Dairy Sci. & Technol.
DOI 10.1007/s13594-012-0067-4
O R I G I N A L PA P E R
Classical enterotoxins of coagulase-positive Staphylococcus
aureus isolates from raw milk and products for raw milk
cheese production in Ireland
Karen Hunt & Jenny Schelin & Peter Rådström &
Francis Butler & Kieran Jordan
Received: 2 December 2011 / Revised: 26 March 2012 / Accepted: 26 March 2012
# INRA and Springer-Verlag, France 2012
Abstract Toxin-producing Staphylococcus aureus can be present in raw milk and
therefore in cheese made from raw milk. To determine the number and type of toxin
producers in raw milk used for raw milk cheese production in Ireland, 117 samples of
raw milk and related products from five raw milk suppliers, to four raw milk
cheesemakers in the South of Ireland, were analysed for coagulase positive S. aureus.
Enumeration, using ISO 688-2 and plating on Baird Parker Rabbit Plasma Fibrinogen
selective agar showed samples were within limits set by EC regulations. Isolates (151
from 81 positive samples) were characterised for production of staphylococcal enterotoxins (SEs) SEA, SEB, SEC and SED by reverse passive latex agglutination (SETRPLA) and by multiplex polymerase chain reaction for the sea, seb, sec, sed and see
genes. The results showed 83.2% of the isolates did not contain the se genes or the
toxin producing capability tested for. From only one supplier, 26 isolates contained
the sec gene and produced SEC. Within these 26 isolates, there were only two PFGE
types. One SEC-producing isolate showed no toxin production when grown in sterile
10% reconstituted skim milk at 10 °C and 12 °C for 96 and 74 h, respectively. Low
concentrations of SEC were produced at 14 °C and 16 °C after 74 and 55 h,
respectively. The results of this survey indicate that milk used for raw milk cheese
production in Ireland poses a limited risk to public health, although further studies on
occurrence of toxin producing S. aureus should be undertaken.
K. Hunt : K. Jordan (*)
Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork, Ireland
e-mail: kieran.jordan@teagasc.ie
K. Hunt : F. Butler
Biosystems Engineering, UCD School of Agriculture, Food Science and Veterinary Medicine,
Belfield, Dublin 4, Ireland
J. Schelin : P. Rådström
Applied Microbiology, Lund Institute of Technology, Lund University, Lund, Sweden
K. Hunt et al.
爱尔兰生乳及其产品中金黄色葡萄球菌毒素和毒素基因的检测
摘要 ' 生乳及由其生产的干酪中可能存在产毒素的金黄色葡萄球菌。为了检测在爱尔兰用
于生产干酪制品的生乳中产毒素菌的金黄色葡萄球菌的类型和数量,从爱尔兰南部地区5个
原料奶的供应商和4个生乳干酪生产厂家收集了117个生乳及其相关产品,采用贝尔德派克兔
血浆纤维蛋白原选择性琼脂平板计数法(ISO 688-2)法测定了凝血酶阳性葡萄球,结果表明,
样品中凝血酶阳性葡萄球菌菌数在欧盟法规规定的范围之内。采用反向被动乳胶凝集试验
(SET-RPLA)测定了金黄色葡萄球菌(从81个阳性样本中分离出的151株菌)产生的肠毒素型
SEA-SED,以及应用多重聚合酶链反应(PCR)对sea、seb、sec、sed和see基因进行扩增,实验结
果表明83.2%的金黄色葡萄球菌株不含有产肠毒素(SE)的基因或者经检测不具有产肠毒素的
能力。仅从1个原料乳供应商所提供的原料乳中分离出的26株金黄色葡萄球菌中检测到了sec
基因和具有产肠毒素C的能力,而且这26株分离菌株属于2个不同PEGE基因型。一株产SEC的
金黄色葡萄球菌在10%的无菌还原脱脂乳培养基中分别在10 °C和12 °C下培养96和74 h时后,
没有肠毒素产生;而在14 °C和16 °C下培养74和55h后,产生了低浓度的SEC。调查结果表明,
尽管爱尔兰用于生产干酪的生乳对公众健康影响的风险较低,但仍有必要进一步研究产肠毒
素的金黄色葡萄球菌对食品安全带来的潜在危害。
Keywords Staphylococcus aureus . Raw milk . Enterotoxins . SE genes . SEC
production
关键词 金黄色葡萄球菌 . 生乳 . 肠毒素 . 葡萄球菌肠毒素基因 – 产葡萄球菌肠毒素C
1 Introduction
Coagulase positive Staphylococcus aureus is a Gram-positive, facultative anaerobe
that is ubiquitous in nature. Some, but not all strains produce pyrogenic, pepsinresistant staphylococcal enterotoxins (SEs) which are potent emetic agents causing
staphylococcal food poisoning (SFP; Pinchuk et al. 2010; Le Loir et al. 2003). SFP
has recently been reported to have a low hospitalisation rate of 6.4% (Scallan et al.
