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International Dairy Journal 30 (2013) 103e107

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International Dairy Journal

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Production of staphylococcal enterotoxin C in milk

Lukas Valihrach*, Babek Alibayov, Katerina DemnerovaInstitute of Chemical Technology, Faculty of Food and Biochemical Technology, Department of Biochemistry and Microbiology, Technicka 5, Prague 166 28, Czech Republic

a r t i c l e i n f o

Article history:Received 12 June 2012Received in revised form12 January 2013Accepted 14 January 2013

* Corresponding author. Tel.: þ420 220445198.E-mail address: valihral@vscht.cz (L. Valihrach).

0958-6946/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.idairyj.2013.01.003

a b s t r a c t

Staphylococcus aureus is a common pathogen contaminating milk-related environments where it is ableto produce thermostable enterotoxins that can cause gastrointestinal diseases. The aim of this study wasto investigate production of enterotoxin C (SEC) in milk. SEC production decreased significantly(P < 0.0001) in milk compared with production in a laboratory media. Different types of milk varying infat content or heat treatment (powdered, pasteurised and ultrahigh temperature treated milk) did notinfluence SEC production. Minor variances were observed when milk of different origins was used (cows’,goats’ and ewes’ milk). Sub-optimal growth temperature reduced SEC production. At 10 �C the pro-duction was almost ceased. Overall, SEC production in milk environments was very low. Under commonstorage conditions (25 and 10 �C), none of the fourteen tested strains were able to produce amounts ofSEC that would be sufficient to cause food poisoning.

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1. Introduction

Staphylococcus aureus is an important pathogen producingsubstances that define its pathogenicity. Production of thermo-stable enterotoxins causing gastrointestinal diseases is, in terms offood microbiology, the most important feature of some S. aureusstrains. In total 21 different types of enterotoxin have been iden-tified (Schelin et al., 2011). Prevalence of enterotoxin genes inS. aureus strains is variable and depends often on the environmentfrom which a particular strain was isolated. Concerning milk-related environments, the gene encoding enterotoxin C (sec) wasreported several times as the most frequent gene for enterotoxinpresent in S. aureus (Foschino, Invernizzi, Barucco, & Stradiotto,2002; Guitierrez, Menes, Garcia, Moreno, & Bergdoll, 1982; Hajek,1978; Scherrer, Corti, Muehlherr, Zweifel, & Stephan, 2004;Stephan, Annemuller, Hassan, & Lammler, 2001).

Although the existence of S. aureus strains capable of producingenterotoxin C (SEC) in milk is known, there is lack of informationdescribing its actual production. The vast majority of studiesdescribing SEC production were conducted in laboratory mediausing only one or two S. aureus strains, mostly well-characterisedones like FRI 137 and 361. The influence of the following factorswas examined: types of laboratory media, incubation temperatureand time, pH, NaCl content, Mg2þ and Fe2þ concentration, presenceof glucose, incubation atmosphere, shaking conditions or presence

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of competitive microorganisms (Smith, Buchanan, & Palumbo,1983). Only a few studies examine SEC production in food matri-ces including various processed meats, various salads and dress-ings, shrimp, Burgos cheese and cooked egg-noodles (Beckers,Vanleusden, & Tips, 1985; Genigeorgis, Savoukidis, & Martin,1971; Gockler, Notermans, & Kramer, 1988; Morita & Woodburn,1983; Otero, Garcia, Garcia, Prieto, & Moreno, 1988; Scheusner &Harmon, 1973). There is only one report describing SEC produc-tion in milk (Otero, Garcia, Garcia, & Moreno, 1987). However, thisstudy focused on ewes’milk, the use of which is limited in the foodindustry.

The objective of this study was to gain information on SECproduction in milk environments. We analysed a large number ofstrains of S. aureus, including two frequently used strains FRI 137and 361. As a growth medium we used different types of milkvarying in origin, heat treatment or fat content. The influence oftime and temperature was investigated as well.

2. Material and methods

2.1. Staphylococcus aureus strains

Fourteen S. aureus strains isolated from different sources wereused in this study (Table 1). There were bacteria obtained from TheCzech Collection of Microorganisms (Brno, Czech Republic) and TheNational Institute of Public Health in Brno, Czech Republic (kindlyprovided by Dr. Renata Karpiskova). Strains were characterised byspecies-specific PCR (Martineau, Picard, Roy, Ouellete, & Bergeron,1998) and genes encoding enterotoxins AeJ were determined

Table 1S. aureus strains used in this study to determine enterotoxin C production in variousdairy products.

