Effects of Temperature Abuse on the Growth and Staphylococcal Enterotoxin A Gene ( sea ) Expression of Staphylococcus aureus in Milk

Abstract

The aim of this study was to determine the effects of different temperatures and storage time on Staphylococcus aureus growth, sea gene expression, and synthesis of staphylococcal enterotoxin A (SEA) in the pasteurized and UHT-pasteurized milk. Pasteurized and UHT-pasteurized milk were inoculated with 3.98 log₁₀ CFU/mL of S. aureus (ATCC 13565). Inoculated milk samples were stored at 8°C, 15°C, and 22°C for 24, 48, and 72 h, respectively. SEA synthesis was detected with a fully automated miniVIDAS instrument using the Enzyme-Linked Fluorescent Assay (ELFA) technology. The patterns of gene regulation were detected by quantitative reverse transcriptase PCR. The 2^−ΔΔCT method has been used as a relative quantification strategy for gene expression responses data analysis. The results indicated that growth rate, sea gene expression, and SEA synthesis were influenced by type of milk, storage time, and temperature. Incubation of milk at different temperatures (15°C and 22°C) and times was used to simulate inadequate transport and storage conditions. Storage of pasteurized milk at 22°C for 24 h significantly upregulated the expression of sea gene compared with milk stored at 8°C, which coincides with the achieved S. aureus number of 10⁵ CFU/mL and detected amount of SEA. In addition, storage of UHT-pasteurized milk at 22°C for 24 h and at 15°C for 48 h significantly upregulated the sea gene expression compared with milk stored at 8°C, which coincides with the detected amount of SEA and the dynamics of S. aureus number change. It can, therefore, be concluded that implementing good hygiene practices to avoid pre- and post-heat treatment milk contamination and maintaining the cold chain at temperature <8°C throughout the entire dairy production chain are of paramount importance to decrease the risk of staphylococcal food poisoning.

Introduction

Staphylococcal food poisoning (SFP) is one of the most frequently reported foodborne illnesses resulting from the consumption of food in which coagulase-positive Staphylococcus aureus have massively grown and produced enterotoxins (SEs) (Lin et al., 2009, 2018; Tsutsuura et al., 2013; Denayer et al., 2017; Schelin et al., 2017). S. aureus can produce a wide range of different extracellular protein toxins, such as Toxin-1, which causes toxic shock syndrome in humans, and staphylococcal enterotoxins, which play an important role in staphylococcal diseases (Srinivasan et al., 2006; Robert et al., 2011; Pajić et al., 2016; Denayer et al., 2017; Babić et al., 2018). Of the SEs, enterotoxin A (SEA) appears to be commonly responsible for SFP worldwide (Tsutsuura et al., 2013; Denayer et al., 2017).

According to the European Food Safety Authority (EFSA) report, among the causative agent group in foodborne outbreaks, bacterial toxins were ranked second in 2016 (17.7% of all outbreaks), whereby the vast majority (86.0%) of the outbreaks were associated with toxins by Clostridium, Staphylococcus, and Bacillus cereus, indicating that the number of SFP outbreaks is rising in the European Union (EFSA, 2017). Furthermore, Centers for Disease Control (CDC) reported 240,000 cases of SFP each year in the United States, which resulted in 1000 cases of hospitalization and 6 deaths (CDC, 2010; Scallan et al., 2011; Johler et al., 2015). SFP is characterized by a rapid onset (2–7 h) of symptoms after the consumption of food containing SEs. Patients have symptoms such as nausea and violent vomiting, which is often accompanied by watery diarrhea, abdominal cramps, prostration, and moderate fever (Schelin et al., 2017). Illness is typically self-limiting, with only 10% of sensitive patients, who belong to the YOPI (young, old, pregnant, immunosuppressed) group, needing to be hospitalized (Denayer et al., 2017).

SFP is often associated with protein-rich food, such as milk and milk products, which may be contaminated with a high number of S. aureus as a result of improper handling, followed by storage under conditions that allow the growth of S. aureus and the production of enterotoxins (Denayer et al., 2017; Lin et al., 2018). In addition, S. aureus can also enter the dairy chain as it is also a likely contaminant of milk when dairy animals are affected by mastitis (Denayer et al., 2017). It is generally accepted that a high number of typical colonies of S. aureus (>10⁵ CFU per g or mL of food) is needed for the production of sufficient amount of enterotoxins to cause illness (EU Regulation 853/2004; Duquenne et al., 2010).