2011); however, due to the nature of the toxin the effect of symptoms are rarely
severe, leading to high levels of under-reporting.
S. aureus strains produce a wide range of toxins, and at present 21 SEs or
enterotoxin-like proteins have been identified (Schlievert and Case 2007; Thomas
et al. 2006). Of these toxins, SEA and SED are most frequently associated with food
(De Buyser et al. 2001; Kérouanton et al. 2007), and are therefore most clinically
relevant, while SEC is associated with animal origins (Pinchuk et al. 2010); more recently
SEE was associated with SFP (Ostyn et al. 2010). Outbreak investigations in Austria
with pasteurised milk had SEA and SED (Schmid et al. 2009), and in Japan SEA was
the cause of an outbreak associated with pasteurised milk (Asao et al. 2003). Other
examples of SFP caused by milk and dairy products are listed by Cretenet et al.
(2011).
One of the major causes of bovine mastitis is S. aureus, which can then easily
contaminate raw milk, particularly in cases of sub-clinical mastitis (Boynukara et
al. 2008; Fagundes et al. 2010). With no heat treatment prior to manufacture, and milk
being an ideal growth medium for bacteria, bacterial counts of S. aureus can increase
(Charlier et al. 2009; Even et al. 2009). With subsequent growth opportunities during
cheesemaking, it is possible that S. aureus counts can be relatively high in cheese.
Staphylococcal enterotoxins in raw milk
Approximately 10% of cheese in Europe is made from raw milk (Beuvier and Buchin
2004), presenting a considerable potential risk to public health. In Ireland, a recent
study showed that 96% of farmhouse cheese was within EU specifications for S.
aureus (O'Brien et al. 2009). In Scotland, S. aureus was found to be the most frequent
pathogen in raw milk cheese (Williams and Withers 2010). In France, a study of foodborne
disease outbreaks from 1992 to 1997 found that S. aureus was the most frequent
pathogen associated with milk related outbreaks (De Buyser et al. 2001). Other S.
aureus studies of raw milk and raw milk products showed that the majority of raw
milk supplied to artisan cheesemakers had high microbiological quality (D'Amico
and Donnelly 2010).
Bovine raw milk cheese is considered a high-risk food in relation to the pathogen
S. aureus (André et al. 2008). Where raw milk and cheese are concerned, there is a
need for good hygiene both in the dairy and in the cheesemaking facilities (Little et al.
2008). Bacterial counts of S. aureus need to reach 105 to 108 cfu.mL−1 before
sufficient toxin is produced to cause illness (Pinchuk et al. 2010). As SE toxins are
heat resistant (and not affected by pasteurisation), bacterial counts of S. aureus in raw milk
cheese can be misleading, where counts of S. aureus are high. Toxin can be produced,
and if the S. aureus cells are inactivated, by pasteurisation for example, the toxin can
remain in the milk and can cause SFP at levels of between 20 and 100 ng.mL−1 (Asao
et al. 2003).The need for measurement of these toxins produced from the S. aureus in
dairy products has been identified (Cretenet et al. 2011).
