Reduced Enterotoxin D Formation on Boiled Ham in Staphylococcus Aureus Δagr Mutant.

Staphylococcal food poisoning (SFP) is a common cause of foodborne illness worldwide, and enterotoxin D (SED) is one of the most frequent Staphylococcus aureus enterotoxins associated with it. It has been reported that the expression and formation of SED in S. aureus is regulated by the quorum sensing Agr system. In this study, the effect of agr deletion on sed expression in S. aureus grown on boiled ham was investigated. Growth, sed mRNA and SED protein levels in an S. aureus wild type strain and its isogenic Δagr mutant were monitored for 14 days at 22 °C. The results showed that although deletion of the agr gene did not affect the growth rate or maximum cell density of S. aureus on boiled ham, it had a pronounced effect on SED formation during the first 5 days of incubation. The SED concentration was not reflected in the amount of preceding sed transcripts, suggesting that sed transcription levels may not always reflect SED formation. The expression of RNAIII transcript, the regulatory signal of the Agr system, was also monitored. Similar transcription patterns were observed for RNAIII and sed. Surprisingly, in the Δagr mutant, sed expression was comparable to that in the wild type strain, and was thus unaffected by deletion of the Agr system. These results demonstrate that the Agr system appears to only partially affect SED formation, even in a real food environment.

Effect of food-related stress conditions and loss of agr and sigB on seb promoter activity in S. aureus.

Staphylococcal enterotoxin B (SEB) causes staphylococcal food poisoning and is produced in up to ten times higher quantities than other major enterotoxins. While Staphylococcus aureus growth is often repressed by competing flora, the organism exhibits a decisive growth advantage under some stress conditions. So far, data on the influence of food-related stressors and regulatory mutations on seb expression is limited and largely based on laboratory strains, which were later reported to harbor mutations. Therefore, the aim of this study was to investigate the influence of stress and regulatory mutations on seb promoter activity. To this end, transcriptional fusions were created in two strains, USA300 and HG003, carrying different seb upstream sequences fused to a blaZ reporter. NaCl, nitrite, and glucose stress led to significantly decreased seb promoter activity, while lactic acid stress resulted in significantly increased seb promoter activity. Loss of agr decreased seb promoter activity and loss of sigB increased promoter activity, with the magnitude of change depending on the strain. These results demonstrate that mild stress conditions encountered during food production and preservation can induce significant changes in seb promoter activity.

Sequence Variability in Staphylococcal Enterotoxin Genes seb, sec, and sed.

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.

Temporal expression of the staphylococcal enterotoxin D gene under NaCl stress conditions.

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.

Validation of reference genes for normalization of qPCR mRNA expression levels in Staphylococcus aureus exposed to osmotic and lactic acid stress conditions encountered during food production and preservation.

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.

Microarray-based characterization of Staphylococcus aureus isolates obtained from chicken carcasses.

A total of 34 Staphylococcus aureus isolates from flock-wise pooled chicken neck skin samples collected at two abattoirs during slaughter were characterized with DNA microarray analysis and spa typing. The 20 isolates from abattoir A all belonged to clonal complex (CC) 12 and spa type t160. Of the 14 isolates from abattoir B, 7 belonged to CC5-t3478, 5 to CC12-t160, 1 to CC45-t040, and 1 to CC101-t056. Of the various resistance-associated genes tested, only blaZ/R/I (6 isolates of CC12 and CC101 from abattoir B), sdrM (n = 34), fosB (n = 33), and qacC (n = 22) were detected. None of the isolates harbored genes conferring methicillin resistance. In terms of genes encoding enterotoxins, seb (all isolates of CC12), egc (seg, sei, selm, seln, selo, selu; all isolates of CC5 and CC45), and sea (14 isolates of CC12 and 1 isolate of CC5) were found. In addition, all isolates harbored genes for intracellular adhesion proteins (icaA/C/D) and were positive for cap5 or cap8 (capsule type 5 or 8). Comparison of DNA microarray profiles identified four categories comprising (i) all isolates of CC12, (ii) all isolates of CC5, (iii) the CC45 isolate, and (iv) the CC101 isolate. The high similarity of the isolates from abattoir A could indicate contamination of chicken carcasses with S. aureus persisting on the slaughter equipment, but further investigations are required to elucidate potential contamination routes.

Outbreak of Staphylococcal food poisoning due to SEA-producing Staphylococcus aureus.

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.