Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene

    Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene Justyna Schubert, Magdalena Podkowik, Jarosław Bystro´n, Jacek Bania PII: DOI: Reference:

S0168-1605(16)30346-4 doi: 10.1016/j.ijfoodmicro.2016.06.043 FOOD 7290

To appear in:

International Journal of Food Microbiology

Received date: Revised date: Accepted date:

24 March 2016 25 May 2016 29 June 2016

Please cite this article as: Schubert, Justyna, Podkowik, Magdalena, Bystro´ n, Jaroslaw, Bania, Jacek, Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus aureus strains harboring seh gene, International Journal of Food Microbiology (2016), doi: 10.1016/j.ijfoodmicro.2016.06.043

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ACCEPTED MANUSCRIPT Production of staphylococcal enterotoxins in microbial broth and milk by Staphylococcus

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aureus strains harboring seh gene

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Justyna Schubert, Magdalena Podkowik, Jarosław Bystroń, Jacek Bania

Department of Food Hygiene and Consumer Health Protection, Wrocław University of

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Environmental and Life Sciences, Wrocław, Poland

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Address correspondence to J. Bania, [email protected]

Abstract

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Twenty S. aureus strains harboring seh gene, including one carrying also sec gene and 11 sea gene, were grown in BHI+YE broth and milk and were tested for SEA, SEC and SEH

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production. All strains decreased pH of BHI+YE broth at 24 hours and increased them at 48 hours. Seventeen S. aureus strains grown in milk changed pH for no more than 0.3 unit until

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48 hours. Three other S. aureus strains significantly decreased pH during growth in milk . All S. aureus produced SEH in BHI+YE broth in amounts ranging from 95 to 1,292 ng/ml, and from 170 to 4,158 ng/ml at 24 and 48 hours, respectively. SEH production in milk by 17 strains did not exceed 23 ng/ml at 24 hours and 36 ng/ml at 48 hours. Three S. aureus strains able to decrease milk pH produced 107-3,029 ng/ml and 320-4,246 ng/ml of SEH in milk at 24 and 48 hours, respectively. These strains were grown in milk and BHI+YE broth with pH stabilized at values near neutral leading to a

significant decrease of SEH production .

Representative weak SEH producers were grown in milk at reduced pH resulting in moderate increase in SEH production . SEA was produced in milk by 10 S. aureus strains at 24-151 ng/ml at 24 hours, and 31-303 ng/ml at 48 hours. SEA production in milk was higher or

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ACCEPTED MANUSCRIPT comparable as in BHI+YE broth in 3 strains and lower for remaining strains. Production of SEC by sec-positive S. aureus strains was lower in milk than in BHI+YE broth, ranging from

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131-2,319 ng/ml at 24 and 48 hours in milk and 296-30,087 ng/ml in BHI+YE at 24 and 48

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hours. Both lacE and lacG transcripts involved in lactose metabolism were significantly up-

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regulated in milk in strong SEH producers. In these strains hld, rot and sarA transcripts were up-regulated in milk as compared to weak SEH producers. Stabilization of milk pH at a value of raw milk significantly down-regulated hld, rot and sarA RNA in strong SEH producers.

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Milk was generally found unfavourable for enterotoxin production. However, certain S.

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aureus strains were not restricted in SEH and SEA expression in milk, unlike SEC which remained down-regulated in this environment. Therefore, low safety risk related to S. aureus producing SEC in milk, as suggested previously, may not pertain to certain SEA and SEH-

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producing strains.

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Key words: Staphylococcus aureus, staphylococcal enterotoxins, expression, milk

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1. Introduction

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Milk was found a favorable environment for Staphylococcus aureus growth. Genome

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sequence analyses revealed the presence of lactose phosphotransferase systems that enabled

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growth of S. aureus in milk (Fujikawa and Morozumi, 2006; Kuroda et al., 2001). Staphylococcal enterotoxin (SE) production by S. aureus was identified as a factor of Staphylococcal Food Poisoning (SFP) (Le Loir et al., 2003). Dairy products have already

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been associated with SFP. SEA was involved in large outbreaks in USA in 1985 and Japan in

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2000 caused by consumption of contaminated chocolate milk and dairy products, respectively (Asao et al., 2003; Evenson et al.,1988). Six SFP outbreaks in France in 2009 were caused by SEE present in soft cheese made from unpasteurized milk (Ostyn et al., 2010).

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Generally, the amount of enterotoxin necessary to cause intoxication can be small. The lowest emetic dose was reported by Evenson et al. (1988), where the concentration of SEA was 0.5

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ng/ml. Only the presence of SEA, SEB, SEC, SED and SEE enterotoxins is currently investigated during routine food analyses (European Regulation 1441/2007). To date, 23 SEs

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have been described. Most of SEs were shown to induce emesis in animal models (Omoe et al., 2013). Although the new SEs possess relatively weaker emetic activity than the classical SEs, S. aureus strains harboring only new SE genes have been isolated from SFP cases

(Jørgensen et al., 2005; Kérouanton et al., 2007). Staphylococcal enterotoxin H, a gastrointestinal toxin with emetic properties, was first described by Su and Wong (1995). SEH was implicated in SFP outbreaks associated with reconstituted milk and raw milk used to prepare mashed potatoes (Ikeda et al., 2005; Jørgensen et al., 2005). Enterotoxin expression is coordinated in S. aureus by several regulatory elements, such as two-component systems (e.g. agr, arlRS, saeRS) and DNA-binding proteins (e.g. sarA

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ACCEPTED MANUSCRIPT family, sigB). Also, environmental signals such as pH, temperature, O2, carbon sources, salts, metal ions and short peptides might have effects on transcription of virulence genes (Fournier,

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2008; Pragman and Schlievert, 2004; Ray et al., 2009).

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It has been suggested that a specific set of genes becomes expressed when S. aureus cells are

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grown in milk. These group includes genes involved in carbohydrate metabolism, transcriptional regulation, nucleotide synthesis and cell-wall synthesis (Lammers et al., 2000). Recently, regulation of SEH expression was investigated in S. aureus producing both SEH

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and SEA, belonging to lineage accounting for majority of SFP cases in Japan (Sato'o et al.,

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2015). Unexpectedly, SEH expression was found to be positively regulated by Rot, previously shown to act as enterotoxin repressor (Sato'o et al., 2015). All data on SEH expression is based on experiments conducted in microbiological broths.

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There is practically no information on its production in food, nor on conditions favoring and limiting its expression in food. Our goal was to study the ability of 20 S. aureus strains to

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medium.

