Following the ban of stilbene and other hormonal-type growth promoters, interest has focused on alternative compounds for promoting live weight gain in food-producing animals. The beta-adrenergic agonists constitute such group of compounds, clembuterol being the main one. It has been reported that consuming calf liver in Spain and France containing clenbuterol residues has induced muscular tremors, tachycardia, muscular pain, nervousness, headache, vertigo, nausea, vomiting and fever. It has also been used as an anabolizant steroid; clenbuterol is used as a tocolytic in cows, thereby supposing an

A controversy has arisen around these events regarding whether to accept or prohibit using clenbuterol in animal production. This drug increases channel performance, it is not potentially oncogenic or mutagenic and is only embryotoxic in large doses whilst its adverse effects on consumers becomes evident when recommended withdrawal times are not respected and when excessive doses are used, whether through inadequate management or

The foregoing has led to clenbuterol being a highly controlled drug today in many countries which have developed programmes and mechanisms for monitoring it and its follow-up. However, in spite of these controls and warning signs, unfortunate events involving adverse reactions continue to be presented, as happened in November 2005 in Jalisco, México, when about 225 people experienced trembling, headaches and discomfort after having consumed

Arsanylic acid, peptide antibiotics and quinoxaline-1,4-dioxides are non-steroidal compounds used as growth promoters in different animal species. Arsanylic acid and its sodium salt are most commonly used, particularly in pigs. They are also efficacious in the egg-producing industry and were previously approved for use in egg-laying hens. However, their use in animals is generally rather limited and the risk–benefit ratio is questionable because these drugs can produce toxicosis, known as peripheral nerve

aimed at increasing animals' weight gain even more (Brambilla et al., 2007).

beef containing residues of this type (Gojmerac, 2002).

therapeutic doses has led to the induction of hepatic cystitis (Durlinger et al., 2002).

which endocrine effects are produced in animals (Mikus et al., 2001).

illegal practice (Dickson, 2003).

growth promoters are permitted.

additional risk.

demyelination.

Mycotoxins are secondary metabolites from fungi, mainly from the species *Aspergillus*, *Fusarium* and *Penicillium*, aflatoxins, ochratoxins, zearalenone, trichothecenes and fumonisins having been the most studied to date. The foodstuffs fundamentally contaminated by these toxins are grains and cereals constituting the main source of contamination for human beings. However, farm animals consuming contaminated foodstuffs may generate residues in meat, viscera, milk and eggs. Residuality is determined by contamination by high concentrations in foodstuffs ingested by animals, this being very uncommon, and also by theway in which the xenobiotic becomes metabolised in the organism. Mycotoxins do not become totally destroyed during cooking or industrialisation of foodstuffs due to their heat-stability.

The types of mycotoxicosis (disease resulting from consuming mycotoxins) in human beings are mainly chronic. These would include Balkan endemic neuropathy in Russia caused by the consumption of ochratoxin A which generates nephrotoxicosis, alimentary toxic aleukia in the former Soviet Union associated with dermatitis, vomiting and hematopoietic alterations caused by trichothecenes (diacetoxiscirpenol and T-2 toxin), possible endocrinal alterations related to reduced masculine fertility caused by consuming zearalenone (such toxin acting as an xenoestrogen), hepatic cancer caused by aflatoxin B1 and possible esophagic and renal cancer caused by fumonisin and ochratoxin A, respectively. The IARC

Chemical Residues in Animal Food Products: An Issue of Public Health 179

ZEA is frequently implicated in reproductive disorders in animals and occasionally in hyperestrogenism syndromes in humans. ZEA becomes biotransformed in the intestine by the mucosa or bacterial flora and involves the formation of α- and β-zearalenol and α- and β-zearalanol. Alpha-zearalanol and β-zearalenol have greater estrogenic power than ZEA since they bind with greater force to their corresponding receptors (Zinedine et al., 2007). Alpha-zearalanol has been employed as growth promoter in cattle. Studies orientated towards determining residuality through experimentation have suggested that residues are not present in meat or eggs, even at high doses. However, a recent study has show that the presence of α-zearalenol in meat-based foodstuffs for infants reached levels of 30.5 µg/kg; the same study demonstrated the presence of mycoestrogens (ZEA, α-zearalenole and β-

FUM properties suggest that their presence in animal meat does not represent an important source of contamination, since they are poorly absorbed. FUM produce liquefaction of the brain in horse and pulmonary oedema in pigs; ruminants and birds are more resistant. They have been correlated with oesophageal cancer in humans in some parts of the world and it has also been presumed that they may cause neural tube alterations. Their presence has been demonstrated and in the liver and kidneys of turkeys fed with the maximum levels

The main TCT of interest in producing animals are T-2 toxin, HT-2 toxin, diacetoxiscirpenol and deoxinivalenol. The TCT do not usually represent a risk of contamination in food of

Around 75 species of marine micro-algae, belonging to the dinoflagellate group, produce secondary metabolites which represent potent toxins, generically called phycotoxins. These organisms form part of the marine plankton and therefore the aquatic food chain leading to filtrator mollusks, gastropods, crustaceans and fish which can accumulate toxins being

The microalgae population may increase suddenly and generate an algal bloom which has increased in frequency, intensity and geographical distribution during recent years. Amongst the explanations put forward to explain for this phenomena has been the increased use of coastal waters for aquaculture, eutrophication caused by domestic, industrial and agricultural residues, the mobility of trace metals and humic substances due to deforestation and/or acid rain and changes in climatic conditions (Erdner et al., 2008). Reports of phycotoxins poisoning have increased during the last few years, perhaps due to the scientific community's greater knowledge and interest in the matter or due to the increase in algal bloom. Such poisoning is mainly acute course; however, there is interest in evaluating the effects being triggered by chronic consumption. The lack of studies on animals which are continuously exposed to phycotoxins and the scarce availability of certified reference materials have led to difficulties in evaluating risk, developing analytical

Studying toxins produced by algae has classically been approached according to the type of poisoning which they have caused. Four groups of toxins can thus be distinguished, causing paralytic shellfish poisoning, diarrhoeic shellfish poisoning, amnesic shellfish

zearalenole) in infants' cow milk formulas (Meucci et al., 2011).

animal origin due to their rapid metabolism (Bailly & Guerre, 2009).

permitted in Europe (Tardieu et al., 2008).

methodologies and regulating these substances.

**6.2 Phycotoxins**

consumed (FAO, 2004).

has classified aflatoxin B1 within group 1 (proven carcinogenic effect on humans) and fumonisin B1 and ochratoxin A within group 2B (possibly carcinogenic to humans) (IARC, 1993). The evidence from *in vitro* studies has shown that zearalenone is a probably implicated in cancer of the reproductive system (Khosrokhavar et al., 2009).

Aflatoxins and ochratoxin A are the main mycotoxins which can generate residuality and attention concerning them as being animal subproduct contaminants has mainly been focused on their presence in milk; however, it has been demonstrated that they can also generate residuality in meat and eggs.

#### **6.1.1 Aflatoxins**

Aflatoxins (AF) B1 B2 G1 and G2 are produced by fungi from the genera *Aspergillus*. AFB1 may be bioactivated through CYP450 enzymes to become an epoxide which is able to form adducts with DNA, meaning that it has been considered that AFB1 undergoes bioactivation in the organism. AFB1 may also become hydroxylised to AFM1 and be excreted in milk. It has been estimated that 1% to 6% of AFB1 ingested by a milk-producing cow could be excreted as AFM1 in milk, depending on bovine productivity. AFM1, like AFB1, may form an epoxide and alter DNA sequences. IARC is considered to be an AFM1 in group 2B (IARC, 1993). MRL regulated in different countries ranges from 0.05 to 0.5 µg/L; MRL has also been established for AF consumed by ruminants (FAO, 2003). Experimental studies have shown that when animals consume foodstuffs contaminated by high levels of AF, that it is difficult to find naturally, the liver and kidneys are the organs where most toxins become accumulated, and their presence in muscle is scarce (Bailly & Guerre, 2009). These types of studies have also demonstrated the presence of AF in eggs from different avian species.

#### **6.1.2 Ochratoxin A**

Ochratoxin A (OA) is produced by fungi from the genera *Aspergillus* and *Pencillium*, the former being from tropical regions and the latter from temperate regions. OA may thus be widely distributed throughout the world. OA may become biotransformed through hydrolysis reactions in which metabolites become less toxic by the opening of the lactone ring which occurs during bioactivation. Detoxification may occur in ruminants through digestive flora action before absorption, thereby limiting the possibility that OA might be found in milk and/or beef (Bailly & Guerre, 2009). However, a recent study evaluating the presence of OA in cows' milk formulas for infants found contamination in 72% of the samples analysed, levels around 690 ng/L being found (Meucci et al., 2010). It has been shown that OA may become accumulated in pigs' kidneys. In countries such as Denmark, OA levels in these organs are regulated since porcine ochratoxicosis is common.

#### **6.1.3 Fusariotoxins**

The fusariotoxins are mycotoxins which are produced by fungi from the genera *Fusarium*, zearalenone (ZEA), the fumonisins (FUM) and the trichothecenes (TCT) being the most important for public health.

ZEA is frequently implicated in reproductive disorders in animals and occasionally in hyperestrogenism syndromes in humans. ZEA becomes biotransformed in the intestine by the mucosa or bacterial flora and involves the formation of α- and β-zearalenol and α- and β-zearalanol. Alpha-zearalanol and β-zearalenol have greater estrogenic power than ZEA since they bind with greater force to their corresponding receptors (Zinedine et al., 2007). Alpha-zearalanol has been employed as growth promoter in cattle. Studies orientated towards determining residuality through experimentation have suggested that residues are not present in meat or eggs, even at high doses. However, a recent study has show that the presence of α-zearalenol in meat-based foodstuffs for infants reached levels of 30.5 µg/kg; the same study demonstrated the presence of mycoestrogens (ZEA, α-zearalenole and βzearalenole) in infants' cow milk formulas (Meucci et al., 2011).

FUM properties suggest that their presence in animal meat does not represent an important source of contamination, since they are poorly absorbed. FUM produce liquefaction of the brain in horse and pulmonary oedema in pigs; ruminants and birds are more resistant. They have been correlated with oesophageal cancer in humans in some parts of the world and it has also been presumed that they may cause neural tube alterations. Their presence has been demonstrated and in the liver and kidneys of turkeys fed with the maximum levels permitted in Europe (Tardieu et al., 2008).

The main TCT of interest in producing animals are T-2 toxin, HT-2 toxin, diacetoxiscirpenol and deoxinivalenol. The TCT do not usually represent a risk of contamination in food of animal origin due to their rapid metabolism (Bailly & Guerre, 2009).

#### **6.2 Phycotoxins**

178 Public Health – Methodology, Environmental and Systems Issues

has classified aflatoxin B1 within group 1 (proven carcinogenic effect on humans) and fumonisin B1 and ochratoxin A within group 2B (possibly carcinogenic to humans) (IARC, 1993). The evidence from *in vitro* studies has shown that zearalenone is a probably

Aflatoxins and ochratoxin A are the main mycotoxins which can generate residuality and attention concerning them as being animal subproduct contaminants has mainly been focused on their presence in milk; however, it has been demonstrated that they can also

Aflatoxins (AF) B1 B2 G1 and G2 are produced by fungi from the genera *Aspergillus*. AFB1 may be bioactivated through CYP450 enzymes to become an epoxide which is able to form adducts with DNA, meaning that it has been considered that AFB1 undergoes bioactivation in the organism. AFB1 may also become hydroxylised to AFM1 and be excreted in milk. It has been estimated that 1% to 6% of AFB1 ingested by a milk-producing cow could be excreted as AFM1 in milk, depending on bovine productivity. AFM1, like AFB1, may form an epoxide and alter DNA sequences. IARC is considered to be an AFM1 in group 2B (IARC, 1993). MRL regulated in different countries ranges from 0.05 to 0.5 µg/L; MRL has also been established for AF consumed by ruminants (FAO, 2003). Experimental studies have shown that when animals consume foodstuffs contaminated by high levels of AF, that it is difficult to find naturally, the liver and kidneys are the organs where most toxins become accumulated, and their presence in muscle is scarce (Bailly & Guerre, 2009). These types of studies have also demonstrated the presence of AF in eggs from different avian

Ochratoxin A (OA) is produced by fungi from the genera *Aspergillus* and *Pencillium*, the former being from tropical regions and the latter from temperate regions. OA may thus be widely distributed throughout the world. OA may become biotransformed through hydrolysis reactions in which metabolites become less toxic by the opening of the lactone ring which occurs during bioactivation. Detoxification may occur in ruminants through digestive flora action before absorption, thereby limiting the possibility that OA might be found in milk and/or beef (Bailly & Guerre, 2009). However, a recent study evaluating the presence of OA in cows' milk formulas for infants found contamination in 72% of the samples analysed, levels around 690 ng/L being found (Meucci et al., 2010). It has been shown that OA may become accumulated in pigs' kidneys. In countries such as Denmark, OA levels in these organs are regulated since porcine ochratoxicosis is

The fusariotoxins are mycotoxins which are produced by fungi from the genera *Fusarium*, zearalenone (ZEA), the fumonisins (FUM) and the trichothecenes (TCT) being the most

implicated in cancer of the reproductive system (Khosrokhavar et al., 2009).

generate residuality in meat and eggs.

