Growth and Enterotoxinproduction by B. Cereus and S.Aureus in the Ready-To-Eat Lasagna Under the Influence of Different Processing Conditions

Faculteit Bio-ingenieurswetenschappen

Academiejaar 2011 – 2012

Growth and enterotoxinproduction by B. cereus and S.aureus in the ready-to-eat lasagna under the influence of different processing conditions.

Eveline Panis

Promotor(en): Prof. dr. ir. Mieke Uyttendaele en Prof. Dr. Andreja Rajkovic (tutor)

Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen: Levensmiddelenwetenschappen en voeding

Faculteit Bio-ingenieurswetenschappen

Academiejaar 2011 – 2012

Growth and enterotoxinproduction by B. cereus and S.aureus in the ready-to-eat lasagna under the influence of different processing conditions.

Eveline Panis

Promotor(en): Prof. dr. ir. Mieke Uyttendaele en Prof. Dr. Andreja Rajkovic (tutor)

Masterproef voorgedragen tot het behalen van de graad van Master in de bio-ingenieurswetenschappen: Levensmiddelenwetenschappen en voeding

Acknowledgement

An education is a process of trial and error with this master thesis as final result. Many people in my environment were there to support me during this process for whom I want to express my gratitude.

First I would like to thank my promoter Prof. dr. ir. Mieke Uyttendaele of the University of Ghent. Furthermore I would like to thank my jury members Prof. dr. ir. Frank Devlieghere and Prof. dr. Marc Heyndrickx who were willing to read and evaluate my master thesis.

My gratitude also goes to Dr. Msc. ing. Andreja Rajkovic, who guided me through the entire process and was always enthusiastic in providing information and never complained when I asked too much questions.

I would also like to thank the members of the Laboratory of Food Microbiology and Food Preservation of the University of Ghent for the help and the company on long days in the lab. Special thanks to Elien Verguldt, Ann Dirkx, Danny Pauwels and the members of the Accreditation Laboratory, whom I could always ask anything.

Special thanks goes to my parents. They supported me from the beginning of my studies and never stopped. Without them I would never been able to finish my studies. I know it has not always been easy but I will always be grateful for giving me this opportunity.

I would also like to thank Saskia for the great help she has been.

Last but not least I would like to thank Tim, who kept on encouraging and stimulating me at all time, also when it was less obvious. He was there when I needed him and this would have been a lot harder without him. This is my dream and I could not have done this without you, thanks for being my tower of strength.

Abstract

Both B. cereus and S. aureus are well described food poisoning organisms, however no literature is found on the consequences of co-presence of both pathogens in food. The occurrence of both organisms in the same food is realistic though due to the fact that they are found in the same type of foods. Within this thesis the effect of different heat treatments of B. cereus spores and different MAP conditions on growth and enterotoxin production of B. cereus and S. aureus together in a food system, ready- to- eat lasagna, was investigated. Results showed that (i) the heat treatment of B. cereus spores had no negative effect on growth and enterotoxin production of B. cereus and S. aureus under the conditions tested. Moreover, as expected heat treatments had and activating role of the used spores, (ii) storage at 12°C and MAP with 3.7% O2 or 100% N2 was effective in terms of enterotoxins inhibition, although numbers of B. cereus and S. aureus still reached up to 4-5 log which is a possible issue for de novo toxin production in the gut, (iii) S. aureus were less suppressed when B. cereus spores were treated at lower temperatures, (iv) storage at 22°C gave faster growth and enterotoxins detection, (v) temperatures in the lasagna during preparation did not reach levels high enough to destroy or inactivate B. cereus spores or staphylococcal enterotoxins. Based on these findings it can be concluded that good hygiene has an important role at all times, from the producer to the consumer, and that lasagna needs to be kept at refrigerator temperatures to remain a safe product.

Key words: B. cereus – S. aureus – lasagna – enterotoxin – MAP

Samenvatting

Zowel B. cereus als S. aureus zijn voedselintoxicanten die uitgebreid beschreven zijn in de literatuur, desondanks is er geen literatuur beschikbaar over de gevolgen van de aanwezigheid van beide pathogenen in levensmiddelen. Het voorkomen van beide organismen in hetzelfde levensmiddel is realistisch doordat ze in hetzelfde type levensmiddelen terug gevonden worden. Binnen deze thesis werd onderzocht wat de invloed van verschillende hittebehandelingen van B. cereus sporen en MAP verpakkingen is op de groei en de enterotoxine productie van zowel B.cereus als S. aureus in een levensmiddel, kant en klare lasagne. Resultaten tonen aan dat (i) de hittebehandeling van de B. cereus sporen geen negatieve invloed heeft op de groei en enterotoxine productie van B. cereus en S. aureus onder de geteste condities. Zoals verwacht had de hittebehandeling een activerende rol op de

B. cereus sporen, (ii) bewaring bij 12°C en een MAP met 3,7% O2 of 100% N2 toonde effectief te zijn voor de inhibitie van enterotoxinen, alhoewel er nog steeds aantallen van 4-5 log bereikt worden voor B. cereus en S. aureus wat voor problemen kan zorgen. Er kunnen bij deze aantallen eventueel enterotoxines gevormd worden in het gastro-intestinaal stelsel, (iii) S. aureus wordt minder onderdrukt door B. cereus wanneer de sporen bij lagere temperaturen werden behandeld, (iv) bewaring van de lasagne bij 22°C geeft een snellere groei en een snellere detectie van de enterotoxines, (v) de temperatuur in de lasagne tijdens de bereiding reikt niet hoog genoeg om in te staan voor de inactivatie van B. cereus sporen of staphylococcus enterotoxinen. Vanuit deze bevindingen kan besloten worden dat goede hygiëne ter aller tijden moet gehandhaafd worden, van de producent tot bij de consument en de lasagne moet steeds bij voldoende lage temperatuur bewaart worden om een veilig product te garanderen.

Kernwoorden: B. cereus – S. aureus – lasagne – enterotoxinen – MAP

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Content Acknowledgement……………………………………………………………………………………………………………………………….i Abstract………………………………………………………………………………………………………………………………………………ii Samenvatting…………………………………………………………………………………………………………………………….………iii Content…………………………………………………………………………………………………………………………………..…………iv List of figures..……………………………………………………………………………………………………………………………………vi List of tables……………………………………………………………………………………………………………………………….…….vii Acronyms…………………………………………………………………………………………………………………………………………viii Part I – Introduction………………………………………………………………………………………………………………………..…1 Part II – Literature review……………………………………………………………………………………………………………….…4 1. Enterotoxins of B. cereus and S. aureus………………………………………………………………………………….6 1.1 B. cereus……………………………………………………………………………………………………………………..…..6 1.2 S. aureus……………………………………………………………………………………………………………………..…..7 2. Prevalence of B. cereus and S. aureus in food commodities……………………………………………….….8 2.1 B. cereus…………………………………………………………………………………………………………...... ……8 2.1.1 Effect of atmosphere…………………………………………………………………………………….……9 2.1.2 Effect of temperature…………………………………………………………………………………………9

2.1.3 Effect of aw………………………………………………………………………………………………………….9 2.1.4 Effect of pH……………………………………………………………………………………………………….10 2.1.5 Stability of the B. cereus spores…………………………………………………………………………10 2.2 S. aureus ……………………………………………………………………………………………………………………….11 2.2.1 Effect of atmosphere ……………………………………………………………………………………….11 2.2.2 Effect of temperature……………………………………………………………………………….………12

2.2.3 Effect of aw ……………………………………………………………………………………………………….12 2.2.4 Effect of pH……………………………………………………………………………………………………….12 2.2.5 Effect of food substrate……………………………………………………………………………….……13 2.2.6 Competitive microorganisms…………………………………………………………………………….13 3. Screening of toxins in foods: need, methods, input for risk assessment……………………………...13 3.1 Screening of B. cereus enterotoxins: commercially available and most commonly used methods…………………………………………………………………………………………………………………..…..14 3.1.1 Reverse passive latex agglutination (RPLA) ………………………………………………………14 3.1.2 Visual immunoassay………………………………………………………………………………………….14 3.1.3 Immunochromatographic based……………………………………………………………………....15 3.2 Screening of SEs: commercially available and most commonly used methods………….…..15 3.2.1 Enzyme linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA) …..15 3.2.2 RPLA………………………………………………………………………………………………………………….15 3.2.3 Radioimmunoassay (RIA)…………….………………………………………………………….………..15 Part III – Methodology…………………………………………………………………………………………………………….…….…19 1. Bacterial strains and inoculums conditions…………………………………………………………………..………20

2. Media…………………………………………………………………………………………………………………………………..20

3. pH and aw measurements…………………………………………………………………………………………….………21 4. Gasmixing and packaging unit………………………………………………………………………………………………21 5. Toxin tests…………………………………………………………………………………………………………………….……..21 6. Temperature measurements……………………………………………………………………………………………….23 7. Growth of B. cereus and S. aureus in TSB………………………………………………………………………..……23 8. Growth of B. cereus and S. aureus in lasagna……………………………………………………………….………24 9. Temperature measurement…………………………………………………………………………………………...……27 Part IV – Results……………………………………………………………………………………………………………………………….28 1. Growth of B. cereus and S. aureus in TSB………………………………………………………………………..……29 2. Water activity and pH measurement…………………………………………………………………………….…..…31 3. Growth of B. cereus and S. aureus in lasagna…………………………………………….…………………………31 3.1 Growth of B. cereus 312 and S. aureus 356……………………………………………………………………31

3.2 Gradient of O2 and CO2 levels in the package……………………………………………………..…………43 4. Temperature gradient in the lasagna………………………………………………………………………………..…44 Part V – Discussion……………………………………………………………………………………………………….………………….45 Part VI – Conclusion…………………………………………………………………………………………………………..…………….49 Part VII – References…………………………………………………………………………………………………………….………….52 1. Literature…………………………………………………………………………………………………………………………….53 2. Internet…………………………………………………………………………………………………………………………….…59 3. Books………………………………………………………………………………………………………………………………..…59 Part VIII – Appendixes Appendix 1: Extraction protocol of staphylococcal enterotoxins Appendix 2: Spore production Bacillus cereus

List of figures Figure 1: Mean temperature frequency distribution of domestic refrigerators………………………….……….3 Figure 2: Growth rate of B. cereus 144 at 25°C at various pH values……………………………………………..….10 Figure 3: The possible results of a SET-RPLA test…………………………………..………………………………………….14 Figure 4: The basic principle of sandwich ELISA………………………………………..………………………………………14 Figure 5: Illustration of the cfu’s on the different selective agars…………………..…………………………….…..21 Figure 6: S. aureus enterotoxin testing…………………………………………………………………………………………..…22 Figure 7: Illustration of results obtained by the VIDAS SETII…………………………………………..…………………22 Figure 8: An example of a positive result with the Duopath®……………………………………………..…………….23 Figure 9: The temperature probe…………………………………………………………………………………….……………….23 Figure 10: Flushing of the bottles……………………………………………………………………………………..………………24 Figure 11: Lasagna in petri plates and packaging the petri plates…………………………………….……………….25 Figure 12: Working schedule…………………………………………………………………………………………………..………..26 Figure 13: Illustration of the temperature measurements in the lasagna……………………………….…………27 Figure 14: Growth of the different B. cereus and S. aureus strains in TSB at 12°C……………………………..29 Figure 15: Growth of the different B. cereus and S. aureus strains in TSB at 22°C…………………..…………30 Figure 16: The changing gas composition in the TSB bottles during storage at 22°C………………...... …30 Figure 17: Growth curves of B. cereus with different heating conditions of the spores different MAP packaging, storage at 12°C…………………………………………………………………………………………………………….…33 Figure 18: Growth curves of B. cereus with different heating conditions of the spores different MAP packaging, storage at 12°C……………………………………………………………………………………………………………….34 Figure 19: Growth curves of S. aureus with different heating conditions of the B. cereus spores different MAP packaging, storage at 12°C……………………………………………………………………………………..…38 Figure 20: Growth curves of S. aureus with different heating conditions of the B. cereus spores different MAP packaging, storage at 22°C…………………………………………………………………………………………39 Figure 21: Comparison between the growth of B. cereus and S. aureus after different heating conditions of B. cereus spores……………………………………………………………………………………………………..……41 Figure 22: Comparison between the growth of B. cereus and S. aureus under different MAP conditions. …………………………………………………………………………………………………………………...... …42 Figure 23: : Illustration and comparison of the growth of B. cereus and S. aureus and the progress of the gas concentration in the package……………………………………………………………………...... …43 Figure 24: Temperatures measurements on two places in the lasagna during preparation………………44

