Effect of sodium nitrite and regulatory mutations Δagr, ΔsarA, and ΔsigB on the mRNA and protein levels of staphylococcal enterotoxin D

Abstract

Staphylococcal food poisoning results from ingestion of enterotoxins produced by Staphylococcus aureus. Staphylococcal enterotoxin D (SED) is one of the most common toxins detected in S. aureus strains associated with intoxications. The effect of sodium nitrite on enterotoxin production has been only partly investigated, despite its wide usage in meat products. In addition, the factors influencing SED regulation are unclear. The aim of this study was to determine the effect of sodium nitrite on sed transcription and SED production, as well as the effect of regulatory mutations on SED protein levels. Temporal sed mRNA and SED protein levels were compared in LB and LB supplemented with 150 mg/L nitrite, and SED protein levels between wild type (wt) and isogenic regulatory mutants (Δagr, ΔsarA, ΔsigB) under control and sodium nitrite conditions. Relative sed mRNA levels of wt strains were higher in late stationary phase in the presence of nitrite compared to control conditions. However, SED protein levels were decreased in the presence of nitrite. In LB, Δagr mutants showed SED levels similar to the wt, while ΔsarA mutants exhibited reduced and ΔsigB mutants increased SED levels compared to the wt. In LB with sodium nitrite, SED levels of mutant strains were reduced similar to the wt strains, except for two Δagr mutants, in which SED levels were increased in the presence of nitrite. Overall, strain-specific variation with regard to the effect of regulatory mutations was observed. In addition, the data suggests that SED regulation may not be as tightly dependent on Agr as previously described.

Reduced Enterotoxin D Formation on Boiled Ham in Staphylococcus aureus Δagr Mutant

“…The accumulation of SED was clearly noticeable in the early to mid-stationary phase, from day 2 to day 6. This observation also corroborates previous reports of higher SED production during the post-exponential growth phase, a typical characteristic of toxins regulated by the Agr system [ 19 , 23 ]. The SED concentration formed by RKI1 increased to above 150 ng × cm −2 (>8.9 µg per ham slice) from day 3 and onwards, and that produced by RKI1Δ agr from day 5.…”

“…Initial characterization of the RKI1 wild type and RKI1Δ agr strains at optimal growth conditions 37 °C, Luria Bertani (LB) medium [ 26 ] for 24 h has been performed previously by Sihto and coworkers. They established that the strains grew similarly; both reaching a maximum cell density of approximately 10 8 –10 9 CFU × mL −1 in the stationary phase after 10 h of growth with an initial inoculum of 5 × 10 3 CFU × mL −1 [ 23 ].…”

“…RNAIII indirectly induces sed expression by repressing the repressor of toxins (Rot) [ 21 ]. The influence of the global Agr regulatory system and numerous food-related stresses including NaCl, nitrite, glucose, and lactic acid on sed expression has so far been studied exclusively using planktonic growth in cultivation broth [ 14 , 16 , 17 , 18 , 19 , 21 , 22 , 23 , 24 , 25 ].…”

Characterization of Staphylococcus aureus isolated from tilapia and utensils used in the commerciali- zation of tilapia in the street markets of a semi-arid Brazilian municipality [pdf]

“…The gene encoding enterotoxin D can be carried by plasmids (Sihto et al, 2016). As previously reported, the structure of the biofilm enhances the pathogenicity of S. aureus strains, as it enables the sed gene to be easily transferred between cells.…”

Characterization of Staphylococcus aureus isolated from tilapia and utensils used in the commerciali- zation of tilapia in the street markets of a semi-arid Brazilian municipality

“…Among the enterotoxins produced, the classic staphylococcal enterotoxins (A, B, C, D, and E) are responsible for 95% of staphylococcal food poisoning (Kroning et al, 2016). Additionally, enterotoxin D is the most common serotype in S. aureus strains associated with food poisoning (Sihto et al, 2016). Small amounts of enterotoxins (approximately 1 μg of toxin per 100 g of food) are sufficient to produce clinical manifestations.…”