Strains Origin Enterotoxin genesa

SA 720 Faecesb sea, sec, seeSA 740 Pork hamb secSA 817 Spinachb secSA 1117 Confectionery productb sec, seg, seiSA 1141 Sausageb secSA 1178 Camembertb sec, seg, seiSA 1189 Ewes’ milkb secSA 1247 Chicken salamib sea, sec, seg, seiSA 1442 Fish productb sec, sed, seg, sei, sejSA 1607 Fish filletsb secSA 1612 Hard cheeseb sea, seb, sec, seeSA 1737 Cows’ milkb sec, seg, seiFRI 137 Leg abscessc sec, seg, seh, seiFRI 361 Cooked chickend sec, sed, seg, sei, sej

a Genes encoding enterotoxins AeJ (i.e., enterotoxin A ¼ sea etc.) detected usinga protocol by Lovseth et al. (2004).

b Isolated in the Czech Republic and provided by The National Institute of PublicHealth in Brno, Czech Republic.

c Isolated in Albert Merritt Billings Hospital, University of Chicago, USA in 1933(Bergdoll, Borja, & Avena, 1965).

d Isolated in England in 1962 (Bergdoll et al., 1965).

Table 2SECa production by various strains of S. aureus in TSBb and milk (culture at 25 �C for48 h).

Strains SEC production (ng mL�1 � SD) Ratioc

TSB (pH 6.8) Cows’ milk (UHT processed, 1.5% fat, pH 6.8)

SA 720 57.60 � 2.15 0.40 � 0.05 144SA 740 73.25 � 7.66 0.12 � 0.03 612SA 817 14.42 � 1.59 0.22 � 0.05 65SA 1117 6.41 � 2.77 0.21 � 0.00 30SA 1141 46.51 � 3.09 0.13 � 0.05 346SA 1178 1.16 � 0.17 0.61 � 0.01 2SA 1189 5.37 � 0.47 0.09 � 0.01 59SA 1247 65.76 � 5.63 0.06 � 0.01 1102SA 1442 10.16 � 3.65 0.16 � 0.01 63SA 1607 7.76 � 0.03 0.43 � 0.03 18SA 1612 22.55 � 5.86 0.06 � 0.00 393SA 1737 0.23 � 0.01 0.02 � 0.00 10SA 137 5.05 � 1.56 0.26 � 0.06 19SA 361 6.30 � 0.56 0.16 � 0.00 39

a Enterotoxin C.b Tryptic Soya Broth, Merck, Darmstadt, Germany.c Ratio is SEC production in TSB/SEC production in milk.

L. Valihrach et al. / International Dairy Journal 30 (2013) 103e107104

using a method previously published by Lovseth, Loncarevic, andBerdal (2004). Strains carrying the gene for SEC were further con-firmed by real-time PCR (Letertre, Perelle, Dilasser, & Fach, 2003).

2.2. Culture conditions

In general, 50 mL of pre-warmed or pre-cooled media wasinoculated with a specific volume of pre-culture to reach about5 � 105 as starting number of cells per mL. As pre-culture 18-h-oldculture incubated in 5 mL of TSB (Tryptic Soya Broth, Merck,Darmstadt, Germany) at 37 �C with shaking (130 rpm) was used.Before inoculation, the pre-culture was washed twice with a salinesolution to remove all residual enterotoxins (repeated cen-trifugation at 12,500 � g for 5 min and resuspension in a salinesolution). Samples for SEC detection were collected during eachexperiment; 1 mL of a medium was harvested, centrifuged(12,500 � g for 5 min) and supernatant stored at �80 �C untilanalysed. The number of cells permL (cfumL�1) was determined byperforming serial dilution and plating on TSA (Tryptic Soya Agar,Merck). All experiments were carried out in duplicate.

In the first experiment, SEC production in milk and laboratorymedium was compared. Ultrahigh temperature (UHT) processedcows’milk,1.5% fat (purchased retail in the Czech Republic), and TSBas a common type of laboratory mediumwas used for this purpose.Cultures were incubated in stationary mode at 25 �C for 48 h.

The influence of various types of milk on SEC production wasexamined in (i) UHT treated cows’milk with different fat content e0.5, 1.5 and 3.5%, (ii) in cows’ milk with 1.5% fat content varying inmethod of heat treatment, (iii) reconstituted powdered, pas-teurised and UHT treated milk, and (iv) in milk from differentproducers: organic cows’ milk, goats’ milk and ewes’ milk (allpurchased retail in the Czech Republic). Cultures were incubated instationary mode at 25 �C for 48 h.