List of tables Table 1: The characteristics of diarrheal toxins of B. cereus and the toxins of S. aureus……………..……..5 Table 2: Available commercial kits for detection of B. cereus diarrheal enterotoxins with their characteristics………………………………………………………………………………………………………………………………..…17 Table 3: Available commercial kits for detection of S. aureus enterotoxins with their characteristics………………………………………………………………………………………………………………………………..…18 Table 4: The composition of the selective media MYP and Baird-Parker…………………………….…….………20 Table 5: The overage nutritional value of the used lasagna…………………………………………………..……..…..24 Table 6: The combination of heat treatment of the B. cereus spores and the MAP conditions during the 5 experiments………………………………………………………………………………………………………………….…………25

Table 7: Results of aw and pH measurements…………………………………………………………………………….….….31 Table 8: Summary of the results of the growth and enterotoxin production of both B. cereus and S. aureus under varying heating of B. cereus spores and storage under MAP conditions with 8% O2……35 Table 9: Summary of the results of the growth and enterotoxin production of both B. cereus and S. aureus under varying MAP conditions ……………………………………………………………………………………………..35 Table 10: Comparison of the onset of enterotoxins of B. cereus and S. aureus at 12°C……………….…….37

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Acronyms

BDE-VIA B. cereus diarrheal enterotoxin visual immunoassay BP Baird Parker cfu Colony forming units cAMP Cyclic AMP CytK Cytotoxine K EIA Enzyme Immunoassay ELISA Enzyme linked immunosorbent assay HBL Hemolytic BL LOD Lower limit of detection MAP Modified atmosphere package MYP Manitol Egg Yolk Polymyxin Agar Nhe Non-hemolytic enterotoxin PPS Peptone physiological solution RFV Relative fluorescence value RPLA Revers passive latex agglutination SE Staphylococcal enterotoxin SEl Staphylococcal like enterotoxin TSA Tryptone soya agar TSB Tryptone soya broth TSST Toxic shock syndrome toxine YOPI Young, old, pregnant and immunosuppressed

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-Introduction-

Part I -Introduction-

-Introduction-

Home-cooked meals have become less important in terms of consumption than ready-to-eat meals nowadays. The consumer has an increased demand for food with minimal processing during preparation. Meanwhile the consumer expects at the same time a product which is safe and has a high sensorial, textural and nutritional quality (Nissen et al., 2002). This increased demand, together with the higher levels of immuno suppressed people, causes an increased frequency and severity of food poisoning by Bacillus cereus (Mahakarnchanakul and Beuchat, 1999). Because B. cereus and Staphylococcus aureus both occur in quite similar products, the possibility exists that both bacteria appear at the same time. The food poisoning mechanism of both B. cereus and S. aureus are extensively described in the scientific literature. A study of Rosenquist et al. (2005) in Denmark reported that B. cereus can be isolated from many different ready-to-eat food products. An average of 0.5% of the samples contained more than 10,000 cfu/g. A study conducted in Austria revealed that 3.8% of the tested food contained coagulase positive S. aureus (EFSA, 2012). Even higher numbers of S. aureus were found in Korean ready-to-eat food, 5.98% of the investigated food were contaminated with S. aureus (Kim et al., 2011). While the appearance of both bacteria separately is well described, the appearance of both B. cereus and S. aureus together is not yet investigated.

Modern ready-to-eat meals undergo a relatively mild heating process to ensure the high quality of the product. Both the vegetative cells of B. cereus and S. aureus are largely eliminated by this mild heat processing, though the spores of some heat-stable B.cereus are able to survive (Samapundo et al., 2011) as well as with the pre-formed heat-stable enterotoxins of S. aureus. The spores are then able to germinate and form enterotoxins in the small intestine of the host (Wijnands et al., 2002). The pre-formed enterotoxins of S. aureus are able to survive the conditions of the stomach and cause food poisoning. This is probably not the case with the enterotoxins of B. cereus (opposite to its heat stable emetic toxin) which will in most cases be inactivated prior to inflicting toxic effect.

Within this master thesis the objective was to monitor survival and growth of both the B. cereus and S. aureus and their enterotoxin production in lasagna as possible example of ready-to-eat foods under different storage conditions. The experimental setup included the control of modified atmosphere packaging (MAP) and storage temperature. The MAP conditions used are based on the results of Daelman et al. (Unpublished results, 2012), who found that in ready-to-eat meals the average gas composition in the packaging was 6.37% O2, 12.78% CO2 and 80.85% N2. For fresh lasagna the resulting gas composition was 0.79% O2, 19.8% CO2 and 79.41% N2 . Only one result for lasagna was available. Results within this research showed a gas composition of 0.955% O2, 10.5%

CO2 and 88.54% N2. Hui et al. (1994) listed the main causes of food poisoning outbreaks as the most important cause improper holding temperatures. In 94% of the foodborne outbreaks this has been the cause. This was followed by contaminated equipment (53%), inadequate cooking (32%), poor personal hygiene (24%) and an unsafe source of food (5%). Hedberg et al. (2008) showed similar results, though with other figures. Research has shown that 5 to 20% of the food items were stored above 10°C (Marklinder et al., 2004) and a study by Kennedy et al. (2005) revealed that temperatures of the refrigerators at home are in 59% of the cases above the recommended 5°C. A study conducted

-Introduction- in 1992 by Orla et al. showed that 3 to 4% of the refrigerators at home had a temperature around 12°C, Fig. 1. A more recent research by Carpentier et al. (2012) revealed an average temperature of 6.6°C and 8.5°C at respectively the shelf above the vegetable compartment and at the vegetable compartment itself. The temperatures reached up to 11.0°C on the shelf above the vegetable compartment and even 12.8°C at the vegetable compartment.

Fig. 1: Mean temperature frequency distribution of domestic refrigerators (Orla et al., 1992).

This, together with sometimes extended period between the refrigerator in the supermarket and the refrigerator at home with usual temperature fluctuations in the zone of temperature abuse, allows possible outgrowth of B. cereus and S. aureus (Mahakarnchanakul and Beuchat, 1999; Thorsen et al., 2009).

-Literature review-

Part II -Literature review-

-Literature review-

B. cereus is a Gram-positive, aerobe or facultative anaerobe bacterium that is widely spread in the environment. It has the ability to form spores wherefore they can survive in a wide range of stress conditions (Senesi and Ghelardi, 2010). Because of the passive spreading of the spores, due to adhesion, they can be found outside their natural environment and can easily be transferred from the environment to food (Senesi and Ghelardi, 2010; Stenfors Arnesen et al., 2008). B. cereus causes a number of infections such as meningitis, brain abscesses, endophtalmitis, pneumonia, etc. (Bottone, 2010) and two different types of food poisoning. Theretofore the bacterium has two types of toxins with different symptoms: the emetic toxin and the diarrheal toxin. The importance of both types of toxins in foodborne outbreaks has been well established. The emetic toxin (cereulide) causes vomiting and has been linked to lethal food poisonings (Mahler et al., 1997; Dierick et al., 2005) but within this master thesis the focus will be on the more prevalent diarrheal toxins (Stenfors Arnesen et al., 2008). In table 1 the characteristics of food poisoning by the diarrheal toxins of B. cereus are given. In a number of ways these toxins show some similarities with enterotoxins of S. aureus and therefore these are comparatively given in the same table.

Table 1: The characteristics of diarrheal toxins of B. cereus and the toxins of S. aureus (Stenfors Arnesen et al., 2008; Lund et al., 2000; Le Loir et al., 2003; Evenson et al., 1988; Park et al., 1992; Hui et al., 1994; Jay, 1992; Drobniewski, 1993; ICMSF, 1996; Hennekinne et al., 2011). B. cereus – diarrheal toxins S. aureus Type of toxin Enterotoxins: HBL, Nhe and Cyt K SEs Infective dose 105 - 108 cells or spores 105-106 cells/g food Incubation time 8-16 h 30 min-8 h Duration of 12-24 h 24 h illness Symptoms Abdominal pain, watery diarrhea, rectal Abdominal cramps, nausea, vomiting tenesmus and occasionally nausea. There (sometimes followed by diarrhea, but have been cases of death reported. never diarrhea alone). Toxin production Toxin produced in small intestine of the Toxin produced in food host Associated foods Meat, milk, vegetables, fish, soups, Meat and meat products, milk and milk puddings and sauces products, poultry, etc. Toxin production pH range 4.9-9.3 5.1-9.6 (aerobic) Temperature 30°C - 37°C 10°C- 46°C range Min aw 0.92 0.86 (aerobic)

S. aureus is a Gram-positive, facultative anaerobe bacterium (Lund et al., 2000) and as most vegetative cells it is not heat-stable but the formed toxins are not easily destroyed by heat, possibly causing food safety problems (Argundín et al., 2010). In contrast to B. cereus, S. aureus is not able to form spores (Le Loir et al., 2003). Almost all strains secrete a group of enzymes and cytotoxics: α-, β -, γ-, δ- hemolysins, nucleases, proteases, lipases, hyaluronidase and collagenase. Quite some strains are able to produce additional exoproteins, which include staphylococcal enterotoxins (SEs). Only

-Literature review- one third of the coagulase positive S. aureus strains are able to produce SEs but one strain can produce multiple enterotoxins (Kim et al., 2011; Sospedra et al., 2012). The enterotoxins are produced by S. aureus in the food and then ingested by the human. These SEs are mainly produced in the mid-exponential phase of the growth of S. aureus (Balaban and Rasooly, 2000). But there are reports sharing earlier onset of SE production (Rajkovic et al., 2006) and different times of production according to the type of SE (Can and Celik, 2011). Apart from food poisoning, S. aureus can also cause infections such as pimples, furuncles, sepsis and toxic shock syndrome. These infections depend on different virulence factors while food poisoning only depends on one virulence factor: SEs (Le Loir et al., 2003).

1.Enterotoxins of B. cereus and S. aureus

1.1 B. cereus

Several enterotoxins are produced by B. cereus such as hemolytic enterotoxin hemolysine BL (HBL), non-hemolytic enterotoxine (Nhe), cytotoxine K (CytK) and enterotoxin T. Enterotoxin T is classified as enterotoxin because of the genetic and structural relationship with enterotoxins but it has not been involved in food borne diseases (Wehrle et al., 2012). The most important food poisoning toxins are the three component enterotoxins Nhe and HBL (Wijnands et al., 2002). All B. cereus strains are able to produce Nhe but HBL and CytK can only be produced by 45-65% of the strain (Stenfors Arnesen et al., 2008; Senesi and Ghelardi, 2010). Therefore Nhe is the most dominant enterotoxin. It has to be mentioned that the relative importance of the enterotoxins depends on the strain (Ceuppens et al., 2011).

HBL consists of three proteinaceous subunits: L1, L2 and B. With subunits L1 and L2 representing the cytolytic section and subunit B representing the binding section. They are produced independently of one another but all three are needed for maximal hemolytic activity (Stenfors Arnesen et al., 2008). HBL shows cytotoxic, aemolytic, dermonecrotic and vascular permeability activity. It also has been shown that HBL causes fluid accumulation in ligated rabbit ileal loops (Beecher et al., 1995). The optimal ratio for maximal toxicity of L1, L2 and B is 1:1:1 (Ceuppens et al., 2011). After association with the cell membrane, pore formation will occur and osmotic lyses takes place (Beecher and Wong, 2000; Ceuppens et al., 2011).

Just like HBL, Nhe consists of three proteinaceous subunits: Nhe A, Nhe B and Nhe C. With Nhe A representing the lytic section and Nhe B and Nhe C representing the binding sections. It only shows maximal activity when all three subunits are present (Banerjee et al., 2011) and the optimal concentration ratio of Nhe A, Nhe B and Nhe C is 10:10:1 for maximal toxicity (Senesi and Ghelardi, 2010; Ceuppens et al., 2011).

