Katholieke Universiteit Leuven

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1 Katholieke Universiteit Leuven FACULTY OF BIOSCIE CE E GI EERI G I TERU IVERSITY PROGRAMME MASTER OF FOOD TECH OLOGY (IUPFOOD) I FLUE CE OF BUTA EDIOL FERME TATIO I SPOILAGE OF FRESH PRODUCE BY E TEROBACTERIACEAE Promoter : Prof. Dr. Ir. Chris Michiels Department: Microbial and Molecular Systems Division or Centre: Laboratory of Food Microbiology Master dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Food Technology By: Waldir Desiderio Estela Escalante May 2

2 This dissertation is part of the examination and has not been corrected for eventual errors after the defense. Use as a reference is only permitted after consulting the promoter, stated on the front page.

3 ACK OWLEDGEME T First of all, I would like to thank the Belgian Technical Cooperation for offering me the scholarship as financial support for my master degree study. This master degree gave me a great chance to improve my knowledge in the field of Food Science and Technology. I am very grateful to my promoter Prof. Chris Michiels for offering me an opportunity to perform my dissertation in the Laboratory of Food Microbiology and for his suggestions during my working time. I would like to thank my supervisor, Bram Vivijs, for his great supervision with a lot of help and invaluable support to my work, both on practicing and on writing. I would also like to thank all members in the laboratory as well as PhD students for their helps, sharing and, laughing, which made my stay in the lab much more pleasant and joyful. Thanks to all professor and seniors of the course IUPFOOD for keeping in touch and sharing during all time of studying and researching. I would like to send my big thank to my parents, especially to my mother Ms. Sabina Escalante Chavez, also to my sisters and brothers who always support me during the whole time of my education. I

4 ABSTRACT The family of Enterobacteriaceae can be divided into two groups based on the type and proportion of fermentation products produced by fermentation of glucose. Two broad patterns are recognized, the mixed-acid fermentation and the 2,3-butanediol fermentation. There are several indications that the latter fermentation pathway may play an important role in the growth or survival of enterobacteria in or on sugar containing foodstuffs. Moreover, there is an increased demand for fresh produce in many industrialized countries and these products are increasingly being recognized as important vehicles for the transmission of human pathogenic enterobacteria through the food chain. Therefore, the influence of the butanediol fermentation on the capacity of some members of the Enterobacteriaceae family to grow on and spoil fresh produce commodities has been studied in this thesis. Studies realized with several enterobacteria, such as Escherichia coli MG, Salmonella Typhimurium LT2, Salmonella Senftenberg LMM2, Salmonella Enteritidis ATCC3, Shigella flexneri LMG2, Cronobacter sakazakii LMG and Serratia plymuthica RVH, in acidic synthetic media showed that the budab genes, which encode for the first two steps of the butanediol pathway, play an important role in the growth of enterobacteria. Presence of these genes enhances the growth rate and the maximum cell number, based on the optical density. The synthesis of acetoin or 2,3-butanediol would be a complex phenomenon that involves sugar consumption and acidification of the medium. On the other hand, the ph increase during fermentation was only observed in strains containing budab genes and would be a result of deacidification of the medium, provided that a source of amino acids is available. Experiments in fruit juices demonstrated that the budab genes do not give an evident advantage on the growth or tolerance to low ph fruit juices. On the contrary, in some cases it can limit bacterial growth. In addition, the growth or survival of the tested enterobacteria would depend on the type of fruit juice since other detrimental factors than the ph are involved. The type of microorganism and strain is also a very important factor. Final experiments with red pepper and cucumber slices showed the potential spoilage activity of S. plumythica RVH and C. sakazakii LMG depending on the temperature. Thus, increase of temperature from C to 2 C accelerates their spoilage activity. The budab genes appear to play an important role in accelerating the spoilage of fresh produce as compared with their respective budab mutants, nevertheless this is dependent on the type of vegetables. II

5 LIST OF ABBREVIATIO S ADP ATP P i NAD + NADH NADP + NADPH FAD + FADH AMP GMP DNA RNA EMP PP TCA GRAS LB VP ITPG HOMOPIPES PPB CFU OD GMP GAP TSI KCN ONPG EHEC EPEC ETEC EAEC EIEC DAEC VTEC ATR MIC Adenosine diphosphate Adenosine triphosphate Inorganic phosphate Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide reduced Nicotinamide adenine dinucleotide phosphate Nicotinamide adenine dinucleotide phosphate reduced Flavin adenine dinucleotide Flavin adenine dinucleotide reduced Adenosine monophosphate Guanosine monophosphate Desoxyribonucleic acid Ribonucleic acid Embden-Meyerhof-Parnas Pentose Phosphate Tricarboxylic acid cycle Generally recognized as safe Luria Bertani Voges-Proskauer Isopropyl-β-D--thiogalactopyranoside Homopiperazine-N,N'-bis-2-(ethanesulfonic acid) Potassium phosphate buffer Colony forming unit Optical density Good Manufacturing Practices Good Agricultural Practices Triple Sugar Iron Agar Potassium cyanide o-nitrophenyl-ß-d-galactopyranoside Enterohaemorrhagic E. coli Enteropathogenic E. coli Enterotoxigenic E. coli Enteroaggregative E. coli Enteroinvasive E. coli Diffusely adherent E. coli Verocytotoxigenic E. coli Acid-tolerance response Minimal inhibitory concentration III

6 LIST OF TABLES Page Table.: Habitat of some members of Enterobacteriaceae related to human and food interactions. Table.2: Characterization of some members of Enterobacteriaceae based on routinary biochemical tests. Table.3: Fermentation products (in moles per mol of glucose fermented) in mixed acid and in butanediol fermentation. 8 Table 2.: Colonization of fresh fruit and vegetables by enteric pathogens. 2 Table 2.2: ph values and main organic acids found in some fruit and vegetables 3 Table 3.: General chemical composition of the fruit juices used in this study. Values were obtained from the information given on the package. Table 3.2: Bacterial strains used in this study, growth medium and optimal temperature of growth. Table 3.3: Plasmids used in this study, characteristics and antibiotic resistance. Table 3.: Antibiotics used in this study. 9 IV

7 LIST OF FIGURES Page Figure.: Major metabolic pathways used by Enterobacteriaceae under aerobic and anaerobic conditions. 2 Figure.2: Mixed acid fermentation. 3 Figure.3: 2,3-butanediol fermentation. Figure 2.: Schematic illustration of factors that can contribute to the contamination of fruit and vegetables with human enteric pathogens in the field. 22 Figure 2.2: Chemical structure of the main organic acids found in fruits and vegetables. 38 Figure 2.3: Interaction of weak acids on the microbial cell. 2 Figure.: Growth of Serratia plymuthica RVH and budab mutant, Cronobacter sakazakii LMG and budab mutant, Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab in LB broth +.% glucose (ph.). Figure.2: Voges-Proskauer test realized during cultivation of S. plymuthica RVH and budab mutant and, C. sakazakii LMG in LB +.% glucose (ph.). 2 Figure.3: Voges-Proskauer test realized during cultivation of E. coli MG ptrc99a and pbudab and, S. Typhimurium LT2 ptrc99a and pbudab in LB +.% glucose + mm ITPG (ph.). 2 Figure.: Growth of Shigella flexneri LMG2 ptrc99a and pbudab and, Salmonella Senftenberg LMM2 ptrc99a and pbudab, at 3 C in LB broth +.% glucose + mm ITPG (ph.). 3 Figure.: Voges Proskauer test realized during cultivation of S. flexneri LMG2 ptrc99a and pbudab in LB +.% glucose + mm ITPG (ph.). 3 Figure.: Growth of Escherichia coli MG pbuda and pbudb and, Salmonella Typhimurium LT2 pbuda and pbudb, at 3 C in LB broth +.% glucose + mm ITPG (ph ). Figure.: Voges Proskauer test realized after 2 hours of cultivation at 3 C in LB broth +.% glucose + mm ITPG (ph ). : Escherichia coli MG pbuda, 2: E. coli MG pbudb, 3: Salmonella Typhimurium LT2 pbuda, : S. Typhimurium LT2 pbudb. Figure.8: Growth curves of Serratia plymuthica RVH, Serratia plymuthica RVH budab mutant, Salmonella Enteritidis ATCC3 pbudab and, Salmonella Enteritidis ATCC3 ptrc99a, cultivated at 3 C in LB broth +.% glucose, adjusted at different ph values with HCl. 8 V

8 Figure.9: Growth curves of Salmonella Typhimurium LT2 pbudab, Salmonella Typhimurium LT2 ptrc99a, Escherichia coli MG pbudab and, Escherichia coli MG ptrc99a, cultivated at 3 C in LB broth +.% glucose + mm IPTG, adjusted at different ph values with HCl. Figure.: Growth curves of Shigella flexneri LMG2 pbudab, Shigella flexneri LMG2 ptrc99a, Salmonella Senftenberg LMM2 pbudab and, Salmonella Senftenberg LMM2 ptrc99a, cultivated in LB broth +.% glucose + mm IPTG, adjusted at different ph values with HCl. 2 Figure.: Cultivation of S. Typhimurium LT2 ptrc99a and, S. Typhimurium LT2 pbudab in LB broth +.% glucose + mm IPTG, without and with mm of buffer HOMOPIPES, adjusted to ph. with HCl. Figure.2: Growth curves and ph change of Serratia plymuthica RVH and Serratia plymuthica RVH budab mutant, cultivated at 3 C in LB broth + glucose (ph.). Figure.3: Cultivation of Serratia plymuthica RVH, Serratia plymuthica RVH budab mutant, Escherichia coli MG pbudab and, Escherichia coli MG ptrc99a, in M9 minimal medium +.% glucose, adjusted at different ph values with HCl. 8 Figure.: Cultivation of Serratia plymuthica RVH in M9 minimal medium +.% glucose (ph.). 9 Figure.: Growth curves and ph change of Serratia plymuthica RVH and, S. plymuthica RVH budab mutant, cultivated at 3 C in M9 minimal medium +.% glucose + mm amino acid, adjusted at ph. with HCl. 8 Figure.: Growth curves and ph change of Serratia plymuthica RVH and Serratia plymuthica RVH budab mutant, cultivated at 3 C in M9 minimal medium +.% glucose + casamino acids, adjusted at ph. with HCl. 83 Figure.: Growth curves of Serratia plymuthica RVH and budab mutant, Salmonella Typhimurium LT2 ptrc99a and pbudab cultivated in: LB broth +.% glucose, LB broth +.% glucose + mm acetoin, LB broth and LB broth + mm acetoin, adjusted at ph. with HCl. 8 Figure.8: Growth curves of S. plymuthica RVH and budab mutant, S. Typhimurium LT2 ptrc99a and pbudab cultivated at 3 C in: LB broth +.% glucose, LB broth +.% glucose + mm acetoin, LB broth and, LB broth + mm acetoin, adjusted at ph. with HCl. 8 Figure.9: Growth curves of Serratia plymuthica RVH and budab mutant, Salmonella Typhimurium LT2 ptrc99a and, pbudab cultivated at 3 C in: LB broth +.% glucose, LB broth +.% glucose + mm acetoin, LB broth and, LB broth + mm acetoin (ph.). 89 Figure.2: Cultivation of Serratia plymuthica RVH and budab mutant and, Salmonella Typhimurium LT2 ptrc99a and, pbudab in: LB broth +.% glucose + mm acetoin (ph.). 9 VI

9 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab in grape juice + mm ITPG, at 2 C, adjusted at different ph values with NaOH. 9 Figure.2: Cultivation of Escherichia coli MG ptrc99a and pbudab in grape juice + mm ITPG, at 3 C, adjusted at different ph values with NaOH. 9 Figure.3: Cultivation of Salmonella Typhimurium LT2 ptrc99a and pbudab in grape juice + mm ITPG, at 2 C, adjusted at different ph values with NaOH. 98 Figure.: Cultivation of Salmonella Typhimurium LT2 ptrc99a and pbudab in grape juice + mm ITPG, at 3 C, adjusted at different ph values with NaOH. Figure.: Cultivation of Cronobacter sakazakii LMG at 2 C and 3 C in grape juice, adjusted at different ph values with NaOH. 2 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 2 C, under anaerobic condition in worldshake juice + mm ITPG, adjusted at different ph values with NaOH. Figure.: Cultivation of Cronobacter sakazakii LMG at 2 C, under anaerobic condition in worldshake juice, adjusted at different ph values with NaOH. Figure.8: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 3 C, under anaerobic condition in Nectar Multifruit juice + mm ITPG, adjusted at different ph values with NaOH. 8 Figure.9: Cultivation of Cronobacter sakazakii LMG at 3 C, under anaerobic condition in Nectar Multifruit juice, adjusted at different ph values with NaOH. 9 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 2 C in apple juice + mm ITPG, adjusted at different ph values with NaOH. 2 Figure.: Cultivation of Cronobacter sakazakii LMG at 2 C in apple juice, adjusted at different ph values with NaOH. 3 Figure.2: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 2 C, under anaerobic condition in apple juice + mm ITPG, adjusted at different ph values with NaOH. Figure.3: Cultivation of Cronobacter sakazakii LMG wild type and budab mutant at 2 C, under anaerobic condition in apple juice, adjusted at different ph values with NaOH. 8 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 3 C in apple juice + mm ITPG, adjusted at different ph values with NaOH. 2 VII

10 Figure.: Cultivation of Serratia plymuthica RVH and budab mutant and, Cronobacter sakazakii LMG at 3 C in apple juice, adjusted at different ph values with NaOH. 22 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab under anaerobic conditions at 3 C in apple juice + mm ITPG, adjusted at different ph values with NaOH. 2 Figure.: Cultivation of Serratia plymuthica RVH and budab mutant under anaerobic conditions at 3 C in apple juice, adjusted at different ph values with NaOH. 2 Figure.: Cultivation of Serratia plymuthica RVH and budab mutant on red pepper slices surrounded by liquid at 3 C. 28 Figure.2: Spoilage activity of Serratia plymuthica RVH and budab mutant cultivated on red pepper slices surrounded by liquid at 3 C. 29 Figure.3: Growth curves of Serratia plymuthica RVH and budab mutant on red pepper slices at C and 2 C. 3 Figure.: Spoilage activity of Serratia plymuthica RVH and budab mutant cultivated on red pepper slices at C and 2 C. 32 Figure.: Growth of Serratia plymuthica RVH and budab mutant cultivated on cucumber slices at C and 2 C. 3 Figure.: Spoilage activity of Serratia plymuthica RVH and budab mutant cultivated on cucumber slices at C and 2 C. 3 Figure.: Growth curves of Cronobacter sakazakii LMG and budab mutant cultivated on red pepper slices at C and 2 C. 3 Figure.8: Spoilage activity of Cronobacter sakazakii LMG and budab mutant cultivated on red pepper slices at C and 2 C. 38 VIII

11 TABLE OF CO TE TS Page ACK OWLEDGEME T I ABSTRACT II LIST OF ABREVIATIO S III LIST OF TABLES IV LIST OF FIGURES V TABLE OF CO TE TS IX I TRODUCTIO PART I: LITERATURE STUDY 3 Chapter : Enterobacteriaceae.. Taxonomy and classification of Enterobacteriaceae.2. Habitat and importance of Enterobacteriaceae.3. General and biochemical characteristics of Enterobacteriaceae.. Metabolism of Enterobacteriaceae 8... Embden-Meyerhof-Parnas pathway Pentose phosphate pathway..3. The tricarboxylic acid (TCA) or Krebs cycle... Mixed acid fermentation ,3-butanediol fermentation.. ph homeostasis in Enterobacteriaceae.. Some representative enteric bacteria related with foodborne diseases... Escherichia coli..2. Salmonella Shigella 9... Yersinia 2... Cronobacter sakazakii 2 Chapter 2: Microbial ecology of enteric bacteria on fresh produce Sources of contamination Preharvest contamination Postharvest contamination 2 IX

12 2.2. Mechanisms of attachment and colonization Enteric pathogens as epiphytes Enteric pathogens as endophytes Prevention of contamination and sanitation methods for fresh produce Behaviour of some pathogenic enterobacteria on fresh produce Pathogenic Escherichia coli Salmonella Shigella Yersinia enterocolitica Cronobacter sakazakii Importance of the ph and organic acids in fruit (juices) and vegetables The effect of ph on the growth of microorganisms The effect of organic acids on the growth of microorganisms Inhibitory mechanisms of ph and organic acids on microbial growth PART II: EXPERIME TAL WORK 3 Chapter 3: Material and Methods 3.. Material 3... Growth media 3... Luria Bertani (LB) broth M9 minimal medium Fruit juices 3... Vegetables Media for stock and count plates Luria Bertani (LB) agar Bacterial strains and plasmids 3... Buffers and solutions 3... Antibiotics Equipments Multiskan RC Multiskan Ascent 9 X

13 3.2. Methods Maintenance of strains Transfer of plasmids by electroporation Voges Proskauer (VP) test Inoculum preparation Growth of Enterobacteriaceae in synthetic medium at different conditions Growth of enterobacteria in LB medium with neutral ph value Growth of enterobacteria in LB medium with low ph values Growth of enterobacteria in LB medium with different glucose concentrations Growth of enterobacteria in LB medium with mm of acetoin Growth of enterobacteria in M9 minimal medium Growth of enterobacteria in fruit juices Growth of enterobacteria on vegetables Preparation of vegetable slices Preparation of bacterial suspension Vegetable spoilage assay Cell count and spoilage activity PART III: RESULTS A D DISCUSSIO 8 Chapter : Growth of Enterobacteriaceae in synthetic media 9.. Growth of enterobacteria in synthetic medium with neutral ph 9.2. Growth of enterobacteria in synthetic medium with low ph.2.. Growth of enterobacteria at low ph based on optical density.2.2. Growth of enterobacteria in buffered medium at low ph 3.3. Influence of glucose concentration on growth of enterobacteria.. Growth of enterobacteria in minimal medium... Growth of enterobacteria in minimal medium at different ph values XI

14 ..2. Growth of enterobacteria in minimal medium containing amino acids 9.. Growth of enterobacteria in presence of acetoin 8 Chapter : Growth of Enterobacteriaceae in fruit juices 92.. Cultivation of enterobacteria in grape juice under oxygen limited conditions Cultivation of enterobacteria in worldshake juice under oxygen limited conditions 3.3. Cultivation of enterobacteria in ectar Multifruit juice under anaerobic conditions.. Cultivation of enterobacteria in apple juice... Cultivation of enterobacteria in apple juice under oxygen limited conditions..2. Cultivation of enterobacteria in apple juice under anaerobic conditions Cultivation of enterobacteria in apple juice based on optical density Oxygen limited conditions Anaerobic conditions 23 Chapter : Growth of Enterobacteriaceae on vegetables 2.. Growth of Serratia plymuthica RVH on fresh sliced vegetables 2... Growth of Serratia plymuthica RVH wild type and budab mutant on red pepper slices surrounded by liquid Growth of Serratia plymuthica RVH wild type and budab mutant on red pepper slices Growth of Serratia plymuthica RVH wild type and budab mutant on cucumber slices Growth of Cronobacter sakazakii LMG wild type and budab mutant on red pepper slices 3 GE ERAL DISCUSSIO A D CO CLUSIO 39 REFERE CE LIST XII

15 Introduction The Enterobacteriaceae family includes a wide range of microorganisms classified in different genera of which some pathogenic representatives are directly involved in foodborne infectious diseases. The most important route of infections caused by enterobacteria is contaminated food, like animal products, but also fresh fruit (juices) and vegetables are now increasingly recognized as a major route of entry into the food chain. Traditionally, fresh fruit juices are considered safe to consume directly. This is in part due to the high acidity of some fruit juices that would inhibit the growth of pathogenic bacteria. In fact, the low ph values of most fruit juices influence their susceptibility to microbial growth. However, some strains of enteropathogens can present specific response mechanisms that enable their growth or survival during exposure in such environments. Enterobacteria are divided into two groups, mixed acid fermenters and 2,3-butanediol fermenters, based on the production of end products during fermentation of glucose. It is believed that the butanediol pathway plays an important role for the growth or survival under low ph environments. To investigate the role of the butanediol pathway in tolerance to low ph environments, a pbudab plasmid, containing two essential genes (budab operon) of the butanediol pathway, was inserted into certain mixed acid fermenters. The bacteria considered in this study were Escherichia coli MG, Salmonella Typhimurium LT2, Salmonella Senftenberg LMM2, Salmonella Enteritidis ATCC3 and Shigella flexneri LMG2, known as mixed acid fermenters and, Cronobacter sakazakii LMG and Serratia plymuthica RVH, known as butanediol fermenters. This study included cultivations of the wild type strains, pbudab strains and budab mutant strains in synthetic medium under different conditions. The following conditions were evaluated, i: the growth at neutral and low initial ph values and, ii: the influence of different concentrations of glucose, amino acids, casamino acids and acetoin on their growth. In order to investigate the effect of ph per se on the above mentioned enterobacteria, experiments have been done using synthetic media adjusted to the required ph with HCl. Subsequently, cultivations in different fruit juices adjusted to different ph values were carried out in order to evaluate the growth and survival of these bacteria at different temperatures. Even when microorganisms are unable to multiply in acidic conditions, they

16 may be able to survive for a prolonged period of time. In case of foodborne pathogens this may have important consequences for food safety. For example, many outbreaks of food poisoning have been attributed to the survival of Salmonella spp. in unpasteurized fruit juices (Parish, 99; Harris et al., 23; Lima Tribst et al., 29) and to the survival of Escherichia coli O:H in unpasteurized apple juice (CDC, 99; Harris et al., 23; Lima Tribst et al., 29). Indeed, the intention of this study was on one hand to evaluate the influence of the budab genes on the growth or survival of enterobacteria on fresh produce commodities and, on the other hand to evaluate the risk of consuming contaminated or unpasteurized fruit juices. Finally, we focused our study on the spoilage activity of Serratia plymuthica RVH and Cronobacter sakazakii LMG on red pepper and cucumber slices at different storage temperatures. At present, the demand for fresh-cut vegetables has increased enormously. These products can be packaged in pouches with and without modified atmosphere or in trays covered with a polymeric wrap and stored at refrigeration temperatures. The cut and exposed surfaces of the vegetables are a main concern because spoilage and pathogenic microorganisms can grow there (Nguyen-The and Carlin, 99; Beuchat, 99; Harris et al., 23). The high water activity and nutrient content of fresh-cut produce can support the growth of a variety of spoilage microorganisms (Sumner and Peters, 99). Thus, deterioration of fresh-cut vegetables can occur after packaging, during retailing, or in some cases during storage under refrigeration temperatures. The spoilage activity of microorganisms causes a decrease of the product shelf life. This study was divided into three parts, the first is concerned with the growth in synthetic medium, the second deals with growth in fruit juices and the third one is concerned with growth on vegetables. Although the results of these experiments will contribute greatly to our understanding about the importance of the butanediol fermentation in Enterobacteriaceae, further research will be necessary to carry out in the future in order to answer the questions emerging in this thesis. 2

17 PART I: LITERATURE STUDY

18 . Enterobacteriaceae.. Taxonomy and classification of Enterobacteriaceae The name Enterobacteriaceae was first proposed in 93 by Rahn to enclose the genus Enterobacter and other bacteria that shared the ability to ferment glucose with the production of gas (Rahn, 93). At that time the name Enterobacteriaceae was referred to as bacteria which inhabit the enteron or intestinal tract of animals. After some decades many members of Enterobacteriaceae have been defined by molecular techniques based on DNA relatedness (Brenner, 992a,b; Fox et al., 992; Stackebrandt and Goebel, 99) and afterwards the high-resolution polyacrylamide gel electrophoresis of proteins became a useful taxonomic tool to identify and type bacteria. Nowadays several molecular techniques based on fragment analysis, techniques using polymerase chain reaction (PCR) and sequencing of S rrna genes emerged for the identification, characterisation and determination of relationships between bacterial groups (Christensen et al., 998; Sproer et al., 999). There are about recognized genera and named species of bacteria belonging to the family of Enterobacteriaceae and undoubtedly in the future the unnamed published and unpublished genomospecies and presently undescribed groups will be included as new genera and species (Garrity et al., 2). Furthermore, as a consequence of the introduction of new molecular methods, reclassification of the current taxonomy and generation of new genera and species might be necessary in some cases, like for example in Salmonella (Brenner et al., 2) or Klebsiella (Drancourt et al., 2). The genera of this family are defined and classified based on genetic and antigenic properties and, also more traditionally, on biochemical characteristics and pathogenicity as follows: Arsenophonus, Brenneria, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cronobacter, Edwarsiella, Enterobacter, Erwinia, Escherichia, Ewingella, Hafnia, Klebsiella, Kluyvera, Lelercia, Leminorella, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Photorhabdus, Pragia, Proteus, Providencia, Rahnella, Raoultella, Salmonella, Serratia, Shigella, Tatumella, Xenorhabdus, Yersinia, Yokenella, and others (Garrity et al., 2; Baylis, 2).

19 .2. Habitat and importance of Enterobacteriaceae The Enterobacteriaceae family is distributed worldwide. The habitats of the most common genera related to humans and food products are listed in table.. The members of this family are found in soil, water, fruits, meats, eggs, vegetables, grains, flowering plants and trees, insects and in the intestinal tract of animals including humans. Some species are pathogenic to humans, whereas others are pathogenic to plants and animals. Many members of this family are of clinical or economic importance and others are important in food spoilage causing substantial losses in some sectors of the food industry. For example, Erwiniae and Pectobacteria can cause disease in potatoes, apples and many other crops (Perombelon, 22; Tsukamoto et al., 2; De Haan et al., 28). Many members of this family can also cause problems in the animal husbandry. Salmonellosis in poultry and eggs is a worldwide problem, both for poultry farmers and as a vehicle for human disease (Mishu et al., 99; Guard-Petter, 2). Enterotoxigenic Escherichia coli strains are primarily responsible for infections in lambs, pigs and calves (Janke et al., 989), while Klebsiella pneumoniae, Escherichia coli and Enterobacter aerogenes are causes of bovine mastitis (Bannerman et al., 23). The most common routes and sources of contamination during food production include: soil, contaminated water, equipments of the processing line and by indirect contact of human and animal faeces. Many species of Enterobacteriaceae can contaminate animal foods such as milk and dairy products, raw fresh meat and poultry and processed meat products, by a variety of sources (Cousin, 982; Gustavsson and Borch, 993; Castaño et al., 22; Iversen and Forsythe, 2; Hudson et al., 28). Fresh fruit and vegetables and related products can also be contaminated by members of the Enterobacteriaceae family (Everis, 2). Good agricultural practices (GAP), good manufacturing practices (GMP) and hygiene control during handling and processing of food products help to reduce and avoid contamination with pathogenic enterobacteria. Some species of this family are used as indicator organisms; the presence of these bacteria in food indicates poor hygiene of food handlers, inadequate processing or contamination after processing. E. coli and the coliform group are used as index of fecal contamination. E. coli is considered as a better indicator of a possible fecal contamination (Gras et al., 99).

20 Table.: Habitat of some members of Enterobacteriaceae related to human and food interactions (Baylis, 2). Genus Citrobacter Enterobacter Erwinia Escherichia Hafnia Klebsiella Kluyvera Moellerella Morganella Proteus Salmonella Serratia Shigella Yersinia atural habitat and isolation sources Human faeces, sewage, soil, water, food. Soil, fresh water, plants, vegetables, sewage, animal and human faeces. Plants as pathogens, saprophytes or normal flora. Gastrointestinal tract of humans and other warm-blooded animals (water, food and soil via faecal contamination). Human and other animal faeces (including birds), sewage, soil and dairy products. Gastrointestinal tract and respiratory tract of human and other animals, faeces, soil, water, fruits and vegetables, grain, etc. Respiratory tract, food, milk, water, soil and sewage. Human faeces and water. Faeces of humans, dogs and other mammals. Intestines of humans and a wide range of animals, soil, manure and polluted waters. Humans and other warm-blooded animals, foods, water and environmental sources. Plant surfaces, soil, water and environmental sources, digestive tracts of rodents and insects. Intestinal pathogen of humans and other primates. Humans, animals (especially birds and rodents), dairy products and other foods, soil and water. Among the most common foodborne pathogenic enterobacteria are the toxigenic species of Escherichia, Salmonella, Enterobacter, Shigella, Salmonella, Yersinia and Klebsiella. On the other hand, the presence of some strains of Enterobacteriaceae in foods seems to have a beneficial effect due to the production of sensory important compounds such as in the process of cheese-making. The produced compounds include aldehydes, ketones, sulphur compounds, alcohols, and aromatic compounds (Morales et al., 2; Chaves-Lopez et al., 2; Deetae et al., 29).

21 .3. General and biochemical characteristics of Enterobacteriaceae The bacteria belonging to this family are Gram-negative, straight rods, of.3. x.. µm in size, non-spore-forming and facultatively anaerobic (Farmer, 99). They can be motile by peritrichous flagella or nonmotile. Most of them grow well at 22 3 C; optimal growth and maximal biochemical capacity of a number of genera (Yersinia, Hafnia, Xenorhabdus, Photorhabdus, and many Erwiniae) occurs at 2 28 C (Farmer, 99; Garrity et al., 2). They are chemoorganotrophic; having both a respiratory and a fermentative metabolism. Acids and visible gas are often produced during fermentation of D-glucose and other sugars. Most species are catalase-positive and oxidase-negative (Garrity et al., 2) and can reduce nitrate to nitrite. All ferment glucose, but those which ferment lactose are grouped together as coliform bacteria (e.g. Citrobacter, Escherichia, Enterobacter and Klebsiella). This group can be defined as enterobacteria which can ferment lactose with production of acid and gas within 8 hours at 3 C. Genera and species of the Enterobacteriaceae family have traditionally been differentiated based on biochemical tests. Biochemical reactions for Enterobacteriaceae include: indole test, methyl red test, Voges-Proskauer test (production of acetoin), growth on citrate as the sole carbon source by plating on Simmons citrate agar, hydrogen sulfide production by plating on TSI agar, growth in KCN broth, utilization of malonate, tartrate utilization by plating on Jordan s tartrate agar and acetate utilization (Farmer et al., 98; Farmer, 99; Farmer, 999). These metabolic properties are useful in characterization and distinction of members of the Enterobacteriaceae family (table.2). During fermentation, the production of gas (CO 2 ) is a tool to differentiate between Escherichia coli and pathogens like Shigella and Salmonella, which do not produce gas (Sawers, 2). Similarly, because they possess formic hydrogenlyase, members of Enterobacter are vigorous gas-producers but paradoxically Serratia does not produce it (Kurokawa and Tanisho, 2).

