Staphylococcus aureus represents the most prevalent cause of food-borne intoxications worldwide. While being repressed by competing bacteria in most matrices, this pathogen exhibits crucial competitive advantages during growth at high salt concentrations or low pH, conditions frequently encountered in food production and preservation. We aimed to identify reference genes that could be used to normalize qPCR mRNA expression levels during growth of S. aureus in food-related osmotic (NaCl) and acidic (lactic acid) stress adaptation models. Expression stability of nine housekeeping genes was evaluated in full (LB) and nutrient-deficient (CYGP w/o glucose) medium under conditions of osmotic (4.5% NaCl) and acidic stress (lactic acid, pH 6.0) after 2-h exposure. Among the set of candidate reference genes investigated, rplD, rpoB,gyrB, and rho were most stably expressed in LB and thus represent the most suitable reference genes for normalization of qPCR data in osmotic or lactic acid stress models in a rich medium. Under nutrient-deficient conditions, expression of rho and rpoB was highly stable across all tested conditions. The presented comprehensive data on changes in expression of various S. aureus housekeeping genes under conditions of osmotic and lactic acid stress facilitate selection of reference genes for qPCR-based stress response models.

In 2008, 150 people gathered for a wedding celebration in Baden-Württemberg, Germany. Three hours after ingestion of a variety of foods including pancakes filled with minced chicken, several guests exhibited symptoms of acute gastroenteritis such as vomiting, diarrhea, fever, and ague. Twelve guests were reported to have fallen ill, with nine of these seeking medical care in hospitals. At least four patients were admitted to the hospital and received inpatient treatment, among them a 2-year-old child and a woman in the 4th month of pregnancy. Within 24 h of the event, an investigative team collected a variety of samples including refrigerated leftovers, food in the storage unit of the caterer, nasal swabs of the caterer, as well as 21 environmental swabs. Five stool samples from patients were provided by the hospitals. Staphylococcus aureus isolates were gathered from eight samples, among them nasal swabs of the caterer, food samples, and one stool sample. Fourier transform-infrared spectroscopy was used for species identification and for primary clustering of the isolates in a similarity tree. The isolates were further characterized by spa typing and pulsed-field gel electrophoresis, and a DNA microarray was used to determine the presence/absence of genes involved in virulence and antimicrobial resistance. We were able to match an enterotoxigenic strain from the stool sample of a patient to isolates of the same strain obtained from food and the nasal cavity of a food handler. The strain produced the enterotoxin SEA and the toxic shock syndrome toxin-1, and was also found to exhibit the genes encoding enterotoxins SEG and SEI, as well as the enterotoxin gene cluster egc. This is one of only a few studies that were able to link a staphylococcal food poisoning outbreak to its source. In 2008, 150 people gathered for a wedding celebration in Baden-Wü rttemberg, Germany. Three hours after ingestion of a variety of foods including pancakes filled with minced chicken, several guests exhibited symptoms of acute gastroenteritis such as vomiting, diarrhea, fever, and ague. Twelve guests were reported to have fallen ill, with nine of these seeking medical care in hospitals. At least four patients were admitted to the hospital and received inpatient treatment, among them a 2-year-old child and a woman in the 4th month of pregnancy.Within 24 h of the event, an investigative team collected a variety of samples including refrigerated leftovers, food in the storage unit of the caterer, nasal swabs of the caterer, as well as 21 environmental swabs. Five stool samples from patients were provided by the hospitals. Staphylococcus aureus isolates were gathered from eight samples, among them nasal swabs of the caterer, food samples, and one stool sample. Fourier transforminfrared spectroscopy was used for species identification and for primary clustering of the isolates in a similarity tree. The isolates were further characterized by spa typing and pulsed-field gel electrophoresis, and a DNA microarray was used to determin...

