* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download MICROBIOLOGICAL ASPECTS OF FOOD - Szak-nyelv
Survey
Document related concepts
Hospital-acquired infection wikipedia , lookup
Bacterial cell structure wikipedia , lookup
Magnetotactic bacteria wikipedia , lookup
Germ theory of disease wikipedia , lookup
Microorganism wikipedia , lookup
Bacterial morphological plasticity wikipedia , lookup
Disinfectant wikipedia , lookup
Gastroenteritis wikipedia , lookup
Listeria monocytogenes wikipedia , lookup
Traveler's diarrhea wikipedia , lookup
Marine microorganism wikipedia , lookup
Human microbiota wikipedia , lookup
Bacterial taxonomy wikipedia , lookup
Transcript
Készült a TÁMOP-4.1.2.D-12/1/KONV-2012-0008 azonosító számú „Szak-nyelv-tudás” – Az idegen nyelvi képzési rendszer fejlesztése a Debreceni Egyetemen pályázat keretében. MICROBIOLOGICAL ASPECTS OF FOOD QUALITY AND SAFETY Erzsébet Karaffa, Ph.D. – Ferenc Peles, Ph.D. Debrecen, 2014 Contents 1. Taxonomy, Role, and significance of microorganisms in food..........................................................3 2. Intrinsic and extrinsic parameters of foods that affect microbial growth .............................. 11 2.1. Moisture content ................................................................................................................................................ 11 2.2. pH ............................................................................................................................................................................. 14 2.3. Oxidation–reduction potential ..................................................................................................................... 16 2.4. Nutrient content ................................................................................................................................................. 19 2.5. Salinity.................................................................................................................................................................... 20 2.6. Physical and biological structures .............................................................................................................. 20 2.7. Temperature of storage .................................................................................................................................. 21 2.8. Osmotic pressure ............................................................................................................................................... 22 2.9. Relative humidity of environment .............................................................................................................. 23 2.10. Presence and concentration of gases in the environment ............................................................. 24 2.11. Processing and preserving operations ................................................................................................... 24 3. Foodborne diseases ............................................................................................................................................... 25 3.1. Staphylococcus aureus and staphylococcal gastroenteritis .................................................... 28 3.2. Listeria monocytogenes and foodborne listeriosis ...................................................................... 32 3.3. Food Poisoning Caused by Gram-Positive Spore-forming ....................................................... 38 3.3.1. Bacillus cereus gastroenteritis............................................................................................................. 38 3.3.2. Clostridium perfringens food poisoning.......................................................................................... 40 3.3.3. Clostridium botulinum food poisoning (botulism) ..................................................................... 43 3.4. Salmonella genus and foodborne gastroenteritis caused by Salmonella ........................ 47 3.5. Escherichia coli and foodborne gastroenteritis caused by Escherichia coli ................... 52 3.6. Shigella genus and shigellosis ................................................................................................................. 58 3.7. Yersinia genus and yersiniosis ................................................................................................................ 61 3.8. Vibrio genus and vibriosis ......................................................................................................................... 63 3.9. Campylobacter genus and campylobacteriosis .............................................................................. 66 3.10. Mycotoxigenic fungi and mycotoxins ............................................................................................... 69 3.11. Foodborne Viruses ...................................................................................................................................... 75 3.12. Foodborne parasites .................................................................................................................................. 78 4. Microorganisms in animal products............................................................................................................. 87 5. Microorganisms in plant products ................................................................................................................ 96 References ..................................................................................................................................................................... 104 1. TAXONOMY, ROLE, AND SIGNIFICANCE OF MICROORGANISMS IN FOOD Microbiology is the science study of the occurrence and significance of microscopic cellular organisms (bacteria, fungi, protozoa and algae) and viruses. Microbes have fundamental roles in the food chain either as producers or as decomposers. Microbes are all around us: present within and on the bodies of all living creatures, and can be found in soil and water, even on the air. Consequently foods are rarely sterile. However this microflora does neither change the physical or chemical characteristic of the food, nor cause disease in most cases. The microbial presence is detectable when they cause spoilage, or foodborne illness. Microbial activity not only decreases the quality of the food, but can even be harmful for the consumers. The safety of a given food product based on total microbial numbers (per gram or milliliter), and the type of microbes as well. We can then predict the general types of microorganisms to be expected on a particular food, in case we know the types of microorganisms associated with plant and animal foods in their natural states. The microbiological safety and quality of a food is determined by the property of the food, the microbial contamination of the raw materials, the production technology, and the storage and packaging conditions. Microbes can also play a positive role in food. They can be consumed as foods in themselves as in the edible fungi, mycoprotein and algae. They can also effect desirable transformations in a food during fermentation processes, changing its properties in a way that is beneficial. Bacterial taxonomy Bacterial taxonomy has changed substantially in the past few decades. Several new taxa have been created as a result of the employment of molecular genetic methods. Three kinds of methods are used for the identification and description of bacteria: (i) phenotypic, (ii) genotypic, and (iii) phylogenetic. Phenotypic and genotypic analysis are group organisms based on their similarities, Phylogenetic analysis is place organisms within an evolutionary framework, and complement the previous methods. Phenotypic analysis examines the morphological, metabolic, physiological, serological and chemical characteristics of the cell. Morphological characters include colony morphology; Gram reaction; cell size and shape; pattern of flagellation; presence of spores, inclusion bodies; capsules, S-layers or slime layers. The Gram-staining based characterization is one of the oldest and most widespread methods for grouping bacteria. The study of bacterial metabolic characteristics in food microbiology refers to utilization of individual carbon, nitrogen, or sulfur compounds; fermentation of sugars; nitrogen fixation; growth factor requirements. Temperature, pH, and salt ranges for growth; response to oxygen (e.g. aerobic, facultative, anaerobic); presence of catalase or oxidase; production of extracellular enzymes belong to studied physiological characteristics. Cellular fatty acid profile identification (FAME: fatty acid methyl ester) is widespread in laboratories where human pathogens routinely must be identified (clinical, public health, food and water-inspection). The fatty acid composition (chain length, presence or absence of double bonds, rings, branched chains, or hydroxy groups) of Bacteria varies from species to species. Genotypic analysis considers characteristics of the genome with methods like DNA hybridization and G + C content of DNA; 23S, 16S, and 5S rRNA sequence similarities, and multilocus sequence typing, multigene and whole genome analysis. DNA–DNA hybridization is useful for differentiating very similar organisms, as it is a sensitive method for detecting subtle differences in the genomes of two organisms. The GC ratio refers the percentage of guanine (G) and cytosine (C) in an organism’s genomic DNA. If GC ratios differ by more than about 5%, two organisms’ are unlikely to be closely related. Taxonomic information can be obtained from the determination of rRNA sequence similarities. The prokaryotic ribosome is a 70S (Svedberg) unit, which is composed of one 50S and one 30S subunits, containing proteins and ribosomal RNA (rRNA). The 50S subunit is composed of 23S (large) and 5S RNA whereas the 30S subunit is composed of 16S (small) RNA (Figure 1.1). Figure 1.1: The structure of prokaryotic ribosomes (Source: http://barleyworld.org/sites/default/files/figure-09-13.jpg (Barley World), download time: 11.12.2014.) The 16S subunit is highly conserved and is considered to be an excellent chronometer of bacteria. 16S rDNA may be sequenced after amplification of specific genomic regions by polymerase chain reaction (PCR)-based methods, or using reverse transcriptase starting from RNA source. The sequencing of rRNA amplification than allow the determination of precise phylogenetic relationships. Carl Woese establishment of three domain of life (Eukaryotes, Archaebacteria, and Prokaryotes) based on 16S rRNA sequences of organisms (Fig. 1.2). Figure 1.2: The phylogenetic tree of life, based on 16/18S rRNA sequences (Source: http://plus.maths.org/content/reconstructing-tree-life (Huson et al.: Reconstructing the tree of life 2011. Plus magazine), download time: 11.12.2014.) The Procariotes domain includes eubacteria, the bacteria of importance in foods. Sequence similarities of 16S rRNA are widely employed, and some of the new foodborne taxa were created primarily by its use along with other information. The sequencing of 23S rDNA is also used in bacterial taxonomy. As only one gene is used for rRNA gene sequence analysis, it may not provide sufficient information for the discrimination of bacterial strains. Sequence comparisons of particular genes can provide valuable insight for taxonomy as well as phylogeny. Single-gene analyses of rRNA and other highly conserved genes, such as recA (encodings a recombinase protein), and gyrB (encoding a DNA gyrase protein) are widespread for distinguishing bacteria at the species level. However those analyses give only very limited genomic information. Moreover the horizontal gene flow of some genes could lead to incorrect taxonomic conclusions. Multigene and whole genomic analyses may circumvent those limitations, and are becoming popular in microbial systematics. A more representative sampling of the genome can be obtained by sequencing several functionally unrelated genes, than is possible with a single one. Analyses of whole genome sequences also provide information about the genome structure (size and number of chromosomes, their GC content, and whether the chromosomes are linear or circular), and comparative analysis of gene content (presence or absence of genes) and the order of which also may have taxonomic significance. Multilocus sequence typing (MLST) is a powerful technique for characterizing strains within a species. We can compare the approximately 450-base-pair sequences of different “housekeeping” genes from different strains of the same organism using MLST. Housekeeping genes encode essential functions in cells and are located on the chromosome. Each nucleotide along the sequence is compared and differences are noted. Each sequence variant is called an allele and is assigned a number. Finally an allelic profile or multilocus sequence type is defined. Being very sensitive method, MLST cannot use, for grouping taxa above species, such as genera and families. As Figure 1.3 shows, some major linages (phyla) of Bacteria based on 16S ribosomal RNA gene sequence comparisons. Species belonging to two phyla has importance in food microbiology: Gram-positive bacteria and Proteobacteria. Figure 1.3: Some major phyla of Bacteria based on 16S ribosomal RNA gene sequence comparisons (Source: http://202.204.115.67/jpkch/jpkch/2008/wswx/chapter%202/picture/11.jpg (Quazoo: Bacterial phyla), download time: 11.12.2014.) The Gram-positive bacteria are a large group of primarily chemoorganotrophic Bacteria. The ones with cell wall can be separated into two subgroups, based on their GC content: Firmicutes with GC <50.53% 55, and the Actinobacteria, with GC > 55%. The later includes the genera Streptomyces, Propionibacterium, Micrococcus, Bifidobacterium, Corynebacterium, Brevibacterium. The Firmicutes group include the genera Clostridium, Bacillus, Staphylococcus, Lactobacillus, Pediococcus, Leuconostoc, and Listeria. The Gram-negative Proteobacteria is the other phylum with importance in food microbiology. Based on 16S rRNA gene sequences, the phylum Proteobacteria can be divided into six classes, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, and Zetaproteobacteria (only one, marine organism). The most important food-related Proteobacteria genera are the following ones: Alphaproteobacteria: Acetobacter, Asaia, Brevundimonas, Devosia, Gluconobacter, Paracoccus, Pseudoaminobacter, Sphingomonas, Xanthobacter, Zymomonas. Betaproteobacteria: Acidovorax, Alcaligenes, Burkholderia, Chromobacterium, Comamonas, Delftia, Hydrogenophaga, Janthinobacterium, Pandoraea, Pseudomonas (plant pathogens), Ralstoni, Telluri, Víriovora, Vogesella, Wautersi, Xylophilus Gammaproteobacteria: Enterobatreiaceae family (Escherichia, Citrobacter, Salmonella, Shigella, Proteus, Raoultella, Klebsiella, Edwardsiella, Yersinia, etc.), Acinetobacter, Aeromonas, Alteromonas, Azomonas, Bacteroides, Carnimonas, Flavobacterium, Halomonas, Moraxella, Plesiomonas, Pseudoalteromonas, Pseudomonas, Psychrobacter, Photobacterium, Shewanella, Stenotrophomonas, Vibrio, Xanthomonas, Xylella. Deltaproteobacteria: Campylobacter, Helicobacter Most foodborne bacteria (especially foodborne pathogens) belong to the γ –subclass. Fungal taxonomy Fungi are a large, diverse, and widespread group of organisms, consisting of the moulds, mushrooms, and yeasts. Yeasts grow as single-celled forms. Moulds are multicellular, forming a network of filaments from tubular cell, surrounded by walls, called hyphae (Fig. 1.4). Figure 1.4: The schematic draw of a hypha Septate fungal hyphae have cross-walls, which are separating cells. There are no cross-walls in the coenocytic hypha. In this case vegetative cell of a fungal hypha contains more than one nucleus (Fig. 1.5). Each hyphal filament grows mainly at the tip by extension of the terminal cell. Figure 1.5: Schematic draw of the septate and coenocytic hypha of fungi (Source: http://www.chesterfield.k12.sc.us/cheraw%20intermediate/DaveEvans/BiologyII/Hyphae%20Diag.jpg (Chesterfield: Biology II), download time: 11.12.2014.) Hyphae typically grow together forming a visible tuft called a mycelium. From the mycelium growing over and into the organic material, hyphal branches form aerial hyphae above the surface, and asexual spores are formed on these branches (Fig. 1.6). Asexual spores are formed without fusion of gametes or meiosis, and have several types and function to disperse the fungus to new habitats. Figure 1.6: Hyphae with aerial mycelia and asexual spores (Source: http://www.atsu.edu/faculty/chamberlain/Website/Lects/Fungi.htm#gen (Dr. Tritz: Medical Microbiology Fall 2000), download time: 11.12.2014.) Conidia are formed on conidiophores and they are often pigmented (black, blue green, red, yellow, or brown), and give the mycelium a dusty appearance. Sporangiospores are formed inside sporangium. Some fungi form macroscopic reproductive structures called fruiting bodies. Fungi may also produce sexual spores as a result of sexual reproduction. Depending on the group, different types of sexual spores (ascospores, zygospores, basidiospores) are produced. Fungal taxonomy has markedly changed in the past decades, similarly to the bacterial one. The earlier predominant morphological identification has been refined with DNA-based methods. The most important phylogenetic molecular marker is the rRNA sequence for fungi; however multigene analysis is preferred in species identification. Figure 1.7: The major phylogenetic groups of fungi (Source: http://www.nature.com/nature/journal/v443/n7113/images/443758a-f1.2.jpg (Bruns (2006) Evolutionary biology: A kingdom revised), download time: 11.12.2014.) The Fig 1.7 shows the latest, accepted phylogenetic tree of Fungi, based on multi gene sequence analysis. The former Zygomycota taxon has split into different groups. The Rhizopus, Mucor and Thamnidium genus with food microbiology interests are grouped in the newly formed Mucoromycotina subphyllum. It can be characterized with coenocitic hyphae and asexual sporangiospores, and sexual zygospores. They are widespread on soil, dead plant materials and foods. Some Rhizopus species cause “black spot” of beef, and may be found on bacon and other processed meats. R. stolonifer is one of the most common species in foods. It is also referred to as “bread molds”, and produce watery soft rot of fruits (apples, pears, stone fruits, grapes, figs). R. oligosporus is important in the production of special fermented foods (oncom, bongkrek, and tempeh). A new subkingdom, Dikarya contains Ascomycota and Basydiomicota phyla. The former Deuteromycota (or Fungi Imperfecti) taxon of asexual fungi does not exist anymore; species has been integrated to the Ascomycota and Basidiomycota groups, and referred as conidial fungi. Their hyphae have septae, and dikaryotic cell forms can be detected prior to karyogamy and meiosis. The ascomycetes, species produce sexual ascospores in asci. In some ascomycetes, the asci are formed within a fruiting body, called an ascocarp. They also produce asexual spores, called conidia. There are three subphyla in Ascomycotina: Taphrynomycotina, Saccharomycotina and Pezizomycotina. The yeasts genus Schizosaccharomyces belong to Taphrynomycotina subphyllum, and divide by lateral fission of cross-wall formation. The most prevalent species, S. pombe, is osmophilic and resistant to some chemical preservatives. Figure 1.8: The budding of Saccharomyces cerevisiae (Source: http://scienceforkids.kidipede.com/biology/cells/yeast.htm (Yeast for kids) Evolutionary biology: A kingdom revised), download time: 11.12.2014.) The majority of yeast is in Saccharomycotina subphylum, which contains budding yeasts. They multiply by budding (Fig. 1.8). Most important genera are: Kluyveromyces, Saccharomyces, Torulaspora, Zygosaccharomyces in Saccharomycetaceae family, moreover Hanseniaspora, Debaryomyces, Pichia. The most important asexual (conidial) yeast genera are Brettanomyces, Candida, Cryptococcus, Rhodotorula, and Trichosporon. The bakers’, brewers’, wine, and champagne yeasts are S. cerevisiae. They are found in kefir grains and can be isolated from a wide range of foods, such as dry-cured salami and numerous fruits. Perizimycotina subhyllum contains the moulds with septate hyphae. The most important genera in food microbiology are: Byssochlamys, Emericella, Eupenicillium, Eurotium (Eurotiales ordo, Trichocomaceae family). Conidial genera are: Alternaria, Aspergillus, Aureobasidium, Botrytis, Cladosporium, Fusarium, Geotrichum, Monilia, Penicillium, Trichothecium. The shape of the conidiophores and the way of conidia production are characteristic to genera (Fig. 1.9). Alternaria Fusarium Aspergillus Geotrichum Aureobasidium Monilia Botrytis Cladosporium Penicillium Trichothecium Figure 1.9: The characteristic asexual spore formation of some important mitosporic fungi (Source: http://mycota-crcc.mnhn.fr/site/genre.php?lang=eng (Mycota: fungal contaminats of cultural heritage), download time: 11.12.2014.) 2. INTRINSIC AND EXTRINSIC PARAMETERS OF FOODS THAT AFFECT MICROBIAL GROWTH Intrinsic parameters Intrinsic parameters are an inseparable part of plant and animal tissues. Intrinsic parameters are moisture content, pH, reduction potential, nutrient content, salinity, physical and biological structures. Each of these factors is discussed below, and we placed the emphasis on their effects on microorganisms in foods. 2.1. MOISTURE CONTENT Life is largely dependent on the presence of water. The water requirements of microorganisms are often described in terms of the water activity (aw) in the environment. Water activity is a measure of the available water in a food. Normally the moisture content can be exchanged between the product and the environment. There is a non-linear relationship between the water activity and water content. This relationship is also known as a moisture sorption isotherm curve. This isotherm is specific to temperature and substance. It can help to estimate the stability of products in different storage conditions as time passes. Water activity is defined by the ratio of the water vapor pressure of food substrate (p) to the vapor pressure of pure water (p0) at the same temperature: aw = p/p0, where p is the vapor pressure of the solution, and p0 is the vapor pressure of the solvent (usually water). Drying or desiccation is one of the oldest methods of preserving foods. The effects of lowering water activity (aw) below optimum are: - increase the length of the lag phase of growth, - decrease the growth rate, - decrease the size of final population. Water activity is influenced by other environmental parameters (e.g. temperature of growth, pH, and Eh). Water activity is a colligative property, because it depends on the number of molecules or ions present in solution, rather than their size. For example sodium chloride (which dissociates into two ions in solution), is more effective at reducing the water activity than a compound like sucrose. The water activity of pure water is 1.00, while the aw of 22% NaCl solution (w/v) is 0.86 (Table 2.1.1). The water activity of saturated NaCl solution is 0.75. Table 2.1.1: Relationship between water activity and concentration of salt solutions (Jay et al., 2005) NaCl concentration % (w/v) Water activity 0,9 0,995 1,7 0,99 3,5 0,98 7 0,96 10 0,94 13 0,92 16 0,90 19 0,88 22 0,86 Water activity is related to relative humidity. The relative humidity of air in equilibrium with a sample is called the Equilibrium Relative Humidity (ERH): ERH (%) = aw × 100 Water potential (ψ) quantifies the tendency of water move from an area to another. Move can be caused by osmosis, gravity, mechanical pressure, or matrix effects (e.g. capillary action). Water potential: ψ = -RT / Vw × log aw R: gas constant (8,314 J × mol-1 × K-1) T: absolute temperature (K) Vw: molar volume of water aw: water activity. The dimension of water potential (ψ) is pressure. Water activity of 0.9 (at 25 oC) is equivalent to a water potential of -143 atm or -14.5 MPa. Most of the microorganisms are not able to grow on the water activity value below 0.6. Below aw value of 0.6, there is no microbiological spoilage in foods. Various microbes have different water activity requirements. Moulds and yeasts are able to grow over a wider water activity range than bacteria. Gram-negative species usually require the highest water activity (higher than Gram-positives). Most Gram-negative bacteria usually grow above aw of 0.97. General we can state that, bacteria require higher values (except for halophilic bacteria) of water activity for growth than fungi. Most of the spoilage-causing bacteria are not able to grow below aw of 0.91. The minimum aw value for most spoilage bacteria is 0.90, for most spoilage yeasts is 0.88, for most spoilage moulds is 0.80, and for halophilic bacteria is 0.75. Osmophilic yeasts (such as Zygosaccharomyces rouxii, Z. baillii, and Z. bisporus) and Xerophilic moulds such as Eurotium spp., Xeromyces bisporus (syn. Monascus bisporus) have been reported to grow at water activity 0.61 (Table 2.1.2). Table 2.1.2: Minimum water activities at which active growth can occur (Jay et al, 2005; Adam–Moss, 2008) Group of microorganisms minimum aw value Most Gram-negative bacteria 0.97 Most Gram-positive bacteria 0.90 Most spoilage bacteria 0.90 Most spoilage yeasts 0.88 Most spoilage moulds 0.80 Halophilic bacteria 0.75 Osmophilic yeast 0.61 Xerophilic moulds 0.61 Staphylococcus aureus is unique among foodborne pathogens, because some strains can grow at a minimum water activity of about 0.86, but toxin production may require more favourable conditions. Most spore-forming bacteria do not grow below water activity of 0.93. Spore germination and outgrowth of Bacillus cereus is prevented at water activity of 0.97 to 0.93. The minimum water activity for Clostridium perfringens spore germination and growth is between 0.97 and 0.95. The minimum water activity for growth of Clostridium botulinum is above 0.94 (Table 2.1.3). Table 2.1.3: Approximate minimum aw values for growth of microorganisms (Jay et al., 2005) Microorganisms Minimum aw values Escherichia coli 0,96 Bacillus subtilis 0,95 Clostridium botulinum 0,94-0,97 Staphylococcus aureus 0,86 Penicillium spp. 0,79-0,83 Aspergillus spp. 0,64-0,70 Zygosaccharomyces rouxii 0,62 Xeromyces bisporus 0,61 Water activity of most fresh food (e.g. vegetables, fruit, meat, fish, milk, etc.) is above 0.98. Dried products (e.g. milk powder, dry pasta, dried fruit, etc.), as well as high-sugar products (e.g. jams) have low water content (Table 2.1.4). Table 2.1.4: Minimum aw values of some foods (Rajkó, 1997; Laczay, 2008) Foods minimum aw value Fresh, raw vegetables, fruit, meat, fish, milk 0.98< Cooked meat, bread 0.95-0.98 Salted meat, ham, cheese 0.91-0.95 Salami, dry cheeses, syrups 0.87-0.91 Sausage, raw ham, flour, rice, beans, peas 0.80-0.87 Jams 0.65-0.80 Dried fruits 0.60-0.65 Milk powder, spices, dry pasta 0.20-0.60 2.2. PH pH is the negative logarithm of the hydrogen ion activity. pH 7 indicate neutral environment, pH values below 7 indicate acidic, and those above 7 indicate alkaline environment. The pH of the environment has influence on the growth and metabolism of microorganisms. Effect of pH is very strong and differentiated. The pH has an effect on the electric charge of the plasma membrane proteins, and it can alter the permeability of the plasma membrane. The pH tolerance of microorganisms can be characterized by three parameters (minimum, maximum and optimum pH value). The microorganisms are able to grow in the pH range from 1.5 to 11.0. Moulds can grow in the widest pH range (1.5 to 11.0), but yeast has also a wide spectrum of pH (2.2 to 8.5) (Table 2.2.1). The optimum pH range for most bacteria is 6.0–8.0, for yeasts is 4.5–6.0, and for moulds is 3.5– 4.0. Table 2.2.1: pH range of microorganisms’ growth (Deák, 2006; Szabó, 2008) Microorganisms Minimum Optimum Maximum Most bacteria 4.0 6.5 8.5 Lactic acid bacteria 3.2 5.0 7.8 Acetic acid bacteria 2.8 4.0 5.5 Yeasts 2.2 5.0 8.5 Moulds 1.5 5.0 11.0 Several microorganisms which may cause foodborne disease or food spoilage are able to grow at low pH values (Table 2.2.2). The minimum pH for the growth of Zygosaccharomyces bailii is 1.8, for the growth of Alicyclobacillus acidocaldarius and Botrytis cinerea is 2.0, for the growth of Staphylococcus aureus and salmonellae is 4.0. Table 2.2.2: Minimum pH values for the growth of some microorganisms (Jay et al., 2005) Microorganisms Minimum pH value Shigella sonnei 5.0-4.5 Clostridium perfringens 5.0 Bacillus cereus 4.9 Vibrio parahaemolyticus 4.8 Clostridium botulinum I. csoport 4.6 Escherichia coli 0157:H7 4.5 Lactococcus lactis 4.3 Yersinia enterocolitica 4.2 Listeria monocytogenes 4.1 Salmonella spp. 4.0 Staphylococcus aureus 4.0 Lactobacillus brevis 3.1 Lactobacillus sakei 3.0 Penicillium roqueforti 3.0 Botrytis cinerea 2.0 Alicyclobacillus acidocaldarius 2.0 Zygosaccharomyces bailii 1.8 On the basis of their pH tolerance microorganisms can be classified into neutrophilic, acidophilic, and alkalophilic group. Optimum pH for neutrophilic microorganisms is between 5.5 and 8.0. They occur in nature widely (includes most bacteria, e.g. Escherichia coli). The acidophilic microorganisms grow optimally below pH 5.5 (e.g. lactic acid and acetic acid bacteria, Alicyclobacillus species, Rhodopila globiformis; Acidithiobacillus ferrooxidans, Picrophilus oshimae, etc.). The alkalophilic microorganisms grow optimally above pH 8.0 (e.g. Bacillus firmus, ureadegrading bacteria, denitrifying bacteria) (Table 2.2.3). Table 2.2.3: Grouping of microorganisms based on their pH tolerance (http://www.bmekornyesz.hu/sqlatm/dl.php?dir=silo%2F4.+f%C3%A9l%C3%A9v%2FMikrobi ol%C3%B3gia%2F&a=dl&id=558&f=silo/4.