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produce SEH in UHT cow`s milk in comparison to culture in BHI, a widely used microbial

2. Materials and methods 2.1. Bacterial strains and growth conditions From the 20 seh-positive S. aureus strains investigated, 8 strains were isolated from food and 12, including FRI137 reference strain, were from humans (Table 1). Enterotoxigenic reference S. aureus strains, were kindly provided by Prof. Gerard Lina of the Centre National de Référence des Toxémies Staphylococciques, Faculté de Médecine, Lyon, France. Frozen stock culture was resuscitated by plating on brain heart infusion (BHI) (Biocorp, Warsaw, Poland) agar and incubated at 37C overnight. Single colony from agar plates was transferred to 5 ml of BHI broth supplemented with 1% yeast extract (YE) (Biocorp) in test tubes for 18

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ACCEPTED MANUSCRIPT hours (37C, 230 rpm). One hundred microliters of overnight culture was inoculated into 5 ml of fresh BHI+YE broth (pre-culture) and incubated under the same conditions for 2-3 hours.

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The media, 100 ml BHI+YE broth or 100 ml UHT 0.0% fat cow`s milk (Mlekpol, Grajewo,

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Poland), was inoculated with pre-culture to attain an optical density at 600 nm (OD600) of 0.02

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(2.6 × 107 CFU/ml). Prior to inoculation, the pre-culture was washed twice with phosphatebuffered saline to remove residual BHI broth and enterotoxins (repeated centrifugation at

37C with constant agitation at 230 rpm.

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12,000 × g for 5 min and resuspension in the saline solution). Cultures were conducted at

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Stabilization of pH in BHI+YE broth and milk cultures was performed using 1.5 M Tris-HCl (pH 7.0) or 2 M NaOH, respectively. Reduction of pH in milk cultures was achieved by

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adding 25% HCl at values noted for S. aureus α41 strain, shown to decrease the milk pH

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during the culture. The actual pH was determined in samples sterilely-collected from the cultures before and after addition of reagent used to alter pH. Milk pH control started at 2

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hours of culture when initial milk pH of 6.80 was adjusted to 6.50. Then, pH was adjusted at following time points: 5 hours to pH 6.10, 8 hours to pH 5.80, 24 hours to pH 5.30, and 30

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hours to pH 5.10. Volumes of each agent stabilizing or decreasing pH were previously determined on cultures of each investigated strain. Samples for RNA extraction, ELISA, determination of bacterial concentration and pH were collected at specified time points during each experiment. Cell concentration was determined by plating serial dilutions of bacteria onto BHI agar. pH was measured by FE20-FiveEasy™ pH-meter (Mettler-Toledo, Greifensee, Swiss). All experiments were carried out in triplicates.

2.2. Preparation of bacterial DNA Two milliliters of bacterial cell suspension from an overnight culture grown in BHI broth were centrifuged for 5 min at 12,000  g and resuspended in 100 l of 100 mM Tris-HCl

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ACCEPTED MANUSCRIPT buffer, pH 7.4, containing 10 g of lysostaphin (A&A Biotechnology, Gdańsk, Poland). After 30-minute incubation at 37C, 15l of 10% SDS was added and the sample was incubated for

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20 min at 37C. Two hundred microliters of 5 M guanidine hydrochloride were added and the

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sample was mixed and incubated at room temperature for 20 min. The DNA was extracted by

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a mixture of phenol: chloroform: isoamyl alcohol (25:24:1), precipitated with isopropanol, washed with ethanol and dissolved in water.

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2.3. PCR for enterotoxin genes

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Detection of sea-see was performed according to Sharma et al. (2000). The selu gene was detected using the method described by Letertre et al. (2003). For the detection of tst gene, the

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primers and conditions were described according to Monday and Bohach (1999). Detection of

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sei, sem, sen, seo, seh, selj, sek, sel, and sep was done as previously described (Bania et al.,

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2006; Lis et al., 2009). S. aureus reference strains served as PCR controls.

2.4. Sandwich ELISA

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Recombinant SEH (rSEH) was obtained previously by Lis et al. (2012) by cloning seh gene into a pET-22b plasmid vector and expressed in E. coli Rosetta cells (Merck, Darmstadt, Germany). Expression of rSEH was performed using IPTG (Sigma-Aldrich, St. Louis, MI) induction and purification was conducted on His-Select Cobalt Affinity Gel (Sigma-Aldrich), with on-column refolding. Antisera preparation was done as described by Lis et al. (2012). Recombinant SEC (rSEC) was obtained by cloning sec gene from S. aureus FRI913 strain into a pET-22b plasmid vector, produced in E. coli Rosetta cells and affinity-purified on HisSelect Cobalt Affinity Gel. Rabbit polyclonal anti-SEA and anti-SEC antibodies were purchased from Acris Antibodies (Herford, Germany). Samples for SEH, SEA, and SEC detection were collected after 24 and 6

ACCEPTED MANUSCRIPT 48 hours of growth and stored at -20C until analyzed. Supernatants were pre-incubated with 20% normal rabbit serum, in order to bind protein A, and diluted in phosphate-buffered saline

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containing 0.1% Tween-20 (Sigma-Aldrich). ELISA was performed according to the protocol

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described by Freed et al. (1982), with modifications. 3,3′,5,5′-Tetramethylbenzidine

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supersensitive (Sigma-Aldrich) was used as a substrate for horseradish peroxidase, which was coupled either to the anti-SEH antibody (Abcam, Cambridge, UK) or streptavidin (SigmaAldrich), which detected biotinylated anti-SEA and anti-SEC antibodies. Biotinylation was

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performed with biotin N-hydroxysuccinimide ester (Sigma-Aldrich).

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The specificity of the ELISA was assessed using culture supernatants of S. aureus reference strains as controls for enterotoxins SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SElJ, SEK,

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SEL, SEM, SEN, SEO, SEP, and SER (validation data for ELISA and antisera preparation

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method were presented in Supplementary Table 1). The concentration of the enterotoxin in samples was measured with rSEH, SEA (Sigma-

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Aldrich), and rSEC as standards, using a 4-parameter logistic curve fit. Data analysis was

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carried out using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA).

2.5. RNA extraction and RT-qPCR Samples for RNA isolation were collected from BHI+YE broth after 4.5, 6.5, 9 and 24 hours of growth and from milk after 5, 8 and 24 hours. Bacterial suspensions from milk cultures were clarified with sodium citrate added to milk to obtain 100 mM solution (Chen and Novick, 2009). Samples were then centrifuged for 5 min at 12,000  g. One millilitre of TRI Reagent (Sigma-Aldrich) and 50 mg of glass beads, 150-212 µm (Sigma-Aldrich) were added to each pellet. Three cycles of beating of 2 min each, with 1 min incubation on ice within cycles, were carried out in the Tissue Lyser LT (Qiagen, Hilden, Germany). RNA extraction was conducted according to TRI Reagent manufacturer’s instruction. After precipitation with

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ACCEPTED MANUSCRIPT isopropanol RNA was dissolved in 50 µl of RNase free water and purified with Blood/Cell RNA Mini kit (Syngen, Wrocław, Poland) and RNase-Free DNase Set (Qiagen). cDNA was

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synthesized using iScript™ cDNA Synthesis Kit (Bio-Rad, Herkules, CA). Real-time

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quantitative PCR (RT-qPCR) was carried out on CFX Connect™ Real-Time System (Bio-

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Rad), using SsoFast EvaGreen Supermix (Bio-Rad). The reaction mixture contained 1 µl of template cDNA, 0.5 µM of each primer (listed in Table 2), and 10 µl of SsoFast EvaGreen Supermix and water up to 20 µl. The reaction protocol was: 95°C for 30 s; followed by 35

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cycles of 95°C for 10 s and 62°C for 15 s. Specificity of PCR was evaluated by melt curve

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analysis in a temperature range 95-58°C, performed for each reaction. Residual DNA contamination was checked in each RNA sample by running no-RT controls. rpoB, a housekeeping gene found to be stably expressed in milk was used for cDNA normalization

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(Valihrach et al., 2014). Duquenne et al. (2010) found rpoB as one of the four most stably expressed gene in S. aureus during growth in milk under different conditions of pH,

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temperature, and aeration.