Staphylococcus aureus is one of the most osmotolerant food-borne pathogens. While its growth is repressed by competing bacteria, the organism exhibits a growth advantage at increased salt concentrations. Staphylococcal enterotoxin D leads to vomiting and diarrhea upon ingestion. To date, the effect of NaCl on both sed expression and its regulatory control are unclear. We determined the impact of NaCl stress on sed expression and the influence of agr, sarA and sigB on sed expression under NaCl stress. The temporal expression of sed in LB and LB with 4.5% NaCl was compared, as well as sed expression of wild-type (wt) strains and isogenic Δagr, ΔsarA and ΔsigB mutants. In general, NaCl stress led to decreased sed expression. However, one strain exhibited a trend towards increased sed expression under NaCl stress. No significant effect of agr on sed expression was detected and only one ΔsigB mutant showed a significant decrease in sed expression in the early stationary phase under NaCl stress. One ΔsarA mutant showed decreased sed expression in the early stationary and another increased sed expression in the stationary growth phase under NaCl stress. These findings suggest high strain-specific variation in sed expression and its regulation under NaCl stress.

Ingestion of staphylococcal enterotoxins preformed by Staphylococcus aureus in food leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. There are five major staphylococcal enterotoxins: SEA, SEB, SEC, SED, and SEE. While variants of these toxins have been described and were linked to specific hosts or levels or enterotoxin production, data on sequence variation is still limited. In this study, we aim to extend the knowledge on promoter and gene variants of the major enterotoxins SEB, SEC, and SED. To this end, we determined seb, sec, and sed promoter and gene sequences of a well-characterized set of enterotoxigenic Staphylococcus aureus strains originating from foodborne outbreaks, human infections, human nasal colonization, rabbits, and cattle. New nucleotide sequence variants were detected for all three enterotoxins and a novel amino acid sequence variant of SED was detected in a strain associated with human nasal colonization. While the seb promoter and gene sequences exhibited a high degree of variability, the sec and sed promoter and gene were more conserved. Interestingly, a truncated variant of sed was detected in all tested sed harboring rabbit strains. The generated data represents a further step towards improved understanding of strain-specific differences in enterotoxin expression and host-specific variation in enterotoxin sequences.

Ingestion of the staphylococcal enterotoxin D (SED) leads to staphylococcal food poisoning, the most prevalent foodborne intoxication worldwide. Patients suffer from acute signs of gastroenteritis such as violent vomiting, diarrhea, cramps, and fever. As the symptoms result in pronounced electrolyte imbalances and dehydration, the intoxication is particularly dangerous to children and the elderly. SED is formed during growth of Staphylococcus aureus in food. While growth of S. aureus is repressed by competing bacteria in most food matrices, the organism exhibits a crucial competitive growth advantage in foods with low pH or a low aw value (e.g. through high sugar concentrations). To date, the effect of these stress conditions on sed expression is unclear. The objective of this study was to determine sed mRNA expression levels of S. aureus exposed to glucose and lactic acid stress conditions similar to food production and preservation. To this end, temporal sed mRNA expression levels of three S. aureus strains grown at control conditions, glucose stress conditions (30% glucose), and lactic acid stress conditions (pH 6.0) were determined using quantitative Real-Time PCR. Under both glucose and acid stress conditions, the mean lag phase duration was prolonged and maximum cell density in late stationary phase was decreased. In addition, glucose stress slightly increased the growth rate of the tested strains and led to decreased sed expression in late stationary phase. Lactic acid stress had no statistically significant effect on sed expression. Our study provides data on the effect of critical food-related stressors on growth and SE expression of S. aureus, which can be used for risk assessment. ABSTRACT 26Ingestion of the staphylococcal enterotoxin D (SED) leads to staphylococcal food poisoning, 27 the most prevalent foodborne intoxication worldwide. Patients suffer from acute signs of 28 gastroenteritis such as violent vomiting, diarrhea, cramps, and fever. As the symptoms result 29 in pronounced electrolyte imbalances and dehydration, the intoxication is particularly 30 dangerous to children and the elderly. SED is formed during growth of Staphylococcus 31 aureus in food. While growth of S. aureus is repressed by competing bacteria in most food 32 matrices, the organism exhibits a crucial competitive growth advantage in foods with low pH 33 or a low a w value (e.g. through high sugar concentrations). To date, the effect of these stress 34 conditions on sed expression is unclear. The objective of this study was to determine sed 35 mRNA expression levels of S. aureus exposed to glucose and lactic acid stress conditions 36 similar to food production and preservation. To this end, temporal sed mRNA expression 37 levels of three S. aureus strains grown at control conditions, glucose stress conditions (30% 38 glucose), and lactic acid stress conditions (pH 6.0) were determined using quantitative Real-39 Time PCR. Under both glucose and acid stress conditions, the mean lag phase duration was 40 prolonged and ...