Bacillus Cereus Cereulide and Staphylococcus Aureus Enterotoxin Production in Lasagna

Faculteit Bio-ingenieurswetenschappen Academiejaar 2011-2012

Bacillus cereus cereulide and Staphylococcus aureus enterotoxin production in lasagna

Hanne ALBOORT

Promotor: Prof. dr. ir. Mieke Uyttendaele Co-Promotor en Tutor: Prof. dr. Andreja Rajkovic

Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen:LEVENSMIDDELENWETENSCHAPPEN EN VOEDING

Woord vooraf

Nu deze thesis naar het einde loopt had ik graag enkele mensen bedankt die er mede voor gezorgd hebben dat deze thesis tot stand is kunnen komen. Hierbij had ik graag Prof. dr. ir. Mieke Uyttendaele bedankt voor het opnemen van het promoterschap. Een zeer grote dank gaat uit naar mijn co-promotor en begeleider, Prof. dr. Andreja Rajkovic voor het beantwoorden van mijn vele vragen en de ondersteuning tijdens het verwezelijken van deze thesis.

Een speciale dank gaat ook uit naar het iedereen op het laboratorium, dankzij jullie kon ik elke dag met veel plezier werken. Ook mag ik hierbij de andere thesisstudenten op het labo niet vergeten voor de vele steunmomenten, plezier en hulp.

Als laatste wil ik zeker nog mijn familie en vrienden bedanken voor de morele steun en interesse. Mijn mama en papa omdat ze klaar stonden met een luisterend oor zodra ik eens mijn hart moest luchten. Mijn broer en zus waarbij ik echt voor alles terecht kan. Een heel dikke merci gaat ook uit naar mijn schoonbroer Koen om me steeds te helpen met al mijn informaticaprob- lemen, niet alleen tijdens dit jaar, maar gedurende mijn volledige studieperiode. Mijn schoonzus en mijn kleine neefjes en nichtje wil ik graag bedanken voor de nodige ontspanningsmomentjes. Tot slot wil ik ook nog mijn vrienden danken voor de vele steunberichtjes net op de momenten dat ik het nodig had.

Hanne Alboort, Gent, juni 2012

i Toelating tot bruikleen

De auteur en de promotoren geven de toelating deze scriptie voor consultatie beschikbaar te stellen en delen ervan te kopi¨erenvoor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting de bron te vermelden bij het aanhalen van resulaten uit deze scriptie.

The author and the promotors give the permission to use this thesis for consultation and to copy parts of it for personal use. Every other use is subject to the copyright laws; more speciﬁcally the source must be speciﬁed when using results from this thesis.

Gent, juni 2012

De auteur, De promotor, De co-promotor,

Hanne Alboort Prof. dr. ir. Mieke Uyttendaele Prof. dr. Andreja Rajkovic

ii Abstract

In this thesis it was the goal to investigate the safety of ready-to-eat meals, exemplified by lasagna. These foods can be stored for a prolonged period of time at home in the refrigera- tor. As these types of foods are gaining in popularity, the safety concerning two relevant toxin producing bacteria was investigated. The bacteria investigated in this thesis were the emetic toxin (cereulide) producing B. cereus and the staphylococcal enterotoxin producing S. aureus. The toxins produced from these two bacteria cannot be removed anymore by a subsequent heat process once they are formed. Furthermore, B. cereus is a sporeforming bacteria and can thus survive the processes in the factory and remain as spore in these ready-to eat foods. On the other hand, S. aureus is often an issue of postheating contamination due to its presence on hair and skin of the workers in the processing environment. The possible growth and toxin production of these two bacteria was investigated during their storage. Two storage temperatures of 12°C and 22°C were chosen; 12°C indicating a severe temperature abuse at home and 22°C, the temperature if storage is at room temperature. These lasagnas were packaged in a modified atmosphere packaging, as ready-to-eat meals are conserved in a reduced oxygen environment. Additionally some lasagna was also stored at ambient atmosphere conditions to distinguish the influence of this reduced oxygen content on the growth and toxin production of both bacteria.

Before the food based experiments started, the possible growth and toxin production of B. cereus and S. aureus was investigated in laboratory media at modiﬁed atmosphere. The laboratory media chosen were Tryptone Soya Agar and Tryptone Soya Broth, both general growth media. In these media, the growth and subsequent toxinproduction was very fast. Staphylococcal enterotoxins were found after 20 hours and cereulide could be found after 43 hours.

In the lasagna, staphylococcal enterotoxins could be found after 43 hours at 22°C. At 12°C, the staphylococcal enterotoxins were detected from day 10. At 12°C a signiﬁcant decrease in growth could be seen if the lasagna was stored at modiﬁed atmosphere conditions, which resulted in a delay of onset of staphyloccal enterotoxins production.

Cereulide could be found in lasagna at 22°C at day 3, if stored ambient. If the lasagna was stored in a modified atmosphere, the onset of toxin production was delayed with one day, showing the influence of the reduced oxygen on the growth and subsequent toxin production. Moreover, the reduced oxygen content had an influence on the critical density necessary to start the cereulide toxin production, resulting in a higher critical density necessary in the modified atmosphere stored lasagna. At 12°C, no cereulide could be detected in the lasagna.

iii Contents

Woord vooraf i

Toelating tot bruikleen ii

Abstract iii

1 Introduction 1

2 Literature review 2 2.1 Bacillus cereus ...... 2 2.1.1 General characteristics and background information ...... 2 2.1.2 B. cereus detection ...... 3 2.1.3 The emetic toxin cereulide ...... 3 2.1.4 Food sources ...... 7 2.1.5 Cereulide detection ...... 9 2.1.6 Prevalence ...... 10 2.2 Staphylococcus aureus ...... 10 2.2.1 General characteristics and background information ...... 10 2.2.2 S. aureus detection ...... 12 2.2.3 S. aureus enterotoxins ...... 12 2.2.4 Food sources ...... 15 2.2.5 Staphylococcal enterotoxin detection ...... 16

3 Materials and methods 18 3.1 S. aureus and B. cereus strains used in the experimental setup ...... 18 3.2 Propagation of the strains ...... 18 3.3 B. cereus spore production ...... 18 3.4 Bacterial enumeration ...... 19 3.5 General experimental setup ...... 19 3.5.1 General setup with Tryptone Soya Broth ...... 19 3.5.2 General setup with Tryptone Soya Agar ...... 19 3.5.3 General setup with ready-to-eat lasagna ...... 20 3.5.4 Bacteria enumeration from samples ...... 21 3.6 Toxin tests ...... 22 3.6.1 Staphylococcal enterotoxin test ...... 22 3.6.2 B. cereus cereulide toxin test ...... 22 3.7 Result interpretation ...... 24 3.8 Overview of the diﬀerent experimental setups ...... 24

iv CONTENTS v

4 Results and discussion 26 4.1 Maximum cell densities of tested strains in TSB-broth ...... 26 4.2 Spore forming by B. cereus ...... 26 4.3 Staphylococcal enterotoxin production by S. aureus strain ...... 27 4.4 Cereulide production by B. cereus strains ...... 27 4.5 Cereulide extraction from lasagna ...... 27 4.6 Growth of S. aureus and B. cereus strains in modiﬁed atmosphere in TSB . . . . 27 4.7 Experimental setup with TSA ...... 29 4.7.1 Growth of S. aureus and B. cereus on TSA ...... 29 4.7.2 Toxin production during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores on TSA ...... 30 4.8 Experimental setup with Lasagna ...... 32 4.8.1 pH and aw-value lasagna ...... 32 4.8.2 Measured gas concentration in fresh lasagna ...... 32 4.8.3 Experiment with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 434 spores in lasagna ...... 32 4.8.4 Experiment with B. cereus LFMFP 434 vegetative cells in lasagna . . . . 36 4.8.5 Experiment with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 436 spores in lasagna ...... 38 4.9 Discussion ...... 42 4.9.1 Growth and toxin production in TSB-broth ...... 42 4.9.2 Growth and toxin production on TSA ...... 42 4.9.3 Growth and toxin detection in lasagna at 22◦C ...... 43 4.9.4 Growth and toxin detection in lasagna at 12◦C ...... 45 4.9.5 Eﬀect of the combination of low residual oxygen content and temperature on the toxin production ...... 47

5 Conclusions 48

6 List of abbreviations 50

Bibliography 51 Chapter 1

Introduction

Bacillus cereus is an omnipresent spore forming bacterium and therefore it is almost impossi- ble to completely avoid it in the food processing area or on raw materials. These spores are furthermore very heat resistant, so pasteurization heat treatment will not be able to eliminate the spores. The spores can thus survive processing conditions and if there is subsequent time / temperature abuse where the spores can germinate, they can multiply to alarming numbers. B. cereus has moreover the ability to form toxins during the growth phase. One of the important toxins of B. cereus is the emetic toxin named cereulide. This toxin is very heat and acid resistant and once it is formed, it cannot be eliminated by a heat treatment. Another important bacterium is Staphylococcus aureus. This bacterium can come in a product as a cross contamination because it is present in 20-30% of the population. This bacterium has also the possibility to produce diﬀerent toxins. Food relevant are the Staphylococcal enterotoxins which are also very heat and acid resistant, but to a smaller extent then cereulide. With the properties of these two bacteria species in mind it is important to investigate the safety of ready-to-eat meals. These are meals prepared with a mild heat treatment and can be stored for a long time at home at refrigerated conditions. These meals are gaining in popularity because the consumer nowadays doesn’t want to spend a lot of time in preparing meals. So these ready-to-eat meals where the consumer only has to heat up at home are very convenient. These products are made from a wide variety of ingredients and thus there are many sources where the bacteria can come from.

The consumer is also a very important link in the food safety. The cold chain can be maintained at the producer and retailer side, but the consumer has also the responsibility to maintain the cold chain at home. Frequent temperature abuses at homes are reported (James et al., 2008).

In this thesis it was the goal to investigate the potential of B. cereus and S. aureus to produce their selected toxins in ready-to-eat meals, exemplified by lasagna. Food was stored at 12◦C; this is a temperature abuse at home where the fridge is not set cold enough. The other storage temperature was 22◦C, to see how fast the bacteria can grow and produce toxins when the products are held at room temperature. The same was done with laboratory media to evaluate the effect of food matrix. This can be important to see if results from laboratory media can be used as an information source. The lasagna’s and laboratory media were also stored in a package with modified atmosphere with some oxygen and the remainder nitrogen. These results could be compared with lasagna stored at ambient atmosphere to see the influence of the modified atmosphere on the growth and onset of toxin production.

1 Chapter 2

Literature review

2.1 Bacillus cereus

2.1.1 General characteristics and background information Bacillus cereus is a gram positive pathogen. It is omnipresent in nature, soil, dust and plants. B. cereus can even survive in the human intestinal tract. It can survive harsh environmental conditions, because of its spore forming properties (Stenfors Arnesen et al., 2008). Some strains of B. cereus are mesophilic, while others are more psychrotrophic and can grow at temperatures of 4-6℃ (Granum and Baird Parker, 2000a). Some generally accepted physical characteristics of the mesophilic B. cereus vegetative cells are represented in Tabel 2.1.

On microscopic examination they look like large rods and are motile because of their ﬂagella. They have a diameter of 1-1.2 µm and length of 3.0 to 5.0 µm (Bennett, 2001a). B. cereus can grow both aerobically and anaerobically (Granum and Barid Parker, 2000a). The spores are resistant towards heat, acid, dehydratation and other physical stresses. Some spores can survive D121°C of 0.03 minutes. Other spores are more heat resistant and are able to survive D121°C of 2.35 minutes. As soon as the conditions are more ideal, the spore germinates (ICMSF, 1996a). B. cereus sporulates easily after 2 to 3 days in most media (Granum, 2005).

Table 2.1: Physical properties B. cereus vegetative cells

Physical property Value Source Temperature Growth 10-48°C Carlin et al. (2006) range (mesophilic strain) Optimal growth range 28-40°C Notermans and Batt (1998) (mesophilic strain) pH growth range 5-8.8 ICMSF (1996a) Optimal pH growth range 6-7 ICMSF (1996a) Min aw 0.93 ICMSF (1996a)

B. cereus can enter the food chain as a spore or vegetative cell. It can come into the production area, because it can attach to the incoming materials. Besides, the spore of B. cereus has characteristics such as hydrophobicity, exospores, appendages and pili. In this way, the spore attaches good at the materials in the production area. It has also the potential to form a bioﬁlm, whereby the vegetative cells and spores are protected towards the cleaning and disinfection materials used to clean the installations (Stenfors Arnesen et al., 2008).

2 CHAPTER 2. LITERATURE REVIEW 3

Other ways which B. cereus can come into the food chain are the contamination of the udders of cows. Soil has been shown to contain 105 to 106 spores/g. So contamination during grazing or because the bedding is contaminated can be the cause. This way, milk can be contaminated with B. cereus (Stenfors Arnesen et al., 2008).

2.1.2 B. cereus detection Knowing that two types of B. cereus strains exist (globally spoken diarrheal and emetic toxin producers), it is important to note that there are some speciﬁc characteristics by which the emetic B. cereus strains can be distinguished from other B. cereus strains. The emetic strain have no or weak haemolysis and have the inability to hydrolyse starch and salicin. They have a rather elevated upper growth temperature limit of 10°C. The spores show to have a higher D-value and a higher survival after 120 min at 90°C. The spores are thus more heat resistant at 90°C and also have a lower germination at 7°C (Carlin et al., 2006) .

Selective plating media can be used to find the amount of B. cereus in contaminated foods. Some of the commonly present features of B. cereus are used for identification purposes. MYP (Mannitol Egg Yolk Polymyxin) agar consists of mannitol, egg yolk, phenol red and polymyxine B. Phenol red is a pH indicator and mannitol is used because B. cereus cannot ferment this carbohydrate and consequently there will be no acid production. Polymyxine B is a selective antibiotic active mainly against G- bacteria. Another property of B. cereus is the lecithinase production. Because of the presence of lecithin in egg yolk and the lecithinase production of B. cereus, a precipitation zone will occur. B. cereus has also B-haemolysis properties, so a confirmation test can be performed on sheep blood agar (Stenfors Arnesen et al., 2008).

2.1.3 The emetic toxin cereulide B. cereus food poisoning B. cereus can cause, in addition to the foodborne diseases, septicemia, meningitis and ocular infections (Drobniewski, 1993) Two food borne diseases, an emetic type and a diarrhoeal type can be associated to B. cereus. These syndromes are related to the toxins produced by B. cereus. The diarrhoeal type is caused by enterotoxins. There are four toxins that are important, namely the poreforming cytotoxins haemolysin BL (Beecher et al., 1995), nonhaemolytic enterotoxin (Lindb¨ack et al., 2004), cytotoxin K (Lund et al., 2000) and hemolysin II (Baida et al., 1999). The emetic disease is caused by a small ring-shaped peptide called cereulide. The intoxication because of the emetic type of B. cereus is caused by a high concentration of preformed cereulide in food (Ehling-Schulz et al., 2004). The toxico-intoxication caused by the diarrheal type of B. cereus is caused by the ingestion of viable cells or spores in food, which can grow and produce enterotoxins in the intestines (Granum et al., 1993).

