VYSOKÉ UČENÍ TECHNICKÉ V BRNĚ

Fakulta chemická
Ústav potravinářské chemie a biotechnologie

Ing. Vendula Hrušková

Foodborne Staphylococcus aureus: Identification

and enterotoxin production in milk and cheese
Zkrácená verze dizertační práce
Key words
Staphylococcus aureus, real-time PCR, enterotoxin production, milk, cheese

Klíčová slova
Staphylococcus aureus, PCR v reálném čase, produkce enterotoxinu, mléko, sýr

Místo uložení práce

Ústav potravinářské chemie a biotechnologie, FCH VUT, Purkyňova 118, Brno 612 00

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CONTENTS

1. INTRODUCTION .................................................................................................................... 4

2. THEORETICAL PART .......................................................................................................... 5

2.1 STAPHYLOCOCCUS AUREUS ................................................................................................. 5

2.1.1 The organism and staphylococcal food poisoning ........................................................ 5
2.1.2 The enterotoxins ............................................................................................................ 5
2.1.3 Environmental factors ................................................................................................... 6
2.1.4 Milk and cheeses ........................................................................................................... 7
2.1.5 Principles of detection ................................................................................................... 8

3. THE AIMS .............................................................................................................................. 12

4. RESULTS AND DISCUSSION............................................................................................. 13
4.1 DETECTION OF S. AUREUS IN FOOD BY PCR ..................................................................... 13
4.2 MILK EXPERIMENTS ......................................................................................................... 14
4.2.1 The effect of temperature on S. aureus enterotoxin D production in milk and BHI ... 14
4.2.2 The combined effect of 12°C and background flora ................................................... 17
4.3 CHEESE EXPERIMENTS ..................................................................................................... 21
4.3.1 Experiment 1: Semi-soft cheese, 13°C ........................................................................ 22
4.3.2 Experiment 2: Semi-soft cheese, 20°C ........................................................................ 24
4.3.3 Experiment 3: Cream cheese, 13°C............................................................................. 26
4.3.4 Experiment 4: Cream cheese, 20°C............................................................................. 28

5. CONCLUSIONS..................................................................................................................... 32
6. REFERENCES ....................................................................................................................... 34
7. LIST OF ABBREVIATIONS ................................................................................................ 37
8. CURRICULUM VITAE ........................................................................................................ 38
9. ABSTRACT ............................................................................................................................ 39

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1. INTRODUCTION
Modern food production chains are evolving to very complex systems that provide greater
opportunities for contamination and growth of pathogens. As a direct consequence,
preventing foodborne diseases (FBDs) becomes a difficult task. FBDs are defined by the
World Health Organization as “diseases of infectious or toxic nature caused by, or thought to
be caused by the consumption of food or water”. More than 250 different FBDs have been
described and bacteria are the causative agents of two thirds of FBD outbreaks 4.
In 2008, a total of 5,332 food-borne outbreaks were reported within the EU. 45,622 human
cases, 6,230 hospitalizations and 32 deaths were related to the reported outbreaks . In the
USA, it is estimated that FBDs affect 6 million to 80 million people each year, causing up to
9000 deaths, and cost about 5 billion US dollars 35. The largest number of reported food-
borne outbreaks in the EU was caused by Salmonella (35.4% of all outbreaks), followed by
viruses (13.1%), bacterial toxins (9.8%) and Campylobacter (9.2%). The most important food
vehicles in the outbreaks were eggs and egg products (23.1%), pig meat and products thereof
(10.2%) and mixed or buffet meals (9.2%) .
Staphylococcus aureus is considered the third most important cause of disease in the world
among the reported FBDs. The growth of S. aureus in foods presents a potential public health
hazard because many strains of S. aureus produce enterotoxins (SEs) which are the causative
agents of staphylococcal food poisoning (SFP) 35.
Rapid methods of pathogen testing have been gaining increasing interest in the food
industry 43. Polymerase chain reaction (PCR)-based methods provide a powerful tool for
highly specific and sensitive identification of pathogenic bacteria in foods and are considered
reliable alternatives to traditional microbiological methods.
Milk and cheeses are foods that have frequently been associated with staphylococcal food
poisoning. When enterotoxigenic strains of S. aureus replicate to numbers exceeding 105
cfu/ml, they may produce staphylococcal enterotoxins. Above this threshold (105 cfu/ml or g
of a dairy product), there is an obligation to screen for SEs. If SEs are detected, products have
to be destroyed, recalled or withdrawn from the market 38.
To improve the production of microbiologically safe food, data about food-borne pathogen
virulence is required to complement already existing knowledge about the growth and
survival ability of pathogenic bacteria. Recent research has shown that there are significant
differences in the behaviour of bacteria in laboratories, i.e. in a controlled environment and in
actual food products. New knowledge about relation between bacterial growth and virulence
expression under adverse environmental conditions will give rise to new approaches in the
prevention of foodborne diseases and enable the advancement of quantitative risk
assessments.

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2. THEORETICAL PART
2.1 Staphylococcus aureus

2.1.1 The organism and staphylococcal food poisoning

Currently, 49 different species of staphylococci exist, out of them 9 are coagulase positive.
Staphylococcus aureus remains to be the predominant pathogen 2.
Staphylococcus aureus is a Gram-positive, non-motile, oxidase negative, catalase and
coagulase positive coccus about 1 m in diameter dividing in more than one plane to form
irregular three-dimensional clusters of cells 1, 3, 4. S. aureus is a facultative anaerobe and
uses glucose by glycolysis and use of the hexose monophosphate pathway. It usually ferments
mannitol and, in the presence of air, uses a wide range of hexoses, pentoses, and sugar
alcohols; lactic acid and acetoin are the main end products of glucose metabolism 1. The
cell wall of S. aureus contatins three main components: peptidoglycan, teichoic acid and
protein A 1, 3. Staphylococcus aureus is found in the nostrils, and on the skin and hair of
warm-blooded animals 4. It lives as a commensal of the human nose in 3070% of the
population.
Staphylococcal food poisoning (SPF) is caused by staphylococci (principally S. aureus)
growing in food and forming enterotoxins as a product of their metabolism 1. SFP presents
as a self-limiting gastrointestinal illness with emesis following a short incubation period (ca. 4
h) after ingestion of food containing preformed enterotoxin(s). Vomiting is the hallmark
symptom of SFP 6. The severity of the illness depends on the amount of food ingested, the
amount of toxin in the ingested food and the general health status of the victim 7. The intake
of as little as 20 – 100 ng of enterotoxin can cause intoxication 8. Ingested bacteria do not
produce toxin, therefore the symptoms normally wear off within 24 h 7.