**6.1.1 Aflatoxins**

species.

common.

**6.1.3 Fusariotoxins**

important for public health.

**6.1.2 Ochratoxin A**

Around 75 species of marine micro-algae, belonging to the dinoflagellate group, produce secondary metabolites which represent potent toxins, generically called phycotoxins. These organisms form part of the marine plankton and therefore the aquatic food chain leading to filtrator mollusks, gastropods, crustaceans and fish which can accumulate toxins being consumed (FAO, 2004).

The microalgae population may increase suddenly and generate an algal bloom which has increased in frequency, intensity and geographical distribution during recent years. Amongst the explanations put forward to explain for this phenomena has been the increased use of coastal waters for aquaculture, eutrophication caused by domestic, industrial and agricultural residues, the mobility of trace metals and humic substances due to deforestation and/or acid rain and changes in climatic conditions (Erdner et al., 2008). Reports of phycotoxins poisoning have increased during the last few years, perhaps due to the scientific community's greater knowledge and interest in the matter or due to the increase in algal bloom. Such poisoning is mainly acute course; however, there is interest in evaluating the effects being triggered by chronic consumption. The lack of studies on animals which are continuously exposed to phycotoxins and the scarce availability of certified reference materials have led to difficulties in evaluating risk, developing analytical methodologies and regulating these substances.

Studying toxins produced by algae has classically been approached according to the type of poisoning which they have caused. Four groups of toxins can thus be distinguished, causing paralytic shellfish poisoning, diarrhoeic shellfish poisoning, amnesic shellfish

Chemical Residues in Animal Food Products: An Issue of Public Health 181

This poisoning is also known as domoic acid (DA) poisoning since memory loss is not always present. It was described for the first time in Canada (Prince Edward Island) in 1987 when 105 people became poisoned after consuming blue mussels. There have also been several reports of poisoning involving effects on wild life, demonstrating that the toxin forms part of the food chain; the toxin responsible for this has been DA which is produced

The DA mechanism of action acts on excitatory amino acid receptors (L-glutamate, Laspartate) and/or synaptic transmission. DA activates specific excitatory amino acid L glutamate receptors producing an excessive accumulation of calcium resulting in cell death. The kainate receptor is DA's primary target. Recent interest in DA has been centred on recognising that effects can result following chronic exposure to it at low concentrations,

Intestinal absorption is limited (5%-10% of the dose administered to experimental animals). It has high distribution in the blood compartment and scarcely penetrates the hematoencephalic barrier. There is no evidence that DA may become metabolised. Elimination occurs via the kidneys. Poisoning in humans produces gastroenteritis which may be accompanied by headache, confusion and permanent loss of short-term memory

Neurotoxic shellfish poisoning, which is endemic on the Gulf of México and the eastern coast of Florida, is caused by brevetoxin (BTX) produced by the dinoflagellate *Gymnodinium breve* (synonyms: *Ptychodiscus breve, Karenia brevis*) present in red-tides. This alga has the special feature of being able to form aerosols due to wave action thereby constituting a risk

BTX are liposolube toxins consisting of around 14 different substances, leading to depolarisation opening sodium channels in cell membranes and increasing the inflow of sodium causing persistent and repetitive activation. Symptoms caused by oral exposure to BTX occur within the first 30 minutes to 3 hours after consuming contaminated organisms and include vomiting, diarrhoea, shivering, sweating, conflicting perception of temperature, hypotension, arrhythmias, numbness, paraesthesia of the lips, face and extremities, cramps, bronchoconstriction, paralysis, convulsions and coma. There have been no reports of lethality. Respiratory difficulty and irritation of the mucosa are the most common symptoms

Ciguatera fish poisoning is caused by ciguatoxin (CTX) which is produced by dinoflagellates from the genera *Gambierdiscus.* CTX becomes accumulated through the food chain, from small herbivorous fish up to large carnivorous fish. This poisoning has passed from being a problem limited to insular regions which affected local communities to being a global matter, given the worldwide consumption of seafood and international tourism. This is the most common poisoning caused by seafood and may affect up to 50,000 people annually

given the discovery that chemical route alteration leads to neurological disturbances.

**6.2.3 Amnesic shellfish poisoning**

(FAO, 2004).

of aerial exposure.

by diatoms from the genera *Pseudo-nitzschia*.

**6.2.4 Neurotoxic shellfish poisoning**

when inhalatory exposure occurs.

**6.2.5 Ciguatera fish poisoning**

poisoning and neurotoxic shellfish poisoning (FAO, 2004). Another group of phycotoxins of interest due to their accumulation in sea fish are the ciguatoxins causing ciguatera fish poisoning.

#### **6.2.1 Paralytic shellfish poisoning**

The toxins responsible for this poisoning are mainly produced by algae from the genera *Alexandrium, Gymnodinium* and *Pyrodinium*. Chemically, they correspond to tetrahydropurin molecules, saxitoxin (STX) having the most importance. PSP incidence and geographical distribution has increased since the 1970s; this poisoning was initially confined to temperate waters in Europe, North America and Japan, but is now considered to be worldwide problem (FAO, 2004).

STX becomes rapidly absorbed through the gastrointestinal tract and equally has rapid distribution, metabolism and excretion. STX selectively blocks (and with high affinity) sodium-dependent channels present in nerves, skeletal muscular fibres and most cardiac muscular fibres, thereby reducing or eliminating the action of propagation potential (Etheridge, 2010).

Paralytic shellfish poisoning symptoms begin in human beings within the first 30 minutes following consumption of contaminated foodstuff and the onset of numbness and/or pins and needles around the lips, gradually extending to the face, neck, arms and legs. Headaches, nausea, lack of muscular coordination and, occasionally, temporary blindness occur. There may be paraesthesia in the arms and legs, motor inability and difficulty in talking in moderate cases and paralysis of respiratory muscles leading to death may occur in severe cases.

#### **6.2.2 Diarrhoeic shellfish poisoning**

Diarrhoeic shellfish poisoning toxins are produced by dinoflagellates from the genera *Dinophysis* and *Protoceratium*, having worldwide distribution. Okadaic acid (OA) and its analogues (dinophysis toxins , pectenotoxin and yessotoxin are included within this group. However, yessotoxin and pectenotoxin produce different toxicological effects in experimental animals. Yessotoxin is related to lesions in the cardiac muscle, liver, pancreas and cerebral neurons and pectenotoxin is clearly hepatotoxic. The effect which yessotoxin and pectenotoxin may have on human beings remains unknown (Dominguez et al., 2010).

OA was initially reported in Japan and Europe, areas in which diarrhoeic shellfish poisoning has had greater importance. OA and its analogues are potent protein phosphatase inhibitors (serine/threonine phosphatases PP1 and PP2A) which dephosphorylate molecules closely related to metabolic processes. It has been postulated that OA induces diarrhoea due to an alteration in hydric balance in the intestines via one of the two following mechanisms: stimulating the phosphorylation of proteins controlling sodium secretion in enterocytes or promoting the phosphorylation of intercell binding proteins regulating solute permeability. Diarrhoeic shellfish poisoning is characterised by diarrhoea, nausea, vomiting and, in some cases, abdominal pain which can begin within the first 3 or 12 hours after having consumed contaminated organisms. No lethal effects have been reported concerning human as having been caused by OA or its analogues.

#### **6.2.3 Amnesic shellfish poisoning**

180 Public Health – Methodology, Environmental and Systems Issues

poisoning and neurotoxic shellfish poisoning (FAO, 2004). Another group of phycotoxins of interest due to their accumulation in sea fish are the ciguatoxins causing ciguatera fish

The toxins responsible for this poisoning are mainly produced by algae from the genera *Alexandrium, Gymnodinium* and *Pyrodinium*. Chemically, they correspond to tetrahydropurin molecules, saxitoxin (STX) having the most importance. PSP incidence and geographical distribution has increased since the 1970s; this poisoning was initially confined to temperate waters in Europe, North America and Japan, but is now considered to be worldwide

STX becomes rapidly absorbed through the gastrointestinal tract and equally has rapid distribution, metabolism and excretion. STX selectively blocks (and with high affinity) sodium-dependent channels present in nerves, skeletal muscular fibres and most cardiac muscular fibres, thereby reducing or eliminating the action of propagation potential

Paralytic shellfish poisoning symptoms begin in human beings within the first 30 minutes following consumption of contaminated foodstuff and the onset of numbness and/or pins and needles around the lips, gradually extending to the face, neck, arms and legs. Headaches, nausea, lack of muscular coordination and, occasionally, temporary blindness occur. There may be paraesthesia in the arms and legs, motor inability and difficulty in talking in moderate cases and paralysis of respiratory muscles leading to death may occur in

Diarrhoeic shellfish poisoning toxins are produced by dinoflagellates from the genera *Dinophysis* and *Protoceratium*, having worldwide distribution. Okadaic acid (OA) and its analogues (dinophysis toxins , pectenotoxin and yessotoxin are included within this group. However, yessotoxin and pectenotoxin produce different toxicological effects in experimental animals. Yessotoxin is related to lesions in the cardiac muscle, liver, pancreas and cerebral neurons and pectenotoxin is clearly hepatotoxic. The effect which yessotoxin and pectenotoxin may have on human beings remains unknown (Dominguez

OA was initially reported in Japan and Europe, areas in which diarrhoeic shellfish poisoning has had greater importance. OA and its analogues are potent protein phosphatase inhibitors (serine/threonine phosphatases PP1 and PP2A) which dephosphorylate molecules closely related to metabolic processes. It has been postulated that OA induces diarrhoea due to an alteration in hydric balance in the intestines via one of the two following mechanisms: stimulating the phosphorylation of proteins controlling sodium secretion in enterocytes or promoting the phosphorylation of intercell binding proteins regulating solute permeability. Diarrhoeic shellfish poisoning is characterised by diarrhoea, nausea, vomiting and, in some cases, abdominal pain which can begin within the first 3 or 12 hours after having consumed contaminated organisms. No lethal effects have been reported concerning human as having

poisoning.

problem (FAO, 2004).

(Etheridge, 2010).

severe cases.

et al., 2010).

**6.2.1 Paralytic shellfish poisoning**

**6.2.2 Diarrhoeic shellfish poisoning**

been caused by OA or its analogues.

This poisoning is also known as domoic acid (DA) poisoning since memory loss is not always present. It was described for the first time in Canada (Prince Edward Island) in 1987 when 105 people became poisoned after consuming blue mussels. There have also been several reports of poisoning involving effects on wild life, demonstrating that the toxin forms part of the food chain; the toxin responsible for this has been DA which is produced by diatoms from the genera *Pseudo-nitzschia*.

The DA mechanism of action acts on excitatory amino acid receptors (L-glutamate, Laspartate) and/or synaptic transmission. DA activates specific excitatory amino acid L glutamate receptors producing an excessive accumulation of calcium resulting in cell death. The kainate receptor is DA's primary target. Recent interest in DA has been centred on recognising that effects can result following chronic exposure to it at low concentrations, given the discovery that chemical route alteration leads to neurological disturbances.

Intestinal absorption is limited (5%-10% of the dose administered to experimental animals). It has high distribution in the blood compartment and scarcely penetrates the hematoencephalic barrier. There is no evidence that DA may become metabolised. Elimination occurs via the kidneys. Poisoning in humans produces gastroenteritis which may be accompanied by headache, confusion and permanent loss of short-term memory (FAO, 2004).

#### **6.2.4 Neurotoxic shellfish poisoning**

Neurotoxic shellfish poisoning, which is endemic on the Gulf of México and the eastern coast of Florida, is caused by brevetoxin (BTX) produced by the dinoflagellate *Gymnodinium breve* (synonyms: *Ptychodiscus breve, Karenia brevis*) present in red-tides. This alga has the special feature of being able to form aerosols due to wave action thereby constituting a risk of aerial exposure.

BTX are liposolube toxins consisting of around 14 different substances, leading to depolarisation opening sodium channels in cell membranes and increasing the inflow of sodium causing persistent and repetitive activation. Symptoms caused by oral exposure to BTX occur within the first 30 minutes to 3 hours after consuming contaminated organisms and include vomiting, diarrhoea, shivering, sweating, conflicting perception of temperature, hypotension, arrhythmias, numbness, paraesthesia of the lips, face and extremities, cramps, bronchoconstriction, paralysis, convulsions and coma. There have been no reports of lethality. Respiratory difficulty and irritation of the mucosa are the most common symptoms when inhalatory exposure occurs.