The effect of incubation temperature and time was investigatedin UHT processed cows’milk (1.5% fat) at 37, 25 and 10 �C for 168 h.Cultures were incubated in stationary mode. Samples were taken at0, 24, 48, 72 and 168 h.

2.3. Enzyme linked immunosorbent assay

SEC detection was performed in cultivation media according toan enzyme linked immunosorbent assay (ELISA) protocol described

by Wallin-Carlquist, Marta, Borch, and Radstrom (2010). Commer-cially available antibodies and enterotoxin were used (SLCI111,SBCC111, CT111; Toxin Technology, Inc., Sarasota, Florida, USA).Serial dilution of a SEC standardwas performed in the samemediumaswas used in given experiment. The range of the serial dilutionwasfrom 10 to 2500 pg mL�1. Positive controls and blank samples wereincluded in each analysis. The detection limit was determined as5 pg mL�1 and the limit of quantification as 10 pg mL�1.

2.4. Statistical analysis

The basic statistical analysis was performed in Microsoft Excel(calculation of means, standard deviations, regression analysis etc.).Advanced statistical tasks were carried out with help of softwareGraphPad InStat (GraphPad Software, La Jolla, California, USA).Hypotheses were tested by following statistical test: ManneWhitney test (comparison of SEC production in TSB and milk) andrepeatedmeasures one-way analysis of variance (ANOVA; influenceof different types of milk on SEC production).

3. Results

Species-specific PCR confirmed all strains to be S. aureus. Mul-tiplex PCR detected heterogeneous distribution of enterotoxingenes in tested strains (Table 1). The most common genotype wassecþ (in 5 strains), followed by sec, seg, seiþ (in 3 strains) and sec,sed, seg, sei, sejþ (in 2 strains).

SEC production was measured in the 14 S. aureus strains in milkand laboratory medium after 48 h of cultivation at 25 �C (Table 2).Results showed a significant decrease in SEC production in milkcompared to TSB (P < 0.0001). However, the level of decrease(expressedas a ratio¼ SECproduction inTSB/SECproduction inmilk)varied among strains e 1102 � lower by SA 1247, versus 2 � by SA1178. Various levels of SEC production among strains in particularmediumwere also observed (ranging from 0.23 to 73.25 ng mL�1 ofSEC in TSB and 0.02e0.61 ng mL�1 in milk). Strains grew in bothmedia and achieved high cell density (Supplemental Table S1). Celldensity was on average higher in TSB: 2.07 (�0.71) � 109 cfu mL�1

compared with 7.24 (�2.94) � 108 cfu mL�1 in milk.The influence of cultivation medium was tested using a limited

number of S. aureus strains during cultivation at 25 �C for 48 h. Thestrains from a dairy environment with different levels of SEC pro-duction in milk (achieved in the previous experiment) were chosen,

Table 3S. aureus enterotoxin C (SEC) production by selected strains in various types of milk (cultivation at 25 �C for 48 h).

Medium SEC production (ng mL�1 � SD) by different strains

SA 1178 SA 1189 SA 1737 FRI 137 FRI 361

UHT treated cows’ milk e 0.5% fat 0.47 � 0.01 0.04 � 0.01 0.02 � 0.01 0.30 � 0.01 0.14 � 0.01UHT treated cows’ milk e 1.5% fat 0.61 � 0.01 0.09 � 0.01 0.02 � 0.01 0.26 � 0.06 0.16 � 0.01UHT treated cows’ milk e 3.5% fat 0.70 � 0.27 0.03 � 0.01 0.02 � 0.01 0.35 � 0.05 0.11 � 0.01Reconstituted powdered cows’ milk e 1.5% fat 0.07 � 0.03 0.07 � 0.02 0.05 � 0.04 0.05 � 0.01 0.05 � 0.01Pasteurised cows’ milk e 1.5% fat 0.32 � 0.11 0.09 � 0.03 0.02 � 0.01 0.19 � 0.05 0.08 � 0.01UHT treated cows’ milk e 1.5% fat 0.61 � 0.01 0.09 � 0.01 0.02 � 0.01 0.26 � 0.06 0.16 � 0.01UHT treated cows’ milk (organic)a 0.15 � 0.01 0.02 � 0.01 0.02 � 0.01 0.30 � 0.02 0.09 � 0.01UHT treated goats’ milka 0.96 � 0.14 0.01 � 0.01 0.02 � 0.01 0.20 � 0.03 0.05 � 0.01UHT treated ewes’ milka 1.52 � 0.16 0.15 � 0.08 0.03 � 0.01 0.50 � 0.12 0.07 � 0.01

a Fat content unstandardised or undefined.