2.1.2 The enterotoxins

To date, 21 staphylococcal enterotoxins (SEs) or enterotoxin-like proteins (SEls) have been

identified and designated SEA to SElV 7. SEs are short, extracellular proteins that are
soluble in water 1, 4, 14. They are heat resistant (depending on the SE type, SE
concentration and food matrix) and highly stable to proteolytic enzymes, such as pepsin,
trypsin chymotrypsine, rennin and papain 1, 4, 15, 16. Enterotoxins stimulate the relase of
serotonin in the gut. The serotonin acts on neuron receptores in the gut, stimulating the
vomiting center in the brain via the vagus nerve 17.
The SEs belong to a family of the so-called pyrogenic toxins. Pyrogenic toxins include
SEs, TSST, exfoliatins A and B and streptococcus pyrogenic toxins. These toxins share some
structure, function and sequence similarities 15. Although pyrogenic toxins are involved in
distinct pathologies, they have common biological activities, i.e. pyrogenicity, immune

5
suppression, and nonspecific T-cell proliferation. These activities are referred to as
superantigen activity 4.
The enterotoxins are located on and spread by different mobile genetic elements, i.e.
pathogenicity islands (SAPIs), prophages, plasmids, enterotoxin gene cluster (egc) and
staphylococcal cassette chromosome (scc) 4, 5, 6, 9, 15, 19, 20. Most of reported SFP
outbreaks are associated with the classical enterotoxins, SEA-SEE 7.
SEA is the most common toxin implicated in SFP. The sea gene is carried by a temperate
bacteriophage 15 and the expression peaks at late exponential phase of S. aureus growth
21. It has not yet been fully understood how sea expression is regulated. The second most
common enterotoxin associated with SFP is SED. The sed gene is located on a 27.6-kb
penicillinase plasmid designated pIB485 15. The expression of sed peaks in the late
exponential growth phase, a consequence of the regulation by the Agr system 22.
As for Staphylococcus aureus, several global regulators have been reported to regulate the
production of virulence-associated exoproteins and cell wall components. Among these
regulatory systems, the accessory gene regulator (agr) system has been the best characterized.
The agr system is a quorum-sensing system and a two-component regulatory system which
responds to an autoinducer peptide 20.

2.1.3 Environmental factors

Environmental factors such as temperature, nutrients, pH, presence or absence of oxygen

and water activity, disturb homeostasis of a cell and affect the cell’s growth and toxin
production. The most important environmental factors affecting the growth and enterotoxin
production of S. aureus are shown in Table 2.1 and Table 2.2.

Table 2.1 Environmental factors affecting the growth of S. aureus 11

Minimum Optimal Maximum

Temperature (oC) 7 35-37 48
pH 4.0 6.0-7.0 9.8
NaCl (%) 0 0.5-4.0 20
Water activity 0.83 0.98->0.99 >0.99

Table 2.2 Environmental factors affecting enterotoxin production 11

Minimum Optimal Maximum
Temperature (oC) 10 35-40 45
5.3-6.8 (Ent. A)
pH 4.8 9.0
6-7 (Others)
NaCl (%) 0 0.5 20
Water activity 0.86 >0.99 >0.99

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2.1.4 Milk and cheeses

Milk and milk products have always been vehicles for staphylococcal food poisoning 30.
In 2000, a mass outbreak of SFP caused by consumption of reconstituted milk occurred in
Japan, and more than 10,000 cases were reported 31. Other recently reported outbreaks were
associated with mashed potatoes made with raw milk in Norway 32, pasteurized milk
products in Austria 33, and Minas cheese in Brazil 34.
S. aureus can gain access to milk either by direct excretion from udders with clinical or
subclinical staphylococcal mastitis or by contamination from the environment during handling
and processing of raw milk. S. aureus is responsible for approximately 30% to 40% of all
mastitis cases 35. There are many other possible sources of S. aureus contamination for
processed milk including humans themselves. The potentially adverse effects of S. aureus on
human health are reduced through pasteurization. It is widely believed that the majority of
food poisoning cases associated with pasteurized milk are due to improper pasteurization or
post pasteurization contamination 30.
S. aureus is a poor competitor against the normal microflora in unpasteurized dairy
products. However liquid milk, raw or pasteurized, is a good substrate for growth of S. aureus
and for enterotoxin production if it is held at temperatures higher than 10°C and the natural
bacterial flora is low. In fact pasteurized milk is a better substrate than raw milk for the
growth of S. aureus and for enterotoxin production because the competitive natural flora has
been partially eliminated 14, 30.
Cheeses comprise a huge number of varieties and thus the composition also varies 37.
For each cheese variety, different technological parameters such as starter culture, milk and
curd heating temperature or stirring conditions, are applied throughout the cheese-making
process and induce different environmental conditions (i.e. pH, temperature, NaCl
concentration or oxygen disponibility) that affect S. aureus growth and enterotoxin production
37, 38. Above a threshold of 105 coagulase-positive staphylococci per gram of a dairy
product, European legislation stipulates the obligation of testing for the presence of
enterotoxins 39. If SEs are detected, products have to be destroyed, recalled or withdrawn
from the market resulting in substantial economic loss. It has been demonstrated that S.
aureus growth occurs mainly during the first 24 hours of cheese-making process 29, 38.
However, SEs are not always detected in the final product even if the population of S. aureus
reaches a value above 105 cfu/g of cheese 38, 13.

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2.1.5 Principles of detection

2.1.5.1 Detection of S. aureus

2.1.5.1.1 Microbiological methods

There are three broad types of agar media that may be used for the isolation and
enumeration of S. aureus in foods from which Baird-Parker agar remains the medium of
choice (EN ISO 6888-1). The good performance of this medium depends on: a balance of
selective agents (lithium chloride, glycine and potassium tellurite) to inhibit Gram-negative
and Gram-positive bacteria; the inclusion of sodium pyruvate to protect S. aureus damaged
cells and the presence of egg yolk, which, in addition to providing one of the main diagnostic
features of the medium, also assists in the recovery of damaged cells. Potassium tellurite is
metabolized by S. aureus with the release of tellurium, resulting in the formation of black
colonies, which is a further useful diagnostic feature .
Giolitti-Cantoni broth and Baird-Parker broth are selective media used in Most Probable
Number (MPN) techniques for detection of small numbers of S. aureus and when necessary,
for selective enrichment before subculture to agar media. The growth of Gram-negative
lactose-fermenting bacilli is inhibited by lithium chloride and Gram-positive bacilli are
inhibited by potassium tellurite in combination with glycine. Anaerobic cultivation inhibits
the growth of micrococci. The growth of S. aureus causes blackening 40.