22 Table.2: Characterization of some members of Enterobacteriaceae based on routinary biochemical tests (Farmer, 99; Farmer, 999; Pandey et al., 2; Garrity et al., 2). Member Indole test a Methyl red test b Voges- Proskauer test c Citrate utilization Escherichia coli + + Shigella +/ + Salmonella Typhimurium + + Citrobacter freundii + + Klebsiella pneumoniae +/ +/ +/ Enterobacter aerogenes + + Yersinia enterocolitica v v +/ d a split indole from tryptophan. b acid production to bring ph below,. c acetoin production. d Voges-Proskauer test for Y. enterocolitica is negative at 3 C, but positive at 2 28 C. V: between % and 9% of strains showing a positive reaction. Enzymatic tests are also commonly used: hydrolysis of urea, gelatin or esculin, phenylalanine deaminase, lysine decarboxylase, arginine dihydrolase, ornithine decarboxylase, tartrate, lipase, DNase, oxidation of nitrate to nitrite, Kovacs oxidase test, ONPG (β-galactosidase) test, yellow pigment production, acid and gas production from D- glucose (Farmer et al., 98; Farmer, 999; Farmer, 23). Many genera and species of Enterobacteriaceae also have typical patterns of resistance and susceptibility to antibiotics; thus, the antibiogram of an isolate can also be used as an aid of identification. The following antibiotics are considered in the antibiogram analysis: polymyxins, ampicillin, cephalothin, carbenicillin, colistin, tetracycline and nitrofurantoin (Farmer et al., 98; Farmer, 999)... Metabolism of Enterobacteriaceae The minimal nutritional requirements of the members of the Enterobacteriaceae family are often very simple. Growth under aerobic condition is easily achieved but, under anaerobic conditions, growth is severely dependent on the availability of fermentable sugars. On the other hand, bacterial cells consist of a wide variety of chemical substances which have to be synthesized or taken up from outside the cell, but these processes require a lot of energy (Fuchs, 999). Each cell has to provide the necessary energy and different possibilities for its supply were developed. Most members of Enterobacteriaceae obtain energy from the oxidation of chemical compounds. Chemicals used as sources for energy are metabolized 8

23 and energy is conserved either by substrate-level phosphorylation or by building up an electrochemical gradient across the cytoplasmic membrane (Fraenkel, 98; White, 99; Fuchs, 999). The most common metabolic pathways in the family of Enterobacteriaceae by which energy is converted, especially from glucose to ATP (Fraenkel, 98; Fuchs, 999) are the followings:. Embden-Meyerhof-Parnas (EMP) pathway, also called glycolysis, 2. Pentose phosphate (PP) pathway, 3. Tricarboxylic acid cycle or Krebs cycle and,. Fermentative pathways. In E. coli and other members of Enterobacteriaceae the EMP and PP pathways are two central and constitutive routes of intermediary carbon metabolism (Fraenkel, 98; Sprenger, 99; Fuchs, 999). In these two pathways, the same reactions occur whether oxygen is present or not. Both pathways convert glucose to glyceraldehyde 3-phosphate (by different routes) and the latter compound is converted to pyruvate via reactions that are the same in both pathways.... Embden-Meyerhof-Parnas pathway In most species of Enterobacteriaceae the breakdown of hexoses is carried out through glycolysis (Fraenkel, 98). The enzymatic reactions of a glycolytic pathway will form pyruvate coupled to ATP synthesis by substrate-level phosphorylation (figure.). Although the initial series of reactions of the EMP pathway require the input of two ATP, the overall reaction is exergonic, so that finally a net gain of two ATP will result from one glucose degraded in glycolysis. The overall reaction is: Glucose + 2 ADP + 2 P i + 2 NAD + 2 Pyruvate + 2 ATP + 2 NADH + 2H + Glycolysis is the most used pathway in microorganisms, in some cases it is the main route to produce energy useful for the growth and other activities of the cells (Fraenkel, 98; White, 99; Fuchs, 999). This route also provides intermediary compounds for many other important reactions depending on the cultivation conditions. 9

24 ..2. Pentose phosphate pathway Many members of Enterobacteriaceae such as Escherichia coli and Salmonella Typhimurium use this pathway together with the EMP pathway (Fraenkel, 98; Sprenger, 99; Enos-Berlage and Downs, 99; Fuchs, 999). Under aerobic conditions, about 8% of the glucose is degraded via the EMP pathway and about 2% enters the PP pathway for generation of ATP, regeneration of NADPH and the synthesis of precursors for nucleotide and aromatic amino acid biosynthesis (figure.). So many variations of the PP pathway are possible, depending on the need of the growing cell (Sprenger, 99). The oxidative cycle of the PP pathway is more commonly used for pentose and NADPH formation by oxidizing C of hexoses to CO 2 (Sprenger, 99; White, 99; Fuchs, 999). The overall reactions of the PP pathway are divided into three stages:. Oxidation and decarboxylation reactions, 2. Isomerization reactions, 3. Sugar rearrangement reactions (transketolase and transaldolase). In the oxidative version of the PP pathway, glucose is converted into ribulose -phosphate and CO 2, a process that requires one ATP and generates two NADPH (figure.). When there is a further need for more NADPH, the excess pentose phosphates have to be removed by a non-oxidative cycle, thus, the enzymes transketolase and transaldolase can convert pentose phosphates back into hexose phosphates (Sprenger, 99; White, 99; Fuchs, 999). This pathway seems to be less important in the glucose metabolism in Escherichia coli. Mutants blocked in -phosphogluconolactonase, an important enzyme of the oxidative PP pathway, still grow with glucose as the carbohydrate source (Kupor and Fraenkel, 99)...3. The tricarboxylic acid (TCA) or Krebs cycle In facultative anaerobic bacteria such as the enteric bacteria, oxygen is the most important regulatory signal and the enzymes of the aerobic metabolism are only synthesized under aerobic conditions (Spencer and Guest, 98; Fuchs, 999). In facultative anaerobic bacteria pathways with the highest ATP yield are preferred. The presence of oxygen represses the anaerobic respiration and also the fermentation pathways (Gunsalus and Park, 99; Fuchs, 999). The Krebs cycle operates only when oxygen is present in the medium.

25 In bacteria this cycle takes place in the cytosol and begins when pyruvate is transformed to acetyl-coa by the pyruvate dehydrogenase complex (Gest, 98; White, 99; Fuchs, 999). In order to finish the respiratory metabolism of glucose, acetyl-coa enters the TCA cycle to produce carbon dioxide, reduced coenzymes and ATP (figure.). The overall reactions are: Acetyl-CoA + ADP + P i + FAD + 2 H 2 O + NADP NAD + 2 CO 2 + ATP + FADH 2 + NADPH + 2 NADH + 3 H + + CoASH Under anaerobic conditions the TCA cycle no longer functions as such because the links to terminal respiration are required to maintain the activities and synthesis of succinate dehydrogenase and the α-ketoglutarate dehydrogenase complex (White, 99; Fuchs, 999). There are several physiological changes which take place in enteric bacteria in the absence of oxygen as part of adaptation to anaerobic growth:. Terminal reductases replace the oxidases in the electron transport chain 2. The TCA cycle is modified to become a reductive pathway. α-ketoglutarate dehydrogenase (enzyme ) and succinate dehydrogenase (enzyme 8) are missing or occur at low levels, the latter being replaced by fumarate reductase. 3. Pyruvate formate lyase (enzyme 29) is substituted for pyruvate dehydrogenase (enzyme ). So pyruvate is oxidized to acetyl-coa and formate, rather than to acetyl-coa, CO 2 and NADH.. They carry out a mixed acid (..) or butanediol (..) fermentation Fumarate reductase activity is increased, providing a mechanism for continued succinate synthesis. Succinate is an important fermentation end product of the mixed acid fermentation. Thus, the TCA cycle functions as a branched biosynthetic pathway: one branch operating as a reductive pathway reversing the sequence from succinate to oxaloacetate and the other branch continuing to operate oxidatively to convert oxaloacetate to α-ketoglutarate (Spencer and Guest, 98; White, 99; Fuchs, 999; Unden, 999).

26 Glucose 9 Malate Fumarate 8 2 Succinate 2 9 Phosphoenolpyruvate Pyruvate Oxaloacetat Succinyl CoA Acetyl-CoA 2 Citrate 3 lsocitrate 2-Oxoglutarate -Phosphoglucono--lacton -Phosphogluconate Glycerol Fructose -P Erythrose -P Xylulose -P Ribulose -P Glycerol 3-P Fructose,-biP Ribose -P Sedoheptulose -P 2 Dihydroxyacetone Glyceraldehyde 3-P phosphate Synthesis: Adenosine,3-biphosphoglycerate (AMP), guanosine (GMP) Acetic acid DNA, RNA. Ethanol 33 3-phosphoglycerate Synthesis of aromatic aminoacids 3 Acetyl-P 8 Acetaldehyde 2-phosphoglycerate 3 Acetyl-CoA Formic acid 3 H 2 + CO Glucose -P Figure.: Major metabolic pathways used by Enterobacteriaceae under aerobic and anaerobic conditions: glycolysis (black lines), Krebs cycle (blue lines), pentose phosphate pathway (red lines) and fermentative pathways (pink lines). Enzymes: : hexokinase, 2: phosphoglucose isomerase, 3: phosphofructokinase, : fructose,-bisphosphate aldolase, : triosephosphate isomerase, : glyceraldehyde 3-phosphate dehydrogenase, : phosphoglycerate kinase, 8: phosphoglycerate mutase, 9: enolase, : pyruvate kinase, : pyruvate dehydrogenase complex, 2: citrate synthase, 3: aconitase, : aconitase, : isocitrate dehydrogenase, : 2-oxoglutarate dehydrogenase, : succinyl-coa synthetase, 8: succinate dehydrogenase, 9: fumarase, 2: malate dehydrogenase, 2: glucose - phosphate dehydrogenase, 22: -phosphogluconolactonase, 23: -phosphogluconate dehydrogenase, 2: ribulose -phosphate isomerase, 2: ribulose -phosphate 3-epimerase, 2: transketolase, 2: transaldolase, 28: lactate dehydrogenase, 29: pyruvate formate lyase, 3: acetaldehyde dehydrogenase, 3: alcohol dehydrogenase, 32: phosphotransacetylase, 33: acetate kinase, 3: formate-hydrogen lyase complex, 3: α-acetolactate synthase, 3: α-acetolactate decarboxylase, 3: acetoin dehydrogenase, 38: 2,3-butanediol dehydrogenase, 39: glycerol 3-P dehydrogenase, : glycerol 3-phosphatase (Fraenkel, 98; Fuchs, 999; White, 99; Unden, 999; Buckel, 999) Lactic acid cis-aconitate 3 CO 2 O 2 Diacetyl 22 Amino acid synthesis α-acetolactate 3 3 CO 2 CO 2 Acetoin 38 2,3-Butanediol 2

27 ... Mixed acid fermentation Under anaerobic conditions and in the absence of alternative electron acceptors, some members of the Enterobacteriaceae (e.g. Escherichia, Salmonella, and Shigella) ferment glucose to a mixture of acetic, formic, lactic and succinic acid and ethanol (mixed acid fermentation, figure.2) (Clark, 989; Becker et al., 99; Buckel, 999). The production of acetate and formate as major products is notable. As much as 8 8% of the glucose fermented by E. coli is metabolized via the EMP pathway. Other pathways must contribute to the products to some extent. These reactions involve the transfer of electrons and hydrogen from NADH to organic compounds. In fermentation, energy gain is very low and occurs as a result of substrate-level phosphorylation. The synthesis of ATP in fermentation is mainly formed during glycolysis (White, 99; Fuchs, 999; Buckel, 999). There may be a large quantitative variation in the end products formed among different species and even within strains under different fermentation conditions as end products are formed by independent pathways. Figure.2: Mixed acid fermentation. Enzymes: glycolytic enzymes, 2 pyruvate kinase, 3 pyruvate formate lyase, lactate dehydrogenase, formate hydrogen lyase, acetaldehyde dehydrogenase, alcohol dehydrogenase, 8 phosphotransacetylase, 9 acetate kinase, phosphoenolpyruvate carboxylase, malate dehydrogenase, 2 fumarase, 3 fumarate reductase (White, 99). 3

28 ... 2,3-butanediol fermentation The remarkable metabolic characteristic of Enterobacter, Klebsiella, Serratia and some species of Erwinia and Yersinia is, besides the production of acids, the production of neutral compounds such as acetoin and its oxidation (diacetyl) or reduction (2,3- butanediol) product (figure.3) (Mayer et al., 99). The first step is the conversion of pyruvate to α-acetolactate by α-acetolactate synthase. Decarboxylation of α-acetolactate is carried out by α-acetolactate decarboxylase. Finally, acetoin is reduced by 2,3-butanediol dehydrogenase, also called acetoin reductase, to obtain 2,3-butanediol. Acetate induces the production of α-acetolactate decarboxylase, acetoin dehydrogenase and 2,3-butanediol dehydrogenase (Bryn et al., 93; Mayer et al., 99). The conversion of pyruvate to neutral compounds seems to prevent overacidification of the intracellular compartment and these products can also serve as carbon sources when glucose is depleted in the medium (Johansen et al., 9; Blomqvist et al., 993; Mayer et al., 99; Xiao and Xu, 2). Figure.3: 2,3-butanediol fermentation. Enzymes: glycolytic enzymes, 2 pyruvate formate lyase, 3 formate hydrogen lyase, acetaldehyde dehydrogenase, alcohol dehydrogenase, and α-acetolactate synthase, 8 α-acetolactate decarboxylase, 9 2,3-butanediol dehydrogenase, lactate dehydrogenase (White, 99).

29 Among the parameters that influence the production of 2,3-butanediol, the high glucose concentration seems to have a positive effect, improves the yield and productivity, but decreases the cell growth (Qureshi and Cheryan, 989). The production of 2,3-butanediol is also dependent on the type of fermentable sugar, pentoses and hexoses are better substrates compared to disaccharides (Champluvier et al., 989). Finally, the oxygen supply is a critical factor in the production of 2,3-butanediol, low oxygen supply increases its yield (Jansen et al., 98; Ramachandran and Goma, 98; Converti et al., 23). Table.3 gives a comparison between the fermentation end products produced during mixed acid or 2,3-butanediol fermentation of glucose. Table.3: Fermentation products (in moles per mol of glucose fermented) in mixed acid and in butanediol fermentation (Stanier et al., 9). Product Mixed acid 2,3-Butanediol fermentation fermentation Carbon dioxide 88 2 Hydrogen 3 Formic acid 2. Acetic acid 3. Lactic acid 9 3 Succinic acid Ethanol 2,3-butanediol Total moles of acids produced ph homeostasis in Enterobacteriaceae The regulation of the cytoplasmic ph in response to the variation of the external ph is an important factor for growth of bacteria. It depends on the type of bacteria (acidophiles, neutrophiles or alkalophiles). Bacteria have to maintain their internal ph within a narrow ph range, in case of acidophiles ph.., neutrophiles ph. 8., and alkalophiles ph (Lowe et al., 993; White, 99). This regulation must be achieved over a relatively short time to prevent inhibition of the cell growth or even cell death. Many species require homeostasis to regulate the internal ph for growth (Slonczewski et al., 98; Zilberstein et al., 98).

30 The transmembrane ph difference ( ph) is a component of the proton motive force, which drives the process of proton transport from inside to outside the cell and vice versa if necessary. The intracelullar concentration of potassium and sodium ions is important for carrying out the transport of protons (White, 99). Most members of Enterobacteriaceae, including Escherichia coli and Salmonella Typhimurium are neutrophiles; they grow very well at approximately neutral ph, but can also grow in moderate acid or base. In acidic environments they have to pump protons outside of the cell in order to maintain the original ph, otherwise the internal ph falls down until equilibrium with the external ph and as a consequence the metabolic processes stop (White, 99; Booth, 999). Pathogenic strains of E. coli, S. Typhimurium and, Shigella flexneri, encounter extreme ph both within and outside human hosts. During pathogenesis, cells are exposed to low ph in the stomach (Wilder-Smith et al., 992; Martinsen et al., 2). Consequently, low ph induces virulence factors that contribute to pathogenesis or acid shock proteins (Abshire and Neidhardt, 993a,b). External ph can be detected directly by proteins containing a periplasmic domain or the lysine decarboxylase regulator (Miller et al., 98; Watson et al., 992), whereas internal ph might be detected by a cytoplasmic protein. External ph could also be sensed indirectly, mediated by changes in internal ph (Slonczewski et al., 98). In Escherichia coli, ph homeostasis requires transport of K + when the external ph <. (Bakker and Mangerich, 98; Dosch et al., 99; White et al., 992). The rapid recovery of internal ph upon a shift of external ph by one or two units indicates that some aspects of ph homeostasis must be constitutive (Slonczewski et al., 982; Zilberstein, et al., 98), although some inducible components may exist as well (Foster and Bearson, 99). Escherichia coli and Salmonella Typhimurium can survive several hours (without growth) with internal ph decreased below ph (Foster and Hall, 99; Small et al., 99)... Some representative enteric bacteria related with foodborne diseases... Escherichia coli The genus Escherichia contains five species. Apart from E. coli, the other members of the genus are E. hermanii, E. fergusonii, and E. vulneris, all isolated from various human

31 sources both intestinal and extra-intestinal, and E. blattae, only ever isolated from cockroaches (Ewing, 98; Cowan et al., 99; Holt et al., 99; Richard, 989). E. coli has an optimum growth temperature of 3 C but can grow over a wide temperature range from C to C. E. coli ferment glucose with the production of lactic, acetic and formic acid. Part of the formic acid is split by a complex formate hydrogen lyase system into equal amounts of CO 2 and H 2 (Garrity et al., 2). The ability to ferment other carbohydrate sources is dependent on the conversion of relevant carbohydrate substrates to glucose or glucose derivatives. E. coli is categorized in serotypes according to particular virulence factors. Serotyping is based on the identification of bacterial antigens; the somatic O antigen, the capsular K antigen, and the flagellar H antigen. Currently, it is considered necessary only to determine the O and the H antigens to serotype strains of E. coli associated with diarrheal disease. The successful combination of virulence factors persist to become pathotypes of E. coli which are capable of causing disease in humans. Typical clinical syndromes caused by infection with these pathotypes are: enteric/diarrhoeal disease, urinary tract infections and sepsis/meningitis (Kaper et al., 2). Six well-described categories of intestinal pathogenic E. coli are recognized: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and diffusely adherent E. coli (DAEC) (Nataro and Kaper, 998; Kaper et al., 2). The pathogenic mechanisms of EPEC are linked to their ability to adhere and invade epithelial cells (Donnenberg et al., 989). Food and water have been implicated as vectors of this pathogen (Williams et al., 99). In case of ETEC, this strain colonise the small intestine of humans. ETEC is thought to be a common cause of traveller s diarrhoea resulting from consumption of contaminated water or food (Daniels et al., 2). The pathogenicity of EIEC is related to the ability to invade and multiply within epithelial cells in the colon. Some reports indicate that food and water may be vectors of EIEC transmission (Snyder et al., 98). The most important characteristic of the enteroaggregative E. coli (EAEC) is the production of a heat stable enterotoxin called EAST (Savarino et al., 99). EAEC derive their name from their pattern of adherence to HEp-2 cells in tissue culture (Nataro et al., 98). EAEC pathogens have been associated

32 with persistent diarrhea in young children (Savarino et al., 99; Kaper et al., 2). Enterohaemorrhagic group of E. coli (EHEC) causes an illness characterised by severe bloody diarrhoea. The key virulence factor for EHEC is Stx, which is also known as verocytotoxin (VT). This group of pathogens is more commonly referred to as verocytotoxigenic E. coli (VTEC) as they produce a toxin which has a cytotoxic effect on vero cell lines (Kaper et al., 2). The pathogenicity of this group is related to the ability of the organism to adhere and colonise the human large intestinal epithelial tissue destroying the microvilli. E. coli O:H is the most representative serotype of this group (Riley et al., 983; Kaper et al., 2). It is now recognised as an important cause of food and waterborne illness in industrialised countries. The infectious dose for E. coli O:H is estimated to be cells (Teunis et al., 2). Diffusely adherent E. coli (DAEC) strains are defined by a pattern of diffuse adherence (DA), in which the bacteria uniformly cover the entire surface of HEp-2 cell monolayers (Scaletsky et al., 98). DAEC have been implicated as a cause of diarrhoea in several studies, particularly in children older than 2 months of age (Nataro and Kaper, 998; Scaletsky et al., 22)...2. Salmonella Salmonella is a foodborne pathogen and an etiological agent for human typhoid fever and salmonellosis. The Salmonella genus consists of two species: S. enterica including six subspecies (enterica, salamae, arizonae, diarizonae, houtenae, and indica) and S. bongori. No fewer than 2,22 serovars are recognized within this genus (Popoff et al., 99; Jay et al., 2). Salmonella enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are mainly transmitted by contaminated food products and are the most important causes of gastroenteritis. The improper handling and inadequate cooking of foods are the principal causes of foodborne outbreaks (Todd, 99). The principal habitat for Salmonella is the intestinal tract of humans and animals (Minor, 992; Jay et al., 2). However, this bacterium has often been isolated from soil, water and animal feed (Anderson and Ziprin, 2). Infection with Salmonella spp. includes the attachment to specialized M cells in Peyer s patches of the small intestine and penetration into the cytoplasm of epithelial cells resulting in mucosal inflammation. The release of bacterial enterotoxins provokes a diarrheagenic 8

33 fluid secretion into the intestinal lumen. After a serie of events polymerization of actin and cytoskeleton rearrangement, and the formation of membrane ruffles on the surface of intestinal epithelial cells and of M cells in Peyer's patches takes place (Francis et al., 993). Evidences suggest that a single Salmonella cell may constitute a human infectious dose (D Aoust, 99). As with most Enterobacteriaceae, Salmonella may harbor plasmids that may code for virulence factors, antibiotic resistance, bacteriocins, metabolic characteristics and antigenic molecules (Minor, 992). Virulence plasmids occur in a limited number of serovars and play an important role in the pathogenicity of Salmonella spp. (D Aoust, 99)...3. Shigella Shigella is a genus of highly adapted human pathogens that cause bacillary dysentery (shigellosis), a disease that provokes severe bloody and mucous diarrhea (Keusch and Bennish, 989). The genus Shigella consists of four species: S. dysenteriae, S. flexneri, S. boydii and S. sonnei which can be differentiated from each other by the fermentation of sugars or sugar alcohols, production of indole, and the synthesis of ornithine decarboxylase or arginine dehydrolase (Lampel et al., 2; Garrity et al., 2). Shigella produces pyruvate from the fermentation of glucose and other sugars and converts it primarily by mixed acid fermentation to formic acid, acetic acid, and ethanol. In contrast to E. coli strains, shigellae do not produce gas from the fermentation of sugars, because they lack the formate hydrogen lyase system (enzyme in figure.2) that splits formic acid into CO 2 and H 2 (Lampel et al., 2). In addition, members of the genus Shigella do not produce detectable amounts of acetoin or 2,3-butanediol (Voges-Proskauer negative) (Lampel et al., 2; Garrity et al., 2). Members of the genus Shigella are genetically, biochemically, and serologically similar to E. coli (Ochman et al., 983; Lampel, 2). A significant difference between EIEC and Shigella is the infective dose, being bacteria for EIEC and to 2 organisms for shigellae (Du Pont et al., 989). To produce clinical symptoms, shigellae must survive the acidic environment of the stomach. Cells attach to the epithelial cells of the colon and invade. They multiply intracellularly, and disseminate intercellularly through adjacent colonic epithelial cells. Shigella species have a preference for M cells of the colon (Perdomo et al., 99; 9

34 Zychlinsky et al., 99). Temperature plays a critical role in controlling the expression of the virulence genes of Shigella spp. Thus, at 3 C, Shigella is not pathogenic; by shifting the growth temperature to 3 C, this enteric organism becomes virulent (Maurelli and Sansonetti, 988).... Yersinia Yersiniae are non-spore-forming straight rods or coccobacilli responsible for self-limiting gastroenteritis and yersiniosis, restricted to the intestinal tract and the intestinal lymphoid system. The genus Yersinia is composed of species, of which only Y. enterocolitica, Y. pseudotuberculosis and Y. pestis are known pathogens for humans and animals (Kapperud and Bergan, 98). Y. enterocolitica and Y. pseudotuberculosis are enteropathogenic organisms that share common modes of transmission mainly through food and water. Yersinia species are classified into serotypes based on biochemical, O-antigenic and virulence properties (Kapperud, 99; Smego et al., 999; Bogdanovich et al., 23). A special characteristic is that different serotypes can be positive to the Voges-Proskauer reaction when cultivated between 22 C to 2 C and negative between 3 C to 3 C (Kapperud and Bergan, 98). Bacteria currently classified as Y. enterocolitica do not constitute a homogeneous group. Within this species, there is a wide spectrum of biochemical variants; such variations form the basis for dividing Y. enterocolitica into biovars (Bercovier et al., 98; Kapperud and Bergan, 98; Swaminathan et al., 982). Infection due to Y. enterocolitica is mostly by the oral route and the clinical symptoms are dependent on the patient s age (Wormser and Keusch, 98). The minimal infectious dose seems to be 3. x 9 organisms (Szita et al., 93). The incubation period has been estimated as being between 2 and days (Szita et al., 93; Ratnam et al., 982). Infection with Y. enterocolitica includes the proliferation of cells in the lumen and in the lymphoid tissue of the intestine. Adherence and penetration into the epithelial cells of the intestinal mucosa are essential factors in the pathogenesis (Bliska and Falkow, 99; Miller et al., 988). Pathogenic Y. enterocolitica possess a special plasmid which is essential for virulence (Brubaker, 99; Portnoy and Martinez, 98). The presence of this virulence plasmid has been associated with several properties, most of which are 2

35 phenotypically expressed only at elevated growth temperatures of 3 C to 3 C (Portnoy and Martinez, 98).... Cronobacter sakazakii Enterobacter sakazakii was recently reclassified into a new genus Cronobacter. This new genus consists of six species, including C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, C. dublinensis and C. genomospecies, to accommodate the biogroups of the hitherto known E. sakazakii (Iversen et al., 28). C. sakazakii ferments glucose with production of acid and gas and is Voges-Proskauer positive (Farmer et al., 98; Farmer and Kelly, 992). C. sakazakii is usually a commensal of the human intestine, but it has occasionally been isolated as an extraintestinal pathogen (Grimont and Grimont, 992). Nevertheless, C. sakazakii should be viewed as a potential agent of foodborne illness. Commonly, Cronobacter spp. are considered to be opportunistic pathogens, implicated in particularly severe foodborne diseases in neonates and infants (Farber, 2; Mullane et al., 2; Townsend et al., 28). Symptoms include bacteraemia, necrotizing enterocolitis and meningitis, with fatality rates as high as 8% (Lai, 2). The infectious dose for neonates has been determined tentatively to range from 3 to 8 CFU (FAO/WHO, 2); such populations may develop only if reconstituted powder infant formulae were subject to time temperature abuse during storage. WHO (2) recommends that powdered infant formula should be reconstituted with water at temperatures > C to reduce microbial risks, and that such feeds should be used within 2 h of preparation. Surveys reported isolation of Cronobacter spp. from a diversity of foods including cheese products, meat, rice and other grains, vegetables, herbs and spices, fermented breads, poultry, ultra-heat-treated milk, spoiled tofu and kefir (Iversen and Forsythe, 2; Friedemann, 2). 2

36 2. Microbial ecology of enteric bacteria on fresh produce 2.. Sources of contamination Contamination of fruits and vegetables with pathogenic microorganisms such as some enterobacteria is possible during growth of the crops. Contamination before harvest can occur from the soil, organic matter, fertilization of farmlands with untreated animal wastes, spray or canal irrigation of growing fields with contaminated water, infected wildlife such as insects or other animals and human contact. Figure 2. gives an overview of the possible contamination routes of fruit and vegetables with human enteric pathogens. On the other hand, contamination can also be the result of postharvest practices, including rinsing or cooling of harvested products with contaminated water, trimming, and multiple manual handling of fruit and vegetables during processing, and packaging (Hotchkiss and Banco, 992; Hurst and Schuler, 992; Bastos and Mara, 99; Beuchat, 99). Figure 2.: Schematic illustration of factors that can contribute to the contamination of fruit and vegetables with human enteric pathogens in the field (Brandl, 2). 22