%20f%C3%A9l%C3%A9v/Mikrobiol%C3%B3gia/el ojegyzet1.pdf.) Groups pH optimum Example Acidophilic < 5.5 pH lactic acid bacteria, acetic acid bacteria, some fungi Neutrophilic 5.5-8.0 most bacteria Alkalophilic > 8.0 pH urea-degrading bacteria, denitrifying bacteria Table 2.2.4 shows that, the pH of some foods (e.g. vinegar, soft drinks, fruit juices, and wines) is below the point at which bacteria can grow. We can also see that, pH is about 5.6 and above in case of meats and seafoods, which makes these products susceptible to spoilage. Table 2.2.4: Approximate pH values of some foods (Biro, 1993; Jay et al., 2005, www.mtk.nyme.hu/~food/int-hu/mikro/segedletek/.../eloadas2.ppt) Food pH value Vinegar, lemon juice 2.3 Soft drinks 2.0-3.0 Fruit juices (cherry) 2.7 Wine 2.8-4.0 Sauerkraut 3.1-3.7 Sour cherry 3.4 Apples, apple juice, orange juice, grape, plum 3.0-4.5 Tomatoes 3.8-4.3 Yogurt 3.8-4.5 Curdled milk 4.4-4.5 Soft cheese 4.5 Beer 4.0-5.0 Coffee 5.0 Tea 5.5 Fresh raw meat 5.7-6.2 Canned vegetable 5.4-6.5 Poultry meat 6,2-6,7 Fresh raw milk 6.4-6.8 Fish 6.6-6.8 Rotten eggs 9.0 2.3. OXIDATION–REDUCTION POTENTIAL Basic definitions Reduction: gain of electrons. Oxidation: loss of electrons. The two processes occur simultaneously. Oxidant (oxidizing agent): is a compound which forces the reaction partner for oxidation, while itself is reduced (e.g. O2, O3, F2, Cl2, Br2, KMnO4, K2Cr2O7, H2O2, organic peroxides, etc.). Reductant (reducing agent): is a compound which forces the reaction partner for reduction, while itself is oxidized (e.g. alkali and alkaline earth metals, hydrogen, most metals, compounds of some non-metallic element, sugars, alcohols, vitamins). Redox reaction: process with electron transfer (reduction + oxidation) Redox potential or oxidation-reduction potential (Eh, ORP): the most important parameter of the reduction-oxidation (redox) reactions. The redox potential is feature of the electron release or electron uptake (oxidation-reduction) capability of a substrate or medium. Redox reaction occurs as the result of a transfer of electrons between atoms or molecules. The most general form of redox reactions: [Oxidant] + H+ + ne ↔ [Reductant] where n is the number of electrons (e) transferred During the reduction-oxidation reaction one agent (reductant) emits electrons, and the other agent (oxidant) accepts electrons. When an element or compound loses electrons, the substrate is oxidized, while the electron receiving substrate becomes reduced. Oxidation: Cu → Cu + e- or 2Cu + O2 → 2CuO Reduction: Cu ← Cu + eA substance that easily loses electrons is a good reducing agent, and one that easily takes up electrons is a good oxidizing agent. When electrons are transferred from one compound to another, a potential difference is generated between the two compounds. This difference can be measured, and expressed as millivolts (mV). The more highly oxidized a substance, the more positive will be its electrical potential; the more highly reduced a substance, the more negative will be its electrical potential. The electrical potential is zero, when the concentration of oxidant and reductant is equal. Redox potential is measured against an external reference by an inert metal electrode (e.g. platinum, gold, silver) immersed in the medium. The redox potential of foods is the result of numerous factors (e.g. pH, redox couples present, ratio of oxidant to reductant, poising capacity, availability of oxygen, microbial activity, etc.) The high positive value of redox potential indicates that, the oxidized species of the couple is a strong oxidant and the reductant only weakly reducing. A large negative value indicates the reverse. The Nernst equation expresses the redox potential, which is a reversible oxidation-reduction relation: Eh = E0’ + RT/nF × ln [oxidant] [H+] / [reductant] Where Eh: redox potential of normal hydrogen electrode, E0’: standard electrode potential, R: gas constant (R=8,314 J × mol-1 × K-1), T: absolute temperature (K), F: Faraday constant (F=9,648×104 J × V-1 × mol-1), n: the number of electrons transferred in the process. Redox potential is strongly dependent on the pH of the substrate. Therefore, the pH of a substrate should be stated when redox potential is given. Redox potential is normally taken at pH 7.0 (expressed Eh). When taken at pH 7.0, 25◦C, and with all concentrations at 1.0 M, Eh = E0’ (simplified Nernst equation). In nature, redox potential tends to be more negative under progressively alkaline conditions. From the Nernst equation, it is clear that the hydrogen ion concentration has great effect on the redox potential. Every unit decreases in the pH, increases the redox potential by 58 mV. The measured electrode potential in a system is proportional to the partial pressure of hydrogen gas which can be found in the system. Since the redox potential is dependent on the pH, therefore the concept of redox value (rH) is introduced. rH is the negative logarithm of the hydrogen gas partial pressure which corresponding the measured electrode potential. rH = -lg p H2 The extent of the rH scale is 0 and 42. Values between 0 and 28 indicate reducing, value 28 indicates neutral, and values from 28 to 42 indicate a system for oxidizing. Has the larger value of rH, the medium has more powerful oxidizing effect. Table 2.3.1 shows the oxygen demand of microorganisms specified in rH value. Table 2.3.1: Oxygen demand of microorganisms specified in rH value (http://www.mtk.nyme.hu/~food/int-hu/mikro/segedletek/szoveg/II.ev/Gazdasagi_ 4gyakorlat.doc) Aerob microorganisms rH > 14 Microaerophil microorganisms 7,4 < rH < 14 Anaerob microorganisms rH < 7,4 Microorganisms (especially aerobes) can lower the redox potential of their environments during growth. When aerobes grow in a media, they use the oxygen content of the media; thereby the redox potential of media will decrease. The redox potential of a medium can be also reduced by microorganisms by their production of certain metabolic byproducts (e.g. hydrogen, H2S). H2S has the capacity to lower redox potential to -300 mV, because it reacts readily with oxygen. It can accumulate only in anaerobic environments. The decline in redox potential as a result of microbial activity (growth and production of reducing compounds) is the basis of some rapid tests. Redox dyes (e.g. methylene blue, resazurin) long been used for milk and dairy products. Redox potential has a great effect on the food microflora. On the basis of the redox range (and their relationship to oxygen), microorganisms can be classified into some groups. Aerobic microorganisms (e.g. moulds, genus Bacillus) require positive redox potential values (oxidized) for growth, whereas anaerobes (e.g. genus Clostridium) reduced conditions (about -200 mV) for growth initiation. Obligate or strict aerobes (e.g. pseudomonads) are respiring, and they require oxygen and high redox potential for grow. If foods are exposed to air they will easily proliferate on the surface of foods. Obligate anaerobes (e.g. clostridia) can grow in the absence of oxygen, and at low or negative redox potentials. They can grow in deep in meat tissues and stews, or in vacuum packs, and canned foods causing spoilage or poisoning. Microaerophiles (e.g. lactobacilli and campylobacters) grow better under slightly reduced conditions. Facultative anaerobes (e.g. Escherichia coli) are able to grow under aerobic and anaerobic conditions. Aerotolerant anaerobes (e.g. many lactic acid bacteria) are not capable of aerobic respiration, but they can grow in the presence of air. In general, plant products has positive redox potential (+300 – +400 mV) and animal products (e.g. meat, cheese, etc.) has negative (-20 – -200 mV). The high positive redox potential values (from +300 to 400 mV) of fruit juices are mainly due to their low pH values (Table 2.3.2). Due to the high redox potential values, aerobic bacteria and moulds are the most common cause of spoilage of these products. Oxygen has a remarkable influence on the redox potential of foods. It is a powerful oxidizing agent. If there is enough air in a food, it will result high positive potential. Many food processing technology operations (e.g. chopping, grinding, or mincing) increase the access of air to a food material, and it also increase the redox potential. For example the redox potential of raw meat is –200 mV, and in case of minced meat is +225 mV (Table 2.3.2). By contrast vacuum packing or canning is reducing the redox potential. There are some reducing agents (with the notable exception of oxygen) which naturally occurring are ascorbic acid and reducing sugars in vegetables and fruits, furthermore glutathione, cysteine, and sulfhydryl (–SH) groups in meats. Table 2.3.2: Redox potentials and pH of some foods (Adam–Moss, 2008; Bíró, 1993; Laczay, 2008; www.mtk.nyme.hu/~food/int-hu/mikro/segedletek/.../eloadas2.ppt) Foods Eh (mV) pH Raw meat (post-rigor) -200 5.7 Raw minced meat +225 5.9 Cooked sausages and canned meats -20 – -150 6.5 Cheeses -20 – -200 5.0 – 5.6 Wheat (whole grain) -320 – -360 6.0 Potato tuber -150 6.0 Spinach, spinach juice +74 6.2 Milk +200 – +340 6.4 – 6.8 Pear, pear juice +436 4.2 Grape, grape juice +409 3.9 Lemon, lemon juice +382 2.2 2.4. NUTRIENT CONTENT Like humans, microorganisms can use foods as a source of nutrients and energy. Food is necessary for them because of the following reasons: • because of the chemical elements, which constitute microbial biomass, • molecules which essential for growth and they are unable to synthesize, • substrates that can be used as an energy source. Microbial growth needs the following: • water, • source of energy, • source of nitrogen, • vitamins and related growth factors, • minerals. Taking account of the requirements listed above, moulds have the lowest requirement, followed by Gram-negative bacteria, yeasts, and Gram-positive bacteria. In case of foodborne microorganisms, the most common sources of energy are sugars, alcohols, and amino acids. Some microorganisms can also utilize complex carbohydrates (e.g. starches and cellulose) as sources of energy. Only a few microorganisms can use fats as sources of energy. A large number of nitrogenous compounds can be used as nitrogen sources by microorganisms. The primary nitrogen sources of microorganisms are amino acids. But some microorganisms can utilize free amino acids, peptides, proteins, and nucleotides. Growth factors are organic substances, which are necessarily needed to the growth of microorganisms. They are unable to produce them, so they must pick up them from the environment. The growth factors may be chemically classified in the following groups: • amino acids (e.g. valine and glutamic acid for lactobacilli) • vitamins (e.g. for streptococci and lactobacilli) • branched-chain fatty acids (for rumen bacteria) • cholesterol (for mycoplasma). Most of the microorganisms require B vitamins in low quantities. Gram-negative bacteria and moulds can synthesize B vitamins most of their requirements. But Gram-positive bacteria are needed to supply with one or more B vitamins. 2.5. SALINITY The salt concentration is closely related to water activity, but it is discussed separately because of special significance. High salt concentration is special case of the low water activity. Most of the pathogenic microorganisms grow optimally under 1-2% NaCl concentration. A higher salt concentration has specifically inhibitory effect. 7.5% NaCl concentration is the upper limit of most bacteria growth. Those microorganisms, which are able to grow under NaCl concentration from 7.5 to 15%, called halotolerant (salt-tolerant). They are able to grow in the absence of salt and also in the presence of relatively high salt concentration. An example for salt-tolerant yeasts is Debaryomyces hansenii. This group also includes some pathogens such as Staphylococcus aureus and Bacillus cereus. Microorganisms, which are able to grow above 15% NaCl concentration called halophiles (salt lovers), such as Actinopolyspora halophila. Among them, from a food hygiene aspect, Vibrio parahaemolyticus has outstanding importance. V. parahaemolyticus may cause food poisoning mainly through the consumption of raw, salted seafood. 2.6. PHYSICAL AND BIOLOGICAL STRUCTURES Biological structure of fruits and other parts of plants is natural barrier against microbial ingress. In general, epidermis and cuticle of plants, testa of seeds, outer shell of crops, shell of nuts, as well as the outer covering and wax of fruits provide significant protection against the penetration of microorganisms into the inner tissues. Like plants, the biological structure of raw materials of animal origin protects them (e.g. skin, fascia on meats, connective tissue membrane, outer shell and membranes of eggs, etc.). This natural protection usually lasts only as long as the biological structure is intact and unharmed. Plant materials, which are damaged during picking, shipping or storage, deteriorate faster than those are not damaged. Slaughtering and processing of animals also cause significant changes compared to the living condition. The cutting and boning of animal body, trimming the skin, furthermore removing the internal organs reveal new surfaces for the colonization of microorganisms. Extrinsic parameters The extrinsic parameters are not substrate dependent. These parameters are representative of the storage environment. These properties have effect on the foods and as well as on their microorganisms. These parameters are temperature of storage, osmotic pressure, relative humidity of environment, presence and concentration of gases, as well as processing and preserving operations. 2.7. TEMPERATURE OF STORAGE Temperature is the most important environmental factor which determining the growth of microorganisms. Microorganisms can grow over a very wide range of temperatures. The lowest temperature at which a microorganism has been reported to grow is −34◦C. The highest is somewhere in excess of 100◦C. Microorganisms are classified into different groups (psychrophiles, psychrotrophs, mesophiles, thermotrophs and thermophiles) according to how temperature influences their growth (Table 2.7.1). The values in table are approximate average values. Table 2.7.1: The grouping of microorganisms on the basis of temperature need (Biró-Szita, 2000; Deák, 2006; Laczay, 2008; Szabó, 2008) Temperature (oC) Group Minimum Optimum Maximum Psychrophiles –10 - +5 10-15 15-20 Psychrotrophs –5 - +5 20-30 30-40 Mesophiles 5-15 25-40 40-45 Thermotrophs 10-20 40-45 50 Thermophiles 25-45 50-80 60-90 In food microbiology mesophiles and psychrophiles have the greatest importance. Psychrophiles (cold-loving): their optimum temperature is between 10–15◦C, and they are unable to grow above 20◦C. They are mainly confined to polar regions and to the marine environment. Psychrotrophs (facultative psychrophiles): their optimum temperature is between 20◦C and 30◦C. Their minimum growth temperature almost the same as psychrophiles (they also can grow below 7◦C), but the optimum and maximum growth temperature is higher. Because of the wider range of temperature, psychrotrophs have greater significance in the spoilage of refrigerated foods, than psychrophiles. Psychrotrophs grow well at refrigerator temperatures and cause spoilage of refrigerated foods (e.g. meats, poultry, fish, eggs, etc.). The most common psychrotrophic bacteria genera are the following: Pseudomonas, Enterococcus, Alcaligenes, Shewanella, Psychrobacter, Brochothrix, Corynebacterium, Flavobacterium, Lactobacillus, Micrococcus, Pectobacterium, etc. Many moulds can grow at refrigerator temperatures (e.g. some strains of Cladosporium, Aspergillus, and Thamnidium genera). Mesophiles: their optimum temperature is between 25◦C and 40◦C. They cause rapid spoilage of foods stored at room temperature. Their origin is frequently human or animal. The most common mesophilic foodborne pathogens are: Salmonella, Staphylococcus aureus and Clostridium perfringens. Thermotrophs: their optimum temperature is between 40◦C and 45◦C. The most common thermotrophs are thermotolerant coliforms, salmonellas and campylobacters. Thermophiles: their optimum temperature is between 50◦C and 80◦C. The most important representatives of thermophiles are thermophilic lactic acid bacteria (e.g. Streptococcus salivarius subsp. thermophilus) and the endospore forming Bacillus and Clostridium genus. Other thermophilic bacteria genera, which have importance in foods, are Alicyclobacillus, Geobacillus, Paenibacillus, and Thermoanaerobacter. Thermofilic bacteria (particularly spore formers) have considerable importance in the canning industry. 2.8. OSMOTIC PRESSURE The more dilute solution will flow toward the more concentrated until concentrations are equal. The driving force of the equalization through the cell wall results the osmotic pressure. The osmotic pressure is expressed in bar. The medium could be hypo-, iso- and hypertonic. The medium is hypotonic, when the solution concentration of the environment more dilute than the solution concentration of the cell. In this case microorganisms take up water and nutrients from the environment, and thus able to grow. The medium is isotonic, when the concentration of the two solutions is equal. At this time there is no water uptake or grow. The medium is hypertonic, when the solution concentration of the environment more concentrated than the solution concentration of the cell. Then the microbe loses water content and dies. This occurs in more concentrated saline and sugar solution. Plasmoptysis: in case of sudden large amount of water absorption, the cell pressure increased in the cell, causing the rupture of the cytoplasmic membrane and cell wall, as well as the protoplasm is ejected. Plasmolysis: the hypertonic solutions withdraw water from the bacterial cell (cell shrinks), causing the cell protoplasm separates from the cell wall. Table 2.8.1 show the relationship between the osmotic pressure of foods and the equilibrium relative humidity (ERH %). Table 2.8.1: The relationship between the ERP value and osmotic pressure (Bíró, 1993; Biró-Szita, 2000) ERH % Osmotic pressure (bar) Osmophilic 60 570 65 564 70 466 Xerophilic 75 377 80 292 Xerotolerant 85 212 90 138 Hydrophilic 95 67 100 0 The osmotic pressure of food can be increased by reducing the water content. The partial removal of water content called concentration (e.g. tomato puree), and the complete removal is called desiccation or drying (e.g. milk powder, egg powder). On the basis of the reaction to the osmosis, the following groups can be separated: • Osmotolerant: they survive but not grow in hypertonic medium. • Facultative osmophilic: their maximum growth is in hypotonic medium, but they can also grow in hypertonic medium. • Obligate osmophilic: they grow only in hypertonic medium. 2.9. RELATIVE HUMIDITY OF ENVIRONMENT Relative humidity and water activity are interrelated. Relative humidity (RH) is a measure of the water activity of the gas phase. The relative humidity of the storage environment is important in two aspects: 1. in terms of water activity inside the foods, and 2. in point of view the growth of microorganisms at the surfaces. When low water activity food is stored in high RH environment, the food will pick up moisture from the air until equilibrium has been established. If microorganisms can start to grow, they usually produce water as an end product of respiration. Thereby they increase the water activity of their own immediate surroundings. Because of the increasing water activity, the waterconsuming microorganisms are also able to grow and spoil food. High water activity foods will lose moisture when placed in low relative humidity environment. The storage of fresh vegetables and fruit needs very mindful control of RH. If the RH is very low during the storage, in this case fruits and vegetables will give off water and become flaccid. If the RH it is too high, in this case condensation may occur on the surface of fruits and vegetables, furthermore microbial spoilage may be initiated. Modifying the gaseous atmosphere, it is possible to slow down the surface spoilage of food, and no need to reduce the relative humidity. 2.10. PRESENCE AND CONCENTRATION OF GASES IN THE ENVIRONMENT Changing the composition of the air (reducing oxygen content, increasing carbon dioxide content) allows a preferred method of storage. The so-called controlled atmosphere storage, which is often combined with cooling, is advantageous in two ways. On the one hand, because it inhibits the respiration of fruits and vegetables (slow maturation), on the other hand, it inhibits the growth of spoilage-causing microorganisms (mainly aerobic moulds). Similar conditions may also occur in the vacuum-packaged foods. In this case increase the possibility of deterioration caused by lactic acid bacteria. Main atmospheric gases, which are used to regulate microorganisms in foods, are carbon dioxide (CO2) and ozone (O3). In case of modified atmosphere packaged (MAP) foods carbon dioxide (CO2) and oxygen (O2) are used. Ozone (O3) has antimicrobial properties, but it is a strong oxidizing agent. Ozone should not be used in case of high-lipid-content foods, because it causes rancidity of them. Carbon dioxide (CO2) is most commonly used in modified atmosphere packaged foods, in carbonated mineral waters, carbonated beverages, and soft drinks. The effect of CO2 on microbes is not uniform. In general, the Gram-positive bacteria (especially lactobacilli) are the most resistant; however the oxidative Gram-negative bacteria and moulds are the most sensitive. Growth inhibition of carbon dioxide is usually greater under aerobic conditions, and low temperature. 2.11. PROCESSING AND PRESERVING OPERATIONS The raw materials are always microbiologically contaminated before processing, and this contamination is modified by several internal and external environmental factors. Such external factors are the various operations that are included in the food processing. Some of the processing operations (e.g. sorting, washing, peeling) significantly reduce, while others (e.g. shredding, chopping, spices, additives) are inevitably increase food contamination. Equipment can also be sources of pollution if their surfaces are contaminated by microorganisms. From this perspective it should be emphasized the importance of hygiene operations (cleaning, cleaning, disinfection) as an integral part of the technological processes, as well as the maintenance of good manufacturing practices. 3. FOODBORNE DISEASES The definition of foodborne disease by the World Health Organization (WHO): “Any disease of an infectious or toxic nature caused by, or thought to be caused by, the consumption of food or water”. The definition includes not only diseases with the enteric symptoms (diarrhoea and/or vomiting), but also other symptoms, like those caused by toxic chemicals, moreover botulism and listeriosis. In spite of the general regards, microbial hazards still have the highest priority of becoming ill connected to food consumption. Several microbial diseases may be originated from foods, but only some of them contracted exclusively (e.g. hemorrhagic colitis and listeriosis). Others, like botulism and staphylococcal food poisoning are predominantly from the consumption of food products. Some of the previously important foodborne diseases like anthrax and brucellosis rarely if ever contracted via the foodborne route nowadays. Foodborne pathogens include the following most important groups and taxa: Bacteria Protozoa Gram-positive Giardia Staphylococcus Entamoeba Bacillus cereus Toxoplasma B. anthracis Sarcocystis Clostridium botulinum, Cryptosporium C. argentinensis Cyclospora C. perfringens Fungi—mycotoxin producers Listeria monocytogenes Aspergillus Mycobacterium avium subsp. Fusarium paratuberculosis Penicillium Gram-negative Alternaria Salmonella Multicellular animal parasites Shigella Flatworms Escherichia Flukes Yersinia Fasciola Vibrio Fasciolopsis Campylobacter Paragonimus Brucella Clonorchis variant form) Tapeworms Viruses Diphyllobothrium Hepatitis A Taenia Noroviruses (Norwalk, etc.) Roundworms Rotaviruses Trichinella Prions Ascaris Anisakis Creutzfeldt-Jakob disease (new Toxigenic Phytoplanktons Pseudoterranova Paralytic shellfish poison Toxocara Ciguatoxin Foodborne disease can only enter through the moth, and must be ingested. All foodborne agents may be contracted via the fecal–oral route, except for botulinal toxins, the mycotoxins, and the phytoplankton toxins, and not so common for staphylococcal food poisoning. However this route is the primary route of infection for the foodborne viruses and enteropathogenic protozoa and bacteria. The fecal–oral route means, foodborne pathogens are transmitted from contaminated feces either via the fingers or cloths of food handlers (because of unsatisfactory hygienic conditions), or by insects, or from water (Figure 3.1). Figure 3.1: The fecal–oral routes of transmission of foodborne intestinal pathogens (Based on Jay et al., 2004) Following the enter of the microbes into the gastro-intestinal tract, pathogen must fulfil several requirements in order to cause illness. (1) First of all it must survive the extremely acidic environment of the stomach. (2) Then colonization of the intestinal walls or attach is necessary for enabling the multiplication of the pathogens. The intestinal mucosa lining the intestinal epithelial surface is defence but, Listeria monocytogenes, is able to overcome the mucus barrier by removing mucus through the aid of listeriolysin O (LLO). Some pathogens pathogen, such as C. perfringens, however does not need to attach to the intestinal tissues. (3) Intestinal pathogens must defend themself against host defense mechanisms in the gut associated lymphoid tissue. (4) The harmless intestinal microbiota is able to exclude pathogens. That means, pathogens microbes have to be able to compete with the heterogeneous and large microbiota of the gut, and the the low-O2 environment gastrointestinal tract. (5) Finally, the organisms need to be able to either elaborate toxic products (e.g., Vibrio cholerae) or cross the epithelial wall and enter phagocytic or somatic cells (e.g., L. monocytogenes). Intestinal pathogens have different site of action. Helicobacter is the only bacterium that colonizes stomach walls, but its foodborne origin has not been proved. Small intestine is colonized by several pathogens: viruses (Astroviruses, Hepatitis A, Rotaviruses), bacteria (Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, Escherichia coli – EPEC and ETEC strains, Listeria monocytogenes, nontyphoid Salmonellae, S. Typhi, Shigellae, Vibrio cholera, V. parahaemolyticus, Yersiniae), protozoa (Cryptosporidium parvum, Cyclospora cayetanensis, Giardia lamblia, Toxoplasma gondii) and helmints (Tapeworms). Several pathogens can colonoze the large intestine as well: Campylobacter, Escherichia coli – EHEC and EPEC strains, Entamoeba histolytica, Salmonella Enteritidis, Shigella dysenteriae. Liver can be colonized by liver flukes, moreover Listeria monocytogenes and viruses (foodborne Hepatitis A and E). Finally Trichinella spiralis grow and encyst in skeletal muscles. The pathogenesis of the foodborne pathogens is very versatile. The helminths are contracted by ingesting infected meat or fish. Following their entry, the may passage to different organs (liver, skeletal muscles) or remain in the gastrointestinal tract. The foodborne protozoa remain in the gut, with the notable exception of Toxoplasma gondii. It can cross the intestine, even the placental barrier, resulting severe damage to a fetus. The mycotoxins are ingested preformed, accumulated in different organs, or cell components (e.g., aflatoxins attach to DNA). The pathogenic mechanisms of the foodborne bacteria are more complex. Gram-positive pathogens produce exocellular virulence factors. For example, Staphylococcus aureus virulent strains are produce a number of exotoxic factors, but only enterotoxin producing strains cause the gastroenteritis syndrome. Also produced exotoxins are responsible for the diseases caused by Clostridium botulinum, C. perfringens, and Bacillus cereus. Although Listeria monocytogenes is also a Gram positive bacterium, it has a different mechanism as a pathogen. Virulent strains of L. monocytogenes can breach the mucous barrier and enter epithelial cell with the help of different factors, like listeriolysin O (LLO). It is an intracellular pathogen. The pathogenesis and virulence properties of Gram-negative bacteria are different and more complex than for Gram-positive bacteria. S. enterica serovars carry pathogenicity islands 1 and 2 (SPI-1, SPI-2), containing the majority of the pathogenicity genes. These genes may be acquired via horizontal transfer by extrachromosomal, mobile genetic elements (plasmids or phages) between the closely related salmonellae, E. coli and shigellae. Different factors play role in the pathogenesis of these bacteria. The first requirement that an intestinal invasive pathogen must meet is that of intestinal adhesion. Mobile genetic elements plays role in the transfer of adherence/adhesive genes between avirulent and virulent strains. Fimbriae enhance the bacterial attachment to the intestinal cells (e.g. fimbrial adhesins in the virulent strains of S. enterica). They may also produce different enterotoxins, usually coded on extrachromosomal elements. One xamle of the horizontal gene transfer is the EHEC strains evolved from EPEC via acquisition of phage-encoded Shiga toxins. 3.1. STAPHYLOCOCCUS AUREUS AND STAPHYLOCOCCAL GASTROENTERITIS Scientific classification Domain: BACTERIA Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Staphylococcaceae Genus: Staphylococcus Species: Staphylococcus aureus Staphylococcus genus Staphylococci were first described by Sir Alexander Ogston (1844-1929), who was a Scottish surgeon. In 1882, Ogston gave them the name staphylococcus (Greek: staphyle means bunch of grapes; coccus means grain or berry), based on the microscopic morphological properties. Staphylococci are facultative anaerobe, catalase-positive, small, spherical, Gram-positive bacteria. Most of the species are able to grow in the presence of bile salts. Their cells have diameters ranging from approximately 0.5 to 1.5 µm. They are chemoorganotrophs with a DNA composition of 30 to 40 mol% guanine + cytosine content. The genus Staphylococcus includes over 40 species and subspecies. Most of them are harmless, and they are usually found on the skin and mucous membranes of humans and other organisms (e.g. warm blooded animals). Many of them are present in food as a result of human, animal, or environmental contamination. Staphylococci often cause pyogenic infections in humans. Numerous species can be linked to hosts, such as S. hyicus with pigs, and S. gallinarum with chickens. The coagulase producing ability is the most important phenotypical characteristic of the species, which is commonly used in the classification of genus Staphylococcus. Coagulase is a protein enzyme, which clotting of the blood. Coagulase-positive staphylococci usually produce thermostable nuclease (TNase). Several species of Staphylococcus can produce Staphylococcal enterotoxins (SEs). Although several species, including some coagulase-negative staphylococci, have the potential to cause gastroenteritis, nearly all cases of Staphylococcal food poisoning (SFP) are attributed to S. aureus. This is a reflection of the relatively high incidence of SE production by S. aureus in comparison to that of other staphylococcal species. Characteristics of Staphylococcus aureus Staphylococcus aureus is a catalase-positive, oxidase-negative, facultative anaerobe, Grampositive coccal bacterium. The cells about 0,5-1,5 µm in diameter, and regularly form irregular clusters which resemble a bunch of grapes. S. aureus is a mesophilic bacterium, which has a growth temperature range between 6 and 48◦C. Their optimum temperature is 37◦C. They can produce enterotoxins between 10◦C and 45◦C, with the optimum between 35◦C and 40◦C. The heat resistance (D62 = 20–65 s, D72 = 4.1 s) of S. aureus is exceptional. The optimum pH for growth is between 6.0 and 7.0. The minimum pH for growth is 4.0, and the maximum pH is 9.8. The pH range of the enterotoxin production is between pH 4.5 and 9.0, and the optimum pH is between 6.0 and 7.0. S. aureus can well tolerate the high salinity and low water activity of media. It can grow well in media which containing 5–10% NaCl, and some strains can grow in 20% NaCl. Staphylococci can grow at lower water activity (minimum aw is 0.83) values than any other salt-loving (nonhalophil) bacteria. The minimum water activity of the enterotoxin production is 0.86 (Table 3.1.1). Table 3.1.1: Ecological parameters of growth and enterotoxin production of Staphylococcus aureus (Laczay, 2008) Growth Enterotoxin production Ecological parameters Range Optimum Range Optimum o Temperature ( C) 6-48 35-37 10-45 35-40 pH 4.0-9.8 6.0-7.0 4.5-9.0 6.0-7.0 NaCl (%) 0-20 0.5-4.0 0-20 0.5 Water activity 0.83-0.99 0.98-0.99< 0.86-0.99< 0.99< Redox potential (mV) -200 - +200< +200< +200< On the basis of their ability to ferment glucose they can be distinguished from the Micrococcus. Majority of S. aureus strains are coagulase-positive, so they produce coagulase enzyme, but some of them do not produce coagulase. Pathogenicity of Staphylococcus aureus S. aureus is the most common human bacterial pathogen. The degree of pathogenicity is determined by the biochemical activities of S. aureus (coagulase, hemolysis, DNase, phosphatase, mannitol fermentation and gelatin hydrolysis). S. aureus have many virulence factors, including protein-, lipid-, carbohydrate-, nucleic acid-degrading enzymes, toxins, and factors which destroy phagocytes. Pathogens produce plasma clotting coagulase and thermostable nuclease enzymes. Cell-associated virulence factors: e.g. capsular polysaccharide, microcapsules, mucus, teichoic acids, lipoteichoic acids, protein A, specific adhesion proteins (fibrinogen, fibrin, fibronectin, thrombin, collagen). Extracellular nontoxic pathogenicity factors (enzymes): • Coagulase (free and bound): it is a protein enzyme which enables the conversion of fibrinogen to fibrin (clots blood plasma) and form a protective layer around the cells, which protects them from the phagocytosis. • Hyaluronidases (also known as spreading factor): they are family of enzymes that degrade hyaluronic acid. They destroy tissues, thereby aiding the spread of bacteria (invasion). • Staphylokinase (fibrinolysin): it is an amino acid enzyme. It activates plasminogen, which is an inactive precursor of plasmin. When plasminogen becomes activated it is converted to plasmin. Plasmin is a trypsin-like serine protease, which digest fibrin clots. This enzyme • • • • attacks and inactivates (dissolve) fibrin molecules; therefore it helps the spread of bacteria (invasion). Deoxyribonuclease (DNase): it is an enzyme, which catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA (degrading DNA). Phosphatase: this enzyme removes a phosphate group from a protein (dephosphorylation). Lipase: it helps to digest lipids. Beta-lactamases: enzymes which provide resistance to β-Lactam antibiotics (e.g. penicillins), because they break the structure of antibiotics. Pyrogenic toxin superantigens (PTSAgs): Superantigens are molecules, which are able to stimulate a much higher percentage of T cells than conventional antigens. • Toxic shock syndrome toxin (TSST-1) • Staphylococcal enterotoxins (SEs) - 5 classical types: SEA, SEB, SEC, SED and SEE - New types and enterotoxin-like (SEl) toxins: SEG, SEH, SEI, SER, SET, SES, SElJ, SElK, SElL, SElM, SElN, SElO, SElP, SElQ, SElU and SElX - They are resistant to heat and trypsin. - Enterotoxin B production is the most common. - The enterotoxigenic strains belong to the phage group III. Exfoliative toxins: Exfoliative (EF) toxins (A and B): which are implicated in the disease staphylococcal scalded skin syndrome (SSSS). This syndrome most commonly occurs in infants and young children. Other toxins: Toxins are thermolabile, antigenic, and antitoxins are produced against them. • α-hemolysin (α-toxin): this toxin is the major cytotoxic agent, which form pores in the cellular membrane and cause the death of cell. • β-hemolysin (β-toxin, sphingomyelinase C) • γ-hemolysin (bicomponent) • δ-hemolysin (δ-toxin): it has a wide spectrum of cytolytic activity. • Leukocidins: they are pore forming cytotoxins, which kill leukocytes. Occurrence of Staphylococcus aureus Among the non-spore-forming human pathogen, S. aureus is one of the most resistant. It is able to survive for extended periods in a dry state. In case of the human disease-causing staphylococci, the main reservoir is the humans. Most of the staphylococci are normal inhabitants of the outer regions of the body. S. aureus occurs most frequently on the skin of higher primates, and in the nasal tract of the humans (20–50% of healthy individuals). However it is also a human pathogen. People can be carriers and sources of transferring bacteria to other humans and to food. Dissemination can occur by direct contact, indirectly by skin fragments, or through respiratory tract droplet nuclei. S. aureus is commonly found in faeces and occasionally from soil, marine and fresh water, plant surfaces, dust, and air. Most sources of staphyloccal food poisoning are humans who can contaminate food during preparation, furthermore contaminated equipment (e.g. meat grinders, knives, storage utensils, cutting blocks, saw blades etc.) used in food processing. Animals are also an important source of staphyloccal food poisoning. They are often highly colonized with staphylococci. Mastitis is a very serious and common problem in the dairy industry. It is an infectious disease of mammary tissue, which often caused by S. aureus. It is one of the most costly diseases in animal agriculture, because of the combined losses and expenses associated with the disease. Raw milk and dairy products can be contaminated with S. aureus by mastitic cow’s milk or during processing. In addition, S. aureus is often found in small numbers in a variety of foods (e.g. poultry, and other raw meats). S. aureus can be eliminated from the product by cooking or pasteurization. Common vehicles of S. aureus: • poultry products, • cold, cooked meats, • salted meats (e.g. ham, corned beef), • canned foods, • raw milk, • hard cheeses, • cold sweets, • cream-filled bakery products, • ice cream. Food poisoning caused by Staphylococcus aureus Food poisoning caused by S. aureus have short incubation period (typically 2–4 h), which is characteristic of an intoxication, where illness is the result of ingestion of a pre-formed toxin in the food. Though S. aureus toxins are often described as enterotoxins, they are strictly neurotoxins. Huge numbers of bacteria (above 106 cfu/g) can produce so many toxins, which cause illness. Usually contamination is not enough for an outbreak, it also necessary appropriate bacterial growth conditions (temperature and time). The most common symptoms are nausea, vomiting, stomach cramps, retching, prostration, and diarrhea. Recovery is normally complete within 1–2 days. In case of severe cases (e.g. dehydration, marked pallor and collapse) may be necessary intravenous infusion treatment. The following factors are help to prevent the staphylococcal food-poisoning: • produce foods with low numbers of staphylococci, • keep foods below 7◦C or above 60◦C until consumed. Following conditions most often associated with food poisoning: • inadequate refrigeration; • preparing foods far in advance of planned service; • infected persons’ practicing poor personal hygiene (e.g., not washing either hands or instruments properly); • inadequate cooking or heat processing of food; prolonged use of warming plates (at bacterial growth temperatures) when serving foods, a practice that promotes staphylococcal growth and staphylococcal enterotoxins. 