It is however diﬃcult to know how many cases of food borne illnesses are attributed to B. cereus. One reason is that only cases of outbreaks are reported and no individual cases, which leads to an underestimation. This is related to the generally mild and self-resolving symptoms and people don’t go to the doctor, or the doctor doesn’t recognize the right food intoxication. Fur- thermore does the cereulide intoxication really resembles other foodborne diseases. For example, the intoxication caused by staphylococcal enterotoxins produced by Staphylococcus aureus can give similar symptoms. The only reported cases are outbreaks where a lot of people are involved and the source could be found (Bennett, 2001a). CHAPTER 2. LITERATURE REVIEW 4

In a report by Scallan et al. (2011) an attempt was made to estimate the number of illnesses, hospitalizations and deaths caused by 31 pathogens. In this report they estimated that yearly 63400 of the 9.4 million (0.67 %) domestically acquired food borne diseases in the United States are caused by B. cereus. In the EFSA journal (2007), they reported that B. cereus was responsible for 1.3 % of the total outbreaks reported in 2006 in Europe and responsible for 1.7 % of the reported human cases. The results from diﬀerent countries may not be compared because they all use diﬀerent methods of surveillance of these food borne diseases (Granum, 2001).

Cereulide Cereulide is a small lipophilic cyclic dodecadepsipeptide, with a molecular mass of 1.2 kDa. It consists of three repeats of 4 amino/oxy amino acids with structure [D-O-Leu-D-Ala-D-O-Val-D- Val]3 (Agata et al., 1994; Ehling-Schulz et al., 2004). It is an emetic exotoxin and is illustrated in Figure 2.1. An exotoxin is a toxin excreted by microorganisms, and as such (and on the basis of chemical nature) is to be diﬀerentiated from endotoxins, which are a component of the cell wall of certain bacteria.

Figure 2.1: structure of cereulide (Biesta-Peters et al., 2010)

Cereulide is very similar to valinomycine. They have the same chemical structure, weight and mitochondrial toxicity, but valinomycin cannot cause emesis (Isobe et al., 1995). They are both potassium(K)-selective ionophores and can cause potassium speciﬁc transport (Hoornstra et al., 2003). It facilitates the movement of the K+ ions through the lipid membrame following the electrochemical potential gradient (Ceuppens et al., 2011). Valinomycin is obtained from Strep- tomyces strains and is used as a standard to express the toxicity of unknown samples. But this CHAPTER 2. LITERATURE REVIEW 5 must be interpreted with care; a study done by Biesta-Peters et al. (2010) showed however that valinomycin is 15 times less toxic towards HEp-2 cells (human carcinoma of the larynx) than cereulide.

The chemical structure of cereulide is very typical for non-ribosomal synthesized peptides. This suggests that the genes of the non-ribosomal peptide synthetase are responsible for the cereulide production. They could ﬁnd the genes on a plasmid called pCERE01 (Hoton et al., 2005).

Symptoms After cereulide consumption, the symptoms occur already after 1-5 hours. This is because the cereulide toxin is already produced in the food. When the diarrheal type of food poisoning is involved, it takes occasionally more than 8-16 hours before the symptoms occur, because the toxins need to be formed in the intestines during the vegetative growth of B. cereus in the intestines. With cereulide food intoxication the symptoms are nausea, vomiting and stomach cramps (Stenfors Arnesen et al., 2008). It resembles Staphylococcus aureus intoxication, but the symptoms of S. aureus intoxication are sometimes additionally accompanied with diarrhea. The diﬀerences and similarities between B. cereus cereulide and S. aureus enterotoxin intoxications are represented in Table 2.2 (Bennett, 2001a). Most often, the symptoms of the cereulide intoxication will disappear after 6 to 24 hours (Granum, 2005).

Table 2.2: Diﬀerences and similarities between B. cereus emetic and S. aureus enterotoxin food poisoning

B. cereus S. aureus cereulide toxin enterotoxin Onset of the 1-5 2-6 symptoms (h) Duration of 6-24 6-24 illness (h) Diarrhea, Fairly common Common abdominal cramps Nausea, vominting Predominant Predominant Pathogenese Preformed toxin Preformed toxin Principal food vehicle Cooked rice and Cold cooked meat pasta and poultry, dairy products

The mechanism by which cereulide can cause emesis is not yet clarified. Animal tests indicated that it is caused by a receptor mediated mechanism. When cereulide enters the intestines, it binds to serotonin 5-HT3 receptor and in this way it stimulates the vagus afferent nerves. This will eventually lead to vomiting in Suncus murinus (Agata et al., 1995). A well known biologic effect is the ionophoric effect of the toxin. This leads to an ionophoric K+ uptake and subsequently a dissipation of the transmembrane potential. Eventually this results in swelling of the mitochondria and their inactivation (Mikkola et al., 1999). Cereulide can be lethal if it causes brain edema and fulminate liver failure due to the inhibition of the mitochondrial fatty-acid oxidation (Dierick et al., 2005; Mahler et al., 1997). Although only several fatal intoxications were reported, a girl of 7 years died 13 hours after eating a pasta salad. Also a 1 year old boy died of acute encephalopathy after 6 hours eating reheated fried rice. The other CHAPTER 2. LITERATURE REVIEW 6 children could survive because they were treated with plasma exchange and fluid therapy (Shiota et al., 2010). Some experiments with mouses could show a degeneration of the liver cells caused by cereulide (Yokoyama et al., 1999). Cereulide has also an effect on the Natural Killer (NK) cells because of the inhibition of the cytotoxicity and cytokine production of these NK cells. The mechanism is that the mitochondria of the NK cells swell and the apoptose of these NK cells are induced (Paananen et al., 2002).

Cereulide production conditions The production of cereulide is regulated by the toxin gene expression. This expression is tightly regulated by internal and environmental signals (Ceuppens et al., 2011). Cereulide is produced during the end of the logarithmic phase, with the biggest production during the start of the stationary phase. B. cereus produces 0.004 to 0.13 µg cereulide per 106 cells in the exponential growth phase (Jääskeläinen et al., 2004). The production of cereulide continues when the stationary phase is reached (Delbrassinne et al., 2011). A density of 105 -108 CFU B. cereus /g are necessary to produce enough cereulide to cause symptoms; this will result in a dose of 8-10 µg/kg body weight (BW) which is enough to cause emesis as obtained by animal models (Agata et al., 1994; Shinagawa et al., 1995). Even lower intoxication levels of 10-1280 ng/g are found in foods implicated in vomiting-type of food poisoning due to B. cereus, reported by Agata et al. (2002). Counts ranging from 200 to 109 B. cereus cells / g or ml have been reported in foods implicated in outbreaks. Generally, foods containing > 103 B. cereus cells / g are considered not safe for consumption (Granum, 2001). Some obtained toxic doses in animals from the literature are in Table 2.3. Although, some foods implicated in food intoxications contained a lower dose of cereulide than obtained by the animal models. This suggests that the obtained dose through animal models should be transferred via a safety factor into a dose for humans (Delbrassinne et al., 2011).

Table 2.3: Toxic dose of cereulide in animal models

Test animal Dose Source Suncus murinus ED50 = 16 µg/kg BW Isobe et al. (1995) Suncus murinus 8 µg/kg BW Agata et al. (1994) Rhesus monkey 10 µg/kg BW Shinagawa et al. (1995) ED50 = the eﬀective dose for 50 % of the population

Low Na+ concentration or a high ratio [K+ ]:[Na+ ] seem to stimulate cereulide production by B. cereus. This means that lowering the salt content in foods doesn’t seem a useful tool to prevent the cereulide production (Ceuppens et al., 2011). Temperature has also an important inﬂuence on the toxin production. A study done by Finlay et al. (2000) showed that cereulide toxin production is possible between 12 and 37°C. Below and above these temperatures, B. cereus can grow, but there is no cereulide production. The optimal temperature for cereulide toxin production is between 12 and 30 °C (Finlay et al., 2000; H¨aggblom et al., 2002). Some more psychrotrophic strains seem to have the greatest toxin production between 12 and 15°C. This despite of the lower B. cereus counts at these temperatures. At 12 °C, there was cereulide detection at day 4, with a bacterial count of 4.40 log. At 15 °C, the cereulide could be detected from day 2 by some strains, by other strains from day 3. The bacterial count was 6 log. The maximum amount of toxin could also be reached at the lowest temperature of 12 °C, at day 12 (Finlay et al., 2000). But these experiments were performed under relative limited type of conditions and with only a few strains, so this information has to be used with care (Granum, CHAPTER 2. LITERATURE REVIEW 7

2005). Some bacterial characteristics are also important; this is the type of B. cereus strain and the growth phase. A study done by Carlin et al. (2006) showed that diﬀerent strains, grown on TSA medium at the same conditions gave diﬀerent cereulide contents. The content ranged from 0.5 to 1600 ng cereulide/mg biomass fresh weight.

There are a lot of environmental factors, besides the food composition and temperature that have an influence on the cereulide production. For instance, the oxygen content, pH, aw -value and the amino acid composition are of interest. When vinaigrette and mayonnaise are used on rice and noodles, B. cereus cannot grow to the critical concentration to produce cereulide. It is because of the lower pH of these modified foods. There is also shown that the lower the pH and aw -value, the slower the cereulide production (Agata et al., 2002). There are three amino acids essential for the production of cereulide, namely valine, leucine and threonine. Adding L-leucine and L-valine seems to stimulate the cereulide production 10- to 20-fold (Jääskeläinen et al., 2004). This has to be taken into consideration because leucine and valine are frequently added to food as a flavor enhancer (Ceuppens et al., 2011). The oxygencontent seems to be also very important. There is no cereulide production when there is less than 1.6 % oxygen. This was seen in a test with B. cereus on TSA plates (Rajkovic et al., 2006b). Moreover showed a study by Jääskeläinen et al. (2004) that there is a major difference in cereulide production at air conditions (3-6 µg/ml) in comparison with no or very little (0.002 to 0.02 µg/ml) cereulide production at exclusively nitrogen atmosphere (>99,5 % N2). At 10 % oxygen, there was a little bit of cereulide production (0.04 to 0.06 µg/ml), but very low compared to the content obtained with air. In this study, the test was performed in TSB broth.

Cereulide stability The problem with cereulide food intoxication is that once cereulide is produced in the food, it cannot be removed anymore. This is because cereulide is highly resistant towards heat, acids and proteolytic enzymes. Only with very high pH and very long exposure to heat, it can be inactivated. In a study of Rajkovic et al. (2008) it was found that the time/temperature ranges needed to inactivate cereulide decreased with increasing pH. To inactivate a dose of 6 µg/ml, 80 minutes at 121◦C and pH 9.5 were necessary. But such a high pH value and high time/temperature range does not occur in foods. At the pH value of 7 they could see that no inactivation could occur, only with a heat treatment of 2 hours at 121◦C, which is not realistic. Mostly one chooses a short time/low temperature treatment to make sure the foods look fresh. Because cereulide is also very resistant towards proteolytic enzymes, it can survive the passage through the stomach when ingested. Cereulide is moreover hydrophobic, so it can attach to contact materials. This means that cereulide cannot be easily eliminated in industrial installations. Cereulide can thus not be eliminated with usual cleaning protocols and when the heat sterilization of 15 minutes at 121◦C is not strict enough (Rajkovic et al., 2008).

2.1.4 Food sources Every food type with a pH > 4.8 can be a possible source of food contamination with cereulide producing B. cereus. This is because B. cereus is omnipresent, has the ability to form spores and is not selective for food substrates (Stenfors Arnesen et al., 2008). But non-acidic foods with a high water and starch content seem to be the food sources that give the highest cereulide content. Although the emetic B. cereus strains cannot hydrolyze starch, food products containing starch seems to be most often implicated in cereulide related outbreaks (Rajkovic et al., 2006a). CHAPTER 2. LITERATURE REVIEW 8

It occurs mainly on fried or cooked rice, pasta or noodles. This was conﬁrmed by a study by Agata et al. (2002) where the cereulide content was determined of some food samples suspected to be the cause of food intoxication. The results are shown in Table 2.4. It is even so that in the same study they could ﬁnd cereulide after 16, 8 and 4 hours on inoculated rice at temperatures of 20, 30 and 35◦C respectively.

Table 2.4: Toxin detected in foods implicated in food intoxication

Foodtype Toxin quantity(ng/g) Fried rice 1280 Cooked rice 640 Chow mein 640 Cooked rice 320 Fried rice 160 Cooked rice 160 Curry and rice 80 Spaghetti 80 Cooked rice 80 Spaghetti 40 Noodles 20 Boiled rice 10

This means that if an adult of 70 kg eats a dose of 100 g contaminated food, 0.014 to 0.18 µg cereulide per kg BW is enough to cause emesis, which is much lower than the dose obtained through animal models (Tabel 2.3). Some other food types where cereulide is detected are in Table 2.5 (Rajkovic et al., 2007).

Table 2.5: Cereulide detection by CASA from food samples from production sites and restaurants

Food type pH Aw Time(s) till PMOT%≤101 Smoked salmon 6,2 0,965 10 Fruit yoghurt 4,5 0,98 60 Black olives on Greek 6,8 NA2 60 manner Pre-roasted turkey 6,5 NA 40 ﬁlet Raw veal meat 5,9 0,994 60 Camembert cheese 7,6 0,961 50 Pasta salad (chicken 4,9 0,981 20 + vegetables) Canned mushroom 6,2 NA 20 soup Bacon 5,8 0,952 30 Emmental cheese 5,3 0,971 20 1: Cereulide content expressed as time needed for the motility of boarsemen to drop below 10% (see 2.1.5) 2: NA= Not available CHAPTER 2. LITERATURE REVIEW 9

On eggs, fish and meat the toxin production was rather low. The same for liquid sources like milk and soymilk. Furthermore, if the amount of B. cereus would be high enough to cause illness, the proteases from B. cereus would produce off flavors and the consumer probably wouldn’t consume the milk anymore (Granum, 2001).

In a study of Rajkovic et al. (2006c) they have inoculated rice, potatopuree and pasta with the emetic type of B. cereus. In this study they could see that the cereulide production was lower in the rice in comparison with the other two food sources, despite of the cell density of B. cereus that was the same in all three sources.

2.1.5 Cereulide detection There are several possibilities to detect cereulide. In earlier days, Suncus murinus or Rhesus monkey feeding trials were used. These values are already indicated in Table 2.3.

Unfortunately, an immunologic testkit cannot be made for this type of toxin. This is because cereulide has low antigenic properties and no antibodies against cereulide are so far reported. Other possible tests are chemical assays, instrumental analyses, and PCR. Some cell culture systems can also be useful. Of these, HEp-2 cells (human carcinoma of the larynx) can be used. If cereulide is present, there will be vacuole formation because of mitochondrial swelling (Jay, 2000a). A very useful chemical method is the detection through high-performance liquid chromatography (HPLC), connected to ion trap mass spectrometry (MS). This LC-MS method has a limit of detection (LOD) of 1 ng/ml and limit of quantification (LOQ) of 5 ng/ml (Häggblom et al., 2002). This method is useful because it can give a quantification of the exact cereulide content, but it is very laborious and expensive. PCR (polymerase chain reaction)-assay can be used to detect the emetic toxin producing strains in a sample (Ehling-Schulz et al., 2004).

Another possibility is the application of a bio-assay using boar semen. This test is based on the motility inhibition of boar semen because of the failure of the mitochondria in the semen. Cereulide works as an ionophore for the potassium ions, so cereulide can consequently bring these potassium ions into the mitochondria through an ion-carrier system. When the mitochondria are damaged, they will no longer perform oxidoreductive functions. The action of cereulide resembles valinomycine. The decrease in speed of the semen because of valinomycine can be used as a semi-quantitative system to detect the cereulide concentration. The system can be com- puterized and a graphic can be obtained by measuring the change in percentage of progressive motility (% PMOT) in function of the time. The system can quantify cereulide concentration in a range of 20 - 400 ng/ml. At higher concentrations sample needs to be ﬁrst diluted (Rajkovic et al., 2006b).