#### **6.2.5 Ciguatera fish poisoning**

Ciguatera fish poisoning is caused by ciguatoxin (CTX) which is produced by dinoflagellates from the genera *Gambierdiscus.* CTX becomes accumulated through the food chain, from small herbivorous fish up to large carnivorous fish. This poisoning has passed from being a problem limited to insular regions which affected local communities to being a global matter, given the worldwide consumption of seafood and international tourism. This is the most common poisoning caused by seafood and may affect up to 50,000 people annually

Chemical Residues in Animal Food Products: An Issue of Public Health 183

probable risk of exposure for humans; however, food of animal origin can also contain pyrrolizidine alkaloids. It has been presumed that more than 6,000 plant species contain AP, mainly those belonging to the families *Asteraceae* or *Compositae* (genera *Senecio* and *Eupatorium*), *Boraginaceae* (genera *Heliotropium* and *Echium*) and *Fabaceae* or *Leguminosae*

APs are heterocyclic compounds which are mainly derived from four necine bases: retronecine, heliotridine, otonecine and platynecine. AP can become enzimatically hydrolised or oxidised; the resulting N-oxide is slightly toxic and may also be found in plants. AP becomes bioactivated to a toxin pyrrole through CYP450 which is electrophilic and unstable and reacts rapidly with endogenous macromolecules (particularly with DNA forming adducts). DNA adducts could be a continuous source of carbon ions originating new adducts, meaning that the total elimination of AP derivates may take months (Edgar et

AP levels in foodstuffs are rarely so significant that they can cause acute diseases; however, low levels and continuous exposure could lead to the presentation of chronic diseases which could hardly be attributable to the toxin in foodstuffs. Pyrroles cause thickening and occlusion of the hepatic vessels resulting in veno-occlusive disease and cirrhosis. They can also alter the pulmonary vessels, causing pulmonary hypertension and congestive cardiac failure. The IARC has classified lasiocarpine, monocrotaline and riddelliine AP within group 2B (probably carcinogenic for humans, given the pertinent evidence regarding how they are

It has been demonstrated that 0.1% to 4% of AP in foodstuffs for lactating cows and sheep could be excreted in milk (Hoogenboom, 2011). The meat and viscera of cattle fed on APrich plants may contain the toxin and its derivates at levels reaching 250 mg/kg in muscle and 2,500 mg/kg in liver (Fletcher et al., 2011). AP can potentially be present in eggs when fowl are fed on AP-rich diets, thereby constituting a risk for human health (Eröksüz et al., 2008). In spite of potential contamination in milk, meat, viscera and eggs, honey is the only foodstuff of animal origin which has been shown to be naturally contaminated with AP; a large number of plants habitually used in apiaries could be a source of significant levels of

Other alkaloids of interest due to their potential residual effect on animal subproducts are the indolizidinics (especially swainsonine) producing lysosomal storage disease and piperidine (coniin and gamma-conicein) which in acute form can produce muscular paralysis and (chronically) teratogenesis (Panter & James, 1990). These last named alkaloids seem to be responsible for coturnism, a human disease which occurs following the consumption of migrating wild European quail; it is characterised by weakness, muscular pain, paralysis of the legs, vomiting and myoglobinuria. It has been postulated that these symptoms occur due to the accumulation of coniin in birds' tissues following the

Glucosinolates, known as mustard oil glucosides as they confer the characteristic flavour on black mustard (*Brassica nigra*), are secondary metabolites produced by plants belonging to the order *Brassicales*, mainly in the family *Brassicaceae (*or *Cruciferae)*. Many plants which are

(genera *Crotalaria*).

al., 2011).

the toxins.

**6.3.2 Glucosinolates**

cancerigens in animals) (Edgar et al., 2011).

consumption of *Conium maculatum* (López & Bianchini, 1999).

(FAO, 2004). CTX are liposoluble toxins having 13 to 14 rings fused in a rigid structure. CTX bind to sodium channels causing them to open during cell membranes' potential repose altering bioenergetic mechanisms. CTX acts on the same receptor as BTX does but with greater affinity (Lehane & Lewis, 2000).

*Gambierdiscus toxicus*, the alga specie most commonly related to CTX production, is distributed throughout the tropical region of the Pacific Ocean, western Indian Ocean and the Caribbean where CFP is endemic. Many coral fish species are involved, including herbivores and carnivores. The latter constitute the main vector for poisoning in humans, particularly *Muraenidae* (moray eels), *Lutjanidae* (snappers), *Carrangidae* (carrangs), *Scombridae* (mackerels) and *Sphyraenidae* (barracuda) (FAO, 2004).

CTX become rapidly absorbed through the intestine and are mainly excreted in the faeces via the bile. The symptoms are gastrointestinal or neurological in nature, the former include vomiting, diarrhoea, nausea and abdominal pain. The neurological symptoms which can begin later include pins and needles in the lips, hand and feet, disturbances in perception of temperature, severe pruritus and fatigue. Some patients can experience pain (muscular, articular and dental) and anxiety. There may be hypotension and bradycardia in severe cases and death may occur, though this is not very common. The neurological symptoms can persist for years in some cases; it seems that the toxin may become accumulated in fat and be released in certain circumstances or also produce an immunological response (Shoemaker et al., 2010).

Other phycotoxins of interest due to their residuality are the azaspiracida*,* discovered in 1998, and whose symptoms resemble those of diarrhoeic shellfish poisoning; the cyclic imins (gymnodimine, spirolids, pinnatoxins, prorocentrolide, spirocentrimine and pteriatoxin), still have no associated effects in humans, but their residuality does interfere with the analytical methodologies used for determining the presence of marine toxins and cianotoxins (nodularin and cylindrospermin) by producing hepatoxicity and inhibiting protein synthesis.

#### **6.3 Phytotoxins**

Plants have secondary metabolites with which they defend themselves from the aggression of herbivorous animals. Many of these are toxic for humans and animals, causing numerous pathologies. Given that the diverse compounds present in plants are degraded during digestion and/or the metabolism of xenobiotics in animals of livestock interest, only some manage to contaminate products of animal origin. Milk is the main subproduct which has been studied in which toxins from plants may be present; however, their presence has also been demonstrated in muscle, viscera and eggs. Regulations regarding these toxins are scarce and are mainly orientated towards their presence in botanical products having pharmacological uses; however, there is growing interest in making advances in this field. The regulating entities have shown the greatest interest in pyrrolizidine alkaloids amongst the plant toxins due to their abundance and proven toxic effects.

#### **6.3.1 Pyrrolizidine alkaloids**

Pyrrolizidine alkaloids (AP) are present in a large variety of plants and are perhaps the most widely distributed toxins. Foodstuffs or botanically-based remedies represent the most

(FAO, 2004). CTX are liposoluble toxins having 13 to 14 rings fused in a rigid structure. CTX bind to sodium channels causing them to open during cell membranes' potential repose altering bioenergetic mechanisms. CTX acts on the same receptor as BTX does but with

*Gambierdiscus toxicus*, the alga specie most commonly related to CTX production, is distributed throughout the tropical region of the Pacific Ocean, western Indian Ocean and the Caribbean where CFP is endemic. Many coral fish species are involved, including herbivores and carnivores. The latter constitute the main vector for poisoning in humans, particularly *Muraenidae* (moray eels), *Lutjanidae* (snappers), *Carrangidae* (carrangs),

CTX become rapidly absorbed through the intestine and are mainly excreted in the faeces via the bile. The symptoms are gastrointestinal or neurological in nature, the former include vomiting, diarrhoea, nausea and abdominal pain. The neurological symptoms which can begin later include pins and needles in the lips, hand and feet, disturbances in perception of temperature, severe pruritus and fatigue. Some patients can experience pain (muscular, articular and dental) and anxiety. There may be hypotension and bradycardia in severe cases and death may occur, though this is not very common. The neurological symptoms can persist for years in some cases; it seems that the toxin may become accumulated in fat and be released in certain circumstances or also produce an immunological response

Other phycotoxins of interest due to their residuality are the azaspiracida*,* discovered in 1998, and whose symptoms resemble those of diarrhoeic shellfish poisoning; the cyclic imins (gymnodimine, spirolids, pinnatoxins, prorocentrolide, spirocentrimine and pteriatoxin), still have no associated effects in humans, but their residuality does interfere with the analytical methodologies used for determining the presence of marine toxins and cianotoxins (nodularin and cylindrospermin) by producing hepatoxicity and inhibiting

Plants have secondary metabolites with which they defend themselves from the aggression of herbivorous animals. Many of these are toxic for humans and animals, causing numerous pathologies. Given that the diverse compounds present in plants are degraded during digestion and/or the metabolism of xenobiotics in animals of livestock interest, only some manage to contaminate products of animal origin. Milk is the main subproduct which has been studied in which toxins from plants may be present; however, their presence has also been demonstrated in muscle, viscera and eggs. Regulations regarding these toxins are scarce and are mainly orientated towards their presence in botanical products having pharmacological uses; however, there is growing interest in making advances in this field. The regulating entities have shown the greatest interest in pyrrolizidine alkaloids amongst

Pyrrolizidine alkaloids (AP) are present in a large variety of plants and are perhaps the most widely distributed toxins. Foodstuffs or botanically-based remedies represent the most

*Scombridae* (mackerels) and *Sphyraenidae* (barracuda) (FAO, 2004).

the plant toxins due to their abundance and proven toxic effects.

greater affinity (Lehane & Lewis, 2000).

(Shoemaker et al., 2010).

protein synthesis.

**6.3 Phytotoxins**

**6.3.1 Pyrrolizidine alkaloids**

probable risk of exposure for humans; however, food of animal origin can also contain pyrrolizidine alkaloids. It has been presumed that more than 6,000 plant species contain AP, mainly those belonging to the families *Asteraceae* or *Compositae* (genera *Senecio* and *Eupatorium*), *Boraginaceae* (genera *Heliotropium* and *Echium*) and *Fabaceae* or *Leguminosae*  (genera *Crotalaria*).

APs are heterocyclic compounds which are mainly derived from four necine bases: retronecine, heliotridine, otonecine and platynecine. AP can become enzimatically hydrolised or oxidised; the resulting N-oxide is slightly toxic and may also be found in plants. AP becomes bioactivated to a toxin pyrrole through CYP450 which is electrophilic and unstable and reacts rapidly with endogenous macromolecules (particularly with DNA forming adducts). DNA adducts could be a continuous source of carbon ions originating new adducts, meaning that the total elimination of AP derivates may take months (Edgar et al., 2011).

AP levels in foodstuffs are rarely so significant that they can cause acute diseases; however, low levels and continuous exposure could lead to the presentation of chronic diseases which could hardly be attributable to the toxin in foodstuffs. Pyrroles cause thickening and occlusion of the hepatic vessels resulting in veno-occlusive disease and cirrhosis. They can also alter the pulmonary vessels, causing pulmonary hypertension and congestive cardiac failure. The IARC has classified lasiocarpine, monocrotaline and riddelliine AP within group 2B (probably carcinogenic for humans, given the pertinent evidence regarding how they are cancerigens in animals) (Edgar et al., 2011).

It has been demonstrated that 0.1% to 4% of AP in foodstuffs for lactating cows and sheep could be excreted in milk (Hoogenboom, 2011). The meat and viscera of cattle fed on APrich plants may contain the toxin and its derivates at levels reaching 250 mg/kg in muscle and 2,500 mg/kg in liver (Fletcher et al., 2011). AP can potentially be present in eggs when fowl are fed on AP-rich diets, thereby constituting a risk for human health (Eröksüz et al., 2008). In spite of potential contamination in milk, meat, viscera and eggs, honey is the only foodstuff of animal origin which has been shown to be naturally contaminated with AP; a large number of plants habitually used in apiaries could be a source of significant levels of the toxins.

Other alkaloids of interest due to their potential residual effect on animal subproducts are the indolizidinics (especially swainsonine) producing lysosomal storage disease and piperidine (coniin and gamma-conicein) which in acute form can produce muscular paralysis and (chronically) teratogenesis (Panter & James, 1990). These last named alkaloids seem to be responsible for coturnism, a human disease which occurs following the consumption of migrating wild European quail; it is characterised by weakness, muscular pain, paralysis of the legs, vomiting and myoglobinuria. It has been postulated that these symptoms occur due to the accumulation of coniin in birds' tissues following the consumption of *Conium maculatum* (López & Bianchini, 1999).