Not all coagulase positive S. aureus produce toxin. In Italy, 55% of food isolates
were positive for classical SEs (Normanno et al. 2007), while in Norway 48% of
isolates from bovine raw milk and raw milk products were identified as SE producers
(Loncarevic et al. 2005). Studies of cheese from dairy farms in Sweden (Rosengren et
al. 2010), including both bovine and ovine milk sources, showed that about
70% of cheese isolates carried one or more se genes, where sec and sea were the
most common, therefore highlighting a risk with cheese produced by on-farm dairies.
The phenotypic characterisation of S. aureus SE producing isolates includes the
production of its specific toxin, while their genotypic characteristic includes the
presence of the specific toxin genes. Polymerase chain reaction (PCR) methods have
been developed to study gene occurrence in correlation with toxin production
(Boerema et al. 2006).
In Ireland, there are approximately 60 farmhouse cheese-producing facilities, which
use milk from a variety of mammals including cows, sheep and goats. The farmhouse
cheese production is approximately 1,000 tonnes per annum, of which approximately
110 tonnes are from raw milk. In this study, the suppliers produce approximately 80
tonnes of bovine raw milk cheese per annum, representing over 70% of the national
farmhouse bovine raw milk cheese production.
The aim of this study was to determine the number of toxin-producing coagulase
positive S. aureus in raw bovine milk and raw milk cheese products made from that
raw milk. Strains isolated were characterised further with regard to the type of SE
produced, the identity of the toxin genes present, and the comparison of the strains
using pulsed field gel electrophoresis (PFGE). SE production by SE-producing
isolates grown in 10% reconstituted skim milk (RSM) at low to moderate temperatures of 10–16 °C was measured. Relatively low temperatures were used in order to
identify the risk associated with toxin production at these temperatures.
K. Hunt et al.
2 Materials and methods
2.1 Sample collection
A total of 117 samples at various stages in cheese manufacture were aseptically collected
from five bovine raw milk suppliers and from four raw milk cheese producers in the
south of Ireland, from February to April 2010 (Table 1). Three of the cheese producers
used raw milk from their own dairy herd, while the remaining cheese producer
obtained milk from suppliers 4 and 5. The samples included raw milk from the bulk
storage tank, the same raw milk after it had been added to the cheese vat and samples
of the cheese (curds, fresh and ripened cheese) made from that milk. Raw milk and
curds and whey were sampled from the farm dairy bulk tank or the cheese vat using a
50-mL sterile dipper and transferred to a sterile container. Fresh and ripened cheese
samples were placed directly into a sterile container. All samples were kept at 4 °C,
transported to the laboratory and analysed within 24 h of receipt.
2.2 Enumeration of S. aureus from raw milk and raw milk products
Coagulase positive S. aureus were enumerated using the method for raw milk and raw
milk cheese, ISO 688-2-1999 on Baird Parker Rabbit Plasma Fibrinogen selective
agar (BP-RPF; Oxoid). The limit of detection for raw milk is 10 cfu.mL−1 and for
curds and cheeses is 100 cfu.g−1.
2.3 Isolates of S. aureus from raw milk and raw milk products
When samples were positive for coagulase positive S. aureus, at least two colonies
were selected and purified by streaking on tryptic soy agar (TSA). The isolates were
frozen at −80 °C in cryovials containing 15% glycerol.
2.4 Analysis of staphylococcal enterotoxin production by the isolates
After growth in tryptone Soya broth (TSB), the 151 isolates were analysed in duplicate
by reverse passive latex agglutination (SET-RPLA; Oxoid product Code: SET TD0900)
Table 1 Numbers of raw milk and associated product samples from five raw milk suppliers to four raw
milk cheesemakers
Product
Bulk raw milk
Supplier/
manufacturer 1
Supplier/
manufacturer 2
Supplier/
manufacturer 3
Supplier/
manufacturer 4
Supplier 5
10 (10)
10 (10)
10 (10)
10 (3)
11 (9)
Vat raw milk
0
10 (6)
2 (1)
0
0
Curds
0
10 (9)
6 (6)
2 (2)
0
Whey
0
10 (0)
6 (0)
2 (2)
0
Fresh cheese
4 (2)
5 (5)
2 (0)
5 (4)
0
Ripe cheese
0
0
0
2 (0)
0
The number of coagulase positive S. aureus samples is shown in brackets
Staphylococcal enterotoxins in raw milk
following the manufacturer’s instructions. This SE test is for detection of presence or
absence of SEA, SEB, SEC and SED. Since toxin production in food can be different
than toxin production in laboratory media (Schelin et al. 2011), strains positive for
enterotoxin in TSB were also analysed for toxin production in RSM.