To detect the qualitative toxicity of samples, it is enough to analyze a not diluted sample. Hereby it is enough to look whether the semen stops moving during 10 minutes of evaluation (Rajkovic et al., 2006b). If the semen is still moving after 10 minutes, the cereulide concentration can be considered below 1 ng/ml (Rajkovic et al., 2007).

The bio-assay using semen has some disadvantages. Other toxins present in the food can also have an inhibiting effect on the semen, so a confirmation with other tests needs to be obtained if specific cereulide determination is required. Another thing is that cereulide is not detected specifically. To do this, methods like HPLC-MS can be used. However, earlier results with CHAPTER 2. LITERATURE REVIEW 10 comparative tests between the methods showed that the results obtained with the bio-assay cor- relates well with those obtained with HPLC-MS (Jääskeläinen et al., 2003). Moreover, a study done by Jääskeläinen et al. (2003) showed the similarity between results with the bio-assay and human cell lines. They had the same sensitivity towards cereulide. In this study four human cell lines (HeLa, CaCo-2, Calu-3 and Paju) were used to detect the threshold value of cereulide at which there was a loss of mitochondrial activity. The obtained concentrations were compared and semen cells had the same sensitivity towards cereulide as human cells. Besides, the sensitivity of boar semen was compared with bull semen. The boar semen seemed a hundred times more sensitive than the bull semen.

Another study looked at the possible influencing factors, which could possibly influence analyze of cereulide through semen (Rajkovic et al., 2007). In this study authors reported that other toxins of B. cereus (HBL and NHE) and the enterotoxins of S. aureus don’t have an influence on the mobility of the semen. Some mycotoxins had an influence on the motility, but the amount of mycotoxins was far above the legal limit of mycotoxins. Preservatives, dioxins, TCDD and acrylamides all didn’t had any influence on the semen motility. Overall, one can conclude that the test analyze with boar semen is the method to be preferred because of the fast analyze and it is much cheaper.

2.1.6 Prevalence Because cereulide is more present in rice and starchy foods, it is to an extent not surprising that the emetic food intoxication is more prevalent in Japan, China and India (Shinagawa et al., 1995; Granum and Baird Parker, 2000a). But also a lot of cases are reported from the UK, involving fried rice in Chinese restaurants (EFSA, 2005). And to a lesser extent, some pasta dishes were also reported to be involved in outbreaks (EFSA, 2005). In the EFSA report of 2011, the distribution of food vehicles in veriﬁed outbreaks caused by Bacillus toxins shows that mixed or buﬀet meals (27.1 %) followed by cereal products inclusive rice and seeds/pulses (11.9 %) were mostly involved (EFSA, 2011).

2.2 Staphylococcus aureus

2.2.1 General characteristics and background information Staphylococcus aureus is a gram positive pathogen. It is a spherical bacterium (coccus). Micro- scopic evaluation shows that they appear in pairs, short chains, or in bunched, grapelike clusters. S. aureus is not motile and is aerobe or facultative anaerobe. They are catalase and coagulase positive and use a wide variety of carbohydrates. Amino acids are required as nitrogen source (Bennett, 2001b). Some common features are listed in Table 2.6. S. aureus is one of the most resistant non spore-forming pathogen as it can survive in dry conditions and can be isolated from dust, sewage, water and air very easily (Jablonski and Bohach, 2001).

It is a pathogen that can cause a wide range of diseases and intoxications in warm-blooded animals. It can cause skin infections such as ulcers, carbuncles, impetigo, and epidermidis necrosis (scalded skin syndrome). It can furthermore cause infections of the internal organs such as bacteremia, endocarditis, pericarditis, osteomyelitis, meningitis and mastitis. Finally, also intoxications such as toxic shock syndrome and food poisoning can be caused by S. aureus (Bergdoll, 1983). CHAPTER 2. LITERATURE REVIEW 11

Table 2.6: Physical properties S.aureus cells

Physical property Value Source Temperature growth range 7-48,5°C Schmitt et al. (1990) Optimal Temperature growth 30-37°C Schmitt et al. (1990) range pH growth range 4,2-9,3 Bergdoll (1989) Optimal pH growth range 7-7,5 Bergdoll (1989) Min aw 0,83-0,86 Troller and Stinson (1978)

They have a D-value of 1 to 6 minutes when heated at 60°C at a high aw such as milk, meat and vegetables (Granum and Baird Parker, 2000b). In phosphate buﬀer, the D60°C value ranges from 1 to 2.5 minutes (Wilson et al., 1994). Moreover, the heat resistance rises when the NaCl content is higher (Bean and Roberts, 1975). But lowering or raising the pH from neutral will reduce the heat resistance (Stiles and Witter,1965). The cells are more resistant towards heat at lower aw-values. However, towards food preservations it is less resistant, with the exception of his osmotolerance. Because of this property, S. aureus will survive conditions of 20 % NaCl and water activities of 0.83 - 0.86. This is possible because of an osmoprotectant system. Proline and glycine betaine can accumulate in the cell, so the intracellular water activity can be lowered towards the same level as outside the cell (Jablonski and Bohach, 2001).

S. aureus can be transferred into food through air, dust and food contact materials. However among the most important transmission routes is a human route, this is because 20 - 30 % of the human population carries S. aureus on their skin and nares (Kluytmans and Wertheim, 2005). S. aureus can thus be spread by direct contact to food, through skin fragments, or through respiratory droplets produced when people cough or sneeze (Jablonski and Bohach, 2001). Next to people, the knives, meat grinders, cutting blocks, and storage containers may be a source of contamination with S. aureus. Other high risk factors causing possible S. aureus intoxication are inadequate refrigeration during storage or a prolonged use of warming plates when the food is served. The latter is the case when the food is hold below 60◦C or the food is prepared too much in advance (Jablonski and Bohach, 2001). Another way to transfer S. aureus is through animals, especially in raw foods. The skin or feathers are regularly contaminated with S. aureus and contamination of dressed carcasses is common and is unavoidable (Bennett, 2001b). S. aureus can cause mastitis (the infection of cow teats) in bovines. In this way S. aureus can contaminate milk and dairy products. In raw bulk milk S. aureus occurs at low number (< 100/ml), but if the milk comes from a mastitis cow, the numbers may exceed 1 million/ml (Gilmour and Harvey, 1990).

Because S. aureus is not a good competitor with other micro organisms in raw foods, it will mainly contaminate food which is poorly treated by the food handler (Granum and Baird Parker, 2000b). In the cases where pasteurization was not correctly done, present S. aureus may survive and if the storage conditions are not good, S. aureus will grow to high numbers and can produce enterotoxins as a product of their metabolism. It are these enterotoxins which cause the disease symptoms. But the fact that S. aureus is signiﬁcant present in food should be carefully interpreted because not all strains of S. aureus can produce enterotoxins (see further). However, absence of S. aureus does not mean absence of the staphylococcal enterotoxins either. CHAPTER 2. LITERATURE REVIEW 12

2.2.2 S. aureus detection The method for S. aureus enumeration is based on a selective plating media and uses some characteristics of S. aureus. In Baird Parker agar, selective agents are present such as lithium chloride, glycine and potassium tellurite. A good balance between these will act on Gram negative and Gram positive bacteria, other than S. aureus. Pyruvate and egg yolk can improve the recovery of stressed or damaged cells. Tellurite can be metabolized by S. aureus with the release of tellurium, which will result in black colonies. Also egg yolk is added as a diagnostic agent. S. aureus uses the lipoprotein lipovitellenin. This will result in precipitation zones around S. aureus colonies (Granum and Baird Parker, 2000b).

2.2.3 S. aureus enterotoxins Staphylococcal Enterotoxins Staphylococcal enterotoxins (SEs) are bacterial proteins with a length of approximately 220-240 amino acids. Their molecular weights are about 26 to 30 kDa and have an isoelectric point between 5.7 and 8.6 (Jay, 2000b). S. aureus can produce a wide variety of enterotoxins. The amount made is strain dependent. SEA is considered to be involved in most outbreaks, followed by SED and SEC (Stewart, 2005). However, the severities of the symptoms are heavier with SEB intoxication. Moreover, the amount produced is diﬀerent. SEB and SEC production may be more than 100 µg/ml, while SEA and SED will only be produced in amounts of 1-10 µg/ml (Tranter and Brehm, 1990). It has been generally accepted that SEs are produced during the logarithmic growth phase or during the transition of the exponential to the stationary phase. In a study done by Rajkovic et al. (2006c), it was noticed that SEB production already started after 4 hours of incubation at temperatures of 22, 37 and 42◦C. This was the ﬁrst half of the exponential growth phase.

Studies suggest that 105 to 108 cells are required to produce enough SEs to cause illness. In a study by Raj and Bergdoll (1969) where humans ingested purified SEs, they could see that 20 - 25 µg of SEB was necessary to cause vomiting (0.4 µg/kg BW). Monkeys are less susceptible to toxins and the 50 % emetic dose is 1 µg/kg BW to start vomiting (Jablonski and Bohach, 2001). Analyses of foods involved in foodborne outbreaks can give information about the toxic dose required. Many outbreaks are caused by 1 to 5 µg of ingested toxin per person (Jablonski and Bohach, 2001). Another analyze of some foods from outbreaks gave results of a level of 1 to 10 µg SEA/100 g (Wieneke and Gilbert, 1985). But the amount necessary to cause symptoms can vary by several factors, such as personal susceptibility, amount of food ingested and the person’s overall health status. A dose of 100-200 ng can be enough for highly sensitive people (Bennett, 2001b). Deaths caused by staphylococcal food poisoning are rare, although there have been cases among elderly, infants and severely debilitated people. So it is of major concern for YOPI’s (Young, Old, Pregnant and Immune deficient). The emetic dose seems to vary also by type of enterotoxin. This together with the fact that the environment with temperature and food source all play a roll, it is difficult to know the exact amount of S. aureus that will cause SE food poisoning . Some of the emetic doses reported for different SEs are in Table 2.7 (Stewart, 2005).

But there has to be noted that not all S. aureus strains have the possibility to produce enterotoxins. The enterotoxin genes are accessory genetic elements, meaning that it are genes which are added to the basic genetic capacity. The genetic genes can be found on plasmids, bacterio- phages, and as a part of the pathogenicity islands (Stewart, 2005). CHAPTER 2. LITERATURE REVIEW 13

Table 2.7: Estimated doses for diﬀerent enterotoxins (from Stewart, 2005)

Toxin Estimated ED50 Species Reference SEA < 25 µg/kg Rhesus monkey Harris et al. (1993) 10 µg/kg Rhesus monkey Stelma and Bergdoll (1982) 144 ± 50 ng Humans Evenson et al. (1988) SEB 0,4 µg/kg Humans Raj and Bergdoll (1969) 0,9 µg/kg Rhesus monkey Schantz et al. (1965) SEC 1 0,1-1,0 µg/kg Pigtail monkey Schlievert et al. (2000) SEG ± 80 µg/kg Rhesus monkey Munson et al. (1998) SEI >150 µg/kg Rhesus monkey Munson et al. (1998)

The staphylococcal enterotoxins act on the gut and the emetic response result from the stimulation of the neural receptors. The pulses are transmitted through the nerves and stimulate the brain vomiting center (Jablonski and Bohach, 2001). There are suggestions that the enterotoxins have the ability to bind to the major histocompatability complex (MHC) class II antigen. This causes T cell stimulation and subsequent release of cytokines. These cytokines can stimulate the neuroreceptors in the intestinal tract and triggers the vomiting center in the brain (Grossman et al., 1992).

Different SE types are indentified, which share structure and sequence similarities. They are rich in lysine, aspartic acid, glutamic acid and tyrosine residues (Le Loir et al., 2003). They are named by letter in order of their discovery. Staphylococcal enterotoxins A, B, C, D and E are the major types. These are all staphylococcal enterotoxins (SEs) which have an emetic effect. There are also staphylococcal enterotoxin likes (SEls). They have the same structure, but are not yet examinated for their emetic potential, or don’t have an emetic effect. The repertoire of S. aureus SEs/SEls comprises 22 members (Argudin et al., 2010). Some properties of some studied enterotoxins are given in Table 2.8 (Jay, 2000b).

Table 2.8: Properties studied enterotoxins

Enterotoxin

A B C1 C2 C3 D E G H I Emetic 5 5 5 5-10 <10 20 10-20 <30 dose(ED50) (monkey, µg/animal) Molecular 27,8 28,366 34,1 34 26,9 27,3 29,6 27,043 28,5 24,928 weight Year 1960 1959 1967 1967 1965 1967 1971 1992 1994 1998 identiﬁed CHAPTER 2. LITERATURE REVIEW 14

Symptoms Food intoxication because of SEs is a global problem. The symptoms of illness appear 2-8 hours after food consumption containing preformed SEs. The mean incubation period is 4.4 hours. Sometimes the vomiting can already start within 30 minutes. The symptoms are nausea, vomiting, abdominal cramping, headaches and sometimes diarrhea. The symptoms disappear after 24-48 hours. Because the illness dissolves fast, there are only limiting cases of hospitalization. The amount of reported cases is an underestimation because people recover fast, diagnose is sometimes wrong and only outbreaks affecting a big amount of people on the same moment are reported. Also an inadequate collection of samples for laboratory analyses and improper laboratory examination can be the reason for underestimation (Bennett, 2001b). But according to estimates done by the Centers for Disease Control and Prevention (CDC, 2011), is S. aureus on the fifth place in the top five of pathogens contributing to domestically acquired foodborne illnesses. There are approximately 241148 numbers of illnesses every year in the United States. It accounts for 3 % of the total cases every year. But death because of S. aureus poisoning is rare. These estimates done by Scallan et al. (2011) show that only 0.44 % of the total deaths because of food poisoning are due to S. aureus. In the EFSA report (2007) it is given that S. aureus is responsible for 4 % of the outbreaks reported and responsible for 3.7 % of the human cases reported in 2006 in Europe. There is also a seasoning trend, with more cases in the summer because the temperatures are warm and food is stored improperly. A second trend is in December, probably because leftover holiday food is handled and stored incorrect (Jablonski and Bohach, 2001). Mostly the reported cases are from outbreaks where a lot of people are involved. This because the food was not good handled by the food catering. But sometimes also individual home cases or an industrial product that is mishandled during the initial food processing operation can be the case. This was the subject when contaminated dry lasagna caused an international outbreak. The pasta was contaminated in the factory because of the use of an unpasteurized egg and a subsequent slow drying at warm temperatures. This dry lasagna contained more than 104 cells/g (Woolaway et al., 1986).

But it is surely of big social and economical concern, because disease causes a loss of working days, productivity is lower and there are costs of hospitalization and consultation. It is of economical concern because S. aureus contaminated food needs to be thrown away. Annually the costs from S. aureus foodborne cases can be estimated and is $ 1.2 billion (Buzby et al., 1996).

Staphylococcal enterotoxin production conditions The optimal temperature where SE production takes place is the same as the optimal growth temperatures and is thus 30-37◦C. The limiting temperature for the SE production is 10◦C. But at this temperature, the rate of enterotoxin production is rather slow. The maximum tempera- ◦ ture for SE production is 45-46 C. The limiting aw -value where the S. aureus cells can grow is much lower than the limiting aw -value for SE production (ICSMF, 1996b).