-Literature review-

The enterotoxins of B. cereus are proteins which are heat-labile and their activity is considerably reduced above 45°C (Eley, 1992).The enterotoxins are inactivated by protease activity of pepsin, trypsin and chemotrypsin (Banerjee, 2011; Ceuppens et al., 2011). The diarrheal toxins are more pH sensitive than the emetic toxin and their inactivation starts at pH under 4.0 and above 10.0 (Pexara and Govaris, 2011; Ankolekar et al., 2009).

The contact with enterotoxins with host-cells is in a great deal facilitated by the adhesion of B. cereus. After adhesion of the enterotoxin on a specific membrane receptor there will be a transmission of the signal across the membrane wall and this will stimulate an intracellular messenger. At last the water and electrolyte exchange in the small intestine will be disturbed and cause diarrhea (Belaiche, 2000). The perturbation of the electrolyte transport by B. cereus has been suggested to be through the cyclic AMP ( cAMP) (Wijnands et al., 2002). As mentioned before, the B. cereus spores are highly adhesive because of their hydrophobicity. This means that the spores are able to attach to solid surfaces and foods but also to epithelial cells (Andersson et al., 1998). The hydrophobicity is higher when the cells are grown at a pH 4.0, than at a pH 7.0 or 10.0 (Lindsay et al., 2002). Because adherence to the epithelial cell is necessary for colonization, the attachment contributes to the pathogenicity of the bacterium. Within this area more research is required to understand the contribution and mechanism of adhesion.

1.2 S. aureus

Staphylococcus and Streptococcus species produce pyrogenic toxins: SE, toxic shock syndrome toxine (TSST), exfoliations A and B, and streptococcus pyrogenic toxins. All these toxins have superantigen activity but only the SE has an emetic activity as well. The superantigen activity is expressed as immune suppression and nonspecific T-cell proliferation. The mechanism of the emetic toxin is not yet completely understood. According to Arbuthnott et al. (1990) the toxin has a direct effect on the intestinal epithelium and on the vagus nerve (a brain nerve). This causes the stimulation of the emetic center and the gut transit. Which can explain the symptoms of emetic toxins. The superantigen and emetic activity are both located on two different domains of the protein (Le Loir et al., 2003) and do not correlate (Hui et al., 2008).

Up to now already more than 22 different kinds of SEs and Staphylococcals like enterotoxins (SEl) are identified (Argundín et al., 2010; Hennekinne et al., 2011). Although there are 6 SEs that are most important with respect to food poisoning: SEA, SEB, SEC (with SEC1, SEC2 and SEC3), SED, SEE and SEH. Newly identified SEs and SEls still have an unclear role in SE food poisoning (Can and Celik, 2011; Kim et al., 2011). These different SEs have some important properties in common: (i) ability to cause emesis and gastroenteritis in a primate model, (ii) superantigen activity, (iii) intermediate resistance to heat and pepsin, and (iv) similarities in the tertiary structure (Dinges et al., 2000). The resistance depends on the type of toxin and on the environmental conditions. SEA, alone or in combination with other SEs or SEls, is the enterotoxin that is most often involved

-Literature review- with a staphylococcal food poisoning. This is probably due to the extreme high resistance to proteolytic enzymes (Argundín et al., 2010). Approximately 75% of the food poisonings were due to SEA (Eley, 1992), though Jay et al. (2000) reported that in 23% of the food poisoning incidence SEA was responsible. This is followed by 14% for SED and 11% for SEB. During the food outbreaks between 1969 and 1990 in the United Kingdom (UK), SEA was responsible for 56.9% of the food poisoning (Argundín et al., 2010). The combination of SEA with SED, SEB, SEC or SEB and SED was respectively 15.4%, 3.4%, 2.5% and 1.1%. Other cases of food poisoning due to S. aureus all showed that SEA was the main contributor (Argundín et al., 2010).

All the staphylococcal enterotoxins are simple proteins which are resistant to proteolytic enzymes such as trypsin, chympotrypsin, rennin and papain, though they are sensitive to pepsine at a pH of 2. Besides this the enterotoxins have a high heat stability and are water soluble (Can and Celik, 2011).

2.Prevalence of B. cereus and S. aureus in food commodities

2.1 B. cereus

The natural environment of B. cereus mainly consists of decaying organic material, fresh water, marine water, vegetables and the intestinal tract of invertebrates. Due to this the soil and food is contaminated with B. cereus and the colonization of the human intestine is possible (Bottone, 2010). The enterotoxins are sensitive to heat and to the gastrointestinal passage. Therefore preformed enterotoxins do not play a role in the food poisoning (Ceuppens et al., 2011). Although it could be theoretically possible to form enough enterotoxins in the food to cause food poisoning, such food would not be acceptable for human consumption because of the spoilage. For example, high levels of B. cereus have been found in milk, but there is insufficient growth before spoilage (Lund et al., 2000). Because of the low pH of the stomach the majority of the vegetative cells will be modified and will subsequently be harmless. The pH of the stomach can be temporarily altered to the type of consumed food and the age of the individual. It is thus possible that the pH can reach higher values in order for the vegetative cells to survive the stomach better. Therefore attention has to be paid to people with a weakened immune system such as elderly (Stenfors Arnesen et al., 2008). These conditions of elevated pH could also protect preformed enterotoxins and cause food poisoning. The spores of B. cereus are capable to survive extreme environmental conditions such as heat, freezing, drying and radiation (Bottone, 2010) and even the lower pH of the stomach, which makes the spores very persistent. After contact with the epithelial cells and adhesion, the spores will germinate and grow. Meanwhile the formed vegetative cells will form a cell monolayer on top of the epithelial cells and form enterotoxins (Wijnands et al., 2002; Bottone, 2010; Lund et al., 2000). The health status of the host will determine how severe the symptoms will be. These symptoms will be more severe with young, old, pregnant and immune suppressed people (YOPI’s), making them into a risk group (Lund et al., 2000).

-Literature review-

The growth of B. cereus and the enterotoxins depend on many factors such as temperature, atmosphere, pH, oxidation-reduction potential, agitation, consistency, carbohydrate availability and relative ratios and cation concentrations and its relative ratios (Ceuppens et al., 2011). Only a few are briefly mentioned below.

2.1.1 Effect of atmosphere

B. cereus is a facultative anaerobe meaning it can grow under both anaerobic and aerobic conditions.

Jääskeläinen et al. (2004) observed no difference of growth between 99.5% N2 and ambient air. A study conducted by Koseki and Itoh (2002) confirmed these results with a research on lettuce at 5°C.

Though Bennik et al. (1995) found out that 100% CO2 is able to extend the lag phase and generation time. The effect of the CO2 increases with lower temperatures, which is due to the higher solubility of

CO2 at lower temperatures (Hui et al., 1994).The safety of ready-to-eat food can be ensured by MAP with high levels, above 40%, of CO2. It is also seen that a lower amount of O2 prolongs the lag phase. The longest lag phase is observed with vacuum packaging (Samapundo et al., 2011; de Sarrau et al., 2012). It has to be mentioned that the germination and growth under MAP conditions is strain dependent (Thorsen et al., 2009).

2.1.2 Effect of temperature

Within the food poisoning B. cereus strains there are both mesophilic and psychrotrophic strains. Both have an optimum temperature of 30°C for enterotoxin production (Ceuppens et al., 2011). Similar results are found by Drobniewski (1993), who gives an optimum temperature for enterotoxin production between 32°C and 37°C and the ICMSF (1996) states that temperatures between 30°C- 40°C are optimal for growth. There will be a higher yield of enterotoxin concentration for both mesophilic and psychrotrophic strains with higher incubation temperatures. This temperature dependence of the enterotoxin production is not applicable for all strains: some strains produce equal amounts of enterotoxins at low and high temperatures (Ceuppens et al., 2011) and some strains are able to grow at temperatures between 4 and 10°C, typical refrigerator temperatures (de Sarrau et al., 2012). These strains form a problem for ready-to-eat foods.

2.1.3 Effect of aw

The minimum water activity (aw) for growth of B. cereus is 0.92, though it has been indicated that foods with low aw prevent the recovery and growth of heat treated spores (Martínez et al., 2007). This is probably due to the fact that the protein inactivation, which is the cause of death by heat of the spores, is higher at lower aw (Mazas et al., 1999). After heat treatment an aw lower than 0.98 inhibits the germination and growth of B. cereus (Martínez et al., 2007). The effects of the aw on the

D-value depends on the nature of the compound that is used to control the aw, the strain and the temperature of heating (Mazas et al., 1999). When the humectant is NaCl B. cereus can still grow at

-Literature review-

aw 0.92-0.93. However when glycerol is used, growth is not possible anymore at aw 0.92 (ICMSF, 1996).

2.1.4 Effect of pH

Different sources give different optimum pH values for enterotoxin production but overall the pH for growth ranges between 4.9 and 9.3 (Robinson et al., 2000). The optimum pH depends on growth rate, nutrient availability and the acidulant used (Ceuppens et al., 2011). When acidulated with HCl growth does not occur below pH 4.8. But when the media is acidulated with lactic acid growth stops already at 5.6 (ICMSF, 1996). It is generally seen that the growth rate of the vegetative cells decreases and the lag phase extends with lower pH (Mols and Abee, 2011). Lindsay et al. (2000) investigated the growth of a B. cereus strain in a milk medium. There was no growth lower than pH 4.0 and higher than pH 10.0, figure 2. So it can be concluded that the pH range for growth and thus enterotoxin production is between pH 4.0 and 10.0. Foods with a low pH are not associated with enterotoxins, though this is probably due to the fact that B. cereus will not grow anymore at this pH, so no enterotoxins are produced (Ceuppens et al., 2011).

Fig. 2: Growth rate of B. cereus 144 at 25°C at various pH values (Lindsay et al., 2002).

The heat resistance of B. cereus spores decreased with lower pH (Casedei et al, 2001; Mols and Abee,

2011). The relation between the pH and the heat resistance (expressed as log10 of the D-value) is linear.

2.1.5 Stability of the B. cereus spores

Spores from B. cereus are resistant to wet heat, dry heat, radiation (both ɣ and UV), chemicals (acids, halogen gases, phenols, peroxides), osmotic stress, multiple cycles of freezing and thawing, enzymes

-Literature review- and extremely high pressures. The higher the optimal temperature for growth, the more heat resistant the spores are probably due to the intrinsic heat resistance of the proteins. It is also noticed that when the spores are formed at a higher temperature, they show a higher resistance to high temperature (Setlow et al., 2006). The fat content of the food also seems to have a protective effect on the spores (Drobniewski, 1993) and a high nutrient medium is required for spores to repair after heat treatment (Martínez et al., 2007). According to Setlow et al. (2000) the resistance properties of the spores are due to (i) the relative dehydration of the protoplast (Moir et al., 2002), (ii) the mineralization of the protoplast, (iii) the protection of the spore DNA by spore specific proteins, (iv) DNA repair, (v) the decrease of permeability of the core to chemicals and (vi) the encasement of the spore in the sporecoats. When the environmental conditions are suitable, the spore starts to germinate and ultimately converts into growing cells (Sethlow, 2003; Berthold-Pluta et al., 2011a). These vegetative cells are then able to produce enterotoxins and cause food poisoning during gastro intestinal passage.

2.2 S. aureus

S. aureus is found on 30 to 50% of the human population, especially in the nostrils, on the skin and hair (Di Giannatale et al., 2012). Because it is able to grow in a wide range of temperatures (7°C to 48°C), pH (4.0 to 10.0) and sodium chloride concentrations (up to 20% NaCl) it can be found in a wide range of food products (Le Loir et al., 2003; Hennekinne et al., 2011). In a review by Hennekinne et al. (2011) foodborne outbreaks associated with S. aureus are listed with foods such as chicken salad, eclairs, dessert cream pastry, cheese curd, pasta salad, roosted beef, etc. The growth of S. aureus and the formation of SEs depend on many different factors: temperature, aw, pH, gases, preservatives, irradiation, disinfectants, competitive micro-organisms, level of contamination, etc. Within the objectives of this master thesis some factors will be mentioned. It is also important to remember the fact that S. aureus is sensitive to microbial competition.