2.5 Analysis by multiplex PCR
The primers used for gene detection of the four classical S. aureus enterotoxins (sea, seb,
sec, sed) and see were all used previously in a PCR multiplex assay (Gonano et al.
2009; Mehrotra et al. 2000). The DNA extract from four S. aureus strains (from Lund
University Sweden), known to contain these five enterotoxins was used as a positive control.
DNA from the 151 isolates sampled was extracted using Prepman ultra™ (Applied Biosciences code 4318930) and tested in a multiplex PCR assay. The PCR master mix
contained the following: 1 μL of a 0.5 μM solution of each primer set, 2 μL DNA, 12.5
μL ImmoMix™ Red (Bioline BIO25021) made up to 25 μL total reaction volume with
PCR grade water. Amplicons were produced by subjecting samples to the following
amplification conditions; initial heat treatment of 95 °C for 10 min; 30 cycles of 94 °C for
30 s, 54 °C for 45 s and 72 for 30 s; a final extension step of 72 °C for 10 min. The amplicons
were separated by electrophoresis conditions at 80 V for 40 min and visualised in UV light
with the addition of SyBr® Green (Invitrogen™). For comparative purposes, a 1-kb
molecular weight marker (Bioline Hyperladder IV) and a positive control (DNA from four
S. aureus strains containing all five genes) were used in all gels.
2.6 Analysis by pulsed field gel electrophoresis
Enterotoxin producing strains were analysed by PFGE using a PulseNet Method
(CDC PulseNet), as previously described (McDougal et al 2003). This method was
modified by adding 2 μL bovine serum albumen (BSA) to the digest mix. The
addition of 50 μmol.L−1 thiourea to both the agarose gel and the running buffer for
PFGE improved band resolution.
2.7 SEC production
An SEC-producing isolate (isolated from this study) was inoculated into RSM at a
concentration 107 cfu.mL−1.This inoculated milk was incubated at 10 °C, 12 °C, 14 °C
and 16 °C for 96, 74, 74 and 55 h, respectively, to determine the minimum
temperature at which toxin will be produced and thus assess the risk of toxin in the
milk prior to cheesemaking. As a control, an SEAD strain (isolated from boiled ham
in a food poisoning outbreak; SA45 obtained from Swedish Institute for Food and
Biotechnology, Gothenburg, Sweden) was tested under the same conditions. During
incubation, five samples at 10 °C and ten samples at 12 °C, 14 °C and 16 °C were
enumerated for S. aureus, using TSA plates (Merck). Initial and final samples of
inoculated milk were collected and analysed for SE using 3 M™ Tecra™ Staph
Enterotoxin Visual Immunoassay (SETVIA96; NZFSA validated) according to the
manufacturer’s instruction. SETVIA96 is an enzyme-linked immunosorbent assay
(ELISA) method which relates SE concentration to absorbance (measured using a
Synergy HT Multi-Mode Microplate Reader Gen 5).
K. Hunt et al.
Table 2 Enumeration results of S. aureus in samples of milk and products manufactured from the milk
Product
Supplier/
Supplier/
manufacturer 1 manufacturer 2
Bulk raw
milk
80–900 (260)
Supplier/
Supplier/
manufacturer 3 manufacturer 4
<10–35 (8)
10–110 (20)
Supplier 5
140–700 (300)
15–330 (80)
–
Vat raw milk
–
<10–60 (14)
<10
–
Curds
–
<100–3,100 (700)
17–800 (300)
780–7,200 (4,000)
–
Whey
–
<1
<1
24–38
–
Fresh cheese
<100(40)
<100–6,700 (4200)
–
1,800–14,000 (2,200)
–
Ripe cheese
–
–
–
<10
–
The range of values (in cfu.mL−1 or cfu.g−1 ) is shown with the average number in brackets
3 Results
3.1 Enumeration of S. aureus from raw milk and raw milk products
Table 1 shows the number of samples taken from each of the five raw milk suppliers. The
bacterial count of coagulase positive S. aureus samples is shown in brackets. Supplier 1
only made cheese occasionally during the sampling period and therefore only four
fresh cheeses were obtained for analysis. A total of 151S. aureus isolates were
obtained from the 81 positive samples. Table 2 shows enumeration results in cfu per
millilitre. All samples were within the regulatory limits of <105 cfu.g−1 for cheese set by the
EU (Commission Regulation [EC] No. 2073/2005; Commission Regulation [EC] No.