The pH conditions where toxin production takes place are stricter than those for growth of S. aureus. The limit is pH 5.0 and the optimal pH is between 7.0 and 8.0. The production thus decreases with acidic pH. For example, acetic acid and lactic acid have an inhibitory eﬀect on the SE production. Furthermore, if sodium chloride is added, the inhibitory eﬀects of the acidic pH increases (Notermans and Heuvelman, 1983). As S. aureus grows faster at aerobically conditions, also the toxin is produced faster. S. aureus is very sensitive towards sorbic acid as a preservative. This will result in an inhibitory growth and consequently less SE production CHAPTER 2. LITERATURE REVIEW 15

(Smith and Palumbo, 1980).

Staphylococcal enterotoxin stability SEs are very heat and acid resistant. When a food that is involved in food poisoning doesn’t contain S. aureus, it doesn’t necessary mean that there are no toxins present. The S. aureus cells could have produced toxins and these could have survived the subsequent heat treatment. It can survive pasteurization and even some can survive the sterilization process of canned foods (Bennett and Berry, 1987). In whole milk, SE remained active after a heat treatment of 20 minutes at 121◦C. The heat resistance reduces with a more acid pH (Tatini, 1976). An important remark is that because of the heat treatment, SE may lose his antigenitic activity and will thus not be detected anymore. However, the biological activity remains. To solve this, urea treatment of the samples, prior of immunological testing can be used to restore the antigeniticity (Akhtar et al., 1996). They are also resistant towards freezing and are moreover very resistant towards gastrointestinal proteases like pepsin, trypsin and rennin. This means that they can retain their activity after ingestion. Staphylococcal enterotoxin B can be destroyed by pepsin digestion at pH 2 but it is pepsin resistant at higher pH’s, which are normal conditions in the stomach after food ingestion (Bergdoll, 1983).

2.2.4 Food sources Food sources mainly contaminated with S. aureus are meat and meatproducts (chicken, egg- products, milk and milkproducts, salads such as egg, tuna, chicken patato and macaroni) and bakery products (creamﬁlled pastries, cake, chocolate eclairs) (Tamarapu et al., 2001; Wieneke et al., 1993). There is a case where canned mushrooms were involved. Also salted products like ham have already been a source of staphylococcal enterotoxins (Qi and Miller, 2000). This is because S. aureus can survive and grow at very low aw -values (aw =0.83-0.86). In Table 2.9 the prevalence of S. aureus in several food products is exampliﬁed.

Table 2.9: Prevalence of S. aureus in some food products (Jablonski and Bohach, 2001)

Product No. of samples % positive for CFU / g * tested S. aureus Ground beef 74 57 ≥ 100 Pork sausage 67 25 100 Ground turkey 50 6 >10 Salmon steaks 86 2 >3,6 Oysters 59 10 >3,6 Blue crab meat 896 52 ≥ 3 Peeled shrimp 1468 27 ≥ 3 Lobster tail 1315 24 ≥ 3 Assorted cream pies 465 1 ≥ 25 Tuna pot pies 1290 2 ≥ 10 Delicatesse Salads 517 12 ≥ 3 *Determined either by direct plate count or most propable numer technique

In a study done by Koluman et al. (2011), 300 diﬀerent food products were analyzed on the presence of S. aureus and its SEs. The study showed that 112 of the 300 samples were contaminated. Minced beef appeared to be the most contaminated product, with 70 % of the minced beef samples contaminated. Thereafter 52 % of the chicken meat, 48 % of the turkey meat, 36 % of the CHAPTER 2. LITERATURE REVIEW 16 beef, 20 % of the Turkish white cheese, 12 % of the cheddar cheese, 4 % of the pasteurized milk and nothing in the creamcheese. The contamination of the milk and cheese products was rather low because there is a lot of competitive ﬂora in the starter culture. In milk and milk products the food intoxication with SEs will not occur often because of the critical density of 105 cells cannot be reached because of the competitive bacteria. In the 112 contaminated samples, a total of 221 S. aureus strains were found. They accounted for an average S. aureus contamination of 4.5 log CFU/g. SEs were also found in 74 of the 112 samples. The most found enterotoxin was SEA.

In an EFSA report of 2011, the distribution of food vehicles in veriﬁed outbreaks caused by staphylococcal toxins in EU in 2009 showed that cheese (21.6 %) followed by mixed or buﬀet meals (15.9 %) were mostly involved. Poultry meat and products thereof accounted for 5.7 %, the same percentage for pig meat and products thereof. The bakery products accounted for 4.5% (EFSA, 2011).

2.2.5 Staphylococcal enterotoxin detection Prior to the serological identiﬁcation of SEs, biological assays were used. In these assays, the toxins were detected by the emetic response of monkeys. The monkey received the sample in the stomach by a catheter and subsequently observed during 5 hours to see if vomiting occurs. If vomiting occurred in this period, the sample was considered positive (Bennett, 2001b).

Other possible methods are micro-slide gel double diffusion test or numbers of commercialized immunological assays such as the visual ELISA (enzyme-linked immunosorbent assay), the VI- DAS set 2 (BioMérieux),reversed passive latex agglutination (RPLA) from Oxoid or Transia immunoenzymatic testkit (Bennett, 2011). These serological methods are based on the use of a specific antibody to each of the SEs. This is possible because SEs have antigenic properties. The most commonly used method to identify enterotoxins of S. aureus are different modifications of enzyme-linked immunosorbent assays (ELISA). Sensitivity of the commercial assays is in the range of 0.25 to 1.0 ng enterotoxin per gram food (Bennett, 2011; instruction manuals VIDAS Set2 and Transia). The double-antibody sandwich ELISA seems to be the most popular for routine toxin identification. In this assay appropriate antibodies are absorbed on a solid support e.g. microtiter wells, plastic tubes. The antibody absorbed onto the solid-phase support is the capture antibody. The SE with its antigenic properties can consequently bind to the capture antibody and it can be detected via a secondary enzyme-linked antibody that is added and can bind to the captured SE. When a substrate, depending on the enzyme used is added, the enzyme will produce a color reaction. The intensity of the color reaction is proportional to the amount of toxin in the sample (Bennett, 2011). A more automated assay is the enzyme-linked fluorescent immunoassay (ELFA). It is labor-saving because the sample is added and the analyzer auto- matically completes the ELFA steps and gives print-out data after 80 minutes. Example of this is a VIDAS Set2 automatized system (Biomérieux). This assay is AOAC approved. In this assay, the prepared sample is injected in the first well of a reagent strip. The reagents are in the other wells. The last well is a cuvette in which the fluorometric reading can be performed. A Solid Phase Receptacle (SPR) serves as the solid phase, as well as the pipetting device. The interior of this SPR is coated with anti-staphylococcal enterotoxin antibodies. The sample can come into the SPR and the antigens binds at the antibodies. Afterwards it is washed several times. In the next well, alkaline phosphatase-labeled antibodies are cycled in and out the SPR and will bind at the SEs which are themselves bound at the antibodies of the SPR. In the last well, 4-methyl-umbelliferyl phosphate is added. The bound enzyme will catalyze the hydrolysis of the last added product and a fluorescent product (4-methyl-umbelliferone) is produced, of CHAPTER 2. LITERATURE REVIEW 17 which the fluorescence can be measured at 450nm (Figure 2.2). This value can be compared to internal references (Instruction manual VIDAS set 2). It is a polyvalent assay and can determine enterotoxins A to E (Bennett, 2001b). With the micro-slide gel double diffusion test, they work with 5 wells. In the center well, an antiserum is used. In the two other wells, known enterotoxins are present. In the other two wells, the sample is placed. With the developing lines, decision can be made if the sample is positive or not (Casman et al., 1969). But this is a rather old system.

Figure 2.2: ELFA- enterotoxin detection (Bennett, 2011)

Another modiﬁcation of ELISA is trough an immunoquantitative real-time PCR (iq-PCR). In this method detection antibody is linked to a DNA segment, which can be ampliﬁed in a subsequent real-time PCR. This was developed by Rajkovic et al. (2006a) for the detection of SEB. They could see that there was no cross-reactivity by other SEs. The major advantage of this method is the higher sensitivity. The iq-PCR seems to be 1000 times more sensitive than ELISA using same antibodies and quantities between 10 pg/ml to 30000 pg/ml of SEB can be detected. To compare, the VIDAS kit, based on ELFA has a detection limit of 0.25 ng/ml (Instruction manual VIDAS set 2). The lower detection limit can be an advantage, because some lower intoxication doses of 100-200 ng were reported (Evenson et al., 1988). This means that it can be very important for the more susceptible population, namely the YOPI’s. Alternative to actual toxin detection is detection of staphylococcal enterotoxin-encoding genes by PCR. This method can be very useful because the newer SEs are not detected with the existing antibodies. A multiplex PCR, where all the existing SE determinants are detected can screen a food isolate fast to see if the isolate harbors any of the known SEs (Monday and Bohach, 1999). Chapter 3

Materials and methods

3.1 S. aureus and B. cereus strains used in the experimental setup

This study comprised two strains of Staphylococcus aureus and two of Bacillus cereus. They all came from the culture collection of the University of Ghent, Laboratory of Food Microbiology and Food Preservation (LFMFP). The strains of S. aureus were staphylococcal enterotoxin producers and were S. aureus LFMFP 356 and 362. The B. cereus strains were both cereulide producers and the strains chosen were B. cereus LFMFP 434 and 436. The strains were all food isolates and their toxicity was characterized in previous studies.

3.2 Propagation of the strains

To grow the strains, Tryptone Soya Broth (TSB) was used. A small amount was taken from the culture collection and was put in a 10 ml TSB tube. This was held for approximately 24 hours at 37◦C. After this ﬁrst growth period, 100 µl of the grown culture was sub-cultured in fresh TSB followed by another 24 hour incubation at 37◦C. From this second culture an amount was taken and spread on a TSA slant to prepare a working stock. This slant was held for another 24 hours at 37◦C and could thereafter be stored in the fridge of 7◦C for approximately 2 months. The purity of the culture was veriﬁed by isolation of single colonies on TSA.

3.3 B. cereus spore production

B. cereus spores were used in this study. To make the spores, a loopfull biomass from the B. cereus slant was taken and released in Brain Heart Infusion (BHI) broth. This was incubated for approximately 24 hours at 37◦C. From this grown culture 100 µl was taken and spread on NAMgCl2CaCl2 agar plates. These plates consisted of Nutrient agar (28 g/L) supplemented with MgCl2 (0,04 g/L) and CaCl2 (0,10 g/L). This was done on 6 plates and the plates were incubated at 30◦C for 5 days. After this period, the spores could be harvested with sterile water and a sterile spatula. The harvested spores were placed in a 50 ml sterile falcon. This falcon was subsequently centrifuged for 15 minutes at 10000 g. The resulting pellet was thereafter washed with a salt solution (0.85 % NaCl). After centrifugation for two times, the resulting pellet was resuspended in a 50 % ethanol solution and held for minimum 12 hours at 4◦C to eliminate the remaining vegetative cells. Afterwards there were 4 centrifugation steps and washing steps with the salt solution to remove the ethanol. The resulting spore suspension could be stored for 2 months at 2◦C.

18 CHAPTER 3. MATERIALS AND METHODS 19

3.4 Bacterial enumeration

Enumerations were performed using spread plate technique and when necessary 10-fold dilution series in Peptone Physiological Saline (PPS with 0.01 % peptone + 0.85 % NaCl in sterile demineralized water) were made. With the spore suspension, the amount was calculated after applying a heat treatment of 10 minutes at 90◦C to inactivate present vegetative cells.

3.5 General experimental setup

3.5.1 General setup with Tryptone Soya Broth To see if S. aureus and B. cereus could grow in a modified atmosphere, preliminary experimental setup with Tryptone Soya Broth (TSB) in bottles was used. This bottle is illustrated in Figure 3.1. Bottles filled with 300 ml TSB where autoclaved and afterwards flushed with a concentration of ± 8 % oxygen balanced with nitrogen. The bottles were hereafter inoculated with S. aureus or B. cereus cells to have approximately 1000 cells/ml. Inoculated bottles were incubated at 12°C for 5 days and at 22°C for 2 days.

Figure 3.1: Bottle ﬁlled with TSB

From these bottles the gas concentration was measured at sampling days at which 1 ml sample was taken and used for enumeration.

3.5.2 General setup with Tryptone Soya Agar To determine the growth and toxin production on general growth media (mimicking solid food) at modified atmosphere, Tryptone Soya Agar (TSA) was used. For this purpose 33 ml TSA was taken by a pipette and was put in petri dishes. Standardized volume was used in order to accordingly determine growth and toxin production on the medium. From an appropriate dilution of bacterial cultures 100 µl was taken to have approximately 100 cells/g (S. aureus) and 100 spores/g (B. cereus) on the agar surface. These plates were placed in EVOH (Ethylene Vinyl Alcohol) trays, covered by a film ( 430 mm, from Decapac NV, Herentals) and packaged in MAP (Modified Atmosphere Packaging) of approximately 8 % O2 and the remainder N2. The packaging machine used was Tray-sealer MECA 900 (Decatechnic, Herentals). The packages were stored at 22◦C and samples for enumeration and toxin detection were daily taken. CHAPTER 3. MATERIALS AND METHODS 20

3.5.3 General setup with ready-to-eat lasagna Lasagna was bought at a local supermarket. It was regular lasagna bolognaise with the ingredients listed in decreasing amount: milk, pork, water, wheat semolina (pasta), cheese, tomato concentrate, tomato’s, oil, wheat flour, modified corn starch, onions, eggs, carrots, zucchinis, salt, sugar, herbs, spices, garlic, aroma’s and beta carotene as a pigment. The composition as declared by the producer is in Table 3.1. The lasagna was firstly mixed to warrant homogeneous inoculation and growth, and non-biased sampling for enumerations and toxin determination. Af- terwards, this mixed lasagna was put in jars and received a heat treatment of 15 min at 121◦C. The lasagna was sterilized to eliminate effect of background flora for the purpose of easier and more reliable enumeration. Afterwards, the jars were closed and could be hold for a night at room temperature to be used the next day. From these lasagna samples were taken to measure aw-value and pH. Before the packages with fresh lasagna from the store were opened, the gas composition was measured with a PBI Dansensor, Checkmate 9900 (PBI-Dansensor A/S, Ring- sted, Denmark). This was done to obtain background information about the gas compositions in lasagna packages.

Table 3.1: Composition lasagna

Compound Amount (g per 100 g lasagne) Proteins 8,2 Carbohydrates 11 Sugars 1,6 Fats 8,2 Fibers 1 Salt 0,8

Approximately 40 gram of the mixed and autoclaved lasagna was put on a sterile manner in sterile petri dishes. This amount was chosen to have about 15 gram for the bacteria enumeration and 10 gram for every toxin test. In this experimental setup was aimed to have approximately 100 vegetative S. aureus cells/gram lasagna and 100 spores B. cereus / gram lasagna . To do this, 1 ml of the appropriately diluted S. aureus cell culture and B. cereus spore suspension was added in the lasagna petri dish. To mimic the conditions when the lasagna is prepared in a company, the spore suspension received a heat treatment. This was done by taking 1 ml of the spore suspension in a test tube. This test tube and a tube with 1 ml water were placed in a hot water bath at 90◦C. In the tube with 1 ml water, a thermometer was placed and when the thermometer showed that the temperature was ± 90◦C, the 10 minute heat treatment started. For both pathogens 1 ml of the appropriate dilution was taken and distributed over the lasagna and mixed with a sterile spatula for a uniform distribution. The two dilution series of the both bacteria were also spread on TSA plates to know the exact amount that was inoculated. These TSA plates were incubated at 37◦C for 24 hours and the exact amount could be counted.