However, some authors (Jablonski and Bohach, 2001) deem that staphylococcal enterotoxins were detected in food, although the count of S. aureus was <10⁵ CFU/g. There are also scientific reports claiming the same issue. For example, Vernozy-Roland et al. (1998) measured the concentrations of SEA recovered after salting of raw goat milk lactic cheese (saturated brine solution at 13°C for 7 min). At S. aureus concentrations of 10⁵ or 10⁶ CFU/mL of milk, the quantity of SEA detected after the salting step was 1 μg/kg, but lower SEA was detected when 10⁴ CFU/mL of milk was used as an inoculum. Next, in raw milk soft red smear cheese, SEA was detected at 0.01–0.04 μg/kg cheese, and SEC was detected at 0.04–0.05 μg/kg, while S. aureus was detected at a concentration <1 × 10⁴ CFU/g (de Reu et al., 2002).

Despite the fact that, for the occurrence of SFP, contamination is necessary, other conditions such as temperature and time must also be adequate to allow the microorganism to grow and produce enterotoxins (Tsutsuura et al., 2013). Although heat treatment of food eliminates S. aureus cells, SEs are heat-stable and, therefore, will remain in the food and can cause SFP (Janštova et al., 2013; Tsutsuura et al., 2013; Schelin et al., 2017).

Given these considerations, the aim of this study was bivalent: firstly, to determine the influence of different temperatures and storage times on S. aureus growth and synthesis of staphylococcal enterotoxin A in the pasteurized (72°C, 15 s) and UHT-pasteurized (135°C, 1–2 s) milk; and secondly, to use the transcriptomic approach and test whether variability in sea gene expression correlates with growth and SEA synthesis.

Materials and Methods

Inoculum preparation

S. aureus strain producing SEA was obtained from the American Type Culture Collection (ATCC 13565). A 50 mL overnight culture was grown in a 250 mL baffled flask (16 h, 250 rpm, 37°C) in Brain-Heart Infusion (BHI) broth (Oxoid, Basingstoke, Hampshire, England). An inoculum culture was prepared by transferring 1 mL of the overnight culture in 50 mL of pre-warmed (37°C) BHI media in a 250 mL baffled flask. The inoculum culture was grown for a total of 9 h. Spectrophotometer (Eppendorf, Germany) was blanked using BHI broth. Every 60 min, a 0.1 mL aliquot was sampled from the inoculum culture. A 0.1 mL aliquot of undiluted overnight culture was transferred to a 10 mm cuvette, and its optical density (OD) at 600 nm was measured. A second 10 mm cuvette was prepared with an aliquot of the inoculum culture diluted in BHI medium. This second measurement was to ensure that the OD of the culture remained within the dynamic OD range of the spectrophotometer. At each 60-min interval, an aliquot of the sample was used to perform serial dilutions. Dilutions were plated on BHI agar plates and incubated at 37°C overnight. Colonies were counted to determine bacterial cell count (CFU/mL) at each time point. Growth curve was designed plotting OD₆₀₀ values versus plate count at exact time points. An appropriate bacterial suspension was used in further experiments to yield initial count of ≈4 log₁₀ CFU/mL of milk.

Experimental conditions

Pasteurized and UHT-pasteurized (1.6% fat) milk samples, previously tested negative for S. aureus, were inoculated with an average of 3.98 log₁₀ CFU/mL of the above-mentioned strain. Inoculated milk samples were stored at 8°C (calibrator-untreated control sample), 15°C, and 22°C (room temperature) for 24, 48 and 72 h, respectively, to simulate inadequate transport and storage conditions. Experiments were conducted in parallel. Subsequently, 0.01 mL of each milk sample was inoculated in duplicate on Baird-Parker Agar (Merck, Germany) plates and incubated under aerobic conditions at 37°C ± 1°C for 24 ± 2 h and 48 ± 2 h. Enumeration of S. aureus colonies was performed using the Baird-Parker drop plate count method (EN ISO 6888-1). The number of S. aureus colonies was determined at a 24-h interval, and the average value was calculated from the results of parallel and repeated experiments.