PCR efficiencies for each primer pair were determined on genomic S. aureus DNA from

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respective reference strains by running serial 5-fold dilutions of the template. Relative transcript levels were calculated according to Pfaffl (2001). Data analysis was carried out using Bio-Rad CFX Manager software.

2.6. Statistics Statistical significance of the results was assessed using the U Mann-Whitney test. p<0.05 was considered statistically significant. Statistical analyses were performed using Statistica version 12 (StatSoft Inc., Kraków, Poland).

3. Results

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ACCEPTED MANUSCRIPT 3.1. Growth of S. aureus strains in BHI+YE broth and milk The tested 20 S. aureus strains reached 7.3-9.2 log CFU/ml after 24-hours culture and from

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8.1 to 9.6 log CFU/ml after 48-hours culture in BHI+YE broth. During growth in milk they

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reached 8.2-9.3 log CFU/ml after 24-hours culture and 8.3-9.5 log CFU/ml following 48-

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hours of culture. Bacterial counts determined at 24 hours were significantly higher in cultures conducted in milk than in BHI+YE broth (p<0.005), whereas no significant differences were noted in strains cultured both in BHI+YE broth and milk at 48 hours (p>0.05) (the growth

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pattern of selected S. aureus strains was presented in Supplementary Figure 1). The pH value

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of every BHI+YE broth culture consequently decreased from 7.06 at the beginning of culture reaching 5.32-6.09 at 24 hours of growth and then went up to 6.35-8.14 at 48 hours. In 17 S. aureus strains cultured in milk pH changed from initial value of 6.81 to 6.43-6.65 at

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24 hours, then remained unaltered (6.42-6.65) until 48 hours of growth. Three S. aureus strains, i.e., α41, 183, and reference S. aureus FRI137 strain were able to decrease milk pH to

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5.80-6.36 at 24 hours, continuing the pH drop to 5.10-5.96 at 48 hours (Table 3).

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3.2. Production of SEH in BHI+YE broth and milk All tested S. aureus strains produced SEH in BHI+YE broth with mean levels of 334 ng/ml, and 1,668 ng/ml after 24 and 48 hours, respectively SEH production in milk by 17 strains did not exceed mean level of 11 ng/ml and 14 ng/ml after 24 and 48 hours, respectively (Table 4).. Levels of SEH produced by these strains in milk was shown to be statistically lower than in BHI+YE broth both at 24 and 48 hours of cultures (p<0.05). S. aureus strains α41, FRI137, and 183 were able to produce SEH in milk with mean level of 1155 ng/ml, and 1820 ng/ml at 24 and 48 hours, respectively (specific amounts of SEH formed (ng/log CFU) were presented in Supplementary Table 2). Significant increase of SEH production in milk as compared to BHI+YE broth was found in S. aureus α41 and FRI137 strains at 24 and 48

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ACCEPTED MANUSCRIPT hours (p<0.05). No difference in SEH production in milk and BHI+YE broth was noted for S. aureus 183 strain at 24 hours (p>0.05).

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To evaluate whether milk can interfere with SEH detection resulting in observed reduction of

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SEH level in milk, we studied stability of both rSEH and native SEH in milk. For this, rSEH

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was added to BHI+YE broth and milk at 1.5 to 100 ng/ml and incubated for 24 hours at 37C with agitation (230 rpm). Also, BHI+YE broth culture supernatants from S. aureus 613 corresponding to 386 ng/ml of native SEH were mixed with milk and BHI+YE broth in the

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ratio 1:10 and incubated as above. No reduction of SEH levels were found during incubation

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of both rSEH and native SEH in milk as referred to their levels simultaneously incubated in

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BHI+YE broth (p<0.05).

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3.3. Contribution of milk and BHI+YE broth on SEH production Three representative S. aureus strains, i.e., 576, 613, and 637, randomly selected from 17

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strains producing low levels of SEH in milk were cultured in BHI+YE broth supplemented with 1, 5, and 10% milk. We found that milk addition did not cause a significant change in

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SEH production as compared to cultures carried out in BHI+YE broth (p>0.05), except S. aureus 637 + 10% milk in which SEH level increased as compared to BHI+YE broth (p<0.05) (Figure 1A). Bacterial counts at 24 hours in BHI+YE broth supplemented with milk were not changed as compared to BHI+YE broth, except strains 613 + 10% milk, 637 + 5% and + 10% milk in which bacterial number increased (p<0.05). After 48 hours culture in strains 576 + 5% milk, 613 + 1% and + 5% milk and 637 + 5% and + 10% milk a decrease of bacterial count was noted (p<0.05), whereas in remaining cultures bacterial counts were stable as compared to controls (p>0.05). Representative weak SEH producers, i.e., S. aureus 576, 613, and 637 strains were cultured in milk containing 1, 5, 10, and 20% BHI+YE broth. Supplementation with BHI+YE broth

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ACCEPTED MANUSCRIPT resulted in higher production of SEH as compared to cultures carried out in milk. Addition of BHI+YE broth resulted in a 2.0-2.6 (1%), 18.3-50.2 (5%), 13.1-35.2 (10%), and a 10.7-17.6

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(20%)-fold increase of SEH after 24 hours, as well as a 2.7-3.0 (1%), 27.4-47.8 (5%), 11.5-

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25.3 (10%), and a 7.7-13.8 (20%) fold rise in SEH production after 48 hours (Figure 1B).

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Bacterial counts at 24 and 48 hours were higher in all strains in milk supplemented with 1% BHI+YE broth than in milk itself (p<0.05). Increase of BHI+YE broth in milk to 5% resulted in higher bacterial counts than for 1% BHI+YE broth (p<0.05). Increase of BHI+YE broth to

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10 and 20% had no further effect on bacterial counts when compared to effect of 5% BHI+YE

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broth. Only in strain 613 bacterial counts were not altered at 48 hours of culture irrespective of increase of BHI+YE broth concentration.