#### **6.3.2 Glucosinolates**

Glucosinolates, known as mustard oil glucosides as they confer the characteristic flavour on black mustard (*Brassica nigra*), are secondary metabolites produced by plants belonging to the order *Brassicales*, mainly in the family *Brassicaceae (*or *Cruciferae)*. Many plants which are

Chemical Residues in Animal Food Products: An Issue of Public Health 185

contaminated by chemical substances could lead to chronic exposure leading to the presentation of diseases lacking an apparent cause and being difficult to diagnose. Foods of animal origin presuppose the risk of contamination, whether from drugs and growth promoters used for optimising livestock production systems, or with biological toxins present in food ingested by animals. It is thus necessary to control these substances in foods,

Sanitary authorities must thus promulgate and ensure compliance with standards and guidelines concerning the production of harmless foodstuffs. Achieving such objective represents a great challenge for underdeveloped and developing countries due to institutional difficulties and the limited availability of equipment and qualified personnel. All nations must make it a priority to try to ensure the safe consumption of foodstuffs by their populations, exercising strict sanitary control aimed to avoid problems of health in the population and preventing the appearance of new problems affecting the development of

Alonso-Amelot, ME.; Castillo, U.; Smith, BL. & Lauren, DR. (1996). Bracken ptaquiloside in

Asociación Española de Farmacéuticos de la Industria. (March 2001). *Validación de Métodos*

Bailly, JD. & Guerre P. (2009). Mycotoxins in Meat and Processed Meat Products In: *Safety of*

Botsoglou, N. & Fletouris, D. (2000). *Drug Residues in Foods, Pharmacology, Food Safety and Analysis,* (First Edition), Marcel Dekker Inc., ISBN: 0-8247-8959-8, New York, USA Blondin, P. & Sirard, M. (1998). Oocyte quality and embryo production in cattle. *Canadian* 27 *Journal of Animal Science,* Vol. 78, (October 1998), pp. 513–516, ISSN 0008-3984 Brambilla, G.; Di Beza, S.; Pietraforte, D.; Minetti, M.; Campanella, L. & Loizzo, A. (2007). *Ex*

Brogden, KA.; Ackermann, M.; McCray, PB Jr. & Tack, BF. ( 2003). Antimicrobial peptides in

Buchmann, K.; Roepstorff, A. & Waller, P.J. (1992). Experimental selection of mebendazole

Dickson, L. C.; Macneil, J.D.; Reid J. & Fesser A. (2003). Validation of Screening Method for

*Diseases,* Vol. 15 No. 5, (September 1992), pp. 393-400, ISSN 1365-2761 Danaher, M.; De Ruyckb, H.; Crooks, S.; Dowling, G. & O'Keeffe, M. (2007). Review of

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*meat and processed meat,* Fidel Toldrá, pp (1-699) Springer, ISBN 978-0-387-89025-8,

*vivo formation* of gastric metabolites of clenbuterol: Preliminary characterisation of their chemical structure. *Analytica Chimica Acta*, Vol. 586, (July 2006), pp. 426–431,

animals and their role in host defences. *International Journal of Antimicrobial Agents*,

resistant gill monogeneans from European eel *Anguilla anguilla*. *Journal of Fish*

methodology for the determination of benzimidazole residues in biological matrices. *Journal of Chromatography B*, Vol. 845 No. 1, (July 2006), pp. 1–37, ISSN

Residues of Diethylstilbestrol, Dienestrol, Hexestrol, and Zeranol, in Bovine Urine Using Immunoaffinity Chromatography and Gas Chromatography/Mass

milk. *Nature*, Vol. 382, No.6592, pp 587, ISSN 0028-0836

Vol. 22, (February 2003), pp. 465-478, ISSN 0924-8579

thereby supposing technological and institutional efforts.

the agro-food industry and global trade in foodstuffs.

**8. References**

España

New York, USA

ISSN 0003-2670

1570-0232

common in the human diet belong to this family (broccoli, Brussels sprouts, cabbage and cauliflower) and several genera (*Brassica*, *Crambe*, *Sinapis* and *Raphanus*) including crops used for producing vegetal oils. The plant *Camelina sativa* (false flax) has recently aroused interest due to biofuel production. When the oils are extracted, the glucosinolates remain in the seed; the resulting press cake is used in animals diet (EFSA, 2008).

The glucosinolates become hydrolysed by enzymatic action giving place to isothiocyanates, thiocyanates, oxazolidinethiones and nitriles. The thiocyanates interfere with iodine capture and the oxazolidinethiones with thyroid (T3 and T4) hormone synthesis, leading to hypothyroidism and thyroid gland enlargement. Consequently, metabolism in all tissues, including the reproductive organs, may become affected.

It has been shown that high glucosinolate consumption in lactating cows reduces iodine levels and increases thiocyanates in milk, the liver and the kidneys. The residues in milk account for around 0.1% of the dose received by animals; the residues in muscles and viscera are even lower. Residuality has also been found in eggs after rapeseed has been administered to egg-laying birds; these also acquire a disagreeable flavour (EFSA, 2008).

#### **6.3.3 Ptaquiloside**

Bracken fern (genus *Pteridium)*, considered one of the five most abundant plants in the world, contains a norsesquiterpene glucoside called ptaquiloside. It has been proved that ptaquiloside may cause tumours in the urinary bladder, mammary glands, intestine and other organs in laboratory rodents. It causes degeneration of the retina in sheep and causes urinary bladder cancer known as bovine enzootic haematuria in cattle. There is epidemiological evidence relating the consumption of bracken fern in humans (Japanese population) suffering from esophagic and gastric cancer, possibly caused by ptaquiloside. Around 8.6% of the ptaquiloside present in *P. aquilinum*, consumed by lactating cows, is excreted in milk, thereby making contaminated raw milk a risk for human health (Alonso-Amelot et al., 1996).

#### **6.3.4 Tremetol**

This is a liposoluble compound mixture of terpene, sterols, tremetone, hydroxytremetone and dehydrotremetone; the last three are present in a ketonic fraction. It is present in the perennial plants white snakeroot (*Ageratina altissima*, previously called *Eupatorium rugosum*) and rayless goldenrod (*Haploppapus heterophyllus*). This poisoning reached an epidemic proportion during the 18th and 19th centuries in the USA (Indiana, Illinois and Ohio), producing high mortality, and its aetiological agent was only discovered in 1910. Cows accumulate the toxin in fat and excrete it in milk. The toxin becomes diluted in milk reception tanks, making such poisoning not very common in modern milk production systems. Tremetol produces acidosis, hyperglycaemia and ketonaemia, as a consequence in the Krebs cycle inhibitor. The symptoms of poisoning are anorexia, listlessness, weakness, stiffness of muscles, vomiting, constipation and coma. Marked acidosis and ketosis may lead to death (Lewis & Elvin-Lewis, 2003).

#### **7. Conclusions**

Interest in diseases caused by food has mainly been orientated towards the acute presentation principally produced by microbiological agents; however, consuming food contaminated by chemical substances could lead to chronic exposure leading to the presentation of diseases lacking an apparent cause and being difficult to diagnose. Foods of animal origin presuppose the risk of contamination, whether from drugs and growth promoters used for optimising livestock production systems, or with biological toxins present in food ingested by animals. It is thus necessary to control these substances in foods, thereby supposing technological and institutional efforts.

Sanitary authorities must thus promulgate and ensure compliance with standards and guidelines concerning the production of harmless foodstuffs. Achieving such objective represents a great challenge for underdeveloped and developing countries due to institutional difficulties and the limited availability of equipment and qualified personnel. All nations must make it a priority to try to ensure the safe consumption of foodstuffs by their populations, exercising strict sanitary control aimed to avoid problems of health in the population and preventing the appearance of new problems affecting the development of the agro-food industry and global trade in foodstuffs.

#### **8. References**

184 Public Health – Methodology, Environmental and Systems Issues

common in the human diet belong to this family (broccoli, Brussels sprouts, cabbage and cauliflower) and several genera (*Brassica*, *Crambe*, *Sinapis* and *Raphanus*) including crops used for producing vegetal oils. The plant *Camelina sativa* (false flax) has recently aroused interest due to biofuel production. When the oils are extracted, the glucosinolates remain in

The glucosinolates become hydrolysed by enzymatic action giving place to isothiocyanates, thiocyanates, oxazolidinethiones and nitriles. The thiocyanates interfere with iodine capture and the oxazolidinethiones with thyroid (T3 and T4) hormone synthesis, leading to hypothyroidism and thyroid gland enlargement. Consequently, metabolism in all tissues,

It has been shown that high glucosinolate consumption in lactating cows reduces iodine levels and increases thiocyanates in milk, the liver and the kidneys. The residues in milk account for around 0.1% of the dose received by animals; the residues in muscles and viscera are even lower. Residuality has also been found in eggs after rapeseed has been administered to egg-laying birds; these also acquire a disagreeable flavour (EFSA, 2008).

Bracken fern (genus *Pteridium)*, considered one of the five most abundant plants in the world, contains a norsesquiterpene glucoside called ptaquiloside. It has been proved that ptaquiloside may cause tumours in the urinary bladder, mammary glands, intestine and other organs in laboratory rodents. It causes degeneration of the retina in sheep and causes urinary bladder cancer known as bovine enzootic haematuria in cattle. There is epidemiological evidence relating the consumption of bracken fern in humans (Japanese population) suffering from esophagic and gastric cancer, possibly caused by ptaquiloside. Around 8.6% of the ptaquiloside present in *P. aquilinum*, consumed by lactating cows, is excreted in milk, thereby

This is a liposoluble compound mixture of terpene, sterols, tremetone, hydroxytremetone and dehydrotremetone; the last three are present in a ketonic fraction. It is present in the perennial plants white snakeroot (*Ageratina altissima*, previously called *Eupatorium rugosum*) and rayless goldenrod (*Haploppapus heterophyllus*). This poisoning reached an epidemic proportion during the 18th and 19th centuries in the USA (Indiana, Illinois and Ohio), producing high mortality, and its aetiological agent was only discovered in 1910. Cows accumulate the toxin in fat and excrete it in milk. The toxin becomes diluted in milk reception tanks, making such poisoning not very common in modern milk production systems. Tremetol produces acidosis, hyperglycaemia and ketonaemia, as a consequence in the Krebs cycle inhibitor. The symptoms of poisoning are anorexia, listlessness, weakness, stiffness of muscles, vomiting, constipation and coma. Marked acidosis and ketosis may lead

Interest in diseases caused by food has mainly been orientated towards the acute presentation principally produced by microbiological agents; however, consuming food

making contaminated raw milk a risk for human health (Alonso-Amelot et al., 1996).

the seed; the resulting press cake is used in animals diet (EFSA, 2008).

including the reproductive organs, may become affected.

**6.3.3 Ptaquiloside**

**6.3.4 Tremetol**

**7. Conclusions**

to death (Lewis & Elvin-Lewis, 2003).

Chemical Residues in Animal Food Products: An Issue of Public Health 187

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**9**

*University of Messina*

 *Italy*

**Viable but Nonculturable Bacteria in Food**

Marco Sebastiano Nicolò and Salvatore Pietro Paolo Guglielmino

"Dis-moi ce que tu manges, je te dirai ce que tu es." (Tell me what you eat and I will tell you what you are - Anthelme Brillat-Savarin, *Physiologie du Goût, ou Méditations de*

"Ninety per cent of the diseases known to man are caused by cheap foodstuffs. You are

In every time, availability of food has been a struggle for human survival. In particular, food storage techniques and maintaining have represented one of the most important milestones in the evolution and development of human society. In that sense, the use of fire for cooking and salt and spices for conservation have been important discoveries in food processing,

Nowadays, food resources remain a primary objective for human society, specifically in

Infectious foodborne diseases are caused by the consumption of food contaminated by

Contamination may occur as consequence of incorrect practices at different steps of food

The rapid globalization and trade among countries with different hygienic standards have increased the possibility of food contamination. Hence, outbreaks of foodborne diseases that were once contained within a small community may now take place on global dimensions. The fundamental strategy for outbreaks monitoring is the traceability, that is the possibility

One of the most critical problems in traceability of bacterial pathogens is represented by a state of latency of most foodborne bacterial species, called "viable but nonculturable (VBNC) state", induced by environmental stresses, such as low temperature, high osmolarity, and

Following environmental stimuli, as temperature shift or replenishment of nutrients, VBNC bacteria can "resuscitate", so restoring their ability to grow on common culture media.

In such a state, bacteria show a discrete metabolic activity, but are not able to replicate.

processing, such as handling of feedstock, decontamination, packaging and storage.

to identify the pathogen(s), the ways of food contamination and the spreading.

In fact, up to date more than 250 foodborne diseases have been described.

**1. Introduction**

*Gastronomie Transcendante,* 1826)

terms of safety and control.

nutrient starvation.

what you eat." (Victor Lindlahr, 1923)

with immediate consequences on human habits and life.

pathogen bacteria, viruses, parasites and prions.