Detection of SEA synthesis

The synthesis of SEA was detected with a fully automated miniVIDAS (Biomerieux, France) instrument using the ELFA (Enzyme-Linked Fluorescent Assay) technology. For enterotoxin production, each of the Staphylococcus isolates was inoculated in the BHI broth and incubated overnight at 37°C. An overnight culture of S. aureus was inactivated at 95°C for 15 min. After cooling, 500 μL of the concentrated protein extract or 500 μL of the controls (positive C1 and negative C2) was placed in the sample well of a VIDAS SET2 reagent strip, and detection was carried out using the VIDAS automated system. The system automatically measures the fluorescence of the newly formed compound of 4-methyl umbelliferone at 450 nm, compares the results with the internal reference, and interprets the results as positive or negative. The detection limit is <0.25 ng SEA/mL.

RNA extraction and quality determination

RNA extraction was performed according to the manufacturer's instructions using the PureLink RNA Mini Kit (Invitrogen), which uses enzymatic lysis and silica membrane extraction. The optimization of RNA extraction was performed using 25 mg/mL of lysozyme (Sigma) for 60 min at 37°C, which increased the yield of total RNA by two- to four-fold (data not shown).

Briefly, bacteria were resuspended in 100 μL RNase-free water and transferred to a tube containing 0.4 g of acid-washed 150–212-mm silica beads (Sigma), 400 μL Lysis buffer R (provided by the kit), and 400 μL 90% phenol solution (Appli-Chem, Germany) vortexed for 20 s before using the BeadBeater cell disruptor (Biospec Products) with setting 6.5 for 60 s. The samples were cooled on ice and the beat-beading step repeated.

Subsequently, samples were centrifuged for 5 min at 16,000 × g and supernatants were mixed with an equal volume of 100% ethanol (Sigma). The samples (including any remaining precipitate) were transferred to the silica columns and centrifuged for 15 s at 13,000 × g at room temperature (RT). Columns were washed using 700 μL of Wash buffer and centrifuged for 15 s at 13,000 × g at RT. Second phase of washing was performed using 500 μL of Wash buffer II using the same centrifugation procedure. Finally, total RNA was obtained by suspending the RNA in 40 μL of RNase-free water, incubated for 1 min, and centrifuged for 1 min at 12,000 × g. All steps were done at room temperature, except where indicated otherwise.

To digest genomic DNA, RQ DNase I (Promega, Madison, WI) treatment was performed by adding 3 μL of DNase I and 3 μL of reaction buffer to the extracted RNA and incubating the mixture at 37°C for 30 min. Afterward, DNase I enzyme was inactivated by adding 5 μL of 25 mM EDTA to the RNA sample and incubating for 10 min at 65°C.

The concentration and purity of the total RNA were spectrometrically assessed with NanoDrop 1000 (Thermo Scientific, Waltham, MA). The absorbance ratios A260/A280 were used as indicators of protein contamination and A260/A230 as indicators of polysaccharide, phenol, and/or chaotropic salt contamination. The integrity of total RNA was assessed by the visualization of the 23S/16S banding pattern. Electrophoresis was carried out at 80 V for 60 min using 1.5% agarose gel. The gel was stained with ethidium bromide (0.5 μg/mL) and visualized with GelDoc2000 (Bio-Rad). RNA was stored at −80°C until further use.

Real-time PCR

cDNA synthesis was performed using commercially available Super Script VILO (Invitrogen, Carlsbad, CA) kit according to manufacturer's instructions. To determine whether the extracted RNA was functional, the same amount of total RNA (500 ng/20 μL) was reverse-transcribed in two reaction volumes, 20 and 10 μL. To determine the possibility of genomic DNA carry-over, control reactions were performed under the same conditions but lacking the reverse transcriptase enzyme (no-RT control). All RNA extracted was absent of significant genomic DNA, as determined by an average cycle threshold difference of 18.5 ± 3.5, equivalent to a maximum quantification error of 0.0003%.