3.2. LISTERIA MONOCYTOGENES AND FOODBORNE LISTERIOSIS Scientific classification Domain: BACTERIA Phylum: Firmicutes Class: Bacilli Order: Bacillales Family: Listeriaceae Genus: Listeria Species: Listeria monocytogenes Listeria genus The listeriae are Gram-positive, non-spore-forming, and non-acid-fast rods. Earlier this genus was classified in the family Corynebacteriaceae, but in 2004, it was moved into the newly created family Listeriaceae. There are two genera in this family (Listeria and Brochothrix). The two genera is similar in many ways (e.g. both genera are catalase-positive, and the mol% G + C is less than 50). The genus contains the following species: L. monocytogenes, L. ivanovii, L. innocua, L. fleischmannii, L. grayi, L. seeligeri, L. marthii, L. rocourtiae, L. weihenstephanensis and L. welshimeri. Within the genus Listeria, pathogen species are: L. monocytogenes (in humans and animals), L. ivanovii and L. seeligeri (especially in animals). Within the L. monocytogenes species, thirteen serotypes are able to cause disease. The most common serovars are 1/2 and 4. More than 90% of human isolates belong to three serotypes: 1/2a, 1/2b, and 4b. Historical overview of Listeria monocytogenes Listeria monocytogenes was first described by Everitt Murray in 1926 as Bacterium monocytogenes, as an intracellular pathogen, which cause infection of laboratory rabbits. In the infected rabbits, the infection was associated with increased number of monocytes in peripheral blood. Listeriae were originally classified as Listerella. However, Listerella was already in use for a slime mould and a protozoan. The generic name was changed to Listeria in 1940, in honor of English pioneer of antiseptic surgery Joseph Lister (1827-1912). The species name refers to that, in case of infection the monocytes accumulate in blood. L. monocytogenes was first reported as cause of infection in humans by Nyfeldt in 1929. L. monocytogenes has been associated with the meningitis by M. Rodler (Hungarian) in 1965. The CAMP (Christie–Atkins–Munch–Petersen) test is considered by many to be the definitive test for L. monocytogenes. Characteristics of Listeria monocytogenes L. monocytogenes is Gram-positive, non-spore-forming, facultative anaerobe, oxidase-negative and catalase-positive. The cells are coccoid to rod shaped (0.4–0.5 µm, 0.5–2.0 µm). If the bacteria cultured at 20–25◦C, they have peritrichous flagella and a characteristic tumbling motility. L. monocytogenes can grow over a wide range of temperature from 0–45◦C with an optimum between 30 and 35◦C. The growth of bacterium is extremely slow below 5◦C. L. monocytogenes is preserved or moderately inactivated under 0°C. The resistant of bacteria against freezing is one of the largest between the foodborne pathogens. L. monocytogenesis can be inactivated by exposure to temperatures above 50°C. The heat resistance of L. monocytogenes is not so large (D60 = a few minutes, D70 = a few seconds. In case of contaminated milk, the bacterium cells inside the milk leukocytes are protected from heat. Most of the strains are unable to grow at pH values below 5.6. The pH range for the growth of L. monocytogenes is between 5.6 and 9.6. However, the bacterium can initiate growth in media at pH values as low as 4.4. L. monocytogenes can survive this low pH (4.4) value, but it is unable to grow. The incubation temperature and the type of acid affect the growth at low pH values. L. monocytogenes grows optimally at water activity of 0.97. For most strains, the minimum water activity for growth is 0.90–0.93. L. monocytogenes is able to survive for long periods at water activity value of 0.83. This bacterium is salt tolerant as well. It is able to grow in 10–12% sodium chloride. The bacterium survives for long periods at high salt concentrations. Most of the pathogenic L. monocytogenes strains cause beta-hemolysis on blood agar, and ferment rhamnose but not xylose. L. monocytogenes produce listeriolysin O (LLO) toxin, which is a hemolysin. This is a virulence factor of the bacterium. LLO is a non-enzymatic, cholesterol-dependent, thiol-activated, cytolytic, pore-forming toxin protein. All strains of L. monocytogenes (also some non-hemolytic) produce listeriolysin O. Occurrence of Listeria monocytogenes L. monocytogenes is present everywhere in the environment. The bacteria can be found in fresh and salt water, soil, sewage sludge, decaying vegetation, silage, furthermore in the faeces of pigs, sheep, cattle, chickens, turkeys and ducks. Listeriae generally live there, where the genus Brochothrix, lactic acid bacteria, and some coryneform bacteria occur. Most of the animal or plant origin food products are containing L. monocytogenes. The bacteria is generally found in raw milk, dairy products (e.g. soft unripened and surface-ripened cheeses), fresh and frozen meat, frankfurters, delicatessen meats (e.g. salami, ham, corned beef, brawn and paté), poultry, smoked fish, seafood products, furthermore on fruits and vegetable products (Figure 3.2.1). L. monocytogenes can grow in the refrigerated stored ready-to-eat (RTE) foods during manufacture, transportation, and storage. L. monocytogenes can be getting into the food processing plant by contaminated animals, raw food of animal origin, raw plant tissue, furthermore by healthy human and with their soil polluted shoes or clothes. Listeriae can grow in the presence of high humidity and nutrients. L. monocytogenes is most often recovered from moist areas such as floor drains, condensate, stagnant water, floors, and residues on processing equipment. L. monocytogenes can attach to various types of surfaces including stainless steel, glass, plastic, and rubber and form biofilms as has been described in meat and dairy processing environments. The bacteria are able to survive the hand-washing. L. monocytogenes can be found in the faeces in different human populations without symptoms (e.g. healthy people, pregnant women, patients with gastroenteritis, slaughterhouse workers, food handlers, and laboratory workers. etc.). L. monocytogenes can be found in 2 to 6% of fecal samples from healthy people, whereas listeriosis patients often excrete higher numbers. Figure 3.2.1: Proportion of single samples at processing and retail in non-compliance with EU L. monocytogenes criteria in 2011 and 2012 (EFSA – ECDC, 2014) Listeriosis L. monocytogenes is a facultative intracellular pathogen (such as Brucella and Mycobacterium) which able to survive and proliferate in the cells of the monocyte–macrophage system. Listeriosis is caused by L. monocytogenes. It has emerged as a major foodborne disease during the past 30 years. Listeriosis is not a typical foodborne illness. It has a major public health concern because of the following reasons: - seriousness of the disease (meningitis, spontaneous abortion, and septicemia), - high mortality rate (about 20–30%), and - long incubation period (up to 80 days). The usual incubation period is a few weeks, but it can vary between 1 and 90 days. This makes it difficult to explore the causes of the disease. A seasonal pattern was observed in the listeriosis cases reported in the EU in the period 2008 and 2012. There was a statistically increasing trend over this period, though only slowly increasing (Figure 3.2.2). The number of confirmed cases of human listeriosis in 2012 was 1642, which means a 10.5% increase over the previous year. Figure 3.2.2: Trend in reported confirmed cases of human listeriosis in the EU between 2008 and 2012 (EFSA – ECDC, 2014) Control of L. monocytogenes in foods represents a significantly greater challenge than most foodborne pathogens. The reasons are the following: - this bacterium is widely distributed in the environment, - it is resistant to diverse environmental conditions (e.g. low pH, high NaCl concentrations), - it is facultatively anaerobic, - it is psychrotrophic. The following reasons show why this bacterium has major concern in the agri-food industry: - there are various ways L. monocytogenes can enter food processing plants, - its ability for prolonged survival in the environment (soil, plants, and water), on foods, and in food processing plants, - its ability to grow at low temperatures (2 to 4°C), - it can survive in biofilms, furthermore on foods and food contact surfaces for a long time under unfavourable conditions. Listeriosis is more common in individuals, which are exposed to a greater risk (such as pregnant women, neonates, immunocompromised adults, and the elderly). In 2012, the highest notification rates of listeriosis were reported in persons aged below one and those aged 65 years and above. In the latter group, the rates increased by age. Major differences in notification rates were also observed in terms of gender. Female cases dominated in the age groups 15-24 and 25-44 years and 71.3 % of these cases were related to pregnancy. Higher incidence rates were observed in male cases compared to female cases in all age groups above 45 years. In these age groups, the male-to-female rate ratio increased by age. In the oldest age group (85 years or above) the male-to-female rate ratio was 1.7 (Figure 3.2.3). Figure 3.2.3: Notification rates of human listeriosis by age and gender in the EU in 2012 (EFSA – ECDC, 2014) Symptoms of the disease can vary from a mild, flu-like illness to meningitis and meningoencephalitis. In case of pregnant women, the most common symptoms of the influenzalike illness are the fever, headache and sometimes gastrointestinal symptoms. During infection transplacental foetal infection may occur, which may cause abortion, stillbirth, or premature labour. Listeriosis in the neonates can be an early-onset syndrome (occur at birth or shortly later) or a late-onset syndrome (occur several days or weeks after birth). The early onset syndrome can be caused by intrauterine infection. Most common syndromes are the sepsis, pneumonia, and granulomas. Syndromes of late-onset syndrome are meningitis and sepsis. The healthy people are very resistant to infection caused by L. monocytogenes. Syndromes of listeriosis in case of non-pregnant adults are septicemia, meningitis, meningoencephalitis, febrile gastroenteritis, and endocarditis. The mortality rate of endocarditis is about 20–30%. The gastrointestinal listeriosis infection requires high dose (105–109 cfu/g) of L. monocytogenes. Most cases of human disease caused by L. monocytogenes occur sporadically. Only three (1/2a, 1/2b, and 4b) of the fourteen known serovars of L. monocytogenes, account for more than 90% of human and animal cases of listeriosis. 3.3. FOOD POISONING CAUSED BY GRAM-POSITIVE SPORE-FORMING The two most important genera of gram-positive bacteria are Bacillus and Clostridium. Bacillus species are aerobic. Clostridia however lack respiratory chain. They obtain energy (ATP) only by substrate-level phosphorylation (fermentation). They are found in nature primarily in soil, where living as saprophytic soil organisms and infect animals only incidentally. Foodborne diseases caused by Gram-positive spore-forming rods are due to their exotoxin production. There are at least three important foodborne pathogen species: Clostridium perfringens, C. botulinum, and Bacillus cereus. A potent neurotoxin is responsible for the symptoms in botulism, which is produced by cells growing in susceptible foods. The C. perfringens enterotoxin is produced during sporulation of bacterial cells in the gastrointestinal tract. B. cereus may cause two types of food-associated illness. The emetic disease is a food intoxication, but the diarrhoeal syndrome of is an infection caused by ingested vegetative cells or spores. The incidence of food poisoning caused by each of these organisms is related to certain specific foods. 3.3.1. BACILLUS CEREUS GASTROENTERITIS Taxonomy Domain: Phylum: Class: Order: Family: Genus: Species: Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus B. cereus The ‘B. cereus group’ contains six genetically highly similar species: B. cereus sensu stricto, B. anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. Bacillus cereus is a facultatively anaerobic, Gram-positive, rod-shaped bacterium (bacillus means small rod) with 1.0–1.2 µm diameter and 3.0–5.0 µm length. Its greyish colonies are large (3–8 mm diameter), rather flat, often with irregular borders (cereus means wax-like). It is able to grow on common agar media, and the colonies are surrounded by β-haemolysis zones on blood agar. Its ellipsoidal endospores are central in the cell and do not cause swelling (Fig. 3.3.1.1). Figure 3.3.1.1: Bacillus cereus cell morphology (left) and colony morphology (right) on commom agra medium and on blood surrounded by β-haemolysis zones agar (Source: http://www.ppdictionary.com/bacteria/gpbac/cereus.htm (Pathogen Profile Dictionary), download time: 14.12.2014.) It is ubiquitous and normally present in soil, dust, water, and vegetation; moreover it is also a common component of the transient gut flora in humans. It cannot tolerate low pH (< 5.0), or water activity. B. cereus sensu stricto is mesophilic, growing between 10 and 50 ◦C. The psychrotolerant B. cereus strains were separated and described as B. weihenstephanensis. This species is characterized by the ability to grow below 7 ◦C but not at 43 ◦C, and also has special genetic characteristics. B. anthracis, B. cereus and B. thuringiensis genetically are closely related. The distinguishing features between the species are encoded by genes located on the highly mobile genetic elements (plasmids). Bacillus cereus can be isolated from different foods and food ingredients: rice, dairy products, spices, dried foods and vegetables. Cross-contamination can distribute spores or cells to other foods, like meat products. B. cereus spores can adhere to the surfaces, moreover able to form biofilms. It may cause hygienic problem in food industry equipment, as the biofilm protects spores and vegetative cells against inactivation by sanitizers. It is frequently isolated from dairy products and lightly heat-treated foods with extended refrigerated storage, but any food with pH>4.8 possess a risk of food spoilage or foodborne disease by B. cereus. An early report associating food poisoning with Bacillus spp. in 1906, when spore forming aerobic bacillus was isolated from meatballs in a sanatorium where 300 people developed symptoms of profuse diarrhoea, stomach cramps and vomiting. The first described food poisoning by B. cereus was described in hospitals in Norway in 1947–1949. Two distinct foodborne disease types are associated with B. cereus: emetic and diarrhoeal, and both syndromes are caused by enterotoxins (Table 3.3.1.1). The diarrhoeal syndrome of B. cereus is an infection caused by vegetative cells, ingested as viable cells or spores. The onset of symptoms of the diarrhoeal syndrome is about 8–16 h after consumption of the food, lasts for between 12 and 24 h, and is characterized by abdominal pain, profuse watery diarrhoea and rectal tenesmus. Nausea and vomiting are less frequent. Vegetative cells of the bacterium are producing heat sensitive protein enterotoxins in the small intestine in the late exponential/early stationary phase of growth. The toxins are also sensitive to proteolytic enzymes, like trypsin and pepsin. Three pore-forming cytotoxins are currently associated with diarrhoeal disease: haemolysin BL (Hbl), nonhaemolytic enterotoxin (Nhe) and cytotoxin K (CytK). The Hbl and the non-haemolytic Nhe are consisting of three proteins, while CytK has only one component. Some strains produce both HBL and NHE though others contain the genes for only one. Table 3.3.1.1: Characteristics of the two types of Bacillus cereus foodborne disease (Adapted from Stenfors Arnesen et al., 2008) Characteristics Diarrhoeal disease Emetic disease Type of toxin Protein enterotoxin(s): Hbl, Nhe, CytK implicated Cyclic peptide: emetic toxin (cereulide) Location of toxin production In the small intestine of the host Preformed in foods Infective dose 105–108 cfu (total) live cells are not required for intoxication Incubation time 8–16 h (occasionally ~24 h) 0.5–6 h Duration of illness 12–24 h (occasionally several days) 6–24 h Symptoms Abdominal pain, watery diarrhoea and occasionally nausea. Lethality has occurred. Nausea, vomiting and malaise A few lethal cases (possibly due to liver damage) Foods most frequently implicated Proteinaceous foods; meat products, soups, vegetables, puddings, sauces, milk and milk products Starch-rich foods; Fried and cooked rice, pasta, pastry and noodles The emetic syndrome was first identified in the early 1970s. This disease is an intoxication caused by the emetic toxin, named cereulide. The incubation period is between 0.5 and 6 hours, and the duration is normally 6–24 h. Vomiting is not accompanied by diarrhoea. It can be severe even lethal. Cereulide, is a 1.2 kDa cyclic cyclic dodecadepsipeptide that is acid and heat resistant. It is produced by a nonribosomal peptide synthetase, encoded by the 24-kb cereulide synthetase (ces) gene cluster which is located on a megaplasmid related. The toxin is produced in the food in the late exponential to stationary phase of growth and is thought to act by binding to and stimulating the vagus nerve. 3.3.2. CLOSTRIDIUM PERFRINGENS FOOD POISONING Taxonomy Domain: Phylum: Class: Order: Family: Genus: Species: Bacteria Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium C. perfringens Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. C. perfringens is a Gram-positive, rod-shaped anaerobe. Though a catalase-negative anaerobe, but survive and occasionally grow in the presence of oxygen. Its endospores are oval and situated subterminal in the cells. It differs from most other clostridia in that the relatively large rods (1 µm diameter and 3 µm length) are encapsulated and nonmotile (Fig. 3.3.2.1). Figure 3.3.2.1: Coloured scanning electron micrograph (SEM) picture of Clostridium perfringens endospores (left) and its colonies on tryptose sulphite cycloserine agar (right). (Source: http://www.medillsb.com/images/artistimages/images/6686_108579.jpg (Medical Illustration Sourcebook) and http://www.grasssickness.org.uk/wp-content/uploads/2013/10/Cperfringens.jpg (The Equine Grass Sickness Fund), download time: 14.12.2014.) C. perfringens is mesophilic, growing over the temperature range 12 to 50 ◦C, with optimum at 43–47 ◦C. Its growth is very slow below 20 ◦C. The generation time of C. perfringens is only 7.1 min at 41 ◦C. It has a pH optimum of 6.0–7.5, and vegetative cells cannot tolerate acidic environment below pH 5. Its minimum aw for growth is 0.95–0.97, but spore production appears to require higher aw values than the above minima. Growth is inhibited by around 5% NaCl. It is heterofermentative, and a large number of carbohydrates are attacked. The microorganism requires for 13 essential amino acids for growth. Food poisoning as well as most other strains of C. perfringens grows well on a variety of media if incubated under anaerobic conditions or if provided with sufficient reducing capacity. The most commonly employed selective plating media used to enumerate C. perfringens employ antibiotic(s) as the selective agent and sulfite reduction to produce black colonies as the differential reaction (Fig. 3.3.2.1). The endospores of food-poisoning strains differ in their resistance to heat. Differences in heat sensitivity among C. perfringens strains are associated with the carriage of the cpe gene. Those strains has higher D100◦C values (> 40) where the cpe gene is chromosomal, while strains cpe carried on plasmids has lower D100◦C (typically <2). Outbreak strains are typically the previous ones, although plasmid-borne cpe-carrying C. perfringens strains has been prooved also cause food poisonings. Antibiotic-associated and sporadic diarrhea, also associated with plasmidborne cpe-positive strains, may be food-related as well. C. perfringens is widely distributed in nature. The species is classified into five types (A–E). Distribution of type A C. perfringens is widespread in the environment. In soil, where it can be found at levels of 103–104 g-1, it persists much longer than types B, C, D, and E. It can be isolated from water, sediments, dust, raw and processed foods and is a common inhabitant of the human gastrointestinal tract. The heat-resistant, non-haemolytic strains can be isolated from 2% to 6% in the general population, while the heat sensitive types are common in the intestinal tract of all humans. C. perfringens gets into meats directly from slaughter animals or by the subsequent contamination of slaughtered meat from containers, handlers, or dust. Because it is a spore former, it can withstand the adverse environmental conditions of drying, heating, and certain toxic compounds. The species is classified into five types (A–E) based on the production of four major exotoxins (α, β, ε, and ι). C. perfringens type A which is responsible for food poisoning and gas gangrene produces only the a major a toxin which has lecithinase (phospholipase C) activity. It is a sporespecific protein, being its production occurs together with that of sporulation. C. perfringens food poisoning is characterized by nausea, abdominal pain, diarrhoea and, less commonly vomiting. The incubation period is usually 8 to 24 h. Consumption of food containing large numbers of enterotoxin positive vegetative C. perfringens cells (>105 g-1, altogether 106–108 cfu) are necessary for food poisoning. Food poisoning due to type A strains has been fatal only in elderly or otherwise debilitated persons, while medical treatment is not usually required in otherwise healthy individuals, and recovery is complete within 1–2 days. Ingested vegetative cells that survive the stomach’s acidity pass to the small intestine where they grow and sporulate. The enterotoxin is synthesized by the sporulating cells, although low levels of production have been observed in vegetative cultures. The toxin is closely associated with the spore coat, and is released into the intestinal lumen following the lysis of the sporangium. The enterotoxin is a 35 kDa protein, and can be inactivated by heating in saline at 60 ◦C for 10 min and is sensitive to some proteolytic enzymes. It reverses the flow of Na+, Cl-, and water across the gut epithelium by from absorption to secretion, similarly to cholera toxin. It acts at the cell membrane by binding to specific protein receptors then inserting into the epithelial cell membrane, and changing cell permeability, produces pores in the membrane, and inhibiting synthesis of macromolecules. Epithelial cells eventually die as a result of membrane damage. The foods involved in C. perfringens outbreaks are often meat dishes prepared one day and eaten the next, when heat preparation inadequate to destroy the heat-resistant endospores. Endospores may germinate and grow during the slow cooling or prolonged storage at room temperature. Accumulated vegetative cells is not destroyed is food is either served cold or reheated insufficiently to kill them. Some of the ingested cells survive through into the small intestine where they sporulate and produce enterotoxin. Cured meats are rarely involved in C. perfringens food poisoning, because salt content, nitrite level and heat processing effectively control growth of C. perfringens in combination. Most outbreaks occur in connection with institutional catering such as schools, old people’s homes and hospitals. The following precautions would help to prevent C. perfringens food poisonings: (i) cook or roast meat until the internal breast temperature reaches at least 74◦C, (ii) prevent cross-contamination of prepared meat with pathogens from the raw one (sanitize all containers and equipment that previously had contact with raw, wash hands and use disposable plastic gloves), (iii) chill the prepared meat as rapidly as possible after cooking, (iv) heat stored meat at least 74◦C just prior to serving. Some type C strains produce enterotoxin and may cause a food-poisoning syndrome enteritis necroticans, a more severe (with a mortality rate of 35–40%), but rare, enteric disease. It is caused by C. perfringens produces α and β toxins. The later one damages the intestinal mucosa causing necrosis. Illness is preventable by active immunization against the β toxin. Symptoms of abdominal pain and bloody diarrhoea develop several days after a high-protein meal. Low levels of intestinal proteases are a predisposing factor in victims. Outbreaks were reported in Germany in 1946 and 1949. Nowadays it occurs mainly in Papua New Guinea following festive occasions, when pork is consumed with sweet potatoes, containing protease inhibitors. 3.3.3. CLOSTRIDIUM BOTULINUM FOOD POISONING (BOTULISM) Taxonomy Domain: Phylum: Class: Order: Family: Genus: Species: Bacteria Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium C. botulinum Botulism is a severe, often fatal, food poisoning. The symptoms of botulism are caused by the ingestion of a highly toxic, soluble exotoxin produced by the organism while growing in foods. It is the most poisonous substance known. Ingesting even a small amount of this neurotoxin can be dangerous. Among the earliest references to human botulism was the order by Emperor Leo VI (rulers of the Byzantium ad 886–912), forbidding the eating of blood sausage because of its harmful health effects. The first documented “sausage poisoning” with 13 cases and 6 deaths, occurred in 1793 in Wildbad Württemberg, in South Western Germany. It was traced to blood sausage (pig gut filled with blood and other ingredients). Justinius Kerner, a local district medical officer Wildbad Württemberg studied of the disease which became known as “sausage poisoning” or botulism (Latin: botulus=sausage). He has noted, that heating was an essential precondition for the development of toxicity in sausages and that small sausages or those containing air pockets were less likely to become toxic. Finally Emile Pierre van Ermengem of the University of Ghent discovered the botulinum toxinproducing bacteria in 1896, and named Bacillus botulinus in 1896, when he studied the outbreak in a music club originated from raw salted ham, when 23 of the 24 members became ill, and 3 died. The causative bacterium was reclassified as Clostridium botulinum in 1923. Botulism is caused by certain strains of C. botulinum, which is Gram-positive, and obligately anaerobic. The cells are straight or slightly curved rods with 2–10 µm long, and form central or subterminal oval spores. Vegetative cells are motile with peritrichous flagella (Fig. 3.3.3.1). Figure 3.3.3.1: Microscopic (left) and coloured SEM (right) picture of Clostridium botulinum (Source: http://www.ppdictionary.com/bacteria/gpbac/botulinum.jpg (Pathogen Profile Dictionary) and http://coraxit.com/wp-content/uploads/2013/08/Clostridium-botulinum-sem-360x212.jpg (Neogeek), download time: 14.12.2014.) On the basis of the serological specificity of their toxins, seven types are recognized: A, B, C, D, E, F, and G. Types A, B, E, F, and G cause disease in humans; type C causes botulism in birds, cattle, mink, and other animals; and type D is associated with forage poisoning of cattle, especially in South Africa. The types are also differentiated on the basis of their proteolytic activity. Types A, G and some types B and F are proteolytic, while nonproteolytic strains are C, D, E and some B and F strains. The proteolytic activity of type G is slower than that for type A, and its toxin requires trypsin potentiation. All strains that produce the Type G toxin are placed in the species C. argentinense. Botulinal toxin type F has been detected in Clostridium baratii, and a neurotoxin that is antigenically similar but not identical to type E is elaborated by Clostridium butyricum. C. botulinum is has been divided into four groups, based on physiological diversity and confirmed by molecular studies based on DNA homology and ribosomal RNA sequences. Group I organisms are proteolytic, producing A, B, or F toxins, and grow optimally at 35 to 40◦C. Group II organisms are not proteolytic with B, E, or F toxins producing and grow optimally at 18 to 25◦C. Group III organisms proteolytic activity range from being slightly to nonproteolytic, produce C or D toxins, and grow optimally at 37 to 40◦C. Group IV organisms are proteolytic, produce G toxin, and grow at 25 to 45◦C. All four groups form subterminal ellipsoidal spores. Most cases of botulism in humans are due to toxin types A, B or E. Group III strains producing toxin types C and D are usually associated with illness in animals and birds. C. botulinum is essentially a soil saprophyte and indigenous to waters too. It occurs widely, although the geographical distribution is not uniform. In European soils type B tends to be more common than type A, which is the most common in the Western States of USA. The Type G toxin producing C. argentinense has been recovered from soils in Argentina, Switzerland, and the United States. The psychrotrophic type E has been particularly associated aquatic muds in regions such as western North America, Japan and the Baltic Sea coasts. As a consequence, type E is often responsible for outbreaks of botulism where fish is the vehicle. The human mortality rate is usually high (20–50%), and type A toxin usually produces a higher mortality than B or E. The minimum pH at which C. botulinum will grow is pH around 4.7. Non-proteolytic strains have a lower acid tolerance and are generally inhibited at pH 5.0–5.2, and the maximum pH for growth is 8.5–8.9, moreover the toxin is unstable at alkaline pH values. It can be inactivated by heating at 80◦C for 10 min, or boiling temperatures for a few minutes. Six types of botulism are recognized, from which foodborne botulism is exclusively bound to food, and infant may connected to food. Foodborne botulism is food poisoning, resulting from the ingestion of an exotoxin produced by Clostridium botulinum growing in the food. The botulinum toxins are neurotoxins. Ingested toxin is absorbed in the upper part of the small intestine and reaches the bloodstream. After botulinal toxins are absorbed into the blood stream, they enter the peripheral nervous system. It binds to the nerve ending at the nerve– muscle junction, blocking release of the acetylcholine responsible for transmission of stimuli, thus producing a flaccid paralysis (Fig. 3.3.3.2). Figure 3.3.3.2: Blockade of neurotransmitter relases by botulinum toxin (Source: http://www.biocarta.com/pathfiles/h_botulinPathway.gif (Biocarta), download time: 14.12.2014.) () The botulinal neurotoxins (BoNT) are the most toxic substances known (lethal dose for an adult human in the order of 10-8 g). They are high molecular mass (150 kDa) proteins and can are produced during logarithmic growth as complexes and released into the surrounding medium on cell autolysis. BoNT is synthesized as a single polypeptide chain that is posttranslationally activated by proteolytic cleavage nicked to form a di-chain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a disulfide bond. It is composed of three domains: binding, translocation, and catalytic. Protoxin can be activated by the gut enzyme trypsin, or by the proteolytic toxin itself. The heavy chain (100 kDa) is responsible for specific binding to neuronal cells and cell penetration. The light chain is a zinc endopeptidase which cleaves components of the docking and fusing complex of the synaptic vesicle, containing the neurotransmitter acetylcholine (Fig. 3.3.3.2). Initial symptoms of botulism occur from 8 h to 8 days, most commonly 12–48 h, after consumption of the toxin-containing food. Symptoms include vomiting, constipation, urine retention, double vision, difficulty in swallowing, dry mouth and difficulty in speaking. The progressive weakness finally results in respiratory or heart failure in fatal cases, 1–7 days after the onset of symptoms. Survival is critically dependent on early diagnosis and treatment, principally by alkaline stomach washing to remove any remaining toxic food, intravenous administration of specific or polyvalent anti-toxins to neutralize circulating toxin, and mechanical respiratory support where necessary. Surviving patients may take as long as 8 months to recover fully. Infant botulism was first described in 1976 and is most frequently reported in the United States in the age of 2 weeks to 6 months (around the time that non-milk feeds are introduced). It differs from the classical syndrome in that it results from colonization of the infant’s gut with C. botulinum and production of toxin in situ. At this stage the infant’s gut microflora is not fully developed and is less able to outcompete. Infants over one year of age tend not to be affected by this syndrome because of the establishment of a more normal intestinal biota. Infants get viable spores from infant foods and possibly from their environment. Vehicle foods are those that do not undergo heat processing to destroy endospores; the two most common products are syrup and honey. There are common features are discernible in outbreaks of botulism: (1) The food has been contaminated from soil or mud at source or during processing with spores or vegetative cells of C. botulinum. (2) The food receives some heat treatment that restricts the competitive microflora. (3) Conditions in the food (temperature, pH, Eh, aw) are suitable for the growth of C. botulinum. (4) The food is consumed cold or after a mild heat treatment insufficient to inactivate toxin. Canning industry has to introduce stringent process control to ensure safety of the lowacid canned foods, which can fulfil all the above criteria. The greatest hazards of botulism come from home-prepared and home-canned foods that are improperly handled or given insufficient heat treatments to destroy botulinal spores. Such foods are often consumed without heating. The best preventive measure is the heating of suspect foods to boiling temperatures for a few minutes, which is sufficient to destroy the neurotoxins. 