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3.4. Effect of pH on SEH production in BHI+YE broth and milk cultures Production of SEH in milk by α41, FRI137, and 183 strains significantly exceeded SEH

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amounts elaborated by the remaining strains in the same environment. These 3 strains were also able to decrease pH during growth in milk and in BHI+YE broth, therefore they were

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cultivated in milk and BHI+YE broth with stabilized pH in order to assess the contribution of pH decrease on SEH production. Milk pH could be stabilized at values of 6.68-6.88 at 24 hours, what led to a 7.7-12.7-fold decrease in SEH production, and at 5.57-6.49 at 48 hours what resulted in a 4.6-17.8-fold drop of SEH concentration. During growth of α41, FRI137, and 183 strains in BHI+YE broth pH could be kept on the level of 7.27-7.50 at 24 hours and 7.57-7.71 at 48 hours of culture, what resulted in a 1.8-4.2 and a 3.3-9.0-fold decrease in SEH secretion at 24 and 48 hours, respectively (Figure 2). Stabilization of pH in milk cultures had no effect on bacterial counts at 24 and 48 hours for S. aureus α41 and 183 (p>0.05), whereas bacterial counts for S. aureus FRI137 at 24 and 48 hours were higher than in untreated milk (p<0.05). Stabilization of pH in BHI+YE broth had

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ACCEPTED MANUSCRIPT no effect on bacterial counts at 24 and 48 hours for S. aureus α41, as well as for S. aureus FRI137 and 183 at 48 hours (p>0.05), whereas bacterial count for S. aureus FRI137 and 183

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at 24 hours of culture was higher than in untreated BHI+YE broth (p<0.005).

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To check if decreased pH had an effect on SEH production by weak SEH producers we

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cultivated S. aureus 576, 613, and 637 strains in milk at pH values noted for S. aureus α41 strain cultured in milk. The pH value measured at 5 hours of culture was not significantly altered as compared to the value set at 2 hours. At 24 hours of culture the actual pH increased

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from 5.80 (as adjusted at 8 hours) to 6.00, when at 30 hours pH was 5.40, instead of 5.30 as

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adjusted at 24 hours of culture. At 48 hours the pH value increased from 5.10 (as adjusted at 30 hours) to 5.20.

Reduction of milk pH to 6.00-6.10 at 24 hours and to 5.10-5.30 at 48 hours resulted in a 1.8-

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2.7 and a 1.7-1.9-fold increase in SEH production at 24 and 48 hours of culture, respectively. Experimental decrease of pH in milk had no effect on bacterial counts at 24 hours in all 3

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strains, at 48 hours bacterial counts were lower in all 3 tested S. aureus strains (p<0.05).

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3.5. Production of SEA in BHI+YE broth and milk cultures Production of SEA in BHI+YE broth was detected in 10 out of 11 tested S. aureus seapositive strains. In S. aureus 637 strain SEA production could not be detected using ELISA (Table 4). The amounts of SEA secreted to BHI+YE ranged from 24 to 151 ng/ml at 24 hours, with mean level of 43 ng/ml, and from 31 to 303 ng/ml at 48 hours, with mean level of 109 ng/ml, respectively. In both BHI+YE broth and milk S. aureus α41 strain was the strongest SEA producer. It secreted SEA to milk in the amount of 244 ng/ml at 24 hours and 283 ng/ml at 48 hours of growth. The SEA production in milk by remaining strains ranged from 5 to 37 ng/ml at 24 hours, with mean level of 16 ng/ml, and was between 7 and 59 ng/ml at 48 hours, with mean level of 30 ng/ml, respectively. S. aureus α41 secreted statistically more SEA into

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ACCEPTED MANUSCRIPT milk than into BHI+YE broth at 24 hours (p<0.005) with no significant difference at 48 hours. No statistical difference was noted for SEA production in milk and BHI+YE broth by S.

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aureus 576 and 613 strains at 24 and 48 hours (p<0.05). Remaining sea-positive strains

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produced less SEA in milk than in BHI+YE broth at 24 and 48 hours (p<0.05), but SEA

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reduction in milk was not higher than 16-fold, ranging from 1.3 to 13 at 24 hours and from 1.2 to 15.9 at 48 hours of culture.

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3.6. Production of SEC in BHI+YE broth and milk cultures

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We found that SEC levels secreted to milk by S. aureus FRI137 strain were lower (p<0.05), accounting for 131 ± 15 and 2,319 ± 76 ng/ml at 24 and 48 hours of cultivation, that these elaborated in BHI+YE broth, ranging from 296 ± 41 to 30,087 ± 621 ng/ml at 24 and 48 hours

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of growth. As S. aureus FRI137 strain belonged to a group of strong SEH producers in milk, a culture of a food sampled, S. aureus strain designated 175, carrying sec, sel and sep, but not

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seh gene, was conducted. This strain turned out to produce moderate amounts of SEC in BHI+YE broth reaching 236 ± 47 ng/ml at 24 hours and 371 ± 36 ng/ml at 48 hours of

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cultivation. Whereas, in milk 175 strain produced significantly less SEC at 24 and 48 hours of growth (p<0.005), accounting for 2 and 8 ng/ml, respectively.

3.7. Expression of genes involved in lactose metabolism in selected S. aureus strains We used RT-qPCR to check whether enzyme II (EII), encoded by lacE gene and phospho-βgalactosidase, encoded by lacG gene were expressed in tested media. Both lacE and lacG mRNA were detectable at a low level in all strains growing in BHI+YE broth, except 613 strain which lacE RNA could not be detected in BHI+YE. In turn, lacE and lacG transcripts were detected in all strains growing in milk. Both transcripts were significantly up-regulated

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ACCEPTED MANUSCRIPT in milk cultures between 8 and 24 hours in S. aureus α41, FRI137, and 183 as compared to S.

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aureus 576, 613 and 637 (p<0.005) (Figure 4).

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3.8. Expression of seh RNA and genes involved in its regulation in selected S. aureus

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strains

To assess how SEH expression in milk is controlled, the RNA from 3 strains secreting low levels of SEH in milk and 3 strains known to produce above 100 ng/ml of SEH in milk were

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analyzed using RT-qPCR. For S. aureus α41, 576, 613, and 637 strains cultivated in BHI+YE

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broth, there was an increase of seh transcript concentration at 6 hours of growth, followed by the drop at 9 hours, and subsequent rise of seh RNA lasting until 24 hours (except 576 and 613 strains, for which the level of seh RNA was stable after 9 hours of cultivation). Whereas,

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in S. aureus FRI137 strain the level of seh RNA constantly increased, and in S. aureus 183 was stable at all time points.

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In all tested S. aureus strains seh gene transcript was detectable in milk at 5 hours of culture. A rise of relative level of seh RNA in milk for strong SEH producers was observed from 8 to

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24 hours of growth, whereas seh expression decreased between 8 and 24 hours in weak SEH producers (p<0.05).