Real-time PCR (qPCR) was performed to determine sea gene expression. Oligonucleotide primers for the detection of ftsZ (housekeeping gene) and SEA encoding gene (sea) were designed using the Primer3 software having S. aureus ATCC 13565 genome as a template (Table 1).

Table 1.

Primers Used in sea Gene Expression Analysis

Gene	Primer sequence	Amplicon size
ftsZ	F: 5′-ATCCAAATCGGTGAAAAATTAACAC-3′	121 bp
ftsZ	R: 5′-CCATGTCTGCACCTTGGATTG-3′	121 bp
sea	F: 5′-TCAATTTATGGCTAGACGGTAAACAA-3′	93 bp
sea	R: 5′-GAAGATCCAACTCCTGAACAGTTACA-3′	93 bp

qPCR analysis was performed with Maxima SYBR Green Master Mix (Fermentas, Lithuania). For transcript detection, 2 × SYBR Green I (Invitrogen) was added to a standard PCR mix. The 20 μL reaction volume contained 2 μL of diluted cDNA or no-RT control, 10 pmol of each primer, 6 μL nuclease-free H₂O, and 10 μL of the respective 2 × master mix. Primer efficiencies were determined by the dilution method as well as performing a temperature gradient reaction from 50°C to 65°C. At 60°C, both sets of primers had the best and more similar efficiency values. qPCR run was performed on MX3005P (Agilent, CA) with the following cycle parameters: 95°C for 30 s, 40 cycles of 95°C for 10 s, 60°C for 15 s, and 68°C for 15 s. qPCR products were evaluated by melting curves to ensure the absence of unspecific products and primer dimer formation. Relative fold increase of specific mRNA transcripts in milk compared with planktonic cultures was determined using the delta Ct method (2^ΔCt), a variation of the Livak method, where ΔCt = Ct (reference gene)–Ct (target gene). The data analysis was based on three independent experiments.

Statistical analysis

Statistical analysis of the results was conducted using GraphPad Prism version 7.00 for Windows (GraphPad Software, San Diego, CA). One-way analysis of variance (ANOVA) was performed to identify conditions when sea gene was altered by at least a 2.0-fold increase or decrease in expression between groups. The numbers of significantly up- and downregulated genes in each comparison group were identified by applying a p value <0.05, together with fold change cut-offs of two-fold decrease and 2.0-fold increase.

Results

The effect of temperature and storage time on growth kinetics of S. aureus strain producing SEA in pasteurized and UHT-pasteurized milk is shown in Figure 1. Increasing the temperature and storage time of pasteurized and UHT-pasteurized milk significantly enhanced (p < 0.05) the growth rate of S. aureus strains producing SEA.

FIG. 1.

Growth kinetics of Staphylococcus aureus strain producing SEA in pasteurized (A) and UHT-pasteurized (B) milk.

The effect of temperature and storage time on SEA synthesis by S. aureus strain ATCC 13565 in pasteurized and UHT-pasteurized milk is shown in Figure 2. The synthesis of SEA in pasteurized milk was detected after 48 h of storage at 15°C, while toxin synthesis in pasteurized milk at 22°C was detected after 12 h of storage. In UHT-pasteurized milk stored at 15°C, SEA synthesis was detected after 48 h, while toxin synthesis in the same milk sample was detected after 24 h of storage at 22°C.

FIG. 2.

SEA synthesis by S. aureus strain ATCC 13565 in pasteurized (A) and UHT-pasteurized (B) milk.

The effect of temperature and storage time on sea gene expression by S. aureus strain ATCC 13565 in pasteurized and UHT-pasteurized milk is shown in Figure 3. The expression of sea gene in pasteurized milk was significantly (p < 0.05) upregulated by 1.94-fold after initial 24 h of storage at 22°C (compared with milk stored at 8°C), while in pasteurized milk stored at 15°C, sea gene expression was significantly (p < 0.05) upregulated by 2.11-fold after 72 h (compared with milk stored at 8°C). sea gene expression in UHT-pasteurized milk was significantly (p < 0.05) upregulated by 1.87-fold after 48 h (compared with milk stored at 8°C). The expression of sea gene in UHT-pasteurized milk was significantly (p < 0.05) upregulated by 2.45-fold after initial 24 h of storage at 22°C (compared with milk stored at 8°C).