L. Valihrach et al. / International Dairy Journal 30 (2013) 103e107 105

overall, five strains were tested e high-producing strain SA 1178,medium producing strain SA 1189, low producing strain SA 1737, aswell as two most studied strains FRI 137 and 361. SEC production(Table 3) as well as cell growth (Supplemental Table S2) was com-parable among various types of milk. No significant effect of fat con-tent, type of heat treatment or origin of milk on SEC production wasobserved (P > 0.05). It was noted some trends in Table 3 e elevated

Fig. 1. Growth (closed symbols, solid lines) of selected strains of S. aureus and production of25 �C (A,>) and 10 �C (:,6). The six strains shown in separate frames had SEC productionare not indicated due to their low values and for better readability of different marks used

SEC production in milk with higher fat content (SA 1178, FRI 137, FRI361) or higher SEC production in goats’ and ewes’ milk compared tocows’ milk (SA 1178). However, all these differences are statisticallyinsignificant (P > 0.05).

The effect of temperature was investigated in all strains at 37, 25and 10 �C for 168 h. Results are divided into two graphs: six strainswith SEC production higher than 500 pg mL�1 are shown in Fig. 1

staphylococcal enterotoxin C (SEC; open symbols, dashed lines) in milk at 37 �C (-, ,),higher than 500 pg mL�1. All experiments were performed in two replicates. Error barsin graphs.

L. Valihrach et al. / International Dairy Journal 30 (2013) 103e107106

and eight strains with SEC production of less than 500 pg mL�1 areshown in Supplemental Fig. S1. In general, highest SEC productionwas at 37 �C, although several strains (SA 817, 1141, 1442, 1607 and1737) produced a comparable or even higher amount of SEC at25 �C. In most cases, SEC concentration in samples incubated at10 �C did not reach the ELISA detection limit (5 pg mL�1) even after168 h of culture. After further culture at 10 �C for an additional 14days (in total 504 h); SEC productionwas still not detectable or wasvery low (less than 100 pg mL�1; data not shown). The most pro-ductive strain was FRI 137, where SEC production increased almostlinearly during incubation period (2294, 4833, 7656 and12,671 pg mL�1 after 24, 48, 72, 168 h of incubation at 37 �C,respectively). In general, all strains were capable of growing at alltested incubation temperatures. The only exception was the strainSA 1612 which showed no growth at 10 �C (Fig. 1). Fastest growthwas observed at 37 �C (the optimal temperature for this bacterium),the lowest at 10 �C.

4. Discussion

Several recently published studies emphasised the necessity ofusing food matrices instead of laboratory media to test the capa-bility of S. aureus strains to produce enterotoxins (Cretenet et al.,2011; Marta, Wallin-Carlquist, Schelin, Borch, & Radstrom, 2011;Wallin-Carlquist et al., 2010). Although milk is commonly con-taminated by S. aureus, studies of its behaviour (especially enter-otoxin production) are scarce; three reports describe enterotoxin Aproduction in cows’ milk (Donnelly, Leslie, & Black, 1968; Fujikawa& Morozumi, 2006; Tatini, Jezeski, Olson, & Casman, 1971), onereport describes SEC production in ewes’ milk (Otero et al., 1987)and one report describes enterotoxin D production in cows’ milk(Medvedova, Valik, Sirotna, & Liptakova, 2009). These studies wereconducted with limited number of strains (maximum of three byOtero et al., 1987).

In our study we used fourteen strains of S. aureus, almostentirely isolated from food sources (Table 1). When we comparedSEC production in a laboratory medium and milk (Table 2), theresults obtained were undisputable. SEC production in milk wassignificantly decreased (P < 0.0001), which supports the currentopinion that enterotoxin production is substantially altered bya food matrix (Schelin et al., 2011). A similar observation waspublished by Otero et al. (1987), when comparing SEC productionin a laboratory medium and ewes’ milk.