37 2... Preharvest contamination Fruits and vegetables can become contaminated in the field during growth (figure 2.). The microflora may arise from inside the plant, such as the seed or tuber, or from the environment. Seeds can be a source of foodborne pathogens. Thus, the first leaves emerging from contaminated seeds will also be contaminated (Maud, 983; Nguyen-The and Carlin, 2). For example, Morris and Lucotte (993) reported a total population of 3 cfu/cm 2 on the first leaves of a green endive plant. Contamination occurs mostly on the surface of plants, although in some fruits and vegetables, the inner tissues may be invaded in the early stages of fruit development (ICMSF, 998). Domestic animals may disseminate spoilage or pathogenic organisms ingested with plant fodder (Nguyen-The and Carlin, 2). Insect activity is encouraged by the presence of decaying organic matter remaining in fields, and the insects may disseminate microorganisms to other crops, resulting in contamination (Lund, 983). Fruit and raw vegetables might also be contaminated with faeces from wild animals or from the use of animal or human wastes as fertilizer, and may constitute a health risk (DeBoer et al., 98). The use of untreated organic fertilizers and direct application of human fecal material to growing crops may result in contamination with pathogens (ICMSF, 998). Several reports of pathogen contamination of fruits indicate manure from grazing cattle as an important source of pathogens, like Salmonella and Escherichia coli O:H (Tauxe et al., 99). Furthermore, wind may also transfer dust contaminated with bacterial spores to the surface of plants. The use of wastewater or water polluted with fecal material can also be a cause of contamination. During an evaluation of methods for irrigating crops, Sadovski et al. (98) recorded fecal coliform counts of 3 cfu/g on cucumbers taken from sewage-irrigated plots. They found that the irrigation methods can significantly influence the extent of contamination (Sadovski et al., 98). Information about bacterial indicators of fecal contamination on raw vegetables is abundant. For instance, E. coli, and mainly Enterobacter aerogenes, E. amnigenus, E. sakazakii, and Klebsiella pneumoniae were identified on carrots (Torriani and Massa, 99). 23

38 2..2. Postharvest contamination Fresh produce can also be contaminated during harvest or postharvest operations, contamination with pathogens can occur due to handlers with poor personal hygiene or from polluted wash water, work surfaces, packaging crates or pallets and trucks during transport (Lund, 992; ICMSF, 998). Improper hygiene practices may influence the microbial safety of produce during harvest. Washing the harvested crop can increase microbial populations, particularly if wash water is not clean (ICMSF, 998). During harvest and postharvest storage, microbial numbers are believed to be influenced by the temperature, the hygiene of storage, transport facilities and the degree of damage of the produce during harvest (Lund, 992; Nguyen-The and Carlin, 99; ICMSF, 998; Brackett, 999; Nguyen-The and Carlin, 2). Contamination of fruit and vegetables and related products might also occur in a food processing plant. The main sources of contamination during the processing of fruits and vegetables are most probably the general factory environment and the processing equipment. A study conducted by Garg et al. (99) during the processing of vegetables such as cabbage, lettuce and onions established that the shredders and slicers were major sources of contamination. Other studies have suggested that biofilm formation on processing equipment may provide contamination points (Carmichael et al., 999) Mechanisms of attachment and colonization Attachment is a prerequisite for colonization and subsequent transmission of pathogens via the edible parts of plants. Colonization of host plants by enteric bacteria can be epiphytally (on external surfaces) and endophytally (in internal tissues). Human pathogens interact with plants/produce by more than simply physical and nonspecific ways. Thus, the number and type of microorganisms present on freshly harvested fruit depend on the weather, the season, the time of harvest within a season, the type of fruit and its proximity to the ground, irrigation, and preharvest treatment (Postmaster et al., 99; Jha et al., 2). The number and type of bacteria also vary among the various kinds of vegetables; counts are also extremely variable among samples from the same vegetables (Garcia-Villanova Ruiz et al., 98). This variability may reflect the great diversity of conditions prevailing during 2

39 cultivation and postharvest life of vegetables (Senter et al., 98). Table 2. shows the different mechanisms of attachment and colonization of fruits and vegetables used by enteric bacteria. Table 2.: Colonization of fresh fruit and vegetables by enteric pathogens (Critzer and Doyle, 2). Sources of enteric foodborne pathogens Mechanisms of attachment for epiphytic colonization Mechanisms of endophytic colonization Contaminated water Feces Contaminated manure/compost Contaminated soil Insects Contaminated seeds Biofilms Fimbriae Flagella Natural openings (stomata) Damaged tissue of rhizosphere or phyllosphere Chemotaxis to metabolites within plants or found in plant exudates Enteric pathogens as epiphytes Specific mechanisms are involved in colonization of external surfaces of fruit and vegetable plants. The mechanisms of adhesion differ among the different species and serovars of enteric bacteria and also depend greatly on the type of host plant (Berger et al., 29). The process whereby bacteria interact with plant surfaces is mediated by attachment factors. Extracellular structures such as multiple types of fimbriae, pilus curli, flagella, and bacterial polymers such as O antigen capsule, poly-β-,-n-acetyl-d-glucosamine, colanic acid, cellulose, capsular polysaccharide and lipopoly-saccharides represent the major known attachment factors in enteric bacteria (Jeter and Matthysse, 2; Barak et al., 2; Barak et al., 2; Wang et al., 23; Giron et al., 99; Muller et al., 99; Utsunomiya et al., 992; Malcova et al., 28). Attachment factors are encoded by specific genes in enteric pathogens. Nevertheless, deletion of a specific gene encoding for an attachment factor does not prevent attachment completely, indicating that other adhesins likely play a role (Barak et al., 2; Jeter and Matthysse, 2). It was found that E. coli O:H utilizes some mechanisms to attach to the surface of spinach leaves, roots of alfalfa sprouts and tomato skins. These mechanisms include 2

40 adhesion mediated by curli (Jeter and Matthysse, 2), and in some cases by the filamentous type III secretion system (T3SS) (Shaw et al., 28). Adhesion of enteroaggregative E. coli (EAEC) to epidermis is reported to be mediated by pilus (Berger et al., 29) while aggregation around the stomata is mediated by flagellae. On the other hand, one study reported that some strains of enterotoxigenic E. coli (ETEC) use flagella as the main adhesin to attach to the epidermis of lettuce leaves (Shaw et al., 2). Studies realized with E. coli O:H on lettuce reported that this serotype can attach to different regions of lettuce leaves by means of hydrophobic interactions, surface carbohydrates (capsular polysaccharide, lipopolysaccharides), neutralization of ionic charges or bridging of anionic moieties by divalent cations (Hassan and Frank, 2; Hassan and Frank, 2). Flagella have also been identified as a mechanism by which pathogens can attach to produce surfaces. An increase in expression of flagellin-encoding gene (flic), was observed in E. coli O:H when inoculated on Romaine lettuce and incubated at C (Carey et al., 29). In case of Salmonella, different mechanisms of attachment were observed depending on the serovar. The most frequent mechanisms include adhesion by curli (encoded by agfb), O antigen capsule (encoded by yiho) and cellulose synthesis (encoded by bcsa) (Barak et al., 2; Barak et al., 2). Flagellae also play an important role in adherence of some serovars (Berger et al., 29). Curli and cellulose have been consistently found to form a cellular matrix, which allows biofilm formation (Jonas et al., 2). Biofilm formation on plant surfaces may enable foodborne pathogens to survive in the harsh phyllosphere and may decrease the efficacy of commonly used sanitizers (Danhorn and Fuqua, 2). Another study confirmed the capability of S. enterica to survive and grow on tomato plants and fruit (Guo et al., 22). Salmonella Montevideo has been reported to survive better at the stem scar and in growth cracks on tomato skin, and multiply on puncture wounds and on tomato slices at 2 C (Wei et al., 99). In apples, pathogenous bacteria attach on the stem and calyx cavity areas and on the skin (Liao and Sapers, 2). Studies suggested that synthesis of cellulose by Salmonella during produce colonization may partly explain why treatment of produce with chlorine does not eliminate pathogens completely (Beuchat et al., 2; Felkey et al., 2; Weissinger et al., 2). 2

41 Enteric pathogens as endophytes Foodborne pathogens usually colonize plant areas where water and nutrient content may enhance survival or growth and, where they are protected from UV light (Lund, 992). Endophytic invasion includes movement of enteric bacteria into intercellular spaces of tissues between host cell walls but not inside the vegetal cells (Dong et al., 23). This process seems to be genetically mediated and depends also of external conditions. Thus, not all serovars of a bacterial species have the ability to internalize in leaves, roots, stems and hypocotyls. This behaviour has been observed in E. coli O:H and Salmonella (Zhang et al., 29; Mitra et al., 29; Dong et al., 23). Internalization of enteric pathogens in fruit and vegetables can occur through natural openings such as stomata, pores, lenticels or damaged regions of the phyllosphere (Sapers, 2). Physical damage during harvest or handling, and exposure to stress conditions increase the susceptibility of the fruit. In tomatoes, bacteria were most frequently found in the stem scar and central core, and there was evidence that the bacteria could enter the fruit in the vicinity of the sepals (Shi et al., 2; Xuan Guo et al., 2). Viable bacteria have been detected in the interior tissues of cucumbers and tomatoes, locations that had been assumed to be sterile (Lund, 992). Internalization through stomata is controlled by guard cells. Thus, plants can sense bacterial surface molecules and trigger stomatal closure. However, this was not oberved during Salmonella contamination, indicating that guard cells do not recognize Salmonella and do not trigger this innate response (Kroupitski et al., 29). On the other hand, it was also observed that stomatal openings alone seem not to promote internalization of Salmonella in iceberg lettuce. Photosynthesis is implicated in driving chemotaxis of Salmonella towards stomatal openings where sugars produced during photosynthesis may serve as chemoatractants (Kroupitski et al., 29) Prevention of contamination and sanitation methods for fresh produce Contamination of fresh produce with pathogenic bacteria can occur in different stages of the food production chain. Food producers try to avoid contamination through implementation of good agricultural practices (e.g. control of irrigation water, manure quality, field and facility sanitation, packing, and transportation), washing and sanitizing treatments, good manufacturing practices (e.g. hygiene practices of food handlers, use of 2

42 non-contaminated water, etc.) and plant sanitation. Freshly harvested fruit and vegetables are generally washed to remove microorganisms from the surface. Washing with pure water achieves a small reduction (about log) in microbial numbers (Nguyen-The and Carlin, 99; Cherry, 999). Chlorinated water is widely used for washing and sanitizing minimally processed fruits and vegetables (Ahvenainen, 99), although the effect of chlorine on microorganisms attached to surfaces is limited. The efficacy of disinfection treatments depends on the disinfectant used, its concentration, the time taken to disinfect, the temperature during disinfecting, the type of commodity and the anticipated microbial load (Dychdala, 99; White, 998). The use of chlorine for washing fruits and vegetables is regulated and does not have to exceed.2% when followed by a potable water rinse (FDA-CFSAN, 2a,b; FDA, 2a). Chlorine is less effective for inactivating bacteria attached to produce surfaces or embedded within the product (Zhuang et al., 99; Wei, et al., 99; Beuchat, et al., 998; Sapers et al., 999). Chlorine dioxide is considered to be efficacious against many classes of microorganisms (Dychdala, 99). This sanitizing agent is approved by the FDA for use on fresh produce, but it is not permitted for use on fresh-cut products (FDA, 2a). Also, the effect of chlorine dioxide depends on the type of produce (Lukasik et al., 23; Costilow et al., 98). Ozone is effective in killing food-related microorganisms (Restaino et al., 999), and has been approved for use on foods (FDA, 2b). Ozone is effective in reducing bacterial populations in wash water and may have some applications as an alternative for chlorine to reduce microbial populations on produce (Kim et al., 999). Hydrogen peroxide is a highly effective antimicrobial agent against bacteria (Block, 99). The efficacy of dilute hydrogen peroxide was demonstrated in sanitizing fresh produce including apples (Sapers et al., 2; Sapers et al., 22), melons (Sapers et al., 2; Ukuku and Sapers, 2), eggplant, and sweet red pepper (Fallik et al., 99). In comparison, concentrations of hydrogen peroxide washes ( to %) were at least as effective as 2 ppm chlorine (Sapers et al., 999; Sapers and Sites, 23). Finally, organic acids, such as lactic and acetic acid, are also effective antibacterial agents (Foegeding and Busta, 99). Lactic acid rinses might have applications for the decontamination of fruit and vegetables. They are classified by the FDA as GRAS (FDA, 98; FDA, 22). 28

43 2.. Behaviour of some pathogenic enterobacteria on fresh produce Contamination of fruit and vegetables by human pathogens can occur anywhere in the food production chain including contamination of seed stocks, during production, harvesting, postharvest handling, storage, processing, distribution and retail display. Produce contaminated with human pathogens can not be completely disinfected by washing or rinsing the product in an aqueous solution, and low sporadic levels of human pathogens can be found on produce (Brackett, 98; Seo and Frank, 999). In the following sections the behaviour of several pathogenic enteric bacteria on fresh produce will be discussed in more detail Pathogenic Escherichia coli Data on the incidence of EPEC, EIEC, ETEC and VTEC in fruit and vegetables are very incomplete. Information about survival of enterobacteria into inoculated fruit and vegetables are available in some extent. One important survey was realized in 22 by the USDA Microbial Data Program, which analyzed a total of.3 samples of five raw agricultural commodities: cantaloupe, celery, leaf lettuce, romaine lettuce, and tomatoes. Samples were collected in commerce at wholesale and/or distribution centers. Results showed the presence of E. coli with a virulence factor in cantaloupe (.9%), celery (.%), leaf lettuce (.2%), romaine lettuce (.3%), and tomatoes (.%) (USDA- MDP, 22). Another survey realized in mixed raw vegetables and in herbs, such as cilantro and coriander, showed that 9 2% of the samples were contaminated with pathogenic E. coli O:H (Beuchat, 99; Nguyen-The and Carlin, 2). E. coli O:H has also been isolated from raw melon (Del Rosario and Beuchat, 99) and fruit juices like unpasteurised apple juice (CDC, 99; Cody et al., 999; Singh et al., 99). The most important factors reported for survival and growth of pathogenic E. coli are ph, temperature and water activity. The low infectious dose of E. coli O:H implies that growth of this organism in food may not be a prerequisite for disease. Studies reported acid tolerance of VTEC, EPEC and EIEC strains, which refers to their ability to survive a stress condition provoked by low ph values. Because of its acid tolerance, E. coli O:H poses a higher risk in low ph fruits and fruit juices than other foodborne pathogens (Zhao et al., 29

44 993; Janisiewicz et al., 999). For instance, the cell count of E. coli O:H remained almost the same when inoculated into samples of ground apples with different ph values (from 3. to.9) and stored at different temperatures ( C, C and 2 C) during 8 days (Fisher and Golden, 998). In apple cider (ph ) stored at C, numbers of E. coli O:H declined from. to 2. log cfu/g in two weeks (Roering et al., 999). Less acid fruits such as cantaloupe and watermelon favour the growth of E. coli O:H (Del Rosario and Beuchat, 99). Temperature is another factor that influences the growth of pathogenic E. coli. E. coli O:H can multiply rapidly between 3 C and 2 C (Doyle and Schoeni, 98). However, growth has also been observed between. C and.2 C under laboratory conditions (ACMSF, 99). Regarding to water activity, E. coli O:H is able to grow at a w of.9 (Glass et al., 992). Infection outbreaks related to consumption of contaminated fruit and vegetables and related products are scarse. Several outbreak investigations indicated that E. coli O:H infections had been associated with the consumption of acidic foods that generally had been regarded as low risk products (Besser et al., 993, CDC, 99). An outbreak with VTEC was reported after consumption of unpasteurized apple juice (Besser et al., 993). This drink, because of its acidity, was previously not considered particularly hazardous. It appeared that the apples were contaminated with cow manure in the orchard. Many other cases of VTEC infection caused by drinking unpasteurized commercial apple juice were also observed (CDC, 99; Cody et al., 999). Around outbreaks due to consumption of unpasteurised apple juice were reported in the USA in 99 (Hilborn et al., 2). Nine cases of infection caused by E. coli O:H were also reported in Oregon-USA in 993 due to consumption of cantaloupe melon, the fruit was possible contaminated with raw beef (Del Rosario and Beuchat, 99). Severe infections (2 cases) caused by E. coli O:H were reported in Japan in 99 due to consumption of radish (Taormina et al., 999). Enterotoxigenic E. coli (ETEC) were also responsible for infection ( cases) due to consumption of fresh orange juice sold by roadside vendors in India (Singh et al., 99). Vegetables and salads were also involved in infections with E. coli O:H, outbreaks of diseases were related to consumption of raw lettuce, shredded carrots and, salads made from iceberg and romaine lettuce, endive, and shredded carrots (Ackers et al., 998; CDC, 99; Beuchat, 99). 3

45 2..2. Salmonella The widespread occurrence of Salmonella spp. in the natural environment, coupled with the intensive husbandry practices used in the animal industry and with the use of contaminated water in irrigation of vegetable and fruit crops during farming have favored the continued prominence of this bacterial pathogen in the global food chain (Minor, 992; Portillo, 2; D Aoust et al., 2). Most reports about incidence of Salmonella spp. were dedicated to raw and processed meat products. Although fruit and vegetables are less frequently implicated with Salmonella spp., fresh produce is nowadays considered as a potential vehicle for human diseases, such as salmonellosis. The presence of Salmonella in raw fruits is often reported. Fruits commonly contaminated with this pathogen include cantaloupes, oranges and strawberries (FDA-CFSAN, 2a; Pao and Brown, 998; Madden, 992). Vegetables like lettuce, cabagge, cauliflower, celery and spinach and mixed raw vegetables are also involved in contamination with Salmonella (Rude et al., 98; Garcia-Villanova et al., 98; FDA-CFSAN, 2a,b; Little et al., 999). Salmonella can multiply on the surfaces of fruits such as tomatoes (Wei et al., 99; Zhuang et al., 99) and cut melons (Escartin et al., 989; Golden et al., 993) and on fresh vegetables manually or mechanically moisturized during their retail display at ambient temperature (Brackett, 992). The temperature, ph and water activity of the environment can influence the growth kinetics of salmonellae. Salmonella has been reported to grow in the temperature range of 2 C to C (D Aoust, 99; Droffner and Yamamoto, 992a; Droffner and Yamamoto, 992b). The minimum temperature for growth prevails at neutral ph and increases sharply as ph deviates from neutrality (Matches and Liston, 92). Salmonella can survive at C without significant decrease in cell number when inoculated in fresh-cut fruits such as cantaloupe and honeydew (Golden et al., 993). Studies realized to evaluate bacterial growth in a natural medium (tomatoes) and in a defined medium with different concentrations of different acids showed that Salmonella spp. possess a great adaptability to extreme environmental conditions due to their ability to proliferate at ph values ranging from 3. to 9.. It was suggested that the concentration of inocula, the type of bacteria and also the acid molecule itself are the most important factors that contribute to the adaptability (Asplund and Nurmi, 99; Chung and Goepfert, 9; Ruzickova, 99). 3

46 Laboratory reports also revealed that Salmonella can grow in damaged, chopped or sliced tomatoes (ph..) stored at 2 C to 3 C (Harris, et al., 23). Foods with a w values of.93 and neutral ph do not support the growth of salmonellae (Portillo, 2; Jay et al., 2). The proliferation at a w >.93 varies between strains and depends on the composition of the food (D Aoust, 989). Approximately, cases of salmonellosis per year were reported in the USA according to the Center for Disease Control and Prevention (CDC, 28), data of illnesses or outbreaks of foodborne salmonellosis in other countries are scarse. The CDC estimates that Salmonella infection causes approximately. million foodborne illnesses annually (Lynch et al., 2). Among bacterial pathogens, Salmonella Enteritidis accounts for the largest overall number of foodborne illnesses and outbreaks (CDC, 28). Several fruits, including tomatoes and cantaloupe, have been implicated in Salmonella-related human illnesses (Minor, 992, USDA-MDP, 2). Many cases of infection caused by consumption of fresh-cut melon, watermelon or cantaloupe contaminated with Salmonella were reported in countries such as the USA and Canada, (Lund and Snowdon, 2; Blostein, 993; Mohle-Boetani et al., 999). The most probable causes were crosscontamination, infection by food handlers and contamination during production or harvest. Fourteen cases of infection caused by Salmonella Enteritidis due to consumption of unpasteurized citrus juice obtained at retail and food services were also revealed in the USA (Butler, 2). Consumption of raw tomatoes in the USA in 99 and 993 caused infection with Salmonella serovars (Tauxe, 99; Beuchat, 99; Lund and Snowdon, 2; Wei et al., 99) Shigella The major routes for contracting shigellosis are person-to-person contact and ingestion of contaminated water or food (Lampel et al., 2). Fresh raw vegetables and vegetable salads are good vehicles for spreading shigellae from infected food handlers to unsuspecting consumers (Smith, 98); contact with contaminated equipment, inadequate cooking, and unsafe food sources are other contributing factors. Incidence of Shigella in raw cantaloupe was reported by FDA-CFSAN (2a,b), these fruits were imported from the USA, samples of nine countries were collected and 2% reported presence of this 32

47 pathogen. A survey realized on green and mixed salads collected from retail and food services also reported contamination with Shigella spp.,.8% of the samples contained this pathogen (Saddik et al., 98). Vegetables and herbs are often implicated with Shigella spp., presence on lettuce, celery, parsley and green onions was reported (Martin et al., 98; Kapperud et al., 99; FDA-CFSAN, 2a,b). Detection of S. dysenteriae, S. boydii and S. flexneri in unpasteurized, freshly squeezed orange juice was also reported (Castillo et al., 2). The ability of shigellae to survive in fruit and vegetables depends on a number of factors. The temperature at which the produce is held, the ph, and the presence of organic acids are critical for the survival of this pathogen. Shigellae can survive over a range of temperatures. In acidic foods, such as apple juice, Shigella spp. survived for up to days stored at C (Bagamboula et al., 22). S. flexneri was also tested for survival in grape juice. After 8h, a reduction of three logarithmic units was observed at high inoculum size ( cfu/ml) (Nicolo et al., 2). S. flexneri survival was also tested in several vegetables, either sterile or non-sterile. In either case, the level ( to cfu/g) remained constant for several days at both ambient and refrigerated temperature (Rafii et al., 99). Reports showed that S. flexneri and S. sonnei can grow from 2. to 9. log cfu/g in hours in fresh-cut watermelon when incubated at 2 C (Fredlund et al., 98). Similar studies were realized on papaya cubes at ph.; S. sonnei, S. flexneri and S. dysenteriae grew from 2. to.2 log cfu/cube when stored at 2 2 C during hours (Fernandez Escartin, 989). Shigella can survive at room temperature up to days in acidic foods, e.g. orange and tomato juice. Acid resistance helps to explain one of the unique characteristics of Shigella pathogenesis, the ability to cause infection and illness with a very small inoculum (Jennison and Verma, 2). It was also showed that Shigella spp. are more acid tolerant than are Salmonella spp. and E. coli. The high acid tolerance of Shigella spp. may contribute to their relatively low infective dose (Gorden and Small, 993). Several types of food have been associated with outbreaks of shigellosis; contaminated raw vegetables are a leading source (FDA-CFSAN, 2a,b). In 999, of the, cases of Shigella infections causing illnesses reported at FoodNet Surveillance sites in the USA, % were due to S. sonnei and 29% due to S. flexneri (CDC, 999). In 983, S. sonnei was related to gastroenteritis outbreaks associated with consumption of lettuce saldas in two 33

48 university campuses in Texas, USA (Martin et al., 98). Another outbreak of shigellosis occurred after consumption of lettuce. The coliform count found in these samples was up to 8 cfu/g indicating fecal contamination, most likely due to irrigation practices using poorly treated sewage water (Kapperud et al., 99). Similar cases of infection with S. flexneri and S. sonnei were observed after consumption of green onion and parsley, contamination during harvest and use of contaminated water were implicated (Tauxe, 99; CDC, 999). A few outbreaks of shigellosis caused by consumption of fruit juices were reported. Orange juice was considered to be vehicle for transmission of shigellosis among visitors to a South African reserve, the pathogen implicated was S. flexneri (Thurston et al., 998) Yersinia enterocolitica Yersinia enterocolitica is considered as a foodborne pathogen mostly related to raw pork meat and pork products. Pathogenic strains are frequently isolated from the oral cavity, especially from the tonsils, submaxillary lymph nodes, intestines and faeces of pigs (Lee et al., 99; Ostroff et al., 99; Tauxe, 98; Fredriksson-Ahomaa and Korkeala, 23). Fruit and raw vegetables might be contaminated with Y. enterocolitica during pre- and postharvest processes. By using real-time PCR, this species was detected in grated carrots and lettuce (Fredriksson-Ahomaa and Korkeala, 23; Thisted-Lambertz et al., 28). A survey realized in minimally processed lettuce purchased from retail supermarkets or provided by a salad production facility over an 8-month period reported contamination of about 9% of samples with Y. enterocolitica (Szabo et al., 2). Yersinia contamination was also reported when prepacked salad vegetables and mixed raw vegetables (lettuce, spinach, watercress and chicory) were analyzed, in most cases more than % of analyzed samples were positive to this species (Brocklehurst et al., 98; Dos Reid Tassinari et al., 99; Catteau et al., 98; Beuchat, 99; Darbas et al., 98). Several factors are important for growth and survival of pathogenic Yersinia species. An important property of this genus is its ability to multiply at temperatures near to C and, therefore, in many chilled foods. Members of the genus Yersinia are psychrotrophic organisms and some strains of Y. enterocolitica can even grow at temperatures as low as C, although growth is very slow below C (Bergann et al., 99). The ability of Y. 3

49 enterocolitica to multiply at low temperatures is of considerable concern to food producers. The optimum growth temperature for Yersiniae is 28 C 29 C (Bercovier and Mollaret, 98; Gill and Reichel, 989; Bottone et al., 2). Yersinia species can grow in a ph range of.-.. The optimum ph for growth is.2-. (Bottone et al., 2). The minimum ph for growth has been reported as being between.2 and. (Kendall and Gilbert, 98), but the presence of organic acids will reduce the ability of Y. enterocolitica to multiply at low ph (Brocklehurst and Lund, 99). Results showed that, in a food with a neutral ph stored at C, Y. enterocolitica counts may increase from ml - to 2.8 x ml - in days. Y. enterocolitica and Y. pseudotuberculosis emerged as important agents of foodborne gastroenteritis outbreaks in the USA and Japan, respectively (Bottone, 99; Vincent et al., 2). Y. enterocolitica and Y. pseudotuberculosis infections in humans are usually acquired by fecal-oral spread via ingestion of contaminated food products or water. Globally, fresh produce has increasingly been identified as a source of outbreaks of different foodborne pathogens. In Japan, vegetable salads containing apples, cucumbers, ham, potatoes, carrots, and mayonaise were implicated in infections caused by Y. enterocolitica in a nursery school (Sakai et al., 2) Cronobacter sakazakii In general, powdered infant formulae products have been identified as common and highrisk foods for the growth of Cronobacter sakazakii (Farmer et al., 98). Fruit and vegetable products were also reported to be contaminated with Cronobacter spp. A survey realized on different food products to detect the presence of Cronobacter spp. reported incidence of C. sakazakii, C. turicensis, and C. muytjensii in of 2 tested samples of fresh vegetables. Fresh fruit and derived products did not indicate contamination with these bacteria (Turcovsky et al., 2). Similar results were also found during analysis of mixed salad vegetables, fresh lettuce, fruit powder, and deep-frozen vegetables at retail level (Geiges et al., 99; Osterblad, et al., 999; Galli et al., 99; Francis and O'Beirne, 998; Soriano et al., 2; Lehner et al., 2). In another survey Cronobacter spp. were isolated from of 23 samples of sprouts and fresh herbs/salads (Baumgartner et al., 29). 3

50 Published information about factors affecting its growth and survival is most related to powdered formulae. Nevertheless, Cronobacter can be present in a wide spectrum of food and food ingredients. C. sakazakii is able to grow in the range of. C, with an optimum at 39. C (Kandhai et al., 2). The generation time is hours at C, min at 23 C (Lambert and Bidlas, 2) and 2 min at optimum temperature. The minimum ph for growth is 3.89 (Lambert and Bidlas, 2) and the optimum between 9. Regarding to water activity, the maximum salt concentration permitting growth is 9.% (Lambert and Bidlas, 2). C. sakazakii can survive at freezing temperature and, also at ph 3. at 3 C (Edelson-Mammel et al., 2). This pathogen can also survive under dry conditions (a w =.2) (Gurtler et al., 2; Beuchat et al., 29). Temperatures of pasteurization can easily destroy it (Nazarowec-White and Farber, 99b; Breeuwer et al., 23; Iversen et al., 2). Acidification reduce the concentration of C. sakazakii in vegetable-based food products (Joosten and Lardeau, 2; Richards et al., 2; Coulin et al., 2). In juices of vegetables, the reduction of ph after 8 h was correlated with a reduction of the numbers of C. sakazakii, but with increasing numbers of C. sakazakii in juices of different fruits (Kim and Beuchat, 2). C. sakazakii produces a protective capsule and also biofilm (Zogaj et al., 23; Iversen et al., 2; Lehner et al., 2), these mechanisms protect them against UV-light treatment, high osmotic pressure, heat, dry conditions and acids (Lehner et al., 2; Iversen et al., 2). Other foods than powdered infant formulae, such as fruit and vegetables or related products, are not known to cause C. sakazakii infections. Some strains of C. sakazakii have been shown to produce an enterotoxin (Pagotto et al., 23), however, its relevance to pathogenesis has not been established yet. The infectious dose relationship for humans has not been clearly determined yet. The minimum infectious dose of Cronobacter spp. for infants is extrapolated from animal models. It was estimated that high levels of the organism (> cfu/feeding) are necessary to cause illness (Pagotto et al., 23). On the other hand, FAO/WHO (2), has determined tentatively the oral infectious dose for human neonates ranging from 3 to 8 cfu. 3

