Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus

BMC Microbiology RAesceaercthiacrtiaclecid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus Nina Wallin-Carlquist 0 Rong Cao 0 Dra Mrta 0 2 Ayla Sant'Ana da Silva 0 1 Jenny Schelin 0 Peter Rdstrm 0 0 Applied Microbiology, Lund Institute of Technology, Lund University , Lund , Sweden 1 Dept. of Biochemistry, Chemistry Institute, Federal University of Rio de Janeiro , Rio de Janeiro , Brazil 2 Dept. of Microbiology and Biotechnology, Faculty of Food Science, Corvinus University of Budapest , Budapest , Hungary Background: The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Results: The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. Conclusions: Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the seaencoding prophage, linking SEA production to the lifecycle of the phage. - Background Staphylococcal enterotoxins (SEs) are extracellular proteins, produced mainly by Staphylococcus aureus, causing food intoxication when ingested. Staphylococcal food poisoning (SFP) was the fourth most common causative agent in food-borne illness within the EU in 2008 [1]. It is associated with food, generally rich in protein, which requires extensive manual handling, often in combination with inadequate heating and/or inappropriate storage of the food [2,3]. To date, 21 staphylococcal enterotoxins or enterotoxin-like proteins (SEA-SEE, SEG-SEV), excluding variants, have been identified. These SE genes are widely disseminated by several mobile genetic elements leading to variations in the SE expression behavior among enterotoxigenic staphylococci [2-5]. The expression of a number of the enterotoxins including SEB, SEC, and SED is to some extent known to involve regulatory systems such as the accessory gene regulator (Agr), the staphylococcal accessory regulator (Sar) and the repressor of toxin (Rot) [6]. However, we still have limited information about SEA, the toxin considered to be mainly responsible for staphylococcal food poisoning outbreaks [7-11]. The SEA gene is carried in the bacterial genome by a polymorphic family of temperate bacteriophages [12-14]. Recent studies of S. aureus strain MSSA476 have shown that mitomycin C (MC) induction of Sa3ms, resulted in increased transcription of enterotoxins SEA, SEG, and SEK and the fibrinolytic enzyme staphylokinase (Sak) carried by the prophage [14]. Although, it is still unclear if the increased transcription of these virulence determinants lead to increased amounts of SE proteins. Furthermore, identification of the environmental parameters that control the expression of SEA in food, and the mechanism by which these signals are transduced to bring about changes in gene expression, are very limited. This knowledge is crucial for understanding the potential of S. aureus to cause food poisoning. Acetic acid is a weak organic acid often used in the food industry as a preservative due to its antagonistic effect on bacterial pathogens [15]. Weak acids have the ability to pass through the cell membrane in the undissociated form. Once inside the cell, the acid dissociates in the more alkaline interior, lowering the intracellular pH of the cell. A decrease in intracellular pH can lead to the damage of macromolecules (e.g. proteins and DNA) and the cell membrane, and have a negative effect on cell maintenance [16,17]. Also, the anion of the acid is accumulated intracellularly, increasing turgor pressure [18]. Acetic acid has been found to be more inhibitory to the growth of S. aureus than lactic acid, citric acid, phosphoric acid and hydrochloric acid, respectively [19]. Also, acetic acid has been found to almost completely inhibit SEA formation in brain heart infusion (BHI) broth when added gradually over time [20]. In the present study, the effects of acetic acid on S. aureus growth, sea expression and SEA production were investigated in four growth phases. Furthermore, the relationship between SEA production and the lifecycle of the phage carrying the toxin gene was determined. Finally, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Batch cultures of S. aureus Mu50, harboring the sea-containing 42-like prophage Mu50A [21], were carried out at controlled pH levels of 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5 (Figure 1A). Acetic acid was used to set the pH to investigate the effects of acetic acid on growth, relative sea expression and extracellular SEA levels during all stages of growth. The maximal growth rate of S. aureus Mu50 was highest at pH 7.0 and decreased with decreasing pH (Figure 1A). Batch cultivations performed at lower pH values showed that pH 5.0 was highly growth-inhibitory, with only a modest increase in optical density, OD, and viable cells in the late stationary growth phase, and that pH 4.5 was too toxic; < 1% of the starting inoculum was viable after 24 h. The relative sea expression pattern was similar at all tested pH levels that allowed expression analysis (Figure 1B); the highest relative levels of sea mRNA were found in the transitional phase and fell during the stationary growth phase. Small increases in sea expression were found in the transitional phase at pH 7.0 and 6.5. However, relative sea expression in the transitional phase at pH 6.0 (...truncated)