1441/2007): there are no EU regulations for raw milk. From supplier 4, two of the
curd samples resulted in higher S. aureus counts, than the associated fresh cheeses;
when the fresh cheese was ripened, bacterial counts were below the limit of detection.
3.2 Characterisation of isolates using SET-RPLA and multiplex PCR
All isolates were grown in TSB and tested for toxin production by SET-RPLA.
Twenty-six of the isolates produced SEC. No other toxins were produced by any of
the strains (Table 3). The SEC-producing isolates were also tested for SEC production
Table 3 Phenotypic and genotypic characterisation of 151S. aureus isolates
No samples tested
Phenotypic (SET-RPLAa)
Genotypic (multiplex PCRb)
SEA
151
0
0
SEB
151
0
0
SEC
151
26
26
SED
151
0
0
SEE
151
Not determined
0
Enterotoxin
SET-RPLA staphylococcal enterotoxin reverse passive latex agglutination, PCR polymerase chain reaction
Staphylococcal enterotoxins in raw milk
Table 4 Sample type from which staphylococcal enterotoxin C (SEC) producing isolates were obtained
Total no. samples testeda
SEC-producing isolatesb
Bulk raw milk
51
1
Vat raw milk
12
6
Curds
18
14
Product
Whey
18
0
Fresh cheese
16
5
Ripe cheese
2
0
a
As described in Table 1
b
All positive isolates were from the same source, supplier/manufacturer 2
at 37 °C in RSM, and all were shown to produce SEC (data not shown). All SEC
producing isolates were obtained from the same supplier/manufacturer (Table 4).
Figure 1 is a representative gel of the results obtained. Of the isolates tested, 26
had only the sec gene while the remaining isolates had none of the toxin genes tested
for (data not shown).
3.3 Analysis of toxin-producing isolates by PFGE
The 26 SEC-producing isolates (which all came from the same supplier/manufacturer) were characterised as two distinct PFGE types, A and B, with three band differences between the two types (Fig. 2). There were nine isolates of type A and 17
isolates of type B. Type A was found in milk and subsequently in the cheese products
associated with that milk; however, type B was only found in the vat milk and the
cheese products.
3.4 Analysis of SEC production at different temperatures
The SEC-producing isolates were grown in RSM for 4 days at 10 °C, 3 days at 12 °C
and 14 °C and 55 h at 16 °C. Figure 3 shows the results of end-point analysis for
toxin. No absorbance increase was seen at 10 °C and 12 °C (indicating no toxin
production), while low absorbance values were measured at 14 °C and 16 °C
(indicating some toxin production). Results are compared to the SEAD control strain,
which showed production of enterotoxin at all four temperatures, under the same
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
Fig. 1 Agarose gel electrophoresis profile, showing multiplex PCR of 14S. aureus isolates. Lanes 1 and 18
represent the 1-kb molecular weight marker; lanes 8, 11, 12 and 15 are strains showing staphylococcal
enterotoxin C (sec) gene. Lane 16 negative control containing no DNA; lane 17 positive control containing
DNA from four S. aureus strains containing sea (102 bp), seb (164 bp), sec (451 bp), sed (278 bp) and see
(209 bp) genes. All other lanes contained strains with no enterotoxin genes
K. Hunt et al.
A
B
C
Fig. 2 PFGE profiles of two staphylococcal enterotoxin C PFGE types found (A, nine strains; B, 17
strains) showing three band differences (arrows showing A has one less band and two additional bands
when compared to B) and Salmonella standard strain C
conditions. Higher absorbance values were observed for the SEAD producing strain,
indicating the possible production of higher concentrations of toxin, although this
would need to be measured by a quantitative assay.
