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 model

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

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 stres

The expression of rstB, evgS and the genes coding for the σE- and σS-factors in Yersinia pseudotuberculosis at 30 °C and 5 °C

Y. enterocolitica and Y. pseudotuberculosis are the third most common reported zoonoses after Campylobacter and Salmonella. Y. pseudotuberculosis has caused 10 outbreaks in Finland since 1997. The symptoms of yersiniosis include fever, right-sided abdominal pain and diarrhea. Post-infectious complications are common. Refrigeration is the most common modern food preservation method, however, as psychrotrophs, enteropathogenic Yersinia are able to grow at refrigerator temperatures. However, the mechanism behind psychrotrophy is still unknown. The aim of this study was to determine the expression levels of rstB, evgS and the genes coding for the σE- and σS-factors of Y. pseudotuberculosis IP 32953 at 30 °C and after temperature drop to 5 °C. Total RNA had been extracted before and 30 minutes, 3, and 7 hours after the temperature downshift for previously performed DNA microarray gene expression studies. Low temperature had been found to increase the expression levels of these genes in these studies. The gene expression levels were determined using real-time quantitative PCR (RT-qPCR). RNA-samples were reverse transcribed into complementary DNA (cDNA), which was used as a template in the qPCR-reactions. The gene expression levels at different time points were determined by measuring the signal emitted by the fluorescent dye. THE ∆∆Ct-method was used for relative quantification. The 16S rRNA (locus tag YPTB_RNA_102) was used for normalization. The expression levels of all the studied genes were approximately doubled 30 minutes after the cold shock demonstrating the significance of the genes in the immediate cold shock response. The expression levels were at highest 3 hours after the cold shock: 2,4-fold for rstB, 4,4-fold for evgS, 6,6-fold for sigmaE and 5,6-fold for rpoS compared with the expression level before the cold shock. The expression levels of all the four genes were decreased 7 hours after the cold shock illustrating adaptation to the lower temperature. The results confirm the findings of the DNA microarray gene expression experiments done previously in the research group. In the future the knowledge of the genes involved in the cold shock response can be utilized in controlling the growth of Y. pseudotuberculosis in foodstuffs.Yersinia enterocolitica ja Y. pseudotuberculosis ovat kolmanneksi yleisimpiä suolistoinfektiota aiheuttavia bakteereita kampylobakteerien ja salmonellojen jälkeen. Vuoden 1997 jälkeen Y. pseudotuberculosis on aiheuttanut Suomessa 10 epidemiaa. Y. pseudotuberculosiksen aiheuttaman yersinioosin yleisimpiin oireisiin kuuluvat kuume, oikeanpuoleinen vatsakipu ja ripuli. Infektioon liittyy usein jälkitauteja. Elintarvikkeiden kylmävarastointi ei estä bakteerin kasvua, koska psykrotrofina Y. pseudotuberculosis pystyy lisääntymään myös jääkaappilämpötilassa (4 - 8 °C). Emme kuitenkaan tiedä, millä mekanismeilla bakteeri pystyy kasvamaan alhaisissa lämpötiloissa. Tutkimuksen tarkoituksena oli määrittää Y. pseudotuberculosiksen histidiinikinaasia RstB, hybridikinaasia EvgS sekä σE- ja σS-tekijöitä koodaavien geenien ilmentymistasot ennen kylmäsokkia 30 °C:ssa ja kylmäsokin (5 °C) jälkeen. Tutkimushypoteesi oli, että ilmentymistasot nousevat kylmäaltistuksessa, koska DNA-mikrosirukokeissa geenien aktiivisuuden oli todettu lisääntyvän kylmässä. Aineistona oli Y. pseudotuberculosis IP 32953 -kanta, josta oli eristetty RNA neljässä eri aikapisteessä: ennen kylmäsokkia eksponentiaalisen kasvun alkuvaiheessa ja kylmäsokin jälkeen 30 minuutin, 3 tunnin ja 7 tunnin kuluttua. Geenien ilmentymistasot määritettiin käyttämällä reaaliaikaista kvantitatiivista PCR-menetelmää (RT-qPCR). RNA-näytteet käännettiin PCR-laitteessa komplementaariseksi DNA:ksi, joka toimi templaattina qPCR-reaktiossa. Geenien ilmentymistasot eri aikapisteissä määritettiin fluoresoivan väriaineen lähettämän signaalin perusteella. Suhteellisessa kvantifikaatiossa käytettiin ∆∆Ct-menetelmää, jossa geenien ilmentymistä eri aikapisteissä verrataan ilmentymistasoon ennen kylmäsokkia. Normalisaatiossa käytettiin 16S rrn –geeniä. Kaikkien tutkittujen geenien ilmentymistasot olivat korkeimmillaan 3 tunnin kuluttua kylmäsokista. rstB-geenin ilmentyminen oli korkeimmillaan 2,4-kertainen, evgS-geenin 4,4-kertainen, sigmaE-geenin 6,6-kertainen ja rpoS-geenin 5,6-kertainen verrattuna geenien ilmentymistasoon ennen kylmäsokkia. Kaikkien geenien ilmentyminen oli noin kaksinkertaistunut 30 minuutin kuluttua kylmäsokista, joten geeneillä on merkitystä välittömässä kylmävasteessa. Havaittu geenien ilmentymistasojen lasku 7 tunnin aikapisteessä kertoo bakteerin alkamisesta sopeutua kylmään 7 tunnin sisällä kylmäsokista. Tulokset vahvistavat aikaisempien tutkimusryhmässä tehtyjen DNA-mikrosirukokeiden havainnot. Tulevaisuudessa tietoa kylmänsietoon liittyvien geenien toiminnasta voidaan hyödyntää etsittäessä keinoja vaikuttaa kylmäsokkivasteen muodostumiseen ja Y. pseudotuberculosiksen kasvun hallitsemiseen elintarvikkeissa

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

Growth behavior and temporal enterotoxin D expression of Staphylococcus aureus strains under glucose and lactic acid stress

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

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

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 sequence

Temporal expression of the staphylococcal enterotoxin D gene under NaCl stress conditions encountered during food production and preservation

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

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