2.2.1 Effect of atmosphere

S. aureus grows best under aerobic conditions, though the survival is better under anaerobic conditions. Under aerobic conditions there is a faster production of enterotoxins and a wider range of pH and temperature is possible to produce SEs. At a concentration of 75% CO2 inhibition of the growth is seen in sliced cooked beef at 12.8°C (Lund et al., 2000). Inhibition of growth occurred at 12°C under aerobic and vacuum packaging during 3 weeks, though there were viable cells apparent. During this period of time no enterotoxins were produced. When stored at higher temperatures both growth and enterotoxins production was observed (Rajkovic, 2012).

-Literature review-

2.2.2 Effect of temperature

The range of temperatures where growth of S. aureus is possible, is between 7°C and 48°C. The lower the temperature, the longer the generation time will be. The optimum temperature is 35°C-40°C (Lund et al., 2000; Hennekinne et al., 2011). The lowest temperature registrated where SEs were produced was 10°C and the maximum temperature was 45°C - 46°C (Hennekinne et al., 2011). The enterotoxin production was reduced at temperatures lower than 20°C - 25°C (Rajkovic, 2012). Attention has to be paid to the fact that once the enterotoxins are formed it is difficult to destroy them. It is thought possible that due to the heating process the enterotoxins lose their antigenicity (Lund et al., 2000).

2.2.3 Effect of aw

S. aureus is one of the most tolerant food poisoning bacteria. It can grow at levels as low as 0.86 (Argundín et al, 2010). Even growth at values of 0.83 is reported (Hennekinne et al., 2011). The limiting aw for growth depends on a number of factors such as the atmosphere, pH, food system, temperature and the mode of adjusting the aw. It is seen that under aerobic conditions the limiting aw is lower than under anaerobic conditions (Hennekinne et al., 2011; Genigeorgis, 1989), while a lower pH and a lower temperature increase the limiting aw.

Enterotoxins are produced at an aw as low as 0.86 when all the other conditions are optimal

(Rajkovic, 2012). There are variations of the minimum aw between the different SEs. SEA and SED can occur at nearly all aw’s where growth can take place, when the other conditions are optimal. SEB on the contrary is sensitive to reduced aw and only produced untill aw 0.93 (Hennekinne et al., 2011).

2.2.4 Effect of pH

The pH range for growth of S. aureus is between 4.0 and 10.0 when the other conditions are optimal (Lund et al., 2000; Hennekinne et al., 2011). At extreme pH’s the growth will occur very slow and at lower pH greater numbers of S. aureus cells are needed to initiate growth (Genigeorgis, 1989). For optimal growth, a pH of 6.0-7.0 is required (Lund et al., 2000; Hennekinne et al., 2011), though the optimum pH depends on factors such as size of inoculum, temperature, food system, atmosphere and the kind of acidifier. Acetic acid for example is more effective in inhibition of growth of S. aureus than lactic acid (Genigeorgis, 1989; Hennekinne et al., 2011). The enterotoxin production takes place in a narrower range. According to Lund et al. (2000) the optimum range is 7.0-8.0. But according to Genigeorgis (1989) the optimum is between 6.5 and 7.3 and according to Rajkovic (2012) optimal enterotoxinproduction is between 6.0 and 7.0. Most strains produced enterotoxins at pH 5.1 under aerobic conditions while under anaerobic conditions most strains failed to produce enterotoxins below 5.7 (Hennekinne et al., 2011). The optimal pH is depending on the strain and type of toxin that is produced. For example, SEA has a wider range for production compared to SEB (Le Loir et al., 2003).

-Literature review-

2.2.5 Effect of food substrate

S. aureus requires amino acids as nitrogen source and the vitamine B source comes of thiamine and nicotinic acid. When the culture is grown anaerobically the bacterium needs uracil (Jay et al., 2000) and a fermentable carbon source.

2.2.6 Competitive microorganisms

Because S. aureus does not compete well with indigenous microbiota in raw foods, contamination is mainly associated with improper handling of cooked or processed foods, followed by storage under conditions which allow growth of S. aureus and production of the enterotoxin(s). Air, dust, and S. aureus is known to be unable to compete with other bacteria present in the food. This is due to the production of organic acid with a pH drop as a result, the production of H2O2 and other antimicrobiological components, competition of essential nutrients or the consumption of O2 by the other bacteria (Genigeorgis, 1989; Lund et al., 2000; Hennekinne et al., 2011). The inhibitory effect by other micro-organisms depends on many different factors such as the ratio of S. aureus to competitors, intrinsic characteristics (pH, aw, etc.) and the temperature and atmosphere of storage (Genogeorgis, 1989; Hennekinne et al., 2011). Noleto et al. (1987) investigated the growth of S. aureus in the presence of other bacteria. They found that the presence of B. cereus in the used medium caused a 10-fold decrease of S. aureus. In a meat medium more than a 10-fold decrease was observed. A suppression of growth of S. aureus automatically causes a suppression of enterotoxin production. Though when S. aureus cells were equal or greater in number than B. cereus, the S. aureus was able to grow in sufficient numbers to produce enterotoxins.

Overall, growth and enterotoxins production is decreased with lower temperature, aw and under anaerobic conditions (Genigeorgis, 1989). The optimal pH for growth and enterotoxin production is 6.0 - 7.0. Though there are variations between the different SEs, for example the SEB production is more sensitive to aw, whereas SEC is sensitive to both aw and temperature (Jay et al., 2000).

3. Screening of toxins in foods; need, methods, input for risk assessment

For the detection of both the enterotoxins of B. cereus and S. aureus there are several commercial detection kits available. Attention has to be paid because these tests often only detect the toxins and not their activity. Sudden difference in activity and virulence may not be detected by these tests. For example the RPLA test for B. cereus shows positive results for enterotoxins after boiling, while this process inactivates the biological activity of these toxins (Robinson et al., 2000).

-Literature review-

3.1 Screening of B. cereus enterotoxins: commercially available and most commonly used methods

3.1.1 Reverse passive latex agglutination (RPLA)

For this rapid test polystyrene latex particles are sensitised with antiserum from rabbits who were immunised with B. cereus enterotoxins. The test is performed in a V-well microtitre plate. A dilution series of the food extract is made in two rows. Thereafter a volume of the appropriate latex suspension is added and after mixing the result is visible. When enterotoxins are present agglutination occurs and a lattice structure is formed. After settling, this structure forms a diffuse layer on the base of the well, view fig. 3. This agglutination is due to the cross-linking of the latex particles by the specific antigen/antibody reaction. The 3 right results: (+), (++) and (+++) are considered to be positive. Each time a control well is performed, this should always be negative. When no enterotoxins are present or in a concentration below the detection limit there is a tight button visible, the two left figures in fig. 3 (Oxoid, 2010).

Fig. 3: The possible results of a SET-RPLA test (Oxoid, 2010).

3.1.2 Visual immunoassay

The B. cereus diarrheal enterotoxin visual immunoassay (BDE-VIA) makes use of a double sandwich enzyme immunoassay (Beecher and Wong, 1994), so it requires two antibodies that bind on different epitopes of the antigen. The capture antibody is bound to a solid phase. The food extract with the antigen is then added and complexes are formed. After washing away the unbound antigens labeled antibodies are added. They complete the complex. The quantification is possible due to colorimetric measurement of the bound labeled antibody. The basic mechanism is showed in fig. 4.

Fig. 4: The basic principle of sandwich ELISA with (a) antibodies coated to the well, (b) antigens present in the food matrix bind with antibody, (c) enzyme labeled antibody binds to antigen and (d) colorimetric measurement of the colored product (Leng et al., 2008).

-Literature review-

3.1.3 Immunochromatographic based

When the extract of the food is put in the sample port the extract will be absorbed by the chromatic paper and move to the reaction zone. In the reaction zone gold-labeled antibodies specific to the enterotoxins Nhe and HBL are located. When the enterotoxin is present the antigen and gold-labeled antibody will migrate to the binding zone. Within this binding zone another antibody for Nhe and another antibody for HBL are located which immobilizes the complex. Because of the gold-labeling a red line appears when enterotoxins are present. So a simple detection is possible. An overview of common commercial kits available for the detection of B. cereus diarrheal enterotoxins is given in table 2.

3.2 Screening of SEs: commercially available and most commonly used methods

3.2.1 Enzyme Linked Immunosorbent Assay (ELISA) or Enzyme Immunoassay (EIA)

The ELISA or EIA kit is based on the agglutination of the antigens in the food extract to the present antibodies. The basic mechanism is described in 3.1.2. In the VIDAS SET II the last step is addition of the substrate 4-methyl- umbelliferyl phosphate. The bound enzyme conjugate will hydrolyze this substrate to 4-methyl umbelliferone, which is a fluorescent product and is measured at 450 nm. The results are automatically analyzed by the instrument and calculates a test value for each sample. Commercial examples of EIA kits are RIDASCREEN and TECRA (Park et al., 1994). An overview of a few common commercial kits is given in table 3. Within ELISA tests many variants are developed: the ELISA-membrane kit, where the solid phase is a membrane attached to a stick with 6 small holes in order to form wells (Wieneke, 1991). Another variant is the ELISA-tube kit, where 20 plastic tubes are coated with a mixture of monoclonal antibodies of the different SEs.

3.2.2 RPLA

This test for enterotoxins produced by S. aureus is performed in V-well microtitre plates and uses polystyrene latex particles. The latex particles are sensitized with antiserum, taken from rabbits who are immunized with the purified SEA, SEB, SEC and SED. A dilution series is made of the food extract in the wells and the appropriate latex suspension is added. After mixing and settling the result is visible, view figure 3.

3.2.3 Radioimmunoassay (RIA)

The RIA is a quantitative test for the detection of enterotoxins and uses radio labeled antigens. These labeled antigens must have the same biological activity than the unlabeled antigens. The labeled and

-Literature review- unlabeled antigens are in competition for the limited binding places on the antibody. With increasing concentration of the unlabeled antigens, a decrease of labeled antigens that bind to the antibody is observed. After separation of the unbounded and bounded labeled antigens the free fraction is counted in an appropriate way. For the labeling of antigens mostly I125 is used, sometimes C14 and H3 are used. By means of a standard curve made by a dilution series of a known concentration of the antigen, the exact amount of antigen of the extract can be revealed (Millipore, 2012).

-Literature review-

Table 2: Available commercial kits for detection of B. cereus diarrheal enterotoxins with their characteristics. Principle Duration of Sensitivity Advantages Disadvantages Source the test TECRA-VIA Immuno- 4 h < 1 ng/ml of -Simple and easy to use -Only detection of the Nhe Noack Group, 2011 assay prepared sample -Automatisation possible for large proteins (Nhe A component) TECRA, 2012 scale testing Beecher and Wong, 1994 Ankolekar et al., 2009 BCET-RPLA RPLA 20- 24 h 2 ng/ml of -Only detection of the HBL Oxoid, 2010 prepared sample proteins (L2 component) Beecher and Wong, 1994

Duopath® Immuno 30 min > 1 B. cereus /g or -Detects both Nhe and HBL Merck, 2011 Sorbent ml of food sample -Easy to use Krause et al., 2010 Assay -Control whether the test is conducted correctly is included -Rapid and sensitive screening -No false positive results due to cross-reactivity with non-toxic exoproteins -No apparatus necessary for reading the result

-Literature review-

Table 3: Available commercial kits for detection of S.aureus enterotoxins with their characteristics. Principle Duration of the test Sensitivity Advantages Disadvantages Source TECRA kit EIA 4 h 1 ng/ml -Semi-automatisation for large -False negative results possible Lund et al., 2000 scale testing is possible so due to the presence of peroxides 3M, 2012 operational flexibility is increased and non - enterotoxins metabolites Patel, 1994 -High convenience in de food -Special equipment necessary -No enterotoxin identification Vidas SETII ELISA 80 min 1 ng/ml -Versatility - Special equipment necessary Lund et al., 2000 kit -Good performance (few false negative and false positive results) Oxoid kit RPLA 20-24 h 0.5 ng/ml -Simple to use -Lower sensitivity, specificity and Lund et al., 2000 extract -No special equipment is required speed Thermo Fisher -Concentration steps is required Scientific Inc, 2010 -Interference with food components leading to false negative and false positive results

RIDASCREEN EIA 3 h 0,5-0,75 -No long extraction or -Special equipment necessary Park et al., 1996 ng/ g of concentration necessary food -High specificity

-Methodology-

Part III -Methodology-

-Methodology-

1. Bacterial strains and inoculums conditions

The bacterial stock cultures used in the thesis were stored in the lab at -75°C on glass beads. They were transferred in trypone soya broth (TSB) (Oxoid, Hampshire, UK) and activated during overnight growth at 30°C for B. cereus and 37°C for S. aureus. Two enterotoxin producing strains were used: strain 312 and 834 for B. cereus and strain 356 and 362 for S. aureus. B. cereus strain 312 produces both Nhe and HBL while S. aureus strain 356 forms only SEA. To save the strain, it can be transferred from the TSB to a Tryptone soya agar (TSA) (Oxoid, Hampshire, UK) slant and kept at 7°C.