Results also revealed a large heterogeneity in the level of SECproduction among the individual strains, which has not beenreported previously. This finding is probably associated with di-versity of SEC. Until now, 7 different SEC subtypes have beenidentified and it is highly probable that there are others (Edwardset al., 1997; Marr et al., 1993). The genes for SEC subtypes arelocated in different loci in the S. aureus genome (Schelin et al., 2011)therefore it can be assumed that there are also different mecha-nisms of its regulation, other than the main Agr-regulatory system.Therefore, different levels of SEC production could also occur.

Further, we tested if various types of milk may affect SEC pro-duction (Table 3). We found no significant influence on SEC pro-duction of fat content, method of heat treatment or origin of milk(P > 0.05). Similar results were observed by Tatini et al. (1971),where the effect of different heat treatments of cows’ milk onenterotoxin A production was investigated. We observed onlya minor change in SEC production among milk with different fatcontent and among milk from different species (cows’, goats’ andewes’ milk). However, these differences were statistically insignif-icant (P> 0.05). On the other hand, it is indisputable that thereweresome strain-dependent differences, which could be more pro-nounced in another experimental setting (higher temperature,

longer cultivation period etc.). Worth mentioning is also the higherproduction (even if still very low) of strain SA 1189 in ewes’ milkcomparedwith other types ofmilk in viewof the fact that this strainwas originally isolated from ewes’milk (Table 1). This may indicatea better adaptation of the strain on this host its environment.

The influence of incubation time and temperature on SEC pro-duction in milk was tested as well (Fig. 1, Supplemental Fig. S1). Asa cultivation medium, we chose a common type of milk, i.e., UHTtreated cows’ milk (1.5% fat). We selected three incubation tem-peratures covering a wide range of possible storage conditions(37 �C w optimal for staphylococcal growth, 25 �C w room tem-perature, 10 �C w poor storage condition). It is known that thetemperature range for possible enterotoxin production is between10 �C and 45 �C (Tatini, 1973).

Our results confirmed that optimal growth conditions coincidewith optimal conditions for enterotoxin production (Tatini, 1973).We observed a broad heterogeneity in SEC production among thetested strains. However, in general, SEC production was very low.Only a limited number of tested strains produced a considerableamount of SEC. None or very low production (approximately10 pg mL�1) was observed in all the tested strains at 10 �C. Thisresult was also confirmed by extended three-week monitoring ofSEC production at 10 �C (data not shown). The interesting fact isthat 3 of 4 strains isolated from a dairy environment which wereused in our study are between 6 most producing strains (Fig. 1: SA1178, SA 1189, SA 1612). This indicates a greater adaptation of thesestrains to a dairy environment.

The minimal emetic dose of SEC necessary to develop food poi-soning symptoms in monkeys was determined to be between 5 and20 mg SEC per animal (Bergdoll, 1988). Human-related data are notavailable, but there is a presumption that the dose would be verysimilar or higher. If we took the most productive strain FRI 137 andcultivated it inmilk for 168h at 37 �C,wewouldhave todrink almost400 mL of such contaminated milk to reach the critical dose 5 mg ofSEC (regardless of the fact that this milk would be coagulated due tostaphylococcalmetabolic activity). In cases of other strains, volumesofmilk (or coagulum)wouldbe evenmuchhigher. Of course, there isa possibility that there are more productive strains. In fact, Oteroet al. (1987) used the strain L2 which was approximately 10 timesmore productive than FRI 137 in ewes’ milk. However, storage ofmilk at 37 �C for 168 h is a rather rare condition. When we wouldconsider cultivation of FRI 137 inmilk at 25 �C for 168 h, the volumeofmilk containing 5 mg SECwould have to be about 4.1 L and at 10 �Cit would be hundreds of litres. With regard to these results, it seemshighly unlikely that someSEC-producing strains couldbe implicatedinmilk-related food poisoning cases. In fact, wewere not able tofindany evidences of such cases in the literature.

5. Conclusions

We performed a comprehensive study describing SEC produc-tion in milk environment. Surprisingly, we found the very low SECproduction compared with production in laboratory media. Ourdata imply two main conclusions: i) S. aureus strains producing SECcould be considered as a relatively harmless milk-contaminatingbacteria, although further studies using a large number of strainswill be necessary to confirm this statement; ii) There is a clear needto study behaviour of food-borne pathogens in their natural envi-ronment instead of using laboratory media.

Acknowledgements

Financial support from specific university research (MSMT No21/2012) and university development fund (FRVS 457/2012). Wethank Ing. Lucie Vondrakova for careful proofreading.

L. Valihrach et al. / International Dairy Journal 30 (2013) 103e107 107

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.idairyj.2013.01.003.

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