3.4. SALMONELLA GENUS AND FOODBORNE GASTROENTERITIS CAUSED BY SALMONELLA Scientific classification Domain: BACTERIA Phylum: Proteobacteria Class: Gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Salmonella Salmonella genus The genus Salmonella was created in 1900 by J. Ligniéres and named in honour of Daniel Elmer Salmon (1850–1914), the American veterinary pathologist who first described Salmonella choleraesuis. Salmonella spp. are Gram-negative, non-spore-forming rods (typically 0.5 µm by 1–3 µm) which are facultative anaerobe, catalase-positive, and oxidase-negative. Although members of this genus are motile by peritrichous flagella, there are non-motile (dysfunctional flagella), and also non-flagellated variants (e.g. Salmonella Pullorum and Salmonella Gallinarum). Salmonellae are chemoorganotrophic, and they are able to metabolize nutrients by fermentative and respiratory pathways. The bacteria grow optimally at 37°C and they can catabolize carbohydrates (e.g. glucose), with the production of acid and gas. Salmonellae can grow on citrate as a sole carbon source, generally produce hydrogen sulfide (H2S), decarboxylate lysine and ornithine, and do not hydrolyze urea. The genus Salmonella consists of two species, and they have numerous serovars. The two species are S. enterica (formerly known as S. cholerae-suis) and S. bongori. S. bongori was formerly subspecies V. S. enterica is divided into six subspecies, which are referred to by a Roman numeral and a name (I: S. enterica subsp. enterica; II: S. enterica subsp. salamae; IIIa: S. enterica subsp. arizonae; IIIb: S. enterica subsp. diarizonae; IV: S. enterica subsp. houtenae; and VI: S. enterica subsp. indica) (Table 3.4.1). Table 3.4.1: Species within the Salmonella genus (Doyle–Buchanan, 2013) Salmonella species and subspecies No. of serovars S. enterica subsp. enterica (I) 1531 S. enterica subsp. salamae (II) 505 S. enterica subsp. arizonae (IIIa) 99 S. enterica subsp. diarizonae (IIIb) 336 S. enterica subsp. houtenae (IV) 73 S. enterica subsp. indica (VI) 13 S. bongori (V) 22 Total 2579 The most useful technique for serotyping salmonellae is the Kauffmann–White scheme. It describes organisms on the basis of their somatic (O), flagellar (H), and capsular polysaccharide (Vi) antigens. Somatic antigens are lipopolysaccharides (LPS), which can be found on the external surface of the bacterial outer membrane. The heat-stable somatic antigens are classified as major or minor antigens. Flagellar antigens are heat-labile proteins, and associated with the peritrichous flagella. Capsular antigens, often occur in the family Enterobacteriaceae, however the Vi antigen is limited to the Salmonella genus. Vi antigen occurs only in S. Typhi, S. Paratyphi C, and S. Dublin. In accordance to the O antigens, Salmonella species and serovars are divided into groups A, B, C, and so on. Flagellar antigens can be used for further classification. There two types of flagellar: specific phase (phase 1), and group phase (phase 2). The flagellar phase 2 is more widely distributed among salmonellae, than phase 1. The phase 1 flagellar antigens are designated with small letters, and phase 2 with arabic numerals. For example the 1,4,5,12:i:1,2 serovar (Salmonella Typhimurium) means the following: O antigens: 1, 4, 5 and 12; H antigens: phase1: i; phase 2: 1 and 2. Salmonella subgroups are defined as serovars. Despite the relatively low number of O, phase 1, and phase 2 antigens, there are numerous (over 2500) serovars. Earlier the salmonella serovars were treated as species, but nowadays it is no longer used. For example, S. typhimurium becomes S. enterica subsp. enterica serovar Typhimurium, or more concisely Salmonella Typhimurium. The serovars can be designated with capitalized and not italicized following the genus. Characteristics of Salmonella genus In general, salmonellae are mesophilic, but some Salmonella strains are able to grow also at high temperatures (above 54°C), and others can grow in foods stored at 2°C to 4°C. Growth has been recorded from temperatures just above 5◦C up to 47◦C with an optimum at 37◦C. Salmonellas are heat sensitive (D60 = 0.1-2 min, D65,5 = 0.02-0.3 min) and are readily destroyed by pasteurization temperatures. Salmonella spp. can grow at pH values ranging from 4.0 to 9.5 with an optimum pH for growth of 6.5 to 7.5. The minimum water activity for growth is around 0.93 but cells survive well in dried foods, the survival rate increasing as the water activity is reduced. Salmonellae cannot tolerate high salt concentrations. Although Salmonella is generally inhibited in the presence of 3 to 4% NaCl, tolerance to salt can increase with increasing temperature in the range of 10 to 30◦C. At 30◦C, Salmonella can grow slowly in the presence of 6% NaCl. Brine above 9% is reported to be bactericidal. In general, salmonellae cannot ferment lactose (with the exception of some serovars) and sucrose, but they ferment glucose while producing gas. Salmonellae generally utilize amino acids as nitrogen sources, but the sole nitrogen sources of S. Typhimurium are nitrate, nitrite, and ammonium. Occurrence of Salmonella genus Salmonellae are widely distributed in nature. The primary reservoirs of salmonellae are humans and animals. The primary habitat of these bacteria is the intestinal tract of animals (e.g. farm animals, birds, reptiles, and insects). Although their main habitat is the intestinal tract, they can also be found in other parts of the body. The bacteria can be transmitted from the faeces to several places by insects and other organisms. They also can be found in polluted water. When polluted water and contaminated foods are consumed by humans and other animals, these organisms are once again shed through faecal matter with a continuation of the cycle. A carrier is defined as a person or an animal that repeatedly sheds Salmonella spp., usually through faeces, without showing any signs or symptoms of the disease. Diseases caused by Salmonella genus Salmonellae are the most important Gram-negative rods, which may cause foodborne gastroenteritis. Salmonellae are responsible for several different syndromes, such as systemic disease and enteritis. Systemic Disease. Host-adapted Salmonella serotypes are more invasive and they are able to cause systemic disease in their hosts. For example in case of humans, the S. Typhi, and S. Paratyphi A, B, and C cause septicaemic diseases and enteric fever. Typhoid fever (enteric fever) is a serious human disease associated with S. Typhi and S. Paratyphi, which are mainly transmitted from human to human via the fecal-oral route and are particularly well adapted for invasion and survival within host tissues. Clinical manifestations of typhoid fever (enteric fever) appear after a period of incubation ranging from 3 to 56 days (usually between 10 and 20 days) and may include diarrhea, prolonged and spiking fever, abdominal pain, headache, and prostration. Enteritis. The most common cause of gastrointestinal infections is serotypes which occur widely in animals and humans. The disease is caused by the consumption of foods which are contaminated with high number non-host-specific salmonellae. The severity of the disease may range from asymptomatic carriage to severe diarrhea. Salmonellosis is the most common type. Most common cause of enterocolitis is the non-typhoid salmonellae, which is often transmitted by food (or water). The newborns, infants, the elderly, and immunocompromised individuals are more sensitive to salmonellosis than healthy adults. The incubation period of the disease is generally between 6 and 48 h. The most common symptoms of the disease are mild fever, nausea, vomiting, abdominal pain, headache, chills and diarrhea. The symptoms are often accompanied by prostration, faintness, muscular weakness, restlessness, and drowsiness. Symptoms usually persist for a few days (2–3 days) but, in some cases, they can persist for a week or more. In this case some patients (up to 5%) may become carriers of the bacteria. In most cases the disease is self-limiting, but in case of susceptible humans (e.g. very young, very old person) it can be more severe. The mortality rate is 5.8% under one year of age, 2% between 1 and 50 year, and 15% over the age of 50 years. The average mortality is 4.1%. The infectious dose of salmonellosis is high (about 106–109 cell/g), but it depends on the virulence of the serotype, the susceptibility of the individual and the food vehicle involved. In case of some outbreaks the infective dose was between 10 and 100 cells. These outbreaks associated with susceptible humans and fatty foods (e.g. cheese, salami, chocolate). It is due to that the high fat content of foods can protect salmonellae form stomach acidity. Salmonellosis is a zoonotic disease, because the main source of human illness is infected animals. The transmission of the bacteria is by the faecal–oral route. In this case the food or water is contaminated by the intestinal contents of infected animals. Factors which are contributing to outbreaks are inadequate cooling, storing and heat treatment conditions, furthermore cross-contamination. Cross-contamination can occur through direct contact or indirectly via contaminated kitchen equipment and utensils. In case of Salmonella transmission, human carriers are usually less important than animals. The primary sources of salmonellosis are foods of animal origin (e.g. poultry, poultry products, meat, eggs, salad dressing, mayonnaise, and unpasteurized milk), but in some cases plant products (e.g. salad vegetables) can also be associated with that. The following measures help to reduce the transmission between animals on the farm: - good animal husbandry, - protection of feeds and water from contamination, - hygienic disposal of wastes, and - maintenance of a generally clean environment. There was a clear seasonal trend in confirmed salmonellosis cases reported in the EU between 2008 and 2012, with most cases reported during summer months. The significant decreasing EU trend observed for several years continued in 2012 (Figure 3.4.1). The number of confirmed cases of human salmonellosis in 2012 was 91034, which means a 4.7% decrease over the previous year (95548 cases in 2011). Figure 3.4.1: Trend in reported confirmed cases of human salmonellosis in the EU between 2008 and 2012 (EFSA – ECDC, 2014) In the European Union, the two most commonly reported Salmonella serovars in 2012 (like in the previous year) were S. Enteritidis (41.3%) and S. Typhimurium (22.1%). Other common serovars in 2012 (in the EU) were monophasic S. Typhimurium (7.2%), and S. Infantis (2.5%) (Figure 3.4.2). Figure 3.4.2: Distribution of the 10 most common Salmonella serovars in humans in the EU between in 2012 (EFSA – ECDC, 2014) 3.5. ESCHERICHIA COLI AND FOODBORNE GASTROENTERITIS CAUSED BY ESCHERICHIA COLI Scientific classification Domain: BACTERIA Phylum: Proteobacteria Class: Gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Escherichia Species: Escherichia coli Characteristics and occurrence of Escherichia coli Escherichia is the type genus of the Enterobacteriaceae family and Escherichia coli is the type species of the genus. E. coli was first isolated from the faeces of children and it was described by the German bacteriologist Theodor Escherich (1857–1911). It is a predominant facultative anaerobe bacterium, which can be found in the gut of humans and other warm-blooded animals. In most cases it is a harmless commensal, which can also be an opportunistic pathogen. It can cause a number of infections in humans such as intestinal gastroenteritis, urinary tract infections, pneumonia in immunosuppressed patients, sepsis, and meningitis in neonates. E. coli as an indicator of faecal contamination and the possible presence of enteric pathogens such as S. Typhi in water because of the following reasons: - common occurrence in faeces, - ready culturability, - generally non-pathogenic character, and - survival characteristics in water. E. coli is a facultative anaerobe, catalase-positive, oxidase-negative, fermentative, short, Gramnegative, non-sporing rod. It is closely related to shigellae. E. coli can ferment lactose and more active biochemically than shigellae. Sugar fermentation and other biochemical test can help in the differentiation of the members of the family Enterobacteriaceae. The most commonly used test is the IMViC tests. IMViC is an acronym that consists of four tests. Bacteria are tested for the ability to produce: - indole from tryptophan (I); - sufficient acid to reduce the medium pH below 4.4, the break point of the indicator methyl red (M); - acetoin (acetylmethyl carbinol) (V); and - the ability to utilise citrate (C). In the IMViC tests, most strains of E. coli are indole and methyl red positive, furthermore VP and citrate negative. E. coli is a typical mesophile growing from 7◦C up to 50◦C with an optimum around 37◦C. Some ETEC strains are able to grow at temperatures as low as 4◦C. It is not resistant to heat (D60 = 0.1 min), and can survive refrigerated or frozen storage for extended periods. The optimal pH for growth is around neutral pH. The minimum pH for growth is about pH 4.4. The minimum water activity for growth is 0.95. In the 1940s Kauffman proposed a serotyping scheme for these bacteria. This scheme is based on somatic (O), flagellar (H), and capsular (K) antigens. The O antigen identifies the serogroup of a strain, and the H antigen identifies its serotype. At present, more than 700 serotypes of Escherichia coli have been identified. Pathogenic E. coli strains are categorized into specific groups (pathotypes) based on their virulence determinants. These virulence determinants include: - controlling adhesions (CFAI/CFAII, type 1 fimbriae, P fimbriae, S fimbriae, and intimin), - invasions (hemolysins, siderophores, siderophore uptake systems, and Shigella-like invasins), - motility (flagella), - toxins (heat-stable and heat-labile enterotoxins, Shiga toxins [Stxs], cytotoxins, and endotoxins), - antiphagocytic surface structures (capsules, K antigens, lipopolysaccharides [LPS]), and - genetic characteristics (genetic exchange through transduction or conjugation, transmissible plasmids, R factors, and drug resistance and virulence plasmids). Virulence groups of Escherichia coli Five virulence groups (pathotypes) of E. coli are recognized: • enteropathogenic E. coli (EPEC), • enterotoxigenic E. coli (ETEC), • enteroinvasive E. coli (EIEC), • enteroaggregative E. coli (EAEC), • enterohemorrhagic E. coli (EHEC). Enteropathogenic E. coli (EPEC) Among the E. coli pathotype, enteropathogenic E. coli was first identified and described. It was first characterized in 1955. EPEC strains cause diarrhea in children generally less than one year of age. They can cause watery diarrhea like ETEC. But EPEC does not have the same colonization factors as ETEC and do not produce enterotoxins (LT or ST toxins). The main somatic (O) serogroups which are associated with illness are the following: O55, O86, O111ab, O119, O125ac, O126, O127, O128ab, and O142. Humans are an important reservoir. The bacteria have adherence factor plasmids which help the adherence to the intestinal mucosa. After adherence they colonize the intestinal mucosa, and then they produce attachment and effacement lesions. It helps them in adhere and invade epithelial cells. Because of the specific patterns of adherence, some types of EPEC are called enteroadherent E. coli (EAEC). Most common symptoms of EPEC infection are malaise, vomiting and diarrhea. The stools are usually containing mucus but rarely blood. Symptoms appear 12–36 h after ingestion of the organism. In case of infants, the disease is more severe than many other diarrheal infections. Furthermore in some cases it can persist for longer than two weeks. Outbreaks caused by EPEC are commonly associated with faecal contamination of water supplies and contaminated food handlers. A number of foods have been involved, including vegetables, potato salad, and sushi. Enterotoxigenic E. coli (ETEC) In developing countries or regions (due to the poor sanitation), ETEC are a major cause of diarrhea in infants (where it can cause serious dehydration). They are often responsible for traveler’s diarrhea. The local adults have developed immunity so ETEC is unable to cause disease. ETEC strains can cause diarrhea in children and also in adults. ETEC colonize the proximal small intestine with the help of fimbrial colonization factors (CFA I-IV) and produce enterotoxins (LT or ST). Enterotoxins are responsible for the fluid accumulation and diarrheal response. The heatlabile toxins (LT) can be inactivated at 60◦C after 30 min and at low pH. The LT enterotoxin is similar to cholera toxin in both structure and mode of action. The heat-stable toxins (ST) are resistant to heating at 100◦C for 15 min, furthermore they are also resistant to acid. The ST enterotoxin is a peptide, which can increase the cyclic GMP in the host cell cytoplasm. Two types of ST (STA and STB) have been recognized. The following ETEC serogroups are often isolated: O6, O8, O15, O20, O25, O27, O63, O78, O85, O115, O128ac, O148, O159, and O167. The main reservoir of the human illness causing ETEC strains are humans. The symptoms of disease caused by ETEC strains appear 12 and 36h after ingestion of the organism. Symptoms may range from a mild diarrhea to a severe cholera-like syndrome. Symptoms of the cholera-like syndrome are watery stools without blood or mucus, stomach pains and vomiting. In case of adult humans high number (about 106–1010 cfu/g) of ETEC strain needed for diarrhea. Bacteria colonize the small intestines and produce enterotoxins. The factors of colonization are generally fimbriae or pili. The most common symptom of the disease is non-bloody diarrhea without inflammatory exudates in stools. The diarrhea is watery and similar to that caused by Vibro cholera. The illness is usually self-limiting, and persisting for 2–3 days. Outbreaks caused by ETEC are commonly associated with faecal contamination of water supplies and contaminated food handlers. A number of foods have been involved. Enteroinvasive E. coli (EIEC) Infection by EIEC has the same symptoms as invasive dysentery caused by shigellae. They are able to enter and multiply in colonic epithelial cells and then spread to adjacent cells (like shigellae). Like Shigella, EIEC invades and multiplies within the colonic epithelial cells causing widespread cell death, ulceration and inflammation. EIEC strains do not produce enterotoxins (LT and ST), or Shiga toxins (Stx). As for shigellae, the invasive capacity of EIEC is associated with the presence of a large plasmid (ca. 140 MDa) that encodes several outer membrane proteins involved in invasiveness. The strains without plasmids are not invasive. Most common symptoms of the disease are fever, severe abdominal pains, malaise and often a watery (non-bloody) diarrhea. The stool is containing blood, mucus, and faecal leukocytes. Dysentery is rare. The most susceptible humans are the very young and very old. The incubation period is between 2 and 48 hours, and the average value is about 18 hours. The infective dose of EIEC strains is higher than for shigellae. This is probably due to the fact that the bacterium has greater sensitivity to gastric acidity. Humans are major reservoir of the disease. The following EIEC serogroups are often isolated: O28ac, O29, O112, O124, O136, O143, O144, O152, O164, and O167. The most commonly occurring serogroup is O124. Outbreaks caused by EIEC are commonly associated with faecal contamination of water supplies and contaminated food handlers. A number of foods have been involved. Enteroaggregative E. coli (EAEC) The enteroaggregative E. coli (EAEC) is also known as enteroadherent E. coli. They are related to EPEC strains, but EAEC strains have a unique aggregative adherence. EAEC adhere to the surface of HEp-2 cells in a characteristic pattern (“stacked-brick-type”). They carry a plasmid (ca. 60 MDa), which is necessary for the fimbriae production. Fimbriae are responsible for the aggregative expression, and for a specific outer membrane protein (OMP). EAEC produce distinctive heat-labile enterotoxin/cytotoxin, called the EAST (enteroaggregative ST) toxin, which is located on the large virulence plasmid. EAEC strains produce a hemolysin, too. The distinguishing clinical feature of EAEC strains is a persistent diarrhea that lasts more than 14 days, especially in children. These strains are not the primary cause of traveler’s diarrhea. EAEC strains are often cause diarrhea in infants and children in several countries worldwide. The following EAEC serogroups are often isolated: O3, O15, O44, O77, O86, O92, O111, and O127. EAEC strains have rarely been associated with in major foodborne illnesses. There was a large outbreak in the European Union in 2011. The centre of the outbreak was in Germany, but various other countries were affected. The outbreak was probably caused by contaminated sprouts. In the outbreak approximately 3,700 people have been infected, and there were more than 50 fatalities. The cause of the outbreak was identified as E. coli O104:H4. It was a Shiga toxin-producing E. coli (STEC) strain, which produced Stx2a toxin. The whole-genome sequencing of the pathogen revealed that it shared 93% genomic homology with an EAEC strain. Genetic analyses revealed that the causative pathogen was a multidrugresistant EAEC strain. Enterohemorrhagic E. coli (EHEC) / Verotoxin-producing E. coli (VTEC) EHEC were first recognized as human pathogens in 1982/1983 when E. coli O157:H7 was identified as the cause of two outbreaks of hemorrhagic colitis. Since then, many other serogroups of E. coli, such as O26, O45, O103, O111, O121, and O145 also have been associated with cases of hemorrhagic colitis and have been classified as EHEC. The O157:H7 serotype is the primary cause of EHEC caused disease. EHEC strains are similar to EPEC in their possession of the chromosomal gene eaeA and in the production of attachment and effacement lesions. EHEC strains (unlike EPEC strains) are confined to only the large intestine and they produce huge amount of Shiga-like toxins (SLT, Stx). EHEC strains produce a plasmid (ca. 60 MDa) that encodes fimbriae that mediate attachment to culture cells, and they do not invade HEp-2 or INT407 cell lines. Some EHEC strains produce curli fimbriae that facilitate attachment of cells to surfaces. EHEC has attracted attention not only because foodborne transmission is more common than with other diarrheagenic E. coli, but because the illness it causes can range from a non-bloody diarrhea, through haemorrhagic colitis, to the life threatening conditions haemolytic uraemic syndrome (HUS) and thrombotic thrombocytopaenic purpura (TTP). Haemorrhagic colitis is typically a self-limiting, acute, bloody diarrhea lasting 4–10 days. Symptoms start with stomach cramps and watery diarrhoea 1–2 (sometimes 3–8) days after eating the contaminated food and, in most cases, progress over the next 1–2 days to a bloody diarrhea with severe abdominal pain. The main difference between the haemorrhagic colitis and inflammatory colitis the absence of fever and there are no leukocytes in the stool. The disease is primarily affects the adults, and for the elderly it can be life-threatening. The incidence of the disease increases during the summer months. EHEC are also known as Verotoxin-producing (Verocytotoxigenic) E. coli (VTEC). EHEC strains produce the verotoxins, which are cytotoxic for Vero (African green monkey kidney) cells and lethal for mice. They produce at least two toxins VTI and VTII which are also called Shiga-like toxins (verotoxin, verocytotoxin), SLTI and SLTII. However, new terminology has been applied, and what was once SLTI is now Stx1 and the former SLTII is Stx2. The genes for Stx1 and Stx2 are encoded by temperate bacteriophages in some EHEC strains. Stx2 appears to be more significant in the etiology of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) than Stx1. Outbreaks caused by EHEC serotype O157:H7 have mostly involved meat, undercooked ground meat products, poultry, seafood products, lettuce, and occasionally raw milk. Cattle seem to be an important reservoir of infection. EHEC strains have relatively low infectious dose (2–2000 cells/g), because they are able to survive at low pH values. EHEC strains have more than 600 serotypes. They have about 160 O serogroups and 50 H types, and the number is still increasing. Only the strains, which cause hemorrhagic colitis are regarded as EHEC. From the human patients about 130 EHEC serotypes have been recovered. Most common serogroups in the EU: - O157 (41.1 %) - O26 (12.0 %) - O91 (3.6 %). Main causes of food-related diseases caused by E. coli: • Improper storage temperatures, • Inadequate personal hygiene, • Contaminated equipment and utensils, • Inadequate cooking, • Food from unsafe sources. There was a clear seasonal trend in the confirmed VTEC cases reported in the EU between 2008 and 2012 with more cases reported in the summer months. A dominant peak in the summer of 2011 was attributed to the large STEC/VTEC O104:H4 outbreak. After the outbreak a declining tendency can be observed (Figure 3.5.1). A statistically significant increasing EU trend of confirmed VTEC cases was observed in the period 2008 and 2010, when the 2011 outbreak data were removed (Figure 3.5.2). Figure 3.5.1: Trend in reported confirmed cases of human VTEC infections in the EU between 2008 and 2012 (EFSA – ECDC, 2014) Figure 3.5.2: Trend in reported confirmed cases of human VTEC infections in the EU between 2008 and 2010 (EFSA – ECDC, 2014) The number of confirmed cases of human VTEC infections in 2012 was 5671, which means a 40% decrease over the previous year (9487 cases in 2011) 3.6. SHIGELLA GENUS AND SHIGELLOSIS Scientific classification Domain: BACTERIA Phylum: Proteobacteria Class: Gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Shigella Characteristics and occurrence of Shigella genus The genus Shigella belongs to the family Enterobacteriaceae. Kiyoshi Shiga (the Japanese microbiologist) in 1898 discovered the genus Shigella, which is the cause of bacillary dysentery. They are non-motile, non-spore-forming, Gram-negative rods which are oxidase-negative, catalase-positive (with the exception of S. dysenteriae serotype 1), and facultative anaerobes. They can ferment glucose while produce acid but usually no gas, but cannot ferment lactose (with the exception of some strains of S. sonnei). Shigellas are sensitive bacteria and they do not survive well outside their natural habitat. The natural habitat of shigellae is the gut of humans and other primates. Shigellas are typical mesophiles, and the growth temperature range between 10 and 45◦C. The heat sensitivity of shigellae is similar to other members of the Enterobacteriaceae. They grow well in the range of pH 6 and 8, but they are not able to survive below pH 4.5. In case of some type of foods (e.g. flour, pasteurized milk, eggs, shellfish, etc.) shigellas have long survival period. The species are distinguished on the basis of biochemical tests. Furthermore, serotyping and phage typing schemes are available for further subdivision of species. On the basis of biochemical, serological, and clinical phenotypic differences, shigellae is subgrouped into four species. Shigellas do not possess flagellar (H) and capsular (K) antigens, so the serologic typing is based only on differences in the O (lipopolysaccharide [LPS]) antigen. Shigella species are classified by four serogroups (based on O antigens): - Serogroup A: S. dysenteriae (15 serotypes), - Serogroup B: S. flexneri (14 serotypes), - Serogroup C: S. boydii (19 serotypes), - Serogroup D: S. sonnei (1 serotype). Groups A, B, and C are physiologically similar. Group D (S. sonnei) can be distinguished on the basis of biochemical metabolism assays. Shigellas are considered as human pathogens, but they differ in the severity of the illness they cause. Shigella dysenteriae is responsible for the severe bacillary dysentery in tropical countries, which is nowadays rare in Europe. In case of S. dysenteriae, as few as 10 cfu/g are known to initiate infection in susceptible individuals. Although S. dysenteriae can be associated with foods, it is not considered to be a food-poisoning organism. In Europe S. sonnei is more common. S. sonnei causes the mildest illness, while that caused by S. boydii and S. flexneri is of intermediate severity. S. flexneri is the most frequently isolated species worldwide. It accounts for 60% of cases in the developing world. S. sonnei causes 77% of cases in the developed world, however it causes 15% of cases in the developing world. S. sonnei, S. flexneri 2a, and S. dysenteriae serotype 1 are the most common species isolated from cases of shigellosis. Shigellas are nearly identical genetically to escherichiae and they are closely related to salmonellae. But shigellae (contrary salmonellae and escherichiae) have no known animal reservoirs. Shigellosis Shigellae are endemic worldwide. The reason of this is the easy transmission of bacteria. In underdeveloped countries shigellosis may cause large-scale epidemics with high mortality rate. Most common cause of the disease is foods and water, which are contaminated with faeces. Shigellas can cause bacillary dysentery in humans and other higher primates. Studies with human volunteers have indicated that the infectious dose is low (10–100 cell/g). The incubation period can vary between 7 hours and 7 days, although foodborne outbreaks usually have shorter incubation periods (up to 36 h). Most common symptoms are: abdominal pain, vomiting, fever and diarrhoea. In case of S. dysenteriae, S. flexneri and S. boydii there is classic dysenteric syndrome with bloody faeces, which contain mucus and pus. In case of S. sonnei there is watery diarrhoea. Shigellosis is more often require hospitalization than many other bacterial diarrheas. It is not usually life-threatening, and mortality is rare. The mortality is higher in case of malnourished children, immunocompromised individuals, and the elderly. The illness persists from 3 days up to 14 days. In some cases it may develop a carrier state, which can last for several months. The milder form of the disease is self-limiting and does not require treatment. In contrast the S. dysenteriae infections often require fluid and electrolyte replacement and antibiotic therapy. Serious complications may arise from the disease such as severe dehydration, intestinal perforation, toxic megacolon, septicemia, seizures, reactive arthritis, and hemolytic uremic syndrome (HUS). Epidemiologic studies suggest that Shiga toxin produced by S. dysenteriae serotype 1 is the cause of HUS. Shigellosis is an invasive infection. The invasive property of the bacterium is encoded on a large plasmid. The consumption of raw or processed food may transmit shigellae. The main factors for food contamination are the poor personal hygiene practices by food workers during the final preparation and food service. In case of foodborne disease the main source is the humans who are involved in the preparation of the food. Infected individuals may spread shigellas by many different ways, such as food, fingers, feces, flies, and fomites. The bacteria may be transferred from human faeces by flies, when the sewage disposal is inappropriate. Inadequate cooking, as well as contaminated equipment and utensils can be contributing factors, which may contaminate foods. If the contaminated foods are not stored under appropriate conditions, they may cause foodborne outbreaks. Most common vehicle foods of shigellosis are shellfish, fruits, vegetables, chicken, and salads. Raw vegetables can be contaminated by sewage (used as fertilizer) or wastewater (used for irrigation). The prominence of these foods is due to the fecal–oral route of transmission. A number of raw or undercooked foods have been linked to shigellosis outbreaks including lettuce, parsley, bean dip, cold sandwiches, potato salad, tofu salad, egg salad, hamburgers, tomatoes, and oysters. 