2. Media

The enumeration was done making use of selective media: MYP (Manitol egg yolk polymyxin Agar) Agar Base (Oxoid, Hampshire, UK) for B. cereus and Baird-Parker Agar Base (Oxoid, Hampshire, UK) for S. aureus. The composition of both selective media are given in table 4. The addition of Polymyxin B (Oxoid, Hampshire, UK) inhibits the growth of Gram negative bacteria on the MYP agar.

Table 4: The composition of the selective media MYP and Baird-Parker (Oxoid, Hampshire, UK). MYP (B. cereus) Baird-Parker Agar (S. aureus) Compositon Meat extract, 1.0 g/l Tryptone, 10.0 g/l Peptone, 10.0 g/l Lab-Lemco’ Powder, 5.0 g/l Mannitol, 10.0 g/l Yeast extract, 1.0 g/l Sodium chloride, 10.0 g/l Sodium pyruvate, 10.0 g/l Phenol red, 0.025 g/l Glycine, 12.0 g/l Agar, 12.0 g/l Lithium chloride, 5.0 g/l Agar, 20.0 g/l Additions 50 ml/450 ml egg yolk emulsion 100 ml/l Egg yolk – Tellurite emlulsion 1 vial Polymyxin B supplement

In figure 5 an illustration of the selective agars used for the spread plate enumeration is shown to visualize and explain the used material and methodology (important information for interpretation of growth on the plating media). B. cereus colonies are bright pink with a zone of egg yolk precipitation on the MYP agar. Although theoretically they should be easy to see and count in practice this medium has number of drawbacks. When over 100 colonies are present the counting is impossible because of the size of the colonies that is fairly large. Regardless of the addition of antibiotics other organisms are still able to grow which makes the counting very difficult to impossible. When the sample is contaminated with Bacillus subtilis, they are able to grow on MYP agar and give a yellow color to the agar but no precipitation zone, fig. 5-b. The BP agar plates used for S. aureus are easier to count and had little disadvantages. The counting is easier due to the absence of a precipitation zone and the smaller colonies, fig. 5-c.

-Methodology-

(a) (b) (c)

Fig. 5: Illustration of the cfu’s on the different selective agars, (a) MYP, (b) MYP with contamination that is also able to grow on the selective agar and (c) BP agar.

3. pH and aw measurements

The water activity (aw) of the lasagna was measured after autoclaving and before inoculation.

Because 2 ml of inoculum is added the aw was also measured after adding 2 ml water to investigate possible increase of aw caused by added inoculum. After comparison the aw showed little differences before and after inoculation. Aw measurements were done by AWK-20 (AWK-20, NAGY Messysteme GmbH, Gäufelden, Germany). The pH before inoculation was measured using the Seven Easy (Mettler Toledo, S20 SevenEasy, Mettler Toledo, Colombo, USA). Both of the measurements were done in triplicate.

4. Gasmixing and packaging unit

Before packaging the gasmixing unit was set at the desired composition with the help of the Checkmate 9900 (PBI Dansensor, Checkmate 9900, Gullimex BV, Ringsted, Denmark). The correct gasmixture was flushed into the package with the packaging unit (MECA 900, DECA® technic, Herentals, Belgium) and the package was automatically sealed. The packages used consisted of polypropylene and EVOH and showed to be a good gasbarrier. The film sealed on the trays by heat was a Opalen HB65AF PP. Each 10 packages the gasmixture was controlled to avoid mistakes.

5. Toxin tests

The extraction protocol used to extract the toxins out the lasagna is added in Appendix 1. For both B. cereus and S. aureus toxins were extracted using the same extraction protocol. For the detection of SEs, 500 µl of the food extract is pipetted in the sample well of the VIDAS SET2 (fig. 6). The VIDAS SET II strips are analyzed by the associated bioMérieux instrument (bioMérieux, VIDAS, bioMérieux, Marcy, France) and the results are instantly available in electronic form after 80 min, fig. 7.

-Methodology-

(a) (b)

(c)

Fig. 6: S. aureus enterotoxin testing with (a) The Biomérieux machine to measure S. aureus, (b) side view of the VIDAS SETII test and (c) top view of the VIDAS SETII, the sample input is shown by the arrow.

The printout gives the type of test preformed, sample identification, date and time, the relative fluorescence value (RFV), test value and the interpreted result for each sample. First a background reading is done of the substrate cuvette before the analytes are introduced into the substrate. The second reading is after incubation of the substrate in the strip. The RFV is calculated by the difference between the background reading and the final reading. From this RFV the test value is calculated and interpreted. A test value greater than or equal to 0.13 is found positive and below 0.13 is negative. The test value can be seen as a value for the amount of enterotoxins present in the sample. Although this is only an estimation and not an exact concentration of enterotoxins. It should be reminded that the VIDAS SET II test tests for all the enterotoxins so it is not know which enterotoxins are present in the sample and at what ratio.

Fig. 7: Illustration of results obtained by the VIDAS SETII.

The entrotoxins of B. cereus are detected by use of the Duopath® (Duopath® Cereus Enterotoxins, Merck KGaA, Darmstadt, Germany): 150 µl is put on the foreseen place and after 30 min at room temperature the result is visible. The extract first has to be filtered to avoid vegetative cells on the

-Methodology-

Duopath® using a filter of 0.20 µm. A positive result for both Nhe and HBL is shown in fig. 8. The color of the stripes indicating the result can vary in intensity. But from the moment a stripe for the enterotoxins is observed, intense or not, this signal was considered as positive. The lower limit of detection (LOD) for the HBL component is 20 ng/ml (Ceuppens et al., unpublished) while the LOD for Nhe is 6 ng/ ml (Krause et al., 2010).

Fig. 8: An example of a positive result with the Duopath®, with C = Control.

6. Temperature measurements

Temperature measurements during preparation of the lasagna were made by use of the DataTrace® Micropack III (Datatrace, Micropack III Temperature Data Logger, Mesa Labs, Colorado, USA), shown in fig. 9. This wireless instrument measured the temperature each 5 seconds during a certain chosen time period. In this research 30 minutes were taken for measurement. Collected data were transferred to Microsoft excel and visualized.

Fig. 9: The temperature probe.

7. Growth of B. cereus and S. aureus in TSB

To achieve the goals of this thesis a first preliminary experimental setup was put in place. Growth and enterotoxin production by B. cereus and S. aureus was tested in TSB. For this purpose the same strains, temperature and MAP conditions were used as planned for the food experiments. The positive results of this preliminary trial had to provide first clues on whether two organisms can grow and produce toxins. This preliminary experiment was conducted in bottles and each bottle was filled with 300 ml TSB and closed with a septum. Subsequently the bottles were autoclaved (121°C, 15 min) and flushed to replace air with gas mixture of MAP (8% O2, 92% N2), fig. 10. Two needles were put in the septum: (i)

-Methodology- a short needle in the headspace to release the present overpressure and (ii) a long needle connected to the gas unit. During 3 minutes gas was flushed through the TSB to ensure uniform gas concentration in the headspace and broth. The gas mixing unit was set on approximately 8% O2 and 3 92% N2. After flushing, the bottles were inoculated with approximately 10 cfu/ml of B. cereus, this by means of a sterile needle through the septum. The inoculum came from a subculture of B. cereus grown in TSB and consist of vegetative cells. 0.3 ml was taken of the TSB with a contamination of approximately 3 *105 B. cereus. After the inoculation one half of the bottles was put at 12°C and the other at 22°C. Each day the gas composition was measured and the enumeration was done by plating on TSA. The experiment was done with each of two B. cereus strains. Exactly the same methods were followed for the two S. aureus strains.

Fig. 10: Flushing of the bottles.

8.Growth of B. cereus and S. aureus in lasagna

The model food system used in this experiment was commercially obtained, ready-to-eat lasagna. The lasagna was blended with a blender (Turbo, Braun) in the lab in order to obtain a homogenous mixture so that later homogeneous inoculation was facilitated. Table 5 represents the average nutritional value of the used lasagna.

Table 5: The average nutritional value of the used lasagna. Energy 153 kcal 638kJ Protein 8.2 g Carbohydrates 11.0g of which sugars 1.6g Fat 8.2 g of which saturated fat 3.9 g Fibers 1.0 g Sodium (salt) 0.3 g (0.8 g)

-Methodology-

The mixture was then autoclaved at 121°C during 15 minutes to remove the background flora for the purpose of easier and less variable enumerations of inoculated pathogens. From this point on the working procedure was carried out in an aseptic manner. The autoclaved lasagna was divided in petri plates each containing 40 ± 0.2 g, figure 11-a. The petri plates were inoculated with approximately 100 cfu/g overnight TSB S.aureus cultures and 100 cfu/g B. cereus spores, this by adding 1 ml of the appropriate 10-fold dilution prepared in peptone physiological solution (PPS) (8.5 g/l NaCl and 1.0 g/l peptone) (Oxoid, Hampshire, UK) to the lasagna and mixing well so a homogenous distribution was obtained. The protocol of the spore production of B. cereus is given in Appendix 2. The B. cereus spores first prior to inoculation were treated during 10 minutes in a hot water bath. The temperature of the treatment varied according to the experiment conducted, table 6.

(a) (b)

Fig. 11: (a) Lasagna into petri plates (aseptic) and (b) packaging the petri plates.

Petri plates with lasagna were subsequently packaged under MAP conditions, figure 11-b. The conditions depended on the experiment conducted, table 6, while all the other parameters remained constant. Half of the packages were incubated at 12°C and the other half at 22°C. For each day and temperature three packages were stored: two for bacterial analyses and one for the toxin test. Samples take for toxin analysis were stored at -20°C, until the moment of analysis.

Table 6: The combination of heat treatment of the B. cereus spores and the MAP conditions during the 5 experiments. Heat treatment B. cereus spores MAP

Temperature – time %O2 %CO2 %N2 1. 90°C – 10 min 8 0 92 2. 80°C – 10 min 8 0 92 3. 70°C – 10 min 8 0 92 4. 90°C – 10 min 3 0 97 5. 90°C – 10 min 0 0 100

-Methodology-

Each day a sample was analyzed: both the gas concentration and the cfu’s of B. cereus and S. aureus were determined. The gas concentration was measured by the Checkmate 9900. For the enumeration of the bacteria ± 25 g of the inoculated lasagna was put in a stomacher bag and 225 ml PPS was added. From this dilution (-1) a 10-fold dilution series is made and of the appropriate dilution 100 µl is plated out on both MYP and BP plates. The MYP plates were incubated at 30°C and the BP plates were incubated at 37°C. After 24 h incubation the growth was measured by counting the colonies on the plates. This general working scheme is illustrated in fig. 12.

Lasagna

Mixing the lasagna

Sterilize (autoclave, 121°C - 15min)

40 g per petriplate

Heat treatment spores (10min)

100 cfu/g S. aureus 100 cfu/g B. cereus spores

MAP

Storage at 12°C Storage at 22°C

MYP Toxintest BP MYP Toxintest BP

Duopath® VIDAS SETII Duopath® VIDAS SETII ® ®

Fig. 12: Working schedule with orange: not sterile working and green: sterile working.

This general scheme was always followed, with little variations in though. Between the different experiments MAP conditions and temperature of the heat treatment of the B. cereus spores varied. Each day a sample was taken and stored at -20°C for toxin analysis. At the end of each experiment the lasagna was tested on toxins of both B. cereus and S. aureus. To minimize the use of resources

-Methodology- the enterotoxin tests were first done on the samples of day 7 and starting from these results other enterotoxin test were performed.