The petri dishes with inoculated lasagna were afterwards stored in a MAP package as previously described. In this study is worked with a concentration of approximately 8 % O2 and the ◦ ◦ remaining N2. The packages were stored at 12 C and at 22 C.

A general scheme is in Figure 3.2. CHAPTER 3. MATERIALS AND METHODS 21

B. cereus spores S. aureus vegetative heat treatment 10 cells minutes @ 90°C

± 40 gram sterile lasagna/petri dish  ±100 spores B. cereus /gram  ± 100 vegetative cells S. aureus /gram

Petri dish in MAP package

± 8%O2, remainder N2

12°C 22°C

Figure 3.2: General scheme of the experimental setup with lasagna

A sample was taken approximately every 24 hours. Two plates from 12◦C and 2 from 22◦C were taken and the gas concentration in the package was measured with a PBI Dansensor. When the samples couldn’t be analyzed on the same moment, the whole package was stored in a fridge of 2◦C until the analyzing moment.

3.5.4 Bacteria enumeration from samples To know the amount of bacteria every day, a spread plate method was used. For S. aureus enumeration, Baird Parker (BP, Oxoid, Hampshire, England) agar was used. To count the amount of B. cereus, Mannitol Egg Yolk Polymyxin (MYP, Oxoid, Hampshire, England) agar was used. The S. aureus colonies are little dark black spots and the B. cereus colonies could be easily counted because they are large pink colonies with a precipitation zone around it. Approximately 15 gram lasagna (or TSA-agar) was taken in a sterile way from the samples and placed in a stomacher bag. The lasagna (or TSA) was subsequently diluted with an amount of PPS to make a 1/10 dilution. This ﬁrst dilution was mixed in a stomacher machine (Stomacher lab-blender 400) for one minute. This basic solution was next diluted in tubes with 9 ml PPS, to have a series of 10-fold dilutions. From the appropriate dilution 100 µl was taken and spread out on BP and MYP agar. Both were incubated for approximately 24 hours at 37◦C and afterwards the Colony Forming Units (CFU) could be counted. The remaining part of the lasagna (or TSA) in the petri dish was put in a freezer of -18◦C. These were stored, so the toxine tests could be performed at the end of the experimental setup, this starting from the last day. In this way test kits could be saved because if the last day was CHAPTER 3. MATERIALS AND METHODS 22 negative, the previous days would also be negative.

3.6 Toxin tests

3.6.1 Staphylococcal enterotoxin test Before the actual experiments could start, the ability of the S. aureus LFMFP 356 strain to produce SEs was veriﬁed. At the same moment the extraction protocol of these enterotoxins from lasagna was checked. From the culture grown overnight in TSB tube, 1 ml was taken (ca. 108 CFU/ml) and put in approximately 30 g lasagna. This lasagna was thereafter incubated at 37◦C overnight. The next day, the general extraction protocol for food samples was performed as indicated by the manufacturer and presence of SEs was tested using VIDAS Set2.

With VIDAS Set2, staphylococcal enterotoxins types SEA to SEE can be detected. Approxi- mately 10 gram of the lasagna or TSA was taken and was placed in a sterile screw cap falcon. The same amount of the extraction buffer from the test kit was added. This was left alone for 15 minutes. Afterwards the falcons were centrifuged for 15 minutes at 5000 g. From these falcons, the supernatant was taken and was inserted in a syringe containing a plug of absorbent cotton. The resulting filtered solution was placed in another screw cap falcon and the pH was adjusted between 7.5 and 8.0 with 1N NaOH. From this, 500 µl could be taken and injected in a test kit. After 80 minutes, the results were printed. On the printed report a test value is given, which is the RFV (Relative Fluorescense Value) of the sample divided by the RFV of a standard (purified enterotoxin A < 1.0 ng/ml). If the tested value was above 0.13, the sample was considered positive.

3.6.2 B. cereus cereulide toxin test To determine the cereulide toxin, a bio-assay with boar semen was used as described by Rajkovic et al. (2006b). Semen was purchased from the company Hypor K.I. in Olsene, Belgium. To know if both B. cereus strains used were cereulide producers, ﬁrstly some toxin tests were performed with cultures grown on laboratory media.

Cereulide detection from TSA B. cereus strains were grown in TSB and from this, 1 ml was taken and spread on TSA plates. After a growth period of 24 hours, the biomass at the surface was scraped oﬀ with a plastic spatula. This biomass was thereafter placed in an Erlenmeyer of 25 ml and methanol was added to have a ratio of 2/3 methanol and 1/3 biomassa.

In the experimental setup with the inoculated TSA-plates, the same method could be used. But the TSA plates were stored in the freezer for a period and the plates were become aqueous. From these samples, the liquid was taken and placed in small eppendorf tubes. These tubes were thereafter centrifuged and the pellet was solved in methanol. This mixture was placed in a little Erlenmeyer of 25 ml. In this Erlenmeyer more methanol was added as described above.

The mixture of biomassa and methanol in the Erlenmeyer was held for 15 minutes in a hot water bath at boiling conditions. When the amount of methanol evaporated, more methanol was added. Afterwards the solution could be examined with the microscope described below in ’Microscopic analyzes’. CHAPTER 3. MATERIALS AND METHODS 23

Cereulide detection from lasagna Before the experiments were set up with lasagna, the extraction protocol of cereulide from lasagna was checked. This was performed to see whether the lasagna matrix does not disturb this extraction. It was checked in two manners. One was done by adding 50 µl of a cereulide containing extract in the lasagna. A second method was by adding the biomass of a 24 hour grown culture of B. cereus LFMFP 434 on TSA (1 ml of the culture spread on TSA) and mixing this in a lasagna sample. This lasagna was held for another 24 hours at 37◦C to make sure the bacteria have produced cereulide. The extraction protocol was applied to the two lasagna samples and they were analyzed on the same method as would be with the experimental setup with lasagna (see below).

In the experiment with lasagna, the other 10 gram of the lasagna from the petri dish was taken and put in a glass conical ﬂask or a 50 ml scott glass bottle. At this amount, 2/3 methanol was added as an extraction solution. It was held for approximately 15 minutes in a hot water bath at boiling conditions. This was performed to make sure all other bacteria are killed, possibly other toxins are destroyed and to facilitate extraction (Rajkovic et al., 2007). In this way, only cereulide remains and other possibly inﬂuencing toxins or bacteria are eliminated. Several times, the bottles were mixed manually and at the end 3 ml extra methanol was added. After 15 minutes, the upper liquid phase was taken in a tube to serve for analyzes.

Microscopic analyzes A camera connected to a microscope (Zeiss, AxioCam Mrm Imager A.1) was used. The image of this camera could be seen on a computer via AxioVision program. On the table of the microscope, a heating block at 37◦C was used, to make sure the semen was in optimal conditions. In general, 5 µl of the solution was mixed with 195 µl of the boar semen in a microtiter well. From this mixture, 5 µl was subsequently injected into two chambered Leja Slides (standard count 2-chambered slide, Leja, Nieuw-Vennep, The Netherlands).

But ﬁrstly the motility of the semen, without methanol or a sample solution was determined (a blanc control). If the semen was motile enough, the motility with methanol was subsequently analyzed. If the boar semen was still motile enough after 10 minutes, the semen could be used for analyze as the methanol didn’t inﬂuenced the semen motility. In general, if the semen stopped moving in < 10 minutes, the sample was considered to be positive. In Figure 3.3 a picture from the semen is showed. CHAPTER 3. MATERIALS AND METHODS 24

Figure 3.3: Green colored: moving semen, Red colored: stopped semen.

3.7 Result interpretation

To analyze the results, Microsoft Oﬃce Excel 2007 was used. To compare if results were significant, the T-test function in Excel was used.

3.8 Overview of the diﬀerent experimental setups

Different experimental setups were performed. The tests were performed on laboratory media and on lasagna. In lasagna two B.cereus strains were used and one S. aureus strain. The tests were performed with and without a modified atmosphere, to have an idea about the difference in growth and toxin production at ambient and modified atmosphere. The experiment in lasagna was also performed with the vegetative cells of B. cereus and the spores to see if this has an influence on the growth and toxin production. The lasagna was stored at 12◦C for approximately 14 days and at 22◦C for approximately 6 days, the time necessary for the bacteria to reach the stationary phase. An overview of the different experimental setups is in Table 3.2. CHAPTER 3. MATERIALS AND METHODS 25

Table 3.2: Overview of the diﬀerent experimental setups

Media Strain B. cereus Map or Ambient Temperature Number of B. cereus S. aureus vegetative days cell (VC) or spore TSB 434 / VC MAP 12°C 5 434 / VC MAP 22°C 2 436 / VC MAP 12°C 5 436 / VC MAP 22°C 2 / 356 / MAP 12°C 5 / 356 / MAP 22°C 5 / 362 / MAP 12°C 5 / 362 / MAP 22°C 5 TSA 434 356 Spore MAP 22°C 4 434 / Spore MAP 22°C 4 434 356 Spore Ambient 22°C 4 Lasagna 434 356 Spore MAP 12°C 14 434 356 Spore MAP 22°C 6 434 356 Spore Ambient 12°C 14 434 356 Spore Ambient 22°C 6 434 356 VC MAP 22°C 6 436 356 Spore MAP 12° C 14 436 356 Spore MAP 22°C 6 436 356 Spore Ambient 12°C 14 436 356 Spore Ambient 22°C 6 Chapter 4

Results and discussion

4.1 Maximum cell densities of tested strains in TSB-broth

In order to investigate the basic growth properties before the food-based experiments, two strains of S.aureus (LFMFP 356 and 362) and two strains of B. cereus (LFMFP 434 and 436) were tested for their growth at optimal temperature of 37◦C in TSB-broth. The cell densities after 24 hour incubation of a loopfull culture are shown in Table 4.1.

Table 4.1: Maximum cell density tested strains

Strain Average maximum Standard deviation cell count after 24 (CFU/ml) hours (CFU/ml) S. aureus LFMFP 356 7.6*108 ±1.8 ∗ 108(n = 18) S. aureus LFMFP 362 7.4*108 ±2.9 ∗ 108(n = 4) B. cereus LFMFP 434 4.2* 106 ±2.5 ∗ 106(n = 5) B. cereus LFMFP 436 2.2 * 107 ±1.4 ∗ 107(n = 2)

4.2 Spore forming by B. cereus

The amount of spores produced for inoculation of lasagna was counted after applying a heat treatment of 10 minutes at 90◦C to eliminate vegetative cells. The average amount from every spore suspension is shown in Table 4.2.

Table 4.2: B. cereus spore concentration after heat treatment

Strain Spore concentration Standard deviation after heat treatment (CFU/ml) (CFU/ml) B.cereus LFMFP 434 7.5*106 ±6.5 ∗ 106(n = 10) B. cereus LFMFP 436 4.2*106 ±7 ∗ 105(n = 2)

26 CHAPTER 4. RESULTS AND DISCUSSION 27

4.3 Staphylococcal enterotoxin production by S. aureus strain

The ability of the selected S. aureus LFMFP 356 strain to produce staphylococcal enterotoxins (SEs) was veriﬁed before the food-based experiments started, at the same moment the extraction protocol ( prescribed by the kit producer) of these enterotoxins from lasagna was checked as described in 3.6.1. The analyzed lasagna sample was positive with a VIDAS Set 2 RF (Relative Fluorescence) value of 2.66.

4.4 Cereulide production by B. cereus strains

Cereulide production by selected B. cereus strains was checked from a sample of 24 hour culture in TSB. This test was however negative. A second test was performed by adding 1 ml of this culture on TSA-plates and incubating at 37◦C for 24 hours. The biomass was examined by boar semen bio-assay after the general extraction protocol from TSA (Rajkovic et al., 2006b). These tests were positive for both B. cereus strains. The semen stopped moving after 30 seconds to 1 minute of exposure to the extract of biomass of B. cereus LFMFP 434 and 436.

4.5 Cereulide extraction from lasagna

The extraction and detection of cereulide from lasagna was checked by 2 manners as described in 3.6.2. These both lasagna mixtures were extracted according to the extraction protocol (Rajkovic et al., 2007). They were both positive, the semen stopped after 4-5 minutes.

4.6 Growth of S. aureus and B. cereus strains in modiﬁed atmosphere in TSB

This experiment was performed to determine whether S. aureus and B. cereus can grow if the ◦ atmospheric conditions are modiﬁed to 8 % O2 / 92 % N2 at storage temperatures of 12 C and 22◦C. The growth curves of S. aureus LFMFP 356 and 362 at 12◦C and 22◦C are in Figure 4.1. The gas composition was also measured at every sampling moment. The results of these are in shown Figure 4.2.

Growth of S. aureus in TSB at 8 % O2 / 92 % N2

8 Strain LFMFP 356, 12°C 6 Strain LFMFP 362, 4 12°C Strain LFMFP 356,

2 22°C Cell count (log CFU/ml) count (log Cell 0 Strain LFMFP 362, 0 50 100 150 200 22°C Time (h)

Figure 4.1: Growth of S.aureus in bottles with TSB at 8 % O2 and 92 % N2 CHAPTER 4. RESULTS AND DISCUSSION 28

Evolution gas composition during growth of S.aureus in TSB

8 Strain LFMFP 356, O2, 12°C 7 Strain LFMFP 356, CO2, 12°C 6 Strain LFMFP 356, O2, 22°C Strain LFMFP 356, CO2, 22°C

evolution(%) 5

2 4 Strain LFMFP 362, O2, 12°C

3 Strain LFMFP 362, CO2, 12°C

andCO

2 2 Strain LFMFP 362, O2, 22°C O 1 Strain LFMFP 362, CO2, 22°C 0 0 50 100 150 200 Time (h)

Figure 4.2: Evolution of the gas composition during the growth of S. aureus in TSB

S. aureus can grow at diﬀerent atmospheric conditions as ﬁgure 4.1 shows, with as expected faster growth at 22◦C and a slower growth at 12◦C. At 22◦C, the maximum cell density of 8.7 log CFU/ml is already reached after 48 hours. At 12◦C, growth goes slower, but after approximately 144 hours (6 days) 8 log CFU/ml is reached. This could also be seen on Figure 4.2 with the gas composition, with a raise in CO2 concentration starting at 28 hours at 22°C. The CO2 slowly begins to rise at 120 hours at 12◦C.

The growth curve and gas composition of B. cereus LFMFP 434 and 436 in TSB are presented in Figure 4.3 and Figure 4.4.