3.7. YERSINIA GENUS AND YERSINIOSIS Scientific classification Domain: BACTERIA Phylum: Proteobacteria Class: Gammaproteobacteria Order: Enterobacteriales Family: Enterobacteriaceae Genus: Yersinia Characteristics and occurrence of Yersinia genus Yersinia genus is a member of the Enterobacteriaceae family. The genus Yersinia is named after the Swiss and naturalized French bacteriologist Alexandre Yersin (1863–1943), who discovered and first described the bacillus (in 1894), which responsible for the bubonic plague (pest). Three species (Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis) of the genus Yersinia recognized as human pathogens. Y. enterocolitica is a pathogen of the versatile intestinal pathogen, which cause predominantly gastroenteritis. Y. pseudotuberculosis is a pathogen in rodents, which occasionally may cause disease also in human. It may cause mesenteric lymphadenitis, septicemia, and immunomediated diseases in humans. Previously it was common in Europe, but nowadays rarely occurs. Y. pestis is the causative agent of bubonic and pneumonic plague. Y. pestis is transmitted to its host via flea bites or respiratory aerosols, whereas Y. enterocolitica and Y. pseudotuberculosis are foodborne pathogens. Y. enterocolitica is Gram-negative, non-spore-forming, rod-shaped (0.5–1.0 by 1–2 µm), facultative anaerobe, oxidase-negative and catalase-positive. It ferments glucose with little or no gas. It is urease positive. Y. enterocolitica is psychrotrophic. It can grow over a wide range of temperature (from -2◦C to 45◦C), with an optimum between 28◦C and 30◦C. It has a number of temperature-dependent phenotypic characteristics. For example, it is non-motile at 37◦C, but motile below 30◦C (due to their peritrichous flagella). Like other psychrotrophs, they can grow slowly at chill temperatures, and rather resistant to freezing. Y. enterocolitica easily resist to freezing and they are able to survive in frozen foods for extended periods. Y. enterocolitica is susceptible to heat (D60 = 1–3 min, D62.8 = 0.7–57.6 s), and pasteurization at 71.8°C for 18 s or 62.8°C for 30 min easily destroy them. It is able to grow over a pH range of approximately pH 4.0 to 10.0, with an optimum pH between pH 7.0–8.0. The minimum pH (in broth at 25◦C) is varying between pH 5.0 and 4.0 depending on the acidulant used. Y. enterocolitica can grow in broth media, which contain 5% salt (but not in 7%). The bacteria may produce a heat-stable enterotoxin (ST), which can survive 100◦C for 20 minutes. Proteases and lipases do not affect the bacterium. Y. enterocolitica may live a longer time in soil, vegetation, streams, lakes, wells, and spring water, especially at low temperatures. The most common environmental sources of the bacteria are soil, fresh water and the intestinal tract of many animals (e.g. swine, cattle, lambs, chickens, fish and oysters). Out of all sources, swine seems to be the main source of human pathogenic strains. Yersiniosis Infections with Yersinia species are zoonotic. Y. enterocolitica and Y. pseudotuberculosis occur in many environments and can be isolated from the intestinal tract of numerous animals (e.g. birds, fish, fleas, flies, and oysters). Yersiniosis often occurs in areas of cooler climates (e.g. northern Europe, North America). In these regions a number of large outbreaks have been reported. In case of gastroenteritis syndrome a seasonal incidence can be observed. The fewest outbreaks occurred during the spring and the greatest number in the autumn and winter. The incidence is highest in children (especially in those less than 5 years of age) and the old. Y. enterocolitica and Y. pseudotuberculosis can get into the gastrointestinal tract by the consumption of contaminated foods or water. Y. enterocolitica can grow at refrigeration temperature (psychrophilic) in numerous foods including raw milk, dairy products, meats (particularly pork), poultry, fish, shellfish, fruits and vegetables. Y. enterocolitica is often isolated from pigs at slaughterhouses. The infective dose of bacteria for humans is likely to exceed 104 cell/g. Symptoms of yersiniosis are diarrhea, abdominal pain, mild fever, vomiting (but it is rare), pharyngitis, and headache. Symptoms of the gastroenteritis syndrome develop several days (about 1-11 days) following ingestion of contaminated foods and are characterized by abdominal pain and diarrhea. The illness generally lasts from a few days to three weeks. In case of some patients chronic enterocolitis may develop, which can persist for several months. After the illness, the bacteria can be present in faeces for up to 40 days. In addition to gastroenteritis, Y. enterocolitica has been associated with human pseudoappendicitis, mesenteric lymphadenitis, terminal ileitis, reactive arthritis, peritonitis, colon and neck abscesses, cholecystis, and erythema nodosum. The incidence of yersiniosis can be eliminated or reduced if people avoid from raw seafood products, raw milk and untreated water, furthermore if they prevent the cross-contamination with contaminated raw materials. 3.8. VIBRIO GENUS AND VIBRIOSIS Scientific classification Domain: BACTERIA Phylum: Proteobacteria Class: Gammaproteobacteria Order: Vibrionales Family: Vibrionaceae Genus: Vibrio Characteristics and occurrence of Vibrio genus Member of the Vibrio genus possessing a curved-rod shape (comma shape). The name Vibrio derives from Filippo Pacini (1812–1883), who isolated bacteria he called "vibrions" (because of their motility) from cholera patients in 1854. The genus Vibrio consists of more than 80 species, including at least 12 capable of causing infection in humans. Of the 12 human pathogens, 8 have been directly associated with foods. Marine and estuarine environment are the natural habitat of vibrios. Most of the Vibrio species are zoonotic. They also cause disease in fish and shellfish. Vibrios are Gram-negative, facultative anaerobe, catalase-positive, oxidase-positive, pleomorphic (curved or straight) short rods, which are motile (with polar flagella with sheaths). They are capable of both fermentative and respiratory metabolism. Most members of the genus are halophilic. It means that, NaCl stimulates the growth of all species and is an obligate requirement for some. The optimum NaCl level for the growth of most species is 1–3% NaCl. Several species of Vibrio are pathogens. Most of the disease-causing Vibrio strains can cause gastroenteritis, but they are able to infect open wounds and cause septicemia. Several marine animals (e.g. crabs or prawns) may carry vibrios, which are able to cause fatal infections in humans. Most common pathogenic Vibrio species are V. cholerae, V. parahaemolyticus, and V. vulnificus. V. cholerae is the causative agent of cholera. It is best known as the cause of human cholera transmitted by contaminated water. It can be found in temperate, sub-tropical, and tropical waters throughout the world. But it disappears from temperate waters during the colder months. The most common strain, which may cause epidemic or pandemic cholera is serovar O group 1. Nonpathogenic (non-01) strains are known to cause gastroenteritis, soft-tissue infections, and septicemia in humans. V. vulnificus is found in seawater and some seafoods. It is isolated more often from oysters and clams than from crustacean shellfish products. V. vulnificus outbreaks generally occur in warm climates. It may cause small outbreaks, which are often lethal. V. parahaemolyticus is more enteroinvasive than V. cholerae. V. parahaemolyticus is able to grow in the presence of 0.5–8% NaCl, but it grows optimally in the range of 2–4% NaCl. It can grow over a range of temperature from 5◦C to 45◦C, with an optimum between 30◦C and 37◦C. It cannot grow at 4◦C, but able to grow between 5◦C and 9◦C at a pH 7.2–7.3 and 3% NaCl, or at a pH of 7.6 and 7% NaCl. V. parahaemolyticus is susceptible to heat (D60 = 0.7-1.0 min), and they are readily inactivated by pasteurization. V. parahaemolyticus and other vibrios grow best at the pH range 4.8–11.0, and the optimum is between pH 7.5 and 8.5. Vibrios are able to grow in alkaline conditions up to a pH of 11.0. In general, vibrios are sensitive to acid, although V. parahaemolyticus may grow down to pH 4.8– 5.0. The minimum water activity for growth of bacteria varies between 0.937 and 0.986 depending on the solute used. Optimum water activity for growth is 0.992. V. parahaemolyticus is primarily associated with oceanic and coastal inshore waters, rather than the open sea. They unable to survive the high pressure in deep water. The numbers of organisms being undetectable until the water temperature rises to around 17–20◦C. Certain strains of V. parahaemolyticus are able to produce extracellular thermostable direct hemolysin (TDH) or Kanagawa hemolysin, which is correlated with virulence. The hemolysin has enterotoxic, cytotoxic and cardiotoxic activity. The production of this hemolysin was originally established on a special blood agar (Wagatsuma agar), in which beta-hemolytic strains were termed Kanagawa phenomenon-positive (KP+) and nonhemolytic strains were Kanagawa phenomenon-negative (KP−). Most of the V. cholerae and V. parahaemolyticus environmental strains are non-pathogenic. The majority of the V. cholerae are non-O1 serotypes, and most of the O1 serotypes are usually nontoxigenic. Vibriosis Cholera, which caused by V. cholera, is regarded primarily as a waterborne infection. However, food which has been in contact with contaminated water can often serve as the vehicle. Several different foods have been concerned in outbreaks. However, the most common products are vegetables and fruits, which are consumed without cooking. Foods coming from a contaminated environment (e.g. sea) may also carry the organism, for example seafoods. The incubation period of cholera is usually between one and three days. It can vary from mild, self-limiting diarrhea to a severe, life-threatening disorder. The infectious dose in normal healthy individuals is large (about 1010 cells/g) when the organism is ingested without food. The infection dose may be reduced if bacteria are consumed with food which protects them from the acidity of the stomach. Cholera is a non-invasive infection. In case of cholera bacteria colonizes the intestinal lumen and may produce enterotoxin. Cholera toxin disrupts the ion transport by intestinal epithelial cells. The severe diarrhea characteristics of cholera are due to the loss of water and electrolytes. If the huge losses of fluid and electrolyte are not replaced, then the volume and pressure of blood will fall, the blood viscosity will increase, may occur renal failure, and the circulatory system will collapse. In fatal cases death occurs within a few days. In untreated outbreaks the death rate is high (about 30–50%). The immediate treatment by intravenous or oral rehydration with electrolyte and glucose solution can reduce the high death rate to less than 1%. Pathogenic vibrios are able to cause foodborne disease. Vehicle foods for vibriosis caused by V. parahaemolyticus are seafoods (e.g. raw fish, oysters, crabs, lobsters, shrimps, and shellfish), which are consumed without processing or cooking. Due to the cross-contamination from seafood products, other foods can also be associated with the disease. The natural habitat of the pathogen is the sea. In addition to its role in gastroenteritis, V. parahaemolyticus is may cause extraintestinal infections in humans. The incubation period for V. parahaemolyticus food poisoning is 4 to 96 hours after ingestion of the bacteria, with a mean of 15 hours. The illness is generally mild or moderate, however some cases need hospitalization. The duration of the illness is about 2-8 days. Symptoms of the disease are profuse watery diarrhea free from blood or mucus, abdominal pain and cramps, nausea, vomiting, headache, fever, and chills. In the most severe cases, watery diarrhea is associated with mucus, blood, and tenesmus. Studies using human volunteers have revealed that ingestion of 105 to 107 cell/g of Kanagawa phenomenon-positive (KP+) V. parahaemolyticus led to the rapid development of gastrointestinal disease. Vibrios are the most common cause of food poisoning in Japan, but some outbreaks also occur in Europe. The reason is the national culinary habit, which means that Japanese often consume raw or partially cooked fish. However illness can also result from cross-contamination of cooked products in the kitchen. The avoidance of raw seafood products and care in preventing cross-contamination with contaminated raw materials will eliminate or drastically reduce the incidence of vibriosis. Postharvest processing methods (e.g. high-pressure treatment, irradiation, quick-freezing, and pasteurization) make the seafood products safer. V. vulnificus is a highly invasive bacterium, which causes principally septicaemia with a high fatality rate (about 50%). Most of the cases occurred in people with pre-existing liver disease, diabetes or alcoholism. Healthy humans are seldom involved, and illness is generally limited to gastroenteritis. Most common symptoms are malaise, fever, chills and prostration. Symptoms occur 16–48 h after consumption of the contaminated food (which usually seafoods). The risk of V. vulnificus infection associated with raw oysters. The risk is most effectively controlled by cooking the product before consumption. Sensitive and the immunosuppressed individuals need to avoid consume uncooked shellfish. 3.9. CAMPYLOBACTER GENUS AND CAMPYLOBACTERIOSIS Scientific classification Domain: BACTERIA Phylum: Proteobacteria Class: Epsilonproteobacteria Order: Campylobacterales Family: Camplylobacteraceae Genus: Campylobacter Species: Campylobacter jejuni Characteristics and occurrence of Campylobacter genus Prior to the 1970s, the campylobacters were known primarily to veterinary microbiologists as bacteria, which caused spontaneous abortions in cattle and sheep and as the cause of other animal pathologies. They were once classified as Vibrio spp. The genus Campylobacter belongs to the family Campylobacteraceae together with the Arcobacter, Sulfurospirillum, and Thiovulum genus. The genus Campylobacter contains more than 25 species, and also some subspecies. These species can be traced to a variety of animal and environmental sources. The most important species are C. jejuni, C. coli, C. lari, and C. upsaliensis. They have significant roles in foodborne and zoonotic illnesses. These four species are thermotolerant, because their optimal growth is between 41 and 43°C. The name Campylobacter is derived from the Greek word “kampylos,” which means curved, because Log-phase cells have a spiral or curved shape. Campylobacters are non-spore-forming, catalase-positive, oxidase-positive, Gram-negative rods. Cells are pleomorphic and may be 0.5–8 µm in length and 0.2–0.8 µm in width. Log-phase cells have a slender curved or spiral shape. As cultures begin to grow old, the spiral or curved bacilli are replaced by round forms. The cells are highly motile by means of one or more polar or amphitrichous flagella, which give them a very characteristic darting, corkscrew-like motility, furthermore they may be an important feature in pathogenesis. In biochemical point of view, campylobacters are not really active. They are unable to ferment or oxidize sugars. They are oxygen-sensitive microaerophiles, which means that, they grow best in an atmosphere containing 5–10% carbon dioxide and 3–6% oxygen. Most Campylobacter species grow at 37◦C. C. jejuni and C. coli have optima at 40–42◦C, and they do not grow under 30°C. They not really survive at room temperature. Campylobacters are sensitive to heat (D55 = 1.1–6.6 min), and they are unable to survive cooking or pasteurization temperatures. Survival of the bacteria in foods is better at chilled (4°C) than ambient temperature (25°C). Their viability decrease during frozen storage, but they are able to survive under these adverse conditions for prolonged periods. They grow well in the range of pH 5.5 and 8.0. They are also susceptible to other unfavourable conditions. They not really survive under dry or acid conditions, either in sodium chloride concentrations above 2%. In contrast to most foodborne pathogen, campylobacters cannot grow in foods during the processing or storage. The gastrointestinal tract of wild and domesticated animals is the main environmental reservoir of campylobacters. They are often found in the intestinal tract of poultry (it is prominent), dairy cattle, sheep, pigs, and wild birds. Its prevalence in fecal samples often ranges from around 30% to 100% (e.g. in chicken intestinal contents 39–83%, in swine faeces 66–87%, in sheep faeces up to 73%). In addition to poultry, the other primary source of campylobacters is raw milk. Asymptomatic carriage of campylobacters in human may also occur. Fecal specimens from humans with diarrhea yield C. jejuni, and it may be the single most common cause of acute bacterial diarrhea in humans. They can be commonly isolated from surface water, too. In case of human diarrhea, Campylobacter coli are the second most frequently isolated Campylobacter species. C. coli were first isolated by Doyle (in 1948) from pigs with infectious dysentery. C. coli are the most frequently isolated species from swine. It can also be found in poultry and cattle. Campylobacter lari can be found in the intestinal tracts of cattle, poultry, wild birds, shellfish, and from untreated water. C. lari occasionally cause diarrhea in humans. Campylobacteriosis Campylobacter is regarded as a leading cause of bacterial foodborne infection in many areas of the world. Campylobacters are the major cause of human diarrheal illness, rivaling or even surpassing Salmonella in importance in many countries. Enteritis caused by campylobacters is characterized by a large number of sporadic cases. Campylobacter enteritis in humans is considered to be mainly (about 90%) foodborne. Campylobacter contamination of foods can occur in different ways, since the bacterium is common inhabitant of the gastrointestinal tract of warm-blooded animals. Campylobacters can contaminate meat (mainly poultry) during slaughter and evisceration. Raw milk can also be contaminated with Campylobacter as a result of fecal contamination, Campylobacter mastitis, or after pasteurization. Acute enterocolitis caused by campylobacters, is difficult to distinguish from disease caused by other pathogens. In addition to C. jejuni, C. coli, C. intestinalis, and several other Campylobacter species are known to cause diarrhea in humans, but C. jejuni is by far the most important. Some strains of C. jejuni produce a heat-labile enterotoxin (CJT). Young children between 1 and 4 years, furthermore young adults between 15 and 24 years are susceptible to campylobacteriosis. Campylobacteriosis may occur throughout the year, but there is a peak during the summertime. C. jejuni may cause illness with an oral dose of 500 – 800 cell/g. The incubation period of campylobacteriosis is very changeable. It is usually 2–5 days, but may be as long as 7–10 days or more. Symptoms of campylobacteriosis are abdominal pain or cramps, diarrhea, malaise, headache, and fever. Symptoms lasted from 1 to 4 days. In the more severe cases, bloody stools, and severe abdominal pain (such as acute appendicitis) may occur. The diarrhea is self-limiting and maintained for 2 and 7 days, but slight relapses often occur. The organisms may be shed for up to 2–3 weeks (or rarely more than 2 months) after the symptoms disappear. Campylobacteriosis can be prevented if humans do not eat undercooked or unpasteurized foods of animal origin. Between 2008 and 2012 there was a clear seasonal trend in confirmed campylobacteriosis cases, reported in the European Union. During this period a significant increasing EU trend was observed (Figure 3.9.1). The number of confirmed cases of campylobacteriosis in 2012 was 214268, which means a 4.3% decrease over the previous year (220209 cases in 2011). Figure 3.9.1: Trend in reported confirmed cases of human campylobacteriosis in the EU between 2008 and 2012 (EFSA – ECDC, 2014) 3.10. MYCOTOXIGENIC FUNGI AND MYCOTOXINS Mycotoxins (“mukes” (Greek): fungi and “toxicum” (Latin): poison) are toxic secondary metabolites of moulds. They can be mutagenic, carcinogenic, and teratogenic or display specific organ toxicity. Aflatoxins are the most toxic compound among them. The mycotoxins may contaminate foods and animal feeds, consumed by humans or domestic animals. However toxins produced in the fruiting bodies of toxic mushrooms (toadstools) are not considered as mycotoxins. The most important mycotoxins are aflatoxins, trichothecenes, ochratoxin, zearalenon, patulin and fumonisins. Consequences of mycotoxin consumption have been described in the Old Testament (ergotism) and several ergotism epidemics occurred between the 8th and 16th centuries referred to as “St. Anthony's fire”. The first described mycotoxin was aflatoxin B1 in connection with the death of more than 100000 turkeys in 1960 in a farm in the UK. Aflatoxins, fumonisins, and ergot alkaloids are associated with acute mycotoxicoses. The important mycotoxins in food and feed: are aflatoxins, ochratoxin, trichothecenes, fumonisins, ZEN, and patulin. Some of them, like aflatoxins, DON, ergot toxin may be produced on field, before harvest. Fumonisin and ochratoxin however are produced mainly during storage. Mycotoxins might be present not only in the fungal mycelia, or spores, but also in the substrate food. The mycotoxins production is influenced by of several factors, like environmental parameters (temperature, humidity), substrate characteristics (moisture content, nutrient content), surrounding and competitive microbiota microorganisms maturity of the fungal colony, physical damage of the plant or plant product (e.g insect activity), etc. Fungi may grow on a broad range of temperature (~ 10 - 40 °C), and pH (4 to 8) range of. The minimum water activity (aw) for their growth is as low as 0.70. Mycotoxicoses may have either acute or chronic forms. Acute toxicoses are known for aflatoxins, fumonisins, and ergot alkaloids. Symptoms may include vomiting, diarrhoea, and other gastrointestinal problems, even death. The chronic forms have a broad range due to the carcinogenic, mutagenic, immuno-suppression effect and estrogenic effect of the toxins. Zearalenon has estrogenic effect, while the suppression of the immune system has been proved for the trichothecenes (DON, T-2). The chronic symptoms may include damage of vital organs, increased incidence of disease, and reduction in animal productivity, and interference with reproductive capacity. Usually more than one mycotoxin can be found on the contaminated products. Their negative impact on health and productivity has been proved to be synergistic (greater compared to the individual effects). Mycotoxins chemically markedly differ from the bacterial toxins associated with food poisoning. The latter ones are usually macromolecules (polypeptides, proteins or lipopolysaccharides). Mycotoxins however are relatively low molecular weight compounds with very complex structures. Although there are several mycotoxins producing moulds, specific strains of filamentous fungi belonging to species of the genera Aspergillus, Penicillium and Fusarium are the most important ones. Mycotoxin-producing fungi may contaminate several plant products (oil seeds, cereal grains, coffee, spices, fruits and juices) and beverages (wine and beer). Animal products (milk, meat, grease) may also contain mycotoxins originate from contaminated feeds and pastures. Good agricultural practices (GAP), good manufacturing practices (GMP) and monitoring, along with an effective Hazard Analysis and Critical Control Point (HACCP) approach may be effective to decrease the risk of mycotoxin contaminated food and feed. Mycotoxins of Aspergillus Aflatoxins Aflatoxins are not only the most toxic, but also the most widely studied of all mycotoxins. Moreover they were the first described mycotoxins. More than 100,000 broiler turkeys died in England in 1959 in the so called turkey X disease. It was proved later, that the birds had been poisoned by a contaminant in the groundnut meal used as a protein supplement in the feed. A mould, Aspergillus flavus was isolated from the poisonous feed, so the produced toxin was designated aflatoxin (Aspergillus flavus toxin—A-fla-toxin). The contaminant was fluoresced intensely under ultra-violet light. Aflatoxins are polyketide secondary metabolites. The six most important forms are B1 and B2 (fluoresce blue), G1 and G2 (fluoresce green and green–blue), M1 and M2. The later ones are the hydroxylated product of aflatoxin B1 and B2, in milk, urine, and feces. Aflatoxin B1 is the most toxic, followed by M1, G1, B2, M2 and G2. The two most important aflatoxin producing mould species are Aspergillus flavus and A. parasiticus, which are common in the tropics and subtropics. Other aflatoxigenic species are A. nomius, A. pseudotamarii and A. ochraceoroseus. High humidity and warm temperatures enhance growth and aflatoxin proguction of fungi in food during poor storage following harvest. The optimum temperature for toxin production has been found to be between 24◦ and 28◦C for Aspergillus flavus and A. parasiticus. It has also been realized that aflatoxins also can be produced in the growing crop by the endophytic aspergilli before harvest when the plant is stressed (e.g. during a drought). It also must be noted, that fungal growth can occur without toxin production. Aflatoxins are highly toxic, mutagenic, teratogenic, and carcinogenic compounds (IARC Group1: confirmed human carcinogen). There are acute and chronic forms of toxicities caused by aflatoxin. The acute form is resulting from the hepatotoxic effect, resulting severe liver lesions, and can be lethal in malnourished persons. Aflatoxin poisoning with fatal outcome have been reported in India in 1974 when approximately 1000 people involved and nearly 100 died in a large outbreak of poisoning. The incriminated food was mouldy maize, like in another large outbreak of aflatoxicosis in Kenya in 2004, resulting in 317 cases and 125 deaths. The estimated LD50 of aflatoxin B1 in humans is between that for the dog (0.5-1 mg/kg body weight) and the rat (5-7 mg/kg body weight). Circumstantial evidence suggests that aflatoxins are carcinogenic to humans. Aflatoxins are persumed to be co-factors in hepatocellular carcinoma along with hepatitis-B virus in tropical Africa, where there is a higher incidence of liver cancer. Aflatoxins bind to the nuclear and to the liver mitochondrial DNA, resulting mainly point mutations. Aflatoxins may contaminate important agricultural commodities. Plant products with high oil content have the highest risk for aflatoxin contamination, like oilseeds (groundnut, soybean, sunflower, cottonseed) and tree nuts (almond, pistachio, walnut, coconut). Maize, is the most important affected cereal, but sorghum pearl millet, rice, wheat toxin contamination has also been reported. Spices, mainly chillies and black pepper may also be affected together with coriander, turmeric and ginger. Aflatoxin contamination in milk (human and animal), and milk products (like butter) is a serious problem, as milk is basal nutrition for the infants’ nutrition, and they are more sensitive to toxins than adult. Aflatoxin might pass into the milk from the contaminated food or feed. The tolerance limit in food products for aflatoxin B1 is 5 μg/kg, while for aflatoxin M1 is only 0.05 μg/kg in milk (FAO/WHO expert committee recommendations). Ochratoxins The ochratoxins consist of several structurally related pentaketide derives, of which ochratoxin A is the best known and the most toxic one. Ochratoxin A is considered the second most important mycotoxin, as it is widespread on different plant products both at the tropical and in the temperate region, and it is among the most frequent mycotoxin detected in human blood. Ochratoxin A is produced by several species of Aspergillus and Penicillium. The most important ones are Aspergillus ochraceus in the tropical and sub-tropical region, and Penicillum verrucosum in the temperate region. Ochratoxin A was isolated first from Aspergillus ochraceus submerged culture in 1965. Aspergillus ochraceus has been spitted recently into two species, A. ochraceus and A. westerdijkiae, both with remarkable ochratoxin production. In Aspergillus section Nigri Aspergillus carbonarius is the most important toxin producer, but Aspergillus niger aggregates, A. lacticoffeatus and A. sclerotioniger, and several other aspegilli (e.g. A. cretensis, A. flocculosus, A. pseudoelegans, A. roseoglobulosus, A. sclerotiorum, A. sulphureus) are also able to produce it. Beyond Aspergillus, two species of Penicillium are play important role in the ochratoxin production and contamination of food and feed: P. verrucosum and P. nordicum. The former produce the toxins growing on stored cereals, while the later one on meat products (like salami and ham). Ochratoxin A has been proved to be nephrotoxic, hepatotoxic, neurotoxic, teratogenic, carcinogenic and immunotoxic on several species of animals. Human epidemiological studies have revaealed ochratoxin A with human nephropathy (Balkan Endemic Nephropathy and Tunisian Nephropathy). It has been classified as possibly carcinogenic to humans (Group 2B) by IARC. (in cereals, coffee, cocoa, dried fruit, spices, and also in pork) and occasionally in the field on grapes. It may also be present in some of the internal organs (particularly blood and kidneys) of animals that have been fed on contaminated feeds. Cereals and cereal products (wheat, maize, rice, oats, barley) seems to be the major source of ochratoxin A. Wines, musts and grape juices, moreover dried grapes (currants, raisins and sultanas) may also contain a considerable levels of this toxin. Ochratoxin A is produced during storage of fresh produce (cereals, millet), on beans (cocoa beans, coffee beans, soya beans), and on several other plant products (beer, citrus fruits, Brazil nuts, mouldy tobacco, herbs, spices, figs, olives and dried fruits) as well. Ochratoxin A also occurs on animal, mainly on pig products (sausages, liver, meat and ham). They either originated from the contaminated food of the animals, or produced by mould on the long-time traditionally ripened hams. Being fat-soluble, ochratoxin A is accumulated in the animal tissues, particularly in pigs. The suggested acceptable daily intake (ADI) of ochratoxin A is 1.5 ng OTA/kg body weight. The maximal residue levels (MRL) in maximal residue levels in the EU is only 5 μg OTA/kg. Mycotoxins of Penicillium Penicillium is more frequent in Europe on fruits (e.g. oranges, lemons, grapefruits, and apples) and foods (e.g. jams, bread and cakes) as a spoilage mould than Aspergillus. Some species (e.g. P. roquefortii and P. camembertii) are important in the production of mould-ripened foods (blue and soft cheeses) Patulin Patulin chemically is a polyketide, described first time as a potential useful antibiotic in 1942. It can be produced be produced by several penicillia (e.g. P. claviforme, P. expansum, P. patulum) and some aspergilli (A. clavatus, A. terreus), by Byssochlamys (B. nivea and B. fulva) and Paecilomyces species. Patulin is the most important and dangerous mycotoxin in apples, pears, and their products (juices and cider) and in other fruits (including bananas, pears, pineapples, grapes, and peaches). Moreover it has been found in moldy bread, sausage, and other products. It is also reported in silage, causing problems in ruminants. P. expansum and P. patulum are only able to grow at aw< 0.83 - 0.81, at a wide temperature range (5–20◦C). It is stable only at the relatively low pH. This toxin affect the functions of gastrointestinal tissue, and the immune system, toxic to kidney, liver and regarded to be genotoxic. It is also associated with typical neurotoxic signs, and death of ruminants, possibly because of its antimicrobial effects on the rumen microflora. The limits on patulin in fruit juices and beverages are 50 µg/ml in the EU. Citrinin Citrinin is produced by Penicillium expansum, Penicillium citrinum, P. viridicatum, and other fungi (Aspergillus and Monascus spp.). It has been recovered from rice (yellow rice disease), moldy bread, country-cured hams, wheat, oats, rye, fruits, barley, maize, cheese, and dietary supplements. It had nephrotoxic and teratogenic effects in animals, and possibly have role on nephropathy in swine and in the Balkan endemic nephropathy in humans. Mycotoxins of Fusarium Fusarium species are not only involved in the post-harvest spoilage of crops in storage, but may also cause devastating diseases of crop plants (blights, root rots wilts, and cankers). Moreover several species are able to produce different mycotoxins both in the field and in storage. There are 2 types of toxins produced (i) similar to the hormone oestrogen (ZEN or F-2 toxin) and (ii) nonoestrogenic toxins. Zearalenone Zearalenone and the corresponding alcohol, zearalenol are oestrogenic compunds, produced by Fusarium graminearum (Gibberella zeae), and other Fusarium species (F. culmorum, F. cerealis, F. equiseti, F. crookwellense and F. semitectum). These species are common soil fungi, and pathogens of cereal plants. Maize is the most affected cereal in Europe, however wheat, oats, barley, sorghum, and soybean products also have been found to be contaminated. Zearalenone cause reproductive disorders of farm animals (pigs, cattle and sheep). Pigs are especially sensitive to this toxin. The vulva and mammary glands become swollen and, there may be vaginal and rectal prolapse in the young ones (gilts). Infertility, reduced litter size is observed in older animals and piglets may be born weakened or deformed. It also may be implicated on hyper oestrogenic syndromes in humans. Beyond reproductive and developmental toxicity, zearalenone and its metabolites also has carcinogenicity, genotoxicity and immunotoxicity. Fumonisins The fumonisins are produced mainly by F. moniliforme (current name: Gibberella fujicuori) and by several other Fusarium spp., (F. sacchari, F. subglutinans, F. thapsinum, F. globosum, F. anthophilum, F. dlamini, F. napiforme, F. nygami, and F. proliferatum). It has been detected in maize, moreover on corn flour, dried milled maize fractions, dried figs, herbal tea, medicinal plants and bovine milk. Certain diseases of humans (oesophageal cancer) and animals (equine leukoencephalomalacia in horses, toxic feed syndrome in poultry, and porcine pulmonary oedema syndrome in swine) are associated with the consumption of grains and grain products that contain high levels of these moulds. It is possibly carcinogenic to humans (IARC group 2B), and hepatotoxic and nephrotoxic in animals. The toxin disturbs the sphingolipid metabolism and cause cardiovascular malfunction. Fumonisins are increasing mortality rates in animals, by impairing the basic immune function, causing liver and kidney damage, heart risk, and reducing weight gains. Trichothecenes Trichothecenes are sesquiterpenoid toxins, and grouped in four types A, B, C and D. They are associated with fusarium head blight of cereals (wheat, maite rice, etc.), with reasonable economic and health impacts worldwide. they are produced by not only Fusarium, but also some Trichoderma, Trichotecium, Myrothecium,and Stachybotrys species. The major type A trichothecenes (T-2 and HT-2) are highly toxic. They are produced mainly by F. sporotrichiodies and F. poae. Type A trichothecenes, like T-2, are approximately ten times more toxic in mammals than the type B Deoxynivalenol (DON) and nivalenon. Hovever DON is the most prevalent toxin associated with fusarium head blight. These types of fusarium toxins have higher phytotoxicity however. They are produced by F. culmorum and F. graminearum. Abdominal pain, nausea, vomiting, diarrhea, dizziness, and headache are the main symptoms of acute trichothecene mycotoxicosis. They also have immunosuppressive effects. Trichothecenes are inhibiting peptide synthesis at 60S ribosomes, disrupt nucleic acid synthesis, mitochondrial function, membrane integrity, and cell division. Trichothecenes are able to induce programmed cell death (apoptosis) in plants and in animal cells. The ingestion of fusarium head blight cereals producing trichotecens cause alimentary toxic aleukia with symptoms of vomiting and diarrhea resulting of intestinal irritation, followed by aleukia and anemia, and ultimately death. More than 100,000 people died in the most devastating outbreak of alimentary toxic aleukia in Russia between 1942 and 1948. Deoxynivalenol, also called DON is a type b trychotecen and the most frequently detected fusarium toxin produced by Fusarium graminearum. It is also called vomitoxin, causing vomiting of pigs fed by contaminated cereals, and detected as the feed-refusal factor. DON has been reported in most parts of the world, and is commonly encountered in food products and feeds prepared from contaminated corn and wheat. Group-A trichothecenes are more toxic than the type B DON. They are suspected to be responsible for reduced feed uptake, vomiting, and immuno-suppression in animals, and resulting changes in the blood cell count and in immune function resulting of chronic poisoning in animals. T-2 toxin is the most important one Group-A trichothecene. Claviceps purpurea /ergot toxins Although the aetiology of ergotism resolved only at the mid-19th, it has been documented since the middle ages as a human disease. The disease is caused by ergot alkaloids produce in the sclerotia (macroscopic, dark brown surviving structure) of the plant pathogenic fungus, Claviceps purpurea. These toxic secondary metabolites are also produced as by fungal Penicillium, Aspergillus, and Rhizopus species. Nowadays ergotism rarely occurs thanks to the improved cleaning and milling processes, which are able to remove most of the ergots. Consumption of claviceps toxins cause delirium, prostration, violent head pain, abscesses, and gangrene. 