Stabilization of pH in strong SEH producers cultured in milk at 6.68-6.88 led to significant down-regulation of seh RNA between 8 and 24 hours of culture, as compared to untreated milk (Figure 3). Reduction of milk pH to 6.0-6.1 at 24 hours resulted in an increase of seh RNA between 5 and 8 hours of culture as compared to untreated milk (p<0.05). The level of rot RNA was highest between 4.5 and 9 hours in all strains grown in BHI+YE broth, then it decreased at 9 hours and increased up to 24 hours of culture. No differences in rot RNA level between strains grown in BHI+YE broth were noted (p>0.05). A rise of a relative level of rot RNA in milk cultures for S. aureus α41, FRI137, and 183 was observed

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ACCEPTED MANUSCRIPT from 8 to 24 hours of growth, whereas rot expression decreased in S. aureus 576, 613, and 637 strains (p<0.05).

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Stabilization of pH at 6.68-6.88 in S. aureus α41, FRI137, and 183 cultured in milk led to

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significant down-regulation of rot RNA between 8 and 24 hours of culture, as compared to

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strains growth in untreated milk (p<0.05) (Figure 5). No decrease of rot RNA at every time point was noted in S. aureus 576, 613, and 637 strains cultured in milk with pH was reduced to 6.00-6.10 at 24 hour as compared to untreated milk (detailed data on rot expression was

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presented in Supplementary Table 3).

Relative level of sarA RNA was higher at 5 and 8 hours in S. aureus 576, 613, and 637 grown

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in milk if compared to S. aureus α41, FRI137, and 183. Then, it was up-regulated between 8 and 24 hours in S. aureus α41, FRI137, and 183 cultured in milk, when in S. aureus 576, 613,

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and 637 strains the level of sarA RNA decreased (p<0.05). Stabilization of pH at 6.68-6.88 in S. aureus α41, FRI137, and 183 cultured in milk led to significant down-regulation of sarA

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RNA at all time points as compared to strains growth in untreated milk, except S. aureus 183 at 8 hours of culture in which sarA level was higher in stabilized milk (p<0.05). No decrease

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of sarA RNA at every time point was noted in S. aureus 576, 613, and 637 strains cultured in milk with pH reduced to 6.00-6.10 at 24 hours as compared to untreated milk (Figure 5). Relative level of sarS RNA was higher at 5 hours in S. aureus 576 and 613 grown in milk than in remaining strains. No significant differences in sarS level between strong and weak SEH producers were found, except 8 hours of culture when higher sarS level was found in weak SEH producers (p<0.05). Both, experimental pH stabilization and decrease resulted in down-regulation of sarS level in all tested strains (Figure 5). sarT expression was not detected in any strain neither in BHI+YE broth nor in milk at any time point.

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ACCEPTED MANUSCRIPT Relative level of saeR RNA was up-regulated between 5 and 8 hours of growth in milk in all strains, except S. aureus 613 in which saeR was not changed. From 8 to 24 hours of milk

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culture saeR level was unchanged in S. aureus α41 and FRI137, increased in S. aureus 183,

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whereas in S. aureus 576, 613 and 637 was decreased. pH stabilization at 6.68-6.88 caused

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down-regulation of saeR level in S. aureus α41 and up-regulation in FRI137 and 183, when compared to untreated milk. No change of saeR transcript was noted in S. aureus 576 and 637 strains cultured in milk with experimentally reduced pH. Whereas, pH reduction resulted in

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down-regulation of saeR RNA in S. aureus 613 (Figure 6).

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No differences in profiles of agrA RNA level were noted in all strains grown in untreated milk and milk with decreased pH, except S. aureus 637 strain, in which the drop of pH caused up-regulation of agrA at 5 hours. Relative level of agrA RNA in S. aureus α41 and 183 was

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higher than in FRI137 at 8 hours when grown in milk with pH stabilized at 6.68-6.88. agrA level was down-regulated in S. aureus α41 and 183 between 8 and 24 hours of cultivation, and

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up-regulated in FRI137, when compared to untreated milk (Figure 6). A rise of relative level of hld RNA in milk cultures for S. aureus α41, FRI137, and 183 was

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observed from 8 to 24 hours of growth, whereas hld expression stayed unchanged in 576, 613, and 637 strains. pH stabilization at 6.68-6.88 led to significant down-regulation of hld RNA between 8 and 24 hours of growth, whereas pH decrease had no effect on hld RNA level (Figure 6).

4. Discussion According to the EC Regulation 1441/2007 on microbiological criteria for foodstuffs only some milk derived products are being examined for the presence of staphylococcal enterotoxins SEA-SEE (http://eur-lex.europa.eu). SEH was already implicated in food poisoning cases involving milk (Ikeda et al., 2005; Jørgensen et al., 2005). In some countries

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ACCEPTED MANUSCRIPT S. aureus lineages harboring sea and seh genes rank among major SFP-associated pathogens (Sato'o et al., 2015).

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Milk was already found unfavourable for enterotoxin expression, although data was obtained

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for SEC and SED only (Hunt et al., 2014; Tollersrud et al., 2006; Valihrach et al., 2013 and

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2014). Some authors argue for low risk of SEC production in milk (Hunt et al., 2014). SEC production in milk was also referred to its expression in microbiological media (Valihrach et al., 2013 and 2014). In a study by Valihrach et al. (2013) production of SEC was assessed in

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14 S. aureus strains grown in milk and microbial broth. The authors found SEC levels

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significantly lower in milk than in microbiological broth in all strains studied. Data obtained in our study confirms in part the trends revealed by Valihrach et al. (2013) since in most S. aureus strains enterotoxin SEA, SEC and SEH production was weaker in milk than in

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microbiological broth. However, we found a significant heterogeneity in ability of enterotoxin production in milk within S. aureus strains producing SEA and SEH. In BHI+YE broth, a

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widely used microbial broth tested here, no such disproportion in SEH production could be observed between our strains. Most of strains tested produced low levels of SEH in milk.

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Similar observation was already done on enterotoxigenic S. aureus MW2 strain where RNA levels of some enterotoxin genes including seh were significantly down-regulated in milk or cheese model as compared to microbial broth (Cretenet et al., 2011; Valihrach et al., 2014). However, three strains investigated here were much less compromised in SEH production in milk. Two of them were found to secrete higher levels of SEH to the milk than to BHI+YE broth and the third one was characterized by a moderate reduction of SEH expression not exceeding 2-fold decrease of SEH level in milk as referred to BHI+YE broth. It was not clear whether low enterotoxin production in milk was related to substances present in milk exerting an inhibitory effect on enterotoxin production or microbial broth favours enterotoxin production to the levels which cannot be observed in milk. This question was

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ACCEPTED MANUSCRIPT addressed here by studying the effect of increased concentration of BHI+YE in milk and milk in BHI+YE. As demonstrated, an increase of milk content in BHI+YE did not affect SEH

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secretion by strains producing low levels of SEH in milk, meaning milk should not contain

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substances inhibiting enterotoxin expression. Thus it could be suggested microbial broth

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includes some factors enhancing enterotoxin production not present in milk or not accessible for some S. aureus strains when grown in milk, as discussed below. To address this question we increased BHI+YE content in milk, showing a significant enhancement of SEH production

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by strains producing low SEH levels in milk. However, bacterial counts in milk supplemented