*control, exposure and risk assessment,* Vol. 27, No. 1*,* (January, 2010), pp 94-71, ISSN: 1944-0049

### **Viable but Nonculturable Bacteria in Food**

Marco Sebastiano Nicolò and Salvatore Pietro Paolo Guglielmino *University of Messina Italy*

#### **1. Introduction**

188 Public Health – Methodology, Environmental and Systems Issues

Mikus, JH., Duff, GC., Krehbie, C.; Hallford, DM.; Walker, DA.; Graham, JD., & Ralphs, M

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Shoemaker, RC.; House, D. & Ryan, JC. (2010). Defining the neurotoxin derived

Tardieu, D.; Bailly, JD.; Skiba, F.; Grosjean, F. & Guerre, P. (2008). Toxicokinetics of

Weissinger, J. (1994). *Animal Drugs and Human Health (*fourth edition*)*, L.M. Crawford, and D.A. Franco, Technomic Publishing Co, ISBN:<PHONE_NUMBER>024, Lancaster, USA World Health Organization, WHO. (1989). In Evaluation of Certain Veterinary Drug

World Health Organization,WHO.(1993). In Evaluation of Certain Veterinary Drug Residues

Zinedine, A.; Soriano, JM.; Moltó JC. & Mañes J. (2007). Review on the toxicity, occurrence,

*Science*, Vo.68, No.3, (March, 1990), pp 892-904, ISSN 0021-8812

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(December, 2010), pp 633-639, ISSN 1872-9738

on Food Additives, Technical Report Series 788

Additives, Technical Report Series 832

ISSN 0278-6915

*control, exposure and risk assessment,* Vol. 27, No. 1*,* (January, 2010), pp 94-71, ISSN:

H. (2001). Effects of an Estradiol Implant on Locoweed Consumption, Toxicity, and Recovery in Growing Beef Steers, *Professional Animal Scientist,* Vol. 17, No. 2, (June

ivermectin (sheep) and doramectin (cattle) residues in muscular tissue from various anatomical locations. *Food Additives & Contaminants: Part A*, Vol. 25, No. 4, (April

Safety, *Institute of Food Technologist*,Vol. 5. No. 3 (August 2006) pp. 71-137 ISNN

illness chronic ciguatera using markers of chronic systemic inflammatory disturbances: a case/control study. *Neurotoxicology and Teratology*, Vol.32, No.6,

fumonisin B1 in turkey poults and tissue persistence after exposure to a diet containing the maximum European tolerance for fumonisins in avian feeds. *Food and Chemical Toxicology*, Vol. 4, No. 9, (January, 2008), pp 3213-3218, ISSN 0278-6915

Residues in Food, Thirty-fourth Report of the Joint FAO/WHO Expert Committee

in Food, Fortieth Report of the Joint FAO/WHO Expert Committee on Food

metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. *Food and Chemical Toxicology*,Vol. 45, No. 1, (January, 2007), pp 1-18 "Dis-moi ce que tu manges, je te dirai ce que tu es." (Tell me what you eat and I will tell you what you are - Anthelme Brillat-Savarin, *Physiologie du Goût, ou Méditations de Gastronomie Transcendante,* 1826)

"Ninety per cent of the diseases known to man are caused by cheap foodstuffs. You are what you eat." (Victor Lindlahr, 1923)

In every time, availability of food has been a struggle for human survival. In particular, food storage techniques and maintaining have represented one of the most important milestones in the evolution and development of human society. In that sense, the use of fire for cooking and salt and spices for conservation have been important discoveries in food processing, with immediate consequences on human habits and life.

Nowadays, food resources remain a primary objective for human society, specifically in terms of safety and control.

In fact, up to date more than 250 foodborne diseases have been described.

Infectious foodborne diseases are caused by the consumption of food contaminated by pathogen bacteria, viruses, parasites and prions.

Contamination may occur as consequence of incorrect practices at different steps of food processing, such as handling of feedstock, decontamination, packaging and storage.

The rapid globalization and trade among countries with different hygienic standards have increased the possibility of food contamination. Hence, outbreaks of foodborne diseases that were once contained within a small community may now take place on global dimensions.

The fundamental strategy for outbreaks monitoring is the traceability, that is the possibility to identify the pathogen(s), the ways of food contamination and the spreading.

One of the most critical problems in traceability of bacterial pathogens is represented by a state of latency of most foodborne bacterial species, called "viable but nonculturable (VBNC) state", induced by environmental stresses, such as low temperature, high osmolarity, and nutrient starvation.

In such a state, bacteria show a discrete metabolic activity, but are not able to replicate.

Following environmental stimuli, as temperature shift or replenishment of nutrients, VBNC bacteria can "resuscitate", so restoring their ability to grow on common culture media.

Viable but Nonculturable Bacteria in Food 191

For example, in 2005, in South Wales an outbreak of *Escherichia coli* O157 occurred, with identification of 157 cases, 31 people hospitalized, and one 5-year old child died, as well as

Recently, in October 2010 an outbreak of cholera occurred in Haiti. On 18 August 2011, the total number of reported cholera cases was 419,511, with 222,359 hospitalized. Overall, data

Moreover, actually it has been recognized that outbreaks may spread on wider geographic areas. In fact, the rapid globalization and trade among countries with different hygienic standards has increased the risk and entity of food contamination. As consequence, many outbreaks that were once contained within a small community may now take place on wider

The Bovine spongiform encephalopathy (BSE), commonly known as **mad-cow disease**, was firstly detected in United Kingdom and represented a world-wide crisis. The cause of global infection was attributed to the use of contaminated bone meal, used in livestock feeding. Only in the United Kingdom, 4.4 millions of cattle were slaughtered in the attempt to eradicate the pathology and limit its spreading; moreover, the European Union banned

In 2011, an outbreak of gastroenteritis caused by *E. coli* O104:H4 in Germany had several

Often, such kind of outbreaks are very difficult to identify while occurring. An example is represented by an outbreak of *Salmonella* which simultaneously happened in Europe, North America and Israel, following the importation of contaminated snack food. The outbreak identification was accidental, because it was caused by a rare strain of *Salmonella* and the

Therefore, there is a strong probability that contaminated foodstuffs distributed in different countries could origin outbreaks which are not readily pointed out by national health authorities if the spreading in each single country is limited. Many sporadic cases

Contamination may occur as consequence of incorrect practices at different steps of food processing, such as feedstock production and handling, food preparation, packaging and

Use of raw manure or sewage for fertilization and lack of hygiene and health controls in fish and cattle breeding are the first cause of contamination. Spinach and lettuce have been linked to *E. coli* O157:H7 outbreaks (Erickson et al., 2010). Untreated food, such as fresh fruit juice and milk, carry risks because they are not subjected to any decontaminating procedure. Similarly, incorrect slaughterhouse practices can lead to contamination, especially when

happening in single countries may be, then, part of a common global outbreak.

in 2008, an outbreak of listeriosis in Canada (57 clinical cases) killed 23 people.

from health facilities indicate that 5,968 people have died (case fatality rate 1.4%).

geographic areas and several examples can be considered.

exports of British beef from March 1996 to May 2006.

number of cases in each geographical entity was not high.

storage. Each stage of food processing shows critical points.

faecal or intestinal matter from cattle mixes with the meat.

**3. The causes of food contamination**

**3.1 Feedstock production**

social, political and economic implications throughout whole Europe.

Still open is the debate about the possibility, for pathogen bacteria in VBNC state, to maintain pathogenicity and trigger disease in their hosts.

The evidence of contrasting results, in fact, indicates the need for a better understanding of such complex phenomenon, particularly about the underlying molecular network.

Several studies have shown that the most of human pathogens, especially foodborne bacteria, may enter VBNC state as survival strategy against environmental stress.

Many chemo-physical characteristics in food (as acidic pH, low content in carbohydrates, etc.), as well as processing, decontamination and storage, may induce VBNC state.

The observation of VBNC forms of foodborne bacteria and the lack of a ultimate information about the possibility for VBNC bacteria to retain their virulence has raised the problem about the necessity of new procedures for VBNC detection in food.

Many systems have been proposed for VBNC detection in water, but their application on food seems to be quite difficult. In fact, factors as food texture and pH, as well as presence of free lytic enzymes and other compounds, may interfere with the chemical reaction(s) required for the assay.

Then, traceability of foodborne VBNC pathogens strongly requires the design of new detecting systems.

#### **2. The causes of infectious foodborne disease outbreaks**

Infectious foodborne diseases are caused by the consumption of food or beverages contaminated by many pathogenic bacteria, viruses, parasites and prions. In addition, contamination can also be due to molecules produced by bacteria, called **toxins**. Toxins can be derived from cell structure, such as lipopolysaccharide (LPS) of Gram negative bacteria (**endotoxins**), as well as they are synthesized inside the cell and secreted in the surrounding environment (**exotoxins**), as botulinum, cholera and Shiga toxins.

After food contamination, the microbe or its toxin enters the organism via the digestive tract, triggering the illness.

In general, an outbreak of foodborne disease occurs when a group of people eats the same contaminated food and two or more of them develop the same illness.

Many outbreaks consist of sporadic cases and are self-limiting, in that they are related to a small quantity of contaminated food which, usually, is totally eaten by few people and involve a specific geographic area. A sporadic outbreak may follow a catered meal or eating a meal at a restaurant on a particularly busy day.

The number of people affected and the extension of geographic areas in which a foodborne outbreak occurs have considerably increased as consequence of the variations in social habits. Commonly, workers and students may have meals at large-scale retail trade structures as canteens, highway stops, fast food chains and refectories. Such structures, usually, look to suppliers which, from the place where food is produced, distribute their products on national scale. Then, contaminated food may be transported in different places, causing many distinct outbreaks at the same time within a country.

Still open is the debate about the possibility, for pathogen bacteria in VBNC state, to

The evidence of contrasting results, in fact, indicates the need for a better understanding of

Several studies have shown that the most of human pathogens, especially foodborne

Many chemo-physical characteristics in food (as acidic pH, low content in carbohydrates,

The observation of VBNC forms of foodborne bacteria and the lack of a ultimate information about the possibility for VBNC bacteria to retain their virulence has raised the problem

Many systems have been proposed for VBNC detection in water, but their application on food seems to be quite difficult. In fact, factors as food texture and pH, as well as presence of free lytic enzymes and other compounds, may interfere with the chemical reaction(s)

Then, traceability of foodborne VBNC pathogens strongly requires the design of new

Infectious foodborne diseases are caused by the consumption of food or beverages contaminated by many pathogenic bacteria, viruses, parasites and prions. In addition, contamination can also be due to molecules produced by bacteria, called **toxins**. Toxins can be derived from cell structure, such as lipopolysaccharide (LPS) of Gram negative bacteria (**endotoxins**), as well as they are synthesized inside the cell and secreted in the surrounding

After food contamination, the microbe or its toxin enters the organism via the digestive

In general, an outbreak of foodborne disease occurs when a group of people eats the same

Many outbreaks consist of sporadic cases and are self-limiting, in that they are related to a small quantity of contaminated food which, usually, is totally eaten by few people and involve a specific geographic area. A sporadic outbreak may follow a catered meal or eating

The number of people affected and the extension of geographic areas in which a foodborne outbreak occurs have considerably increased as consequence of the variations in social habits. Commonly, workers and students may have meals at large-scale retail trade structures as canteens, highway stops, fast food chains and refectories. Such structures, usually, look to suppliers which, from the place where food is produced, distribute their products on national scale. Then, contaminated food may be transported in different places,

such complex phenomenon, particularly about the underlying molecular network.

bacteria, may enter VBNC state as survival strategy against environmental stress.

etc.), as well as processing, decontamination and storage, may induce VBNC state.

about the necessity of new procedures for VBNC detection in food.

**2. The causes of infectious foodborne disease outbreaks**

environment (**exotoxins**), as botulinum, cholera and Shiga toxins.

contaminated food and two or more of them develop the same illness.

causing many distinct outbreaks at the same time within a country.

a meal at a restaurant on a particularly busy day.

required for the assay.

tract, triggering the illness.

detecting systems.

maintain pathogenicity and trigger disease in their hosts.

For example, in 2005, in South Wales an outbreak of *Escherichia coli* O157 occurred, with identification of 157 cases, 31 people hospitalized, and one 5-year old child died, as well as in 2008, an outbreak of listeriosis in Canada (57 clinical cases) killed 23 people.

Recently, in October 2010 an outbreak of cholera occurred in Haiti. On 18 August 2011, the total number of reported cholera cases was 419,511, with 222,359 hospitalized. Overall, data from health facilities indicate that 5,968 people have died (case fatality rate 1.4%).

Moreover, actually it has been recognized that outbreaks may spread on wider geographic areas. In fact, the rapid globalization and trade among countries with different hygienic standards has increased the risk and entity of food contamination. As consequence, many outbreaks that were once contained within a small community may now take place on wider geographic areas and several examples can be considered.

The Bovine spongiform encephalopathy (BSE), commonly known as **mad-cow disease**, was firstly detected in United Kingdom and represented a world-wide crisis. The cause of global infection was attributed to the use of contaminated bone meal, used in livestock feeding. Only in the United Kingdom, 4.4 millions of cattle were slaughtered in the attempt to eradicate the pathology and limit its spreading; moreover, the European Union banned exports of British beef from March 1996 to May 2006.

In 2011, an outbreak of gastroenteritis caused by *E. coli* O104:H4 in Germany had several social, political and economic implications throughout whole Europe.

Often, such kind of outbreaks are very difficult to identify while occurring. An example is represented by an outbreak of *Salmonella* which simultaneously happened in Europe, North America and Israel, following the importation of contaminated snack food. The outbreak identification was accidental, because it was caused by a rare strain of *Salmonella* and the number of cases in each geographical entity was not high.

Therefore, there is a strong probability that contaminated foodstuffs distributed in different countries could origin outbreaks which are not readily pointed out by national health authorities if the spreading in each single country is limited. Many sporadic cases happening in single countries may be, then, part of a common global outbreak.

#### **3. The causes of food contamination**

Contamination may occur as consequence of incorrect practices at different steps of food processing, such as feedstock production and handling, food preparation, packaging and storage. Each stage of food processing shows critical points.

#### **3.1 Feedstock production**

Use of raw manure or sewage for fertilization and lack of hygiene and health controls in fish and cattle breeding are the first cause of contamination. Spinach and lettuce have been linked to *E. coli* O157:H7 outbreaks (Erickson et al., 2010). Untreated food, such as fresh fruit juice and milk, carry risks because they are not subjected to any decontaminating procedure.