3.11. FOODBORNE VIRUSES Viruses are non-cellular microbes, having only one type of nucleic acid (either RNA or DNA). The nucleic acid (viral genome) is coated with protein capsid. Some viruses possess an outer lipid membrane (envelope), however those ones are destroyed by exposure to bile and acidity in the digestive tract consequently cannot be transmitted via food. There are several difficulties with the detection of viruses. Being extremely small (25–300 nm), they cannot recognize with conventional light microscopy, only with electron microscope. Moreover being obligate intracellular parasites, viruses do not grow on culture media, in tissue culture or chick embryo. Extraction and concentration are necessary before detection, as their numbers may be expected to be low. PCR techniques are the most suitable methods presently for the detection of foodborne viruses. Foods serve vehicles only for the intestinal or enteroviruses. Foodborne viruses are mainly originated from fecal contamination. Viruses have special affinity to tissues, for example some shellfish may accumulate them up to 900-fold. There are currently more than 100 human enteric viruses recognized. The pathogenic enteric viruses enter the body via the gut, but they differ in their target tissues. Gastroenteritis viruses remain in the gut (rotaviruses, adenoviruses, astroviruses, and caliciviruses); while others (e.g. polio and hepatitis A and E viruses) cause illness once they have migrated to other organs. People can be infected without showing symptoms. The importance of foodborne transmission of viruses is increasingly recognized, and explained by changes in food processing and consumption patterns. Although the exact number of foodborne and waterborne viral diseases is unknown, viral gastroenteritis is believed to be second most important microbial diseases, after common cold. Foods can be contaminated by enteric viruses either as primary contamination, or as secondary contamination. Primary contamination is from polluted water in the field (mainly for bivalve molluscan shellfish), or salad vegetables fertilized with human excrement or irrigated with sewage in the production site. Secondary food contamination is mainly originated from an infected food handler during handling, preparation and serving. Bivalve molluscan shellfish are the primary source of foodborne viral infections, but several other foods, however, have also been implicated as vehicles of transmission (desserts, fruits, vegetables, salads, sandwiches). Affected foods are those items that are subject to extensive handling in their preparation and are consumed without reheating (e.g. vegetable salads). Norwalk-like caliciviruses and hepatitis A virus are the more frequently detected fecal–orally transmitted viruses. We have to note however, that both can be transmitted directly from person to person, beyond the indirect way, via contaminated food or water. Hepatitis A and E viruses Hepatitis A and hepatitis E are enterically transmitted with food- or water, while other described hepatitis viruses (B, C, D, and G) are parenterally transmitted. Hepatitis A used to be the most prevalent foodborne virus, but improvements in public hygiene and sanitation have reduced the reported cases in the developed world. Hepatitis A virus belongs to the family Picornaviridae (like polio). It is a small, non-enveloped spherical virus, with positive, single-stranded RNA (ss RNA) genome. The virus multiplies in the gut epithelium cells in the first period. Later it is carried by the blood to the liver. The incubation period ranges from 15 to 50 days. And usually lifetime immunity occurs after an attack. Early symptoms are non-specific, including fever, abdominal discomfort, nausea and vomiting. Than 1 or 2 weeks later, signs of hepatitis liver damage symptoms (passage of dark urine and jaundice) may develop in the older children and adults. The mortality rate increases with age. However in children younger than 6 years of age, most infections are asymptomatic. In the developing world, where hepatitis A infection is endemic, the majority of persons are infected in early childhood, and virtually all adults are immune (no epidemic events). However in countries with higher hygienic standard, the hepatitis A epidemics occurs sporadically, usually from an infected common source. Hepatitis A is usually transmitted by the fecal–oral route, and raw or partially cooked shellfish from polluted waters is the most common vehicle food. Outbreaks from milk, fruits (strawberries and raspberries), and salad vegetables, are usually due to contamination by an infected food handler. Hepatitis E is a calici-like RNA virus. It is endemic in a wide geographic area, but rare and usually travel-related in the industrialized countries. In case of pregnant women, it is characterized with a high (15–20%) mortality rate. The primary source appears to be fecally contaminated water. Gastroenteritis Viruses Several viruses have been identified as etiological agents of viral gastroenteritis in humans in the last decades. Foodborne viral gastroenteritis is usually diagnosed only by epidemiologic criteria, and rarely with electronmicroscopic, immunological, or PCR detection. Viral gastroenteritis may be caused by rotaviruses, astroviruses, adenoviruses, and the human enteric caliciviruses. Caliciviruses (Caliciviridae family) include the human infecting Norovirus (formal Norwalk virus) and Sapporovirus genera. They are non-enveloped, positive-sense ssRNA viruses, and cannot grow on cell culture. The Norwalk virus was first recognized in a school outbreak (possibly from drinking water) in Norwalk, (Ohio, USA) in 1968. The virus is more resistant to destruction by chlorine than other enteric viruses. Noroviruses are responsible for the majority of viral gastroenteritis. The onset of symptoms may be very sudden and unexpected, following a 1–3-day incubation period. Characteristic symptoms are projectile vomiting, diarrhea, and headache, lasting usually 2–3 days. Mixtures of symptoms may occur, since contaminated foods may contain multiple agents. The infection root for the caliciviruses are either direct (person-to-person contact) or indirect (via contaminated water, food or environments). The infectious dose can be probably as low as 10–100 virus particles. Rotaviruses belong to the family Reoviridae. They are nonenveloped, double-stranded RNA (dsRNA) viruses. The fecal–oral route is the primary mode of their transmission. Group A rotaviruses are the most common among infants and young children, most susceptible between the ages of 6 months and 2 years. These viruses cause an estimated one-third of all hospitalizations for diarrhea in children below age 5, and the peak season for infection occurs during the winter months. Group B causes diarrhea in adults, and they have been seen only in China. Rotaviruses are transmitted directly among children in day care centres, and by water. 3.12. FOODBORNE PARASITES Numerous parasites can be transmitted by food including many protozoa, flatworms, and roundworms. Many of them also can be transmitted by water, soil, or person-to-person contact. Trichinellosis and echinococcosis are the most important foodborne parasites in Europe. Symptoms of foodborne parasitic infections are varying from gastrointestinal symptoms (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis) to muscle pain, cough, skin lesions, malnutrition, weight loss, and neurological symptoms. Foodborne parasites may have different transmission routes. Waterborne parasites (Cyclospora cayetanensis, Cryptosporidium and Giardia) may be transmitted by contaminated food. Others (e.g. Toxoplasma gondii) may be transmitted through faecal contamination of foods. Raw or undercooked meat infected with cyst (e.g. Trichinella spp. Sarcocystis spp.) of parasites may also be responsible for transmission. Wide variety of food products may be contaminated with one or more parasites. The main main sources are pigs, cattle, fish, crabs, crayfish, snails, frogs, snakes and aquatic plants. Animal parasites require for more than one (a definitive and an intermediate) animal hosts to carry out their life cycles. The definitive host is the animal in which the adult parasite carries out its sexual cycle. The intermediate host is the animal where larval or juvenile forms develop. In some cases, both larval and adult stages reside in the same host (e.g., trichinosis). Parasites do not proliferate in foods, and cannot grow on culture media. Their presence can be detected directly with microscope following enrichment from food or stool. Protozoa Food- or waterborn protozoan enteric infections include toxoplasmosis (Toxoplasma gondii), cryptosporidiosis (Cryptosporidium sp.), cyclosporiasis (Cyclospora cayetanensis), cystoisosporiasis (Cystoisospora belli) sarcocystosis (Sarcocystis sp.). These pathogens are all sporozoids, members of the phylum Apicomplexa. Non apicomplexan protozoa include the flagellate Giardia intestinalis, and the amoeboid Entamoeba histolytica. All, but E. histolytica and C. belli have a domestic or wild zoonotic reservoir. More recently oral transmission for Trypanosoma cruzi, the agent of Chagas disease has been widely detected in Brasil. Giardia lamblia Giardia intestinalis (syn. G. duodenalis, G. lamblia) is the causative agent of giardiasis, which is a diarrheal disease with fecal-oral transmission route. It is a flagellated protozoon with characteristic trophozoit morphology of eight flagella and paired nuclei with kite form. The cysts are pear shaped, with a size range of 8–20 μm in length and 5–12 μm in width (Figure 3.12.1). The organism survives in food and water as cysts. Cysts are killed by boiling or during normal cooking procedures, but cysts are generally resistant to the levels of chlorine used in the water treatment systems. Giardia cysts could occur on any foods (e.g. lettuce, strawberries) which are washed with contaminated water or handled by infected persons with inappropriate hygienic practice. The protozoon cysts are transmitted via contaminated drinking water and food, or contact with infected persons, or animals. Figure 3.12.1: The life cycle of Giardia intestinalis (Source: http://www.cdc.gov/dpdx/images/giardiasis/Giardia_LifeCycle.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) Giardia cysts excyst in the small intestine with the aid of stomach acidity and proteases and give rise to giardiasis. The trophozoites are obtaining their nutrients by absorption. They resides in the lumen of the small intestine, and do not invade tissues. Adhesion of Giardia trophozoites to the intestinal epithelium is via a specialized disc and is crucial to initiate colonization as well as to maintain the infection. The incubation period for clinical giardiasis is 1–4 weeks, and cysts appear in stools after 3–4 weeks. Symptoms are vary between benign manifestation with asymptomatic cyst passage to when clinical giardiasis when, symptoms may last from several months to a year or more. Symptoms include diarrhoea, abdominal cramps and nausea. Amebiasis Amebiasis (amoebic dysentery) is caused by Entamoeba histolytica. This pathogenic amoeba can cause invasive intestinal and extra-intestinal disease worldwide. It is usually transmitted by the faecal–oral route, although transmission by water, food handlers, and foods are also occurring. Infection is endemic in many poor communities in all parts of the world. The organism is an aerotolerant anaerobe lack mitochondria, which survives in the environment in an encysted form. Excystation of the ingested cysts occurs in the intestine, than the parasite encysts in the ileum, and cysts may occur free in the lumen (Figure 3.12.2) . E. histolytica does not invade individual cells, however disrupts the protective mucus layer with enzymes. Adhesion molecules are also important virulence factors of this parasite. Figure 3.12.2: The life cycle of E. histolytica and E. dispar (Source: http://www.cdc.gov/dpdx/images/amebiasis/Amebiasis_LifeCycle.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) The incubation period for amebiasis is 2–4 weeks, and symptoms may persist for several months. Most infections however remain symptomless. Its onset is often insidious, with. In case of illness symptoms are loose stools and generally no fever. Passing of mucous and bloody stools are characteristic, due to ulceration of the colon. Severe diarrhoea, abdominal pain, fever and vomiting may also progress a few weeks following the infection. A recently separated species, E. dispar may develop an invasive form of amebiasis, when trophozoites invade extraintestinal sites (liver, brain, and lungs) through the bloodstream. A person with amoebic dysentery may pass up to fifty million cysts per day. Cysts are not motile, but motile trophozoites cav be identified in stools as containing red blood cells ingested by pseudopodia, while non-pathogenic amoebas does not. Toxoplasmosis This disease is caused by Toxoplasma gondii, which is an obligate intracellular parasite. The ingestion of T. gondii oocysts causes no symptoms in healthy adults, or the infection is selflimiting. In these cases, the organism encysts and becomes latent. When symptoms occur, they consist of fever with rash, headache, muscle pain and pain, and swelling of the lymph nodes. Following the ingestion of oocysts from cat feces (direcly, or via contaminated food, water) or infected meet (mainly pork), motile sporozoites are released with the help of digestive enzymes in the intestine. When freed in the intestines, these forms (called tachyzoites) pass through intestinal walls and multiply rapidly in many other parts of the body, giving rise to clinical symptoms. Later protozoan cell clusters are surrounded by a protective wall forming a tissue cyst intracellular in host cells. The development of a cyst wall around protozoan cells (called bradyzoites) coincides with the development of permanent host immunity. These tissue cysts may persist in the body for the lifetime of an individual, but if the cysts are mechanically broken or break down under immunosuppression, protozoan cells are freed and begin to multiply rapidly bringing on another active infection (Figure 3.12.3). In pregnant mothers with newly contracted toxoplasmosis, the T. gondii protozoan tachyzoites cells are able to cross the placenta, and severe encephalitic disease may also occur among new-borns. Life-threatening toxoplasmosis also results from the breaking out (recrudescence) of the latent infection when patients are in immunocompromised state (e.g. AIDS). The incubation period in adults is 6–10 days while in infants it is congenital. Figure 3.12.3: The life cycle of Toxoplasma gondii (Source: http://www.cdc.gov/dpdx/images/toxoplasmosis/Toxoplasma_LifeCycle_BAM1.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) The definitive hosts of T. gondii are cats (domestic and wild). Normally, the disease is transmitted from cat to cat, but virtually all vertebrate animals are susceptible to the oocysts shed by cats. As few as 100 oocysts can produce clinical toxoplasmosis in humans, and the oocysts can survive over a year. Pigs are the major animal food source as raw or undercooked to humans. Toxoplasmosis in humans can be prevented by avoiding environmental contamination with cat feces, and by avoiding the consumption of meat and meat products that contain viable tissue cysts. The cysts of T. gondii can be destroyed by heating meats above 60◦C. Sarcocystosis Species of Sarcocystis are two-host parasites. The definitive host (place of sexual reproduction of the parasite) is carnivore (e.g. humans). In the tissues of the intermediate host (e.g. cattle, pigs) the asexual cysts are formed. Two species can infect humans: S. hominis, which infects cattle, and S. suihominis from pigs. Although symptoms are usually mild, they can include nausea and diarrhoea. Beef and pork which have been adequately cooked lose their infectivity. When humans ingest a sarcocyst, bradyzoites are released and penetrate the the small intestine. Following the sexual reproduction sporocysts form pass out of the bowel in feces. When sporocysts are ingested by pigs or bovines, the sporozoites are released and spread throughout the body. They multiply asexually and lead to the formation of sarcocysts in skeletal and cardiac muscles (Figure 3.12.4). Figure 3.12.4: The life cycle of Sarcocystis hominis and S. suihominis (Source: http://www.cdc.gov/dpdx/images/sarcocystosis/Sarcocystis_ LifeCycle_v3.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) Cryptosporidiosis The protozoan Cryptosporidium parvum is known to be a pathogen of at least 40 mammals, moreover reptiles and birds. Cryptosporidium meleagridis, previously associated with birds, is also known to infect humans. Cryptosporidiosis appears to be an increasing cause of diarrhoea. In humans, the disease is selflimiting in immunocompetent individuals, diarrhea symptoms typically last 9–23 days following an incubation period of 6–14 days. The symptoms are more sever in the immunocompromised, such as AIDS patients, where diarrhea is profuse and watery, with as many as 71 stools per day and up to 17 l per day. Diarrhea is sometimes accompanied by mucus but rarely blood. The fecal–oral route of transmission is the most important, but indirect transmission by food and milk is known to occur. It is mainly a waterborne disease (tapwater or pools) with the largest documented outbreak of gastrointestinal disease in USA with an estimated 403,000 reported cases in Milwaukee in 1993. Apple cider, raw green onion and chicken salad have been proved to involve in foodborne cryptosporidiosis yet. The whole life cycle of C. parvum can take place in a single host, either human or farm animal (e.g. cattle or sheep). It is an obligate intracellular parasite. The thick-walled oocysts excyst in the small intestine following ingestion, and free sporozoites and penetrate the microvillous region of host enterocytes, where sexual reproduction leads to the development of zygotes. They invade host cells by disrupting their own membrane as well as that of the host. The environmentally resistant oocysts are shed in feces, and the infection is transmitted to other hosts when they are ingested (Figure 3.12.5). Figure 3.12.5: The life cycle of Cryptosporidium parvum (Source: http://www.cdc.gov/dpdx/images/cryptosporidiosis/Cryptosporidium_ LifeCycle.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) High-temperature, short time (HTST) milk pasteurization, moreover temperatures above 60◦C and below −20◦C may kill C. parvum oocysts. However the oocysts are resistant to chlorid treatment used for water. Cyclosporiasis The protozoan that causes this disease, Cyclospora cayetanensis, is closely related to Cryptosporidium in the phylum of protists known as the Alveolata. It has been recognised since the early 1990s as a causative agent of a few gastrointestinal outbreaks associated with unprocessed fresh food products such as soft fruits and vegetables. The C. cayetanensis oocysts measure approximately contain two sporocysts, sand each sporocyst contains two crescentshaped sporozoites (Figure 3.12.6). Figure 3.12.6: The life cycle of Cyclospora cayetanensis (Source: http://www.cdc.gov/dpdx/images/cyclosporiasis/Cyclospora_LifeCycle.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) C. cayetanensis is an intestinal pathogen that parasitizes epithelial cells of the jejunum. The disease symptoms mimic those of cryptosporidiosis. The incubation period ranges between 2 and 11 days. Diarrhea is prolonged (43 ± 24 days), but self-limiting and include non-bloody diarrhoea, loss of appetite, weight loss, stomach cramps, nausea, vomiting, fatigue and fever. Symptomes are more severe in human immunodeficiency virus (HIV)-infected individuals. Helminths and nematodes There are a number of animal parasites amongst the flatworms and roundworms, which can be transmitted to humans via food and water. These complex animals do not multiply in foods and their presence is normally detected by direct microscopic examination often following some form of concentration and staining procedure. Platyhelminths: Liver Flukes and Tapeworms The two most important foodborne parasites among the Platyhelminths (flatworms) are the Fasciola hepatica, and the genus Taenia. These organisms have complex life cycles which may include quite unrelated hosts at different stages. The mature stage of the liver fluke develops in humans, sheep or cattle which may be referred to as the definitive host. It is living in the bile duct after entering and feeding on the liver. Their eggs secreted in the faeces after passing from the bile duct. The embryos from the hatch egg a water snail. Cysts will only develop further if they are swallowed by an appropriate definitive host, usually cattle or sheep in which infection can cause serious economic loss, or more rarely in humans after eating raw or undercooked watercress on which the cysts have become attached. Tapeworms Taenia solium associated with pork and T. saginata associated with beef, are best known. The larval stages of the beef tapeworm have to develop in cattle and finally infect humans through the consumption of undercooked beef. The mature tapeworm of these species can only develop in the human intestine. The effects may include nausea, abdominal pain, anaemia and a nervous disorder resembling epilepsy, as well as mechanical irritation of the gut. Roundworms Perhaps the most notorious of the nematodes in the context of foodborne illness is Trichinella spiralis, the agent of trichinellosis which was first recognised as a cause of illness in 1860. It has no free-living stage but is passed from host to host which can include quite a wide range of mammals including humans and pigs. Thus trichinellosis in the human population is usually acquired from the consumption of infected raw or poorly cooked pork products. Figure 3.12.7: The life cycle of Trichinella spiralis (Source: http://www.cdc.gov/dpdx/images/trichinellosis/Trichinella_LifeCycle.gif (Centers for Disease Control and Prevention), download time: 16.12.2014.) Infection starts by the consumption of raw or undercooked muscle tissue containing encysted larvae, where digestive juices of the stomach enhance the release of the larvae. Deliberated larvae than grow and mature in the lumen of the intestines. The female warms ca produce more hundreds of larvae, which burrow through the gut wall enter the blood stream and reach a of specific muscle tissues (eye, tongue, and diaphragm, sometimes heart) in which they encysting (Figure 3.12.7). The symptoms caused by T. spiralis in the first period, during which the larvae are invading the intestinal mucosa, associated with abdominal pain, nausea and diarrhoea. The second phase of symptoms, which include muscle pain and fever, occurs as the larvae invade and finally encyst in muscle tissue, and accompanied by severe pain, fever. Sometimes, eating heavily infested meat, lead to death from heart failure. The larvae grow to about 1 mm in muscles then encyst by curling up and becoming enclosed in a calcified wall some 6–18 months later. About 75 species of animals can be infected by T. spiralis. Trichinosis can be controlled by avoiding the feeding of infected meat scraps or wild game meats to swine, and by preventing the consumption of infested tissues by other animals. Moreover adequate cooking (reaching at least 76.7 oC) of pork products are the most important precautions. Curing, smoking and the fermentations leading to such products as salami do all eventually lead to the death of encysted Trichinella larvae. 4. MICROORGANISMS IN ANIMAL PRODUCTS The presence of microorganisms in the food is often unrecognizable. However the microbial activity may change the food in an unwanted way (spoilage). Microbiological food spoilage can manifest in different ways, like visible microbial growth (colonies or surface slime), change of normal texture, or production of different chemicals (gas, pigments, polysaccharides, off odours and flavours). The presence of foodborne pathogens in food is a threatening safety risk, which should be avoided. The microbiological safety and quality of a food is determined by the property of the food, the microbial contamination of the raw materials, the production technology, and the storage and packaging conditions. Milk Milk is secreted by mammals. Cow milk is the most important one produced for human consumption, although several animals are used to produce it. Milk has very high water content. Furthermore it contains fat, protein and lactose has moderate pH (6.4–6.6). Milk is ideal medium with its high nutrient content. Milk contains a very adequate supply of B vitamins with pantothenic acid and riboflavin being the two most abundant. The water activity of the milk is high, near 1.0. Overall, the chemical composition of whole cow’s milk makes it an ideal growth medium for heterotrophic microorganisms, including the nutritionally fastidious Gram-positive lactic acid bacteria. However several antimicrobial substances present in the raw milk: lysozyme, lactoferrin, conglutinin, and the lactoperoxidase system (lactoperoxidases and lactoferrin). They are destroyed by pasteurization. The objective of milk pasteurization is the destruction of all disease-causing microorganisms. Most Gram-negative bacteria (especially psychrotrophs) are destroyed along with many Gram-positives. However thermoduric Gram positives belonging to the genera Enterococcus, Streptococcus (especially Streptococcus salivarius subsp. thermophilus), Microbacterium, Lactobacillus, Mycobacterium, Corynebacterium are survive. Endospores of pathogens, like Clostridium botulinum and spoilage organisms, like Clostridium tyrobutyricum, C. sporogenes, or Bacillus cereus are not destroyed. Among the survivors are a number of psychrotrophic species of the genus Bacillus. Microbes in the raw milk may originate from three sources: (i) the udder interior, (ii) the teat exterior and (iii) the surroundings, and the milking and milk-handling equipment. Milk from a healthy cow normally contains low numbers of organisms, so the udder interior is almost sterile. The most commonly isolated microbes are micrococci, streptococci, and Corynebacterium bovis. Mastitis however increase the bacterial number from 102-103 to 108 cfu/ml in the milk. The most important species associated with acute mastitis are Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Pseudomonas aeruginosa, Corynebacterium pyogenes, moreover Salmonella, Listeria monocytogenes, Mycobacterium bovis and Mycobacterium tuberculosis may also present in high number. Several of them are potential human pathogens. The udder exterior is contaminated from the cow’s general environment: bedding, manure, soil and silage. Human pathogens, like E. coli, Campylobacter, Salmonella and Bacillus may contaminate milk though this way. Moreover Clostridium butyricum and Clostridium tyrobutyricum can cause the problem in some cheeses (late blowing). The principal source of the micro-organisms in raw milk is from the milk-handling equipment. Fast growing and heat sensitive bacteria may grow on milk residues which can contaminate subsequent batches of milk. Missing proper cleaning and sanitizing procedures however, leading the development of milk stone. It is a hydrophobic deposit, rich in minerals, where slower growing, thermoduric microbes (like micrococci and enterococci) develop. Milk usually is chilled almost immediately and held and transported at a low temperature following until use. Chilling conditions only allow the proliferation of psychrotroph bacteria: of the genera Pseudomonas, Acinetobacter, Alcaligenes, Flavobacterium (Gram-negative rods), psychrotrophic coliforms, (dominantly Aerobacter spp.), and Gram-positive Bacillus spp. Milk is an important agent in the spread of human disease, which had been recognized in the 19th century. Milk pasteurization was introduced around 1890 to prevent the spread of milkborne disease, like tuberculosis (caused by Mycobacterium bovis and Mycobacterium tuberculosis), and brucellosis (caused by Brucella spp.). Raw milk can be a source of several other human diseases: enterohemorrhagic colitis (caused by E. coli 0157:H7), anthrax (caused by Bacillus antracis), salmonellosis, Q fever (caused by Coxiella burnetii), and campylobacteriosis. Pasteurization is the most effective method for controlling Salmonella, Campylobacter and Listeria contamination of milk. The four types of heat treatment applied to milk: (i) Low Temperature Holding (LTH - 62.8 OC for 30 min), (ii) High Temperature Short Time (HTST - 71.7 OC for 15 sec), (iii) Ultra High Temperature 135 OC for 1 s), and (iv) Sterilization (>100 OC 1 s - 20–40 min). Pasteurization destroys Gram-negative psychrotrophs, but heat resistant extracellular lipases and proteases produced by Pseudomonas spp. may cause rancidity and casein degradation in the processed milk. The Thermoduric Gram-positives sporeforming bacteria and members of the genera Microbacterium, Micrococcus, Enterococcus, Lactobacillus, microbes can survive mild pasteurization treatments. Psychrotrophic post-pasteurization contaminants of Gram-negative rods (Pseudomonas, Alcaligenes, Acinetobacter) are the main spoilage agents of pasteurized milk, resulting odours, fruity or putrid flavours, and clotting. Growth of thermoduric Bacillus spp. associated flavour defects, like bitty cream phenomenon. As milk is the only natural source of the disaccharide lactose, it undergoes microbial spoilage in a unique way. Only a relatively small number of milk-borne bacteria