51 2.. Importance of the ph and organic acids in fruit (juices) and vegetables The ph of many fruits is lower than. and this low ph, combined with the presence of organic acids, generally prevents the multiplication of foodborne pathogenic bacteria. Nevertheless, these bacteria may survive for a sufficient time to cause foodborne disease. Table 2.2 shows the ph values and the main organic acids present in some fruits and vegetables. Table 2.2: ph values and main organic acids found in some fruit and vegetables. Produce ph Organic acids Source Vegetables Cabbage (green).2.3 citric, malic, Lund, 992; Sousa et al., 2 ascorbic Asparagus.. citric, malic, oxalic Lund, 992; Bhowmik et al., 2 Cucumber.. malic, citric, oxalic McFeeters et al., 982. Carrots.9.3 citric, malic, quinic Lund, 992; Schaller and Schnitzler, 2 Potatoes..2 citric, malic, oxalic, ascorbic Lund, 992; Bushway et al., 98. Peppers (several types).. citric, malic, oxalic, quinic Lund, 992; López-Hernandez et al., 99. Fruits Grapefruit citric Splittstoesser, 99 Grape 3.. tartaric, malic Patil et al., 99 Orange 3.. citric, malic Splittstoesser, 99 Kiwifruit 3.. citric, malic, quinic Luh and Wang, 98 Peach 3..2 malic, citric Splittstoesser, 99 Pineapple 3.2. citric, malic Splittstoesser, 99 Strawberry citric, malic Deuel, 99 Apple 3.3. malic, citric Splittstoesser, 99 Mango citric, tartaric Splittstoesser, 99 Pear 3.. malic, citric, quinic Splittstoesser, 99 Tomato, ripe.2. malic, citric, glutamic Watermelon.8. citric, malic Splittstoesser, 99 Melon, cantaloupe.2. citric, malic Splittstoesser, 99 Pinheiro and Almeida 28; Davies, 9 The optimum ph for growth of microorganisms associated with fruit and vegetables is in the range.., but some can multiply in highly acidic conditions. This is the case for spoilage yeasts, such as Candida, Pichia, Rhodotorula, Torulopsis, Saccharomyces, Zysossaccharomyces and Hansenula (Renard et al., 28), acetic acid bacteria: Acetobacter aceti and Acetobacter pasteurianus (Worobo and Splittstoesser, 2), lactic 3

52 acid bacteria: Lactobacillus fructivorans and Leuconostoc spp. (Winniczuk and Parish, 99), obligatory acidophilic bacteria such as Alicyclobacillus acidoterrestris (Walker and Phillips, 2) and some molds such as Byssochlamys fulva and eosartorya spp. (Olliver and Rendle, 93). In general, bacteria show a lesser ability to grow at acid ph. For instance, if watermelons (ph.8-.) are sold as cut pieces, any foodborne pathogen that is present may multiply on the cut surfaces of this fruit. Salmonella spp. and Shigella spp. have been shown to multiply on the cut surface of watermelons, honeydew melons and papaya fruits at 23 C to 2 C (Escartin et al., 989; Golden et al., 993). Moreover, initiation of bacterial soft rot by Erwinia spp. may result in conditions that favor survival and growth of Salmonella in or on fruit (Wells and Butterfield, 99). Organic acids are naturally present in fruit and vegetables. The presence and amount of organic acids depend on the type of fruit or vegetable. Acetic, citric, succinic, malic, tartaric, benzoic and sorbic acid are the most important organic acids that naturally occur in fruit and vegetables (Beuchat, 998). The main organic acids are showed in figure 2.2. In vegetables the major abundant organic acids are malic and citric acid, but others, such as oxalic, ascorbic, fumaric and quinic acid, can also be found in lesser amounts (Ruhl and Herrmann, 98; Haila et al., 992; Nisperos-Carriedo et al., 992). The amount of organic acids and the ph value of fruit and vegetables influence their susceptibility to microbial growth. Organic acids, like acetic or formic acid, can also be produced by fermentation (Clark, 989; Becker et al., 99; Buckel, 999). Citric acid Malic acid Tartaric acid Ascorbic acid Oxalic acid Quinic acid Figure 2.2: Chemical structure of the main organic acids found in fruits and vegetables. 38

53 Many fruit juices are highly acidic. The ph of most fruit juices ranges from 2.. (Hicks, 99). Hexose sugars constitute the largest soluble fraction of fruit juices. The main sugars in fruit juices are fructose, glucose and sucrose. Besides sugars, acids constitute the second largest constituents of fruit juices. Concentrations and types of acids present in juices are characteristic of the fruit. Malic acid predominates in apples, cherries and plums; citric acid in citrus fruits (e.g. orange, lemon and grapefruit) and tartaric acid is characteristic of grape juices. The concentrations of acids in fruit juices are usually of the order of % (Fry, 99). The majority of bacteria will not grow in acidic media. Many are supposed to be rapidly killed in such an environment, but a few are able to survive and grow at low ph (Booth and Kroll, 989). Due to the acidity, pathogenic bacteria are rarely found in fruit juices. This has led to the mistaken assumption that acidity causes rapid death of all bacterial pathogens, and to the idea that less risk for infection exists as juices are consumed without preservative treatment. Some reports indicated the presence of Escherichia coli O:H in fruit juices. E. coli O:H is acid tolerant, showing slight growth in apple juice (ph 3..) in large inocula ( ml - ) at 8 C (Zhao et al., 993). At small inocula ( 2 ml - ), viability was maintained for 2 days and was lost gradually over the next 3 weeks. Research has shown that Salmonella spp. can survive and grow in acidic media, down to ph. (Baik et al., 99; Foster, 99). Salmonella has been shown to remain viable in apple juice at ph 3. for 3 days at room temperature (Dingman, 999). Furthermore, the acid-tolerance response (ATR) of Salmonella Typhimurium has been shown to provide protection against organic acids (Baik et al., 99) The effect of ph on the growth of microorganisms The ability of bacteria to grow and survive at acid ph depends on factors such as temperature, medium composition and mainly on the number of the microorganisms in the inoculum (Lund et al., 98). Bacteria may survive in conditions of low ph, and although growth may have stopped, the cells may still be metabolically active. The energy requirements of a microorganism in a low ph environment are greater than the energy required at optimal ph values (Russel and Cook, 99). This is because an energyrequiring proton pump is in use, with protons being pumped out of the cell. In high ph environments, protons may be pumped into the cell. If the ph is not balanced, the cell is 39

54 unable to synthesize normal cellular components and is unable to divide and grow (Booth and Kroll, 989; Brown and Booth, 99). Strong acids, such as HCl, lower the external ph but are not able to permeate through the cell membrane. These acids exert their antimicrobial effect by denaturing enzymes present on the cell surface and by lowering the cytoplasmic ph due to increased proton permeability when the ph gradient is very large. This can result in a reduced growth rate and can cause an extension of the lag phase (Cheroutre-Vialette et al., 998). ph affects lag time and the rate of microbial growth or death. The effect of ph on the lag time will depend on the previous history of the inoculum (growth phase, growth medium, ph, and temperature of propagation) (George and Lund, 992). There is an approximately linear inverse relationship between growth rate and hydrogen ion concentration (Presser et al., 99). If cells, growing rapidly at ph., are exposed to ph. for one generation, their ability to survive when exposed to even lower ph values increases. This is known as inducible acid tolerance response (ATR) (Bearson et al., 99). The log-phase ATR appeared to involve two stages: i: synthesis of a ph homeostasis system, i.e., inducible amino acid decarboxylases, and ii: formation of about acid-shock proteins, believed to prevent damage or repair damaged macromolecules (Lin et al., 99; Bearson et al., 99) The effect of organic acids on the growth of microorganisms Organic acids naturally present in fruit and vegetables have antimicrobial activity. The antimicrobial effect of these weak organic acids depends on their pka value, concentration, solubility, molecular weight, water activity and sensitivity of the microorganism (Chung and Goepfert, 9; Pethybridge et al., 983; Eklund, 989; Doores, 983; Kabara and Eklund, 99; Eswaranandam et al., 2; Bloomfield, 99; Denyer, 99; Brul and Coote, 999). The order of increase in inhibitory effect of weak acids is very similar to the order of decrease of their lowest pka values (Chung and Goepfert, 9). From all organic acids present in fruit and vegetables, acetic acid is one of the most important when assessed on the basis of total concentration, which would be expected from its pka value (Doores, 993; Russell and Gould, 999). The antimicrobial activity of weak organic acids is attributed to ph reduction, depression of internal ph of microbial cell by ionization of undissociated acid molecules, and disruption of substrate transport by altering cell membrane permeability or reduction of proton motive force (Jay, 2; Jay et al., 2).

55 Therefore, it is important to separate the effect of ph from that of the organic acid. This latter effect is mainly due to the undissociated form of the weak acid. The effect of organic acids on bacteria may also depend on differences in the ability of these microorganisms to metabolize subinhibitory concentrations of these weak acids. This ability may be accompanied by relatively high resistance to the acids. Even when microorganisms are unable to multiply in acidic conditions, they may be able to survive for a prolonged period of time. In the case of foodborne pathogens, especially those with a low infective dose, this may have important consequences for the food safety. For example, outbreaks of food poisoning have been attributed to the survival of Salmonella spp. in unpasteurized apple cider and in orange juice (Cook et al., 998; Parish, 99) and to the survival of E. coli O in unpasteurized apple juice and cider (Besser et al., 993; CDC, 99) Inhibitory mechanisms of ph and organic acids on microbial growth When bacteria are in direct contact with fresh-cut fruits and vegetables or with related products (fruit juices, vegetable salads, etc.), they are exposed to the effect of the weak organic acids. These acids exert their antimicrobial effect partly by reducing the ph of the environment and partly due to the specific properties of the acid itself. The effects of weak acids include the reduction of the growth rate or even complete inhibition. In principle, growth inhibition can be caused by inactivation of, or interference with, the cell membrane, cell wall, metabolic enzymes, protein synthesis system, or genetic material (Eklund, 989). These effects depend on the concentration of the weak acid (MIC, minimal inhibitory concentration). Weak acids also affect the cell yield, ATP levels, and the ability of the cells to maintain ph homeostasis (Cherrington et al., 99a,b; Denyer, 99). Once inside the cell, weak acids generally encounter a higher ph (due to the cell buffers), dissociate tending to cause accumulation of protons and acidify the cell interior (Roe et al., 998; Cherrington et al., 99a,b; Brown and Booth, 99; Denyer, 99). Weak organic acids can dissociate only partially (AH (aq) A (aq) + H + (aq)). The undissociated form (AH) and protons (H +, decrease in ph) both exert the inhibition effect. The lower the ph value of the food, the greater the proportion of the acid in the undissociated form and thus the greater the antimicrobial effect (Brown and Booth, 99; Sofos and Busta, 98). The undissociated form of weak acids, due to a lack of charge and to its hydrophobic character, can easily diffuse through the cell membrane of microorganisms (Cherrington et al.,

56 99a,b; Denyer, 99). Weak acids generally inhibit essential biochemical reactions by increasing the hydrogen ion concentration, which results in a decrease in intracellular ph and consequently growth inhibition (Brown and Booth, 99). Figure 2.3 shows the interaction of weak acids on the microbial cell. Figure 2.3: Interaction of weak acids on the microbial cell. A) Exterior of the cell favors undissociated weak acid. B) Interior of the cell favors dissociation of the acid. C) Proton pumps remove excess H + ions and use energy (Beales, 2). Due to the low permeability of the bacterial cytoplasmatic membrane, the proton influx from outside to inside of the cell is limited. Thus the difference between the internal and external ph ( ph) is relatively small, and the cytoplasm tends to be maintained at a ph near to neutrality (Booth, 98; Hill et al., 99). Maintenance of the internal ph results partly from the very low permeability of the cell membrane to protons and partly from the buffering capacity of the cytoplasm. Active ph homeostasis in neutrophilic and acidophilic bacteria depends on expulsion of protons through the cell membrane and a corresponding movement of potassium ions into the cell (Hill et al., 99). Extreme lowering of the external ph causes some decrease in internal ph; eventually the difference between the internal and external ph ( ph), reaches a maximum, ph begins to collapse, and the cells will die (Hill et al., 99). 2

57 PART II: EXPERIME TAL WORK

58 3. Material and methods 3.. Material 3... Growth media Several media were used during this thesis to cultivate enterobacteria. All culture media were sterilized by autoclaving for 2 minutes at 2 C and psi Luria Bertani (LB) broth LB broth was prepared by dissolving. g/l tryptone (LAB M),. g/l yeast extract (Oxoid) and. g/l NaCl (Fisher Scientific) in distilled water. Medium was autoclaved as described above. In some cases,.% glucose and/or mm acetoin were added to this growth medium after autoclaving. In some cases, the ph of the medium was adjusted using 3% HCl. mm IPTG was added when this medium was used to cultivate strains containing plasmids M9 minimal medium Stock solution of M9 minimal medium ( x concentrated) was prepared by dissolving.8 g/l Na 2 HPO.2H 2 O, 3. g/l KH 2 PO,. g/l NaCl,. g/l NH Cl. Then, the ph was adjusted to. and this stock solution was autoclaved. The working solution was prepared by diluting this stock solution times. After autoclaving of the working solution,.% of glucose, 2 mm MgSO and. mm CaCl 2 were added. In some cases, mm of specific amino acids (lysine, arginine, glutamic acid or ornithine) or.2%,.% or % of casamino acids were added. In some cases, the ph of the medium was adjusted using 3% HCl. mm IPTG was added when this medium was used to cultivate strains containing plasmids.

59 Fruit juices Several fruit juices (grape, apple, worldshake and nectar multifruit juice) were used in the experiments. These juices were bought in a local supermarket in Leuven, Belgium. They were sterilised by filtration before use. The ph of the fruit juices were adjusted to different ph values with NaOH. mm IPTG was added when the juice was used to cultivate strains containing plasmids. General composition of these fruit juices are shown in Table 3.. Table 3.: General chemical composition of the fruit juices used in this study. Values were obtained from the information given on the package. Values per ml Grape juice Apple juice Worldshake* ectar multifruit** ph Energy kcal kcal 8 kcal 2 kcal Proteins.3 g. g < g,2 g Carbohydrates of which sugar Fat g 9.8 g. g. g, g,3 g < g <, g. g of which saturated. g g <, g Fibres. g. g <.2 g,2 g Sodium. g <. g <.3 g <, g *Worldshake: mixture of orange, apple, pear, grapefruit, banana, red beet. **Nectar multifruit: mixture of apple, orange, guava, mango, apricot, passion fruit, pineapple, banana, papaya, lemon, pear, grapefruit 3... Vegetables Fresh vegetables, such as red pepper (Capsicum annuum) and cucumber (Cucumis sativus), were used in spoilage experiments. Vegetables were obtained from a local supermarket in Leuven, Belgium Media for stock and count plates Luria Bertani (LB) agar LB agar was prepared by dissolving. g/l tryptone (LAB M),. g/l yeast extract (Oxoid),. g/l NaCl (Fisher Scientific) and. g/l bacteriological agar (LAB M) in distilled water. After autoclaving, the media were cooled down to C in a water bath

60 before pouring the plates. When necessary, ampicillin or chloramphenicol was added before pouring in plates to get concentrations of µg/ml and 3 µg/ml respectively Bacterial strains and plasmids All bacteria and plasmids used in the experiments are listed in Table 3.2 and 3.3. Table 3.2: Bacterial strains used in this study, growth medium and optimal temperature of growth. Mixed acid fermenters Bacterial strain Growth Growth medium temperature Escherichia coli MG LB 3 C Escherichia coli MG ptrc99a LB + Ap 3 C Salmonella Typhimurium LT2 LB 3 C Salmonella Typhimurium LT2 ptrc99a LB + Ap 3 C Salmonella Enteritidis ATCC3 LB 3 C Salmonella Enteritidis ATCC3 ptrc99a LB + Ap 3 C Salmonella Senftenberg LMM2 LB 3 C Salmonella Senftenberg LMM2 ptrc99a LB + Ap 3 C Shigella flexneri LMG2 LB 3 C Shigella flexneri LMG2 ptrc99a LB + Ap 3 C Serratia plymuthica RVH budab::cm* LB + Cm 3 C Cronobacter sakazakii LMG budab::cm* LB + Cm 3 C Butanediol fermenters Bacterial strain Growth Growth medium temperature Serratia plymuthica RVH LB 3 C Cronobacter sakazakii LMG LB 3 C Escherichia coli MG pbudab LB + Ap 3 C Salmonella Typhimurium LT2 pbudab LB + Ap 3 C Salmonella Enteritidis ATCC3 pbudab LB + Ap 3 C Salmonella Senftenberg LMM2 pbudab LB + Ap 3 C Shigella flexneri LMG2 pbudab LB + Ap 3 C * The budab::cm mutants were constructed by the supervisor.

61 Table 3.3: Plasmids used in this study, characteristics and antibiotic resistance. Plasmid Description Resistance Reference ptrc99a Cloning vector encoding IPTG Ap Amann et al., 988 inducible trc promoter (P trc ) pbudab ptrc99a encoding the budab Ap Moons et al., 2 operon from S. plymuthica RVH fused downstream of P trc pbuda ptrc99a encoding the buda gene Ap This study* from S. plymuthica RVH fused downstream of P trc pbudb ptrc99a encoding the budb gene from S. plymuthica RVH fused downstream of P trc Ap This study* * These plasmids were constructed by the supervisor Buffers and solutions mm potassium phosphate buffer (PPB, ph.): To prepare ml of M PPB stock solution,. ml of a M K 2 HPO (Merck) stock solution and 38. ml of a M KH 2 PO (Riedel-de Haen) stock solution were mixed. The ph of the stock solution was adjusted to ph.. This stock solution was diluted times in deionised water to obtain a working solution of mm. The buffer was sterilized by autoclaving for 2 minutes at 2 C and psi. After autoclaving, this solution was stored at room temperature. 2% glucose: 2% w/v glucose was prepared by dissolving 2 g glucose (Chem Lab) in deionised water until reaching ml of solution. This solution was sterilised by filtration. % α-naphtol: % α-naphtol solution was prepared by dissolving.2 g α-naphtol (Merck) in absolute ethanol (AnalaR NORMAPUR) until reaching 2 ml of solution. This solution was sterilised by filtration. % KOH solution: % KOH solution was prepared by dissolving 2 g KOH (Riedel de Haen) in deionised water until reaching ml of solution. This solution was sterilised by filtration.

62 . M homopiperazine-n,n'-bis-2-(ethanesulfonic acid) buffer (HOMOPIPES, C 9 H 2 N 2 O S 2 ):. M HOMOPIPES buffer was prepared by dissolving 3. g HOMOPIPES (Aldrich) in deionised water until reaching ml of solution. This solution was sterilised by filtration. M acetoin (C H 8 O 2 ): M acetoin solution was prepared by dissolving 8.8 g acetoin (Aldrich) in deionised water until reaching ml of solution. This solution was sterilised by filtration and stored at C. Paraffin oil: It was sterilized by autoclaving for 2 minutes at 2 C and psi. Paraffin oil was supplied by Sigma Aldrich. M amino acid solutions: M of individual amino acid solution (lysine, arginine, glutamic acid or onithine) was prepared by dissolving (.9 g lysine), (.2 g arginine), (.3 g glutamic acid) or (3.2 g ornithine) in deionised water until reaching ml of solution. Amino acids were supplied by Merck. These solutions were sterilised by filtration and stored at room temperature. 2% Casamino acids: 2% casamino acid solution was prepared by dissolving 2 g casamino acids (Merck) in deionised water until reaching ml of solution. This solution was sterilised by filtration and stored at room temperature. M Isopropyl β-d--thiogalactopyranoside (IPTG, C 9 H 8 O S) solution: M ITPG was prepared by dissolving.9 g ITPG (Acros organics) in deionised water until reaching ml of solution. This solution was filter sterilized and stored at -2 C. mm IPTG was added to the growth media to induce the butanediol genes on pbuda, pbudb or pbudab plasmids. Physiological water: It was prepared by dissolving 8. g NaCl in deionised water till reaching liter of solution. This medium was sterilized by autoclaving for 2 minutes at 2 C and psi and stored at room temperature. 8

63 3... Antibiotics Antibiotics used in this study are listed in Table 3.. Table 3.: Antibiotics used in this study. Antibiotic Abbreviation Concentration stock solution (mg/ml) Solvent Final concentra tion (µg/ml) Ampicillin Ap Sterile MilliQ H 2 O Chloramphenicol Cm 3 % Ethanol 3 Antibiotics were stored at -2 C Equipments 3... Multiskan RC This equipment is designed to measure the optical density (turbidity) of solutions contained in wells of a microtiter plate at different wavelengths (2, or nm). Before each measurement, the microtiter plate is shaken in order to homogenize the suspension of each well. The data are collected in an Ascent software installed on a computer connected to the spectrophotometer. The equipment was supplied by ThermoLabsystems, Helsinky, Finland Multiskan Ascent The equipment is basically a spectrophotometer designed to measure the optical density (turbidity) of solutions contained in wells of a microtiter plate at different wavelengths. The equipment can be set up to measure the optical density every defined time in a nonstop way. The Multiskan Ascent is also equipped with a thermostat system in order to keep the temperature to the desired value during the whole cultivation. The data of optical density are collected in an Ascent software installed on a computer connected to the Multiskan Ascent. Data are recorded in graphs in order to visualise the growth based on optical density during cultivations. The equipment was supplied by ThermoLabsystems, Helsinky, Finland. 9

64 3.2. Methods Maintenance of strains Bacterial strains were stored on stock plates at C. Wild type strains were maintained on Luria Bertani (LB) agar, strains containing plasmids were mantained on LB agar containing ampicillin ( mg/l) and the budab mutants on LB agar containing chloramphenicol (3 mg/l) Transfer of plasmids by electroporation Plasmids were inserted by electroporation to Escherichia coli MG, Salmonella Typhimurium LT2, Salmonella Enteritidis ATCC3 and Salmonella Senftenberg LMM2. The electroporation was carried out as follows: each strain was inoculated into Luria Bertani (LB) broth and incubated at 3 o C until reaching the stationary phase. After that, a volume of cell suspension ( µl) was diluted (/) in ml of LB broth and incubated at 3 C for 3 hours. After this time, the test tube was placed on ice for one hour. The cooled cell suspension was divided in four eppendorf tubes ( ml for each one), and then centrifugated ( minutes at x g, C). The pellet was resuspended in ml of cooled milliq water. This was repeated three times. During the last washing step, the pellet was resuspended in µl of cooled milliq water and stored on ice. 2 µl of the plasmid solution was added to the cell suspension and this suspension was brought into an electroporation cuvette. The electroporation was carried out exposing the cell suspension. milliseconds at 2.2 kv. Resuscitation of cells was carried out in LB broth for one hour at 3 o C. After resuscitation, the cell suspension was plated on LB agar containing mg/l of ampicillin and then incubated 2 hours at 3 o C. Selection of cells with incorporated plasmids was possible due to the ampicillin resistance gene on the plasmid. Finally, purification of cells containing the plasmids was carried out by the streaking out method on LB agar containing ampicillin.

65 Voges Proskauer (VP) test VP test was carried out in order to evaluate the production of acetoin by the strains during cultivation. To µl of cell suspension, 3 µl of % α-naphtol followed by µl of % KOH were added. Then, the suspension was left to rest for one hour. After this time, the colour of the solution was observed. A change to red colour is indicative of acetoin production, whereas a yellow brown colour indicates absence of this compound. Acetoin production is an intermediate product during butanediol fermentation Inoculum preparation All strains were propagated in LB broth. For those strains containing plasmids, mg/l of ampicillin and mm of IPTG was added to the medium. The temperature of cell propagation for these bacteria was 3 o C. Bacteria such as, Cronobacter sakazakii LMG (and budab mutant) and Serratia plymuthica RVH (and budab mutant), were propagated at 3 o C. Bacteria were inoculated into ml of LB broth and left to grow until reaching the stationary phase ( 9 cells/ml) during 8 hours. The test tubes were shaken at 2 min - on a rotary shaker in order to create aerobic conditions and promote the growth. After propagation, dilution of cell suspension (/) was done using the corresponding culture medium of each experiment (see section 3..), until reaching an initial cell population about cells/ml Growth of Enterobacteriaceae in synthetic medium at different conditions Different culture media were used in these experiments (see 3..)..% glucose was added as carbon source and mm IPTG was added to media when strains containing plasmids were cultivated. The ph of the media was adjusted with 3% HCl according to the protocol of each experiment. To determine the cell number, the cell suspensions were diluted in PPB solution ( mm, ph.) and plated on LB agar containing ampicillin ( mg/l) for strains containing plasmids, or chloramphenicol (3 mg/l) for the budab mutants, and without antibiotics for the wild type ones (see 3..2). Plates were incubated at 3 o C for the mixed acid fermenters and at 3 o C for the butanediol fermenters. To evaluate the growth in intervals of time, optical density was measured at nm in a Multiskan RC

66 (see 3...). The experiments of non-stop optical density measurements were done in a Multiskan Ascent set up in such a way to measure the optical density (3 nm) every minutes during 8 or 2 hours of cultivation at 3 o C (see 3...2) Growth of enterobacteria in LB medium with neutral ph value Escherichia coli MG (ptrc99a, pbudab, pbuda and pbudb), Salmonella Typhimurium LT2 (ptrc99a, pbudab, pbuda and pbudb), Salmonella Senftenberg LMM2 (ptrc99a and pbudab) and Shigella flexneri LMG2 (ptrc99a and pbudab) were cultured at 3 o C, and Cronobacter sakazakii LMG (and budab mutant) and, Serratia plymuthica RVH (and budab mutant) at 3 o C. Culture medium, inoculum preparation, plate count and optical density measurements were done as described in sections 3..., 3.2. and Cultivation was carried out in microtiter plates containing 3 µl of culture medium covered with a plastic film. Measurements of ph, optical density and cell number were done at, 2,,,,, 8, 9 and 2 hours of cultivation. Voges-Proskauer test was done in order to evaluate the acetoin production during growth (see 3.2.3). Additionally, determination of the cell count of Escherichia coli MG (pbuda and pbudb) and Salmonella Typhimurium LT2 (pbuda and pbudb) was also done by plating on LB agar without ampicillin Growth of enterobacteria in LB medium with low ph values Growth of bacteria based on optical density Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab), Salmonella Senftenberg LMM2 (ptrc99a and pbudab), Shigella flexneri LMG2 (ptrc99a and pbudab), Salmonella Enteritidis ATCC3 (ptrc99a and pbudab) and Serratia plymuthica RVH (and budab mutant) were cultured in LB media adjusted at different ph values (ph range: 3..9). Culture medium and inoculum preparation is described in sections 3... and Cultivations were carried out at 3 o C under oxygen limitation or anaerobic conditions (culture medium in wells 2

67 covered with paraffin oil) in microtiter plates covered with a plastic film. Non-stop optical density measurements were done as explained in section Growth of bacteria in buffered medium Salmonella Typhimurium LT2 (ptrc99a and pbudab) was cultured in LB media of ph.. To one culture medium. M of HOMOPIPES buffer was added in order to maintain the ph constant during the cultivation. Culture medium and inoculum preparation is described in sections 3... and The media were sterilized by filtration using a syringe with a filter membrane (.2 µm). Cultivations were carried out at 3 o C in microtiter plates filled with 3 µl of medium and covered with a plastic film. Measurements of ph, optical density and cell count were done at, 3,,, 8, 9, 2, 2 and 3 hours of cultivation. Plate count and optical density measurements were done as described in section Growth of enterobacteria in LB medium with different glucose concentrations Serratia plymuthica RVH (and budab mutant) were cultured at 3 o C in LB medium containing different concentrations of glucose (%,.%,.3%,.%,.%, 3.% and.%). Optical density and ph were measured at, 2, 3,,,,, 8 and 2 hours of cultivation. Inoculum preparation was done as described in section An experiment of non-stop optical density measurements was also carried out at the same time as explained in section 3.2. in order to evaluate the growth during 8 hours of cultivation Growth of enterobacteria in LB medium with mm of acetoin Serratia plymuthica RVH (and budab mutant) and Salmonella Typhimurium LT2 (ptrc99a and pbudab) were cultivated in a microtiter plate containing 3 µl of LB medium with.% glucose and mm acetoin. mm IPTG was added when strains containing ptrc99a and pbudab plasmids were cultivated. Optical density, ph and cell count were measured at, 2,,,, 8 and 2 hours of cultivation. Inoculum preparation, plate count and optical density measurements were done as described in sections 3.2. and Furthermore, experiments of non-stop optical density measurements were also 3

68 carried out with LB medium with or without.% glucose and/or mm acetoin. These culture media were adjusted to ph values of.,. and.. Non-stop optical density measurements were done as explained in section Growth of enterobacteria in M9 minimal medium Growth in minimal medium at different ph values Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab) and Serratia plymuthica RVH (and budab mutant) were cultivated in M9 minimal medium containing.% of glucose (see 3...2). Inoculum preparation was done as described in section Mixed acid fermenters were cultivated at 3 o C and butanediol fermenters at 3 o C. mm IPTG was added when strains containing plasmids were cultivated. The ph was adjusted to.,. or.. Optical density and ph were measured at, 2,,, 8, 2 and 3 hours of cultivation. Additionally, cell numbers were measured during cultivation of medium of ph.. Plate count and optical density measurements were done as described in section Cultivations were carried out in a microtiter plate containing 3 µl of culture medium covered with a plastic film. Growth in minimal medium containing amino acids Serratia plymuthica RVH (and budab mutant) were cultivated at 3 o C in M9 minimal medium containing.% glucose and mm of different amino acids (lysine, arginine, ornitine or glutamic acid) or.2%,.% or.% of casamino acids (see sections 3...2). Inoculum preparation was done as described in section The ph of the media was adjusted to.. The optical density and ph variation were measured at, 2,,,, 8, 2 and 3 hours of cultivation. Optical density measurements were done as described in section Cultivations were carried out in a microtiter plate containing 3 µl of culture medium covered with a plastic film. An experiment of non-stop optical density measurements was also carried out as explained in section 3.2.., in order to evaluate the growth during 8 hours.