9. Temperature gradient in the lasagna

Measurement of the temperature gradient in the lasagna during home preparation was simulated with the Micropack III Temperature Data Logger. The measurement was done on two different places in the lasagna: on the side and in the middle, visible in fig. 13. According to the preparation instructions on the packaging the lasagna was heated about 9 min at 900 W in the microwave. Because the microwave available in the lab only reached to 800 W the preparation time was prolonged to 10 min.

Fig. 13: Illustration of the temperature measurements in the lasagna.

-Results-

Part IV -Results-

-Results-

1.Growth of B. cereus and S. aureus in TSB

To ensure growth at the temperature and MAP conditions used in the food setup both B. cereus and S. aureus strains were first grown in bottles containing TSB at those conditions. The results showed differences in growth between the two pathogens and between strains of the same pathogen, but the overall trends were the same, fig. 14. At 12 °C all strains grew more than 2 log cfu/ml over the tested time period. It was only with strain B. cereus 834 that this trend is not present in this preliminary study.

8 7 6 B. cereus 312 5 B. cereus 834 4 S. aureus 356 3 S. aureus 362 2

1 Bacterial counts Bacterial(log cfu/ml) 0 0 50 100 150 Time (h)

Fig. 14: Growth of the different B. cereus and S. aureus strains in TSB bottles at 12°C.

All strains were able to grow at 22°C, and naturally for these pathogens this growth at 22°C was faster than at 12°C. Over a time period of about 30 hours the cell counts of all strains increased with 3 log cfu/ml or even more, fig. 15. Numbers of S. aureus reached 8 log cfu/ml after only 14 hours, starting from an inoculation level of 5.5 log cfu/ml (the inoculation level was higher than intended). When 4 log cfu/ml B. cereus was inoculated, the numbers after 28 hours reached 7 to 8 log cfu/ml, which means an increase of 3 to 4 log cfu/ml in 28 hours.

-Results-

8 7 6 5 B. cereus 312 4 B. cereus 834 3 S. aureus 356 2 S. aureus 362

Bacterial counts Bacterial(log cfu/ml) 1 0 0 10 20 30 40 50 60 Time (h) Fig. 15: Growth of the different B. cereus and S. aureus strains in TSB at 22°C.

During the growth enumeration, also the gas concentrations were also measured at every sampling time. At 12°C the percentage of O2 and CO2 varied with a maximum of 2% from the original gas composition while at 22°C the gas composition changed more rapidly and more drastically over time. In fig 16. the gas concentrations of all four strains at 22°C is shown.

(a) (b)

10,00 10,00

2 2

5,00 5,00

andCO andCO

2 2 % O % 0,00 O % 0,00 0 20 40 60 0 20 40 60 Time (in h) TIme (in h)

15,00 15,00

2 2

10,00 10,00

and CO and

2 5,00 5,00 % O % % O % 0,00 0,00 0 50 100 150 0 50 100 150 Time (in h) Time (in h)

Fig. 16: The changing gas composition in the TSB bottles during storage at 22°C with (a) B. cereus 312, (b) B. cereus 834, (c) S. aureus 356 and (d) S. aureus 362.

-Results-

2. Water activity (aw) and pH measurement

Before inoculation both the aw and pH were measured. To make sure that the 2 ml inoculum added had not a big influence on the aw of the lasagna, the aw after adding 2 ml water was measured as well. The results are presented in table 7. The average aw is 0.9832 and the aw after inoculation is

0.9890, showing that there was a minor difference between the aw before and after inoculation. The pH of the lasagna showed an average of 5.65.

Table 7: Results of aw and pH measurements.

aw aw (+2 ml) pH 0.9894 0.9900 5.63 0.9887 0.9900 5.58 0.9716 0.9870 5.74 Average 0.9832 0.9890 5.65

3.Growth of B. cereus and S. aureus in lasagna

3.1 Growth of B. cereus 312 and S. aureus 356

Before the inoculation of the lasagna the B. cereus spores are first treated during 10 min at a pre-set temperature. Because the bacteria are both present in the lasagna there is a possible influence possible of the bacteria on each other in terms of growth and enterotoxin production. Growth and enterotoxin production of B. cereus are clearly influenced by both the MAP conditions and the temperature of storage, fig. 17 and fig. 18. In fig. 17 the growth curves of B. cereus after the different heating conditions of the B. cereus spores and under different MAP conditions are shown, all stored at 12°C. Visual observations of the plotted data show that maximum and fastest growth occurs when the spores are treated for 10 min at 90°C and when the lasagna is packed under MAP conditions with

8% O2. A growth of 6.5 log cfu/g over only 64.5 h is seen. When the heat treatment of the spores is not changed but the MAP conditions are, the growth is reduced to almost complete lack of multiplication. It is seen that the heat treatment of the spores has limited influence on the maximum cell density at the stationary phase at 12°C. Statistical analyses showed a p>0.05 meaning that the maximum cell density of 90°C, 80°C and 70°C is similar with a 95% confidence interval the same. However, it does take longer (lower growth rate) to come to this maximum cell density after heat treatment at 80°C and 70°C. Lowering oxygen level in MAP causes a lower maximum cell density when comparing the effects of 8, 3.7 and 0% O2 on cell that are treated in a similar way. With 3.7%

O2 the cell density even stays under the detection limit of 2 log cfu/g during 142 h. Once passed the detection limit only slow growth is observed. It is seen that the lower the oxygen content of the MAP the lower the final cell density, although no statistically signing difference is observed between 3.7%

O2 or 0% O2 (p>0.05). The enterotoxin production is shown in the figure with an arrow pointing the first sample where enterotoxins was detected. More detailed overview is given in table 8 and 9.

-Results-

Primary detection of enterotoxins after heat treatment of the spores during 10 min at 80°C and 70°C is after respectively approximately 235 h and 241 h. Heat treatment of the spores at 90°C showed already showed detection of enterotoxins after 159 h. Lowered oxygen levels in the MAP inhibits the production of enterotoxins; oxygen levels of 3.7% and 0% completely abolished the enterotoxin production during 14 days storage at 12°C. So a lowered oxygen level and storage at 12°C was sufficient to guarantee a safe product in terms of B. cereus in-food production of enterotoxins.

Storage at 22°C gave completely different results, fig. 18. The heat treatment of the spores does not really seem to have an influence on the growth of B. cereus. When the oxygen level was set on 8% the growth curves of heat treatments of B. cereus spores at 90°C, 80°C and 70°C were equal with a 95% confidence interval (p>0,05). Moreover, the shape of the growth curves at lower oxygen level is less steep compared to 8% O2. But at the end, after approximately 160 h, the cell density of all experiments reaches around 8 log cfu/g independent of the spore heat treatment or oxygen levels in the MAP, though when statistically tested the cell density at the end is different (p<0.05). The final cell density of spores treated at 90°C was the highest (8.12 log cfu/g), while those of 70°C and 80°C treatments were statistically not different from each other (p>0.05). With respect to the enterotoxin detection some difference are seen. When the heat treatment of the spores was only at 70°C the enterotoxins are already formed after 43.5 h, while this is after approximately 65 h with heat treatment of 80°C and 90°C, table 9. At oxygen levels of 3.7% O2 no HBL is detected while Nhe is first observed after 65.5 h. When no oxygen is present at time of packaging Nhe is detected after 67 h and HBL after 90 h.

-Results-

B. cereus

4 Bacterial counts Bacterial(log /g) cfu 3

2 90°C - 8% O2 - 12°C 80°C - 8%O2 - 12°C 70°C - 8%O2 - 12°C 1 90°C - 3.7%O2 - 12°C 90°C - 100% N2 - 12°C 0 0 50 100 150 200 250 300 350 Time (h)

Fig. 17: Growth curves of B. cereus with different heating conditions of the spores different MAP packaging, storage at 12°C. The moment of initial enterotoxin detection is indicated with an arrow in the corresponding colour.

-Results-

B. cereus 10,00

9,00

8,00 Only Nhe, no HBL

7,00

6,00

5,00 HBL

4,00 Nhe Bacterial counts Bacterial(log g) cfu/ 3,00

90°C - 8%O2 - 22°C 2,00 80°C - 8%O2 - 22°C 70°C - 8%O2 - 22°C 1,00 90°C - 3.7%O2 - 22°C 90°C - 100% N2 - 22°C 0,00 0 20 40 60 80 100 120 140 160 180 Time (h)

Fig. 18: Growth curves of B. cereus with different heating conditions of the spores different MAP packaging, storage at 22°C. The moment of initial enterotoxin detection is indicated with an arrow in the corresponding colour.

-Results-

Table 8.: A summary of the results of the growth and enterotoxin production of both B. cereus and S. aureus under varying heating of B. cereus spores and storage under MAP conditions with 8% O2. Time of maximum Average maximum Time of toxin cell density (h) cell density (log) production (in h)

90°C - 8% O2 B. cereus at 12°C 137 8.4 159 B. cereus at 22°C 64.5 8.5 64.5 S. aureus at 12°C 256 6.98 280.5 S. aureus at 22°C 64.5 8.57 40.5

80°C - 8% O2 B. cereus at 12°C 212 7.22 235.25 B. cereus at 22°C 65.25 8.52 65.25 S. aureus at 12°C 307 6.71 329.25 S. aureus at 22°C 65.25 6.78 95.75

70°C - 8% O2 B. cereus at 12°C 210.5 7.72 241 B. cereus at 22°C 67.5 8.61 43.5 S. aureus at 12°C 282 6.31 282 S. aureus at 22°C 67.5 7.31 67.5

Table 9.: A summary of the results of the growth and enterotoxin production of both B. cereus and S. aureus under varying MAP conditions. Time of maximum Average maximum Time of toxin cell density (h) cell density (log) production (in h)

90°C - 8% O2 B. cereus at 12°C 137 8.4 159 B. cereus at 22°C 64.5 8.5 64.5 S. aureus at 12°C 256 6.98 280.5 S. aureus at 22°C 64.5 8.57 40.5

90°C - 3% O2 B. cereus at 12°C 162.5 3.66 / B. cereus at 22°C 118 7.04 Nhe: 65.5 and HBL: / S. aureus at 12°C 332 5.46 / S. aureus at 22°C 92 7.98 65.5

90°C - 0% O2 B. cereus at 12°C 210 4.7 / B. cereus at 22°C 114.5 7.76 Nhe: 67 and HBL: 90 S. aureus at 12°C 284.5 4.53 / S. aureus at 22°C 114.5 6.14 114.5

-Results-

Indirectly heat treatment of B. cereus spores could have an impact on accompanying flora, in this case S. aureus. The growth of S. aureus at 12°C after different heat treatments of B. cereus spores and under different oxygen levels in the package showed a somewhat lower growth rate, fig. 19. There was a very small difference in growth of S. aureus when B. cereus spores were treated at 70°C or 80°C. When B. cereus spores were treated at 90°C, a slightly better growth of S. aureus was observed. Although no significant difference between the maximum cell densities of S. aureus in response to the effect of B. cereus heat treatments at 90°C, 80°C and 70°C was noticed (p>0.05). Overall there is a clear difference between the different MAP conditions (p<0.05): lower oxygen levels result in a lower maximum cell density. While the packaging conditions with 8% O2 has a maximum cell density of more than 7 log cfu/g at the end of the 14 days, packages with 3.7% O2 show maximum cell density at approximately 5.5 log cfu/g and with 100% N2 it is approximately 4.5 log cfu/g. When enterotoxin detection is considered similarities between the heat treatments of B. cereus spores are also visible. After heat treatment at 70°C or 90°C staphylococcal enterotoxins are produced after approximately 280 h while SEs are detected 50 h later for heat treatment at 80°C, table 8. Lowering oxygen levels in the package seem to be effective to inhibit enterotoxin production because no enterotoxins of S. aureus were detected at 3.7% O2 and 100% N2, table 9.

In comparison to the S. aureus growth at 12°C, the growth at 22°C showed, as expected, higher slope on the growth curve, except for the growth at 100% N2 which has a somewhat lower growth rate, fig. 20. As with storage at 12°C a lowered maximum cell density is observed with lowered temperature of the heat treatment of the B. cereus spores. When effects of the oxygen levels of 8% O2 or 3.7% O2 are visually compared from the graphs only slight difference is observed in growth at 22°C, while 100%

N2 clearly caused a slower growth of S. aureus. Effect of the temperature of the heat treatment of B. cereus spores on S. aureus enterotoxin production had similar effect as at 12°C. Enterotoxins were detected first with 90°C heat treatment (40.5 h), then with 70°C (after 67.5) and then at 80 °C (95.75 h). Lowering oxygen concentration in the MAP caused a delay in enterotoxin production. At 8% O2 enterotoxins were detected after 40.5 h, at 3.7% O2 after 65.5 h and with no oxygen after 114.5 h.