can obtain energy from this sugar: Gram-positive lactic acid bacteria and Gram-negative coliform bacteria. The bacterial spoilage of milk is accompany by the production of lactic acid by lactose users, and the reduction of normal pH (around 6.6) to 4.5. That leads to the precipitation of casein (curdling). The thermoduric Streptococcus salivarius subsp. thermophilus strains preferentially use the glucose moiety of lactose and excrete galactose, which is a ready substrate for nonlactose users. The spoilage of UHT milk is caused by Bacillus spp. (B. cereus, B. licheniformis, B. badius, and B. sporothermodurans) that survive the UHT process. The highest percentage of the microbiota of whole milk is found in as the fat droplets rise to the surface of milk, they carry up microorganisms too. The processing of creams to yield butter brings about a reduction in the numbers of all microorganisms Butter contains around 15% water, 81% fat, and generally less than 0.5% carbohydrate and protein. Although it is not a highly perishable product, it does undergo spoilage by bacteria and moulds. Bacteria cause two principal types of spoilage in butter: (i) putridity or surface taint, and (ii) rancidity. The first is caused by Pseudomonas putrefaciens or Chryseobacterium joostei, and accompanied by characteristic odour due to certain organic acids, especially isovaleric acid. Rancidity, the second most common bacterial spoilage of butter is caused by the hydrolysis of butterfat with the liberation of free fatty acids. Lipase both from non-microbial source, as well as microbes (Pseudomonas fragi, P. fluorescens) can cause the effect. Other less common spoilage conditions in butter are “malty flavor” (Lactococcus lactis var. maltigenes), “skunklike odour” (Pseudomonas mephitica), black discolorations (Pseudoalteromonas nigrifaciens). The generally high lipid content and low water content make butter more susceptible to spoilage by moulds than by bacteria. Cladosporium, Alternaria, Aspergillus, Mucor, Rhizopus, Penicillium, and Geotrichum, (especially G. candidum) can be seen commonly growing on the surface of butter and produce colorations by their spores. The yeasts genus Torula also has been reported to cause black discolorations on butter. Hard and semi hard ripened cheeses has low moisture content, which make them insusceptible to spoilage by most organisms, but moulds. Some ripened cheeses however have sufficiently low oxidation-reduction potentials to support the growth of anaerobes, in case of appropriate water activity. Clostridium species (C. pasteurianum, C. butyricum, C. sporogenes, C. tyrobutyricum) have been reported to cause causing the spoilage (gassiness) of cheeses. The aerobic Paenibacillus polymyxa, has also been reported to cause gassiness, producing CO2 from lactic acid. Meat Meat is a term applied almost solely to animal flesh. The meat of cattle, pigs, sheep, goats and poultry has a principal importance in economic terms. Edible animal flesh comprises principally the muscular tissues. Meats are the most perishable of all major foods, being an excellent medium for microbial growth, because of all nutrients required for the growth of bacteria, yeasts, and moulds exists in fresh meats, and no effective levels of antimicrobial constituents are known to occur in. It also has a favourable high water activity. Fresh meats of beef, pork, and lamb, as well as fresh poultry, seafood, and processed meats, have pH values within the growth range of most of the organisms. The redox potential of whole meats is low, enhancing only the growth of strict anaerobes. However redox potential at the surfaces tend to be higher, so strict aerobes and facultative anaerobes also may find conditions suitable for growth. Microorganisms that grow on meat first consume its carbohydrates and non-protein nitrogen content. Proteolysis only occurs only in the later stages of decomposition. The carbohydrate content of muscle has microbiological importance. The main energy storage molecule in the muscle is a glucose polymer, called glycogen. The oxygen supply to the muscles is cut off after death, but the continuing anaerobic glycolytic breakdown of glycogen leading to an accumulation of about 1% lactic acid, which results the decrease in muscle pH from around 7 to 5.4–5.5 in a typical mammalian muscle. If muscle has been exercised or animal had stress or exposure to cold before slaughter, it will have a limited supply of glycogen, and results a higher ultimate pH. That gives rise to dark cutting, known as dry, firm, dark (DFD) meat. The microbial growth is faster so spoilage will occur sooner in DFD meat with higher pH. Pale, soft, exudative (PSE) condition is another meat defect, and associated with post mortem changes in muscle is stimulated just before slaughter leading to a rapid post mortem fall in pH while the muscle is still relatively warm. This has no microbiological consequences. Muscles from a freshly slaughtered animal is relatively free from micro-organisms (< 10 cfu/kg), but can be increased under conditions of stress and higher if the animal is suffering from an infection. However the internal tissues of healthy slaughter can also be infected through different sources and routes. One of them is the stick knife to slit the jugular vein. If the knife is not sterile, organisms are swept into the bloodstream, where they may be deposited throughout the carcass. Organisms may also enter the carcass via the stick knife from the hide, or may be deposited on the dehaired carcass or invade the freshly cut surfaces. Gastrointestinal tract, especially the rumen of ruminant animals, contains high number of microbes. The later one typically contains ∼1010 bacteria per gram. By way of punctures, intestinal and rumen contents along with microbes may be deposited onto the surface of freshly dressed carcasses. The hands of handlers are an important source of human pathogens to freshly slaughtered meats. In case gloves are worn, organisms from one carcass can be passed on to the other. The nonsterile containers for meat cuts may also be source of microbial contamination. This is usually the primary source of microorganisms to ground or minced meats. In general, the most significant source of contamination is the nonsterile containers, when portions handled in high number within one day. The microbial content of handling and storage environment, like air may also be responsible for contamination, although the circulating is not an insignificant source of organisms to the surfaces of all slaughtered animals, as only limited microbial groups (like fungal conidia, bacterial endospores, pigmented Gram-positives) are able to survive in the air for a longer time. Lymph nodes are usually embedded in fat and often contain large numbers of bacteria. If they are cut through or grinded into the minced meat, this biota may easily become prominent. The development of rigor mortis require between 24 and 36 hours following slaughtered at the usual temperatures of holding freshly slaughtered beef (2–5 ◦C). Meanwhile, the normal biota of the meat originates from the animal’s own lymph nodes, the stick knife used for exsanguination, the hide of the animal, intestinal tract, dust, the handlers’ hands, the cutting knives, and the storage bins may also be the source of microbial contamination of meat. During prolonged storage at refrigerator temperatures led to microbial spoilage. In case the internal temperatures are not reduced to the refrigerator range, the spoilage is caused by bacteria of internal sources, mainly by Clostridium perfringens and different species belong to the Enterobacteriaceae family. On the other hand, bacterial spoilage of refrigerator-stored meats is, primary a surface phenomenon, and the spoilage biota reflects external contamination sources. The biota of meat and poultry reflects the slaughtering and processing environment. Typically Gram-negative bacteria are being predominant. Among Gram-positives, the enterococci are often found along with lactobacilli. The Gram-negative genera most frequently found in fresh meats are Acinetobacter, Aeromonas, Moraxella, Pseudomonas, Psychrobacter, while species in the Enterococcus are the most often isolated Gram-positives. Several other Gram-negative (Alcaligenes, Arcobacter, Citrobacter, Enterobacter, Escherichia, Flavobacterium, Hafnia, Pantoea, Pseudomonas, Proteus, Salmonella, Serratia, Shewanella, Yersinia) and Gram-positive (Bacillus, Brochothrix, Carnobacterium, Caseobacter, Clostridium, Corynebacterium, Erysipelothrix, Kocuria, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Microbacterium, Micrococcus Paenibacillus, Pediococcus, Staphylococcus, Vagococcus, Weissella) genera has known to occurs on fresh meet. The microbiota of fresh liver may contain Gram-negative Acinetobacter, Alcaligenes, Escherichia, Flavobacterium, Moraxella, and Gram-positive Micrococcus, moreover Brochothrix, Corynebacterium, Enterococcus, Leuconostoc, Staphylococcus and Weissella. Poultry most frequently contaminated by Gram-negative Acinetobacter, Campylobacter, Pseudomonas and Gram-positive Corynebacterium, Listeria, Micrococcus, Vagococcus species. Member of Aeromonas, Alcaligenes, Citrobacter, Flavobacterium, Moraxella, Pantoea, Proteus, Psychrobacter, Salmonella, Serratia (Gram-negative) and Bacillus, Brochothrix, Clostridium, Enterococcus, Erysipelothrix, Microbacterium, Paenibacillus, Staphylococcus (Gram-positive) genera has also been isolated from poultry. Because of their ubiquity in meat-processing environments, a rather large number of mould genera may be expected on fresh and refrigerated meats, including Penicillium, Mucor, Rhizopus, Sporotrichum, Thamnidium and Cladosporium. However Alternaria, Aspergillus, Eurotium, Fusarium, Monascus, Monilia, Neurospora is also known to occur. The most ubiquitous yeasts found in meats and poultry are members of the genera Candida and Rhodotorula. Other yeast species detected from meats: Cryptococcus, Debaromyces, Pichia, Rhodotorula, Torulopsis and Trichosporon. Chopped meats have been shown to contain higher numbers of microorganisms than no comminuted meats such as steaks, partially because commercial ground meats consisting of cut from various parts. The rarely cleaned meat grinders, cutting knives, and storage utensils enhance the successive accumulation of microbes. One heavily contaminated piece of meat is sufficient to contaminate others, as well as the entire lot, as they pass through the grinder. Moreover ground meat provides a greater surface area, which itself accounts in part for the increased biota. Finally, the greater surface area favors the growth of aerobic, low temperature spoilage bacteria. The addition of 10-30 % soy protein to the ground meats decreased their microbiological quality and spoilage could be detectable earlier than in the soy-free products. The possible reason is that soy-extended meat products have a slightly higher pH (0.3–0.4 unit), and that soy protein may increase the surface area of soy–meat mixtures as well. The livers, kidneys, hearts, and tongues differ from the skeletal muscle parts. These animal parts, especially liver, have higher pH (6.1 - 6.5) and glycogen levels. Usually low numbers of microorganisms can be found on these products. The biota on the fresh livers, kidneys and hearts consist largely of Gram-positive cocci, coryneforms, aerobic sporeformers, Moraxella-, Acinetobacter, and Pseudomonas spp. Meat for human consumption should be produced only from healthy animals; regarding some animal diseases can be transmitted to humans. Meat can be contaminated mainly from the skin (fleece) and gastrointestinal tract of the animal. The greatest opportunity for contamination during slaughter and butchering is the dressing, when head, feet, hides, excess fat, viscera and offal are separated from the bones and muscular tissues. Skinning can spread contamination from the hide, carrying a mixed microbial population of micrococci, staphylococci, pseudomonads, yeasts and moulds as well as organisms derived from sources such as soil or faeces. Large numbers of micro-organisms, including potential pathogens inhabit the animal gastrointestinal tract. Great care must be taken to ensure the carcass is not contaminated with visceral contents either as a result of puncture or leakage from the anus or oesophagus during removal. Surface numbers of bacteria is typically 102–104 cfu/cm2 at the end of dressing. Counts are generally higher in pigs (the skin scalded and dehaired and not being removed from the carcass) and in sheep carcasses than beef. Although there are several genera of bacteria, moulds, or yeasts in non-spoiled meat, interestingly, one or some genera are found to be characteristic of the spoilage of a given type of meat product. The fungal spoilage of fresh meats results several visible characteristic lesions. “White spots” on meat surfaces are the results of the growth of Sporotrichum and Chrysosporium. The Cladosporium, which is common, cause “black spot”, while Penicillium, produces green patches. When white mycelia appear on the surface of whole beef are caused by Thamnidium, Mucor, and Rhizopus, genera. Moulds generally do not grow on meats if the storage temperature is below 5◦C. Among genera of yeasts recovered from refrigerator-spoiled beef with any consistency are Candida. Psychrotrophic organisms represent only a small percentage of the initial microflora, but become predominant subsequently as the meat is held constantly at chill temperatures. Aerobic storage of chilled red meats enhance the growing of psychrotrophic aerobes, predominantly Pseudomonas species (P. fragi, P. lundensis, P. fluorescens), and other non-fermentative Gramnegative Acinetobacter and Psychrobacter. Psychrotrophic Enterobacteriaceae such as Serratia liquefaciens and Enterobacter agglomerans, lactic acid bacteria and the Gram-positive Brochothrix thermosphacta are the minor component of the spoilage microflora, of meats. The first indication of spoilage in fresh meat is the production of off odours. Produced volatile esters, alcohols, ketones and sulfur-containing compounds are responsible for the detected off odours. When microbial numbers reach levels of about 108 cfu/cm2, a visible surface slime on the meat appears. Vacuum and modified-atmosphere packing of meat restrict the growth of pseudomonads, and microflora dominated by Gram-positives, mainly lactic acid bacteria (Lactobacillus, Carnobacterium, Leuconostoc). Meat with high pH (>6.0,) hydrogen sulphide can be produced by Shewanella putrefaciens, and psychrotrophic Enterobacteriaceae. Bigger meat parts with bones, like beef rounds and quarters, are undergo deep spoilage, usually near the bone, where the primary causative agents are Clostridium and Enterococcus. Beef cuts (steaks or roasts) tend to undergo surface spoilage. It is caused either by bacteria or moulds, depends on available moisture. Bacterial spoilage is preferential to mould spoilage in higher humidity. Moulds only tend to predominate in the spoilage of cuts when the surface is too dry for bacterial growth. Visible mould growth is non-existent on ground beef. Exclusively bacteria are responsible for the spoilage of ground meat. The first sign of spoilage of ground beef is the development of off-odours followed by tackiness, which indicate the presence of bacterial slime. Pseudomonas, Alcaligenes, Acinetobacter, Moraxella, and Aeromonas are the most important isolated genera. Those generally agreed to be the primary cause of spoilage are Pseudomonas and Acinetobacter-Moraxella spp., with others playing relatively minor roles in the process. The primary processing of poultry differs from red meat, impacting microbial contamination. Regarding, that the intestinal tract of poultry contain high numbers of pathogens like Salmonella and Campylobacter, the microbial contamination of carcasses by faeces has a high safety threatens. Microbes can be spread between birds by faeces and feathers (i) during transport, (ii) hanging on the plant before killing, (iii) in hot water for scalding, and (iv) mechanical defeathering and evisceration, finally (v) chilling in cooling water. Most of the organisms on poultry are at the surface, and cut-up poultry usually have higher microbial count than whole ones. Salmonella is one of the most important bacterial contaminations of broiler products, which should be checked and controlled. Campylobacter jejuni, the other bacterium causes foodborne diseases in high ratio, is also often found on poultry carcasses. It must be mentioned, that C. jejuni found less often on turkey products than salmonellae. Enterococci are also common on poultry products. The genera of Enterobacteriaceae (E. coli, Enterobacter spp.), and different other bacteria, like Corynebacterium spp., Micrococcus, C. perfringens, S. aureus, L. monocytogenes may also responsible for the different foodborne diseases connected to poultry products. Several yeasts were detected on broiler carcasses stored at 4◦C. The most predominant genus was Candida, followed by Cryptococcus and Yarrowia. The most characteristic feature of poultry spoilage is the appearance of slimes at the outer surfaces of the carcass or cuts. The visceral cavity often displays sour. As poultry undergoes spoilage, off-odors are generally noted before sliminess. The primary spoilage organisms usually belong to the genus Pseudomonas if poultry meats undergo low-temperature spoilage. Genera of Acinetobacter, Flavobacterium, Corynebacterium and yeasts are also common, while Enterobacteriaceae and others can be isolated in lower numbers. Fungi have less importance in poultry spoilage. The poultry spoilage is mainly restricted to the surfaces. The spoilage contaminates the surfaces and hides from water, processing, and handling. The surfaces of fresh poultry are susceptible to the growth of aerobic bacteria such as pseudomonads, which grow well on the surfaces, and form biofilm. The inner portions of poultry tissue are generally sterile, or contain relatively few organisms, which generally do not grow at low temperatures. Chicken breast muscle spoils differently than leg muscles because the pH of the former is typically in the pH range of 5.7–5.9 while the latter is higher (6.3–6.6). The biota of chicken leg muscle stored at low temperature usually contains pseudomonads, Acinetobacter-Moraxella, S. putrefaciens. The latter one produces H2S, methyl mercaptan, and dimethyl sulphide, which are result a sulphide like odours. It is not characteristic in the spoilage of chicken breast muscles. Cured, smoked, or cooked meat products are called processed meats. Spoilage is most frequently occurs on sausages and frankfurters. These products have not only meat components, but spices, which are additional sources of microbes. Milk solids are also added to some products, which are contribute to the lactic acid bacteria and yeasts contamination and proliferation. In the case of pork sausage, natural casings have been shown to contain large numbers of bacteria. The microbiota of frankfurters consists largely of Gram-positive organisms (micrococci, bacilli, lactobacilli, microbacteria, enterococci, leuconostocs) along with yeast. Sausage usually contains a more varied biota than most other processed meats due to the different seasoning agents with typical contamination microbiota. Spoilage of these products is generally of three types: (i) sliminess, (ii) souring, and (iii) greening. Slimy spoilage (i) occurs on the outside of sausages (especially frankfurters), and first may be seen as discrete colonies, which may later form a uniform layer of grey slime, composed of yeasts and lactic acid bacteria (Lactobacillus, Enterococcus, Weissella, B. thermosphacta). Brochothrix thermosphacta has been found by many investigators to be the most predominant spoilage. Moist surface enhance slime formation. Souring (ii) results from the growth of lactobacilli, enterococci, and related organisms generally underneath the casing of these meats and. The usual sources of these organisms to processed meats are milk solids. The souring results from the utilization of lactose and other sugars by the organisms and the production of acids. Two types of greening (iii) occur on processed red meats: one caused by H2O2 and the other by H2S. The former generally appears after an anaerobically stored meat product (frankfurters, vacuum packaged or other cured meats) is exposed to air. The forming H2O2 reacts with nitrosohemochrome results a greenish oxidized porphyrin. H2O2 may also be accumulated from growth of causative organisms (mainly Weissella viridescens, but also Enterococcus faecium, Enterococcus faecalis) in the interior core, where the oxidation–reduction (Eh) potential is low. Other microbes, like Lactobacillus fructivorans and Lactobacillus jensenii can even produce H2O2. In spite of the discoloration, the green product is not known to be harmful if eaten. The second type of greening is caused by H2S production. H2S reacts with myoglobin to form sulphmyoglobin. This type of greening does not usually occur when meat pH is below 6.0. H2S-producing lactobacilli were isolated from vacuumpackaged beef. Bacon and smoked hams are preparing with smoking and brining, make them relatively insusceptible to spoilage by most bacteria. The most common form of bacon spoilage is mouldiness, which may be due to Aspergillus, Alternaria, Fusarium, Mucor, Rhizopus, Botrytis, Penicillium, and other moulds. Vacuum-packaged bacon however may undergo souring due primarily to micrococci and lactobacilli. Fish and Shellfish Although fish has similar composition and structure to meat, it has several distinctive features, contribute to the unique perishability of fish flesh. The fatty fish has high proportion of polyunsaturated fatty acids, which are more susceptible to the development of oxidative rancidity. In most cases though, spoilage is microbiological in origin. Fish flesh naturally contains very low levels of carbohydrate and these are further depleted during the death struggle of the fish, limiting the degree of post mortem acidification of the tissues so that the ultimate pH of the muscle is relatively high: 6.2–6.5. In the absence of carbohydrate, microbes will immediately use the assimilated nitrogenous materials, producing off-odours and flavours far sooner. Elasmobranchs (dogfish and shark) contain high levels of urea, from which bacterial urease rapidly produce ammonia. The muscle and internal organs of healthy, freshly caught fish are usually sterile. The microbial biota of fish is found generally in three places: the outer slime, gills, and the intestines. Fresh or warm-water fish tend to have a biota that is composed of more mesophilic Gram-positive bacteria than cold-water fish, which tends to be largely Gram-negative. The skin, gills carry substantial numbers (102–109 cfu/cm2) of bacteria. These are mainly Gram-negatives (Pseudomonas, Shewanella, Psychrobacter, Vibrio, Flavobacterium, Cytophaga), but some Grampositives (coryneforms and micrococci) may also be exist. The microflora of fish from northern temperate waters is predominantly psychrotrophic or psychrophilic. Several foodborne illnesses are associated with fish: Vibrio cholera, Vibrio parahaemolyticus, Vibrio vulnificus, Clostridium botulinum Type E, enteric viruses (noroviruses, rotaviruses), scombroid fish poisoning, paralytic shellfish poisoning. Regarding the spoilage of fish, fresh iced or cooled fish are spoiled by bacteria, whereas salted and dried fish are more likely to undergo fungal spoilage. Psychotropic Gram-negative, asporogenous rods (Pseudomonas and Acinetobacter-Moraxella types) are dominating in the bacterial biota of spoiling fish. The earliest signs of organoleptic spoilage may be noted by examining the gills, the most susceptible part of fish for the presence of off-odours. Crustaceans spoil rapidly that is crabs and lobsters by keeping alive until cooking, however this is not possible with shrimp or prawns. Shrimp are often contaminated with bacteria from the mud trawled up with them and are therefore subject to rapid microbiological deterioration following capture. Public health problems associated with shellfish arise mainly not from spoilage, but more from their ability to concentrate viruses and bacteria from surrounding waters, with frequent pollution of sewage, and the practice of consuming many shellfish raw or after relatively mild cooking. The microbiota of shellfish is depending on the quality of the water from which these fish are taken and the quality of wash water and other factors. The following genera of bacteria have been recovered from spoiled oysters: Serratia, Pseudomonas, Proteus, Clostridium, Bacillus, Escherichia, Enterobacter, Pseudoalteromonas, Shewanella, Lactobacillus, Flavobacterium. The late stage of spoilage is dominated by Micrococcus, and Pseudomonas and Acinetobacter-Moraxella spp., with enterococci, lactobacilli, and yeasts. Due to the relatively high level of glycogen, the spoilage of molluscan shellfish is basically fermentative. EGGS The hen’s egg normally is well protected by its intrinsic parameters. Externally, the outer waxy shell membrane, the shell, and the inner shell membrane provide effective physical barrier, retarding the entry of microorganisms. Internally, lysozyme content has antimicrobial effect against Gram-positive bacteria. Egg white also contains avidin, which forms a complex with biotin, and conalbumin, which forms a complex with iron thereby making these components unavailable to microorganisms. In addition, egg white has a high pH (~ 9.3). On the other hand, the nutrient content of the yolk material and its pH in fresh eggs (~ 6.8) make it an excellent source of growth for most microorganisms. Freshly laid eggs are generally sterile. However, numerous microorganisms may be found on the outside and, under the proper conditions, may enter eggs, grow, and cause spoilage. The entry of microorganisms into whole eggs is favoured by high humidity. Bacteria grow rapidly in the nutritious medium of yolk, producing by-products of protein and amino acid metabolism such as H2S and other foul-smelling compounds. The effect of significant growth is to cause the yolk to become “runny” and discoloured. Moulds generally multiply first in the region of the air sac, where oxygen favours their growth; however in high humidity, moulds may be seen growing over the outer surface of eggs. Several bacterial species (Pseudomonas, Acinetobacter, Proteus, Aeromonas, Alcaligenes, Escherichia, Micrococcus, Salmonella, Serratia, Enterobacter, Flavobacterium, Staphylococcus), moulds (Mucor, Penicillium, Hormodendron, Cladosporium), and yeast (Torula) have been found in eggs. The most common bacterial spoilage of eggs is a rotting. The different forms are mainly characterized by different colours: green rot (caused by Pseudomonas spp., especially P. fluorescens) black rot (caused by Proteus, Pseudomonas, and Aeromonas), pink rot (caused by Pseudomonas), and red rot (caused by Serratia spp). Pseudomonas, Acinetobacter, and other species may cause colourless rots, while the “custard” rots is caused by Proteus vulgaris and P. intermedium. Mould spoilage with mycelial growth inside the eggs can be detected during candling. The most common causes of fungal rotting in eggs are Penicillium and Cladosporium spp. 5. MICROORGANISMS IN PLANT PRODUCTS Plants provide a considerable part of human food consumption, and not only seeds and fruits, but also root, tuber, stem, leaves, even flowers are used, depending on the particular plant species. Nowadays wide range of plants is cultivated in a large scale. They must be stored for long periods following harvest and may be transported for distant countries. Microbiological problems may occur in the field, where plant pathogens, mainly fungi may colonize them resulting yield loss, decreased quality, and may produce toxic metabolites. Several microorganisms may cause post-harvest spoilage as well. Plants have different physical and chemical barriers to prevent microbial invasion of their tissues. Cuticle is a tough, resistant layer on the outer surface is part of the physical barrier like pericarp or wax and peels on the surface of the fruits. Plant tissues may also contain different antimicrobial agents, like polyphenols (e.g. lignin), essential oils or produce phenolic phytoalexins (e.g. pisatin in peas), in response to the initiation of microbial invasion. The low pH of the fruit tissues provides considerable protection against bacteria. In contrast, many vegetables have higher pH and may be susceptible to bacterial spoilage. The low water activity of cereals, pulses, nuts and oilseeds are restrict the spoilage flora to xerophilic and xerotolerant fungi, in case of using appropriate post-harvest technology (drying and storage). Cereals and bakery products One of the most important sources of carbohydrates in the human diet are cereals, the seed of the grass (Gramineae) family. Wheat, rice and maize are by far the most important cereal crops. The microbial biota of wheat, rye, corn, and related products may be expected to be that of soil, storage environments, and those picked up during the processing of these commodities. Although these products are high in proteins and carbohydrates, their low aw is restricting the growth of all microorganisms if stored properly. If it has higher aw growth of aerobic spore former bacteria (Bacillus genus) and moulds of several genera are usually the only ones that develop. The aerobic spore former bacteria are able to utilize flour by producing amylase. With less moisture, mould growth occurs and may be seen as typical mycelial growth and spore formation. Members of the genus Rhizopus are common and may be recognized by their black spores. The microbiota of cereals, are dominated by the moulds during growth, harvest and storage. They are grouped into two ecological groups: (1) field fungi, and (2) storage fungi. Field fungi require relatively high water activities (aw) for optimum growth, and are well adapted to the rapidly changing conditions on the surfaces of senescing plant material in the field. The fungal species of the Fusarium, Cladosporium, Alternaria and Epicoccum genera belong to that group. The most important fungi on stored cereals with lower aw are belonging to Penicillium and Aspergillus genera. Water activity and temperature are the most important environmental factors influencing the mould spoilage of cereals, and the possible production of mycotoxins. Although xerophilic moulds may grow very slowly at the lower limit of their water activity range (aw = 0.71), if they start growing, an autocatalytic process with producing water and heat during their respiration leading to the rise of the local water activity, allowing them a more rapid growth. Mould growth on cereals leading to a sequence of changing starting with a decrease in germination potential of the grain following by discolouration, the production of mould metabolites (e.g. mycotoxins), increase in temperature (by self-heating), the production of musty odours and caking. Finally the complete decay with the growth of a wide range of microorganisms can be observed. The storage of high moisture cereals, such as barley, can be with the help of enhancing lactic acid fermentation comparable to the making of silage, or by the careful addition of fatty acids such as propionic acid. If this process is not carried out carefully, Aspergillus flavus may produce aflatoxin B1. If to dairy is fed by this contaminated feed, aflatoxin M1 may be secreted in the milk. Properly handled bread generally lacks sufficient amounts of moisture to allow for the growth of microbes, except moulds, but storage of bread under conditions of low humidity retards mould growth. Rhizopus stolonifer, often referred to as the “bread mould” is one of the most common, and this type of spoilage is generally seen only when bread is stored at high humidity or when wrapped while still warm. The rope spoilage of bread is caused by the growth and amylase production of certain strains of Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, and isolates of Bacillus clausii and Bacillus firmus. Those bacteria are originated from the flour. Its growth is favoured by holding the dough for sufficient periods of time at suitable temperatures. Cakes rarely undergo bacterial spoilage due to their high concentrations of sugars, which restrict the availability of water. The most common form of spoilages of these products is caused by mould. Common sources of spoilage moulds are cake ingredients, especially sugar, nuts, and spices. Although the baking process is generally to destroy moulds, it can be infected with toppings, or from handling and from the air. Growth of moulds on the surface of cakes is favoured by high humidity. On some fruitcakes, growth often originates underneath nuts and fruits if they are placed on the surface of such products after baking. Growth of moulds may continue on breads and cakes results in a hardening of the products. Oil rich seeds Groundnuts, soya beans, rapeseed, sunflower seed and different edible nuts are rich in oil. They have higher water activity at a given water content than cereals. For example groundnuts with 7.2% water content have a water activity of about 0.65–0.7 at 25 °C, while in cereals shows same water activity only at about 12% water activity. At moisture contents greater than 7.2% moulds can grow on oil rich seeds, producing mycotoxins (most notably aflatoxins), and different degrading enzymes. Lipolytic enzymes are produced by different species of Aspergillus (A. niger, A. tamari), Penicillium, Paecilomyces, and Rhizopus species. The strong lipolytic activity of moulds increases the free fatty acid content, leading to enhanced rancidity of the extracted oils. Nutmeats, such as pecan, walnuts, filberts, almonds, pistachios and hickory