4 Discussion
There are no EU specifications for bacterial counts of coagulase positive S. aureus in
raw milk, but all of the cheeses tested were within the EU specifications for cheese
(EC regulation 2073/2005, as amended by EC regulation 1441/2007).
The bacterial counts of coagulase positive S. aureus in the raw milk varied from <1
to 900 cfu.mL−1. Of the 51 milk samples, 78% were positive for S. aureus, although
the majority of the samples had <200 cfu.mL−1. Samples in this study were collected
from late winter to early spring — a time period considered ‘worst case’ with respect
to S. aureus counts (Rea et al. 1992). In Vermont, the majority of samples had S.
aureus at <1 cfu.mL−1 (D'Amico et al. 2008). In this current study, there was no
physical indication of mastitis in the herds. S. aureus enumeration of milk samples
from supplier/manufacturer 3 (11 milk samples tested) was relatively lower than other
suppliers tested. This may be as a result of the low pressure milking system used by
this supplier.
Staphylococcal enterotoxins in raw milk
9.00
8.50
log cfu.mL-1
S.aureus (SEC strain)
A
8.00
7.50
7.00
6.50
6.00
0
20
40
60
80
100
Time (h)
S.aureus (SEAD strain)
log cfu.mL-1
B
9.00
8.50
8.00
7.50
7.00
6.50
6.00
0
20
40
60
80
100
Time (h)
Absorbance, 414 nm
C
3.0
2.5
2.0
1.5
1.0
0.5
0.0
10°C
12°C
14°C
16°C
Temperature, °C
Fig. 3 S. aureus growth, measured in log cfu.mL−1 of A an SEC-producing strain and B an SEAD
producing strain, at temperatures of 10 °C (empty square), 12 °C (empty triangle), 14 °C (filled square) and
16 °C (filled triangle). C Absorbance measurements of SEC (empty square) and SEAD (filled square)
production after growth in reconstituted skim milk for 96 h at 10 °C, 74 h at 12 °C, 14 °C and 55 h at 16 °C
The presence of coagulase-positive S. aureus in milk, although it may be an
indicator of a food safety issue, is not the most important factor. Enterotoxinproducing S. aureus must have the opportunity to grow during cheesemaking in
order to produce sufficient toxin to cause illness. In general, cheesemaking does
provide the opportunity for growth, although the manufacturer can take steps to
reduce the amount of growth. For example, coagulase positive S. aureus do not grow
at 4 °C and are poor competitors; therefore, proper temperature control and timely
addition of starter culture can help to control growth (Charlier et al. 2009; Even et al.
2009). In addition, different starter cultures used during cheesesmaking, have been
shown to be an important factor in the survival of S. aureus (Lindqvist et al. 2002;
Bachmann and Spahr 1995). All cheeses in the current study were within the
K. Hunt et al.
specifications of <105 cfu.g−1 established by the EU, indicating that adequate control
measures were taken by the manufacturers. Only the cheese manufactured from
supplier 4 had relatively high S. aureus counts in the fresh cheese. Although only
two ripe cheeses from supplier/manufacturer 4 were tested, this and other studies with
this cheese type (Hunt and Jordan unpublished) have shown that coagulase positive S.
aureus counts decrease as the cheese ages. The study of Williams and Withers (2010)
reported a lower percentage of cheeses having coagulase positive S. aureus; however,
that study was on ripened cheese, compared to the current study where the majority of
cheeses were fresh cheese.
It is considered that in order to pose a threat to food safety, the counts of coagulase
positive S. aureus in food must reach >105 cfu.g−1, and must also have the ability to
produce toxin. Sufficient toxin to cause illness varied depending on the toxin type, as
little as 20–100 ng SEA has been reported to induce a toxic response (Asao et al.
2003). Therefore, the ability and type of strains to produce toxin is a relevant
criterion. In this study, 151 isolates were obtained from the samples collected.
