toxin, this research used 13 S. aureus strains previously determined to contain the SE type R (ser) gene. These S. aureus isolates were evaluated using serological assays for identification of SEA-SEE and PCR for the detection of newly described SE and SE-like enterotoxin genes seg-seu. PCR-based cloning was performed such that the ser gene could be ligated into the pTrc99A plasmid expression vector. Ligation products were used to transform Escherichia coli (DH10Br) strains so that the ser open reading frame (ORF) could be sequenced and expressed for further characterization. Four of the 13 S. aureus strains tested harbored a ser ORF that yielded a PCR-positive result, but contained a frameshift mutation that subsequently introduced a premature stop codon abrogating expression of a full-sized functional protein. In this study, 30% of the PCR-positive ser strains tested were found to carry genes that coded for a nonfunctional SER protein, a finding that clearly illustrates the limited effectiveness of PCR for reliably evaluating enterotoxin potential for ser and, perhaps, other enterotoxin types.

Screening, detection, and serotyping methods for toxin genes and enterotoxins in Staphylococcus strains.

Staphylococcus aureus continues to play a significant role in foodborne outbreak investigations, with numerous individuals sickened each year after ingesting assorted foods contaminated with staphylococcal enterotoxins. The purpose of this study was to evaluate the use of several methods for the screening, detection, and enterotoxin serotyping of staphylococcal bacterial strains for classical staphylococcal enterotoxins (SEs; SEA, SEB, SEC, SED, and SEE) and the newly described SE and SE-like enterotoxin genes (seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, and seu). Inclusivity and exclusivity panels of staphylococcal strains were tested using a multiplex PCR method in addition to three polyvalent commercially prepared ELISA systems for the detection of SEA-SEE and one monovalent assay for the identification of classical SE serotypes. The results indicate an overall agreement between serological detection methods with a few exceptions, and molecular characterization identified an abundance of SE and SE-like enterotoxin genes including several potentially enterotoxigenic isolates that would have otherwise been missed by ELISA-based methods. These findings demonstrate the significance of PCR for future screening purposes and the use of ELISA systems for the detection and enterotoxin serotyping of staphylococcal bacterial strains.

Staphylococcal enterotoxin B-specific electrochemiluminescence and lateral flow device assays cross-react with staphylococcal enterotoxin D.

Guam school children and faculty members experienced symptoms of vomiting, nausea, abdominal cramps, and diarrhea shortly after eating breakfast prepared by contracted caterers. The first illness was reported within an hour after breakfast, affecting 295 students and two faculty members. Local hospitals treated 130 people, and 61 were admitted for further treatment. Reported symptoms were consistent with staphylococcal food poisoning. Initial food testing using a lateral flow device and electrochemiluminescence method incorrectly implicated staphylococcal enterotoxin B as the causative agent, prompting partial activation of Guam's Emergency Response Center. Traditional ELISAs proved that the food poisoning agent was staphylococcal enterotoxin D. More specific and sensitive assays would have alleviated the issues and confusion that surrounded the reporting and investigation of this outbreak.

Efficient isolation and identification of Bacillus cereus group.

Bacillus cereus is a group of ubiquitous facultative anaerobic sporeforming Gram-positive rods commonly found in soil. The spores frequently contaminate a variety of foods, including produce, meat, eggs, and dairy products. Foodborne illnesses associated with toxins produced by B. cereus can result in self-limiting diarrhea or vomiting. Plate enumeration methods recommended by recognized food authorities to detect the presence of B. cereus in potentially contaminated food products do not inhibit other Gram-positive competitive bacteria. This study evaluated the use of Bacara, a new chromogenic agar, as an efficient method to identify and enumerate B. cereus group from food matrixes, even in the presence of background flora. Inclusivity and exclusivity testing was performed using four different selective and differential media for B. cereus, including Mannitol Egg Yolk Polymyxin (MYP), Polymyxin Pyruvate Egg-Yolk Mannitol Bromothymol Blue Agar, Bacillus Chromogenic Media, Brilliance, and Bacara. MYP and Bacara were also used in plate enumeration studies to isolate B. cereus from artificially contaminated foods.

Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology.

The problem of Staphylococcus aureus and other species as contaminants in the food supply remains significant on a global level. Time and temperature abuse of a food product contaminated with enterotoxigenic staphylococci can result in formation of enterotoxin, which can produce foodborne illness when the product is ingested. Between 100 and 200 ng of enterotoxin can cause symptoms consistent with staphylococcal intoxication. Although humans are the primary reservoirs of contamination, animals, air, dust, and food contact surfaces can serve as vehicles in the transfer of this pathogen to the food supply. Foods may become contaminated during production or processing and in homes or food establishments, where the organism can proliferate to high concentrations and subsequently produce enterotoxin. The staphylococcal enterotoxins are highly heat stable and can remain biologically active after exposure to retort temperatures. Prior to the development of serological methods for the identification of enterotoxin, monkeys (gastric intubation) and later kittens (intravenous injection) were used in assays for toxin detection. When enterotoxins were identified as mature proteins that were antigenic, serological assays were developed for use in the laboratory analysis of foods suspected of containing preformed enterotoxin. More recently developed methods are tracer-labeled immunoassays. Of these methods, the enzyme-linked immunosorbent assays are highly specific, highly sensitive, and rapid for the detection of enterotoxin in foods.

Microbial evaluation of selected fresh produce obtained at retail markets.

The microbial quality of five types of fresh produce obtained at the retail level was determined by standard quantitative techniques. These techniques included aerobic plate count (APC), total coliform counts, Escherichia coli counts, and yeast and mold counts. Three different methods were used to determine total coliform counts, which consisted of MacConkey agar plate counts, Colicomplete most probable number counts, and Petrifilm E. coli (EC) plate counts. The mean APCs for sprouts, lettuce, celery, cauliflower, and broccoli were 8.7, 8.6, 7.5, 7.4. and 6.3 log10 CFU/g, respectively. MacConkey agar counts indicated that 89 to 96% of the APCs consisted of gram-negative bacteria. Yeast and mold counts were in a range expected of fresh produce. Fresh produce was also analyzed for human pathogens. Samples were analyzed for Staphylococcus spp., Bacillus spp., Salmonella spp., Listeria spp., and Campylobacter spp. One isolate of Staphylococcus was found to be enterotoxigenic, and one species of Bacillus was also toxigenic. Neither Salmonella spp. nor Campylobacter spp. were detected in any of the produce samples. A variety of Listeria spp., including Listeria monocytogenes, were found in fresh produce.

Atypical Toxigenic Staphylococcus and Non-Staphylococcus aureus Species on the Horizon? An Update †.

Staphylococcal food poisoning is a commonly reported illness caused by the ingestion of preformed staphylococcal enterotoxin in foods, With some exceptions, enterotoxin production is associated with coagulase-positive rather than coagulase-negative staphylococci. Of the coagulase-positive staphylococcal species, S. aureus was historically thought to be exclusively implicated in human foodborne illness. More recently, however, other coagulase-positive and some coagulase-negative staphylococcal species have been associated with foodborne intoxication, Coagulase activity has been used to indicate pathogenicity of a foodborne isolate, and thermostable nuclease is being suggested as a more reliable indictor of enterotoxigenicity. Evidence suggests that the metabolic expressions that are the bases of the tests may not be reliable indicators of pathogenicity. A more useful approach to determine the pathogenicity of a Staphylococcus species is to test directly for enterotoxigenicity with one of the new rapid methods. None of the conventional ancillary identification tests has been conclusively associated with enterotoxin synthesis. Furthermore, evidence exists that enterotoxin production is a characteristic of several species in the genus Staphylococcus .