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with BHI+YE were also increased in most weak SEH producers, thus, the contribution of BHI+YE on SEH expression could not be resolved this way. Interestingly, an increase of BHI+YE content in milk resulted in a significant decrease of milk pH by weak SEH

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producers to the values noted for strong SEH producers in milk. The ability to decrease pH during growth in milk was also a common feature of three S. aureus strains secreting high

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amounts of SEH into milk, and was not observed in any strain producing low levels of SEH in milk. Since our data suggested that pH of the environment contributes to enterotoxin

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expression we tested the effect of pH on SEH production both in BHI+YE and milk. We demonstrated pH reduction in milk to the values noted for strong SEH producers, led to an increase of SEH production by weak enterotoxin producers. Similarly, stabilization of pH in strong SEH producers at value close to that of raw milk resulted in a significant decrease of SEH production. Some authors found neutral pH as optimal for enterotoxin production (Hennekinne et al., 2012). However, a significant number of studies were performed on a single S. aureus strain, what does not allow to draw conclusions on enterotoxin production within S. aureus population. pH-dependence of SEH production was assessed on S. aureus FRI-569 strain by Su and Wong (1998). The highest SEH production was found at pH around 7.00. Interestingly, in a series of broths in which pH was not controlled pH values after 24-

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ACCEPTED MANUSCRIPT hours culture increased to 7.40-8.30 and the SEH levels were lowest (Su and Wong, 1998). Moreover, Cretenet et al. (2011) demonstrate SEH RNA was up-regulated in S. aureus MW2

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in cheese model when the pH was decreased to 5.2-5.3. More is known about pH-dependence

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of expression of enterotoxins other than SEH. According to a study performed on two SEC-

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producing S. aureus strains the optimal pH for SEC production in milk was between 5.5 and 6.0 (Hunt et al., 2014). The effect of pH on SEC production in microbial broth was already determined on S. aureus FRI137, one of strains investigated here. The optimal pH for SEC

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expression was determined to be 5.50-6.50 (Genigeorgis et al., 1971). Thus, our data together

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with some previous observations suggests that pH decrease seems to enhance enterotoxin production both in milk and microbial broths. However, ability to decrease the milk pH was not common to all S. aureus strains. It can be hypothesized that strains able to decrease milk

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pH should have more efficient mechanisms of lactose utilization. We assessed two elements of S. aureus lactose operon, i.e., lacE encoding EII enzyme involved in lactose transport and

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phosphorylation and lacG, encoding staphylococcal 6-phospho-β-galactosidase that hydrolyses lactose into glucose and galactose-6-phosphate (Staedtler et al., 1995). We found

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that both enzymes were significantly up-regulated during growth of S. aureus strains in milk as compared to BHI+YE where their expression could be barely detected. In three strong SEH producers both lacE and lacG RNA were significantly up-regulated as compared to strains producing low levels of SEH in milk. Bacteria respond to the environment by altering their gene expression, what is achieved by interaction of global regulatory loci such as agr, sar, sigB, rot, arlR/S, svrA and saeR/S (Goerke and Wolz, 2004; Hennekinne et al., 2012). It has been shown, that growth in milk alters some S. aureus properties, including phagocytosis resistance and virulence (Sandgren et al., 1991; Sutra et al., 1990). Recently, Sato'o et al. (2015), using mutational analysis of global regulators, found that repressor of toxin (Rot) unexpectedly functions as a stimulator of SEH

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ACCEPTED MANUSCRIPT production. This report indicates Rot, SaeR, SarT and SarA as SEH expression inducers, and SarS as SEH repressor. Agr acts indirectly repressing Rot and SarT and up-regulating SeaR.

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We investigated the RNA levels of potential SEH expression regulators suggested by Sato'o et

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al. (2015) and elements of Agr system, i.e., hld and agrA in 3 weak and 3 strong SEH

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producers during growth in milk. We found hld, rot and sarA RNAs significantly up-regulated in strong, but not in weak SEH producers, during growth in milk. High level of regulatory genes expression was related to pH decrease by strong SEH producers, since pH stabilization

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at a value of raw milk resulted in a significant decrease of hld, rot and sarA RNAs,

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simultaneously accompanied with a decrease of SEH at both RNA and protein level. Cretenet et al. (2011) studied enterotoxin expression in chemically defined medium and model cheese matrix by S. aureus MW2 strain. They have shown that presence of Lactococcus lactis

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decreased the culture pH to c.a. 5.0, which was related to significant alteration of expression of virulence genes and regulators (Cretenet et al., 2011). Expression of rot and seh genes in S.

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aureus MW2 was found to be up-regulated at low pH what agrees with our results. In turn, Weinrick et al. (2004) using microarray analysis, RT-qPCR and Northern blotting

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demonstrated that rot RNA was down-regulated at pH 5.5 by S. aureus RN6734 strain. hld, agrA and sarA were shown to be down-regulated at low pH in S. aureus MW2 strain (Cretenet et al., 2011). sarA and hld were up-regulated in our high SEH-producing strains, and their level decreased when pH was stabilized. Also in weak SEH producers experimentally-induced pH drop prevented decrease of sarA and hld transcripts. Effect of low pH on agrA was less clear, but Cretenet et al. (2011) suggest that effect of pH on agrA and hld can be independent. Since Rot was shown to be repressed at a level of translation by hld transcript the meaning of simultaneous increase of rot and hld RNA on SEH expression is not clear (Benson et al., 2011). Interestingly, pH reduction in our strains producing low SEH levels in milk could not induce neither SEH nor the regulatory genes to the levels observed in

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ACCEPTED MANUSCRIPT the same strains grown in BHI+YE broth, nor to levels expressed by strong SEH producers. It may be suggested that ability to decrease pH contributes to SEH expression impacting some

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enterotoxin regulatory genes in all strains tested here, but strong SEH producers should have

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additional mechanisms promoting high-level enterotoxin expression.

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Some strains studied here harboured other enterotoxin genes besides seh, creating an opportunity to refer their production patterns to that of SEH. Interestingly, a comparison of SEC production in BHI+YE and milk by S. aureus FRI137 strain revealed disparate trend to

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SEH expression. When SEH level was not changed at 24 hours being up-regulated at 48 hours

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of milk culture, SEC production by the S. aureus FRI137 strain was significantly weaker in milk than in BHI+YE irrespective of culture duration. This seems to support the thesis of different regulation mechanisms of these two toxins, as also suggested by Sato'o et al. (2015).

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In turn, the level of SEA production was not changed significantly between BHI+YE and milk in a part of strains. In strong enterotoxin producer, i.e., S. aureus α41 SEA was up-

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regulated in milk, while in other strains SEA level was equal or lower in milk than in BHI+YE. However, SEA decrease was always much less pronounced than that of SEH.