In table 10 a comparison of the onset of enterotoxin production is given for both B. cereus and S. aureus together with the cell densities of both the pathogens at that time. Attention has to be paid on the fact that the moment of detection is not the same moment of enterotoxin production. It is clear from the table that B. cereus enterotoxins are produced earlier with higher heat treatment of the spores.

-Results-

Table 10: Comparison of the onset of enterotoxins of B. cereus and S. aureus at 12°C, the corresponding cell densities of B. cereus and S. aureus on the moment of detection is given with the standard deviation. Heat treatment Onset of B. cereus enterotoxins Onset of S. aureus enterotoxins of B. cereus (cell density B. cereus; cell density (cell density B. cereus/ cell density spores S. aureus) S. aureus) 90°C 159 h (8.00 ±0.01; 6.30 ±0,34) 280.5 h (7.95 ±0.07; 6.79 ±0.28) 80°C 235.25 h (6.39 ±0.01; 5.45 ±0.32) 329.25 h (7.70 ±0.43; 6.57 ±0.35) 70°C 241 h (8.21 ±0.45; 5.18 ±0.21) 282 h (7.39 ±0.59; 6.31 ±0.21)

-Results-

S. aureus 9

Bacterial counts Bacterial(log g) cfu/ 3

2 90°C - 8%O2 - 12°C 80°C - 8%O2 - 12°C 1 70°C - 8%O2 - 12°C 90°C - 3.7%O2 - 12°C 90°C - 100% N2 - 12°C 0 0 50 100 150 200 250 300 350 Time (h)

Fig. 19: Growth curves of S. aureus with different heating conditions of the B. cereus spores different MAP packaging, storage at 12°C. The moment of initial enterotoxin detection is indicated with an arrow in the corresponding colour.

-Results-

S. aureus 10,00

9,00

8,00

7,00

6,00

5,00

4,00 Bacterialcounts (log cfu/g) 3,00

90°C - 8%O2 - 22°C 2,00 80°C - 8%O2 - 22°C 70°C - 8%O2 - 22°C 1,00 90°C - 3.7%O2 - 22°C 90°C - 100% N2 - 22°C 0,00 0 20 40 60 80 100 120 140 160 Time (h) Fig. 20: Growth curves of S. aureus with different heating conditions of the B. cereus spores different MAP packaging, storage at 22°C. The moment of initial enterotoxin detection is indicated with an arrow in the corresponding colour.

-Results-

The growth of both bacteria is compared for the different heat treatment of the B. cereus spores and for both the storage temperatures in fig. 21. Besides the presented data, a trend becomes visible showing that both B. cereus and S. aureus follow the same shape of growth curve, with a main difference in their cell density for heat treatment at 80°C and 70°C. Although the growth curves after heat treatment at 90°C show almost no difference. Visual observation gives a difference of approximately 0.5 log cfu/g for 80°C and less than 0.5 log cfu/g for 70°C when stored at 22°C. For every heat treatment and both the storage temperatures it is always seen that B. cereus reaches a higher cell density than S. aureus.

When oxygen levels in the package are varied there is no trend visible between the growth curves.

Only at 8% O2 the two growth curves of B. cereus and S. aureus run parallel, fig. 22-a and d. In all the growth curves B. cereus reaches higher cell density at all times during storage, though storage under

3.7% O2 is an exception on this finding. Here, both at 12°C and 22°C, the S. aureus reaches higher cell densities than B. cereus, fig. 22-b and e. It was the purpose to inoculate both B. cereus and S. aureus with equal quantities, although as visible in fig 22-c and f this was not the case for the experiment with MAP conditions of 100% N2. A difference of 1.7 log was observed between B. cereus and S. aureus at inoculation time.

-Results-

(a) (b) (c)

(a)10,0 10,0

10,0

9,0 9,0 9,0 8,0 8,0 8,0 7,0 7,0 7,0 6,0 6,0 6,0 5,0 5,0 5,0 4,0 4,0 4,0 3,0 3,0 3,0 2,0 2,0 2,0

1,0 1,0 1,0

Bacterial counts Bacterial(log /g) cfu Bacerial counts Bacerial(log g) cfu/ 0,0 0,0 counts Bacterial(log g) cfu/ 0,0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 TIme (h) Time (h) Time (h)

(d) (e) (f) 10,0

10,0

10,0 9,0 9,0 9,0 8,0 8,0 8,0 7,0 7,0 7,0 6,0 6,0 6,0 5,0 5,0 5,0 4,0 4,0 4,0 3,0 3,0 3,0 2,0 2,0 2,0 1,0

1,0 counts Bacterial(log g) cfu/

1,0 counts Bacterial(log g) cfu/ Bacterial counts Bacterial(log g) cfu/ 0,0 0,0 0,0 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 Time (h) Time (h) Time (h)

Fig. 21: Comparison between the growth of B. cereus (blue curve) and S. aureus (red curve) after different heating conditions of B. cereus spores. (a) 90°C – 8% O2 at 12°C, (b) 80°C – 8% O2 at 12°C, (c) 70°C – 8% O2 at 12°C, (d) 90°C – 8% O2 at 22°C, (e) 80°C – 8% O2 at 22°C and (f) 70°C – 8% O2 at 22°C.

-Results-

(a) (b) (c)

10,0 10,0 10,0

9,0 9,0 9,0 8,0 8,0 8,0 7,0 7,0 7,0 6,0 6,0 6,0 5,0 5,0 5,0 4,0 4,0 4,0 3,0 3,0 3,0 2,0 2,0 2,0

1,0 1,0 1,0

Bacterial counts Bacterial(log g) cfu/ Bacterial counts Bacterial(log cu/g) 0,0 0,0 counts Bacterial(log g) cfu/ 0,0 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 0 50 100 150 200 250 300 350 TIme (h) Time (h) Time (h)

(d) (e) (f)

10,0 10,0 10,0

9,0 9,0 9,0 8,0 8,0 8,0 7,0 7,0 7,0 6,0 6,0 6,0 5,0 5,0 5,0 4,0 4,0 4,0 3,0 3,0 3,0 2,0 2,0 2,0 1,0 1,0

Bacterial counts Bacterial(log g) cfu/ 1,0

Bacterial counts Bacterial(log g) cfu/ Bacterial counts (log counts(log g) cfu/ Bacterial 0,0 0,0 0,0 0 100 200 0 50 100 150 200 0 50 100 150 200 Time (h) Time (h) Time (h)

Fig. 22: Comparison between the growth of B. cereus (blue curve) and S. aureus (red curve) under different MAP conditions. (a) 90°C – 8% O2 at 12°C, (b) 90°C – 3.7% O2 at 12°C, (c) 90°C – 100% N2 at 12°C, (d) 90°C – 8% O2 at 22°C, (e) 90°C – 3.7% O2 at 22°C and (f) 90°C – 100% N2 at 22°C.

-Results-

3.2 Gradient of O2 and CO2 levels in the package

Each time a sample was taken the gas concentration of the package was measured and registered. In fig. 23 the growth curves and the corresponding progress of the gas concentration in the package is illustrated at both storage temperatures. At 22°C the growth occurs very fast in the beginning. This is also the moment where the gas concentration is changing fast. The % O2 drops from 8% to almost 0% and the CO2 rises almost up to 20% in 95 h. After 95 h a stationary composition of the gas mixture is obtained, and on the growth curve this is also the moment where the stationary phase started. When these results are compared to the results obtained with storage at 12°C a complete other picture is seen. The growth of B. cereus and S. aureus occurs much slower at 12°C than at 22°C and this is also visible in the changes in gas mixture in the package. Here the gas mixture stays steady for a long time at 8% O2 and 0% CO2, but at the end the O2 content starts lowering and the CO2 content starts rising in the package. These changes occur very slow in comparison with those at 22°C.

(a) (b) (a)10,0 10,0

8,0 8,0

6,0 6,0

4,0 4,0

2,0 %and O2 %CO2 2,0 Number (log) Number

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

8,0 15,0 6,0 10,0 4,0

Number (log) Number 5,0 2,0 and%O2CO2 %

0,0 0,0 0 50 100 150 200 0 50 100 150 200 Time (in h) Time (in h)

Fig. 23: Illustration and comparison of the growth of B. cereus and S. aureus and the progress of the gas concentration in

the package. (a) growth of B. cereus and S. aureus: 80°C - 8% O2 at 12°C, (b) gas concentration in the package: 80°C - 8% O2

at 12°C, (c) growth of B. cereus and S. aureus: 80°C - 8% O2 at 22°C, (d) gas concentration in the package: 80°C - 8% O2 at

22°C. With blue line= B. cereus, red line= S.aureus, orange line= % CO2 and green line= % O2.

-Results-

4. Temperature gradient in the lasagna

As visible in fig. 24 the temperature gradient during preparation of the lasagna according to preparation instructions differs between the place of measurement. In the middle of the lasagna the maximum temperature during microwave heating of 10 minutes raises to 93.6°C while this is only 55.0°C at the side of the lasagna. The maximum is reached at a different moment for the side and middle. The temperature in the middle reaches after 10 min in the microwave its maximum and then drops rapidly. The temperature at the side reaches its maximum temperature about three minutes after the end of heating. The temperature at the side, on the moment that the lasagna is taken out of the microwave is only 50.2°C. The temperature drop at the sides is slower than in the middle.

100,00 90,00 80,00

70,00

C) ° 60,00 50,00 40,00

Temperature ( ( Temperature 30,00 20,00 10,00 0,00 0 500 1000 1500 2000 Time (sec)

Fig. 24: Temperatures measurements on two places in the lasagna during preparation, blue line= temperature on the side of the package and red line= temperature in the center of the package.

-Discussion-

Part V -Discussion-

-Discussion-

The experiment with the bottles filled with TSB showed that both B. cereus and S. aureus are able to grow at conditions of 8% O2 and storage temperatures of 12°C and 22°C. Although this does not automatically imply the growth ability and enterotoxin production in food for different reasons, such as aw, pH, food components, background flora etc. The conditions of the lasagna, used in this study as an example of ready-to-eat foods, are so that in theory both the bacteria are able to grow and produce enterotoxins. The aw of the lasagna found in this research was 0.9832 and the min aw for B. cereus and S. aureus was respectively 0.93 (Martínez et al., 2007) and 0.83 (Hennekinne et al., 2011). The pH of the lasagna in this study was 5.65 which also falls in the range of 4.9 - 9.3 for B. cereus (Robinson et al., 2000) and 4.0 - 10.0 for S. aureus (Hennekinne et al., 2011). Some psychrotrophic strains of B. cereus are able to grow and produce enterotoxins up to 4°C (van Netten et al., 1990), while the lowest reported temperature at which S. aureus produces enterotoxins is 10°C (Hennekinne et al., 2011). Therefore, temperatures chosen for this study should be permissible enough to allow growth and enterotoxin production for both organisms (depending on the other conditions). This hypotheses was confirmed in the current thesis. Although not all conditions were equally permissive in growth and enterotoxin production.

It is well accepted that heat treatments can activate spores for germination. Overall, 90°C had the best activation effect on the B. cereus spores, followed by 80°C and 70°C, as seen in this thesis. Although the differences were very little and not significant. It was seen that with lower heat treatment of B. cereus spores (80°C and 70°C) the growth curves of B. cereus and S. aureus were further from each other. This trend of similar growth curves of B. cereus and S. aureus is less visible when stored at 12°C, because this lower storage temperature causes more stress on the pathogens and therefore more variation. Other research showed an activating temperature of 80°C on B. cereus spores (Samapundo, internal communication) here it can be stated that 90°C is activating the B. cereus spores. B. cereus produced enterotoxins in an earlier stage when the B. cereus spores are treated at 90°C. This confirms the hypotheses that the spores are activated by treatment at 90°C during 10 minutes and that lower heat treatments have a less activating effect on the spores. Though no clear trend is seen with the influence of heat treatment of B. cereus spores on the staphylococcal enterotoxin production. During preparation of lasagna in the factory the ingredients of the lasagna only undergo pasteurization, so temperatures of 90°C are not reached (Martins and Germano, 2008). Although 80°C and 70°C are also activating the spores, growth and enterotoxin production by B. cereus is still possible, forming a potential threat to public health (with attention paid to the fact that these enterotoxins are mainly seen as a causative agent of toxin mediated infection).