2.1.5.1.2 Biochemical methods

The principal method used to confirm that an isolate is S. aureus is to apply the coagulase
test. The literature now commonly refers to two forms of coagulase: free coagulase which is
detected in the tube test and bound coagulase (which may be given an alternative name
clumping factor) which is detected in the slide test. Free coagulase is an extracellular protein
that is produced when the organism is cultured in broth. Bound coagulase remains attached to
the cell wall of the organism. The test is positive if clotting of plasma occurs [3, 40].
Thermonuclease test has a useful role in food microbiology because it will survive
conditions, e.g. pasteurization, that would destroy S. aureus but not the enterotoxins. Thus it
can be used as a screening test for possible presence of enterotoxins 1.

2.1.5.1.3 Rapid methods

Rapid methods of pathogen testing have been gaining increasing interest in the food
industry. These methods include antibody-based assays, genetic amplification methods and
newer sensor development. Traditional plating methods following enrichment can take days
to yield results, while newer rapid methods require hours. Genetic amplification methods such
as the polymerase chain reaction (PCR) have made it possible to significantly reduce assay
times while maintaining a high level of sensitivity and specificity. These methods are also
able to distinguish closely related species which most antibody tests could not 43.
Nevertheless, rapid methods are currently used as screening techniques, with negative results

8
accepted as is, but positive results requiring confirmation by the appropriate official method,
which, in many instances, is cultural 44.

Polymerase chain reaction

PCR is a method for enzymatic amplification (multiplication) of a specific DNA fragment
in vitro. PCR consists of repeated cycles, each involving the following steps taking place at a
defined temperature:
1. Denaturation of DNA, i. e. conversion of the double-stranded DNA to single-stranded
DNA at 9495°C,
2. Annealing of the oligonucleotide primers to homological DNA sequences at 3770°C,
3. Polymerization of the second DNA strand from nucleotides by the action of a
thermostable DNA polymerase at 6072°C.
PCR is carried out in a programmable thermocycler. In a conventional version, the PCR
product is analysed by agarose gel electrophoresis and presence/absence of the DNA fragment
of a given molecular weight is evaluated 46. The most commonly used stain for detecting
DNA is Ethidium Bromide (EtBr). EtBr is a DNA intercalator, inserting itself into the spaces
between base pairs of the double helix and forming a complex that emits fluorescence under
UV light. The major drawback to EtBr is that it is a potent mutagen 47.
The main application of PCR is a rapid (15 min – 2 h), highly sensitive (limit of detection
100 molecules) and highly selective detection of a defined DNA fragment, which is
detectable even at an excess (of up to at least 5 orders of magnitude) of other DNA fragments
46.
In summary, several problems that can be encountered when PCR-based methods are used
to detect food pathogens are inhibition of the reaction, the presence of false-positive reactions
caused by contamination, detection of dead bacteria and results that are not quantitative.
However, these problems can be overcome 48.

Real-time PCR
In real-time PCR, the PCR products are detected as they accumulate. In contrast to end
point analysis in which only the plateau phase of the PCR can be detected, real-time PCR
allows monitoring of the exponential phase 48. The presence of amplified DNA fragments is
detected by continuous monitoring of fluorescence. As a source of fluorescence, an
intercalation complex of DNA with a dye SYBR Green I is used, or various types of probes or
primers labelled with fluorescent dyes. When labelled probes or primers are to be used,
various possibilities are available. Out of them, 5-nuclease PCR has become the most widely
used, thanks to its robustness 46. This method utilises so called TaqMan probes. TaqMan
probes are oligonucleotides longer than the primers (20 – 30 bases long with a Tm value of
10°C higher) that contain a fluorescent dye usually on the 5 base, and a quenching dye
(usually TAMRA) typically on the 3 base. When irradiated, the excited fluorescent dye

9
transfers energy to the nearby quenching dye molecule rather than fluorescing (this is called
FRET = Förster or fluorescence resonance energy transfer). Thus, the close proximity of the
reporter and quencher prevents emission of any fluorescence while the probe is intact.
TaqMan probes are designed to anneal to an internal region of a PCR product. When the
polymerase replicates a template on which a TaqMan probe is bound, its 5 exonuclease
activity cleaves the probe. This ends the activity of quencher (no FRET) and the reporter dye
starts to emit fluorescence 52. When a sufficient amount of probe has been cleaved, the
intensity of reporter fluorescence emission increases. A threshold level of emission above the
base line is selected and the point at which the amplification plot crosses the threshold is
defined as CT and is reported as the number of cycles at which the log phase of product
accumulation is initiated 53.
The main advantages of real-time PCR are minimization of the laboratory contamination,
because the entire process is carried out in closed microtubes, and a potential for
quantification of the specific DNA fragment based on amplification curves 46. Furthermore,
real-time PCR is quicker, less laborious, more sensitive and more specific when compared to
conventional PCR.

Reverse Transcription PCR (RT-PCR)

Assessment of toxin genes expression has become crucial to understand the pathogenesis
of staphylococcal infections 10. Reverse-transcription polymerase chain reaction remains
the most sensitive technique for the detection of often-rare mRNA targets, and its application
in a real-time setting has become the most popular method to quantitate steady-state mRNA
levels . Generally two quantification types in real-time RT-PCR are possible: absolute
and relative. The absolute RT-PCR relates the PCR signal to input copy numbers using a
calibration curve, and neither comparisons nor references are needed. The relative RT-PCR
determines the expression level in comparison with a reference sample. It is based on the
relative expression of a target gene versus a reference gene. Normalization of target gene
expression is useful in order to compensate for sample-to-sample and run-to-tun variations
and to ensure the experimental reliability .