Growth of B. cereus in TSB at 8 % O2 / 92 % N2

7 6 Strain LFMFP 434, 5 12°C 4 Strain LFMFP 436, 12°C 3 2 Strain LFMFP 434, 22°C

1 Cell count ( log count log Cell CFU/ml) ( 0 Strain LFMFP 436, 22°C 0 25 50 75 100 125 150 Time (h)

Figure 4.3: Growth of B. cereus in bottles with TSB at 8 % O2 and 92 % N2

As ﬁgure 4.3 shows, were both strains of B. cereus able to grow at 12◦C and 22◦C in modiﬁed atmosphere. At 22◦C a cell count of 6.7 log CFU/ml was observed after 48 hours of incubation for B. cereus LFMFP 434. At the same moment the cell count for B. cereus LFMFP 436 was 7.5 log CFU/g. At 12 °C, slower growth and lower cell counts were seen. CHAPTER 4. RESULTS AND DISCUSSION 29

Evolution of the gas composition during growth of B. cereus in TSB 9

8 7 Strain LFMFP 434, O2, 12°C 6 Strain LFMFP 434, CO2, 12°C

5 Strain LFMFP 434, O2, 22°C

evolution(%)

2 4 Strain LFMFP 434, CO2, 22°C

3 Strain LFMFP 436, O2, 12°C andCO 2

2 Strain LFMFP 436, CO2, 12°C O 1 Strain LFMFP 436, O2, 22°C 0 Strain LFMFP 436, CO2, 22°C 0 25 50 75 100 125 150 Time (h)

Figure 4.4: Evolution of the gas composition during the growth of B. cereus in TSB

A corresponding trend could also be seen on ﬁgure 4.4 with the gas composition, where the CO2 ◦ ◦ ◦ increased faster at 22 C than at 12 C. In the same way, depletion of O2 was faster at 22 C than ◦ at 12 C. The CO2 concentration started to rise after 25 hours.

4.7 Experimental setup with TSA

4.7.1 Growth of S. aureus and B. cereus on TSA The experiments on TSA were performed to know if S. aureus and B. cereus can grow and produce their corresponding toxins when they grow in different atmospheric conditions. An experiment comprised TSA inoculated with S. aureus vegetative cells and B. cereus spores. These TSA plates were packaged with MAP (Modified Atmosphere Packaging) and without MAP. Additional experiment was done with B. cereus alone to know if a competition between B. cereus and S. aureus could possibly have an influence on the cereulide production of B. cereus. The respective growth curves of S.aureus and B. cereus and the onset of respective toxin production at these conditions are shown with an arrow in Figure 4.5 and Figure 4.6.

SE detection Growth of S. aureus LFMFP 356 on TSA at 22°C

4 MAP ambient

2 Cell count CFU/g) Cell (log 0 0 25 50 75 100 125 150 Time (h)

Figure 4.5: Growth of S. aureus LFMFP 356 on TSA with or without MAP

From the presented data it can be seen that S. aureus had a maximum cell count after 2 days at 8.7 log CFU/g. CHAPTER 4. RESULTS AND DISCUSSION 30

Cereulide detection Growth of B. cereus LFMFP 434 on TSA at 22°C

6 MAP

4 MAP, without S. aureus 2

ambient Cell count CFU/g) Cell (log 0 0 25 50 75 100 125 150 Time (h)

Figure 4.6: Growth of B.cereus LFMFP 434 on TSA with or without MAP

B. cereus reached a maximum cell count of 8.7 log CFU/g after 2 days (43 hours). The maximum cell count was independent of the atmosphere in the package. The ﬁgures show that the copresence of B. cereus spores and S. aureus vegetative cells didn’t had an inﬂuence on the mutual growth. The gas concentration was also taken at every sampling moment. The results of these data are shown in Figure 4.7. At day 2, the gas composition was completely changed with no O2 present and high CO2 concentration.

Evolution of the gascomposition in the package during growth on TSA

11 9 7 Both, MAP, O2

evolution (%) 5

2 Both, MAP, CO2 3

Alone, MAP, O2 and CO

2 Alone, MAP, CO2 O -1 0 25 50 75 100 125 150 Time (h)

Figure 4.7: Evolution of the gas composition in the package during the growth of B. cereus LFMFP 434 alone or together with S. aureus LFMFP 356 on TSA

4.7.2 Toxin production during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores on TSA The results of toxin determination are shown in Table 4.3 In the table is indicated from which day the results were positive. For SE detection, the tested relative ﬂuorescence (RF) value is also given. If this value was higher than 0.13, the result was positive (manufacturer instructions). If the value is higher, the SE concentration is also higher. The cereulide detection was positive if the semen stopped moving in a time span of 10 minutes. If the semen stopped faster, the cereulide concentration was higher. The days the toxins were detected are also identiﬁed by an CHAPTER 4. RESULTS AND DISCUSSION 31 arrow on Figure 4.5 and Figure 4.6.

Table 4.3: Toxin detection during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores on TSA

Staphylococcal Enterotoxin detection

Sample Day Positive/ VIDAS Average log cell negative1 set 2 RF count (log CFU/g) S. aureus 356+ 1 + 0.64 6.7 B. cereus 434, 2 + 2.88 8.6 MAP 3 + 2.67 8.7 S. aureus 356+ 1 + 0.41 6.7 B. cereus 434, 2 + 2.47 8.8 ambient 3 + 2.97 8

Cereulide detection

Day Positive/ Time till stop Average log cell negative (seconds) count (log CFU/g) S. aureus 356+ 1 - /2 6.8 B. cereus 434, 2 + 20 8.7 MAP 3 + 20 8.5 S. aureus 356+ 1 - / 7 B. cereus 434, 2 + 20 8.7 ambient 3 + 20 8.2 B. cereus 434, 1 - / 6.8 MAP 2 + 20 8.7 3 + 20 8.7 1 If indicated as ’+’, this means the sample was considered positive. If indicated ’-’, the sample was considered negative. 2 If indicated ’/’, the semen was still motile after 10 minutes

SEs could be detected already from day 1, which corresponded with a cell count of 6.7 log CFU/g. The following day, the VIDAS set 2 RF value rose rapidly, indicating a relative increase in SE amount, but remains than approximately constant during the following day, which indicates a constant RF value during the stationary growth phase.

The results show that cereulide could be produced at modiﬁed atmospheric conditions on TSA agar, this from day 2 (43 hours). This corresponded with a cell count of 8.7 log CFU/g. The same could be seen with the ambient stored TSA plates. The presence of S. aureus didn’t had an inﬂuence on the cereulide production. The semen stopped moving after 20 seconds, indicating the presence of a big quantity of cereulide. CHAPTER 4. RESULTS AND DISCUSSION 32

4.8 Experimental setup with Lasagna

4.8.1 pH and aw-value lasagna

After the lasagna was autoclaved and mixed, pH and aw-value were measured. The aw-value was also measured for the mixture of 40 gram lasagna with 2 ml PPS, because the lasagna was inoculated with PPS containing the spores and cells. In Table 4.4 the results are represented.

Table 4.4: pH and aw-value lasagna

Parameter Value pH 5,6 aw (regular lasagna) 0,9894 aw (lasagna + 2 ml PPS) 0,99

4.8.2 Measured gas concentration in fresh lasagna Before the lasagna was mixed, the gas concentration from the fresh, unopened lasagna package was measured. The results are in Table 4.5.

Table 4.5: Gas concentration fresh lasagna package

Lasagna package O2 concentration CO2 concentration Days till the end of (kg) (%) (%) shelf life* 0,4 0,5 11,9 11 1 5,3 9,9 9 1,6 0,825 9,5 / 0,4 0,829 12,2 8 1 0,223 11,3 8 1,6 0,818 9,9 8 0,4 0,713 14,7 / 1,6 0,79 19,8 / ∗ The days till the end of the shelf life are not indicated from every sample. The ’/’ symbol indicates that the date was not wrote down the moment of analyze.

The O2 concentration was almost always around 1 %. But from the eight investigated samples, one had a value that was around 5.3 % O2. The CO2 concentration ranged from 10 to 20 %.

4.8.3 Experiment with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 434 spores in lasagna Growth S. aureus LFMFP 356 and B. cereus LFMFP 434 spores in lasagna In this ﬁrst experimental setup, S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 434 spores were inoculated in lasagna. These lasagna containing plates were MAP packaged and held at 12◦C and 22◦C. Additionally, a number of samples were stored at ambient atmosphere to see the diﬀerence in growth and toxinproduction at both atmospheric conditions. The growth curves and the onset of respective toxin production of S. aureus LFMFP 356 and B. cereus LFMFP 434 at 12◦C and 22◦C are presented in Figure 4.8 and Figure 4.9. CHAPTER 4. RESULTS AND DISCUSSION 33

SE detection Growth of S. aureus LFMFP 356 in lasagna

6 22°C, MAP 4 22°C, ambient 12°C, MAP 2

Cell count CFU/g) Cell (log 12°C, ambient 0 0 50 100 150 200 250 300 350 Time (h)

Figure 4.8: Growth of S. aureus LFMFP 356 in lasagna (with B. cereus LFMFP 434) with or without MAP

Cereulide detection Growth of B. cereus LFMFP 434 in lasagna

6 22°C, MAP 4 22°C, ambient 12°C, MAP 2

Cell count CFU/g) Cell (log 12°C, ambient 0 0 50 100 150 200 250 300 350 400 Time (h)

Figure 4.9: Growth of B. cereus LFMFP 434 spores in lasagna with or without MAP

At 22◦C, S. aureus grew to a maximum cell count of 8.6 log CFU/g, with or without modified atmosphere. At 12◦C a significant difference of 0.5 log CFU/g (p=0.019<0.05) could be seen between the S. aureus cells growing with or without MAP. The maximum cell count after 14 days at 12◦C reached 7.3 log CFU/g.

B. cereus at 22◦C grew to a maximum cell count of 8 log CFU/g at day 5 with modiﬁed atmosphere. When the lasagna was stored at ambient air conditions, a maximum cell count of 8.5 log CFU/g was achieved at day 6. At 12◦C, growth was observed too, but slower and to lower cell counts. There is almost no growth during the ﬁrst 8 days, but from then the growth slowly started. After 14 days at 12◦C, the maximum cell count at both packaging conditions reached 5 log CFU/g.

At 22◦C, no big diﬀerence between the growth of both bacteria can be seen. At 12◦C, on the other hand, S. aureus started to grow faster than B. cereus. S. aureus had a lag phase of 2 days, while for B. cereus this was 7-8 days.

The gas composition is represented in Figure 4.10. CHAPTER 4. RESULTS AND DISCUSSION 34

Gas composition in lasagna packages during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434

15 22°C, O2

evolution(%)

2 22°C, CO2

5 12°C, O2

andCO

2 12°C, CO2

O 0 0 50 100 150 200 250 300 350 Time (h)

Figure 4.10: Evolution of the gas composition in lasagna packages during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores

The gas composition at 12◦C almost didn’t change during the 14 day period. At 22◦C the composition started to change from day 3, in the way that O2 was depleted and CO2 was formed.

Toxin production during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores in lasagna The results of SE and cereulide detection are presented in Table 4.6 and Table 4.7. The SE detection is indicated as the average value and corresponding average growth. For cereulide the detection and corresponding growth are indicated for the A and B sample separately, because the growth and corresponding semen motility was sometimes diﬀerent between the two samples on the same day. The day that the toxins were detected for the ﬁrst time is also indicated with an arrow on Figure 4.8 and Figure 4.9. CHAPTER 4. RESULTS AND DISCUSSION 35

Table 4.6: Enterotoxin detection during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores in lasagna

Staphylococcal enterotoxin detection

Sample Day Positive/ VIDAS Average log Negative Set 2 RF cell count (log CFU/g) S. aureus 356 1 − 0 3.4 +B. cereus 434 2 + 1.18 7 MAP, 22°C 3 + 2.84 8.3 S. aureus 356 1 − 0 5.3 +B. cereus 434, 2 + 0.25 6.3 ambient, 22°C S. aureus 356 10 − 0.04 5.3 +B. cereus 434, 11 − 0.03 5.4 MAP, 12°C 12 + 1.07 6.4 13 + 1.32 6.4 14 + 2.03 7.3 S. aureus 356 9 − 0.06 5.4 +B. cereus 434, 10 − 0.12 5.8 ambient, 12°C 11 + 0.25 6.4

SEs were detected from day 2 at 22◦C, with or without the modiﬁed atmosphere and corresponding growth of 7 and 6.3 log CFU/g. The RF value from the SE test rose the next day. At 12◦C, SEs were detected from day 12 in the modiﬁed atmosphere packages. The corresponding cell count of S. aureus at that moment was 6.4 log CFU/g. When the lasagna was conserved at ambient atmosphere, the SEs were detected from day 11; the cell count of S. aureus at that moment was 6.4 log CFU/g. CHAPTER 4. RESULTS AND DISCUSSION 36

Table 4.7: Cereulide detection during growth of S. aureus LFMFP 356 and B. cereus LFMFP 434 spores in lasagna

Cereulide detection

Sample Day Positive Time till stop Log cell count

/negative (log CFU/g) ABAB S. aureus 356 3 - / / 6.9 7 +B. cereus 434, 4 + 7m30s 5m 7.6 7.9 MAP, 22°C 5 + 2m30s 3m 7.7 8 S. aureus 356 2 - / / 6 6.2 +B. cereus 434, 3 + 1m40s 2m30 7.2 7.5 ambient, 22°C 4 + 1m30s 1m 7.5 7.5 6 + 40s 40s 8.7 8.4 S. aureus 356 1-14 - / / 5.2 4.4 +B. cereus 434, MAP, 12°C S. aureus 356 1-14 - / / 4.3 5.2 +B. cereus 434, ambient, 12°C

Cereulide was detected at day 4 in the lasagnas conserved at 22◦C with a modiﬁed atmosphere. The corresponding average cell count of B. cereus LFMFP 434 at that moment was 7.75 log CFU/g. In the lasagna conserved with ambient atmosphere at 22◦C, cereulide was detected at day 3 with a corresponding average cell count of 7.35 log CFU/g. The semen motility decreased faster in de ambient stored lasagna than in the MAP lasagna, indicating higher concentration of cereulide corresponding with the higher cell counts. At 12◦C, no cereulide could be detected regardless of the atmosphere condition in the package.

However, in one repetition, cell count of B. cereus LFMFP 434 spores in lasagna at 22◦C in MAP was below 7 log CFU/g after 7 days and no cereulide was found. In another experiment with only B. cereus LFMFP 434 spores (and no S. aureus cells) in lasagna packed in MAP and conserved at 22◦C, the cell count reached 7.4 log CFU/g after 7 days with only one of the two tested samples positive.

4.8.4 Experiment with B. cereus LFMFP 434 vegetative cells in lasagna Growth of B. cereus LFMFP 434 vegetative cells in lasagna In this experiment the possible inﬂuence of B. cereus vegetative cells instead of spores on the growth and toxin production was examined. A similar experimental setup as before was used in lasagna, with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 434, but now the vegetative cells were used. The growth of B. cereus 434 vegetative cells in this experiment is represented in Figure 4.11. CHAPTER 4. RESULTS AND DISCUSSION 37

Growth B. cereus LFMFP 434 vegetative cells Cereulide detection and spores in lasagna at 22°C

8 Vegetative cells, 6 22°C, MAP 4 Spores, 22°C, MAP 2 Cell count Cell (CFU/g) Spores, 22°C, 0 ambient 0 25 50 75 100 125 150 175 Time (h)

Figure 4.11: Growth B. cereus 434 vegetative cells and spores in lasagna at 22°C

On the growth curve the difference between the experiments with B. cereus spores in MAP and without MAP with the B. cereus vegetative cells in MAP can be seen. The vegetative cells had a faster growth, but the maximum cell count was basically the same as the B. cereus spores in a modified atmosphere. The cell count of the B. cereus spores at the ambient conditions was a bit higher at day 7, with a count of 8.5 log CFU/g. This is a significant difference of 0.5 log CFU/g (p=0.024<0.05) with the B. cereus vegetative cells in MAP conditions.

The gas composition is in Figure 4.12.