Similarly, incorrect slaughterhouse practices can lead to contamination, especially when faecal or intestinal matter from cattle mixes with the meat.

Viable but Nonculturable Bacteria in Food 193

Bacteria are single-cell microrganisms colonizing any environment. From an ecological point of view, they play a pivotal role in the biogeochemical cycles, by which chemical elements move from living to non-living matter and vice versa. Some of them produce molecules which improve the quality of human life, such as antibiotics, vitamins, probiotics and other

In food industry, several *Lactobacillus* species are very important for production of cheese,

Several bacteria inhabit the skin and the intestine of animals and humans, protecting them

Pathogen bacteria are responsible for infectious diseases, by colonization of tissues and

Foodborne pathogen bacteria are a group of microrganisms which can contaminate food

Common symptoms of foodborne illness are diarrhoea and/or vomiting, abdominal

A description of the most common known foodborne pathogen bacteria and related illness(es) follows. Intriguingly, for the most of them, the VBNC state has been described.

Gram positive rod-shaped bacterium, able to form endospores under negative conditions, commonly found in soil and vegetation. After pasteurisation or heating, endospores survive, whereas competing microflora is eliminated. During food cooling, endospores germinate and vegetative cells proliferate, producing several toxins, one of which is highly resistant to heat and to pH between 2 and 11. The infection may be almost diarrhoeal, but some cases

Genus of gram negative, aerobic, rod-spherical shaped bacteria which infect animals and humans. Four species are recognised as harmful, that is *B. abortus* (from cattle), *B. melitensis*

It can contaminate raw milk and cheese, but may be acquired also by inhalation, causing **brucellosis**, also known as Malta fever, undulant fever, Rock of Gibraltar fever, and Bang's

The symptoms are non-specific and systemic, with fever, sweats, headache, anorexia, back pain. The chronic form causes suppurative lesions in the liver, spleen, and bone, with a

Gram negative, microaerophilic, spirally curved bacterium, commonly living in the bowel of

(from goats, sheep, and camels), *B. suis* (from pigs), and *B. canis* (from dogs).

yogurt, beer, cider, wine, bread and chocolate, as well as in functional food.

from contamination by hazardous microbes and playing a role in metabolism.

organs, whose physiology is altered by bacterial metabolism and reproduction.

**4. Foodborne bacteria**

nutritional factors.

*Bacillus cereus*

*Brucella spp.*

disease.

*Campylobacter jejuni*

and, after swallowing, cause illness.

cramps, nausea, fever, joint/back aches, and fatigue.

described nausea and vomiting. The illness is self-limiting.

animals, especially in poultry, and spread by faeces and milk.

mortality of 5% in untreated individuals.

#### **3.2 Feedstock handling**

Handling and washing of feedstock before processing are very important sources of contamination. Handling by ill people which have cuts, open sores or skin infections causes spreading of *Staphylococcus aureus*, which is often found on skin with boils and blisters. That is why food handling should be performed by personnel wearing gloves. However, touching contaminated surfaces, coughing into a gloved hand or handling money before food preparation can still spread germs, which is why gloves should be changed often.

#### **3.3 Food preparation**

Beyond the causes of contamination above discussed, in this step cross contamination is a crucial factor. This can happen during food preparation and storage. For example, juices from raw beef and poultry could mix with ready-to-eat food or when kitchen tools are used without distinction for both raw beef and fresh vegetables. Cleaning tools with soap and hot water strongly reduces contamination.

Also undercooking can be cause of food contamination, because heat drastically reduces the presence of bacteria in food.

#### **3.4 Food packaging**

In food packaging, hygiene of handlers and materials employed are critical parameters.

Historically, canned food, if prepared under unsafe conditions, can be susceptible to contamination by toxinogenic microbes, such as *Clostridium botulinum*.

Nowadays, food packaging is mostly based on plastics, employed for vegetables, cheese, fruit. However, even if a correct packaging assures food safety from microbe contamination and remarkably prolongs shelf-life, much attention has to be paid about hygienic requirements. Bacteria can colonize and adhere on plastics, producing biofilms which may reduce shelf life or, for pathogenic bacteria, they may secrete toxins or proliferate on food.

#### **3.5 Food storage**

This is a critical step for perishable food which requires refrigeration or freezing, because low temperature slows down bacterial reproduction, even if some species, called **psycrophiles**, such as *Listeria monocytogenes*, *Yersinia enterocolitica* and *Pseudomonas* sp., are able to develop also at refrigerator temperature.

Food storage has become increasingly important in the perspective of globalization, in that different food tipologies may be transported together. Physical contact and dripping of juices to other foodstuffs are causes of crosscontamination. In this way, one kind of food can be contaminated by unusual bacteria, that become difficult to be identified. Also changes in livestock farming and industrialization of slaughtering of pigs have played an important role. Transportation of live animals for slaughtering has in some studies been proved to be an important factor in dissemination of *Y. enterocolitica* from farm to farm (Nesbakken, 2007*).*

#### **4. Foodborne bacteria**

192 Public Health – Methodology, Environmental and Systems Issues

Handling and washing of feedstock before processing are very important sources of contamination. Handling by ill people which have cuts, open sores or skin infections causes spreading of *Staphylococcus aureus*, which is often found on skin with boils and blisters. That is why food handling should be performed by personnel wearing gloves. However, touching contaminated surfaces, coughing into a gloved hand or handling money before food preparation can still spread germs, which is why gloves should be

Beyond the causes of contamination above discussed, in this step cross contamination is a crucial factor. This can happen during food preparation and storage. For example, juices from raw beef and poultry could mix with ready-to-eat food or when kitchen tools are used without distinction for both raw beef and fresh vegetables. Cleaning tools with soap and hot

Also undercooking can be cause of food contamination, because heat drastically reduces the

In food packaging, hygiene of handlers and materials employed are critical parameters.

contamination by toxinogenic microbes, such as *Clostridium botulinum*.

Historically, canned food, if prepared under unsafe conditions, can be susceptible to

Nowadays, food packaging is mostly based on plastics, employed for vegetables, cheese, fruit. However, even if a correct packaging assures food safety from microbe contamination and remarkably prolongs shelf-life, much attention has to be paid about hygienic requirements. Bacteria can colonize and adhere on plastics, producing biofilms which may reduce shelf life or, for pathogenic bacteria, they may secrete toxins or

This is a critical step for perishable food which requires refrigeration or freezing, because low temperature slows down bacterial reproduction, even if some species, called **psycrophiles**, such as *Listeria monocytogenes*, *Yersinia enterocolitica* and *Pseudomonas* sp., are

Food storage has become increasingly important in the perspective of globalization, in that different food tipologies may be transported together. Physical contact and dripping of juices to other foodstuffs are causes of crosscontamination. In this way, one kind of food can be contaminated by unusual bacteria, that become difficult to be identified. Also changes in livestock farming and industrialization of slaughtering of pigs have played an important role. Transportation of live animals for slaughtering has in some studies been proved to be an important factor in dissemination of *Y. enterocolitica* from farm to farm

**3.2 Feedstock handling**

changed often.

**3.3 Food preparation**

water strongly reduces contamination.

able to develop also at refrigerator temperature.

presence of bacteria in food.

**3.4 Food packaging**

proliferate on food.

**3.5 Food storage**

(Nesbakken, 2007*).*

Bacteria are single-cell microrganisms colonizing any environment. From an ecological point of view, they play a pivotal role in the biogeochemical cycles, by which chemical elements move from living to non-living matter and vice versa. Some of them produce molecules which improve the quality of human life, such as antibiotics, vitamins, probiotics and other nutritional factors.

In food industry, several *Lactobacillus* species are very important for production of cheese, yogurt, beer, cider, wine, bread and chocolate, as well as in functional food.

Several bacteria inhabit the skin and the intestine of animals and humans, protecting them from contamination by hazardous microbes and playing a role in metabolism.

Pathogen bacteria are responsible for infectious diseases, by colonization of tissues and organs, whose physiology is altered by bacterial metabolism and reproduction.

Foodborne pathogen bacteria are a group of microrganisms which can contaminate food and, after swallowing, cause illness.

Common symptoms of foodborne illness are diarrhoea and/or vomiting, abdominal cramps, nausea, fever, joint/back aches, and fatigue.

A description of the most common known foodborne pathogen bacteria and related illness(es) follows. Intriguingly, for the most of them, the VBNC state has been described.

#### *Bacillus cereus*

Gram positive rod-shaped bacterium, able to form endospores under negative conditions, commonly found in soil and vegetation. After pasteurisation or heating, endospores survive, whereas competing microflora is eliminated. During food cooling, endospores germinate and vegetative cells proliferate, producing several toxins, one of which is highly resistant to heat and to pH between 2 and 11. The infection may be almost diarrhoeal, but some cases described nausea and vomiting. The illness is self-limiting.

#### *Brucella spp.*

Genus of gram negative, aerobic, rod-spherical shaped bacteria which infect animals and humans. Four species are recognised as harmful, that is *B. abortus* (from cattle), *B. melitensis* (from goats, sheep, and camels), *B. suis* (from pigs), and *B. canis* (from dogs).

It can contaminate raw milk and cheese, but may be acquired also by inhalation, causing **brucellosis**, also known as Malta fever, undulant fever, Rock of Gibraltar fever, and Bang's disease.

The symptoms are non-specific and systemic, with fever, sweats, headache, anorexia, back pain. The chronic form causes suppurative lesions in the liver, spleen, and bone, with a mortality of 5% in untreated individuals.

#### *Campylobacter jejuni*

Gram negative, microaerophilic, spirally curved bacterium, commonly living in the bowel of animals, especially in poultry, and spread by faeces and milk.

Viable but Nonculturable Bacteria in Food 195

*C. burnetii* secretes a toxin inside the phagolysosome which inhibits its fusion with the cell

In humans it causes the **Q fever**, with fever, severe headache, muscle and joint pains, upper respiratory problems and gastro-intestinal symptoms such as nausea, vomiting and diarrhoea. Life threatening complications are acute respiratory distress syndrome (ARDS),

The chronic form of Q fever is virtually identical to endocarditis. It is usually fatal if

Gram negative, rod-shaped bacterium, inhabitant of mammal bowel. Among the pathogenic

• Enterotoxigenic (ETEC) *E. coli* produces and releases two exotoxins, LT (heat-labile) enterotoxin, similar to cholera toxin, and ST enterotoxin, which causes cGMP accumulation in the target cells and a subsequent secretion of fluid and electrolytes into the intestinal lumen. Both toxins induce watery diarrhoea, similarly to cholera. It is responsible of the

• Enteroinvasive (EIEC) *E. coli* can invade the intestinal wall, giving inflammation, fever

• Among the enterohaemorrhagic (EHEC) *E. coli* strains, the most important is the well known serotype O157:H7.  Sometimes the toxin may spread in kidney, causing a very dangerous complication called **haemolytic uraemic syndrome**.

• Enteroaggregative (EAEC) strains have fimbriae which aggregate tissue culture cells. EAEC strains bind to the intestinal mucosa to cause watery diarrhoea without fever and are not invasive. They produce a haemolysin and a ST enterotoxin similar to that of ETEC

Gram negative, spiral-shaped microaerophilic bacterium, normally colonising the stomach

The ways of transmission are likely to be oral and oro-faecal routes, even if it has been quite

In the stomach, the bacterium secretes the enzyme urease, which degrades urea to ammonia.

It is responsible of chronic gastritis, peptic ulcer disease and stomach cancer. Actually, it is also associated with the gastric MALT lymphoma and its role on other several illnesses, as

majority of "traveller's diarrhoea" and infant diarrhoea in developing countries.

The first symptom is the presence of blood in urine leading to kidney failure. • Enteropathogenic (EPEC) strains use an adhesin known as **intimin** to bind host intestinal cells. Adherence to the intestinal mucosa causes a rearrangement of actin in the host cell, causing significant deformation. EPEC cells are moderately invasive and elicit an inflammatory response. Changes in intestinal cell ultrastructure are likely the

untreated; however, with appropriate treatment the mortality falls to around 10%.

degradation endosomes.

*Escherichia coli*

strains. *Helicobacter pylori*

granulomatous hepatitis and retinal vasculitis.

strains, distinct groups can be identified.

and diarrhoea similar to *Shigella*-like dysentery.

prime cause of diarrhoea in those afflicted with EPEC.

of 30-50% of the human population in developed countries.

difficult its isolation by the faeces (Thomas et al., 1992; Kelly et al., 1994).

Sjögren, Prader-Willy and Raynaud syndromes, is matter of discussion.

Ammonia lowers the pH locally, so permitting the development of the infection.

It can also contaminate incorrectly prepared meat and poultry.

It is one of the first cause of foodborne illness (**campylobacteriosis**) in United States and in United Kingdom. Symptoms of campylobacteriosis are fever, cramping abdominal pain and diarrhoea. Remission follows within a week.