2.1.5.2 Detection of staphylococcal enterotoxins

A number of techniques for the detection of staphylococcal enterotoxins are currently
available. These techniques include radioimmunoassay (RIA) , 51, microslide double
diffusion , enzyme immunoassay (EIA) , MS-based analysis , 42 or methods
including biosensors .
Out of enzyme immunoassay methods, enzyme-linked immunosorbent assay (ELISA),
enzyme-linked fluorescent assay (ELFA) and reverse passive latex agglutination (RPLA) are
widely used 1, 54. Several ELISA methods have been proposed for the identification of
enterotoxins in foods, but, except for a polyvalent ELISA and ELFA, their specificity has not

10
been studied extensively. Among ELISA methods, the "double antibody sandwich" ELISA is
the method of choice, because reagents are commercially available in polyvalent and
monovalent formats for both toxin screening and serotype specific identification. An
automated enzyme-linked fluorescent immunoassay (ELFA) has been developed and is
commercially available. This method has undergone specificity and sensitivity evaluations
and has proven to be an effective serological system for the identification of staphylococcal
enterotoxin in a wide variety of foods .
Methods of SE detection now are available commercially and have reduced preparation
and detection time from several days to a few hours. Among most widely used, currently
available test kits belong TECRA kit (3M), VIDAS SET2 kit (BioMérieux), TRANSIA
PLATE Kit (Diffchamb), SET RPLA (Oxoid), RIDASCREEN (r-Biopharm).
In many cases, purification and concentrations techniques are needed prior to testing to
produce a sample able to be analysed. With regard to dairy products, concentration technique
based on trichloroacetic acid precipitation and dialysis concentration method against 30%
polyethylene glycol are used.
It is well known that one of the main drawbacks associated with EIA kits designed for
detecting SE, is the high frequency of false-positive results depending on the type of food
assayed as a result of cross-reaction with unrelated antigens .

Figure 2.1 Principle of the Sandwich ELISA 

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3. THE AIMS
The dissertation focuses on two main goals. The first one is to present a newly developed
real-time PCR-based method for the detection of Staphylococcus aureus in food and the other
one is to investigate and bring about more information on the behavior of S. aureus in the
dairy chain. In order to improve our understanding about S. aureus virulence in dairy products
we investigated:
 The effect of temperature on S. aureus enterotoxin D production in pasteurized milk
and brain heart infusion
 The combined influence of low temperature and background flora on S. aureus
enterotoxin D production in pasteurized milk
 The growth of S. aureus and enterotoxin A and D production in two types of cheese

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4. RESULTS AND DISCUSSION
4.1 Detection of S. aureus in food by PCR
A rapid and sensitive method for the detection of S. aureus in food based on real-time
polymerase chain reaction was developed. Specific primers and a TaqMan probe targeting a
103-bp DNA sequence of a gene coding for S. aureus acriflavin-resistant protein (AcrB) were
designed using Primer Express software. 83 S. aureus strains from either clinical collections
or as clinical or food isolates gave positive signals in PCR assay (100% inclusivity) and 64
non-S. aureus strains gave negative PCR results confirming 100% exclusivity of the method.
PCR detection limit determined using decimal dilutions of overnight cultures was 6.8  101
cfu/ml and 3.4  101 cfu/ml with 100% and 70% detection probability, respectively. Two
different selective media, modified Giolitti and Cantoni broth and Baird-Parker broth, at two
levels of artificial contamination and oxygen conditions were investigated to find out which
one gives better results. Giolitti and Cantoni medium at 37°C for 18 h under aerobic
conditions was selected for further work on the evaluation of the complete method for
foodborne S. aureus identification. Three DNA extraction methods were compared and since
all gave similar results the simplest and the least expensive one, the lysis by boiling with
Triton X-100, was the method of our choice. Finally, the developed method of selective
enrichment and real-time PCR detection was compared with the standard method (EN ISO
6888-3) by analysis of 112 food samples. 61 samples were found positive for S. aureus by our
method and 53 samples were found positive for S. aureus by the standard method. Ten food
samples were artificially contaminated with S. aureus at the levels of 4  101 and 4  100
cfu/g to evaluate the efficiency of the developed method. All ten samples were detected
positive by our method whereas one and four samples produced negative results using the
standard method at the levels of 101 and 100 cfu/g, respectively. Moreover, 100 cfu/10 g was
detected in all ten artificially contaminated samples by PCR-based method in contrast to
seven false negative by the standard method. All false negative results of S. aureus presence
by the standard detection were obtained in samples with natural background flora higher than
104 cfu/g.
The standard method for the detection of S. aureus in food (EN ISO 6888-3) is based on
selective enrichment and subsequent isolation of colonies with characteristic morphology and
identification by microbiological- and biochemical-based confirmations. This procedure is
time-consuming, labor-intenstive and may yield false-positive or false-negative results.
The methods based on real-time PCR referred to as rapid methods have shown a great
potential to overcome limitations of standard methods relating to sensitivity, specificity and
speed. Almost all methods used to detect specific pathogens in foods require some cultural
enrichment which prolongs assay speed. On the other hand, the enrichment step provides a
benefit in terms of diluting the effects of inhibitors coming from food matrix as well as
differentiation of viable from non-viable cells. The disadvantages that have to be considered

13
if PCR-based methods are to be applied to laboratories are the cost, molecular biology skills
and the implementation of excellent laboratory procedures to prevent cross contamination.
The proposed method can be used for S. aureus detection as a faster, more highly specific,
and more sensitive alternative to the microbiological method with its potential for providing
improved food-processing hygiene control.

4.2 Milk experiments

4.2.1 The effect of temperature on S. aureus enterotoxin D production in milk and BHI

The effect of three different temperatures, 8°C, 12°C and 20°C on S. aureus growth and
SED production in pasteurized milk and on growth, sed gene expression and SED production
in a rich chemically defined medium, Brain heart infusion, was investigated (Figure 4.1). The
amount of SED produced was correlated to bacterial growth to investigate if S. arueus
produces an increase amount of SED under sub-optimal growth conditions. A comparison
was made between the behaviour of S. aureus in a optimal bacgterial growth medium (BHI)
and a highly complex matrix, milk. The experiments were performed in small-scale
fermentors. Two independent growth experiments were performed for each temperature and
type of medium.
The growth pattern in BHI was the same at 20°C and 12°C but delayed in the latter. At 8°C
there was no growth observed. The growth in milk was lower compared to BHI at all
temperatures and the lag phase of the growth increased gradually with decreasing
temperatures. Expression profile of staphylococcal enterotoxin D studied in BHI showed that
transcription and translation patterns were correlated at 20°C and 12°C. sed mRNA was
detected at 20°C and 12°C after 4 and 7 hours respectively. The production of SED occurred
3 and 17 hours later and during the exponential phase of growth which contradicts the
generally quoted agr-dependent expression of sed gene induced during the transition from the
exponential to the stationary phase of growth. At 8°C, a high level of sed expression was
detected at 72 h but no protein synthesis occurred in response. In milk, SED production at
20°C and 12°C occurred earlier in growth but a lower total amount was produced compared to
BHI. At 8°C there was no SED production.
Research has been performed to identify key parameters that prevent or stimulate
enterotoxin production in laboratory media and in different food products and it was found
out that a multifaceted network of environmental and genetic factors is likely to be
responsible for the regulation of enterotoxin production 7.
Temperature is one of the identified environmental factors with impact on production of
c
staphylococcal enterotoxins. The minimal temperature needed to induce enterotoxin
production is 10°C and temperature seems to affect enterotoxin production more than growth.
Our investigation supports the knowledge about the behaviour of S. aureus under low
temperatures since no growth or enterotoxin D production was observed at 8°C.