Evolution of gas composition during growth of B. cereus in lasagna

15 O2, Vegetative cells

evolution(%)

2 10 CO2, Vegetative cells

5 O2, Spores andCO

CO2, Spores 2

O 0 0 25 50 75 100 125 150 175 Time (h)

Figure 4.12: Evolution of gas composition during growth of B. cereus LFMFP 434 spores and vegetative cells in lasagna at 22°C

The start of O2 depletion with vegetative cells and spores started at day 1 and day 3 respectively.

Toxin production of B. cereus LFMFP 434 vegetative cells during growth in lasagna The day the toxins were detected and the amount are in Table 4.8. The day that cereulide was found is also indicated with an arrow on Figure 4.11. CHAPTER 4. RESULTS AND DISCUSSION 38

Table 4.8: Toxin detection during growth of B. cereus LFMFP 434 vegetative cells

Staphylococcal enterotoxin detection Sample Day Positive/ Vidas set 2 RF Average log cell count negative (log CFU/g) S. aureus 356 1 - 0 4.7 +B. cereus 434 2 - 0.09 5.9 vegetative cells, 3 + 0.32 6.5 MAP, 22°C

Cereulide detection Sample Day Positive/ Time till stop Log cell count negative (log CFU/g) ABAB S. aureus 356 3 - / / 6.7 6.9 +B. cereus 434 4 + 9m 6m 7.4 7.6 vegetative cells, 6 + 5m 7m 7.9 8.1 MAP, 22°C

SEs were detected from day 3. This corresponds with a cell count of 6.5 log CFU/g. Cereulide was detected from day 4, which corresponded with an average cell count of 7.5 log CFU/g.

4.8.5 Experiment with S. aureus LFMFP 356 vegetative cells and B. cereus LFMFP 436 spores in lasagna Growth of S. aureus LFMFP 356 and B. cereus LFMFP 436 spores in lasagna To investigate strain dependency, the experiment was additionally performed with another cereulide producing strain under otherwise same experimental conditions. The growth of S. aureus LFMFP 356 and B. cereus LFMFP 436 is shown in Figure 4.13 and Figure 4.14. The corresponding evolution of the gas mixture in the packages is shown in Figure 4.15.

SE detection Growth of S. aureus LFMFP 356 in lasagna

6 22°C, MAP 4 22°C, ambient 12°C, MAP 2

Cell count CFU/g) Cell (log 12°C, ambient 0 0 50 100 150 200 250 300 350 400 Time (h)

Figure 4.13: Growth of S. aureus LFMFP 356 in lasagna (with B. cereus LFMFP 436) with or without MAP CHAPTER 4. RESULTS AND DISCUSSION 39

Cereulide detection Growth of B. cereus LFMFP 436 in lasagna

6 22°C, MAP 4 22°C, ambient 12°C, MAP 2

Cell count CFU/g) Cell (log 12°C, ambient 0 0 50 100 150 200 250 300 350 400 Time (h)

Figure 4.14: Growth of B. cereus LFMFP 436 spores in lasagna with or without MAP

At 22◦C, S. aureus grew fast to the maximum cell count of 8.7 log CFU/g, with or without a modified atmosphere. This maximum was reached after 4 days. As for B. cereus at 22◦C, a significant difference (p=0.078<0.025) in cell count of 1 log CFU/g was found when comparing ambient with MAP. At ambient atmosphere, the maximum was 8.6 log CFU/g after 6 days. At modified atmosphere, the maximum cell count was 7.5 log CFU/g, reached at day 4 and remained constant for the rest of the storage.

S. aureus grew at 12◦C, but the growth was slow. At ambient conditions, the growth was constantly significant 1 log higher (p=0.0371<0.05). After 14 days, the cell count of S. aureus at modified atmosphere was 7.7 log CFU/g. The growth at ambient atmosphere was 8.3 log CFU/g after 14 days. B. cereus grew at 12◦C, but very slow. There was no growth during the first 6 days. After 6 days, the growth started, but was still very slow. After 14 days, a cell count of 5 log CFU/g was reached.

Evolution of gas composition during growth of S. aureus LFMFP 356 and B. cereus LFMFP 436 in lasagna

15 22°C, O2

evolution(%) 10 22°C, CO2

2 5 12°C, O2

12°C, CO2

andCO

2 0 O 0 50 100 150 200 250 300 350 400 -5 Time (h)

Figure 4.15: Evolution of gas composition during growth of S. aureus 356 vegetative cells and B. cereus 436 spores in lasagna at 22°C

At 22◦C, the change in gas composition was very fast and started at day 3. At 12◦C, the change was very slowly, with almost no change during the 14 day period. CHAPTER 4. RESULTS AND DISCUSSION 40

Toxin production during growth of S. aureus LFMFP 356 and B. cereus LFMFP 436 spores in lasagna The enterotoxins and cereulide detected are represented in Table 4.9 and Table 4.10. The day that they were indicated are also indicated with an arrow on Figure 4.13 and Figure 4.14.

Table 4.9: Staphylococcal enterotoxin detection during growth of S. aureus LFMFP 356 and B. cereus LFMFP 436 spores in lasagna

Sample Day Positive/ VIDAS Average log negative set 2 RF cell count (log CFU/g) S. aureus 356 1 - 0 4.4 + B. cereus 436, 2 + 1.43 7.2 MAP, 22°C 3 + 3.03 8.3 S. aureus 356 1 - 0 4.2 + B. cereus 436, 2 + 1.71 7.2 ambient, 22°C S. aureus 356 8 - 0 4.7 + B. cereus 436, 9 - 0.03 5.3 MAP, 12°C 10 + 0.19 6.1 11 + 0.18 6.1 12 + 0.87 6.9 13 + 0.93 6.9 S. aureus 356 8 - 0 5.2 + B. cereus 436, 9 - 0.07 5.8 ambient, 12°C 10 + 0.22 6.4 11 + 0.45 6.5 12 + 1.14 7.2 13 + 2.38 7.5

SEs were already present in great amount at day 2 at 22◦C, with or without a modiﬁed atmosphere package. At 12◦C, SEs were detected from day 10 for both atmosphere conditions. This corresponded with a cell count of 6.14 log CFU/g with modiﬁed atmosphere and a cell count of 6.40 log CFU/g at ambient atmosphere. CHAPTER 4. RESULTS AND DISCUSSION 41

Table 4.10: Cereulide detection during growth of S. aureus LFMFP 356 and B. cereus LFMFP 436 spores in lasagna

Sample Day Positive/ Time till stop Log cell count

negative (log CFU/g) ABAB S. aureus 356 3 - / / 6.1 6.3 + B. cereus 436, 4 + 5m 4m50s 7.8 7.3 MAP, 22°C 5 + 9m 4m 7.0 7.6 6 + 3m15s 5m 7.6 7.1 S. aureus 356 2 - / / 6.0 6.5 + B. cereus 436, 3 + 2m45 3m30s 6.8 6.8 ambient, 22°C 4 + 50s 2m 8.1 8.1 6 + 45s 50s 8.7 8.5 S. aureus 356 1-14 - / / 5.2 5.2 + B. cereus 436, MAP, 12°C S. aureus 356 1-14 - / / 4.8 4.7 + B. cereus 436, ambient, 12°C

At 22◦C, cereulide could be detected from day 3 in lasagna packed with ambient atmosphere, this corresponded with an average cell count of 6.8 log CFU/g. The lasagna with the modiﬁed atmosphere package had cereulide from day 4, which corresponded with an average cell count of 7.55 log CFU/g. At 12◦C, no cereulide was detected in the lasagna with or without modiﬁed atmosphere. CHAPTER 4. RESULTS AND DISCUSSION 42

4.9 Discussion

4.9.1 Growth and toxin production in TSB-broth Both S. aureus and B. cereus were able to grow at modified atmosphere conditions in TSB- broth. The growth was as expected faster at 22◦C than at 12◦C. In TSB-broth a difference in maximum cell count could be seen between S. aureus and B. cereus, with S. aureus reaching a higher cell density at 22°C. Both strains of S. aureus reached similar maximum cell counts if the strains were grown in TSB for 24 hours at 37◦C and in the experiment with growth in TSB at modified atmosphere. But this maximum cell count was reached after 48 hours at 22◦C and after 144 hours at 12◦C. Between the strains of B. cereus however, a difference in maximum cell count could be seen in the experiments at 37◦C and also in the experiment with modified atmosphere at 22◦C. This indicates certain strain variability between tested emetic strains of B. cereus.

In this thesis, no cereulide could be detected from both B. cereus strains grown in TSB for 24 hours at 37◦C. While in other reports, this was the case. In a report of Jääskeläinen et al. (2004), 0.01 µg/ml cereulide could be detected from a B. cereus strain grown in TSB for 16 hours at room temperature. In a study done by Rajkovic et al. (2006b) they report cereulide detection in B. cereus grown in BHI (Brain heart infusion) for 24 hours at 30◦C. Cereulide could be detected from the pellet and the supernatant, however in much smaller quantities in the latter case. Possible reasons why in this thesis no cereulide was produced in the TSB-broth can be that detectable cereulide production started after 24 hours. This was seen in a detailed study by Häggblom et al. (2002). There the authors reported that cereulide production only starts after 16-24 hours, dependent of the strain used and that the final concentration is reached in the subsequent 24 hours. Another possible reason for the undetectable cereulide is that it was grown at 37◦C, while Finlay et al. (2000) reported that the production of cereulide is low or non-detectable at 37◦C in Skim milk while the production is optimal between temperatures of 12-30◦C. The test with the biomass from a TSA plate with 1 ml 24 hour grown B. cereus culture on, gave however positive results. Though, these plates were also incubated at 37◦C. This indicates that the difference between a solid growth medium and the liquid broth as growth medium has a possible influence on the cereulide production, which to a great extent confirms previously published findings.

4.9.2 Growth and toxin production on TSA When both strains were grown on TSA with or without modified atmosphere, no difference in maximum cell count or growth rate could be seen between S. aureus and B. cereus. Even the copresence of S. aureus didn’t have suppressing influence on the growth of B. cereus, or the other way around.

A clear diﬀerence in the onset of toxin production between S. aureus and B. cereus could be seen on TSA plates at 22°C. After 20 hours, an RF value (relative ﬂuorescence in VIDAS Set2 instrument) indicating concentration of staphylococcal enterotoxines of approximately 0.50 is reached, regardless of the atmosphere composition used in the package. The corresponding cell count of S. aureus was 6.7 log CFU/g. If the reached RF-value is compared with the reference value (RF value of 0.13 indicates the positive reaction, i.e. presence of staphylococcal enterotoxins), conclusion can be made that the onset of staphylococcal enterotoxin production will be earlier than 20 hours and at a lower cell count than 6.7 log CFU/g. Detectable cereulide concentration was much later, being 43 hours of incubation at 22°C. However, the cereulide CHAPTER 4. RESULTS AND DISCUSSION 43 concentration at that moment was rather high, judging on the fast stop of semen motility (20 seconds). This indicates that the actual onset of cereulide production will have started earlier, with a corresponding cell count between 6.8 and 8.7 log CFU/g. These counts correspond to the end of the logarithmic phase, beginning of the stationary phase, as also described in the literature. Cereulide could be produced despite of the reduced oxygen content of 8 % O2, which has been also already demonstrated by Rajkovic et al. (2006b).

Looking at the cell counts when toxin production started for both S. aureus and B. cereus, it becomes clear that SEs were produced at a lower cell density than cereulide. But to conﬁrm this, the detection limits of both tests should also be took in account. The detection limit of the VIDAS SET 2 test is ≥ 0.25 ng/ml, which is just below the amount necessary (1-5 µg of ingested SE per person) for a person to get ill (Jablonski and Bohach, 2001). But is enough to make sensitive people sick (Bennett, 2001b).The detection limit with the bio-assay with boar semen is 1 ng/ml, which is just below intoxication levels of 10-1280 ng/g found in a report of Agata et al. (2002). This means that the earlier detection of the SEs at a lower cell density must be interpreted with care, as the detection limit of VIDAS Set 2 is lower. But still, SEs are capable of inducing food intoxications at lower concentrations. This means that the early detection of SEs, using VIDAS Set2, indicates amounts that can upon consumption result in intoxication symptoms.

In this thesis, cereulide on TSA is detected from day 2 (after 43 hours) while in earlier reports, cereulide could be detected already after 24 hours. The earlier reports (Jääskeläinen et al., 2004; Rajkovic et al., 2006b) however used higher inoculation amounts. In this thesis, it took a longer period for the lower inoculation amount to achieve the critical cell density and thus to start cereulide production. Once the stationary phase was reached (around 8.7 CFU/g), the cereulide concentration remained constant (this stationary phase was however only followed during 2 days). Jääskelainen et al. (2004) also described a constant cereulide concentration during the stationary phase, while a recent report of Delbrassine et al. (2011) indicated an increase of cereulide production during the late stationary phase. This was also confirmed by other authors (Agata et al., 2002; Bauer et al., 2010). As already mentioned was the stationary phase only followed during 2 days in this thesis, so a real conclusion about the cereulide concentration to be constant or not can’t really be made. Also the SE concentration remained constant when the stationary phase (around 8.7 CFU/g) was reached, which is because the synthesis is throughout the logaritmic phase of growth or during the transition from exponential to the stationary phase. Although, Wallin-Carlquist et al. (2010) and Marta et al. (2011) indicated a constant increase of SEA and SED respectively during the stationary phase for some meat products held at room temperature during 7 day storage. But again, the stationary phase in this thesis was only followed during 2 days, what is too low to make conclusions. Moreover is the detection using VIDAS Set 2 capable of detecting SEA to SEE and is it thus not known which SE is produced with this tested strain.

4.9.3 Growth and toxin detection in lasagna at 22◦C

The lasagna had a pH of 5.6 and a high aw-value of 0.99. The pH was thus lower than the optimum pH for B. cereus and S. aureus growth. But still, not low enough to prevent growth of both bacteria. Also, the pH was not ideal for toxin production, but still was not low enough to prevent toxin production.

Both bacteria could grow fast at 22°C in lasagna. But comparing both pathogens shows that S. aureus grew earlier to the maximum cell density, while the growth curve of B. cereus is less CHAPTER 4. RESULTS AND DISCUSSION 44 steep, indicating a somewhat slower growth rate. The modified atmosphere did not influence the maximum cell count reached for S. aureus, while for B. cereus a relative small difference could be seen (0.5 log CFU/g difference for B. cereus LFMFP 434 and 1 log CFU/g difference for B. cereus LFMFP 436). The maximum cell count reached at ambient atmosphere packaging conditions at 22°C was the same for B. cereus and S. aureus. For B. cereus a difference in the steepness of the growth curves between the experiment on TSA and in lasagna could be seen, which indicates a faster growth rate for B. cereus growing on TSA. The same can be seen if the growth curves of S. aureus from both experiments are compared. This is as expected, since the pH of the lasagna was lower, which will have influenced the growth rate.

Staphylococcal enterotoxins were detected a day later in the lasagna experiment than in the experiment with TSA. But this is merely because of the slower growth of S. aureus in lasagna compared with the growth on TSA, which can be seen by comparing the cell counts in both experiments. The cell count at day 1 on TSA was approximately the same as the cell count at day 2 (± 6.7 log CFU/g) in lasagna. This indicates that the SE production started in the middle of the logarithmic phase. The modified atmosphere didn’t have an influence on the onset of toxin production, or the amount produced. The amount produced, was only influenced by the corresponding growth phase.