#### *Clostridium botulinum*

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Food contamination can occur due to improperly preserved or home-canned, low-acid food, which allows endospore germination and subsequent toxin secretion. Among the toxins, seven are neurotoxins, that cause the **flaccid muscular paralysis** by inhibition of neuromuscular transmission through decreased acetylcholine release. Death occurs when respiratory mechanics is compromised.

#### *Clostridium perfringens*

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Preferentially, it contaminates meatbased food, because aminoacid content satisfies its nutritional requirements. After cooking, oxygen concentration is lowered and endospores germinate during cooling. Storage time and refrigeration are critical, in that the vegetative cell can duplicate in 20 minutes. After ingestion, in the bowel the bacterium produces an exotoxin which causes abdominal pain and diarrhoea. In the most of cases, the illness is self-limiting.

#### *Corynebacterium ulcerans*

Gram-positive, nonmotile, straight to slightly curved rod-shaped bacterium that causes subacute bovine mastitis, but *C. ulcerans* has increasingly been isolated from domestic animals such as dogs and cats.

Consumption of raw milk and dairy products or contact with cattle may cause infection of humans, causing a disease very similar to diphtheria by secretion of a toxin which inhibits protein synthesis of epithelial cells, leading them to death.

Symptoms are sore throat, low fever, and a pseudomembrane on the upper respiratory tract.

Toxin may spread through the bloodstream and can lead to life-threatening complications in heart and kidneys. It can also cause nerve damage, eventually leading to paralysis. 40% to 50% of those left untreated can die.

#### *Coxiella burnetii*

*C. burnetii* is an obligate intracellular, small Gram-negative bacterium highly resistant to high temperature, osmotic pressure, ultraviolet light.

It can contaminate people after ingestion of raw milk or contact with infected animals.

*C. burnetii* is highly infectious via the respiratory route, but the infectivity via the oral route is poorly understood. It has been proposed that *C. burnetii* can escape the gastrointestinal tract and produce infection sufficient to stimulate systemic immunity (Loftis et al., 2010).

*C. burnetii* secretes a toxin inside the phagolysosome which inhibits its fusion with the cell degradation endosomes.

In humans it causes the **Q fever**, with fever, severe headache, muscle and joint pains, upper respiratory problems and gastro-intestinal symptoms such as nausea, vomiting and diarrhoea. Life threatening complications are acute respiratory distress syndrome (ARDS), granulomatous hepatitis and retinal vasculitis.

The chronic form of Q fever is virtually identical to endocarditis. It is usually fatal if untreated; however, with appropriate treatment the mortality falls to around 10%.

#### *Escherichia coli*

194 Public Health – Methodology, Environmental and Systems Issues

It is one of the first cause of foodborne illness (**campylobacteriosis**) in United States and in United Kingdom. Symptoms of campylobacteriosis are fever, cramping abdominal pain and

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Food contamination can occur due to improperly preserved or home-canned, low-acid food, which allows endospore germination and subsequent toxin secretion. Among the toxins, seven are neurotoxins, that cause the **flaccid muscular paralysis** by inhibition of neuromuscular transmission through decreased acetylcholine release. Death occurs when respiratory mechanics is

Gram positive rod-shaped bacterium, obligate anaerobe, which forms endospores in presence of oxygen or other environmental stresses. Preferentially, it contaminates meatbased food, because aminoacid content satisfies its nutritional requirements. After cooking, oxygen concentration is lowered and endospores germinate during cooling. Storage time and refrigeration are critical, in that the vegetative cell can duplicate in 20 minutes. After ingestion, in the bowel the bacterium produces an exotoxin which causes abdominal pain

Gram-positive, nonmotile, straight to slightly curved rod-shaped bacterium that causes subacute bovine mastitis, but *C. ulcerans* has increasingly been isolated from domestic

Consumption of raw milk and dairy products or contact with cattle may cause infection of humans, causing a disease very similar to diphtheria by secretion of a toxin which inhibits

Symptoms are sore throat, low fever, and a pseudomembrane on the upper respiratory tract. Toxin may spread through the bloodstream and can lead to life-threatening complications in heart and kidneys. It can also cause nerve damage, eventually leading to paralysis. 40% to

*C. burnetii* is an obligate intracellular, small Gram-negative bacterium highly resistant to

*C. burnetii* is highly infectious via the respiratory route, but the infectivity via the oral route is poorly understood. It has been proposed that *C. burnetii* can escape the gastrointestinal tract and produce infection sufficient to stimulate systemic immunity

It can contaminate people after ingestion of raw milk or contact with infected animals.

It can also contaminate incorrectly prepared meat and poultry.

and diarrhoea. In the most of cases, the illness is self-limiting.

protein synthesis of epithelial cells, leading them to death.

high temperature, osmotic pressure, ultraviolet light.

diarrhoea. Remission follows within a week.

*Clostridium botulinum*

compromised.

*Clostridium perfringens*

*Corynebacterium ulcerans*

animals such as dogs and cats.

50% of those left untreated can die.

*Coxiella burnetii*

(Loftis et al., 2010).

Gram negative, rod-shaped bacterium, inhabitant of mammal bowel. Among the pathogenic strains, distinct groups can be identified.

#### *Helicobacter pylori*

Gram negative, spiral-shaped microaerophilic bacterium, normally colonising the stomach of 30-50% of the human population in developed countries.

The ways of transmission are likely to be oral and oro-faecal routes, even if it has been quite difficult its isolation by the faeces (Thomas et al., 1992; Kelly et al., 1994).

In the stomach, the bacterium secretes the enzyme urease, which degrades urea to ammonia. Ammonia lowers the pH locally, so permitting the development of the infection.

It is responsible of chronic gastritis, peptic ulcer disease and stomach cancer. Actually, it is also associated with the gastric MALT lymphoma and its role on other several illnesses, as Sjögren, Prader-Willy and Raynaud syndromes, is matter of discussion.

Viable but Nonculturable Bacteria in Food 197

headache, then diarrhoea appears and fever declines. Differently from salmonellosis, bacteria multiply into phagocytic cells and disseminate in the body. In severe cases,

Today, the mortality rate of typhoid fever is about 1-2%; in the past it exceeded also 10%.

The genus *Shigella* is composed of Gram negative, rod-shaped bacteria, which are normally

Food contamination occurs via handling by unhealthy operators. Four species are pathogenic: *S. sonnei*, *S. dysenteriae*, *S. flexneri* and *S. boydii*. Many cases of "traveller's

*S. dysenteriae* causes severe dysentery and prostration by secreting the "Shiga toxin", which inhibits protein synthesis in epithelial cells of intestinal wall, killing them. Bacteria multiply in the small intestine and spread in the large intestin, entering the epithelial cells. Infection proceeds towards neighbouring cells, via a "cell-to-cell" mechanism, thus avoiding immune system response. As consequence, intestinal mucosa is damaged, causing severe diarrhoea with blood and mucus in the faeces. Additional symptoms may be abdominal cramps and

Only *S. dysenteriae* may reach the bloodstream, causing septicaemia. Its mortality rate is

Gram positive, spherical-shaped bacterium, it is a frequent inhabitant of respiratory tract from which it can contaminate hands and skin. Typically, *S. aureus* and others staphylococcal species are resistant to various stresses. They can survive 60°C for 30 minutes, drying and high osmotic pressures. Such characteristics allow their survival under conditions that, generally, eliminate the most of bacteria. In absence of competitors, then, it

It is able to produce several toxins that improve the virulence or damage tissues, but the toxin produced by serogroup A is responsible for most of the cases. The toxin is heat-stable,

Symptoms of intoxication are vomiting and abdominal cramps followed by diarrhoea.

Genus of gram positive, microaerophilic, spherical-shaped bacteria which occur in chains or pairs. The genus is defined by a combination of antigenic, haemolytic, and physiological

Group A is formed by a single species, *S. pyogenes*, with 40 antigenic types, while Group D

quite significant and may reach 20% in tropical areas, where it is prevalent.

in that maintains its virulence up to 30 minutes of boiling.

perforation of bowel mucosa may occur.

diarrhoea" are supported by *Shigella* spp.

present in the bowel of humans, apes and monkeys.

*Shigella* **spp.**

fever.

*Staphylococcus aureus*

can rapidly proliferate.

*Streptococcus* **spp.**

Remission is reached within 24 hours.

characteristics into Groups A, B, C, D, F, and G.

is represented by the new genus *Enterococcus*.

Groups A and D can be transmitted to humans via food.

#### *Listeria monocytogenes*

Gram positive, facultative anaerobe, rod-shaped intracellular bacterium, commonly spread in soil and water. Vegetables are contaminated by the soil or by manure used as fertilizer. Animals can carry the bacterium asymptomatically and can contaminate food, such as uncooked meat, raw milk and soft cheeses, that are usually prohibited to pregnant women.

It is the causative agent of **listeriosis**. After having entered immune system cells, it becomes septicemic and can grow. The intracellular state in phagocytic cells may permit access to the brain and probably to reach the fetus in pregnant women by crossing the placenta.

Listeriosis may arise as septicaemia, meningoencephalitis, corneal ulcer, pneumonia and during pregnancy (causing abortion within 6-9 months or stillbirth).

When no internalization in cells occurs, the disease is presented as a febrile gastroenteritis.

*L. monocytogenes* is the leading cause of death among foodborne bacterial pathogens, with 20 to 30 percent of clinical infections resulting in death.

#### *Plesiomonas shigelloides*

Gram-negative, rod-shaped bacterium, which has been isolated from freshwater, freshwater fish, and shellfish and from many types of animals including cattle in tropical and subtropical areas.

Common symptoms are fever, shivers, abdominal pain, nausea, diarrhoea (which, in severe cases, may be greenish-yellow, foamy, and bloody) or vomiting and appear 20-24 hours after consumption of contaminated food or water.

*P. shigelloides* gastroenteritis is usually a mild self-limiting disease with remission in healthy people within 1-7 days, described in African countries, but sporadic cases have been reported also in Europe and North America. Severe forms may include sepsis and meningoencephalitis in newborns, arthritis, cholecystitis and osteomyelitis (Terpeluk, 1992).

#### *Salmonella* **spp.**

The genus *Salmonella* consists of Gram negative, rod-shaped facultatively anaerobe bacteria, which live in the bowel of humans and animals.

Meat, poultry and eggs are the most common food contaminated by *Salmonella*. However, food is contaminated by low loads of *Salmonella*, which are quite difficult to be appreciated by standard detection methods.

All species are considered pathogenic and responsible for a gastroenteritis known as **salmonellosis**. Bacteria reach the bowel and duplicate. Sometimes, they may cross the mucosa, entering lymphatic and cardiovascular systems; from there, they may infect other organs, causing septicaemia.

Symptoms are moderate fever, nausea, abdominal pain and cramps, diarrhoea. Average mortality rate is below 1%, but increases in children and old people, occurring as septicaemia.

Among the species, *S. typhi* is the most virulent. It is the aetiological agent of **typhoid fever** and is spread only by human faeces. Symptoms are high fever (40°C) and continued headache, then diarrhoea appears and fever declines. Differently from salmonellosis, bacteria multiply into phagocytic cells and disseminate in the body. In severe cases, perforation of bowel mucosa may occur.

Today, the mortality rate of typhoid fever is about 1-2%; in the past it exceeded also 10%.

#### *Shigella* **spp.**

196 Public Health – Methodology, Environmental and Systems Issues

Gram positive, facultative anaerobe, rod-shaped intracellular bacterium, commonly spread in soil and water. Vegetables are contaminated by the soil or by manure used as fertilizer. Animals can carry the bacterium asymptomatically and can contaminate food, such as uncooked meat, raw milk and soft cheeses, that are usually prohibited to pregnant women. It is the causative agent of **listeriosis**. After having entered immune system cells, it becomes septicemic and can grow. The intracellular state in phagocytic cells may permit access to the

Listeriosis may arise as septicaemia, meningoencephalitis, corneal ulcer, pneumonia and

When no internalization in cells occurs, the disease is presented as a febrile gastroenteritis. *L. monocytogenes* is the leading cause of death among foodborne bacterial pathogens, with 20

Gram-negative, rod-shaped bacterium, which has been isolated from freshwater, freshwater fish, and shellfish and from many types of animals including cattle in tropical and sub-

Common symptoms are fever, shivers, abdominal pain, nausea, diarrhoea (which, in severe cases, may be greenish-yellow, foamy, and bloody) or vomiting and appear 20-24 hours

*P. shigelloides* gastroenteritis is usually a mild self-limiting disease with remission in healthy people within 1-7 days, described in African countries, but sporadic cases have been reported also in Europe and North America. Severe forms may include sepsis and meningoencephalitis in newborns, arthritis, cholecystitis and osteomyelitis (Terpeluk, 1992).