These represented 20–40 isolates from each of the suppliers/manufacturers. Isolates
were characterised for the presence of classical enterotoxins, and associated genes,
including the see gene. None of the isolates from four of the five raw milk suppliers
produced SEA, SEB, SEC or SED. From one manufacturer, 26 of the isolates
produced SEC in milk or laboratory media, but no SEA, SEB or SED were
produced in either medium. Among these toxins, SEC is the least important as it is
rarely implicated in outbreaks of food poisoning (Pinchuk et al. 2010), being mostly
associated with bovine strains (Jørgensen et al. 2005; Stephan et al. 2001). In other
studies, 55%, 53% and 70% of isolates tested positive for SEs (Normanno et al. 2007;
Loncarevic et al. 2005; Rosengren et al. 2010).
SET-RPLA is a recognised frequently used qualitative test for SE analysis by S.
aureus isolates with a sensitivity of 0.25–0.5 ng.mL−1 (Rose et al. 1989; Cretenet et al
2011), equivalent to commercial ELISA or PCR for detection of SEs (McLauchlin et
al. 2000). It has been used extensively in studies of SE production (Cenci-Goga et al.
2003; Wieneke 1991).We used a multiplex PCR assay to support the results of the
SET-RPLA test. There was 100% correlation between the phenotypic and genotypic
methods used. None of the strains tested contained the genes for SEA, SEB or SED
and the SEC-producing strains only had the genes for SEC production. It is possible
that a strain could contain the genes for toxin production but under the conditions of
milk/cheese no toxin was produced. In different conditions toxin could be produced.
That the strains tested did not harbour additional toxin producing genes is an
additional risk reduction factor as toxin could not be produced under any
conditions. In previous studies characterising S. aureus strains isolated from raw
milk and cheese samples, SEC-producing isolates were also prevalent (Jørgensen et
al. 2005; Normanno et al. 2007; Williams and Withers 2010).
From the 26 SEC-producing strains isolated, there were two PFGE types detected:
A and B. Type A was found in the bulk tank milk and subsequently in the cheese vat
milk and the cheese. Type B was only detected in the cheese vat milk and subsequent
cheese. It is possible that type B could have been in the bulk tank milk but not
detected as S. aureus counts may have been below the limit of detection
(10 cfu.mL−1). It is also possible that type B was a contaminant in the milk lines
between the milk transport lines between the bulk milk tank and the cheese vat, or it
Staphylococcal enterotoxins in raw milk
may be a human contaminant during cheesemaking (Williams and Withers 2010).
Further characterisation of these strain types would be required to determine the
source.
The SEC production in RSM is more relevant to assessing the risk of toxin in the bulk
tank environment when compared to that in broth based assays (Schelin et al. 2011). The
guidelines for storage of on-farm bulk milk are <4 °C, and reduction to <4 °C in 3 h
when fresh milk is added to the tank. The results of this study indicate that even if
milk was stored at an abuse temperature of 14 °C, very little toxin (based on the
absorbance values obtained) would be produced even in 3 days. It would be expected
that in raw milk toxin production would be even less as S. aureus are poor competitors (Charlier et al. 2009; Even et al. 2009). This result would need to be validated
with other strains, including strains isolated from human sources and strains
producing other SEs.
5 Conclusions
This study shows that only 17% (26 isolates) of coagulase positive S. aureus isolates
were enterotoxigenic although within these strains there were only two PFGE types.
These 26 strains were isolated from the same supplier, were all SEC producers, and
were found both in milk and cheese samples. From the samples collected, no isolates
harboured the genes encoding SEA, SEB or SED. One of the SEC-producing strains
isolated showed no toxin production at 10 °C or 12 °C in milk. Therefore, the risk to
public health from toxin-producing S. aureus in dairy products can be quantified to
some extent and can be managed via temperature control.
Acknowledgements This work was supported by the EU 6th Framework Programme under the project
BIOTRACER. The authors wish to acknowledge the assistance of CAIS — the Irish Farmhouse Cheesemakers
Association — and the cooperation of all five raw milk suppliers and farmhouse cheesemakers who participated
in this work. Karen Hunt was in receipt of a Walsh fellowship during this study.
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