nuts has extremely high fat and low water content, consequently these products are quite refractory to spoilage bacteria, but moulds can and do grow on them if they are stored under conditions that permit sufficient moisture to be picked up. Common genera are Aspergillus, Rhizopus and Penicillium, and that nuts with fungi are usually colonized by several fungi rather than by single species. Moulds of many genera are picked up by the products during collecting, cracking, sorting, and packaging, so harvest or postharvest handling has a major influence on nut mycoflora. Moulds are often introduced by insect cracking. Cereals and seeds with high oil content must be stored with safe water content, and in low humidity atmosphere, as water would transfer from the high relative humidity gas phase to the food. Consequently water activity may increase in stored seeds slowly. Water condensation may occur more rapidly on surfaces, providing water for germination of mould propagules (conidia) and growth of them. Once the micro-organisms become physiologically active, the water content further increase, being the end product of respiration. Finally micro-organisms requiring a high aw will able to grow, as a result of the increasing water activity, and spoil the food which was initially considered to be microbiologically stable. Temperature differentials in the large storage facilities, like silos, would result similar spoilage. The water content of the air is depending on temperature. If one side of a silo heats up (e.g. exposure to the sun during the day), the relative humidity on that side is reduced, so water molecules migrate from the cooler side to reequilibrate the relative humidity. However, when the same side cools down again (e.g. during night), the relative humidity increases in the cooler air, and local water condensation increase water activity and initialize growth of moulds and subsequent spoilage of the grain. Sugars, Candies, and Spices These products lack of sufficient moisture for growth, consequently rarely undergo microbial spoilage if properly prepared, processed, and stored. However the microbial contamination of cane and beet sugars with bacteria of the genera Bacillus, Paenibacillus, and Clostridium may sometimes cause trouble in the canning industry. The osmophilic strains of Saccharomyces (Zygosaccharomyces spp.), and “Torula” (Candida utilis, former name: Torula utilis) and have been reported to cause inversion of sugar in high-moisture sugars. One of the most troublesome organisms in sugar refineries is Leuconostoc mesenteroides, which synthesizes a dextran. This gummy and slimy polymer sometimes clogs the lines and pipes through which sucrose solutions pass. Fruits and Fruit Products Despite most fruits have high water activity, which is ideal for all microbes, the low pH leads to their spoilage being dominated by fungi, mainly moulds, except of pears, which sometimes undergo Erwinia rot. A variety of yeast genera can usually be found on fruits. Fruits hardly have any natural openings (like lenticels, stomata nectaries on other plant parts), so microbial colonization usually occurs through external damage such as cracks, and punctures. Microbial colonization through wounds and lesion development can be relatively rapid, accompanied proliferation of spoilage microorganisms. Inadequate culling of damaged fruits even with asymptomatic lesions may result serious loss during storage by dangerous wound pathogens, like Penicillium expansum and Botrytis cinerea. Those fungi degrade the wound sites, create lesions, and cross-contaminate adjacent fruits. Yeast often bring about the spoilage of fruit products, especially in the field. Many types of yeast are capable of fermenting the sugars in fruits with the production of alcohol and carbon dioxide. Due to their generally faster growth rate than moulds, they generally precede the latter organisms in the spoilage process of fruits in certain circumstances. The high-molecular-weight constituents of fruits are destructed by moulds. Many moulds are capable of utilizing alcohols, but these organisms proceed to destroy the structural polysaccharides and rinds of the fruits. Each fruit has their specific spoilage microflora. Penicillium italicum and P. digitatum grow on citrus fruits (e.g. oranges and lemons), while Penicillium expansum grow on apples and causes a soft and pale brown rot. The later one is able to produce the mycotoxin patulin, detected apple juices and in cider. Venturia inaequalis the common diseases of apple infect fruits on orchard and causing scrub. Scrubs are reducing commercial value of the fruit, but do not cause extensive rotting of the tissue. The brown rot, caused by Monilia fructigena, however, can lead to extensive rotting either during storage, or even on the tree. The fungal colonies are visible as greyish brown powdery pustules forming rings on the surface of the fruit. Apparently healthy, but infected fruit is finally transformed into a shiny black mummified structure. Botrytis cinerea is able to infest several fruits (grape, strawberry, raspberry, etc.), causing grey rot. This fungus is also an ascomycete, similarly to the previous mentioned Penicillium, Aspergillus, and Monilia species. B. cinerea infection deteriorates wine quality. However infecting matured grapes may enhance sugar content and producing special flavours in berries under special circumstances (e.g. in the Tokaj wine region - Hungary, or in Sauternes - France). Under these circumstances the fungus has been referred to as the noble rot (Pourriture Noble –France, aszú –Hungarian). To avoid excessive mould spoilage of harvested fruit during storage, (i) fruits should be harvested in the right stage of maturity, and transport it is necessary to avoid damage. Fruits with visible mould infection should be removed and destroyed. Good hygiene of containers and packaging equipment is also essential to prevent a build-up of mould propagules. The usage of different pesticides during transportation is widespread during long-time transportation. The combined controlling with cold temperature and modified atmosphere (increased carbon dioxide concentration) is also useful and widespread in mould spoilage during storage and transport. However appropriate conditions need to be established for each fruit, as they have different sensitivity toward low temperatures and enhanced CO2 levels. Low pH of fruits allows the relatively low heat treatment of canned fruits. The ascospores of some members of the Eurotiales are sufficiently heat resistant to survive. Byssochlamys species are important spoilage fungi in pasteurized and canned fruits contaminated soil origin. Their ascospores tolerate 85°C, and their vegetative form (mycelia) can grow under very low oxygen tension. They also produce pectinolytic enzymes, and mycotoxins (e.g. patulin). Vegetables and Vegetable Products The incidence of microorganisms in vegetables reflects the microbiological condition of the raw product at the time of processing and the sanitary quality of the processing steps. Different fresh vegetables have been reported to contain aerobic plate count 106-107 cfu/g, and coliform number often reached 105-106 cfu/g. Lactic acid cocci are associated with many raw and processed vegetables. The nutrient content, vegetables of are capable of supporting the growth of moulds, yeasts, and bacteria. Consequently any or all of these organisms may cause its spoilage. The higher water content (~ 88%) of vegetables favours the growth of spoilage bacteria, and the relatively low carbohydrate (< 10%, except corn, onion, peas, potatoes, sweet potatoes) and fat contents (< 1%, except corn), suggest that much of this water is in available form. The relatively high oxidation–reduction potential of vegetables favours the growth aerobic and facultative anaerobic microbes. The tissues of the vegetable have higher pH values, which provide ideal circumstances for not only fungal, but also bacterial invasion. The usually species with pectinolytic activity of the Gram-negative Pectobacterium, Pseudomonas, Erwinia and Xanthomonas bacteria are involved. However pectinolytic strains of the Gram-positive, spore forming Clostridium can also be important in the spoilage of potatoes. The common spoilage pattern displayed by these organisms is referred to as bacterial soft rot. Many of the post-harvest spoilage microbes are also known as plant pathogens. Phytophthora infestans causes potato blight. It may also cause a rot of the tubers during storage, or a new cycle of disease in the next season’s crop. However, the most frequent agents of spoilage of vegetables are opportunistic micro-organisms, and the plant pathogens. Those may enter through wounds (cracks, insect damage or lesions caused by plant pathogens) into the plant tissue. The natural surface flora of the freshly harvested vegetables contains low numbers of pectinolytic bacteria, and endophytic microorganisms. Harvest is a stress for the plant tissues, as a result of water loss and wilting. Moreover nutrients may release through the wounds, providing nutrients for the microbial growth. This stress may also change the microbial activity of the endophytic microflora. The bacterium Erwinia carotovora subsp. carotovora is a highly effective spoilage microbe that causes soft rot across a broad host range of vegetables and some fruits. One of six known genera of soft-rot bacteria (including Xanthomonas, Pseudomonas, Clostridium, Cytophaga, and Bacillus), E. carotovora subsp. carotovora is one of several species of Erwinia that infect and destroy plant tissues both pre- and postharvest and is the species that causes the greatest damage to harvested vegetables. Soft rot is a form of decay characterized by a watery transparency in infected leafy plant parts Soft rots occur in plants refers to the mushy consistency of the plant or vegetable in contrast to some other spoilage conditions where the product remains firm. The six known genera of softrot bacteria are Erwinia, Xanthomonas, Pseudomonas, Clostridium, Cytophaga, and Bacillus). E. carotovora subsp. carotovora has a broad host range, and causes soft rot on several vegetables and some fruits. Soft-rot erwinia are active only at temperatures of 20◦C and above, but other spoiliging bacteria (i.e., Pseudomonas fluorescens and Pseudomonas viridiflava), can decay plant tissue at temperatures at or below 4◦C. The later ones are referred as fluorescent pseudomonads with broad host range, while Pseudomonas tolaasii causes spoilage of the white mushroom (Agaricus bisporus). P. tolaasii also produces siderophores that fluoresce under ultraviolet light, like P. fluorescens and P. viridiflava. Several distinct enzymes are involved in the degradation of pectin, which is a complex molecule, and is built up from methyl ester of a-1,4-poly-D-galacturonic acid with side chains of Lrhamnose, L-arabinose, D-galactose, D-glucose and D-xylose. The most frequently observed form of spoilage of vegetables is a softening of the tissue due to the pectinolytic activity of microorganisms. When the outer plant barrier has been destroyed by pectinase producers, other microbes may also enter the plant tissues and degrade the simple carbohydrates. Available nutrients, like simple nitrogenous compounds, vitamins (especially the B-complex group), and minerals provide appropriate circumstances to sustain the growth of the invading organisms until the vegetables have been consumed or destroyed. The malodours that are produced are the direct result of volatile compounds (such as NH3 and volatile acids) produced by the biota. The fungal spoilage may be initiated either preharvest or postharvest. Fungi may cause different rots. Most fungal species are polyphagous (Botrytis cinerea, Gaeotrichum candida, Alternaria alternate), attacking several vegetables, and even fruits. Grey mould rot is caused by Botrytis cinerea, which produces a grey mycelium on several plants in case of high humidity. The optimal temperature for its growth is between and 18-22 °C, but B. cinerea is able to grow even as low as 2 °C. The polyphagous fungus may cause grey rot on decayed areas of asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, celery, tomatoes, endives, globe artichokes, lettuce, rhubarb, cabbage, Brussels sprouts, cauliflower, broccoli, radish, rutabagas, turnips, cucumbers, pumpkin, squash, peppers, and sweet potatoes. It can enter vegetables through cuts and cracks, but it may also invade plant tissues through unbroken skin in case of optimal conditions. Sour rot is caused by Geotrichum candidum on asparagus, onions, garlic, beans (green, lima, and wax), carrots, parsnips, parsley, endives, globe artichokes, lettuce, cabbage, Brussels sprouts, cauliflower, broccoli, radishes, rutabagas, turnips, and tomatoes. The opportunistic pathogen fungus, like other fruit-rotting organisms, is widely distributed in soils and on decaying fruits and vegetables. The fungus is windborne or splash borne, and fruit fly (Drosophila melanogaster) may also carry spores and mycelial fragments on its body, inoculating it through cracks and wounds in healthy fruits and vegetables. Rhizopus stolonifer and other species cause rhizopus soft rot, making vegetables soft and mushy. Vegetables are often covers with its cottony mycelia with small black dots of sporangia. The affected vegetables are beans (green, lima, and wax), carrots, sweet potatoes, potatoes, cabbage, Brussels sprouts, cauliflower, broccoli, radish, rutabagas, turnips, cucumbers, cantaloupes, pumpkins, squash, watermelons, and tomatoes. The fungus is widespread and is disseminated by several ways, including by D. melanogaster. Fruit fly lays its eggs in the growth cracks on various fruits and vegetables. Entry usually occurs through wounds and other skin breaks. Phytophthora rot, caused by different Phytophthora spp., occurs mainly in the field as blight and fruit rot of market vegetables. It affects different plants in different ways. Among the vegetables affected are asparagus, onions, garlic, cantaloupes, watermelons, tomatoes, eggplants, potatoes and peppers. Anthracnose is characterized by dark, sunken lesions on leaves, fruit, or seed pods. It is caused by Colletotrichum species. These fungi are considered weak plant pathogens. Their spread is favored by warm, wet, weather. Among the vegetables affected are beans, cucumbers, watermelons, pumpkins, squash, tomatoes, and peppers. The prevention of spoilage during storage and transport of vegetables includes control of the relative humidity and the composition of the atmosphere. The presence of free water on the surfaces of vegetables achieved by temperature control is very important to prevent condensation of water. Phytopathogenic and spoilig bacteria cannot invade intact vegetable tissues, but water on the surface will allow the motile bacteria such as Erwinia, Pectobacterium and pseudomonads to reach cracks, wounds and natural openings, like stomata. On the other hand relative humidity of the surrounding atmosphere however should not be below 90–95%, otherwise the loss of water from vegetable tissues will lead to wilting. A combination of constant low temperature, controlled relative humidity, and a gas phase with reduced oxygen and enhanced CO2 has made it possible to store different vegetables even for several months, resulting the continuous production of these vegetables virtually independent of the seasons. Public health concern is from the contamination vegetables with enteric pathogens, such as salmonella, enterohemorrhagic, or verotoxin producing Escherichia coli (EHEC), and shigella. The transmission of vegetables is possible by direct contamination from farmworkers, the faeces of birds and animals, the use of manure or sewage sludge as fertilizer, or the use of contaminated irrigation water. Vegetables using as salad usually are not cooked before consumption, so it is very important to avoid their contamination during production (e.g. following good agricultural practices). Contamination can be reduced by washing with clean water. Mainly vegetables which edible parts are in soil (celery, carrot), or close to the soil (lettuce, endive, cabbage) and different sprouts have been associated Salmonella infections, including typhoid and paratyphoid fevers, and EHEC. Beyond these microbes with faecal origin, Clostridium botulinum endospores with soil origin also may contaminate vegetables. Products contaminated with soil can be assumed to be contaminated with C. botulinum endospores as well. This would not present a problem in case processing or storage conditions allow spore germination, growth and production of botulinum neurotoxin. It may happen with underprocessed, homemade canned vegetables and mushrooms, contaminated with soils. Special attention must take in case of sealed, vacuum or modifiedatmosphere packs of prepared salads with partly cooked ingredients. Heat treatment activates spore germination and the reduced numbers of the potential competitors, could pose particular problems. Similar risks may occur in film-wrapped mushrooms. Adequate refrigeration appears to be the most effective safety factor, as higher temperature is necessary for botulinum toxin production. Listeria monocytogenes, a psychrotrophic species associated with plant material, soil, animals, sewage and a wide range of other environmental sources has also been associated with different foods prepared from vegetables (coleslaw, chopped cabbage, lettuce and other salad vegetables). Neither low temperatures, nor modified-atmospheres prevent its proliferation. Yersinia enterocolitica and Aeromonas hydrophila are also psychrotrophic organisms which are readily associated with vegetables and could grow to levels capable of causing illness if care is not taken during the growth. The production of pre-cut packaged, ready-to-use (RTU), or ready-to-eat (RTE) fruit and vegetable salads has increased in the past decades. RTU fresh-cut fruits are melon chunks and slices; cored and sliced pineapple; de-capped strawberry; de-stemmed and washed grapes; apple wedges; peeled citrus fruits and segments; sliced kiwifruits, and fruit salads. The RTU vegetables are shredded lettuce, shredded and diced cabbage, washed and trimmed spinach, peeled “baby” carrots, cauliflower and broccoli florets, sliced tomatoes, peeled and sliced potatoes, snapped green beans, trimmed green onions, cleaned and diced onions, and mixed salads. Fresh-cut RTUs are 100% usable product, and always requires processing, refrigeration, and packaging, specifically modified atmosphere (<0.1% CO2, 20.9% O2, 78% N2). RTU products are no microbe-free. Intact vegetables are washed, typically with water that contains chlorine, which reduces microbial numbers. The following cutting and packaging has the potential to recontaminate them. Moreover, the fresh cut vegetables provide a higher level of moisture, more simple nutrients, and a higher surface area, all of which make the RTU product more susceptible to microbial growth than the original. Pseudomonas spp. and Erwinia spp. the two most common Gram-negative spoilage microbe associated with fresh-cut vegetables. Several Gram-positive bacteria, most notably the lactic acid bacteria, have also been isolated from spoilage of fresh-cut fruits and vegetables that are packaged under MA, and stored at 7◦C or above. Fungi, including yeasts and moulds may also cause spoilage of RTU fruits and vegetables. Among yeasts, Saccharomyces, Candida, Torulopsis, and Hansenula, Rhodotorula and Zygosaccharomyces genera have been isolated widely from RTU fruits and vegetable products. Mould spoilage of RTU produce, especially fresh fruit, is caused by species of Penicillium, Phytophthora, Alternaria, Botrytis, Fusarium, Cladosporium, Phoma, Trichoderma, Aspergillus, Alternaria, Rhizopus, Aureobasidium, and Colletotrichum. The symptoms include visible growth, rots and discoloration, such as blue mould rot, grey rot, etc. RTUs are packed in low-O2 permeable packaging, to prevent enzymatic browning. However, it increases the possibility for the growth of microbial pathogens such as C. botulinum and L. monocytogenes. Seed sprouts are produced by the germination of certain plant seeds (e.g., alfalfa, radish, clover, mung beans). Although they are popular, these products have been the vehicle for a number of foodborne illness outbreaks. Since germinating seeds are rich in simple nutrients (to nourish the young plant until it can make its own food by photosynthesis), seed sprouts may contain high numbers (106-1011 cfu/g) of microorganisms, especially bacteria. A number of moulds and yeasts have also been found on sprouts. Certain foodborne pathogens (Salmonella, E. coli 0157:H7) are even enter to vegetable plants from the time of seed germination. Since sprouts are eaten without being heated or cooked, problems arise when the sprout seed stock contains foodborne pathogens such as Salmonella or E. coli 0157:H7. Finally it has to be mentioned, that intestinal viruses are also common on fresh produce, and they often originate from the contaminated wash water used. Among the most common are the noroviruses. Hepatitis A and E along with rotavirus and astrorvirus may be expected when polluted water is used as is the case with any gastrointestinal illness agent. REFERENCES ADAMS, M.R. - MOSS, M.O. (2008): Food microbiology Third Edition. The Royal Society of Chemistry, 478 p. BALLA CS. - SIRÓ I. (2007): Élelmiszer-biztonság és -minőség I. Alapismeretek. Mezőgazda Kiadó, Budapest. 318 p. BANIA, J. - DABROWSKA, A. - BYSTRON, J. - KORZEKWA, K. - CHRZANOWSKA, J. - MOLENDA, J. (2006): Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food. International Journal of Food Microbiology. 108. 36-41 p. BAYMAN, P. - BAKER, J.L. - MAHONEY, N.E. (2002): Aspergillus on tree nuts: incidence and associations. Mycopathologia, 155:161-169. BELÁK Á. (2009): Élelmiszer-biztonsági szempontból jelentős baktériumok kimutatása, PCRalapú molekuláris azonosítása és tipizálása. Doktori értekezés 147 p. BERDANIER D.C. - DWZER T.J. - FELDMAN B.E. (2007): Handbook of nutrition and food. CRC Press, 42-43 p., 48 p. BHAT, R. - RAVISHANKAR, R.V. - KARIM, A.A. (2010): Mycotoxins in Food and Feed: Present Status and Future Concerns. Comprehensive Reviews in Food Science and Food Safety, 1:5781. BÍRÓ G. (1995): A listeriosis hazai és nemzetközi élelmiszer-higiéniai kérdései humán epidemiológiai szempontból. Magyar Állatorvosok Lapja, 50. 282-286 p. BIRÓ G. (1993): Élelmiszer-higiénia. Agroinform Kiadó és Nyomda Kft., Budapest. 668 p. BÍRÓ G. (szerk.): 1998. Élelmiszer-higiénia, Budapest, Agroinform Kiadó és Nyomda Kft., 576 p. BIRÓ G. - BIRÓ Gy. (2000): Élelmiszer-biztonság. Táplálkozás-egészségügy. Agroinform Kiadó, Budapest. 396 p. BÍRÓ G. - SZITA G. (2000): Élelmiszermikrobiológia, élelmiszer-higiénia. Agrárszakoktatási Intézet, Budapest. 172 p. BOEREMA, J.A. - CLEMENS, R. - BRIGHTWELL, G. (2006): Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus. International Journal of Food Microbiology. 107. 192–201. p. BRUNS, T. (2006): Evolutionary biology: A kingdom revised. Nature 443, 758-761. CSAPÓ J. - CSAPÓNÉ KISS ZS. (2002): Tej és tejtermékek a táplálkozásban. Mezőgazda Kiadó, Budapest, 464 p. DAWSON, D. (2005): Foodborne protozoan parasites. Int J Food Microbiol. 103:207-27. DEÁK T. (2006): Élelmiszer-mikrobiológia. Mezőgazda Kiadó, Budapest. 382 p. DÉSI I. (2013): Élelmezés higiéne. Egészségtudomány, LVII. évf., 1. 1-6 p. DOYLE, M.P. - BEUCHAT, L.R. - MONTVILLE, T.J. (2013): Food Microbiology: Fundamentals and Frontiers. 4th edition. ASM Press, Washington. 1139 p. DUDUK V. (1995): Állategészségtan. Mezőgazda Kiadó, Budapest, 276 p. EFSA-ECDC (2013): The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2011. The EFSA Journal. 11. 4. 250 p. EFSA-ECDC (2014): The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2012. The EFSA Journal. 12. 2. 312 p. EL KHOURY, A. - ATOUI, A. (2010): Ochratoxin A: General Overview and Actual Molecular Status. Toxins, 2(4), 461–493. FAO/WHO standards programme (1990): Codex Alimentarius Commission. Alinron 91/29 and 93/12. FARKAS J. - MOHÁCSINÉ FARKAS Cs. (2010): Az állattartás körülményei és az állati eredetű élelmiszerek mikrobiológiai biztonsága. Magyar Élelmiszer Tudományi és Technológiai Egyesület és a Központi Élelmiszer Tudomány Kutatóintézet Szakfolyóirata. 64. 1. 6-8.p. FDA (2007): Food Protection Plan http://www.fda.gov/oc/initiatives/advance/food/plan.html FROST, J.A. (2001): Current epidemiological issues in human campylobacteriosis. Journal of Applied Microbiology. 90. 85-90p. FOROUD, N.A. - EUDES, F. (2009): Trichothecenes in Cereal Grains. International Journal of Molecular Sciences, 10(1), 147–173. GARRITY, G.M. - LILBURN, T.G. - COLE, J.R. - HARRISON, S.H. - EUZÉBY, J. - TINDALL, B.J. (2007): Phylum „Proteobacteria”: the Deltaproteobacteria and Epsilonproteobacteria. Taxonomic Outline of Bacteria and Archaea, Release 7.7. 244-270 p. GASPARIKNÉ REICHARDT J. - KROMMER J. - SZABÓ G. - TÓTH SZ. - INCZE K. (2004): Patogén baktériumok előfordulása vágóhidakon és húsfeldolgozó üzemekben II. A Hús. 1. 40-42 p. GERGELY L. (1999): Orvosi mikrobiológia Semmelweis Kiadó, Budapest. 511 p. HARRIGAN, W.F. - PARK, R.W.A. (1994): Biztonságos élelmiszerek előállítása. Mezőgazda Kiadó, Budapest. 154 p. HATA, E. - KATSUDA, K. - KOBAYASHI, H. - OGAWA, T. - ENDO, T. - EGUCHI, M. (2006): Characteristics and epidemiologic genotyping of Staphylococcus aureus isolates from bovine mastitic milk in Hokkaido, Japan. The Journal of Veterinary Medical Science. 68. 165–170. p. HOLT, J.G. - KRIEG, N.R. - SNEATH, P.H.A. - STALEY, J.T. - WILLIAMS, S.T. (2000): Bergey’s Manual of Determinative Bacteriology, 9th Edition, Lipcott William and Wilkins, 41-60 p. JAY, J.M. – Loessner, M.J. – Golden, D.A. (2005): Modern Food Microbiology. Springer Science + Busieness Media, Inc., New York 782 p. JÁVOR A. - JANKÓNÉ FORGÁCS J. - MOLNÁR GY. - FENYVESSY J. - MEZŐSZENTGYÖRGYI D. (2007): Állati termékek feldolgozása II.. Debrecen, 293 p. JØRGENSEN, H.J. - MØRK, T. - CAUGANT, D.A. - KEARNS, A. - RØRVIK, L.M. (2005). Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk. Applied and Environmental Microbiology. 71. 8352–8361. p. JOZWIAK Á. (2009): A Campylobacterek előfordulása az élelmiszerláncban, valamint környezeti tényezők hatása túlélésükre és pusztulásukra. PhD értekezés, 100 p. KATONA F. - PUSZTAI S. (1975): Élelmiszer-higiéniai vizsgálatok állatorvosi laboratóriumokban. Mezőgazdasági Kiadó, Budapest, 216 p. KISS R. - TIRCZKA T. - SZITA G. - BERNÁTH S. - CSIKÓ GY. (2004): Listeria monocytogenes food monitoring data and incidence of human listeriosis in Hungary. International Journal of Food Microbiology, 71-747 p. KOOPMANS, M. - von BONSDORFF, C.-H. - VINJÉ, J. - de MEDICI, D. - MONROE, S. (2002), Foodborne viruses. FEMS Microbiology Reviews, 26: 187–205. KORCZAK, B.M. - STIEBER, R. - ELMER, S. - BURNENS, A.P. - FREY, J. - KUHNERT, P. (2006): Genetic relatedeness within the genus Campylobacter inferred from rpoB sequences. International Journal of Systematic and Evolutionary Microbiology, 56. 937-945 p. KOVÁCS G. - PÁSZTOR M. (2001): Rezisztenciaváltozás a Gram-pozitív kórokozók körében. Infektológia. LAM 11. 3. 190-195. p. KRISZTALOVICS K. - FEHÉR Á. - IVELINA B.D. - BORBÁS K. (2011): A kampilobakteriózis jelentősége. Élelmiszerbiztonság. 1-2. 10-13 p. LACZAY P. (2008): Élelmiszer-higiénia. Élelmiszerlánc-biztonság. Mezőgazda Kiadó, Budapest. 649 p. LAISNEY, M.J.: 2002. Food hygienie. Poultry and game meat zoonosis (Élelmiszer higiénia. Baromfi és vadhús zoonózisok), 100 p. LÁNYI B. (1980): Járványügyi és klinikai bakteriológia. Országos Közegészségügyi Intézet, Budapest, 588 p. LETERTRE, C. - PERELLE, S. - DILASSER, F. - FACH, P. (2003): Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus. Journal of Applied Microbiology 95. 38–43. p. LINA, G. - BOHACH, G.A. - NAIR, S.P. - HIRAMATSU, K. - JOUVIN-MARCHE, E. - MARIUZZA, R. (2004): Standard nomenclature for the superantigens expressed by Staphylococcus. The Journal of Infectious Diseases 189. 2334–2336. p. LINDSTRÖM, M. – HEIKINHEIMO, A. – KORKEALA, H. (2011): Novel insights into the epidemiology of Clostridium perfringens type A food poisoning. Food Microbiology, 28: 192– 198. MÉBIH (2008): Élelmiszer-biztonsági helyzetelemzés és kockázatértékelés. Agroinform Kiadó, Budapest. 238 p. NACHAMKIN, I. (1997) Campylobacter jejuni. In: Doyle, M.P., Beuchat, L.R., Montville, T.J. (eds.) Food Microbiology. Fundamentals and Frontiers, Chapter 9, 159-170 p. NAGY B. (2009): Szalmonellákról és a szalmonellózisokról dióhéjban. Élelmiszer- Biztonság. 7. 2. 12-14.p. OEK (2012): Sárgadinnye által terjesztett listeriosis-járvány az USA 28 államában, 2011. Epinfo 19. évf. 6., 69-75 p. OMOE, K. - IMANISHI, K. - HU, D.L. - KATO, H. - TAKAHASHI-OMOE, H. - NAKANE, A. - UCHIYAMA, T. - SHINAGAWA, K. (2004): Biological properties of staphylococcal enterotoxin-like toxin type R. Infection and Immunity 72. 3664–3667. p. ON, S.L.W. (2001) Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status, future prospects and immediate concerns. Journal of Applied Microbiology, 90. 1-15 p. ORSZÁGOS EPIDEMOLÓGIAI KÖZPONT (2001): Salmonella surveillance, 2000. Epinfo. 8. 50. 19.p. ORSZÁGOS EPIDEMOLÓGIAI KÖZPONT (2004): Salmonella surveillance, 2003. Epinfo. 11. 50. 28.p. ORSZÁGOS EPIDEMOLÓGIAI KÖZPONT (2006): Salmonella - A tojótyúkállományok salmonellafertőzöttsége az Európai Unió tagállamaiban. Epinfo. 13. 25. 1-5.p. ORSZÁGOS EPIDEMOLÓGIAI KÖZPONT (OEK) (2014): Az Országos Epidemiológiai Központ előzetes jelentése a 2013. évben bejelentett fertőző megbetegedésekről. Epinfo. 21. 27. 309326 p. PELES, F. - WAGNER, M. - VARGA, L. - HEIN, I. - RIECK, P. - GUTSER, K. - KERESZTÚRI, P. KARDOS, G. - TURCSÁNYI, I. - BÉRI, B. - SZABÓ, A. (2007): Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary. International Journal of Food Microbiology 118. 2. 186–193. p. PEPE, O. - BLAIOTTA, G. - MOSCHETTI, G. - GRECO, T. - VILLANI, F. (2003): Rope-producing strains of Bacillus spp. from wheat bread and strategy for their control by lactic acid bacteria. Appl. Environ. Microbiol., 69: 2321-2329. PETZINGER, E. – ZIEGLER, K. (2000): Ochratoxin A from a toxicological perspective. Journal of Veterinary Pharmacology and Therapeutics, 23: 91–98. RALOVICH B. (1991): A listeriosis problémáiról. Orvosi Hetilap 132: 809-814 p. RALOVICH B. - KOVÁCS S. (1991): Élelmiszerek-közvetítette kórokozó baktériumok jellemzése Magyarországon I. A megbetegedések megelőzésének lehetőségei. A HÚS. Budapest. 4. 35-38 p. REICHARDT J. (2009): Élelmiszer közvetítette Listeria-megbetegedések előfordulása. A Hús. 3-4. 140 p. RODLER I. (2007): Élelmezés-higiéné Budapest, Medicina Könyvkiadó. 166 p SALI J. - KRISZTALOVICS K. (2010): Zoonózisok 2008-ban az Európai Unióban és Magyarországon. Élelmiszer-Biztonság. 8. 1. 15-17.p. SPERBER, W.H. - DOYLE, M.P. (2010): Compendium of the Microbiological Spoilage of Foods and Beverages, Food Microbiology and Food Safety, DOI 10.1007/978-1-4419-0826-1_6, C Springer Science+Business Media, LLC 367 p. STENFORS ARNESEN, L.P. - FAGERLUND, A. - GRANUM PE. (2008): From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev., 32:579-606. SZABÓ A. (2008): Bevezetés a mezőgazdasági mikrobiológiába. Center-Print Nyomda, Debrecen. 288 p. SZABÓ M. (2009): Élelmiszer-eredetű lisztériafertőzések megelőzése az élelmiszerláncban. A Hús. 3-4. 205-209 p. SZAKÁLY S. (2001): Tejgazdaságtan. Dinasztia Kiadó, Budapest, 478 p. SZALKA Á. (2004): A sertések Escherichia coli okozta betegségei. Mezőhír, 8. 6. 70. p. SZÁZADOS I. (1995): Az emlős vágóállatok húsvizsgálata. Mezőgazda Kiadó, Budapest. 344 p. SZEITZNÉ SZABÓ M. (2008): Mikrobiológiai élelmiszerbiztonság napjainkban. Élelmiszervizsgálati Közlemények, 54. Különszám. 5-6. p. SZEITZNÉ SZABÓ M. – Krisztalovics K. – Sréterné Lancz Zs. – Fehér Á. – Cseh J. (2008): Magyarország mikrobiológiai élelmiszerbiztonsági helyzete. Élelmiszervizsgálati Közlemények, 54. Különszám. 7-42. p. SZEITZNÉ SZABÓ M. (2009): Élelmiszer eredetű lisztériafertőzések megelőzése az élelmiszerláncban. A Hús. 3-4. 105-109 p. SZEITZNÉ SZABÓ M. (2011): Hazai és nemzetközi stratégiai törekvések a kampilobakteriózis csökkentésére. Élelmiszerbiztonság. 1-2. 6 p. SZENT-IVÁNYI T. - MÉSZÁROS J. (1985): A háziállatok fertőző betegségei. Mezőgazdasági Kiadó, Budapest, 522 p. SZLADECSKÓ GY-né. (1998): Mikrobiológia és táplálkozás-élettan. Budapest, Agrárszakoktatási Intézet, 143 p. THOMAS, D.Y. - JARRAUD, S. - LEMERCIER, B. - COZON, G. - ECHASSERIEAU, K. - ETIENNE, J. GOUGEON, M.L. - LINA, G. - VANDENESCH, F. (2006): Staphylococcal enterotoxin-like toxins U2 and V, two new staphylococcal superantigens arising from recombination within the enterotoxin gene cluster. Infection and Immunity. 74. 4724–4734. p. TUBOLY S. (1998): Állatorvosi járványtan I. Mezőgazda Kiadó, Budapest 612 p. VARGA J. (1993): A zoonosisok járványtana. Mezőgazdasági Kiadó, Budapest 420 p. VARGA J. - TUBOLY S. - MÉSZÁROS J. (1999): A háziállatok fertőző betegségei (Állatorvosi járványtan II.) Mezőgazda Kiadó, Budapest 522 p. VITAS A.I. - AGUADO, V. - GARCIA-JALON I. (2004): Occurence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain). International Journal of Food Microbiology, 349-356. p. WELCH, D.F. (1991): Applications of cellular fatty acid analysis. Clin Microbiol Rev., 4: 422–438. World Health Organization, WHO (1998): WHO Surveillance Programme for Control of Foodborne Infections and Intoxications in Europe - Newsletter 57, 5-6. p. World Health Organization, WHO (2000): The increasing incidence of human campylobacteriosis. Report and proceedings of a WHO consultation of experts. Copenhagen, Denmark 21-25. ZHANG, J.C. - SUN, L. - NIE, Q.H. (2010): Botulism, where are we now? Clinical Toxicology, 48, 867–879. YOSHIDA, N. - TYLER, K.M. - LLEWELLYN, M.S. (2011): Invasion mechanisms among emerging food-borne protozoan parasites. Trends in parasitology, 27: 459-466.