14
 BHI 20C Milk 20C
1e+10 1e+10

300 300 300

1e+9 1e+9

SED [ng/ml]
SED [ng/ml]
200 200 200

cfu/ml
cfu/ml

RE
1e+8 1e+8
100 100 100

1e+7 0 0 1e+7 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

Time (hr) Time (hr)

BHI 12C Milk 12C

1e+10 1e+10

300 300 300

1e+9 1e+9
SED [ng/ml]

SED [ng/ml]
200 200 200
cfu/ml

cfu/ml
RE
1e+8 1e+8
100 100 100

1e+7 0 0 1e+7 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

Time (hr) Time (hr)

15
 BHI 8C Milk 8C
1e+10 1e+10

300 300 300

1e+9 1e+9

SED [ng/ml]

SED [ng/ml]
200 200 200
cfu/ml

cfu/ml
RE
1e+8 1e+8
100 100 100

1e+7 0 0 1e+7 0
0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140

Time (hr) Time (hr)

Figure 4.1 Growth of S. aureus, relative sed levels in BHI and SED concentration when grown at 20, 12 and 8°C in BHI and milk. () Growth curve; (yellow
bars) relative sed mRNA levels in BHI; (red bars) absolute SED concentration. The average of two independent growth experiments for each temperature and
growth media are presented.

16
4.2.2 The combined effect of 12°C and background flora

In these experiments, the combined influence of low temperature (12°C) and the presence of a
competitor (BGF originating from raw milk provided by ARLA Foods) on S. aureus growth
and enterotoxin A and D production in pasteurized milk with 3% fat content was investigated.
The competitor (BGF) was introduced by the addition of defined volumes (5, 1, 0.5 and 0.1 %
of the total volume; 600 ml) of raw milk to the pasteurized milk. In total, 5 runs of
experiments were carried out with two fermentors in parallel in each run, in length of 5 days
for each experiment.
In all experiments with the strain SA 45, very mild growth of S. aureus was observed
regardless the lower amount of BGF or higher starting concentration. If the starting
concentration of S. aureus was between 103 and 104 cfu/ml, the population never reached the
cell density (105 – 106 cfu/ml) to be considered hazardous for enterotoxin production. If the
starting concentration was higher,  106 cfu/ml, the growth was slight and within the same
order of magnitude. S. aureus is a well-known poor competitor. An inhibitory effect of BGF
on S. aureus growth and enterotoxin production was observed in all experiments. Despite the
low temperature as a stressful factor, the control experiments without BGF demonstrated that
the impact of a competitor is higher. The production of enterotoxin D was low and correlated
with the growth. By lowering the amount of competing microflora and increasing inoculation
level of S. aureus, only a slight increase in enterotoxin production occurred. No major strain
variation was observed when testing three different strains with different origin. pH was
measured in these experiments. The bigger the amount of competing microflora the bigger
and earlier in time the drop in pH due to accumulation of acidic products.
S. aureus and/or enterotoxins can enter the pasteurized milk chain on the farm and can
continue to be introduced or exacerbated at any point in the dairy chain until the milk reaches
the consumer. A probabilistic model for the representation of the risks that arise from the
presence of S. aureus, and particularly staphylococcal enterotoxins, in the pasteurized milk
chain, has been developed by the Institute of Food Research, Norwich, United Kingdom
within the EU-funded IP project BIOTRACER. The probabilistic analysis has been
implemented using a Bayesian belief network (BBN) technique. BBNs are a type of expert
system that integrates a graphical representation of a hazard domain with a probabilistic
model of the events. The obtained experimental data set described above, especially the
correlation between S. aureus growth and enterotoxin production kinetics, was incorporated
in the Bayesian belief network in order to update the model.

17
 5% Raw milk Control
1e+10 8,0 3,0 1e+10 8 3,0

1e+9 7,0 7
2,5 1e+9 2,5
6,0 6
1e+8 1e+8
2,0 2,0

SED [ng/ml]

SED [ng/ml]
5,0 5
1e+7
cfu/ml

1e+7

cfu/ml
pH

pH
4,0 1,5 4 1,5
1e+6 1e+6
3,0 3
1,0 1,0
1e+5 1e+5
2,0 2
0,5 0,5
1e+4 1,0 1e+4 1

1e+3 0,0 0,0 1e+3 0 0,0

0 20 40 60 80 100 120 0 20 40 60 80 100 120
Time (hr) Time (hr)

1% Raw milk Control

1e+10 8 3,0 1e+10 8 3,0

1e+9 7 1e+9 7
2,5 2,5
6 6
1e+8 1e+8
2,0 2,0

SED [ng/ml]
SED [ng/ml]
5 5
1e+7 1e+7

cfu/ml
cfu/ml

pH
pH

4 1,5 4 1,5
1e+6 1e+6
3 3
1,0 1,0
1e+5 1e+5
2 2
0,5 0,5
1e+4 1 1e+4 1

1e+3 0 0,0 1e+3 0 0,0

0 20 40 60 80 100 120 0 20 40 60 80 100 120

Time (hr) Time (hr)

18
 0,5% Raw milk
1e+10 8 3,0

1e+9 7
2,5
6
1e+8
2,0

SED [ng/ml]
5
1e+7
cfu/ml

pH
4 1,5
1e+6
3
1,0
1e+5
2
0,5
1e+4 1

1e+3 0 0,0
0 20 40 60 80 100 120

Time (hr)

Figure 4.2 (for all graphs) Growth of S. aureus (SA 45) and background flora coming from raw milk,
pH and SED production in milk at 12°C. () Growth curve for S. aureus; (০) Growth curve for BGF;
(▲) pH; (red bars) absolute SED concentration. No raw milk was added to milk in the control
experiments. One growth experiment was performed for 5% and 1% raw milk and corresponding
control experiments. Two independent growth experiments were performed for 0.5% raw milk
experiment.

a)