Cereulide was detected from the ambient stored lasagna at day 3, corresponding to an average cell density of 7.3 log CFU/g for strain LFMFP 434 and 6.8 for strain LFMFP 436. In a study of Agata et al. (2002) however, cereulide could be found after 16 hours at 20°C in boiled rice with corresponding cell count between 7 and 8 log CFU/g.

In the case of cereulide production, there is a clear difference between lasagna stored at ambient atmosphere conditions in comparison with the lasagna stored with modified atmosphere packaging. The cereulide production was delayed with one day if lasagna was stored at reduced oxygen atmosphere, indicating the influencing role of reduced oxygen content. Reducing the oxygen content not only can have an influence on the growth rate of B. cereus, but it can also have an influence on the onset of cereulide production and the amount of cereulide produced. This can be seen because cereulide was never produced in modified atmosphere below a cell count of 7 log CFU/g (the lowest cell count were cereulide was detected was 7 log CFU/g but the corresponding stop of semen motility was at 9 minutes, just below the detection range of 10 minutes), while at ambient conditions, cereulide could be detected at cell counts of 6.8 log/CFU with corresponding stop of semen motility after 3 minutes. This influencing role of reduced oxygen content was also already indicated by Jääskelainen et al. (2004) and Rajkovic et al. (2006). The authors reported that although B. cereus can grow to same high densities, cereulide production might be inhibited. Another clear difference is the amount of cereulide detected. This could be seen with the semen motility with semen stopping after 5 to 7 minutes if detected at day 4 with the MAP conditions. The lasagna stored ambient gave results of 2-3 minutes for the semen to stop at the first day of detection (day 3). In both atmosphere conditions the rate with which semen motility stopped was higher if the samples were longer stored. This could be clearly seen in Table 4.7. But the motility decrease was higher for the ambient stored lasagna in comparison with the MAP stored lasagna, but this runs parallel with the higher cell counts. For the lasagna stored in MAP, the rate of semen motility decrease became higher when the stationary phase was reached, what corresponds to the findings of Delbrassinne et al. (2010), who indicated a further increase of cereulide concentration during the late stationary phase.

When the experiment was performed with vegetative cells instead of spores of B. cereus, a faster CHAPTER 4. RESULTS AND DISCUSSION 45 growth at the beginning could be seen, but from day 3 cell counts were approximately the same, as represented in Figure 4.11. Also the day cereulide was detected and the corresponding cereulide concentration was the same. This means that B. cereus germinated fast, as indicated by Broussolle et al. (2008) who found that maximum germination for B. cereus strains tested was obtained after 100 minutes at 30°C. Also, the preheating step (10 minutes at 90°C) that was used in the experimental setup had an activating eﬀect on the spores to germinate in the presence of a permissive germination environment (Setlow, 2003).

Between the two strains of B. cereus tested (B. cereus LMFMP 434 and 436) no diﬀerences could be seen between the growth and onset of toxin production at both atmospheric conditions. Even the amount of cereulide produced was more or less the same, when judged using rate of decrease in semen motility. But this is a rough conclusion based on the ability of the investigator to distinguish diﬀerences in the motility of the semen. More computer based methods are developed where the computer provides numerical characterization of the motility of the semen (Rajkovic et al., 2006b) allowing an objective interpretation of the results.

It is interesting to emphasize that in one repetition of the experimental setup, no cereulide could be detected in lasagna stored at 22◦C in MAP conditions. The comparison of cereulide data and corresponding growth data showed that the cell counts in no samples reached 7 log CFU/g, which is as already indicated, the limit for cereulide production in lasagna stored at modiﬁed atmosphere conditions for the strains used in these experimental setups. But it gave an indication that a somewhat high critical density of 7 log CFU/g is necessary for these strains to produce detectable cereulide in lasagna stored in MAP conditions. This is much higher than the safety limits of 3-5 log CFU/g described in the literature (Granum, 2001a). Meaning that in this case a concentration of 5 log CFU B. cereus/g does not necessary mean that the lasagna is not safe regarding cereulide. This indicates the usefulness of evaluation of the safety of foods through toxine tests instead of counting the amount of bacteria present. Moreover, the amount of present bacteria can be lower than the safety limits because of food processing and preservation, but the produced toxins can be still present.

If lasagna was packaged in a modified atmosphere, as ready-to-eat meals mostly are, the onset of cereulide production was delayed with one more day. But still, 3-4 days at 22°C were necessary to make the lasagna a threat concerning cereulide food intoxication, which is somewhat unrealistic. Lasagna after this time/temperature period looked moreover anything but appetizing, indicating that the consumer would probably not opt to consume it. This is different for SEs, which were found after 2 days in a sufficient amount to cause staphylococcal food intoxication, with or without the MAP conditions. This was the case for abusive storage at room temperature of 22◦C; if the storage temperature would be higher, what can be the case in a hot summer, the critical density will be reached earlier and thus the SEs can be produced earlier.

4.9.4 Growth and toxin detection in lasagna at 12◦C At 12◦C, a pronounced difference in growth between B. cereus and S. aureus could be seen. The first difference is between the lag phases. S. aureus had a lag phase of 1 to 2 days, while B. cereus had a lag phase ranging from 6 to 8 days. But this is probably a mere reflection of the lower germination of B. cereus spores at lower temperatures (Carlin et al., 2006). After the lag phase, the cell count steadily raised. For S. aureus a difference could be seen between the MAP and ambient packed lasagnas, indicating that the presence of the reduced oxygen content at 12◦C could slow down the growth, but the reduction was very small. As already indicated by Belay and Rasooly (2002), is S. aureus capable of growing anaerobically, but the growth is CHAPTER 4. RESULTS AND DISCUSSION 46 slower than if grown aerobically. No difference could be seen between the growth of B. cereus in modified and ambient atmosphere conditions. There was also no difference between the growths of both tested strains of B. cereus (LFMFP 434 and 436) at 12◦C.

What is worth mentioning is that sometimes the lasagna packages were conserved in a fridge of 2◦C during one or two days. This was the case if the sample needed to be taken in the weekend and the analyzing moment was not until on Monday. On these days, a remarkable decrease in B. cereus could be seen on the growth curves. If the sample was stored for 2 days, no more B. cereus was present in the sample. If stored for 1 day, the number of viable B. cereus was decreased. This indicates that this mesophilic B. cereus is very sensitive towards low temperatures, which was a novel ﬁnding. On the other hand, the cell count of S. aureus remained the same during the conservation period at 2◦C.

When the cell count of S. aureus was around 6 log CFU/g, SEs could be detected at 12°C. This was just above the detection limit, which thus corresponds with an amount of > 0.25 ng/ml. This amount is already enough for highly sensitive people to become ill (Bennett, 2001b). The corresponding cell count with the start of enterotoxin production, was about 6 log CFU/g, which corresponds to the data in the literature, reporting a cell density of 105 to 108 S. aureus cells that are necessary to start the production (Jablonski and Bohach, 2001). The literature also stated that the production starts during the logarithmic phase or during the transition from the exponential to the stationary phase. In this thesis it can be clearly seen that the toxin production started during the logarithmic phase. The critical density was reached from day 10 in one experiment, while in the other experiment this was from day 11 and 12, depending from the used atmosphere condition in the packaging. But this seems to be a mere reﬂection of the delay in reaching the critical density with the MAP conditions, due to the slower growth rate. The fact that there is 1-2 days diﬀerence between the two experiments is because in the last experiment the lag phase was shorter, and thus the growth could start sooner and the critical density could be reached earlier. This was probably because of the state of the inoculums used.

No cereulide was produced at 12◦C, while in other studies this was the case (Rajkovic et al., 2006a; Finlay et al., 2000). In this experimental setup, the cell counts of B. cereus only reached 5 log CFU/g after 14 days at 12◦C. As already indicated is this below the critical density of 7 log CFU/g. The extended lag phase of B. cereus and germination time needed by the spores was responsible for the low cell count after 14 days. In the experiments by Rajkovic et al. (2006a) and Finlay et al. (2000) vegetative cells were used, which will result in a shorter lag phase. Moreover, Rajkovic et al. (2006a) reported that no cereulide could be found at 12◦C if background flora was present. In this thesis the possible copresence of S. aureus could have had an influence on the growth at lower temperature, although at 22◦C on TSA no influence was seen. But at 12◦C in lasagna, the conditions are less ideal and thus possible additional influences can influence the growth reduction.

However, cereulide production is also linked to the type of strain as Carlin et al. (2006) showed. In this study the authors indicated that the productivity of diﬀerent B. cereus strains grown on TSA ranged from 0.5 to 1600 ng/mg of bacterial biomass. In this thesis only 2 strains were investigated. To make ﬁnal conclusions, more B. cereus strains should be investigated. CHAPTER 4. RESULTS AND DISCUSSION 47

4.9.5 Effect of the combination of low residual oxygen content and temperature on the toxin production The use of reduced atmospheres can be thus a useful tool to reduce the cereulide production in ready-to-eat meals. However, oxygen content needs to be below 8 % O2 to increase this delay of cereulide production. The reduction to oxygen content below 1.6 % O2 can ensure that no cereulide is produced on TSA (Rajkovic et al., 2006b). But a difference in results is obtained if experiments are performed on ideal growth media or on food, as this thesis already showed. This means that the limiting oxygen content for cereulide production needs to be investigated in foods, as the food matrix can have an influence both on growth and toxin production. The reduced atmosphere did not only have an influence on the cereulide production, it had also an influence on the growth rate of S. aureus. The growth rate of S. aureus under MAP was slower and thus the critical cell density was reached later during the storage.

As Table 4.5 already indicated, are most ready-to-eat lasagnas packaged in a modified atmosphere of approximately 1 % O2, 10-20 % CO2 and the rest N2. This low oxygen content will thus normally be low enough to reduce the growth of both S. aureus and B. cereus. This will logically lead to a later onset of toxin production, or even no cereulide production, as low oxygen content works inhibitory. But also the fact that a concentration of 10-20 % CO2 is added in the package will act inhibitory on the growth of both bacteria (Eklund, 1984; Kimura et al., 1999). In this thesis a more permissible gas composition of ± 8 % oxygen balanced by N2 was used to allow sufficient proliferation of inoculates cells which in consequence led to a limited difference in growth and toxin production in comparison to airborne conditions. Such approach covered conditions of industrial packages and also possible errors in packaging, representing more risky scenarios. Namely from the 8 investigated lasagna packages 1 had higher (5 %) oxygen content. This indicates that on market samples may exist that can present conditions in which S. aureus and B. cereus can grow and produce toxins if other conditions are permissible. But the fact that 10-20 % CO2 is additionally present in ready-to-eat lasagna could represent a sufficient hurdle to warrant desired level of food safety. The effect thereof should be investigated.

The emetic type of B. cereus is mesophilic and the limiting growth temperature for this type of B. cereus is 10◦C. It was worth investigating limiting temperature abuses, which are reported in domestically refrigerators (James et al., 2008), on growth of these strains and effective cereulide production. However, this temperature limit of 10◦C was recently questioned as a psychrotrophic emetic B. cereus strain is discovered (Altayar and Sutherland, 2006). Also two strains of Bacillus weihenstephanensis (Thorsen et al., 2006) are discovered. This B. weihenstephanensis species is also psychrotolerant and can grow at refrigeration temperatures of 6°C. Also its ability to produce cereulide at 8◦C was demonstrated. This means that B. weihenstephanensis and the psychrotolerant B. cereus can grow and produce cereulide in Refrigerated Processed Foods of Extended Durability (REPFED) stored at home, as Nauta et al. (2003) indicated that the overall average air temperature in European fridges is 6.64◦C with temperatures ranging from -1 to 12◦C. The safety of these REPFED are thus worth investigating concerning both cereulide producing psychrotolerant species and the influence of a modified atmosphere condition on the growth and cereulide production in REPFED. Chapter 5

Conclusions

In this thesis the safety of ready-to-eat meals, exemplified by lasagna was investigated. The safety was examined concerning the presence of two toxin producing bacteria, namely entero- toxigenic S. aureus and the emetic B. cereus producing cereulide. These both toxins are of concern because they can’t be removed anymore by the subsequent heat treatments, which are used in the processing or home-preparation of these ready-to-eat foods. The growth and toxin production of both bacteria was investigated at 22◦C; which is a storage at room temperature and at 12◦C; which is a temperature abuse at the consumer home. As ready-to-eat meals are packaged in a modified atmosphere package, the possible growth of both bacteria on laboratory media was investigated prior to the food based experiment. Both had the possibility of growing well at 22°C at the reduced atmosphere conditions of 8 % oxygen and the rest nitrogen. No difference could be seen in the growth and toxin production with or without the modified air conditions. Also the growth at 12°C was investigated, this showed that the strains were able to grow, but maximum cell counts were reached later than at 22°C.

In the food based experiment, exemplified with lasagna, no difference in growth and onset of toxinproduction could be seen at 22◦C. At 12°C however, the influence of the reduced oxygen content on S. aureus could be seen. This reduced oxygen content had an influence on the growth rate and could therefore perform an influence on the onset of staphylococcal enterotoxin production. However, the experiments in lasagna were performed with a reasonable permissible oxygen content of 8 %, while in ready-to-eat foods mostly the oxygen content is more reduced, meaning that the influence of the reduced oxygen content will be more suppresive.

A difference in growth and toxin production could be seen for B. cereus at 22◦C. The presence of reduced oxygen content had an influence on the growth rate and the onset of cereulide production as this was delayed with one day. Moreover had the reduced oxygen content an influence on the critical density neccessary to start cereulide production. This density appeared to be higher in the lodified atmosphere stored lasagna. At 12◦C, the critical density was not reached after 14 days of storage and thus no cereulide was detected.

The combination of low temperature and low oxygen content is thus a useful tool to provide safe ready-to-eat foods. The growth is delayed because of the lower temperature and thus toxins are produced later. The same can be said about the inﬂuence of less oxygen in a package. But still, a cold chain temperature below 7◦C needs to be maintained throughout the chain, even at the consumer side to ensure that no growth of the mesophilic emetic B. cereus and S. aureus can occur. The producer has to make sure that a good gas composition is inserted in the package. But at home and at caterers this hurdle is of limited use, so they have to make sure that portions

48 CHAPTER 5. CONCLUSIONS 49 are not stored too long in a temperature range of 10-50◦C. Fast cooling in small portions need to be used. Moreover is it very important that Good Manufacturing Practices (GMP) and HACCP principles are applied at the production side to keep the initial contamination as low as possible. Additional product formulation can be adjusted to provide an additional set of hurdle for microbial contaminants. Chapter 6

List of abbreviations

aw Water Activity BHI Brain Heart Infusion BP Baird Parker BW Body Weight CFU Colony Forming Units ED50 Effective Dose (50% of the population) ELFA Ezyme-Linked Fluorescent Immunoassay ELISA Ezyme-Linked Immunosorbent Assay GC Gas Chromatography HPLC High Performance Liquid Chromatography LFMFP Laboratory of Food Microbiology and Food Preservation LOD Limit Of Detection LOQ Limit Of Quantification MS Mass Spectrometry MYP Mannitol Egg Yolk Polymyxin PMOT % Progressive Motility Percentage PPS Peptone Physiological salt solution PCR Polymerase Chain Reaction REPFED Refrigerated Processed Foods of Extended Durability RF Relative Fluorescense SE Staphyloccocal Enterotoxin TSA Tryptone Soya Agar TSB Tryptone Soya Broth YOPI Young, old, Pregnant and Immune deficient

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