The genus *Salmonella* consists of Gram negative, rod-shaped facultatively anaerobe bacteria,

Meat, poultry and eggs are the most common food contaminated by *Salmonella*. However, food is contaminated by low loads of *Salmonella*, which are quite difficult to be appreciated

All species are considered pathogenic and responsible for a gastroenteritis known as **salmonellosis**. Bacteria reach the bowel and duplicate. Sometimes, they may cross the mucosa, entering lymphatic and cardiovascular systems; from there, they may infect other

Symptoms are moderate fever, nausea, abdominal pain and cramps, diarrhoea. Average mortality rate is below 1%, but increases in children and old people, occurring as

Among the species, *S. typhi* is the most virulent. It is the aetiological agent of **typhoid fever** and is spread only by human faeces. Symptoms are high fever (40°C) and continued

brain and probably to reach the fetus in pregnant women by crossing the placenta.

during pregnancy (causing abortion within 6-9 months or stillbirth).

to 30 percent of clinical infections resulting in death.

after consumption of contaminated food or water.

which live in the bowel of humans and animals.

by standard detection methods.

organs, causing septicaemia.

*Listeria monocytogenes*

*Plesiomonas shigelloides*

tropical areas.

*Salmonella* **spp.**

septicaemia.

The genus *Shigella* is composed of Gram negative, rod-shaped bacteria, which are normally present in the bowel of humans, apes and monkeys.

Food contamination occurs via handling by unhealthy operators. Four species are pathogenic: *S. sonnei*, *S. dysenteriae*, *S. flexneri* and *S. boydii*. Many cases of "traveller's diarrhoea" are supported by *Shigella* spp.

*S. dysenteriae* causes severe dysentery and prostration by secreting the "Shiga toxin", which inhibits protein synthesis in epithelial cells of intestinal wall, killing them. Bacteria multiply in the small intestine and spread in the large intestin, entering the epithelial cells. Infection proceeds towards neighbouring cells, via a "cell-to-cell" mechanism, thus avoiding immune system response. As consequence, intestinal mucosa is damaged, causing severe diarrhoea with blood and mucus in the faeces. Additional symptoms may be abdominal cramps and fever.

Only *S. dysenteriae* may reach the bloodstream, causing septicaemia. Its mortality rate is quite significant and may reach 20% in tropical areas, where it is prevalent.

#### *Staphylococcus aureus*

Gram positive, spherical-shaped bacterium, it is a frequent inhabitant of respiratory tract from which it can contaminate hands and skin. Typically, *S. aureus* and others staphylococcal species are resistant to various stresses. They can survive 60°C for 30 minutes, drying and high osmotic pressures. Such characteristics allow their survival under conditions that, generally, eliminate the most of bacteria. In absence of competitors, then, it can rapidly proliferate.

It is able to produce several toxins that improve the virulence or damage tissues, but the toxin produced by serogroup A is responsible for most of the cases. The toxin is heat-stable, in that maintains its virulence up to 30 minutes of boiling.

Symptoms of intoxication are vomiting and abdominal cramps followed by diarrhoea. Remission is reached within 24 hours.

#### *Streptococcus* **spp.**

Genus of gram positive, microaerophilic, spherical-shaped bacteria which occur in chains or pairs. The genus is defined by a combination of antigenic, haemolytic, and physiological characteristics into Groups A, B, C, D, F, and G.

Groups A and D can be transmitted to humans via food.

Group A is formed by a single species, *S. pyogenes*, with 40 antigenic types, while Group D is represented by the new genus *Enterococcus*.

Viable but Nonculturable Bacteria in Food 199

For the most of the above described foodborne bacteria, the VBNC state has been proven by several studies. Therefore, the meaning of VBNC state as well as inducing and resuscitating factors have to be well understood, in order to plan how to face the difficulties of detection

In their natural environments, bacteria undergo fluctuating chemo-physical conditions, such as nutrient availability, temperature, osmolarity, and pH, which may interfere with their growth and survival. In such a situation, they modulate their gene expression, in order to survive, by activating the **bacterial stress response**, which consists in variations of cell morphology, dimensions, energetic levels, directly related to cell survival. The final effect is an increased global resistance of surviving cells against further stresses, such as exposure to antibiotics, hydrogen peroxide and high osmolarity (Matin, 1991; Nyström et al., 1992).

In such a situation, some genera of Gram positive bacteria, as *Bacillus*, *Clostridium* and few

Endospore is a differentiated bacterial cell in which new structures, required for mechanical and physical resistance, are synthesised. In the endospore, metabolism has been abolished by a controlled process of dehydration. The lack of metabolism confers a global resistance extended for prolonged periods of time. When environmental conditions become favourable, endospore undergoes **germination**, a process by which the metabolic activity is

The most of Gram positive and all Gram negative bacteria are not able to generate endospores. However, they can trigger a stress response with a highly-complex network of

Studies carried on bacterial stress response have started from bacterial physiology in natural

Subsequent studies have focused on the underlying molecular mechanisms, especially regarding a specific chemophysical stress and single-nutrients starvation (Eberl et al, 1996;

One aspect on which little is known is that, in natural environments, nutritional and chemophysical factors inducing bacterial stress response may change simultaneously, with one

Therefore, several mechanisms of bacterial stress responses should be activated. Unfortunately, few data are available about bacterial response to simultaneously-acting multiple stresses.

Bacterial responses to chemophysical stresses, such as cold- and heat-shock,

It has been observed that *E. coli*, when subjected to a temperature decrease, triggers the cold shock response, in which sets of proteins are synthesised, globally indicated as CIPs (Cold

specific factor influencing another and being, in turn, influenced by a third one.

oligotrophic environments (Kurath & Morita, 1983; Morita, 1982).

Matin, 1991; Nakashima et al., 1996; Rockabrand et al., 1995).

**5.1 The bacterial stress response to chemophysical stress**

hyperosmolarity and acid pH, have been investigated.

**5.1.1 The cold shock response**

and make traceability feasible.

**5. The bacterial stress response**

others, differentiate into **endospore**.

restored and cell turns to vegetative life.

molecular mechanisms.

Many types of food may be contaminated by Group A *Streptococcus*, including raw milk, ice cream, ham, potato salad, shrimps salad, steamed lobster via manipulation by ill handlers. Moreover, they may contaminate food with low content of free water, rich in salt or with very acidic pH, which is generally difficult to be contaminated by other bacterial species.

Group A *Streptococcus* may cause several diseases, as bacteraemia, impetigo, erysipelas, pneumonia, osteomyelitis, septic arthritis, meningitis and toxic shock syndrome.

Among complications, acute rheumatic fever may follow respiratory infections as an autoimmune disease, in which antibodies raised against the streptococcal M-protein crossreact with autoantigens of pericardium and synovium.

Enterococci can cause food intoxication through production of biogenic amines, such as histamine and tyramine, mainly by the decarboxylation of amino acids in fish, meat and dairy products, wine, beer, vegetables, fruits, and nuts.

The genus *Enterococcus* is responsible for severe diseases, such as urinary tract infections, bacteraemia, bacterial endocarditis, diverticulitis, and meningitis.

#### *Vibrio cholerae*

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in brackish waters, but can easily spread also in freshwater.

It can contaminate fish and seafood.

It is the aetiological agent of cholera, a serious illness that stroke Europe and North America during the XIX century with different outbreaks. Symptoms are nausea, vomiting, abdominal pain, watery diarrhoea which may induce severe dehydratation, that can be fatal if untreated.

Nowadays, cholera is endemic in India and rarely it causes outbreaks in Western countries. In 1991-1994, Latin America had an epidemic, in consequence of importation of contaminated seafood from Asia, with more than one million cases and 9600 deaths. In 2010, a new outbreak in Haiti has occurred.

#### *Vibrio parahaemolyticus*

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in salt waters.

The related gastroenteritis follows the consumption of contaminated seafood. Symptoms include abdominal pain, vomiting, a burning sensation in the stomach and cholera-like watery faeces. Remission occurs within few days.

#### *Vibrio vulnificus*

As *V. parahemolyticus*, it is found in estuarine waters and contaminates seafood. It represents a serious threat for people with compromised immune system. In people with liver disease, it may cause septicaemia, with mortality rates often exceeding 50%.

#### *Yersinia enterocolitica* **and** *Y. pseudotuberculosis*

Psycrofile Gram-negative bacteria which usually colonise the bowel of domestic animals and are transmitted by milk and meat. Outside their natural habitat, they are able to grow at refrigeration temperature (4°C). The symptoms are diarrhoea, fever, headache and abdominal pain.

Many types of food may be contaminated by Group A *Streptococcus*, including raw milk, ice cream, ham, potato salad, shrimps salad, steamed lobster via manipulation by ill handlers. Moreover, they may contaminate food with low content of free water, rich in salt or with very acidic pH, which is generally difficult to be contaminated by other bacterial species.

Group A *Streptococcus* may cause several diseases, as bacteraemia, impetigo, erysipelas,

Among complications, acute rheumatic fever may follow respiratory infections as an autoimmune disease, in which antibodies raised against the streptococcal M-protein cross-

Enterococci can cause food intoxication through production of biogenic amines, such as histamine and tyramine, mainly by the decarboxylation of amino acids in fish, meat and

The genus *Enterococcus* is responsible for severe diseases, such as urinary tract infections,

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in brackish

It is the aetiological agent of cholera, a serious illness that stroke Europe and North America during the XIX century with different outbreaks. Symptoms are nausea, vomiting, abdominal pain, watery diarrhoea which may induce severe dehydratation, that can be fatal if untreated. Nowadays, cholera is endemic in India and rarely it causes outbreaks in Western countries. In 1991-1994, Latin America had an epidemic, in consequence of importation of contaminated seafood from Asia, with more than one million cases and 9600 deaths. In 2010,

Gram negative, slightly curved rod-shaped bacterium, which lives naturally in salt waters. The related gastroenteritis follows the consumption of contaminated seafood. Symptoms include abdominal pain, vomiting, a burning sensation in the stomach and cholera-like

As *V. parahemolyticus*, it is found in estuarine waters and contaminates seafood. It represents a serious threat for people with compromised immune system. In people with liver disease,

Psycrofile Gram-negative bacteria which usually colonise the bowel of domestic animals and are transmitted by milk and meat. Outside their natural habitat, they are able to grow at refrigeration temperature (4°C). The symptoms are diarrhoea, fever, headache and

pneumonia, osteomyelitis, septic arthritis, meningitis and toxic shock syndrome.

react with autoantigens of pericardium and synovium.

dairy products, wine, beer, vegetables, fruits, and nuts.

waters, but can easily spread also in freshwater.

It can contaminate fish and seafood.

a new outbreak in Haiti has occurred.

watery faeces. Remission occurs within few days.

*Yersinia enterocolitica* **and** *Y. pseudotuberculosis*

it may cause septicaemia, with mortality rates often exceeding 50%.

*Vibrio parahaemolyticus*

*Vibrio vulnificus*

abdominal pain.

*Vibrio cholerae*

bacteraemia, bacterial endocarditis, diverticulitis, and meningitis.

For the most of the above described foodborne bacteria, the VBNC state has been proven by several studies. Therefore, the meaning of VBNC state as well as inducing and resuscitating factors have to be well understood, in order to plan how to face the difficulties of detection and make traceability feasible.

#### **5. The bacterial stress response**

In their natural environments, bacteria undergo fluctuating chemo-physical conditions, such as nutrient availability, temperature, osmolarity, and pH, which may interfere with their growth and survival. In such a situation, they modulate their gene expression, in order to survive, by activating the **bacterial stress response**, which consists in variations of cell morphology, dimensions, energetic levels, directly related to cell survival. The final effect is an increased global resistance of surviving cells against further stresses, such as exposure to antibiotics, hydrogen peroxide and high osmolarity (Matin, 1991; Nyström et al., 1992).

In such a situation, some genera of Gram positive bacteria, as *Bacillus*, *Clostridium* and few others, differentiate into **endospore**.

Endospore is a differentiated bacterial cell in which new structures, required for mechanical and physical resistance, are synthesised. In the endospore, metabolism has been abolished by a controlled process of dehydration. The lack of metabolism confers a global resistance extended for prolonged periods of time. When environmental conditions become favourable, endospore undergoes **germination**, a process by which the metabolic activity is restored and cell turns to vegetative life.

The most of Gram positive and all Gram negative bacteria are not able to generate endospores. However, they can trigger a stress response with a highly-complex network of molecular mechanisms.

Studies carried on bacterial stress response have started from bacterial physiology in natural oligotrophic environments (Kurath & Morita, 1983; Morita, 1982).

Subsequent studies have focused on the underlying molecular mechanisms, especially regarding a specific chemophysical stress and single-nutrients starvation (Eberl et al, 1996; Matin, 1991; Nakashima et al., 1996; Rockabrand et al., 1995).

One aspect on which little is known is that, in natural environments, nutritional and chemophysical factors inducing bacterial stress response may change simultaneously, with one specific factor influencing another and being, in turn, influenced by a third one.

Therefore, several mechanisms of bacterial stress responses should be activated. Unfortunately, few data are available about bacterial response to simultaneously-acting multiple stresses.

#### **5.1 The bacterial stress response to chemophysical stress**

Bacterial responses to chemophysical stresses, such as cold- and heat-shock, hyperosmolarity and acid pH, have been investigated.

#### **5.1.1 The cold shock response**

It has been observed that *E. coli*, when subjected to a temperature decrease, triggers the cold shock response, in which sets of proteins are synthesised, globally indicated as CIPs (Cold

Viable but Nonculturable Bacteria in Food 201