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Cretenet et al. (2011) already suggested environment acidification as a factor accounting for leading role of SEA in SFP epidemiology. Taking together, the difference between weak and strong SEH producers seems to be in part related to the ability to decrease pH of the environment by S. aureus. Microbial broth tested here allowed each S. aureus strain to decrease pH during growth which was related to high level of SEH expression and moderate differences in level of secreted SEH between strains. Milk seems not to offer the same opportunity for a significant part of tested S. aureus strains. Certain S. aureus strains growing in milk seem not to be restricted in SEH and SEA expression, unlike SEC which remained down-regulated in milk. Moreover, the level of SEC seems to be down-regulated in milk also in S. aureus producing high levels of SEH in milk.

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ACCEPTED MANUSCRIPT Therefore low food safety risk related to strains producing SEC in milk as suggested previously may not pertain to certain SEA and SEH-producing strains. Occurrence of

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staphylococci carrying sea and seh genes may pose an increased risk for enterotoxin

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production in milk-related products.

5. Acknowledgements

Project was financially supported by National Science Centre, Poland, on the basis of decision

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DEC-2012/05/B/NZ9/03343.

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ACCEPTED MANUSCRIPT Figure captions Figure 1. A. Effect of increase of milk concentration to 1, 5, and 10% in BHI+YE broth on

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SEH production at 24 and 48 hours of culture by S. aureus 576, 613, and 637 representing

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weak SEH producers in milk. B. Effect of increase of BHI+YE broth concentration to 1, 5,

637 representing weak SEH producers in milk.

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and 10% in milk on SEH production at 24 and 48 hours of culture by S. aureus 576, 613, and

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Figure 2. Effect of pH on production of SEH in milk by S. aureus α41, FRI137 and 183 secreting to milk the highest levels of SEH (strong SEH producers) and S. aureus 576, 613,

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and 637, selected from 17 strains producing low levels of SEH in milk (weak SEH producers). Stabilization of pH in milk cultures of strong SEH producers was performed using 2 M

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NaOH. Reduction of pH in milk cultures of weak SEH producers was achieved using 25%

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HCl at pH of 6.50 (2 hours of culture), pH 6.10 (at 5 hours), pH 5.80 (at 8 hours), pH 5.30 (at

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24 hours), and pH 5.10 (at 30 hours). Effect of pH on production of SEH in BHI+YE broth by strong SEH producers, i.e., S. aureus α41, FRI137 and 183. Stabilization of pH at 7.27-7.50 at 24 hours and 7.57-7.71 at 48-hours of cultures in BHI+YE broth was achieved using 1.5 M

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Tris-HCl pH 7.00.

Figure 3. Effect of pH on expression of seh RNA by strong SEH producers in milk i.e., S. aureus α41, FRI137 and 183, and weak SEH producers, i.e., S. aureus 576, 613, and 637. Stabilization of pH in milk cultures of strong SEH producers was performed using 2 M NaOH. Reduction of pH in milk cultures of weak SEH producers was achieved using 25% HCl at pH of 6.50 (at 2 hours of culture), pH 6.10 (at 5 hours), pH 5.80 (at 8 hours), pH 5.30 (at 24 hours), and pH 5.10 (at 30 hours).

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ACCEPTED MANUSCRIPT Figure 4. Expression of lacE and lacG genes involved in lactose metabolism in strong SEH producers in milk i.e., S. aureus α41, FRI137 and 183, and weak SEH producers, i.e., S.

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aureus 576, 613, and 637.

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Figure 5. Effect of pH on RNA levels of rot, sarA, and sarS genes involved in regulation of

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SEH in strong SEH producers in milk i.e., S. aureus α41, FRI137 and 183, and weak SEH producers, i.e., S. aureus 576, 613, and 637. Stabilization of pH in milk cultures of strong

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SEH producers was performed using 2 M NaOH. Reduction of pH in milk cultures of weak SEH producers was achieved using 25% HCl at pH of 6.50 (at 2 hours of culture), pH 6.10 (at

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5 hours), pH 5.80 (at 8 hours), pH 5.30 (at 24 hours), and pH 5.10 (at 30 hours). Figure 6. Expression of genes involved in regulation of SEH in strong SEH producers in milk

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i.e., S. aureus α41, FRI137 and 183, and weak SEH producers, i.e., S. aureus 576, 613, and

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637.

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ACCEPTED MANUSCRIPT Tables Table 1. S. aureus strains used in this study Enterotoxin gene content

Source

FRI913

sea, sec, see, sek, sel, tst

reference strain

FRI137

sec, seh, sel, egc2

reference strain

CCM5757

seb, sek

A900322

sep, egc

FRI1151m

sed, selj, ser

α41

sea, seh, sek

74

seh

183

seh

184

seh

187

seh

188

seh

189

seh

190

sea, seh, sek

human

218

seh

food

258

sea, seh, sek, sel

human

500

sea, seh, sek

human

567

sea, seh

food

sea, seh, sek

human

sea, seh, sek

human

613

sea, seh, sek, egc

human

637

sea, seh, sek

human

663

sea, seh, sek

human

666

sea, seh, egc, sek, seq

human

675

seh, sek,

human

175

sec, sel, sep

food

587

IP

SC R

reference strain reference strain

NU

reference strain human

CE P

TE

D

MA

food

AC

576

T

Strain

food food food food food

31

ACCEPTED MANUSCRIPT Table 2. Primers used for RT-qPCR. Amplicon Source length

Target Nucleotide sequence (5`-3`) F: CCTCGCAACTGATAATCCTTATG

T

agrA

127 bp

saeR

sarA

sarS

sarT

seh

R: CATCAAATTCACGTTGTCCG

F: TGCAGTATTTCAACCACACAC R: GTATCGTTAATGCGCCAGT

F: GACCTCTGTGCTTAGCTGTAATAGC R: GCGAACATGCAACGTCAAG F: TAGTCATATCCCCAAACTT R: CCATTTACGCCTTAACTTTA F: TTGCTTTGAGTTGTTATCAATGG R: TTTCTCTTTGTTTTCGCTGATGT F: CACCATAAATACCCTCAAACTGTTAGAG R: TCATCTTCAGTTGAGCGTTCTTTT F: AGTATTAACTACATATGAGCTGCG R: CTCTACATTTATTCAAGTAACCCT F: TGAATGTCTATATGGAGGTACAAC R: CTACCCAAACATTAGCACCAA

Valihrach et al., 2014

90 bp

Valihrach et al., 2014

356 bp

Sharer et al., 2003

99 bp

This study

140 bp

Sato'o et al., 2015

121 bp

Duquenne et al., 2010

298 bp

Goerke et al., 2005

124 bp

Valihrach et al., 2014

86 bp

Bæk et al., 2013

215 bp

Xue et al., 2014

80 bp

Derzelle et al., 2009

SC R

NU

F: GAGGTTACGATGGAGAATCTG

MA

rpoB

R: GCTGTTGCGAATGACTAAATCTAA

D

rot

F: CAGGAACGACAGCGAAATCTT

TE

lacG

R: GTGAATTTGTTCACTGTGTCGAT

CE P

lacE

F: TAAGGAAGGAGTGATTTCAATGG

AC

hld

IP

R: ACGAATTTCACTGCCTAATTTGA

32

ACCEPTED MANUSCRIPT Table 3. Growth and pH values of S. aureus strains in BHI+YE broth and milk. BHI+YE broth 48 hours