FIG. 3.

Expression of sea gene in pasteurized (A) and UHT-pasteurized (B) milk.

Discussion

S. aureus is thermolabile and does not compete well with other microorganisms, and therefore, contamination usually occurs as a result of improper handling of processed foods when there is little competition from other microorganisms (Kitagwa et al., 2006; Panneerseelan and Muriana, 2007; Denayer et al., 2017). Therefore, the highest risk of SE production and SFP is associated with secondary contamination of pasteurized and UHT-pasteurized milk, followed by storage under inappropriate conditions (Argudín et al., 2010; Janštová et al., 2013; Denayer et al., 2017).

This study investigated the growth kinetics of S. aureus, SEA synthesis, and sea gene expression in pasteurized and UHT-pasteurized milk contaminated with S. aureus strain producing SEA at different storage temperatures (15°C and 22°C) compared with milk contaminated with S. aureus and held at 8°C for 24, 48, and 72 h. The storage of milk samples at 15°C and 22°C was chosen to simulate inappropriate storage and transport conditions in the milk supply chain, while 8°C was used as maximum allowed temperature for milk storage with little effect on its microbial quality (EU Regulation 853/2004).

In the present study, the number of S. aureus in both pasteurized and UHT-pasteurized milk stored at 8°C remained unchanged during the entire storage period. Conversely, the storage of both pasteurized and UHT-pasteurized milk at increased temperature and for a prolonged period resulted in an elevated number of S. aureus, which is in agreement with the results obtained by Janštová et al. (2013).

The growth data, curves, and parameters of the tested ATCC strain were found to fit the mechanistic modeling technique of Baranyi and Roberts (1994), which has been incorporated in various predictive modeling software (PMP, ComBase, etc.). Satisfactory growth-fitted data were a prerequisite for correlation with SEA synthesis and sea level expression.

In pasteurized milk contaminated with S. aureus producing SEA and stored at 15°C and 22°C for 72 h, an increase in S. aureus count from 3.98 to 7.99 log₁₀ CFU/mL and from 3.98 to 8.47 log₁₀ CFU/mL was observed, respectively (Fig. 1A). Furthermore, we observed an increase in S. aureus count from 3.98 to 7.83 log₁₀ CFU/mL and from 3.98 to 8.70 log₁₀ CFU/mL in UHT-pasteurized milk inoculated with S. aureus producing SEA and stored for 72 h at 15°C and 22°C, respectively (Fig. 1B). The number of S. aureus in pasteurized milk stored at 22°C and UHT-pasteurized milk stored at 15°C and 22°C for 24 h exceeded 10⁵ CFU/mL (Fig. 1), which represents the threshold for enterotoxin synthesis and an increased risk of SFP (Duquenne et al., 2010). In agreement with the results of this study, other researchers have also found an increased number of S. aureus in milk stored at different temperatures, which resulted in increased pathogen virulence (Fujikawa and Morozumi, 2006; Medveďová et al., 2009; Tsutsuura et al., 2013; Babić et al., 2018).

The growth of S. aureus and the production of enterotoxins are affected by several factors, including the population density of S. aureus, the presence and density of competitive microflora, the lactic acid starter culture, the food storage environment, temperature, pH, salt, water activity (aw), and presence of O₂. Temperature remains one of the main environmental factors that influence bacterial growth and SEA production in milk and milk products (Fujikawa and Morozumi, 2006). SEA is produced between 10°C and 45°C, whereby the rate of SEA production increases with increasing temperatures (Schelin et al., 2017), which was also confirmed by the present study. Enterotoxin production is unlikely to occur at temperatures <10°C. Optimum enterotoxin production occurs at a pH of 6–7 and the minimum suitable pH is about 4.8 (Schelin et al., 2017). The presence of SEA was detected in both pasteurized and UHT-pasteurized milk after 48 h of storage at 15°C, as shown in Figure 2. A marked increase in S. aureus count and earlier enterotoxin productions were both observed when pasteurized and UHT-pasteurized milk were stored at 22°C (Figs. 1 and 2). The synthesis of SEA at this temperature was detected (>0.25 ng SEA/mL) as early as 12 h after storage. Several studies have reported that the lowest toxic dose of SEA for humans ranges from 20 to 100 ng (Hennekinne et al., 2012; Tsutsuura et al., 2013; Denayer et al., 2017). This suggests that both pasteurized and UHT-pasteurized milk stored for 12 h at 22°C exceeded the minimal toxic dose reported to cause symptoms in adults and, therefore, can represent a potential health risk for humans. In contrast, the production of SEA was not detected in both pasteurized and UHT-pasteurized milk stored at 8°C for the entire period of storage. This highlights the importance of maintaining the cold chain <8°C from production through to comsumption to ensure the safety of milk and milk products (Janštová et al., 2013).