69 3.2.. Growth of enterobacteria in fruit juices Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab) and Cronobacter sakazakii LMG (and budab mutant) were cultivated in grape, apple, worldshake and nectar multifruit juice (for composition of these fruit juices see Table 3.). The juices were acquired from a local supermarket, Leuven, Belgium. The fruit juices were sterilized by filtration and then adjusted to different ph values (ph range: ) with N KOH. mm IPTG was added to the juices when strains containing plasmids were cultivated. Preparation of inoculum was done as described in 3.2., but in this case, cell suspensions were diluted in physiological water before inoculation into. ml of fruit juice (initial cell number of cells/ml). Cultivations were carried out in test tubes at 2 o C or 3 o C under oxygen limitation or under anaerobic conditions (culture medium covered with 2 ml of sterilized paraffin oil). Cell count and ph were measured every 2 hours. The cell count was determined by plating of µl spots on LB agar containing mg/l of ampicillin, except for Cronobacter sakazakii LMG. Before plating, -fold dilutions of cell suspensions were made in mm of PPB solution in microtiter plates. The agar plates were incubated at 3 o C for C. sakazakii LMG and at 3 o C for the rest of bacteria. Furthermore, experiments of non-stop optical density measurements were done under oxygen limitation and under anaerobic conditions with these strains including Serratia plymuthica RVH (and budab mutant) in apple juice with different ph values in order to evaluate the growth based on optical density at 3nm Growth of enterobacteria on vegetables Serratia plymuthica RVH (and budab mutant) and Cronobacter sakazakii LMG (and budab mutant) were cultivated on red pepper and cucumber slices in order to evaluate the growth and spoilage activity.

70 Preparation of vegetable slices Vegetables were disinfected with % ethanol and then rinsed with sterile water to remove possible traces of ethanol. Red pepper was cut into slices with a sterile special device with a diameter of 2 cm. Cucumber was cut into slices of around. cm thick with a sterile knife. All handlings were performed under a laminar flow cabinet to assure aseptic conditions Preparation of bacterial suspension Preparation of inoculum was done as described in section 3.2., but in this case, dilution of cell suspensions till CFU/ml was done in potassium phosphate buffer ( mm, ph.). In the first experiment, square Petri dishes (2 cm 2 cm) were filled with aliquots of ml of the resulting bacterial suspensions (resulting in a fluid depth of about 2.8 mm) and used in the spoilage assay Vegetable spoilage assay Ten red pepper slices were placed in a Petri dish containing a bacterial suspension, and turned upside down after min in order to bring both sides of the slice in contact with bacteria. In the first experiment, the red pepper slices were only about halfway submerged in the suspension, so that one edge remained exposed to the air. For each strain, five or more such Petri dishes were prepared. Subsequently, Petri dishes with bacterial suspension and red pepper slices were incubated at 3 C under a humid atmosphere to prevent dehydration. In the rest of experiments, red pepper and cucumber slices were submerged in bacterial suspension as explained above. Subsequently, Petri dishes with drained red pepper or cucumber slices (without bacterial suspension) were sealed in sterile plastic bags and incubated at C or 2 C Cell count and spoilage activity To determine bacterial growth on vegetables, the upper surface (that exposed to the air) of one slice was sampled with a sterile wrap previously wet in sterile potassium phosphate

71 buffer ( mm, ph.). For red pepper slices, the whole surface was sampled (area of 3. cm 2 ) and, for cucumber slices only one cm 2. Slices were sampled every two or three days. Cells were -fold diluted in PPB solution and then µl was plated on LB agar. Incubation of petri dishes was at 3 o C. Spoilage activity was considered when exudation or biofilm formation was noticed.

72 PART III: RESULTS A D DISCUSSIO

73 . Growth of Enterobacteriaceae in synthetic media. Growth of enterobacteria in synthetic medium with neutral ph The aim of these experiments was to evaluate the growth of enterobacteria in nutrient rich media with glucose and to follow up the ph change and acetoin production associated with the metabolism of these bacteria. This was done for two butanediol fermenters, Serratia plymuthica RVH and Cronobacter sakazakii LMG, and their respective budab mutant strains, and for four mixed acid fermenters, Escherichia coli MG, Salmonella Typhimurium LT2, Salmonella Senftenberg LMM2 and Shigella flexneri LMG2. Plasmids ptrc99a and pbudab were inserted by electroporation in the latter strains as explained in section Plasmid pbudab was derived from the cloning vector ptrc99a and contains the budab operon from Serratia plymuthica RVH after an IPTG inducible promoter. In this way, acetoin producing strains of mixed acid fermenters were created. Strains containing ptrc99a were used as control strains. Figure. and figure. show the growth curves and ph change during 2 hours of cultivation in LB +.% glucose at 3 or 3 C as explained in section Enterobacteria grow well in Luria Bertani (LB) broth with addition of glucose as carbon source. The nitrogen compounds are used to generate building blocks and glucose as a source of energy. During growth, many by-products are produced, mainly organic acids, which can be harmful for the cells in high amounts. Bacterial growth can stop due to the depletion of the carbon or nitrogen source or due to the inhibitory effect of by-products of the fermentation. In figure. (a and b) can be observed that S. plymuthica RVH and C. sakazakii LMG and their respective budab mutants grow with concomitant acidification of the medium. After certain time (approximately 8 hours) only wild type strains were able to increase the ph. In addition, the Voges-Proskauer (VP) test confirmed the production of acetoin (precursor compound of butanediol) by these strains (see figure.2). The production of acetoin and its relation with the increase of ph still remain unclear and many speculations can be done. Deacidification of the culture medium would be due to the consumption of organic acids produced during growth of butanediol producing bacteria. Acetic acid is one of the most important weak organic acids produced and is in part responsible for the acidification since it provides protons to the medium. Low ph 9

74 environments are not considered as appropriate environments for the growth of most enterobacteria. In addition, the genes which are responsible for the synthesis of acetoin can be expressed under certain conditions. The ph increase of the medium would be an advantage that enables these bacteria to grow or survive in low ph environments. S. plymuthica RVH is a spoilage bacterium whose ability to increase the ph from.3 up to toward the 2 th hour of cultivation has made possible to reach populations of 8.9 log CFU/ml (see figure.a). On the contrary, the growth of the budab mutant strain was affected in part by the inability to increase the ph. C. sakazakii LMG showed some difference in its behaviour, mainly in the change of ph during cultivation. They acidified the medium faster than S. plymuthica RVH reaching values of.2. Thus, the final cell population of C. sakazakii LMG decreased at the end of cultivation (2 hours) to values lower than 8.9 log CFU/ml. The final cell count can be influenced by the initial cell concentration used as inoculum and of course by the ability of the strain to grow in adverse environments. The growth and ph change during cultivation of mixed acid fermenters is shown in figure. (c,d). The neutral plasmid ptrc99a and the pbudab plasmid, containing the budab genes of the butanediol pathway, were inserted by electroporation to these bacteria. It can be observed that, all these bacteria grew and acidified the medium, but only those that received the pbudab plasmid were able to increase the ph after approximately 8 hours of cultivation. The VP-test confirmed the production of acetoin by bacteria harbouring pbudab plasmid (see figure.3). In case of E. coli MG pbudab, this strain has a slight advantage compared with the E. coli MG ptrc99a. The pbudab plasmid seemed to be helpful for the growth under low ph conditions toward the 2 hours (see figure.c). Similar behaviour was noticed in Salmonella Typhimurium LT2 (figure.d). A clear advantage for the pbudab strain compared with the ptrc99a strain was observed.

75 A. Cultivation of Serratia plymuthica RVH at 3 C B. Cultivation of Cronobacter sakazakii LMG at 3 C Log (cfu/ml)... ph Log (cfu/ml). ph Time (hours) ph: S. plymuthica RVH ph: S.plymuthica RVH budab::cm Cell count: S.plymuthica RVH Cell count: S. plymuthica RVH budab::cm Time (hours) ph: C.sakazakii LMG ph: C.sakazakii LMG budab::cm Cell count: C.sakazakii LMG Cell count: C.sakazakii LMG budab::cm C. Cultivation of Escherichia coli MG at 3 C D. Cultivation of Salmonella Typhimurium LT2 at 3 C Log (cfu/ml) Time (hours) ph: E. coli MG ptrc99a Cell number: E. coli MG ptrc99a ph: E. coli MG pbudab Cel number: E. coli MG pbudab ph Log (cfu/ml) Time (hours) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Figure.: Growth of Serratia plymuthica RVH and budab mutant (a), Cronobacter sakazakii LMG and budab mutant (b), Escherichia coli MG ptrc99a and pbudab (c) and, Salmonella Typhimurium LT2 ptrc99a and pbudab (d), in LB broth +.% glucose (ph.). mm ITPG was added to cultivations with strains containing ptrc99a or pbudab. ph

76 S. plymuthica RVH S. plymuthica RVH budab::cm C. sakazakii LMG h 2h h h h h Figure.2: Voges-Proskauer test (positive = red), realized during cultivation of S. plymuthica RVH and budab mutant and, C. sakazakii LMG in LB +.% glucose (ph.). E. coli MG ptrc99a E. coli MG pbudab S. Typhimurium LT2 ptrc99a S. Typhimurium LT2 pbudab h 2h h h h h Figure.3: Voges-Proskauer test (positive = red), realized during cultivation of E. coli MG ptrc99a and pbudab and, S. Typhimurium LT2 ptrc99a and pbudab in LB +.% glucose + mm ITPG (ph.). Interesting results were observed when Shigella flexneri LMG2 pbudab was cultivated (figure.a). This strain was able to keep the ph value around. during at least three hours after hours of cultivation and afterwards the ph started decreasing. This result was not expected. It is clear however that the pbudab plasmid gave some advantage for the growth compared with S. flexneri LMG2 ptrc99a. In this case, the pbudab plasmid seemed to play somehow an important role in keeping the ph at. during at least three hours. Additionally, the VP-test confirmed production of acetoin during cultivation of S. flexneri LMG2 pbudab (see figure.). The growth of Salmonella Senftenberg LMM2 was influenced positively by the pbudab plasmid as shown in figure.b, a slight ph increase was also observed in this case. 2

77 A. Cultivation of Shigella flexneri LMG2 at 3 C Log (cfu/ml).... ph Time (hours) 3. ph: S. flexneri LMG2 ptrc99a Cell number: S.flexneri LMG2 ptrc99a ph: S.flexneri LMG2 pbudab Cell number: S.flexneri LMG2 pbudab B. Cultivation of Salmonella Senftenberg LMM2 at 3 C Log (cfu/ml)... ph Time (hours) 3. ph: S.Senftenberg LMM2 ptrc99a Cell number: S.Senftenberg LMM2 ptrc99a ph: S.Senftenberg LMM2 pbudab Cell number: S.Senftenberg LMM2 pbudab Figure.: Growth of Shigella flexneri LMG2 ptrc99a and pbudab (a) and, Salmonella Senftenberg LMM2 ptrc99a and pbudab (b), at 3 C in LB broth +.% glucose + mm ITPG (ph.). S. flexneri LMG2 ptrc99a S. flexneri LMG2 pbudab h 2h h h h h Figure.: Voges Proskauer test (positive = red), realized during cultivation of S. flexneri LMG2 ptrc99a and pbudab in LB +.% glucose + mm ITPG (ph.). 3

78 The ability to increase the ph of the surrounding medium can be an advantage for butanediol producing enterobacteria. These bacteria can prolong the stationary phase or survive for a longer time when the energy source is almost depleted. This statement is confirmed by observing figures. (c, d) and. where bacteria containing the pbudab plasmid showed a better physiological condition in the stationary phase and in some cases the growth was promoted. Pathogenic strains of Escherichia coli, Shigella flexneri or serovars of Salmonella can potentially incorporate butanediol genes by horizontal gene transfer, obtained from bacteria living in the same ecological niche. In this way, human enteropathogens could acquire an additional advantage such as tolerance to low ph environments, especially in non-termal treated foods. Cultivations of Escherichia coli MG and Salmonella Typhimurium LT2 containing plasmids pbuda or pbudb were also carried out in order to evaluate the presence of the budb (α-acetolactate synthase) or buda (α-acetolactate decarboxylase) gene of the butanediol pathway. During butanediol fermentation, α-acetolactate is formed from pyruvate by α-acetolactate synthase and decarboxylated to acetoin by α-acetolactate decarboxylase as shown in figure. and.3. Data for the cell numbers and ph change during cultivations are shown in figure.. The growth of E. coli MG pbuda and pbudb in LB broth +.% glucose was combined with acidification of the medium (see figure.a). They were not able to increase the ph (final ph was. and.8 respectively). It seemed that pbuda or pbudb plasmids did not have an advantage for the growth, but rather had a disadvantage in case of the pbudb plasmid. Regarding to the ph change, a difference could be noticed in case of pbudb. The acidification of the medium was much slower than in case of pbuda, probably due to a slower increase of the cell number. On the other hand, a positive influence of the pbudb plasmid on the growth of S. Typhimurium LT2 was observed after 8 hours of cultivation in comparison with the pbuda strain. The ph change showed a similar behaviour as observed during cultivation of E. coli. VP-tests confirmed in previous experiments that acetoin is produced when both buda and budb genes are present. In this case, the VP-test was (slightly) positive for strains carrying the pbudb plasmid after 2 hours (see figure.).

79 A. Cultivation of Escherichia coli MG at 3 C Log (cfu/ml) Time (hours) ph: E.coli MG pbuda ph: E.coli MG pbudb Cell count: E.coli MG pbuda Cell count: E.coli MG pbudb ph B. Cultivation of Salmonella Typhimurium LT2 at 3 C Log (cfu/ml)... ph Time (hours) ph: S.Typhimurium LT2 pbuda Cell count: S.Typhimurium LT2 pbuda 3. ph: S.Typhimurium LT2 pbudb Cell count: S.Typhimurium LT2 pbudb Figure.: Growth of Escherichia coli MG pbuda and pbudb (a) and, Salmonella Typhimurium LT2 pbuda and pbudb (b), at 3 C in LB broth +.% glucose + mm ITPG (ph ). 2 3 Figure.: Voges Proskauer test (positive = red), realized after 2 hours of cultivation at 3 C in LB broth +.% glucose + mm ITPG (ph ). : Escherichia coli MG pbuda, 2: E. coli MG pbudb, 3: Salmonella Typhimurium LT2 pbuda, : S. Typhimurium LT2 pbudb.

80 .2 Growth of enterobacteria in synthetic medium with low ph.2. Growth of enterobacteria at low ph based on optical density The aim of these experiments was to evaluate the influence of the budab genes in the ability of some members of the Enterobacteriaceae to grow in synthetic medium adjusted to low ph values. The experiments were carried out as explained in section LB broth +.% glucose was adjusted with HCl. Because this acid is completely dissociated at ph values above about 2., adjustment of the ph with HCl enables the effect of hydrogen ions on microorganisms to be tested. It is known that the ph influences i: the probability of growth of microbes, ii: the growth rate, and iii: survival and death rate. Experiments were done using microtiter plates covered with a plastic film in order to prevent evaporation of liquid during 8 hours of cultivation at 3 C. Growth measurements based on optical density showed that S. plymuthica RVH, which contains budab genes in the genome, was able to grow well at a ph above.3 (figure.8a). Growth at ph. and.2 was rather difficult and, below ph., growth was not possible. ph measurements at the end of cultivation (8 hours) showed a ph increase as observed previously in cultivations carried out in medium with initial ph. Comparisons with the budab mutant (figure.8b) revealed a clear advantage of these two genes in the growth of the wild type strain. In this case, the growth failed at ph values lower than. and only a slight increase of OD was observed above this ph value. ph measurements at the end of cultivation showed a decrease of this parameter in cultivations carried out in media with initial ph of. or higher values. Cultivations of Salmonella Enteritidis ATCC3 also showed a clear advantage of the pbudab plasmid on the growth at low ph (figure.8c,d). This strain was able to grow better at low ph compared with the ptrc99a strain. A ph increase was observed in all cultivations, but growth was only possible at a ph higher than 3.8. The ph increase in cultivations when no growth was observed could be due to the fact that these cells tried as much as possible to increase the ph to a value favourable to start growth. This can lead to an extended lag phase or to the death of the cells. The effect of ph on the lag phase will depend on the previous history of the inoculum, the growth phase during harvesting, the growth medium, the ph itself, and the temperature. Apart from affecting the initiation of

81 growth, ph also affects the rate of microbial growth. In contrast to S. plymuthica RVH, where the ph particularly influenced the growth rate, in this case, the lag phase was seriously affected by the ph. Regarding to the ph change, S. Enteritidis ATCC3 pbudab was able to increase the ph in all conditions compared with the strain containing ptrc99a. This ability could be associated with growth in low ph environments. Presser et al. (99) showed an inverse relationship between the growth rate and the hydrogen ion concentration (ph). Thus, as the ph is reduced, so the yield of cell mass per unit of substrate used falls, reflecting the increased energy needed to maintain a higher and near constant cytoplasmic ph.

82 A. Growth curves of Serratia plymuthica RVH B. Growth curves of Serratia plymuthica RVH budab ::Cm ph after 8h ph after 8h OD (3nm) ph 3.: 3.9 ph 3.: 3. ph 3.: 3. ph 3.8:.89 ph 3.9:. ph.:. ph.:.2 ph.2:. ph.3:.29 ph.:.2 ph.:.8 ph.:.8 ph.:.8 ph.8:.9 OD (3nm) ph 3.: 3. ph 3.: 3. ph 3.: 3. ph 3.8:.88 ph 3.9:. ph.:. ph.:.22 ph.2:. ph.3:.3 ph.:.22 ph.:.29 ph.:.3 ph.:.33 ph.8: Time (hours) Time (hours) ph ph, ph,2 ph,3 ph, ph, ph, ph, ph, ph, ph,2 ph,3 ph, ph, ph, ph, C. Growth curves of Salmonella Enteritidis ATCC3 pbudab D. Growth curves of Salmonella Enteritidis ATCC3 ptrc99a ph after 8h ph after 8h OD (3nm) ph 3.: 3. 3 ph 3.: 3. ph 3.: 3. 3 ph 3.8:. 8 ph 3.9:. 38 ph.:. 3 ph.:. 8 ph.2:. ph.3:. 9 ph.:. 3 ph.:. 2 ph.:. 3 ph.:. 38 ph.8:. 2 OD (3nm) ph 3.: 3.3 ph 3.: 3.3 ph 3.: 3. ph 3.8:.83 ph 3.9:.2 ph.:. ph.:. ph.2:.9 ph.3:.23 ph.:.2 ph.:.3 ph.:.33 ph.:.3 ph.8: Time (hours) Time (hours) ph 3,8 ph 3,9 ph ph, ph,2 ph,3 ph, ph, ph 3,8 ph 3,9 ph ph, ph,2 ph,3 ph, ph, Figure.8: Growth curves of Serratia plymuthica RVH (a), Serratia plymuthica RVH budab mutant (b), Salmonella Enteritidis ATCC3 pbudab (c) and, Salmonella Enteritidis ATCC3 ptrc99a (d), cultivated at 3 C in LB broth +.% glucose, adjusted at different ph values with HCl. mm ITPG was added to cultivations with strains containing ptrc99a or pbudab plasmids. 8

83 Salmonella Typhimurium LT2 (ptrc99a and pbudab) was also cultivated in synthetic medium with low initial ph in order to evaluate their ability to grow. As it can be observed in figure.9 (a,b), S. Typhimurium LT2 pbudab was able to grow at ph 3.8 and above this value the growth was strongly enhanced as compared with S. Typhimuriun LT2 ptrc99a. ph measurements at the end of cultivation showed the importance of the pbudab plasmid. In case of S. Typhimurium LT2 ptrc99a, only a weak growth was observed at a ph above.. The growth was coupled with a ph decrease when cultivated in media of ph. and higher values. The ph increase observed when cultivated in media of lower initial ph than. might be due to the attempt of the bacterial population to shift the ph to a value which is favourable to start growth. The pbudab plasmid has influenced positively the growth of Escherichia coli MG in comparison with E. coli MG ptrc99a as it can be observed in figure.9 (c,d). The minimum ph for growth of E. coli MG pbudab in these experiments was.2, the same value as observed in E. coli MG ptrc99a. But, it is clear that the main advantage of bacteria harbouring the pbudab plasmid is the improvement of the growth rate and in some cases shortening of the lag phase. Furthermore, the ph increase at the end of cultivation was observed in E. coli MG pbudab, while E. coli MG ptrc99a was not able to increase the ph when cultivated in media of ph. and higher values. The improvement of the growth of E. coli MG pbudab was also observed when cultivated in medium of neutral ph (see figure.c, d). The high concentration of hydrogen ions outside the cell promotes the flux of protons throughout the cytoplasmic membrane. This accumulation of protons inside the cell inhibits bacterial growth and can lead to cell death. The rapid adaptation to high concentration of hydrogen ions and the capacity to increase the ph during growth are related to an efficient homeostatic control system of the cells, this characteristic is the main factor that enables bacteria harbouring pbudab plasmids to deal with low ph environments. 9

84 A. Growth curves of Salmonella Typhimurium LT2 pbudab B. Growth curves of Salmonella Typhimurium LT2 ptrc99a OD 3nm ph after 8h ph 3.: 3.8 ph 3.: 3.8 ph 3.: 3.9 ph 3.8:.3 ph 3.9:.33 ph.:.39 ph.:. ph.2:. ph.3:.3 ph.:. ph.:.3 ph.:.2 ph.:.9 ph.8:. ph.9:. OD (3nm) ph after 8h ph 3.: 3.2 ph 3.: 3.3 ph 3.: 3.3 ph 3.8: 3.8 ph 3.9:. ph.:.29 ph.:.3 ph.2:.3 ph.3:.3 ph.:.3 ph.:.3 ph.:.38 ph.:.39 ph.8:.2 ph.9: Time (hours) ph 3, ph 3,8 ph 3,9 ph ph, ph,2 ph,3 ph, ph, Time (hours) ph 3, ph 3,8 ph 3,9 ph ph, ph,2 ph,3 ph, ph, 3 3 C. Growth curves of Escherichia coli MG pbudab D. Growth curves of Escherichia coli M G ptrc99a.2 ph after 8h ph after 8h OD (3 nm).8. ph.:. ph.:. ph.2:.2 ph.3:.38 ph.:. ph.:.2 ph.:.9 OD (3 nm) ph.:. ph.:. ph.2:.3 ph.3:.3 ph.:.3 ph.:.3 ph.: Time (hour) ph. ph. ph.2 ph.3 ph. ph. ph Time (hour) ph. ph. ph.2 ph.3 ph. ph. ph. Figure.9: Growth curves of Salmonella Typhimurium LT2 pbudab (a), Salmonella Typhimurium LT2 ptrc99a (b), Escherichia coli MG pbudab (c) and, Escherichia coli MG ptrc99a (d), cultivated at 3 C in LB broth +.% glucose + mm IPTG, adjusted at different ph values with HCl.

85 Cultivations carried out with Shigella flexneri LMG2 (ptrc99a and pbudab), were also done in order to evaluate its ability to grow in media adjusted to low ph values. The results of growth based on optical density are showed in figure. (a,b). In this case, the effect of the pbudab plasmid on the growth was not as clear as observed in previous experiments realized with other enterobacteria. This correlates with the results from the experiments at neutral ph, where there was no big difference of cell numbers and ph change between strains carrying ptrc99a or pbudab plasmid (see figure.a). The growth improvement of S. flexneri LMG2 does not only depend on the expresion of the genes contained in the pbudab plasmid but also on other, hitherto unkown, factors. The positive effect of the pbudab plasmid in S. flexneri LMG2 was evidenced in the ph increase when cultivated in media of initial ph.2 and higher values. On the contrary, S. flexneri LMG2 ptrc99a was not able to increase the ph during cultivation in media of initial ph. and higher values. A more evident effect of the pbudab plasmid on the growth was observed during cultivation of Salmonella Senftenberg LMM2 (figure.c, d). Here, the minimum ph value for growth observed in both pbudab and ptrc99a strains was 3.8. The positive effect of the pbudab plasmid in S. Senftenberg LMM2 was noticed in the growth rate which was higher than that of S. Senftenberg LMM2 ptrc99a when cultivated under the same conditions. The effect of the pbudab plasmid on the growth of S. Senftenberg LMM2 could be correlated with the ph increase observed during cultivation. The ability of microorganisms to initiate growth at low ph also depends on the number of microorganisms in the inoculum. The resistance of microorganisms and their ability to survive and multiply in adverse conditions is very important and it depends in some extent on the characteristics of the strain itself. A large amount of cells at the beginning of the cultivation can influence the tolerance to a given set of conditions. In addition, the cytoplasm of cells exposed to low ph conditions tends to be maintained at a ph near to neutrality (Booth, 98). But, extreme lowering of the external ph might cause a decrease of internal ph; eventually the difference between the internal and external ph ( ph) reaches a maximum ( ph begins to collapse) and the cells begin to die.

86 A. Growth curves of Shigella flexneri LMG2 pbudab B. Growth curves of Shigella flexneri LMG2 ptrc99a ph after 8h. ph after 8h OD (3nm) ph 3.: 3. ph 3.: 3.9 ph 3.: 3. ph 3.8:.89 ph 3.9:.2 ph.:. ph.:.2 ph.2:.8 ph.3:. ph.:.3 ph.:. ph.:. ph.:. ph.8:.2 OD (3nm) ph 3.: 3.8 ph 3.: 3. ph 3.: 3. ph 3.8: 3.9 ph 3.9:.2 ph.:.2 ph.:.23 ph.2:. ph.3:. ph.:. ph.:. ph.:. ph.:. ph.8: Time (hours) ph ph, ph,2 ph,3 ph, ph, ph, ph, ph,8 ph Time (hours) ph ph, ph,2 ph,3 ph, ph, ph, ph, ph,8 ph OD (3nm) C. Growth curves of Salmonella Senftenberg LMM2 pbudab Time (hours) ph 3, ph 3,8 ph 3,9 ph ph, ph,2 ph,3 ph, ph, ph ph after 8h ph 3.: 3.9 ph 3.: 3. ph 3.: 3.8 ph 3.8:.2 ph 3.9:. ph.:.22 ph.:.28 ph.2:.32 ph.3:.3 ph.:.3 ph.:.3 ph.:. ph.:.9 ph.8:. OD (3nm) D. Growth curves of Salmonella Senftenberg LMM 2 ptrc99a Time (hours) ph 3, ph 3,8 ph 3,9 ph ph, ph,2 ph,3 ph, ph, ph ph after 8h ph 3.: 3.8 ph 3.: 3.9 ph 3.: 3. ph 3.8:.8 ph 3.9:.3 ph.:. ph.:.9 ph.2:.2 ph.3:.2 ph.:.29 ph.:.33 ph.:.32 ph.:.3 ph.8:.3 Figure.: Growth curves of Shigella flexneri LMG2 pbudab (a), Shigella flexneri LMG2 ptrc99a (b), Salmonella Senftenberg LMM2 pbudab (c) and, Salmonella Senftenberg LMM2 ptrc99a (d), cultivated in LB broth +.% glucose + mm IPTG, adjusted at different ph values with HCl. 2

87 .2.2. Growth of enterobacteria in buffered medium at low ph The aim of these experiments was to evaluate the effect of the presence of the ptrc99a and pbudab plasmids on the growth capacity of Salmonella Typhimurium LT2 in sugar containing LB broth medium at low ph with and without the addition of.m homopiperazine-n,n'-bis-2-(ethanesulfonic acid) buffer (HOMOPIPES). HOMOPIPES (pka.) is a good buffer to maintain the ph constant in a ph range between 3.9 and. and can not be used by the bacteria in contrast to organic acids. In this way, the growth at low ph could be observed while the ph remains more or less constant. The ph of the LB medium was adjusted to. with HCl. Figure. (a,b) shows the cell number and ph change during the cultivation. The behaviour of S. Typhimurium LT2 ptrc99a in buffered medium at ph. was severely affected. The cells started dying from the beginning of cultivation and no bacteria were detected after 3 hours of cultivation. In case of cultivations in unbuffered LB medium, the cell number decreased about log unit during the first hours of cultivation with concomitant increase of the ph. Then, once the ph has slightly increased (ph.), the cells started growing until reaching values of.9 log CFU/ml. After 2 hours of cultivation, the cell population started dying, probably due to the effect of both glucose depletion and ph decrease of the medium. Similar effect was observed when S. Typhimurium LT2 pbudab was cultivated in buffered medium. Cells started dying slowly and after 3 hours, the cell number was decreased with approximately 2. log units. In unbuffered medium, the cellular adaptation has taken about hours with concomitant increase of ph until.. After this time, the cells started growing until reaching a cell number of 8.8 log CFU/ml, this was accompanied with a further ph increase to.8. At the end of cultivation the cell population remained almost constant and the ph has diminished to about., probably due to the production of by-products, such as acetic acid, during the fermentation. The influence of the pbudab plasmid as observed in these experiments would be in keeping the stationary phase for longer time compared with the ptrc99a strain and in limiting the death rate to lower values when cultivated in buffered medium. 3

88 A. Cultivation of Salmonella Typhimurium LT2 ptrc99a Log (cfu/ml)... ph Time (hours) ph: S.Typhimurium LT2 ptrc99a (LB) Cell count: S.Typhimurium LT2 ptrc99a (LB) ph: S.Typhimurium LT2 ptrc99a (LB+Buffer) Cell count: S.Typhimurium LT2 ptrc99a (LB+Buffer) B. Cultivation of Salmonella Typhimurium LT2 pbudab Log (cfu/ml)... ph Time (hours) ph: S.Typhimurium LT2 pbudab (LB) Cell count: S.Typhimurium LT2 pbudab (LB) ph: S.Typhimurium LT2 pbudab (LB+B) Cell count: S.Typhimurium LT2 pbudab (LB+B) Figure.: Cultivation of S. Typhimurium LT2 ptrc99a (a) and, S. Typhimurium LT2 pbudab (b), in LB broth +.% glucose + mm IPTG, without and with mm of buffer HOMOPIPES, adjusted to ph. with HCl..3 Influence of glucose concentration on growth of enterobacteria The aim of these experiments was to evaluate the influence of different concentrations of glucose on the growth of Serratia plymuthica RVH wild type and budab mutant. The cells were cultured at 3 o C in LB broth containing different concentrations of glucose as explained in section Cell growth was measured by means of optical density. Additionally, ph changes during the cultivations were measured too. This is shown in figure.2.