24 hours

Log CFU/ml

pH

5.80

9.2 ± 0.2

5.10

8.6 ± 0.4

6.36

9.3 ± 0.3

5.26

8.3 ± 0.1

6.55

8.8 ± 0.1

6.52

9.3 ± 0.4

6.34

9.4 ± 0.2

5.96

pH

Log CFU/ml

pH

Log CFU/ml

α41

9.0 ± 0.7

5.84

9.5 ± 0.3

7.88

9.1 ± 0.4

FRI137

8.4 ± 0.5

5.58

9.3 ± 0.4

7.44

74

8.2 ± 0.8

5.72

8.4 ± 0.1

7.63

183

8.4 ± 0.6

5.93

9.2 ± 0.3

8.14

184

8.3 ± 0.5

6.09

8.5 ± 0.4

187

9.0 ± 0.2

5.54

9.3 ± 0.3

188

8.4 ± 0.7

5.43

8.6 ± 0.4

189

8.2 ± 0.6

5.39

190

7.3 ± 0.1

218

pH

8.7 ± 0.3

6.54

8.8 ± 0.1

6.59

7.6

9.0 ± 0.1

6.58

9.4 ± 0.1

6.53

7.66

8.8 ± 0.1

6.62

9.3 ± 0.1

6.63

8.6 ± 0.5

6.71

8.2 ± 0.5

6.65

9.1 ± 0.1

6.55

5.41

8.4 ± 0.3

7.34

8.9 ± 0.6

6.50

9.1 ± 0.1

6.49

9.2 ± 0.6

5.40

9.6 ± 0.1

7.17

8.8 ± 0.1

6.55

8.9 ± 0.1

6.62

258

8.5 ± 0.2

5.45

9.5 ± 0.2

7.62

8.2 ± 0.3

6.62

8.3 ± 0.2

6.57

500

8.7 ± 0.2

5.45

8.9 ± 0.3

7.79

8.6 ± 0.3

6.60

9.2 ± 0.2

6.57

567

8.1 ± 0.1

5.44

8.9 ± 0.6

7.30

8.6 ± 0.5

6.53

8.7 ± 0.1

6.60

576

8.2 ± 0.4

5.32

8.4 ± 0.4

7.01

8.7 ± 0.4

6.45

9.1 ± 0.2

6.42

587

7.6 ± 0.4

5.37

8.2 ± 0.2

6.35

8.9 ± 0.2

6.48

9.1 ± 0.0

6.49

613

8.0 ± 0.8

5.50

9.2 ± 0.1

7.45

8.4 ± 0.7

6.43

9.5 ± 0.3

6.43

7.6 ± 0.4

5.41

9.6 ± 0.1

7.99

8.8 ± 0.5

6.56

9.1 ± 0.1

6.58

663

AC

CE P

TE

MA

7.81

D

NU

SC R

IP

Log CFU/ml

Strain

48 hours

T

24 hours

Milk

8.2 ± 0.1

5.46

8.5 ± 0.3

7.64

8.7 ± 0.1

6.65

8.7 ± 0.2

6.57

666

8.4 ± 0.2

5.61

9.6 ± 0.4

7.42

8.7 ± 0.2

6.56

8.9 ± 0.2

6.60

675

7.9 ± 0.2

5.38

8.1 ± 0.3

7.78

8.7 ± 0.2

6.58

9.1 ± 0.1

6.65

637

33

ACCEPTED MANUSCRIPT Table 4. Production of SEH and SEA by S. aureus in BHI+YE broth and milk. BHI+YE broth

1292 ± 145 151 ± 28

SEA [ng/ml]

SEH [ng/ml]

2866 ± 135

303 ± 43

3029 ± 253

SEA [ng/ml]

SEH [ng/ml]

SEA [ng/ml]

244 ± 56

4246 ± 282

283 ± 32

328 ± 4

-

894 ± 25

1±1

-

6±3

-

107 ± 22

-

320 ± 40

-

18 ± 3

-

12 ± 4

-

-

8±1

-

9±1

-

-

2±1

-

8±2

-

-

6±1

-

9±1

-

T

SEH [ng/ml]

IP

SEA [ng/ml]

48 hours

289 ± 21

-

406 ± 9

-

74

113 ± 28

-

1687 ± 47

-

183

141 ± 28

-

577 ± 21

-

184

218 ± 16

-

1453 ± 131

-

187

249 ± 17

-

1118 ± 131

188

183 ± 33

-

689 ± 65

189

150 ± 28

-

170 ± 8

190

506 ± 27

35 ± 1

3067 ± 159

126 ± 7

13 ± 2

23 ± 3

13 ± 5

39 ± 2

218

199 ± 29

-

3151 ± 108

-

12 ± 3

-

10 ± 3

-

258

679 ± 52

40 ± 3

2120 ± 96

111 ± 8

1±1

5±1

5±2

7±1

500

575 ± 36

35 ± 2

4158 ± 106

101 ± 16

6±0

20 ± 2

7±2

32 ± 4

567

333 ± 41

26 ± 3

1615 ± 94

96 ± 8

18 ± 3

2±1

19 ± 1

27 ± 4

576

262 ± 21

28 ± 4

294 ± 43

31 ± 6

9±2

26 ± 1

15 ± 2

32 ± 4

587

95 ± 40

24 ± 1

183 ± 46

44 ± 1

20 ± 1

18 ± 3

18 ± 0

37 ± 3

613

306 ± 54

37 ± 7

386 ± 33

58 ± 5

23 ± 6

37 ± 4

36 ± 7

59 ± 5

637

347 ± 26

0

407 ± 43

0

20 ± 4

0

32 ± 6

0

663

359 ± 30

28 ± 1

3986 ± 119

120 ± 6

10 ± 1

8±2

8±0

19 ± 1

666

250 ± 27

29 ± 3

3516 ± 112

104 ± 9

9±1

7±0

15 ± 2

20 ± 1

675

130 ± 14

-

1518 ± 135

-

13 ± 0

-

19 ± 0

-

AC

CE P

D

MA

FRI137

TE

α41

SEH [ng/ml]

24 hours

SC R

Strain

48 hours

NU

24 hours

Milk

-

34

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Figure 1

35

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 2

36

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

Figure 3

37

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

Figure 4

38

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 5

39

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

Figure 6

40

ACCEPTED MANUSCRIPT

T

IP SC R NU MA D TE CE P AC

    

Highlights Most S. aureus express SEH at lower level in milk than in BHI+YE All S. aureus decrease pH in BHI+YE, but only some S. aureus decrease milk pH High SEH levels are secreted to milk only by S. aureus strains decreasing milk pH Lactose metabolism genes are up-regulated in milk in strong SEH producers hld, rot and sarA RNA are up-regulated in milk in strong SEH producers

41