Unlike the majority of exo-proteins that are controlled by global regulatory systems designated as an accessory gene regulator (agr), SEA belongs to the group of prophage-encoded enterotoxins. The sea gene is carried by a polymorphic family of temperate bacteriophages, which are inserted into the bacterial chromosome as a prophage and behaves like part of the bacterial genome (Schelin et al., 2011). In this study, the storage of pasteurized milk at 22°C for 24 h significantly upregulated (p < 0.05) the expression of sea gene (Fig. 3) compared with milk stored at 8°C, which coincides with the achieved S. aureus number of 10⁵ CFU/mL (Fig. 1) and detected amount of SEA (Fig. 2). In addition, the storage of UHT-pasteurized milk at 22°C for 24 h and at 15°C for 48 h significantly upregulated (p < 0.05) the sea gene expression compared with the milk stored at 8°C (Fig. 3), which coincides with the detected amount of SEA (Fig. 2) and the dynamics of S. aureus number change (Fig. 1).

The results of this study indicate that increase in S. aureus count, SEA production, and respective sea gene expression were faster in UHT-pasteurized milk compared with pasteurized milk (Figs. 1 –3). One of the possible explanations is that unlike UHT, low-temperature pasteurization does not kill thermophilic lactic acid bacteria (LAB) (Walstra et al., 2005; Schaechter, 2009), which are able to suppress S. aureus growth and interfere with the expression of S. aureus virulence, including staphylococcal enterotoxin production (Janštová et al., 2014; Nouaille et al., 2014). The antagonistic potential of LAB against S. aureus and SE production involves lowering the milk pH, competition for nutrients, the production of bacteriocins and hydrogen peroxide (Charlier et al., 2009; Janštová et al., 2013, 2014; Nouaille et al., 2014). In addition, it has also been found that LAB cause a decrease in SEA levels through extracellular protease activities (Schelin et al., 2011; Sabike et al., 2014).

Conclusions

In this study, the safety of milk represented by pasteurized and UHT-pasteurized milk samples was investigated. The safety was examined concerning the presence of toxin-producing bacterium, that is, enterotoxigenic S. aureus. This toxin-producing bacterium is of public health concern because it cannot be removed by subsequent heat treatments, which are used in the processing of milk. The growth, toxin production, and respective gene expression of S. aureus was investigated by modeling growth at 8°C (which is a maximum storage temperature of milk), 15°C, and 22°C, respectively, which served as an abuse temperature simulating HACCP non-compliance conditions.

Our results indicated that the critical level of SEA in contaminated milk could be reached <48 h of storage at improper conditions. Knowledge about the effects of key environmental factors on S. aureus responses, such as growth rate, virulence gene expression, lag phase duration, and extracellular virulence formation, is of paramount importance for ensuring the quality and safety of milk. Accordingly, the main factors in the prevention of milk-borne diseases are implementing good hygiene practices to avoid pre- and post-heat treatment contamination, and maintaining the cold chain at temperature <8°C throughout the dairy production chain, which will prevent S. aureus from reaching the critical count of 10⁵ CFU/mL that is necessary for the synthesis of staphylococcal enterotoxins.

Footnotes

Acknowledgment

Funding for research presented in this article was provided through projects of the Ministry of Education, Science and Technological Development of Republic of Serbia (TR-31034 and III46009).

Disclosure Statement

No competing financial interests exist.

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