89 It was observed that glucose concentrations higher than.% w/v have a favourable effect on the growth of S. plymuthica RVH. During the metabolism, glucose is broken down to obtain energy (in form of ATP) and this is used for the growth and other metabolic requirements such as maintenance. Furthermore, it was also observed that the growth in terms of optical density when cultivated in media with glucose concentrations from.3% to % w/v is similar to each other. Bacterial growth in media with high concentrations of glucose (higher than the saturation constant for glucose Ks) is equal or close to the maximum specific rate and follows the Monod relationship provided that cell growth does not undergo glucose repression (catabolite effect). Low concentrations of glucose (lower than Ks) lead to its quick depletion and as a consequence the cells enter the stationary phase rapidly. It was observed that during cultivations under all conditions, the ph diminished in similar pattern except for that carried out in medium without glucose. After approximately hours, the ph started increasing for all conditions. However, interesting observations were pointed out in case of cultivation in media with 3% and % w/v glucose. The ph values at the end of cultivation diminished in this case in contrast to the other conditions. From these results it is posible to suggest that the ph change during the cultivation would be probably related with the depletion of glucose, production of byproducts such as organic acids and the activity of the budab genes present in S. plymuthica RVH. Low concentration of glucose in the culture medium is rapidly depleted with concomitant production of organic acids which exert the ph decrease. The medium without glucose would contain only very low concentrations of carbon source present normally in the peptone or yeast extract (for composition of LB broth 3...). Results for the growth and ph change during the cultivation of S. plymuthica RVH budab mutant under the same conditions are presented in figures.2(c,d). The growth in all conditions was severly limited in comparison with the wild type strain. In addition, the ph decrease was faster and reached lower values compared with the wild type strain, except for the medium without glucose. A ph increase was only observed when cultivated in media without and with.% w/v glucose. The ph increase at this condition would be connected to the utilization of fermentation by-products, such as organic acids, as carbon source to produce energy as their concentrations were not high enough to affect the growth.

90 A. Growth curves of Serratia plymuthica RVH..9.8 B. Change of ph during cultivation of Serratia plymuthica RVH 8. OD (3 nm).... ph Time (hours) % glucose,% glucose,3% glucose,% glucose % glucose 3% glucose % glucose Time (hours) ph: % glucose ph:.% glucose ph:.3% glucose ph:.% glucose ph: % glucose ph: 3% glucose ph: % glucose C. Growth curves of Serratia plymuthica RVH budab::cm..9.8 O D (3 nm) ph D. Change of ph during cultivation of Serratia plymuthica RVH budab ::Cm Time (hours) % glucose,% glucose,3% glucose,% glucose % glucose 3% glucose % glucose Time (hours) ph: % glucose ph:.% glucose ph:.3% gluc ose ph:.% glucose ph: % glucose ph: 3% glucose ph: % glucos e Figure.2: Growth curves and ph change of Serratia plymuthica RVH (a,b) and Serratia plymuthica RVH budab mutant (c,d), cultivated at 3 C in LB broth + glucose (ph.).

91 . Growth of enterobacteria in minimal medium.. Growth of enterobacteria in minimal medium at different ph values The aim of these experiments was to evalute the growth of Serratia plymuthica RVH wild type and budab mutant, Escherichia coli MG (ptrc99a and pbudab) and, Salmonella Typhimurium LT2 (ptrc99a and pbudab) when cultivated in M9 minimal medium with.% glucose adjusted to ph.,. and.. The procedure is explained in section and the results are shown in figures.3 and.. The growth profile of Serratia plymuthica RVH and budab mutant were similar (see figure.3a,b) and it was connected with the decrease of ph during cultivation. Surprisingly, the wild type strain was not able to increase the ph during the cultivation. In all cultivations, the initial ph almost did not change during the first four hours, time corresponding to the delay of the lag phase. The poor growth of the cell population would be connected with the lack of amino acids and proteins necessary for the growth. The inability of the wild type strain to increase the ph during the cultivation could also be related with the lack of nitrogen source in the medium, since organic acids such as acetic acid produced during fermentation could be used as carbon source provided that there are amino acids in the medium necessary for the building blocks of the cellular components. The growth profile of Escherichia coli MG ptrc99a and pbudab is shown in figure.3c and.3d, respectively. The growth of both strains was affected by the lack amino acids. In this case, the pbudab plasmid did not have a positive influence on the growth of E. coli MG. On the other hand, it seemed that the presence of the pbudab plasmid enables E. coli MG to prevent a fast decrease of the ph during the cultivation in M9 minimal medium. Thus, for example in cultivations carried out in medium of ph about, in case of E. coli MG pbudab, the ph decreased from an initial value of.8 to., whereas in case of E. coli MG ptrc99a the ph decreased from an initial ph.88 to. toward the end of the experiment. However, the growth of E. coli MG pbudab was somehow hampered in this case. In order to explain consistently the ph change along the cultivation it is necessary to analyze the synthesis of organic acids and the glucose consumption.

92 A. Cultivation of Serratia plymuthica RVH B. Cultivation of Serratia plymuthica RVH budab::cm OD (nm) Time (hours) ph OD (nm) Time (hours) ph Initial ph:. Initial ph:. Initial ph:. OD: Initial ph. OD: Initial ph. OD: Intial ph. Initial ph:. Initial ph:. Initial ph: ph. OD: Initial ph. OD: Initial ph. OD: Initial ph. C. Cultivation of Escherichia coli MG pbudab D. Cultivation of Escherichia coli MG ptrc99a OD (nm) ph OD (nm) ph Time (hours) Time (hours) Initial ph:. Initial ph:. Initial ph:. OD: Initial ph. OD: Initial ph. OD: Initial ph. Initial ph:. Initial ph:. Initial ph:. OD: Initial ph. OD: Initial ph. OD: Initial ph. Figure.3: Cultivation of Serratia plymuthica RVH (a), Serratia plymuthica RVH budab mutant (b), Escherichia coli MG pbudab (c) and, Escherichia coli MG ptrc99a (d), in M9 minimal medium +.% glucose, adjusted at different ph values with HCl. mm ITPG was added to cultivations with ptrc99a and pbudab strains. 8

93 The growth of Serratia plymuthica RVH and the budab mutant in M9 minimal medium adjusted to ph about. is shown in figure.. It was observed that both strains were able to grow from an initial cell count of about.3 log CFU/ml to 8.8 log CFU/ml in case of the wild type. During the 3 hours of cultivation no increase of ph was noticed in case of the wild type strain. Acidification of medium is in part consequence of the production of organic acids during the fermentation of glucose. From these results, we can state that amino acids would play a very important role in increase of ph at least under these conditions. In addition, the budab genes of Serratia plymuthica RVH would be advantageous in preventing a fast decrease of the ph as compared with the budab mutant L og (c fu/ m l). ph Time (hours) 3 ph: S. plymuthica RVH Cell count: S. plymuthica RVH ph: S. plymuthica RVH budab::cm Cell count: S. plymuthica RVH budab::cm Figure.: Cultivation of Serratia plymuthica RVH in M9 minimal medium +.% glucose (ph.)..2 Growth of enterobacteria in minimal medium containing amino acids The aim of these experiments was to evaluate the effect of different amino acids on the growth of Serratia plymuthica RVH wild type and budab mutant in minimal medium. Cultivations were carried out at 3 o C in minimal medium containing.% of glucose and mm of different individual amino acids (lysine, arginine, ornithine, glutamic acid). These amino acids were chosen because the presence of the respective amino acid 9

94 decarboxylase systems, which are important for acid resistance, has been shown in some enterobacteria (Zhao and Houry, 2) and these systems are related with deacidification. In this way, we tried to find out the reason for the observed ph increase during cultivation. Inoculum preparation was done as described in section Measurements of optical density and ph change were realized during 2 hours of cultivation. Results are shown in the figure.. The growth of S. plymuthica RVH wild type and budab mutant in M9 minimal medium with.% glucose and amino acids was not comparable to that observed in LB broth in previous experiments. Moreover, the growth of both strains was similar in terms of optical density (see figure.a and.c). The growth in complex media enables bacteria to grow better than in media supplied with individual amino acids. Generally, the requirement for nitrogen source is complex for most microorganisms and therefore it is necessary to supply the culture medium with a source of amino acids or proteins such as for example yeast extract or peptone. Regarding to the ph, a ph decrease was observed after 2 hours of cultivation. No increase of ph was noticed even in cultivations with the wild type strain (see figure.b and.d). From these results it is possible to suggest that the growth of butanediol producing bacteria involves on one hand, the breakdown of glucose to generate energy and the concomitant acidification of the medium which causes a decrease of the ph. On the other hand, the use of the produced acidic compounds as carbon source once the glucose is depleted, provided that the medium contains the required aminoacids for the growth, leads to a ph increase and enables the cell population to keep growing until the carbon sources are completely depleted. The presence of the budab genes in Serratia plymuthica RVH would play an important role in preventing a fast decrease of ph during cultivation. Here, the ph in all cultivations reached values higher than. whereas with the budab mutant the ph reached values lower than.. 8

95 OD (3 nm) A. Growth curves of Serratia plymuthica RVH Time (hours) Without AA Lysine Arginine Glutamic acid Ornithine ph B. Change of ph during the growth of Serratia plymuthica RVH Time (hours) ph: without AA ph: with lysine ph: with arginine ph: glutamic acid ph: ornithine C. Growth curves of Serratia plymuthica RVH budab::cm.9 D. Change of ph during the growth of Serratia plymuthica RVH budab::cm OD (3 nm) ph Time (hours) Without AA Lysine Arginine Glutamic acid Ornithine Time (hours) ph: without AA ph: with lysine ph: with arginine ph: with glutamic acid ph: with ornithine Figure.: Growth curves and ph change of Serratia plymuthica RVH (a,b) and, S. plymuthica RVH budab mutant (c,d), cultivated at 3 C in M9 minimal medium +.% glucose + mm amino acid, adjusted at ph. with HCl. 8

96 Cultivations in minimal medium with.% of glucose and different concentrations of casamino acids were also carried out in order to evaluate the influence of a range of amino acids on the ph profile and growth of Serratia plymuthica RVH. Casamino acids are a mixture of amino acids and small peptides derived from casein which can be used by bacteria as nitrogen sources. Figure. (a, b, c and d) shows the growth profile and the change of ph during cultivations of the wild type and budab mutant of S. plymuthica RVH. Here, the growth of both the wild type and the budab mutant strain was greatly improved in presence of casamino acids in comparison with that observed when cultivated in minimal medium with.% glucose and addition of individual amino acids (see figures.a, c). A better growth of S. plymuthica RVH was noticed when higher concentrations of casamino acids were present in the culture medium. Moreover, when comparing the growth of the wild type and budab mutant strain cultivated under the same conditions, there was a remarkable growth advantage due to the presence of casamino acids (figure.a, c). Regarding to the change of ph during the cultivation, a difference between the wild type and budab mutant strain was observed. Contrary to S. plymuthica RVH budab mutant, the wild type strain was able to increase the ph during cultivation in all conditions except when cultivated in medium without casamino acids (figure.b, d). These observations support what was suggested before. Thus, once glucose is depleted, the growth would be somehow related with the concentration and type of amino acids present in the medium, the organic acids produced during fermentation which would be used as carbon source and, the activity of the budab genes of S. plymuthica RVH. As a consequence, an increase of the ph and production of acetoin can be observed. The importance of the butanediol fermentation genes on the growth of this strain is confirmed by comparison with the growth of the budab mutant observed under the same conditions. 82

97 A. Growth curves of Serratia plymuthica RVH. B. Change of ph during the growth of Serratia plymuthica RVH OD (3 nm)... ph Time (hours) Without AA,2% casaa,% casaa % casaa Time (hours) ph: without AA ph: with.% casamino acids ph: with.2% casamino acids ph: with % casamino acids C. Growth curves of Serratia plymuthica RVH budab::cm. D. Change of ph during the growth of Serratia plymuthica RVH budab::cm OD (3 nm)... ph Time (hours) Without AA,2% casaa,% casaa % casaa Time (hours) ph: without AA ph: with.2% casamino acids ph: with.% casamino acids ph: with % casamino acids Figure.: Growth curves and ph change of Serratia plymuthica RVH (a,b) and Serratia plymuthica RVH budab mutant (c,d), cultivated at 3 C in M9 minimal medium +.% glucose + casamino acids, adjusted at ph. with HCl. 83

98 . Growth of enterobacteria in presence of acetoin The aim of these experiments was to evaluate the influence of acetoin, an intermediate product of the butanediol fermentation, on the growth of Serratia plymuthica RVH and budab mutant and, Salmonella Typhimurium LT2 ptrc99a and pbudab. These bacteria were cultivated at 3 C in LB broth containing.% glucose, and mm of acetoin. Culture media were adjusted to ph.,. and. with HCl. Cultivations in media without acetoin were also carried out in order to compare its effect on the growth. The experiments were realized as described in section Results of the growth profile based on optical density are shown in figures.,.8 and.9. The ph was the most important factor that affected the growth of these bacteria. Thus, at ph., the growth of Salmonella Typhimurium LT2 ptrc99a and Serratia plymuthica RVH and budab mutant was completely inhibited (figure.). The pbudab plasmid played an important role on the growth of S. Typhimurium LT2. Thus, measurements of ph at the end of cultivations in all conditions showed an increase of its value, probably caused by the metabolism of this strain. Glucose was the second factor affecting the growth of S. Typhimurium LT2 pbudab. Thus, cultivations in media without glucose affected severely the growth (figure.c, d). Glucose is the main carbon source and its breakdown supplies energy for the growth of microorganisms. LB broth contains only very small amounts of sugars which can be used by the bacteria. Addition of acetoin to the medium apparently did not have a significant effect on the growth of S. Typhimurium LT2 pbudab as observed in figure.b. Acetoin is a neutral, four carbon molecule used as an external energy store by many fermentative bacteria. As stated by Xiao and Xu (2), once superior carbon sources are exhausted, and the culture enters the stationary phase, acetoin can be utilised in order to maintain the culture density. From this statement it is possible to suggest that acetoin was probably used as a carbon source once there was no glucose or other carbon sources. 8

99 OD (nm) A. Cultivation in LB broth +.% glucose ph a fter 8h S. plymuthica RVH: 3.99 S. plymuthica RVH budab::cm: 3.99 S.Typhim urium LT2 ptrc99a:.2 S.Typhim urium LT2 pbudab: Time (hours) Serratia plymuthica RVH SalmonellaTyphimurium LT2 ptrc99a Serratia plymuthica RVH budab::cm SalmonellaTyphimurium LT2 pbudab OD (nm) B. Cultivation in LB broth +,% glucose + mm acetoin ph after 8h S. plymuthica RVH:. S. plymuthica RVH budab::cm:.3 S.Typhimurium LT2 ptrc99a:. S.Typhimurium LT2 pbudab: Time(hours) Serratia plymuthica RVH SalmonellaTyphimurium LT2 ptrc99a Serratia plymuthica RVH pbudab::cm Salmonella Typhimurium LT2 pbudab C. Cultivation in LB broth.9.8. ph after 8h S. plymuthica RVH:. 8 S. plymuthica RVH budab::cm:.8 S.Typhi murium LT2 ptrc99a:.8 S.Typhi murium LT2 pbudab:.3 D. Cultivation in LB broth + mm Acetoin.9.8. ph after 8h S. plymuthica RVH:.8 S. plymuthica RVH budab::cm:.8 S.Typhi murium LT2 ptrc99a:.9 S.Typhi murium LT2 pbudab:. OD (nm)... OD (nm) Time (hours) Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab Time (hours) Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Serratia plymtuhica RVH budab::cm Salmonella Typhimurium LT2 pbudab Figure.: Growth curves of Serratia plymuthica RVH and budab mutant, Salmonella Typhimurium LT2 ptrc99a and pbudab cultivated in: LB broth +.% glucose (a), LB broth +.% glucose + mm acetoin (b), LB broth (c) and LB broth + mm acetoin (d), adjusted at ph. with HCl. mm ITPG was added to cultivations with ptrc99a and pbudab strains. 8

100 Results of cultivations carried out in media adjusted at ph. are shown in figure.8. It was observed that the growth of the strains without butanediol fermentation genes, S. plymuthica RVH budab mutant and S. Typhimurium LT2 ptrc99a, was compromised in comparison with the acetoin producing counterparts when cultivated in the presence of glucose. Apparently, the presence of the budab genes influenced positively the growth of S. plymuthica RVH and S. Typhimurium LT2 pbudab at ph.. On the other hand, addition of acetoin to the culture medium did not have a significant effect on the growth of Salmonella Typhimurium LT2 ptrc99a and pbudab when compared with cultivation carried out in medium without acetoin (see figures.8a and.8b). On the contrary, acetoin seemed to affect negatively the growth of S. plymuthica RVH at ph.. Measurements of ph at the end of cultivations revealed that S. plymuthica RVH and S. Typhimurium LT2 pbudab were able to increase the ph when cultivated in LB containing.% of glucose (figure.8a). In cultivations carried out in LB with.% glucose and mm of acetoin, only a ph increase was observed in cultivation with S. Typhimurium LT2 pbudab. On the contrary, S. plymuthica RVH was not able to increase the ph in this case. From these observations it is possible to suggest that the influence of acetoin on the growth of this strain would depend on the ph of the medium. The mechanisms why acetoin could affect the growth of S. plymuthica RVH at low ph needs to be clarified (figure.8b). In cultivations carried out in LB broth and in LB broth containing mm of acetoin, the growth was severely limited in all strains (figure.8c, d). All strains increased the ph at the end of cultivation, but the ph increase in case of S. Typhimurium LT2 pbudab was the most remarkable. This strain was able to increase the initial ph of. with more than. ph units. As commented before, LB broth contains low amounts of sugars which would be used for the growth of these strains under these conditions. From these observations it is possible to suggest that the rapid depletion of sugars during the cultivations affects the growth and plays an important role in the increase of ph value. 8

101 A. Cultivation in LB broth +.% glucose B. Cultivation in LB broth +.% glucose + mm acetoin.9.8. ph after 8h S. plymuthica RVH:. S. plymuthica RVH budab::cm:.28 S.Typhimurium LT2 ptrc99a:.39 S.Typhimurium LT2 pbudab: ph after 8h S. plymuthica RVH:. S. plymuthica RVH budab::cm:.2 S.Typhimurium LT2 ptrc99a:.38 S.Typhimurium LT2 pbudab:.3 OD (nm)... OD (nm) Time (hours) Serratia plymuthica RVH Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 ptrc99a Salmonella Typhimurium LT2 pbudab Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Time (hours) Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab C. Cultivation in LB broth.9.8. ph after 8h S. plymuthica RVH:.8 S. plymuthica RVH budab::cm:.8 S.Typhimurium LT2 ptrc99a:.9 S.Typhimurium LT2 pbudab:.2 D. Cultivation in LB broth + mm Acetoin.9.8. ph after 8h S. plymuthica RVH:.9 S. plymuthica RVH budab::cm:.9 S.Typhimurium LT2 ptrc99a:. S.Typhimurium LT2 pbudab:.93 OD (nm)... OD (nm) Time (hours) Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Srratia plymuthica RVH budab::cm Salmonella Tyhimurium LT2 pbudab Time (hours) Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab Figure.8: Growth curves of S. plymuthica RVH and budab mutant, S. Typhimurium LT2 ptrc99a and pbudab cultivated at 3 C in: LB broth +.% glucose (a), LB broth +.% glucose + mm acetoin (b), LB broth (c) and, LB broth + mm acetoin (d), adjusted at ph. with HCl. mm ITPG was added to cultivations with ptrc99a and pbudab strains. 8

102 Cultivations carried out in media with ph. were also realized in order to evaluate the growth of S. plymuthica RVH (and budab mutant) and S. Typhimurium LT2 (ptrc99a and pbudab). Figure.9 shows the results of growth based on optical density when cultivated in the same media as before at ph.. At this ph value, the growth in all conditions was greatly enhanced as compared with cultivations carried out at ph. and.. Enterobacteria are neutrophilic microorganisms, thus their metabolism is greatly favoured in environments with neutral ph. The growth of S. plymuthica RVH and S. Typhimurium LT2 pbudab in LB with.% glucose and, in LB with.% glucose and mm acetoin are similar, indicating that acetoin did not exert a significant effect on the growth at this ph value. Another interesting observation was a better growth of S. Typhimurium LT2 ptrc99a in media without glucose compared with the other strains (figure.9c, d). Nonetheless, the growth in all strains was limited compared with cultivations carried out in media containing.% glucose. Measurements of ph at the end of cultivations were also done. It is necessary to point out that the ph varies along the cultivation and the value measured at the end only represents the condition of the metabolism at that time. In cultivations carried out in LB broth with addition of.% glucose and, in LB broth with.% glucose and mm of acetoin a decrease of ph at the end of cultivations was observed (figure.9a, b). One can suggest that the ph has decreased from the beginning as a consequence of the synthesis of acidic compounds such as acetic acid and other organic acids produced during the glucose metabolism. After that, the ph probably has increased by the activity of bacteria that contains the budab genes as observed in previous experiments. The constant value of optical density during stationary phase would not represent necessarily cellular populations but rather turbidity caused by particles released during the cellular death. In other words, optical density does not tell us the amount of viable cells during stationary phase. In cultivations carried out in LB and in LB with mm acetoin, the ph in some cases has decreased towards the end of cultivations. In this case, it is not possible to state whether it has increased during the growth. 88

103 A. Cultivation in LB broth +.% glucose.9.8. B. Cultivation in LB broth +.% glucose + mm acetoin.9.8. OD (nm)... OD (nm) ph after 8h S. plymuthica RVH:. S. plymuthica RVH budab::cm:.8 S.Typhimurium LT2 ptrc99a:. S.Typhimurium LT2 pbudab: Time (hours) Time (hours) ph after 8h S. plymuthica RVH:.8 S. plymuthica RVH budab::cm:. S.Typhimurium LT2 ptrc99a:. S.Typhimurium LT2 pbudab:.3 Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab OD (nm) C. Cultivation in LB broth ph after 8h S. plymuthica RVH:. S. plymuthica RVH budab::cm:. S.Typhimurium LT2 ptrc99a:.92 S.Typhimurium LT2 pbudab: Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Time (hours) Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab OD (nm) D. Cultivation in LB broth + mm acetoin ph after 8h S. plymuthica RVH:. S. plymuthica RVH budab::cm:. S.Typhimurium LT2 ptrc99a:.8 S.Typhimurium LT2 pbudab: Time (hours) Serratia plymuthica RVH Salmonella Typhimurium LT2 ptrc99a Serratia plymuthica RVH budab::cm Salmonella Typhimurium LT2 pbudab Figure.9: Growth curves of Serratia plymuthica RVH and budab mutant, Salmonella Typhimurium LT2 ptrc99a and, pbudab cultivated at 3 C in: LB broth +.% glucose (a), LB broth +.% glucose + mm acetoin (b), LB broth (c) and, LB broth + mm acetoin (d) (ph.). mm ITPG was added to cultivations with ptrc99a and pbudab strains. 89

104 Growth, determined by plating counts, of Serratia plymuthica RVH (and budab mutant) and Salmonella Typhimurium LT2 (ptrc99a and pbudab) was also carried out in LB with.% glucose and mm of acetoin in order to evaluate the influence of acetoin on the growth and ph change profile during cultivations at 3 C and 3 C respectively. Results of growth and ph change are shown in figure.2. The cell number of S. plymuthica RVH was higher than that of the budab mutant after 2 hours as observed in figure.2a. The increase of ph was noticed only in cultivation of the wild type strain. Addition of acetoin did not have a significant influence during the growth of S. plymuthica RVH and budab mutant as compared with results obtained in previous cultivations carried out in media without acetoin (figure.a, b). Results of cultivation of S. Typhimurium LT2 ptrc99a and pbudab are shown in figure.2b. The cell number after 2 hours of the budab containing strain was higher (about two log units) than that of the strain without the budab genes. Regarding to the ph change, S. Typhimurium LT2 pbudab was able to increase the ph toward the end of the cultivation. As stated by Xiao and Xu (2), the production of acetoin by bacteria containing butanediol fermentation genes happens during the exponential growth phase and it would prevent overacidification of the cytoplasm that would result from accumulation of acidic metabolic products such as acetic acid. In addition, acetoin can be used as carbon source once glucose and organic acids are consumed. In our experiments the addition of acetoin to the culture medium did not have a significant effect on the growth of S. Typhimurium LT2 ptrc99a and pbudab as compared with previous results obtained in cultivations carried out in medium without acetoin (see figure.d). 9

105 A. Cultivation of Serratia plymuthica RVH at 3 C Lo g (cfu/ml) ph Time (hours) 3 ph: S.plymuthica RVH Cell number: S. plymuthica RVH ph: S. plymuthica RVH budab::cm Cell number: S. plymuthica RVH budab::cm B. Cultivation of Salmonella Typhimurium LT2 at 3 C Log (cfu/ml) 3.. ph Time (hours) 3 ph: S. Typhimurium LT2 ptrc99a Cell number: S. Typhimurium LT2 ptrc99a ph: S. Typhimurium LT2 pbudab Cell number: S. Typhimurium LT2 pbudab Figure.2: Cultivation of Serratia plymuthica RVH and budab mutant (a) and, Salmonella Typhimurium LT2 ptrc99a and, pbudab (b) in: LB broth +.% glucose + mm acetoin (ph.). mm ITPG was added to cultivations with ptrc99a and pbudab strains. 9

106 . Growth of Enterobacteriaceae in fruit juices Fruit juices are acidic products, the ph of most fruit juices is below.. The majority of Enterobacteriaceae are unable to grow at such a low ph value (Booth and Kroll, 989). Due to the acidity, enteropathogenic bacteria are rarely found on fruit or in fruit juices. However, fruits can be contaminated with fecal material and contamination can also occur during processing of the fruit juices. Coliforms from animal manure have been shown to contaminate apples gathered from the ground in orchards and to persist through juice processing (Goverd et al., 98). The following experiments were done in order to evaluate the effect of the presence of the butanediol genes on the growth capacity of some members of the Enterobacteriaceae in several fruit juices.. Cultivation of enterobacteria in grape juice under oxygen limited conditions The aim of these experiments was to evaluate the growth capacity of Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab) and Cronobacter sakazakii LMG when cultivated in grape juice adjusted to ph.,.2,.,.,.8 and. with NaOH. The change of ph was also evaluated during these cultivations. The experiments were perfomed as explained in section Cultivations were realized in test tubes under oxygen limitation at 2 C and 3 C during days. The results are shown in figure. -.. The results of cultivation of E. coli MG ptrc99a and pbudab at 2 C are shown in figure.. These two strains were not able to grow in grape juice adjusted to ph.,.2 and. when the initial cell count was about. log unit. The cellular population decreased about one log unit after days of cultivation. The survival of cells in acidic products is of considerable importance for food safety since E. coli O:H is capable of causing illness from a very low infective dose, ca. 2 cells (Butler, 99; Smith, 99). Cells cultivated in grape juice are exposed to the effect of the concentration of hydrogen ions (ph) and, concentration of weak organic acids. These acids can dissociate partially in aqueous medium. Thus, combination of these two factors is detrimental for cell growth. Results of ph change showed that, under these conditions, the change of ph was negligible. Moreover, the pbudab plasmid seemed not to be an advantage for the survival of E. coli 92

107 MG. Different results were observed at ph. or higher values. E. coli MG ptrc99a was able to grow from an initial cell concentration of about. log CFU/ml to about. log CFU/ml after days of cultivation and after this time, cells started dying (see figure.e,f). E. coli MG pbudab was able to grow at ph.8 or higher values, but the cell numbers were below these from E. coli MG ptrc99a (see figure.d,e,f). These results were not expected since pbudab plasmid may provide a growth advantage under low ph environments (see section.2. in Chapter ). In this case, not only factors such as ph, temperature and concentration and type of weak organic acids present in grape juice affect the growth, but also the weak organic acids, like acetic and lactic acid, produced during fermentation. The size of the inoculum would also play a very important role. On the other hand, oxygen availability has an important role during the metabolism and it defines in most cases, together with the sugar concentration in the medium, the manner of obtaining energy by the cells. Thus, cultivations in test tubes could be considered as oxygen limited. In static cultivation, the cells at the bottom of the test tube are under more anaerobic conditions since the oxygen is almost depleted and carry out anaerobic fermentation. The cells near the surface fermentate in proportion to the amount of dissolved oxygen. In case of cultivation of E. coli and other enterobacteria, low concentrations of oxygen would enhance the growth and would influence the synthesis of fermentation by-products. In presence of oxygen, the TCA pathway in E. coli and S. Typhimurium would be induced and would operate depending on the amount of dissolved oxygen in the medium (Iuchi et al., 989; Guest and Rusell, 992). The growth of E. coli in fruit juices with ph. is important because it refers to their potentiality to cause infections when fruit juices without thermal treatment are consumed. The ph change was also followed during cultivation. Only E. coli MG pbudab was able to increase the ph as observed after days of cultivation when cultivated in grape juice adjusted to ph.8 or.. This result was already observed when cultivated in synthetic media. In these experiments, the pbudab plasmid did not confer any advantage for E. coli MG. On the contrary, it would be a disadvantage for the growth, at least when cultivated under these conditions during days. 93