Literature research showed that absence of O2 prolonged the lag phase of B. cereus (Samapundo et al., 2011; de Sarrau et al., 2012) which is confirmed in this thesis. Although when oxygen was still present and only reduced in the package, it showed to extend the lag phase of B. cereus as well. At concentrations of 8% O2 and storage at 12°C the lag phase was not visible while no presence of oxygen showed a lag phase still existing after 350 h. The growth at 22°C showed a short lag phase or no lag phase. In literature the longest lag phase was observed with vacuum packaging (Samapundo et

-Discussion-

al., 2011; de Sarrau et al., 2012) when compared to higher oxygen levels. When the same MAP conditions are applied but there is storage at different temperatures, growth of micro-organisms will be slower at lower storage temperature. This implicates that the effect of MAP is depending on the storage temperature (González-Aguilar et al., 2004). This is also confirmed within this thesis. Growth at 12°C was slower than at 22°C in the same MAP conditions. The effect of temperature and MAP also depends on the gas mixture used. For example, CO2 solubility is temperature dependent and it is dissolved CO2 that creates an antimicrobial effect.

Under aerobic conditions S. aureus seems to produce enterotoxins earlier during storage, this both at 12°C and 22°C. At oxygen levels of 8%, 3.7% and 0% the enterotoxins are detected after respectively 40.5 h, 65.5 h and 114.5 h at 22°C. Storage at 12°C inhibits production of enterotoxins over 14 days with 3.7% and 0% O2. In the presence of 8% O2 the enterotoxins are detected after 280 h (more or less 11 days). These results correspond with results found in the literature (Lund et al., 2000). It can also be concluded from of the results that B. cereus and S. aureus are able to produce enterotoxins at any phase of the growth. These findings were also found in the literature (Gzop and Bergdoll, 1974; Balaban and Rasooly, 2000; Rajkovic et al., 2006). Within the setup Nhe and HBL were not always detected at the same time. When the B. cereus enterotoxins are considered production is inhibited at oxygen levels of 3% and 0% and storage at 12°C. Though at 22°C no HBL is detected with 3% O2 and with 100% N2 it is detected after 90 h. While Nhe is at both oxygen levels detected after approximately 65 h. These findings confirm the results of Zigha et al. (2006) that anaerobic conditions are more stimulating on HBL while Nhe encounters less influence. However, an effect of LOD for HBL and Nhe needs to be taken into account. The sensitivity of Duopath® is not the same for HBL and Nhe (Ceuppens et al., 2012). The time of SE production is depending on the type of the SE according to Can and Celik (2011). These findings could not be confirmed in this thesis because of the lack of identification of the VIDAS SET II test but the according strain is only able to produce SEA so identification was not necessary.

In the protocol of the experiment with B. cereus and S. aureus in lasagna it was set that both bacteria are to be inoculated at equal quantities, in this case 100 cfu/g. Though during the experiment with

100% N2 the number of S. aureus was 1.7 log cfu/g lower than B. cereus at the time of inoculation. Knowing that S. aureus is not a competitive organism this could influence the results. In consulted literature (Noleto et al., 1987) growth of S. aureus was possible in sufficient numbers to produce enterotoxins when B. cereus and S. aureus were present in equal numbers or when S. aureus was present in greater numbers. Here B. cereus outnumbered S. aureus with 1.7 log cfu/g though both growth and enterotoxin productions were possible when stored at 22°C. At 12°C no enterotoxins where detected of both B. cereus and S. aureus so no conclusion can be formulated whether the lack of enterotoxins of S. aureus is due to the difference at inoculation time or due to the absence of O2 at packaging time.

-Discussion-

To complete the simulation from manufactory to the consumers’ plate a temperature measurement was done during heating in the microwave according to preparation instruction. It is seen that the temperature reaches maximum 93.6 °C in the centre of the lasagna while this is only 55.0°C at the side of the package. This is probably due to the fact that the distribution of heat in the microwave is not equal at all places. The pre-formed B. cereus enterotoxins are heat-labile (Eley, 1992) and sensitive to protease activity (Banerjee, 2011; Ceuppens et al., 2011) so they most probably get degraded during preparation of the lasagna and gastro intestinal passage (Ceuppens et al., unpublisched). In literature D-values for B. cereus spores are described in different foods but not yet for ready-to-eat lasagna. The D-values in pork meat are D95°C = 2.0 min, D90°C = 10.1 min and D85°C =

29.5 min (Byrne et al., 2006), while in milk D-values are D95°C = 4.4 min and D85°C = 19.45 min (Vijaykumar, 2010). Based on these values it can be concluded that the heating of the lasagna in the microwave will not be sufficient to reduce the present B. cereus spores. Pre-formed SEs are highly heat resistant and are only inactivated by sterilization so the time-temperature combination is not sufficient to inactivate the present SE. The fact that the temperature of the lasagna does not reach high levels at all places and that the period of time of high temperatures is very short there is a chance that pathogens and pre-formed enterotoxins are not inactivated, forming a threat for public health.

-Conclusion-

Part VI -Conclusion-

-Conclusion-

B. cereus and S. aureus are frequently found in many different foods presenting a threat to public health. The number of outbreaks caused by B. cereus and S. aureus in 2009 was respectively 124 and 293 (EFSA, 2011). The B. cereus spores, which are omnipresent, are also very adhesive and therefore difficult to eradicate from the processing environment. They are very heat resistant and not easily inactivated by disinfection processes (Drobniewski, 1993). Nowadays, the consumers prefer high quality ready-to-eat meals with minimal processing. B. cereus spores survive heat treatments with relatively low temperatures and are able to persist during low temperature storage. The spores will probably also survive the heating at the preparation because only relatively low temperatures are reached and maintained for a limited period of time. This way the spores stay in the food and when the conditions are favorable, these spores are able to germinate and possibly produce enterotoxins. However the role of B. cereus enterotoxins, when formed in food, is not clear in food borne diseases. For the majority of cases these enterotoxins will be degraded during gastro-intestinal passage and it will be de novo production in small intestine that will cause disease (toxin-mediated infection). On the other hand many authors suggest that preformed toxins under specific conditions can result into (or contribute) symptoms. In contradiction to B. cereus enterotoxins, staphylococcal enterotoxins are usually formed in food and are also very stable in terms of heat, pH and enzymes so they can cause food intoxications. The food contamination with S. aureus probably happens after heating and before packaging in the factory because the vegetative cells of S. aureus are not able to survive heating. Because the problem of contamination with B. cereus and S. aureus is not eliminated by pasteurization, it is important to apply good hygiene practice in the production plant to minimize the level of contamination. Contamination can also happen at home so proper hygiene should be applied there as well. The results of the experiment with lasagna showed no enterotoxin production of both

S. aureus and B. cereus when stored at 12°C under MAP conditions of 3.7% O2 or 100% N2. So lowered oxygen levels and storage at 12°C are good conditions for the preservation of this kind of ready-to-eat foods. Though attention has to be paid to the fact that once packages are opened, the protective effect of MAP disappears (Thorsen et al., 2009). Another point of attention should be that at 12°C and lowered oxygen levels still numbers of 4 to 5 log of both B. cereus and S. aureus are reached. These figures have already shown to be dangerous. Although S. aureus is not a competitive organism it is still able to grow in significant numbers and produce enterotoxins possibly causing problems. This has been seen even when S. aureus is present in lower numbers than B. cereus, which suggests that the lack of competiveness of S. aureus should be reconsidered and perhaps re-evaluated.

Both of the pathogens are probably more commonly involved in outbreaks than thought because of the self-limiting and moderate symptoms (Senesi and Ghelardi, 2010). Though with the rising numbers of people with a lowered immunorespons and the up-come of ready-to-eat foods these organisms are rightfully often included in the HACCP plans for these foods. Perhaps more stringent official monitoring should be done in order to get a clear view on their prevalence and role in foodborne diseases. Both the producers and the consumers, as well as competent authorities, should be aware of the problem and act appropriately.

-Conclusion-

Further research should be done on the survival of pre-formed SEs, B. cereus spores and B. cereus enterotoxins through the gastro-intestinal system. It is also important to completely understand the germination and enterotoxin production of B. cereus in the small intestine.

-References-

Part VII -References-

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Part VIII -Appendixes-

- Appendix 1 -

Extraction protocol of staphylococcal enterotoxins

General extraction protocol

Sample

25 g or 25 ml

25 ml extraction buffer

15 min / 18-25°C

Centrifugation 15 min at 3000-5000 g 18-25°C

Supernatant

Syringe containing a plug of absorbent cotton

Adjust pH between 7.5 and 8.0 using 1N NaOH

500 µl

VIDAS® SET2 Results within 80 min

- Appendix 2 – Protocol spore production Bacillus cereus

Preparation Salt solution (0,85 % NaCl) -> 200 mL 1. Weigh 8.5 g NaCl per liter distilled water -> 1.7 g NaCl for 200 mL 2. Autoclave

Solution of 50% ethanol (v/v) -> 20 mL 1. Take in a sterile way 10 mL pure ethanol in a sterile falcon 2. Add 10 mL autoclave distilled water 3. Mix well

Sporulation medium: NAMSCaCl2 agar plates 1. Prepare 28 g/L Nutrient Agar (NA), e.g. 5.6 g NA in 200 mL distilled water

2. Add 40 mg/L MnSO4, e.g. 8 mg in 200 mL

3. Add 100 mg/L CaCl2, e.g. 20 mg in 200 mL 4. Boil, autoclave and pour into sterile Petri dishes

Other materials - A few tubes with 10 mL sterile Brain Hearth Infusion broth (BHI) - Pipette tips of 1 mL and 100 µL - At least 14 tubes with 9 mL sterile PPS - At least 12 TSA plates - 100 mL sterile distilled water

Protocol production of spore solution 1. Grow the appropriate B. cereus strain in sterile BHI broth for 24 h at 30°C.

2. Plate out 100 µL of this culture on 6 NAMSCaCl2 agar plates, and incubate minimum 5 days at 30°C.

3. Wash all NAMSCaCl2 plates with sterile water -> 2 falcons with 18 mL (washing water of 3 plates in each falcon)

a. Put 3 mL sterile distilled water on NAMSCaCl2 plate and gently remove the spores with a sterile spatula. b. Bring the water containing the bacteria in a sterile falcon of 50 mL with a pipette.

c. Put 3 mL sterile distilled water on the same NAMSCaCl2 plate and gently remove the spores with a sterile spatula and add this water to the falcon. 4. Centrifuge for 15 min at 10 000 x g. 5. Remove the supernatant with a pipette (careful and destroy it!) and keep the pellet containing the spores. 6. Resuspend the pellet in 10 mL sterile salt solution. 7. Wash the pellet two times: repeat the previous centrifugation and resuspension step twice, except for the last resuspension.

8. The resulting pellet after the second washing is resuspended in 10 mL 50% ethanol (v/v in autoclaved distilled water). 9. Incubate overnight (min 12 h) at 4 °C to eliminate any remaining vegetative ells. 10. Centrifuge for 15 min at 10 000 x g. 11. Resuspend the pellet in 10 mL sterile salt solution/ water. 12. Wash the pellet three times to remove all ethanol: repeat the previous centrifugation and resuspension step three times. 13. Resuspend the resulting pellet in 10 mL pure sterile water. 14. The spore solution must be kept at 2 °C (and can be kept for 2 months). 15. Take 1 mL of the spore solution, make a dilution series in PPS and plate out 100 µL of dilutions -2 until -7 on TSA. 16. Take 1 mL of the spore solution, heat it for 10 min at 80 °C, make a dilution series in PPS and plate out 100 µL of dilutions -2 until -7 on TSA. 17. Incubate the TSA plates at 30 °C and count colonies after 24 h, 48 h and 72 h incubation. 18. Calculate the spore concentration.