0.5% Raw milk

1,E+10 8 161:3 - VC
1,E+09 7
168:1 - VC
1,E+08 6
161:3 - TC
5
cfu/ml

1,E+07
168:1 - TC
pH

4
1,E+06
3 161:3 - pH
1,E+05 2 168:1 - pH
1,E+04 1
1,E+03 0
0 20 40 60 80 100 120 140 160

Time (h)

19
 b)

0.5% Raw milk

3,5
3
[SEA] ng/ml

2,5
2 SA 161:3
1,5 SA 168:1
1
0,5
0
0 12 24 48 72 96 120 144

Time (h)

c)

0.1% Raw milk

1,00E+10 8
7,5
1,00E+09
7 F1-VC
1,00E+08 6,5 F2-VC
6
cfu/ml

1,00E+07 F1-TC
pH

5,5
1,00E+06 F2-TC
5
F1-pH
1,00E+05 4,5
4 F2-pH
1,00E+04
3,5
1,00E+03 3
0 24 48 72 96 120 144 168

Time (h)

Figure 4.3 Growth of S. aureus (SA 161:3 and SA 168:1) and background flora coming from raw
milk, pH and SEA production in milk at 12°C. a) Growth of S. aureus described as viable count (VC),
growth of BGF described as total count (TC), pH; b) absolute SEA concentration; c) Growth of S.
aureus described as viable count (VC), growth of BGF described as total count (TC), pH. Two
independent experiments with the strain 161:3.

20
4.3 Cheese experiments
These experiments were set up to simulate a post-contamination scenario during cheese
production. The growth and enterotoxin A and D production of four S. aureus strains with
different origins were investigated in two types of cheese at two different temperatures.
Four different experiments were performed. 25 g of semi soft cheese or cream cheese were
inoculated with one of four stains of S. aureus and incubated at 13°C or 20°C. Two biological
replicas were made (A+B) for each of the four S. aureus strains tested. In total, 54 cheese
samples were used in each experiment. Samples were collected after 0 hour, 3 days, 6 days,
13 days, 20 days (or 19 days) and 27 days. Growth of S. aureus was followed by viable count
measurements on Baird-Parker agar plates, BHI or TSA plates were used for total cell count
and MRS plates for cell count of lactic acid bacteria. The enterotoxins were extracted and
concentrated using dialysis, and finally the enterotoxin levels were measured with ELISA.

a) b)

Figure 4.4 a) Cream cheese samples inoculated with different strains. b) Example of large
unidentified colonies on BHI.

21
4.3.1 Experiment 1: Semi-soft cheese, 13°C

a)

b)

c)

22
d)

e)

Figure 4.5 a) Growth of S. aureus strains 161:3, 168:1, 45 and 564 in semi-soft cheese at 13°C. Two
independent replicas, A and B. b) Total amount of enterotoxin A found in the 25 g of cheese samples,
measured with ELISA. c) Total amount of enterotoxin D found in the 25 g of cheese samples,
measured with ELISA. d) Total cell count (TCC) and the cell count of lactic acid bacteria (LAB) in the
cheese samples. Average of two independent replicas, A and B, for each sample. e) Total cell count
(TCC), lactic acid bacteria (LAB) cell count and S. aureus cell count in the control samples.

23
4.3.2 Experiment 2: Semi-soft cheese, 20°C

a)

b)

c)

24
d)

e)

Figure 4.6 a) Growth of S. aureus strains 161:3, 168:1, 45 and 564 in semi-soft cheese at 20°C. Two
independent replicas, A and B.  Samples contaminated with mould. b) Total amount of enterotoxin A
found in the 25 g of cheese samples, measured with ELISA. c) Total amount of enterotoxin D found in
the 25 g of cheese samples, measured with ELISA. d) Total cell count (TCC) and the cell count of
lactic acid bacteria (LAB) in the cheese samples. Average of two independent replicas, A and B, for
each sample. e) Total cell count (TCC), lactic acid bacteria (LAB) cell count and S. aureus cell count
in the control samples.

25
4.3.3 Experiment 3: Cream cheese, 13°C

a)

b)

c)

26
d)

Figure 4.7 a) Growth of S. aureus strains 161:3, 168:1, 45 and 564 in cream cheese at 13°C. Two
independent replicas, A and B. Samples contaminated with mould. b) Total amount of enterotoxin A
found in the 25 g cheese samples, measured with ELISA. c) Total amount of enterotoxin D found in the
25 g cheese samples, measured with ELISA. d) Total cell count in the cheese samples. Average of two
independent replicas, A and B, for each sample.

27
4.3.4 Experiment 4: Cream cheese, 20°C

a)

b)

c)

28
d)

Figure 4.8 a) Growth of S. aureus strains 161:3, 168:1, 45 and 564 in cream cheese at 20°C. Two
independent replicas, A and B.  Samples contaminated with mould. b) Total amount of enterotoxin A
found in 25 g cheese samples, measured with ELISA. c) Total amount of enterotoxin D found in 25 g
cheese samples, measured with ELISA. d) Total cell count in the cheese samples. Average of two
independent replicas, A and B, for each sample.

The semi-soft cheese used in the experiments contained a high level of background flora
(LAB). Several studies have demonstrated that the ratio between the inoculation level of S.
aureus and the number of LAB determines the efficiency of the inhibition. For example, with
ratios of 1:1 or 1:10 (S. aureus : L. lactis) which approximately corresponds to ratios (S.
aureus : LAB) in the experiment 1 and 2, respectively, the maximal level which S. aureus can
reach is 105 – 106 cfu/ml, compared to 1010 cfu/ml in a control culture 24. This finding can
be supported by our results with one exception, when population of S. aureus 45 replica B
crossed 107 cfu/g of cheese in the experiment 1. Even in the samples contaminated with mold,
which frequently boosted S. aureus growth, the population of S. aureus did not grow higher
than 106 cfu/g. LAB, similarly to S. aureus, can grow in a wide range of temperature but
prefer higher temperature, above 30°C. The growth of LAB was better at 20°C (exp. 2). The
number of LAB also tended to be higher than the total count at 20°C. The reason could be that
LAB due to limited biosynthetic ability and thus requirements for special nutrients like amino
acids or vitamins do not grow on BHI agar as well as on MRS which was designed to recover
LAB from various food products. In general, no overall increase in growth of any strain
during 27 days in semi-soft cheese was observed. S. aureus 45 despite being isolated from
ham was the best-growing strain, always present over time in higher or lower counts
compared to inoculum in both replicas. Out of strains isolated from dairy products, the strain