108 A: Cultivation in grape juice, ph. Log (cfu/ml) T ime (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a p H ph: E.coli MG pbudab Cell number: E.coli MG pbudab B: Cultivation in grape juice, ph.2 Log(cfu/ml) Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph ph: E.coli MG pbudab Cell number: E.coli MG pbudab Log (cfu/m l) C: Cultivation in grape juice, ph Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph: E.coli MG pbudab Cell number: E.coli MG pbudab ph L og (cfu /m l) D: Cultivation in grape juice, ph Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a p H ph: E.coli MG pbudab Cell number: E.coli MG pbudab Log(c fu/ml) E: Cultivation in grape juice, ph Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph ph: E.coli MG pbudab Cell number: E.coli MG pbudab F: Cultivation in grape juice, ph. Log(cfu/ml) Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph ph: E.coli MG pbudab Cell number: E.coli MG pbudab Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab in grape juice + mm ITPG, at 2 C, adjusted at different ph values with aoh. 9

109 Similar experiments were also carried out at 3 C. The change of cell number and ph at this temperature are shown in figure.2. In all conditions, the behaviour of E. coli was severely affected by increasing the temperature to 3 C as compared with cultivations carried out at 2 C. Cells died rapidly when cultivated in media of ph. and.2, reaching values below the detection limit after the 8 th and th day of cultivation respectively. In this case, significant changes of ph were not observed in both strains. Interesting results were noticed in grape juice of ph. and higher values. Here, the cells (initial cell number about log CFU/ml) started growing from the beginning of cultivation and reached the stationary phase after day, reaching values of -8 log CFU/ml. From that time, the cells started dying slowly toward the th day of cultivation. Then, toward the 8 th day, cells started dying quickly until reaching values below the detection limit. E. coli has an optimal growth temperature of 3 C, but the temperature can influence its growth and metabolism. From the technological point of view, a combination of temperature and time during heat treatment of acidic foods needs to be taken into account in order to destroy the possible human pathogens as for example E. coli O:H. From the point of view of human health, consumption of fresh grape juice stored at refrigeration temperatures without heat treatment could be a risk since it is demonstrated that E. coli can grow or survive for longer times at lower temperatures. The pbudab plasmid did not play a significant positive effect in the ph increase to enhance the growth of E. coli MG. In most cases, this plasmid appeared to influence the growth of E. coli negatively. In comparison with cultivations carried out at 2 C another interesting difference was pointed out when cultivated in grape juice of ph.. At this ph, growth of E. coli at 2 C was not possible compared to that observed at 3 C. From these results one can suggest that, the cultivation at 3 C has provoked a fast growth during the first hours since this temperature was near to the optimal growth temperature. Then, temperature effect combined with the ph and that of the weak organic acids of the grape juice and those produced during fermentation exerted a detrimental effect on the growth and survival. Consumption of grape juice contaminated with pathogenic E. coli which was stored for instance at a temperature of about 3 C during 8 days in tropical countries could cause serious foodborne infection diseases. 9

110 L og(cfu/ml) A: Cultivation in grape juice, ph Time (days) ph: E.coli MG ptrc99a ph: E.coli MG pbudab Cell number: E.coli MG ptrc99a Cell number: E.coli MG pbudab ph B: Cultivation in grape juice, ph.2 Log(cfu /ml) Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a p H ph: E.coli MG pbudab Cell number: E.coli MG pbudab L og(cfu/ml) C: Cultivation in grape juice, ph Time (days) ph: E.coli MG ptrc99a ph: E.coli MG pbudab Cell number: E.coli MG ptrc99a Cell number: E.coli MG pbudab ph Log(cfu/ml) D: Cultivation in grape juice, ph Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph ph: E.coli MG pbudab Cell number: E.coli MG pbudab E: Cultivation in grape juice, ph.8 Log(cfu/ml) Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph ph: E.coli MG pbudab Cell number: E.coli MG pbudab F: Cultivation in grape juice, ph. Log (c fu /ml) Time (days) ph: E.coli MG ptrc99a Cell number: E.coli MG ptrc99a ph ph: E.coli MG pbudab Cell number: E.coli MG pbudab Figure.2: Cultivation of Escherichia coli MG ptrc99a and pbudab in grape juice + mm ITPG, at 3 C, adjusted at different ph values with aoh. 9

111 Cultivations with Salmonella Typhimurium LT2 ptrc99a and pbudab in grape juice were also carried out at 2 and 3 C and the results are shown in figures.3 and. respectively. Here, a positive effect of the pbudab plasmid on the growth of S. Typhimurium LT2 could be observed when cultivated in grape juice adjusted to ph. and.2. These results are contrary to the observations with E. coli MG. Under these conditions, the cell number of S. Typhimurium LT2 pbudab increased from about log CFU/ml to about. log CFU/ml after 8 days of cultivation. Moreover, the survival of S. Typhimurium LT2 ptrc99a in grape juice with ph. was severely affected during the first days, reaching values below to the detection limit. Research has shown that Salmonella spp. can survive or even grow in acidic media, down to ph. (Baik et al., 99; Foster, 99). In cultivations carried out in grape juice with ph., an opposite effect of the pbudab plasmid on the growth of S. Typhimurium LT2 was observed. In addition, S. Typhimurium LT2 ptrc99a and pbudab were able to grow during the first days in cultivations carried out in grape juice adjusted to ph. or higher values. After this time, a decrease of the cell number was observed. The cellular death rate was higher in cultivations with S. Typhimurium LT2 pbudab. From these results it is possible to state that the pbudab plasmid seemed to give some advantage to S. Typhimurium LT2 when cultivated at low ph values such as. to.. On the other hand, at ph. or higher values, there was rather a negative effect on the growth and survival of S. Typhimurium LT2. Regarding to the ph change during cultivation, an increase of ph was observed in all conditions at the end of the cultivations. But this ph increase was not related to the enhancement of the growth of S. Typhimurium LT2 pbudab. S. Typhimurium is a foodborne pathogen which causes infection disease at low cellular concentrations. In this case, consumption of low ph fruit juices stored under these conditions for periods of days or less does not assure their safety. 9

112 Log(cfu/ml) A: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell numbe: S.Typhimurium LT2 ptrc99a ph ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Log(cfu/ml) B: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph ph: S.Typhim urium LT2 pbudab Cell number: S.Typhim urium LT2 pbudab C: Cultivation in grape juice, ph Log(cfu/ml) Time (days) ph: S.Typhimurium LT2 ptrc99a ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 pbudab ph D: Cultivation in grape juice, ph.. E: Cultivation in grape juice, ph.8. F: Cultivation in grape juice, ph.. Lo g(cfu/ml) Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph ph: S.Typhimuirum LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Log(cfu/ml) Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a p H ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Log (cfu/m l) Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Figure.3: Cultivation of Salmonella Typhimurium LT2 ptrc99a and pbudab in grape juice + mm ITPG, at 2 C, adjusted at different ph values with aoh. 98

113 The graphs of the cell number and ph change during cultivations of Salmonella Typhimurium LT2 ptrc99a and pbudab in grape juice at 3 C are shown in figure.. Both strains were able to grow from ph., but the growth was clearly enhanced at higher ph. A negative effect of the pbudab plasmid on the growth of S. Typhimurium LT2 was noticed in all culture conditions. These results were also noticed when cultivated at 2 C. Furthermore, an increase of temperature to 3 C has increased the growth and death rate in all culture conditions as compared with that observed when cultivated at 2 C. The effect of the temperature on the growth rate is due to the fact that this bacterium grows optimally at 3 C thus, temperatures near this value favoured its growth. On the other hand, the increase of the death rate would be connected to the fact that temperature increases the movement of protons and weak acids throughout the cytoplasmic membrane thus, acidification of the cytosol happens rapidly. As a consequence, the energetic metabolism fails and the cells die quickly. Another difference between the growth of both strains is that, S. Typhimurium LT2 ptrc99a reached the maximum cell number toward the 2 nd day of cultivation and, in case of S. Typhimurium LT2 pbudab, it was only 2 hours. The maximum cell number attained by S. Typhimurium LT2 pbudab was 8. log CFU/ml when cultivated in medium of ph., while in case of S. Typhimurium LT2 ptrc99a, the maximum cell number was 8.3 log CFU/ml when cultivated also at ph.. Regarding to the ph change, an increase of ph toward the end of cultivation in all culture conditions was noticed. The increase of ph would be linked to the activity of the pbudab plasmid, but unfortunately, this effect was not enough to keep the cells growing. The temperature is not the only factor affecting the growth of S. Typhimurium LT2, other factors such as the ph, organic acids of the grape juice and those produced during fermentation also contribute to this effect. 99

114 Log(cfu/ml) A: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Log(cfu/ml) B: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Log(cfu/ml) C: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a ph ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Lo g(cfu /ml) D: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a p H ph: S.Typhimuirum LT2 pbudab Cell number: S.Typhimurium LT2 pbudab E: Cultivation in grape juice, ph.8 L og (cfu/ml) ph: S.Typhim urium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a 2 8 Time (days) p H ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Lo g(cfu/m l) F: Cultivation in grape juice, ph Time (days) ph: S.Typhimurium LT2 ptrc99a Cell number: S.Typhimurium LT2 ptrc99a p H ph: S.Typhimurium LT2 pbudab Cell number: S.Typhimurium LT2 pbudab Figure.: Cultivation of Salmonella Typhimurium LT2 ptrc99a and pbudab in grape juice + mm ITPG, at 3 C, adjusted at different ph values with aoh.

115 No investigations about the growth and survival of Cronobacter sakazakii in fruit juices were found. This species belongs to the butanediol fermenters and is considered as an opportunistic pathogen. Therefore, in this study Cronobacter sakazakii LMG was cultivated in grape juice adjusted to ph.,.2,.,.,.8 and. with NaOH. The data are represented in figure.. C. sakazakii LMG was able to grow in all culture conditions at 2 C and 3 C. Comparisons of cultivations carried out at both temperatures showed that C. sakazakii LMG grows better at 3 C in media of ph.,.2 and. than at 2 C, while in grape juice of higher ph values the effect of temperature seemed not to influence the growth significantly. The optimal temperature for growth of C. sakazakii LMG is about 3 C. Lower temperatures can influence the growth rate, especially when other parameters, like ph, also deviate from their optimal value. The maximum cell number of C. sakazakii LMG at 3 C in media of ph.,.2 and. attained values of above 8. log CFU/ml. After reaching this value, the cell number remained almost constant until the end of the cultivation. The growth was in correlation with the ph of the juice so, the higher the ph of the juice, the higher the maximal growth attained. Cultivations in grape juice of ph. and higher values were more favourable. Cells rapidly attained populations of above 8. log CFU/ml in 2 hours. After that time, the cell number hardly increased toward the end of cultivation not exceeding 9. log CFU/ml. Regarding to the change of ph during the cultivations, it was observed that C. sakazakii LMG was able to increase the ph in all culture conditions. The ability to increase the ph was also confirmed when cultivated previously in synthetic medium (see section. in chapter ). C. sakazakii LMG contains the budab genes, which codes for the butanediol pathway, in the genome. Thus, the increase of ph would be connected somehow with the capacity of producing butanediol.

116 A: Cultivation of in grape juice, ph.. B: Cultivation in grape juice, ph.2. C: Cultivation in grape juice, ph.. Log(cfu/m l) p H Log (cfu /ml) ph Log (cfu/ml) ph 2 8 Time (days) ph: Cultivation at 2 C ph: Cultivation at 3 C Cell number: Cultivation at 2 C Cell number: Cultivation at 3 C Time (days) ph: Cultivation at 2 C ph: Cultivation at 3 C Cell number: Cultivation at 2 C Cell number: Cultivation at 3 C Time (days) ph: Cultivation at 2 C ph: Cultivation at 3 C Cell number: Cultivation at 2 C Cell number: Cultivation at 3 C 3. D: Cultivation in grape juice, ph.. E: Cultivation in grape juice, ph.8. F: Cultivation in grape juice, ph.. Log (cfu/m l) Time (days) ph: Cultivation at 2 C ph: Cultivation at 3 C Cell number: Cultivation at 2 C Cell number: Cultivation at3 C ph Log(cfu/ml) Time (days) ph: Cultivation at 2 C ph: Cultivation at 3 C Cell number: Cultivation at 2 C Cell number: Cultivation at 3 C ph Log(cfu/ml) Time (days) ph: Cultivation at 2 C ph: Cultivation at 3 C Cell number: Cultivation at 2 C Cell number: Cultivation at 3 C Figure.: Cultivation of Cronobacter sakazakii LMG at 2 C and 3 C in grape juice, adjusted at different ph values with aoh ph 2

117 .2. Cultivation of enterobacteria in worldshake juice under oxygen limited conditions Fruit juices normally have low ph values as shown in table 2.2. Worldshake juice is a mixture of many fruit juices as shown in the table 3., chapter 3. The low ph values of fruit juices are in part due to the presence of organic acids which supply protons to the medium. Acidic environments are known to be harsh environments for the growth of enterobacteria. Nevertheless, in previous experiments it was demonstrated that Escherichia coli MG, Salmonella Typhimurium LT2 and Cronobacter sakazakii LMG are able to grow at low ph values typically found in many fruit juices. Cultivations in worldshake juice adjusted at different ph values with NaOH were carried out in order to evaluate the capacity of these bacteria to grow and attain cell numbers that could produce an infection disease. The data of the cell numbers and ph change during the cultivations are shown in figures. and.. The growth or survival of E. coli MG ptrc99a and pbudab in worldshake juice adjusted to ph 3.8,.,.2,.,. and.8 are shown in figures.a and.b, respectively. They were able to grow at ph.8, while growth below this value was difficult or even impossible during 3 days of cultivation. In juice of ph 3.8,.,.2 and. the cell number remained almost constant during days and then, cells started dying toward the end of the cultivation. In cultivations carried out in juice of ph., both strains were not able to grow and the cell population remained almost constant during the experiment. The pbudab plasmid did not play any positive role to enhance the growth of E. coli MG pbudab when cultivated under these conditions. Regarding to the ph change during the cultivation, only E. coli MG pbudab was able to increase the ph (see figure.b). Nevertheless, the increase of ph during cultivation apparently did not contribute to the growth of this bacterium at least as cultivated under these conditions. Similar results were found when cultivated these two strains in apple and grape juice (see figures.a,b and.a,b). The growth or survival of Salmonella Typhimurium LT2 ptrc99a and pbudab in worldshake juice is shown in figure.c and.d, respectively. A better growth of S. Typhimurium LT2 ptrc99a in comparison with S. Typhimurium LT2 pbudab was observed. This negative effect of pbudab plasmid in S. Typhimurium LT2 was also 3

118 noticed in previous experiments when cultivated in grape and apple juice under similar conditions. S. Typhimurium LT2 ptrc99a and pbudab were able to grow at ph. and higher values. The growth rates were higher for S. Typhimurium LT2 ptrc99a cultivated in all ph conditions as compared with those observed in cultivations of S. Typhimurium LT2 pbudab under similar conditions. The maximum cell number attained by S. Typhimurium LT2 ptrc99a was. log CFU/ml, while in case of S. Typhimurium LT2 pbudab the maximum cell number has hardly reached. log CFU/ml, in both cases after 2 days when cultivated in worldshake juice adjusted at ph.8. Below ph., growth was rather difficult and even impossible and, in some cases after days the cells under these conditions started dying progressively.

119 A: Cultivation of Escherichia coli MG ptrc99a B: Cultivation of Escherichia coli MG pbudab. Log (cfu/ml) ph Log (cfu/ml) ph Time (days) Initial ph: 3.8 Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph ph. Cell count: Initial ph. Cell count: Initial ph Time (days) Initial ph: 3.8 Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 3. Log (cfu/ml) C: Cultivation of Salmonella Typhimurium LT2 ptrc99a Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Initial ph.8 Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph ph D: Cultivation of Salmonella T yphimurium LT2 pbudab Log (cfu/ml) Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Initial ph.8 Cell count: initial ph 3.8 cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 2 C in worldshake juice + mm ITPG, adjusted at different ph values with aoh ph

120 The growth or survival of Cronobacter sakazakii LMG in worldshake juice is shown in figure.. This strain was able to grow at ph.2. Below this value, growth was not possible. Growth of C. sakazakii LMG was enhanced at higher ph values, thus the higher the ph the higher the growth rate at least as observed under these conditions. The maximum cell number attained by C. sakazakii LMG was about.9 log CFU/ml after 8 days of cultivation at ph.8. After this time, the cell number remained almost constant during the experiment. Cultivation of Cronobacter sakazakii LMG Log (cfu/ml) ph Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Initial ph.8 Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 Figure.: Cultivation of Cronobacter sakazakii LMG at 2 C in worldshake juice, adjusted at different ph values with aoh Cultivation of enterobacteria in ectar Multifruit juice under anaerobic conditions The aim of these experiments was to evaluate the growth of E. coli MG (ptrc99a and pbudab), S. Typhimurium LT2 (ptrc99a and pbudab) and C. sakazakii LMG when cultivated during days, at 3 C, under anaerobic conditions in Nectar Multifruit juice adjusted to ph 3.8,.,.,.3,.,. and. with NaOH. Test tubes partially filled with juice were covered with paraffin oil in order to create anoxic conditions. The results are shown in figures.8 and.9.

121 The minimum ph for growth of E. coli MG ptrc99a and pbudab was.. Below this value, growth was not possible. Under these conditions, a clear advantage of the pbudab plasmid on the growth of E. coli MG was not noticed. The maximum cell number attained by E. coli MG ptrc99a was about.9 log CFU/ml, while in cultivations carried out with E. coli MG pbudab, the maximum cell number attained was 8.3 log CFU/ml when cultivated in juice of ph.. In cultivations under anaerobic conditions, the cells grew until reaching similar cell numbers as observed in cultivations carried out in grape juice at 3 C under oxygen limitation (see figure.2). Enterobacteriaceae are facultative anaerobic microorganisms able to grow in the absence of oxygen. Under anaerobic conditions, the TCA cycle can no longer provide energy and instead fermentative pathways are activated and supplies energy for cell growth. Regarding to the change of ph during cultivation, a ph increase was only observed in E. coli MG pbudab when cultivated in media of ph.3. The minimum ph for growth of S. Typhimurium LT2 ptrc99a and pbudab under these conditions was.. Below this value, growth was not possible. Nevertheless, the growth was enhanced when the initial ph of the juice was higher. Apparently, no growth differences were observed between cultivations carried out with S. Typhimurium LT2 ptrc99a and pbudab in all culture conditions. The effect of pbudab plasmid on the growth of S. Typhimurium LT2 under these conditions could not be clearly demonstrated. Moreover, the maximum cell number of S. Typhimurium LT2 ptrc99a attained was about. log CFU/ml, while in case of S. Typhimurium LT2 pbudab this was about. log CFU/ml when cultivated in medium of ph.. The potential survival of Salmonella under extreme environmental conditions is a major public health concern. Salmonella can survive in low ph environments such as fruit juices (Parish et al., 99). Acid tolerance at ph <. of Salmonella increases with increasing temperature (Humphrey and Hart, 988; Thomas et al., 992).

122 A: Cultivation of Escherichia coli MG ptrc99a B: Cultivation of Escherichia coli M G pbudab Log (cfu/ml) ph Log (cfu/ml) ph 2 Time (days) 3. 2 Time (days) 3 Initial ph: 3.8 Initial ph:. Initial ph:. Initial ph:.3 Initial ph:. Initial ph:. Initial ph:. C.count: Initial ph 3.8 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph.3 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph. Initial ph: 3.8 Initial ph:. Initial ph:. Initial ph:.3 Initial ph:. Initial ph:. Initial ph:. C.count: Initial ph 3.8 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph.3 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph. C: Cultivation of SalmonellaTyphimurium LT2 ptrc99a. D: Cultivation of Salmonella Typhimurium LT2 pbudab Log (cfu/ml) Time (days) Initial ph: 3.8 Initial ph:. Initial ph:. Initial ph:.3 Initial ph:. Initial ph:. Initial ph:. C.count: Initial ph 3.8 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph.3 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph ph Log (cfu/ml) Time (days) Initial ph: 3.8 Initial ph:. Initial ph:. Initial ph:.3 Initial ph:. Initial ph:. Initial ph:. C.count: Initial ph 3.8 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph.3 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph. Figure.8: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 3 C, under anaerobic condition in ectar Multifruit juice + mm ITPG, adjusted at different ph values with aoh ph 8

123 In case of Cronobacter sakazakii LMG, the minimum ph for growth was.. Growth below this value was not possible (see figure.9). The maximum cell number attained when cultivated in medium of ph. was about 8. log CFU/ml. This result also confirms that growth of this strain in fruit juices reaches similar cell numbers as observed when cultivated under oxygen limited conditions. C. sakazakii is considered as an opportunistic pathogen, particularly implicated in severe foodborne diseases in neonates and infants. This bacterium has been found in powder infant formulae. Nevertheless, results from these experiments confirmed that they could be potentially found in acidic beverages such as fruit juices since they are able to grow and survive in low ph fruit juices. On the other hand, fruit juices are normally sold in hermetic packages in order to maintain anoxic conditions. But, this condition would not avoid the growth of this bacterium as observed in this experiment. Moreover, a ph increase of the juice was noticed when cultivated in media of ph. and.3 toward the end of the cultivation. Increase of ph under these conditions could have influenced the growth of C. sakazakii LMG. Cultivation of Cronobacter sakazakii LMG Log (cfu/ml) ph 2 Time (days) 3 Initial ph: 3.8 Initial ph:. Initial ph:. Initial ph:.3 Initial ph:. Initial ph:. Initial ph:. C.count: Initial ph 3.8 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph.3 C.count: Initial ph. C.count: Initial ph. C.count: Initial ph. Figure.9: Cultivation of Cronobacter sakazakii LMG at 3 C, under anaerobic condition in ectar Multifruit juice, adjusted at different ph values with aoh. 9

124 .. Cultivation of enterobacteria in apple juice... Cultivation of enterobacteria in apple juice under oxygen limited conditions The aim of these experiments was to evaluate the growth of Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab) and Cronobacter sakazakii LMG when cultivated in apple juice adjusted to ph 3.8,.,.2,. and. with NaOH. The cultivations were carried out at 2 C during 3 days. The results are represented in figures. and.. Growth of E. coli MG ptrc99a was not possible when cultivated in apple juice with ph lower than.. During cultivations carried out in apple juice of ph 3.8,. and.2, the initial cell number (about. log CFU/ml) remained almost constant (see figure.a). Enteropathogenic E. coli can cause foodborne diseases in concentrations lower than log CFU/ml. Investigations showed that E. coli O:H is acid tolerant, showing slight growth in apple juice in large inocula, ml -, but from small inocula, 2 ml -, viability was maintained for 2 days and lost gradually over the next 3 weeks (Zhao et al., 993). Survival of E. coli in apple juice would be related in part to the ability to maintain ph homeostasis, pumping the hydrogen ions outside the cell. In this case, the obtained energy is almost entirely used to keep the cell population alive during cultivation. On the other hand, survival of cells would also be connected to the ability to resist the effect of weak organic acids normally present in the apple juice. Weak organic acids are known to penetrate the cell membrane easily because they do not dissociate completely in aqueous medium. Acidification of the cytoplasm may prevent growth by inhibition of glycolysis and as a consequence stops the cellular metabolism. In comparison with cultivations carried out in grape juice at 2 C (see figure.), it was observed that E. coli MG ptrc99a was not able to grow in grape juice at ph.. In this case, other factors such as the chemical composition and content and type of organic acids present in the apple juice would be involved. Regarding to the ph during the cultivations, no significant change was noticed toward the end of the experiment. For E. coli MG pbudab, growth was not possible at ph lower than.. On the other hand, a slight growth was observed when cultivated in media of ph. and.. Under these conditions, the maintenance of the cellular population during the cultivation has

125 involved the use of energy. The increase of ph as observed in cultivations carried out in media of ph. and. would be connected with the activity of pbudab plasmid which could have contributed somehow to the slight growth of E. coli MG pbudab under these conditions. Comparisons between cultivations carried out with E. coli MG ptrc99a and pbudab showed that the pbudab plasmid apparently did not play any significant role on the growth of E. coli MG under these conditions. Different results were obtained when Salmonella Typhimurium LT2 ptrc99a and pbudab were cultivated in apple juice (figure.c,d). In these experiments, S. Typhimurium LT2 ptrc99a grew better than the pbudab counterpart in media of ph. and higher values. This observation confirms that pbudab would not necessarily represent a real advantage for the growth of S. Typhimurium LT2 when cultivated in apple juice under these conditions. S. Typhimurium LT2 ptrc99a reached cell numbers of above 8 log CFU/ml when cultivated in medium of ph., while S. Typhimurium pbudab hardly reached log CFU/ml under the same conditions. Moreover, growth of S. Typhimurium LT2 was not possible in apple juice adjusted at ph 3.8. At this ph, the cells died slowly from an initial cell number of. log CFU/ml but the cell numbers remained above the detection limit during 3 days of cultivation. Previous studies showed that Salmonella remained viable in apple juice at ph 3. for 3 days (Goverd et al., 98). The acid-tolerance response of Salmonella Typhimurium has also been shown to provide protection against organic acids (Baik et al., 99). The higher the ph, the higher the growth rate and the higher the maximum cell number of S. Typhimurium LT2 ptrc99a, at least as observed under these conditions. Regarding to the ph change, only S. Typhimurium LT2 pbudab was able to increase the ph of the apple juice. The capacity to increase the ph of the juice did not represent an advantage for growth enhancement in this case. Similar results were observed when cultivated in grape juice under similar conditions (see figure.3). The importance of S. Typhimurium to grow or survive in low acidic environments is the fact that many foodborne diseases caused by Salmonella has been attributed in many cases to consumption of unpasteurized fruit juices such as orange juice (Birkhead et al., 993).

126 A: Cultivation of Escherichia coli MG pt rc99a B: Cultivation of Escherichia coli MG pbudab Log (cfu/ml).. ph Log (cfu/ml)... ph Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. 3. C: Cultivation of Salmonella Typhymurium LT2 ptrc99a D: Cultivation of Salmonella Typhimurium LT2 pbudab. Log (cfu/ml) Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Cell count: Initial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph ph Log (cfu/ml) Time (days) Internal ph 3.8 Internal ph. Internal ph.2 Internal ph. Internal ph. Cell count: Internal ph 3.8 Cell count: Internal ph. Cell count: Internal ph.2 Cell count: Internal ph. Cell count: Internal ph. Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 2 C in apple juice + mm ITPG, adjusted at different ph values with aoh ph 2

127 The ability of Cronobacter sakazakii LMG to grow in fruit juices was already noticed in previous experiments such as in grape juice at 2 C and 3 C. Here, cultivations of C. sakazakii LMG were also done in apple juice in order to evaluate the growth capacity of this strain in this juice. The data for the cell number and ph change during the cultivations are shown in figure.. Results showed that this strain was able to grow in apple juice adjusted to ph. and higher values, but below this value, growth was not possible. The growth rate was influenced by the ph of the apple juice. Thus, ph values higher than.2 increased the growth rate of C. sakazakii LMG when cultivated under these conditions. The maximal cell number (about 9. log CFU/ml) was reached after days when cultivated in media of ph. and.. After that time, the cell number decreased with almost one logarithmic unit and remained almost contant during the rest of the cultivation. Cultivation of Cronobacter sakazakii LMG Log (cfu/ml) ph Time (days) Initial ph 3.8 Initial ph. Initial ph.2 Initial ph. Initial ph. Cell count: intial ph 3.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Figure.: Cultivation of Cronobacter sakazakii LMG at 2 C in apple juice, adjusted at different ph values with aoh Cultivation of enterobacteria in apple juice under anaerobic conditions The aim of these experiments was to evaluate the growth of Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab) and Cronobacter sakazakii LMG when cultivated during days, at 2 C, under 3

128 anaerobic conditions in apple juice adjusted to ph.,.2,.,. and.8 with NaOH. Test tubes partially filled with apple juice were covered with paraffin oil in order to create anoxic conditions. The cell numbers and the ph change during these experiments are represented in figures.2 and.3. Temperature plays an important role in the growth and in the extent of the cultivation as observed in those carried out in grape juice at 2 C and 3 C (see figures. to.). The results of cultivation of E. coli MG ptrc99a and pbudab in apple juice are shown in figures.2a,b. It was noticed that both strains grew in similar pattern when cultivated under similar conditions. No significant difference could be demonstrated. In this case, pbudab plasmid would not represent an advantage for E. coli MG. The maximum cell number attained by E. coli MG ptrc99a and pbudab, when cultivated in apple juice of ph.8, was.2 log CFU/ml and. log CFU/ml respectively. These values remained almost constant during the following 9 days of cultivation. Moreover, the minimum ph for growth of both strains was.. Below this value, growth of E. coli MG was not possible. Nevertheless, the initial cell population of about. log CFU/ml remained almost constant during two weeks. This clearly demonstrates that when microorganisms are unable to multiply in acidic conditions, they may be able to survive for a prolonged period of time. In case of foodborne pathogens this may have important consequences for the food safety. For example, outbreaks of food poisoning have been attributed to the survival of Escherichia coli O:H in unpasteurized apple juice and cider (Besser, 993; CDC, 99; CDC, 99), and to the survival of Salmonella spp. in unpasteurized apple cider (CDC, 9) and in orange juice (Cook et al., 998; Parish, 99). Cultivations carried out with S. Typhimurium LT2 ptrc99a and pbudab showed that both strains were able to grow under all culture conditions (see figure.2c,d). Apparently, no growth difference between these two strains could be noticed in all conditions. From these results, it is possible to state that the pbudab plasmid did not represent an advantage for the growth of S. Typhimurium LT2. In some cases, it could even negatively influence the growth of S. Typhimurium LT2. Similar results were already observed in previous experiments carried out with different fruit juices under similar conditions. In these experiments, the maximum cell number attained by S. Typhimurium LT2 ptrc99a and

129 pbudab, when cultivated in apple juice of ph.8, were 8.38 log CFU/ml and 8.2 log CFU/ml respectively. Furthermore, in all culture conditions the cell population showed to decrease toward the end of the cultivation. It was clearly observed that, the growth rate and also the maximum cell number reached in each culture condition was related with the initial ph of the apple juice. An increase of ph during the cultivation was only noticed when S. Typhimurium LT2 pbudab was cultivated.

130 A: Cultivation of Escherichia coli MG ptrc99a B: Cultivation of Escherichia coli MG pbudab Log (cfu/ml).. ph Log (cfu/ml).. ph Time (days) Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph Time (days) Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 3 C: Cultivation of Salmonella Typhimurium LT2 ptrc99a D: Cultivation of Salmonella T yphimurium LT2 pbudab Log (cfu/ml) ph Log (cfu/ml) ph Time (days) Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph Time (days) Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 Figure.2: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 2 C, under anaerobic condition in apple juice + mm ITPG, adjusted at different ph values with aoh.

131 Figure.3 shows the observations of the cell number and ph change during anaerobic cultivation of Cronobacter sakazakii LMG wild type and budab mutant in apple juice. The wild type strain was able to grow in all culture conditions (see figure.3a). The maximum cell number (8.8 log CFU/ml) was observed when cultivated in apple juice of ph.8. Surprisingly, the cell population in all culture conditions remained at their highest values during the rest of the cultivations. In this case, the maximum cell number attained in each culture condition was clearly in relation with the initial ph value of the culture medium. Thus, the higher the initial ph, the higher the maximum cell number attained. Slight increase of ph was observed in some culture conditions during the cultivations. The increase of ph or the maintenance of its value during the cultivation would be connected with the presence and the activity of the budab genes contained in the genome of C. sakazakii LMG in order to keep favourable conditions for the growth. The influence of the presence of the budab genes was clearly evidenced when the growth of the wild type and the budab mutant was compared (see figures.3a,b). In this case, the budab genes apparently had some effect on the growth of C. sakazakii LMG when cultivated in juices adjusted to low ph values (. and.2). During cultivations carried out in apple juice of higher ph values (ph >.2), the growth of both strains (wild type and budab mutant) showed to attain similar cell populations. Finally, anoxic conditions apparently did not affect the growth of C. sakazakii LMG in terms of maximum cell numbers since they are able to reach populations of about 8.8 log CFU/ml or even more.

132 A: Cultivation of Cronobacter sakazakii LM G 9 8 Log (cfu/ml) ph Time (days) Initial ph:. Initial ph:.2 Initial ph:. Initial ph:. Initial ph:.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 3 B: Cultivation of Cronobacter sakazakii LMG budab ::Cm Log (cfu/ml) ph Time (days) 3. Initial ph. Initial ph.2 Initial ph. Initial ph. Initial ph.8 Cell count: Initial ph. Cell count: Initial ph.2 Cell count: Initial ph. Cell count: Initial ph. Cell count: Initial ph.8 Figure.3: Cultivation of Cronobacter sakazakii LMG wild type and budab mutant at 2 C, under anaerobic condition in apple juice, adjusted at different ph values with aoh...3. Cultivation of enterobacteria in apple juice based on optical density..3.. Oxygen limitated conditions The aim of these experiments was to evaluate the growth of Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab), Serratia 8

133 plymuthica RVH and budab mutant and Cronobacter sakazakii LMG when cultivated in apple juice adjusted to different ph values with NaOH. Growth based on optical density (3 nm) was measured during 8 hours of cultivation in a non-stop multiscan spectrophotometer. The experiments were realized in similar way as described in section , chapter 3, but instead of synthetic medium, here was used sterilized apple juice. The results of these experiments are shown in figures. and.. The growth of E. coli MG ptrc99a and pbudab in apple juice is shown in figure.3a, b. It was observed that the minimum ph for growth of these two strains was.2. Below this ph value, growth was not possible at least as observed after the 8 hours of cultivation. The mechanism of inhibition by low ph concerns the acidification of the cytoplasm which may prevent growth in part by inhibition of ph-sensitive enzymes (Burroughs, 98). Nevertheless, growth at ph.2 of both strains was observed after hours of cultivation. In general, growth of E. coli MG was very difficult in all culture conditions and reached very low values of optical density. Under these conditions, cultivations carried out with E. coli MG pbudab showed somehow a better growth than those carried out with E. coli MG ptrc99a. In cultivations with E. coli MG ptrc99a, a ph decrease was observed at the end of the cultivation. In case of E. coli MG pbudab, the increase of ph observed at the end of the experiment would be related to the activity of the pbudab plasmid. Similar results were observed in cultivations realized with Salmonella Typhimurium LT2 ptrc99a and pbudab (see figure.3c, d). In this case, the minimal ph for growth of S. Typhimurium LT2 ptrc99a and pbudab were. and 3.9, respectively. The growth at ph. was possible after 8 hours of cultivation, and at ph 3.9 after 2 hours. Under these conditions, the growth of S. Typhimurium LT2 ptrc99a and pbudab was difficult and reached very low values of optical density. In cultivations with S. Typhimurium LT2 ptrc99a, no significant decrease of the ph was observed at the end of the cultivation. This would reflect the poor growth of this bacterium under these conditions. In case of S. Typhimurium LT2 pbudab, the increase of ph observed at the end of the cultivations would also be related to the activity of the pbudab plasmid. 9

134 A: Growth curves of Escherichia coli MG ptrc99a B: Growth curves of Escherichia coli MG pbudab. ph after 2h. ph after 2h OD (3 nm) ph 3.: ph 3.2 ph 3.: ph 3.3 ph 3.8: ph 3.8 ph 3.9: ph 3.9 ph.: ph. ph.: ph. ph.2: ph.2 ph.3: ph.3 ph.: ph.38 ph.: ph. ph.: ph. ph.: ph. OD (3 nm) ph 3.: ph 3.2 ph 3.: ph 3.3 ph 3.8: ph 3.8 ph 3.9: ph 3.9 ph.: ph. ph.: ph. ph.2: ph.2 ph.3: ph. ph.: ph. ph.: ph. ph.: ph. ph.: ph Time (hours) Time (hours) ph. ph. ph. ph. ph.3 ph.2 ph. ph. ph. ph. ph. ph.3 ph.2 ph. C: Growth curves of Salmonella Typhimurium LT2 ptrc99a D: Growth curves of Salmonella Typhimurium LT2 pbudab. ph after 2h. ph after 2h OD (3 nm) ph 3.: ph 3.3 ph 3.: ph 3. ph 3.8: ph 3.83 ph 3.9: ph 3.9 ph.: ph. ph.: ph.3 ph.2: ph.22 ph.3: ph.3 ph.: ph. ph.: ph.8 ph.: ph. ph.: ph. OD ( 3 nm) ph 3.: ph 3.3 ph 3.: ph 3.3 ph 3.8: ph 3.8 ph 3.9: ph.8 ph.: ph.2 ph.: ph.32 ph.2: ph.3 ph.3: ph.3 ph.: ph. ph.: ph. ph.: ph.8 ph.: ph Time (hours) Time (hours) ph. ph. ph. ph. ph.3 ph.2 ph. ph. ph 3.9 ph 3.8 ph. ph. ph. ph. ph.3 ph.2 ph. ph. ph 3.9 ph 3.8 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab at 3 C in apple juice + mm ITPG, adjusted at different ph values with aoh. 2

135 The growth curves of Serratia plymuthica RVH and budab mutant are shown in figure.a,b. It was observed that the minimum ph for growth of both strains (wild type and budab mutant) was.. The better growth of the wild type strain was observed at ph. and.. On the contrary, the growth of the budab mutant strain was rather difficult and reached only very low values of optical density. These results clearly show the importance of the budab genes on the growth of the wild type strain at ph. and.. On the other hand, the influence of these genes on the growth at lower ph is less significant. The ph decrease measured at the end of the cultivation would be a result of the metabolic activity of the budab mutant strain, which is unable to increase the ph, contrary to the observed cultivations carried out with the wild type strain. The increase of ph would be related to the acitivity of the budab genes of Serratia plymuthica RVH. The growth curves of Cronobacter sakazakii LMG are shown in figure.c. The minimum ph for growth was.. At higher ph values, the growth was not as expected or compared with those observed in cultivations with Serratia plymuthica RVH in media of ph. and.. Both strains are members of the butanediol producing enterobacteria. The ph increase observed at the end of the cultivations would be related with the activity of the budab genes contained in the genome of this bacterium. 2

136 A: Growth curves of Serratia plymuthica RVH B: Growth curves of Serratia plymuthica RVH budab::cm ph after 2h ph after 2h OD (3 nm) ph 3.: ph 3.2 ph 3.: ph 3.2 ph 3.8: ph 3.8 ph 3.9: ph 3.9 ph.: ph. ph.: ph. ph.2: ph. ph.3: ph.89 ph.: ph. ph.: ph.9 ph.: ph.9 ph.: ph.38 OD (3 nm) ph 3.: ph 3.2 ph 3.: ph 3.2 ph 3.8: ph 3.8 ph 3.9: ph 3.9 ph.: ph. ph.: ph. ph.2: ph.2 ph.3: ph.3 ph.: ph.3 ph.: ph.8 ph.: ph. ph.: ph Time (hours) Time (hours) ph. ph. ph. ph. ph.3 ph.2 ph. ph. ph. ph. ph. ph. ph.3 ph.2 ph. ph. C: Growth curves of Cronobacter sakazakii LMG.9.8 OD (3 nm).... ph after 2h ph 3.: ph 3.3 ph 3.: ph 3.2 ph 3.8: ph 3.83 ph 3.9: ph 3.9 ph.: ph.2 ph.: ph.3 ph.2: ph. ph.3: ph.3 ph.: ph.3 ph.: ph.8 ph.: ph.8 ph.: ph Time (hours) ph. ph. ph. ph. ph.3 ph.2 ph. ph. ph 3.9 Figure.: Cultivation of Serratia plymuthica RVH and budab mutant and, Cronobacter sakazakii LMG at 3 C in apple juice, adjusted at different ph values with aoh. 22

137 Anaerobic conditions The aim of these experiments was to evaluate the growth of Escherichia coli MG (ptrc99a and pbudab), Salmonella Typhimurium LT2 (ptrc99a and pbudab), Serratia plymuthica RVH and budab mutant and, Cronobacter sakazakii LMG when cultivated under anaerobic conditions in apple juice adjusted at different ph values with NaOH. Cultivations were carried out in microtiter plates; the wells were partially filled with culture medium and then covered with a thin layer of paraffin oil. Growth based on optical density (3 nm) was measured during hours in a non-stop multiscan spectrophotometer. The results are shown in figures. and.. The growth curves of Escherichia coli MG ptrc99a and pbudab are shown in figure.a,b. It was observed that the minimal ph for growth of both strains was.. Growth of Escherichia coli MG ptrc99a in all culture conditions was very limited and reached only very low values of optical density. On the other hand, growth of E. coli MG pbudab was favoured under anaerobic conditions as compared with previous experiments. Similar results were observed in cultivations of S. Typhimurium LT2 ptrc99a and pbudab. The minimum ph for growth of S. Typhimurium LT2 ptrc99a and pbudab were.2 and 3.9, respectively. The growth of S. Typhimurium LT2 ptrc99a was severely limited under this condition, while the growth of S. Typhimurium LT2 pbudab has been somehow favoured under anaerobic conditions (see figures.c, d). In anoxic conditions, respiratory pathways are completely inhibited. Instead, fermentative pathways are put on in order to provide energy for bacterial growth. In addition, butanediol producing bacteria would have an advantage compared with the mixed acid fermenters since under anaerobic conditions the butanediol pathway is activated and would contribute to maintain the redox balance of the cells. This could be in part the reason for a better growth observed when S. Typhimurium LT2 pbudab was cultivated. 23

138 A: Growth curves of Escherichia coli MG ptrc99a....3 B: Growth curves of Escherichia coli MG pbudab....3 OD (3 nm) OD (3 nm) Time (hours) Time (hours) ph 3,9 ph, ph, ph,2 ph, ph, ph,8 ph, ph,2 ph 3,9 ph, ph, ph,2 ph, ph, ph,8 ph, ph,2 C: Growth curves of Salmonella Typhimurium LT2 ptrc99a....3 D: Growth curves of Salmonella Typhimurium LT2 pbudab....3 OD (3 nm) OD (3 nm) Time (hours) Time (hours) ph 3,9 ph, ph, ph,2 ph, ph, ph,8 ph, ph,2 ph 3,9 ph, ph, ph,2 ph, ph, ph,8 ph, ph,2 ph 3,8 Figure.: Cultivation of Escherichia coli MG ptrc99a and pbudab and, Salmonella Typhimurium LT2 ptrc99a and pbudab under anaerobic conditions at 3 C in apple juice + mm ITPG, adjusted at different ph values with aoh. 2

139 The growth curves of Serratia plymuthica RVH wild type and budab mutant are shown in figure. (a, b). An enhancement of the growth of the wild type strain was observed in almost all culture conditions as compared with those observed when cultivated under oxygen limitation (see figure. a, b). Furthermore, it was confirmed that the budab operon, normally present in S. plymuthica RVH, play a very important role on the growth of this bacterium as compared with the budab mutant (see figure.b). Under these conditions, the minimal ph for growth of S. plymuthica RVH and budab mutant were.2 and.8 respectively. The growth of S. plymuthica RVH budab mutant was severely limited in absence of the budab operon. A: Growth curves of Serratia plymuthica RVH.9.8. O D (3 nm) Time (hours) ph 3,9 ph, ph, ph,2 ph, ph, ph,8 ph, ph,2 B: Growth curves of Serratia plymuthica RVH budab ::Cm.9.8. O D (3 n m) Time (hours) ph 3,9 ph, ph, ph,2 ph, ph, ph,8 ph, ph,2 Figure.: Cultivation of Serratia plymuthica RVH and budab mutant under anaerobic conditions at 3 C in apple juice, adjusted at different ph values with aoh. 2

140 . Growth of Enterobacteriaceae on vegetables Fresh sliced vegetables can be considered as minimally processed vegetables. In this case, vegetables are usually trimmed, peeled, cut, washed, and sometimes disinfected, and frequently packaged in polymeric films. Raw vegetables regularly harbor spoilage microorganisms. The first step during colonization is penetration of the vegetable tissues. Upon penetration, microorganisms may invade the host tissues or may be kept in check by the defense mechanisms of the vegetable. The predominant microbial species identified in minimally processed vegetables are those found in whole, unprocessed vegetables (Nguyen-the and Carlin, 99). The most frequently identified Enterobacteriaceae are: Enterobacter agglomerans, Erwinia herbicola, and Rahnella aquatilis (Koek et al., 983; Garg et al., 99). During minimal processing, the overall contamination of vegetables is reduced by between and 2 log units (Garg et al., 99; King et al., 99; Torriani and Massa, 99). Serratia species are ubiquitous and can be found in water, soil and plants and therefore can be present on foods of plant origin. Serratia plymuthica is most frequently associated with plants and has been isolated from the edible parts of green onion, carrot and lettuce (De Vleesschauwer and Hofte, 2). The principle environmental sources for Cronobacter sakazakii are water, soil and plant material. This species has been found on courgette, lettuce and tomato and has been demonstrated to grow well on freshcut apple, cabbage, carrot, cucumber, lettuce, and tomato at 2 C and in some types of produce at 2 C (Beuchat et al., 29). This species is known to produce biofilm composed of polysaccharides on surfaces where they grow (Beuchat et al., 29; Hartmann et al., 2). In this chapter the behaviour of Serratia plymuthica RVH, previously isolated from a vegetable washing and cutting machine in an industrial kitchen (van Houdt et al., 2), and Cronobacter sakazakii LMG will be evaluated on red pepper and cucumber slices under different storage conditions. 2

141 .. Growth of Serratia plymuthica RVH on fresh sliced vegetables The aim of these experiments was to evaluate the growth and spoilage activity of Serratia plymuthica RVH wild type and budab mutant on cut vegetables such as red pepper and cucumber slices. Vegetables were first cleaned with % ethanol and rinsed with sterile water. Then, with a sterile device, in case of red pepper, or a knife, in case of cucumber, slices of around. cm thick were obtained. Then, in the first experiment, slices of red pepper were about halfway submerged in a cell suspension, so that one edge remained exposed to the air and the Petri dishes were stored at 3 C. In the rest of experiments, slices were placed in a Petri dish containing a bacterial suspension, and turned upside down after min in order to bring both sides of the slice in contact with bacteria. After that, drained slices were placed in other Petri dishes and sealed in sterile plastic bags and stored at temperatures of C or 2 C. Measurements of cell count were done during storage. Experiments were realized as described in section Growth of Serratia plymuthica RVH wild type and budab mutant on red pepper slices surrounded by liquid The growth curves and ph change during the cultivation of Serratia plymuthica RVH and budab mutant on red pepper slices surrounded by liquid are shown in figure.. In this experiment, the cell number and ph of the surrounding liquid was determined during 3 days at 3 C. Corresponding pictures of the red pepper slices are shown in figure.2. It was observed that S. plymuthica RVH was able to grow from.9 log CFU/ml till 8.2 log CFU/ml after day of cultivation and this value remained almost constant toward the end of the experiment (3 days). In case of the S. plymuthica RVH budab mutant, cells started dying from the beginning of the experiment. Thus, the cell population diminished from. log CFU/ml till.3 log CFU/ml toward the end of the experiment. Differences in ph change of the surrounding liquid during cultivation were observed between both strains. The wild type was able to increase the ph from the 2 nd day of cultivation whereas the budab mutant continued acidifying the medium. The ability to increase the ph was already observed in previous experiments when S. plymuthica RVH was cultivated in synthetic medium and in fruit juices. Measurements of ph change of the control samples 2

142 were also done. A ph decrease of the surrounding liquid was observed during the cultivation and it would be related to the activity of endogenous enzymes which provoked the breakdown of cellular constituents. The survival or growth of microorganisms on vegetables depends on the presence of free moisture, relative humidity and also of temperature. High storage temperatures permit rapid bacterial growth, but at the same time they accelerate spoilage development and reduce shelf life. Pictures of the red pepper slices are shown in figure.2. The spoilage activity of S. plymuthica RVH and S. plymuthica RVH budab mutant was compared with control samples during three days. It was observed that the growth of S. plymuthica RVH was linked to the formation of biofilm, production of exudate and damage of the vegetable structure. Besides, production of a characteristic off-odor was noticed. Spoilage activity of S. plymuthica RVH budab mutant was not clearly evidenced at least during the first two days of storage, but the production of a milky exudate was noticed at the 3 rd day of the experiment. Studies done at 3 C by using carrot slices demonstrated the spoilage activity of S. plymuthica RVH after days with formation of mucoid patches on the surface exposed to the air (Wevers et al., 29), nevertheless some differences could be observed depending on the kind of vegetable. Log (cfu/ml) Time (days) ph ph S.plymuthica RVH ph Blanc cell number S.plymuthica RVH budab::cm ph S.plymuthica RVH budab::cm cell number S.plymuthica RVH Figure.: Cultivation of Serratia plymuthica RVH and budab mutant on red pepper slices surrounded by liquid at 3 C. 28

143 Serratia plymuthica RVH Control Serratia plymuthica RVH budab::cm Day Day Day 2 Day 3 Figure.2: Spoilage activity of Serratia plymuthica RVH and budab mutant cultivated on red pepper slices surrounded by liquid at 3 C. 29

144 ..2. Growth of Serratia plymuthica RVH wild type and budab mutant on red pepper slices The spoilage activity of Serratia plymuthica RVH and budab mutant on red pepper slices at C and 2 C are shown in figures.3 and.. At C, it was observed that for both strains the cell population increased in. log unit from an initial population of about. log CFU/slice (see figure.3a). Furthermore, the cell population toward the end of the experiments (2 days) diminished reaching values up to 8. log CFU/slice in both cases. From these results it is possible to suggest that the growth of both strains was similar. Under these conditions, the presence of budab genes would not play any significant role in the enhancement of the growth. Besides, the spoilage activity of S. plymuthica RVH was observed from the 8 th day and in case of the budab mutant from the th day (see figure.). In this case, spoilage activity was considered when a visible mucoid patch or slime was observed on the surface of the red pepper slice. Refrigeration delays spoilage activity of this bacterium as compared with observations at 3 C. Therefore, the important information is the extent of growth or survival at time of spoilage development. However, appreciation of spoilage is subjective. It is difficult to estimate at which spoilage level a product will be rejected by consumers. The growth and spoilage activity of S. plymuthica RVH and budab mutant at 2 C is shown in figures.3b and.. It was observed that these strains were able to grow from about. log CFU/slice to above. log CFU/slice in case of the wild type and, to above 9. log CFU/slice in case of the budab mutant. However, the cell number declined toward the end of the experiment (2 days) reaching values of 9. log CFU/slice in case of the wild type and. log CFU/slice in case of the budab mutant. The wild type showed a slightly better growth than the budab mutant at 2 C. Moreover, packaging and respiration of minimally processed vegetables provide high relative humidity for bacterial growth. However, growth rates could be variable, even for a single product. An important difference in the spoilage activity was noticed at 2 C in comparison with spoilage at C. At higher temperature, the spoilage activity was faster as shown in pictures of the figure.. S. plymuthica RVH started spoiling after 3 days whereas S. plymuthica RVH budab mutant started producing exudate and slime or biofilm after days. This observation would be another important difference, the fact that the budab mutant strain started 3

145 producing slime or biofilm later than observed in the wild type one. In this case, the absence of these two genes appeared to delay the spoilage activity of S. plymuthica RVH at least under these conditions. The same observation was also noticed when cultivated at C. The spoilage activity was characterized by production of slime or biofilm, loss of texture and production of a characteristic off-oddor. The loss of texture can be in part due to the activity of pectinolytic enzymes. However, previous studies carried out with S. plymuthica RVH confirmed that this strain does not produce pectinolytic enzymes (Wevers et al., 29). From this point of view, it is possible to suggest that the loss of texture could be in part due to the loss of water from the matrix of red pepper pieces caused somehow by this bacterium. A: Growth of Serra tia pl ymuthica RVH at C 2 B: Growth of Serratia plymuthica RVH at 2 C 2 Lo g (c fu /slic e) 8 L og (cfu /slice) Time (days) Time (days) S. plymuthica RVH S. plymuthica RVH budab::cm S. plymuthica RVH S. plymuthica RVH budab::cm Figure.3: Growth curves of Serratia plymuthica RVH and budab mutant on red pepper slices at C and 2 C. 3

146 Serratia plymuthica RVH ( C) Serratia plymuthica RVH budab::cm ( C) Serratia plymuthica RVH (2 C) Serratia plymuthica RVH budab::cm (2 C) Day Day 3 Day Day 8 Day Day 2 Figure.: Spoilage activity of Serratia plymuthica RVH and budab mutant cultivated on red pepper slices at C and 2 C. 32

147 ..3. Growth of Serratia plymuthica RVH wild type and budab mutant on cucumber slices The growth and spoilage activity of Serratia plymuthica RVH and budab mutant on cucumber slices at C and 2 C during days of storage are shown in figures. and.. It was observed that the wild type and the budab mutant strain were able to grow at C from an initial cell population of. log CFU/cm 2 and.32 log CFU/cm 2 till reaching values of 9.8 log CFU/cm 2 and.9 log CFU/cm 2 toward the end of the experiment respectively (see figure.a). The growth of these two strains at this temperature can be considered as similar since some variability in the growth on different slices was observed. Variability of cell population in each cucumber slice depends on the individual condition of each slice such as moisture content and in the amount of cells attached on specific sites on the surface of each slice. Thus, the initial microbial population is important in order to evaluate the spoilage activity. In addition, depending on the type of vegetable and the source of contamination, the amount of spoilage microorganisms can vary. Studies reported that the number of microorganisms found on unstored minimally processed vegetables are of the same order of magnitude as those reported for raw vegetables and range from 3 cfu/g on shredded iceberg lettuce to cfu/g on shredded carrots (Nguyen-the and Carlin, 99). The spoilage activity at C was evident after the 3 rd day. Spoilage was characterized by formation of biofilm, production of exudate, loss of texture and production of off-oddor. Moreover, spoilage was noticed in the core of the cucumber slices where more moisture content is available. Then, a complete damage and softening of the slice structure was evidenced toward the end of the experiment (see figure.). Growth of S. plymuthica RVH and budab mutant at 2 C is shown in figure.b. A better growth of the wild type strain compared with the budab mutant was observed. Nevertheless, some variability of the cell number was observed on each cucumber slice. At this temperature, water evaporation on the surface of each cucumber slice is faster compared with experiments carried out at C and this could affect the growth of cells. In this case, the wild type and the budab mutant strain grew from an initial microbial population of about.3 log CFU/cm 2 till reaching values of. log CFU/cm 2 and 8. log CFU/cm 2 toward the th day of storage, respectively. Spoilage activity of S. plymuthica 33

148 RVH and budab mutant at this temperature was not evident at least toward the end of the experiment ( days) (see figure.). Storage temperature influences the water evaporation on the surface of the cucumber slices and as a consequence can affect the growth and spoilage activity of S. plymuthica RVH. From these observations one can speculate that probably the loss of water on the surface of the cucumber slices during storage at 2 C avoided the spoilage activity of S. plymuthica RVH and budab mutant. As observed in previous experiments, spoilage appears during storage as the number of microorganisms increases, but it may also vary with the kind of vegetables. From comparisons of spoilage at C and 2 C it is possible to point out that, microorganisms may be a cause of spoilage but storage conditions are in part a determining factor. A: Growth of Serratia plymuthica RVH at C B: Growth of Serratia plymuthica RVH at 2 C 2 2 L o g (c fu/ s l ic e ) 8 L o g (c fu /s lic e ) Time (days) 2 Time (days) Serratia plymuthica RVH Serratia plymuthica RVH budab::cm Serratia plymuthica RVH Serratia plymuthica RVH budab::cm Figure.: Growth of Serratia plymuthica RVH and budab mutant cultivated on cucumber slices at C and 2 C. 3

149 Serratia plymuthica RVH ( C) Serratia plymuthica RVH budab::cm ( C) Serratia plymuthica RVH (2 C) Serratia plymuthica RVH budab::cm (2 C) Day Day Day Day 2 Day Figure.: Spoilage activity of Serratia plymuthica RVH and budab mutant cultivated on cucumber slices at C and 2 C. 3

150 .2. Growth of Cronobacter sakazakii LMG wild type and budab mutant on red pepper slices The aim of these experiments was to evaluate the ability of Cronobacter sakazakii LMG and budab mutant to grow on and spoil fresh cut red pepper slices. Information about implication of this bacterium in spoilage of minimally processed vegetables is rather scarce. In this experiment, slices were placed in a Petri dish containing a suspension of C. sakazakii LMG, and turned upside down after min in order to bring both sides of the slice in contact with bacteria. After that, drained slices were placed in other Petri dishes and sealed in sterile plastic bags and stored at temperatures of C and 2 C. Measurements of cell count were done during storage. Experiments were realized as described in section In the experiments the initial microbial population was about. log CFU/slice and, the growth and spoilage activity was evaluated at C or 2 C. The growth and the spoilage activity of C. sakazakii LMG and the budab mutant are shown in figures. and.8. At C, it was observed that the wild type and budab mutant were able to grow until values of 9. log CFU/slice and 9.3 log CFU/slice respectively. The cell population increased in case of the wild type strain, whereas in case of the budab mutant it diminished to values below 8 log CFU/slice toward the end of cultivation. The spoilage activity under these conditions was not clearly observed in case of the budab mutant and, in case of the wild type, production of exudate and soft rot was noticed towar the 9 th day of cultivation. Results of experiments carried out at 2 C are shown in figures.b and.8. A growth difference between both strains was observed. The wild type strain appeared to grow better than the budab mutant. In this case, the former attained a maximum cell number of 9.9 log CFU/slice after days, whereas the budab mutant attained 9.9 log CFU/slice after days. Comparison between cultivations carried out at C and 2 C showed that the growth of the wild type was not affected by decrease in storage temperature to C whereas the growth of the budab mutant appeared to be slightly affected at 2 C than at C (see figure.a,b). 3

151 Spoilage activity at 2 C was noticed from the th day of cultivation with production of exudate, soft rot and loss of texture. Spoilage was more evident in the wild type than in the budab mutant strain. In general, spoilage activity of C. sakazakii LMG at 2 C increased as compared with that observed when cultivated at C (see figure.8). Cutting or slicing of vegetables probably creates favorable conditions for microbial growth. Sliced vegetables would release components useful for the growth of bacteria. A: Growth curves of Cronobacter sakazakii LMG at C 2 B: Growth of Cronobacter sakazakii LMG at 2 C 2 L o g (C F U /m l ) 8 L o g (C F U /m l ) Time (days) C. s akazakii LMG C. sakazakii LMG budab::cm 2 Time (days) C. sakazakii LMG C. sakazakii LMG budab::cm Figure.: Growth curves of Cronobacter sakazakii LMG and budab mutant cultivated on red pepper slices at C and 2 C. 3

152 Cronobacter sakazakii LMG ( C) Cronobacter sakazakii LMG budab::cm ( C) Cronobacter sakazakii LMG (2 C) Cronobacter sakazakii LMG budab::cm (2 C) Day Day 2 Day Day 8 Day 9 Figure.8: Spoilage activity of Cronobacter sakazakii LMG and budab mutant cultivated on red pepper slices at C and 2 C. 38