29
161:3 had rather the tendency to die off, the strain 168:1 seemed to have a greater ability to
compete. The behaviour of the strain 564 varied, with at least one replica always survived till
27 days.
The cream cheese seemed to lack background flora. No colonies were observed on MRS
agar plates when analyzing control samples showing that the cream cheese is void of LAB (or
at least those species of LAB that can be recovered on MRS agar). In both experiments 3 and
4, the total cell count more or less corresponded to the cell count of S. aureus. The growth of
large unidentifed colonies of irregular shape and size on BHI or TSA agar plates used for total
cell count made the counting difficult and less accurate. In the experiment 3, the population of
all S. aureus strains started with high numbers and kept surviving for 13 days and then
declined except in the contaminated samples. In the experiment 4, starting with lower
inoculum of S. aureus strains, despite the higher temperature the situation was similar,
although some strains were not detected even at 13 days. The technology applied to make the
cream cheese seemed to create an unfavourable environment for S. aureus since no growth
was observed despite the absence of competing microflora.
According to commission regulation (EC) No 1441/2007 on microbiological criteria for
foodstuffs, 25 g of cheese must be void of staphylococcal enterotoxins and there is an
obligation to screen for enterotoxins if S. aureus concentration exceeds 105 cfu/g in any food.
In all experiments, SEs could not be detected before 19 days. In the experiment 1 (semi-soft
cheese, 13°C), S. aureus 45 produced quite a high amount of enterotoxins, more SEA than
SED, in response to high cell counts (106 cfu/g in one sample and above 107 cfu/g in the
other). The counts of other S. aureus strains were not high enough (or due to other conditions)
to trigger SE production. In all the other experiments, we encountered a contamination by
mold which created an interesting phenomenon and which is discussed below.
In the experiment 2 (semi-soft cheese, 20°C), two contaminated samples with 106 cell counts
of strains 45 and 564 contained a huge amount of enterotoxins. On the contrary, 2
contaminated samples with the same cell counts (of the strain 564 in one sample) showed no
enterotoxins. In addition, enterotoxin A and D could be detected in two replica samples with
no contamination and with counts of 106 and 104 cfu/g of S. aureus strain 45. The possible
explanation of the last could be a mistake during the analysis. The extraction of enterotoxin
consists of many steps performed manually, such as acidification followed by neutralization
and finally dialysis where especially the recovering of toxins from the dialysis membrane can
result in inaccuracy. The explanation of the detection of enterotoxin in the sample with lower
counts (104 cfu/g) of S. aureus could be that some toxin was formed and stayed but the
number of cells declined over time. In the experiments with cream cheese, enterotoxin could
only be detected after 27 days. In the experiment 3 (cream cheese, 13°C) with high inoculum
of all S. aureus strains, enterotoxin was detected in two contaminated samples, in the sample
with low cell counts (102 cfu/g) and in the sample with no viable cells. Detection of
enterotoxin D in the sample with no viable cells of S. aureus 45 could be a mistake since the

30
strain 45 produces both enterotoxins, A and D and we could detect only D. In the experiment
4 (cream cheese, 20°C) a massive enterotoxin production occurred in two contaminated
samples with high cell counts (106 cfu/g) increased from low initial inoculum level.
TSST was not detected in any of the samples with TSST producing strains, 168:1 and 564.
This might be due to unfavorable experimental conditions for TSST production.
Cheese represents one of the more challenging matrices to work with. In the experiments
with semi-soft cheese, the independent replicas A and B seem to differ slightly more in the
number of S. aureus cells than replicas in the cream cheese experiments. This could be due to
semi-soft cheese being a less homogenous food matrix (with holes, different composition in
different parts of the cheese, etc.) and/or due to the fact that the cheese samples did not obtain
the same amount of inoculum. Despite great effort to reach the same initial inoculum, some
drops might have ended up on the bottom of the plates containing the cheese samples (holes
in cheese). Furthermore, there are several steps in the analysis that might result in loss of
cells. For instance, when transferring the diluted sample from the stomacher bag into new
tubes, some cells might still remain inside the bag.
Many cheese samples got contaminated by mould. The contamination was probably caused
by spores from the environment since moulds are ubiquitous, possibly during preparation and
inoculation of samples. Visible mould was never detected before 19 days. The phenomenon
observed in most contaminated samples was that mould stimulated the growth of S. aureus
and triggered a significant enterotoxin production. A huge enterotoxin production was
accompanied by an increase of the pH to 7.3 in one semi-soft cheese sample and to 5.9 and
6.4 in two cream cheese samples. Moulds have a complex enzyme system and as they
proliferated on the cheese samples they might have consumed lactic acid and have formed
alkaline metabolites due to proteolysis which led to an increase in pH 18, 23. The other
factors coming into play are what type and when mould appeared (was visible) and how it
interacted with other microorganisms present in the cheese including different strains of S.
aureus. More research would be needed to elucidate the encountered phenomenon but it
would have little impact on food safety since a consumer would not eat mouldy cheese.
However in some circumstances, very rare probably, a block of cheese gets contaminated with
S. aureus, is kept under inadequate temperature and mold develops on the surface. The moldy
area is cut away, cheese sold and possible outbreak born. The mould with its high metabolism
was not the only reason contributing to the increase of pH. The overall change in pH was
bigger in semi-soft cheese as a consequence of the biochemical changes occurring during
ripening and continuing over time in the experiments.

31
5. CONCLUSIONS
We are confronted with the ongoing challenge to control foodborne diseases caused by
bacteria. Although we know a great deal about these bacteria, they are still causing significant
problems in the food industry. The development of rapid methods for detecting foodborne
pathogens and the improvement of our understanding of pathogenic virulence is critical to
ensure the food safety.
The work described in this dissertation has provided a new method for rapid and sensitive
detection of S. aureus in food and new information about S. aureus and its enterotoxin
formation in the dairy chain. Here is a brief summary of the conclusions from:

Food pathogen detection

 The developed real-time PCR based method involving overnight selective enrichment
under aerobic conditions facilitated sensitive next-day S. aureus detection. The method
was able to overcome the problematic colony identification on Baird-Parker agar,
particularly in the case of atypical S. aureus colonies.

Milk experiments
 The effect of temperatures 20, 12 and 8°C on S. aureus growth and enterotoxin D
production in milk and BHI showed that growth was lower in milk compared to BHI.
SED production in milk at 20°C and 12°C occurred earlier in growth but a lower total
amount was produced compared to BHI. At 8°C, there was no growth and SED
production.
 The presence of competing microflora frow raw milk created an inhibitory effect on S.
aureus growth and enterotoxin production. The impact of competition was higher than
low temperature (12°C). Low SED production at S. aureus inoculation levels from 103 to
106 cfu/ml was observed. By lowering the amount of competing microflora and
increasing inoculation level of S. aureus, only a slight increase in enterotoxin production
occurred. No major strain variation was observed when testing three different strains
with different origin.
 Studies performed in laboratory media do not necessarily reflect the situation in a real
food product. Food represent a more complex matrix with different types of
microorganisms often present, which interact with each other and the matrix.

Cheese experiments
 The different origin of the strains did not influence the ability to survive, grow or
produce enterotoxin in semi-soft or cream cheese.
 No overall increase in growth of any strain during 27 days was observed.

32
 Enterotoxin production was never detected before 19 and 27 days in semi-soft cheese
and cream cheese, respectively. The amount of enterotoxin produced was sufficient to
cause food poisoning.
 Mould contamination often caused increase in pH and promoted growth of S. aureus
with concomitant enterotoxin production.
 No generalizations should be made about the behaviour of S. aureus in the types of
cheese other than those studied.

33
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7. LIST OF ABBREVIATIONS

AcrB acriflavin-resistant protein

Agr accessory gene regulator
BGF background flora
BHI brain heart infusion
BPA Baird-Parker agar
cfu colony forming unit
egc enterotoxin gene cluster
ELISA enzyme-linked immunosorbent assay
EIA enzyme immunoassay
FBD foodborne disease
LAB lactic acid bacteria
MPN most probable number
PCR polymerase chain reaction
RT-PCR reverse-transcription polymerase chain reaction
SAPI Staphylococcus aureus pathogenicity island
scc staphylococcal cassette chromosome
SE staphylococcal enterotoxin
SEl staphylococcal enterotoxin-like proteins
SEA staphylococcal enterotoxin A
SED staphylococcal enterotoxin D
SFP staphylococcal food poisoning
TCC total cell count
TSA tryptic soy agar
TSST toxic shock syndrome toxin
VC viable count

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8. CURRICULUM VITAE

Name: Vendula Hrušková

Date of Birth: 21.3.1983
Address: Zámecká 143, 747 57 Slavkov, Czech Republic

Education
1998 – 2002 Grammar school in Opava
2002 – 2007 Master student at Faculty of Chemistry, Brno University of Technology
Diploma thesis: Identification of S. aureus in food using polymerase
chain reaction
2007 – PhD student at Faculty of Chemistry, Brno University of Technology
Doctoral thesis: Foodborne S. aureus: Identification and enterotoxin
production in milk and cheese

Abroad research studies

2007 – 2008 Food Research Institute, Bratislava
2008 – 2010 Department of Applied Microbiology, Lund University, Sweden

Publications

Papers

1. Trnčíková, T., Hrušková, V., Oravcová, K., Pangallo, D., Kaclíková, E. Rapid and
sensitive detection of Staphylococcus aureus in food using selective enrichment and
real-time PCR targeting a new gene marker. Food Analytical Methods, 2008, vol. 2,
241-250 p.
2. Hrušková, V., Kaclíková, E. Rapid and sensitive detection of pathogenic Yersinia
enterocolitica strains in food using selective enrichment and real-time PCR. Journal of
Food and Nutrition Research, vol 48, 2009, 100-108 p.

Abstracts – international congresses

3. Schelin, J., Bordignon, S., Hrušková, V., Rådström, P. Production of S. aureus

enterotoxin D in pasteurized milk and Brain Heart Infusion at 8, 12 and 20°C. 3rd FEMS
Congress of European Microbiologists, Gothenburg, Sweden, Book of Abstracts, 2009.
4. Bordignon, S., Hrušková, V. Carlquist Wallin, N., Rådström, P., Schelin, J. Production
of Staphylococcus aureus enterotoxin A and D in cheese. 22nd International IFCMH
Symposium, Food Micro, Copenhagen, Denmark, 2010.

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9. ABSTRACT
Foodborne diseases caused by bacteria are an actual issue worldwide. To produce food, which
is safe for human consumption, data about food-borne pathogen virulence is required to
complement the already existing knowledge about the bacterial growth and survival in food.
There is also a growing need for rapid and sensitive methods to detect these pathogens.
In this dissertation, the real-time PCR-based method for the detection of S. aureus in food
using selective enrichment and the impact of environmental factors on S. aureus growth and
enterotoxin production in milk and cheese are described.
We developed a rapid and sensitive method for the detection of S. aureus in food using
selective enrichment and a new species-specific real-time PCR. The method facilitated the
detection of S. aureus on the next day after the sample reception. This method can be used for
S. aureus detection as a faster, highly specific, and more sensitive alternative to the
microbiological method.
We investigated the effect of three different temperatures, 8°C, 12°C and 20°C on S.
aureus growth and SED production in pasteurized milk and on growth, sed gene expression
and SED production in Brain heart infusion. The experiments were performed in small-scale
fermentors for six days and gene expression was followed by qRT-PCR. SED production was
measured using Enzyme-Linked ImmunoSorbent Assay (ELISA). In BHI the growth pattern
was the same at 20°C and 12°C but delayed in the latter. At 8°C there was no growth. In milk,
growth was lower compared to BHI. sed mRNA was detected at 20°C and 12°C after 4 and 7
hours respectively in BHI and the production occurred during the exponential phase of
growth. In milk the SED production at 20°C and 12°C occurred earlier in growth but a lower
total amount was produced compared to BHI. At 8°C, there was no SED production like in
BHI. The combined effect of low temperature, 12°C, and the presence of competing
background microflora derived from raw milk on the growth of S. aureus and SED production
in pasteurized milk was further investigated. An inhibitory effect on S. aureus growth and
enterotoxin production was observed and the impact of competition was greater than the
impact of low temperature. The enterotoxin production was low and correlated with the
growth. By lowering the amount of competing microflora and increasing the inoculation level
of S. aureus, only a slight increase in enterotoxin production occurred. In the next stage, two
different cheese matrices were inoculated with S. aureus to simulate a post-contamination
scenario in cheese manufacture. Samples were collected over period of 4 weeks. Critical food
factors, like competing microflora and pH, which are responsible for down- and up-regulation
of the virulence of S. aureus, were monitored. We tried to indentify if there are situations in
which: (i) no growth but enterotoxin formation is observed, and (ii) growth and no
enterotoxin formation occurs.

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