**1. Introduction**

46 Salmonella – A Dangerous Foodborne Pathogen

Van Cauteren, D., Jourdan-da Silva, N., Weill, F.X., King, L., Brisabois, A., Delmas, G.,

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Wong, T.L., Nicol, C., Cook, R. & MacDiarmid, S. (2007). *Salmonella* in uncooked retail meats

Yang, B., Qu, D., Zhang, X., Shen, J., Cui, S., Shi, Y., Xi, M., Sheng, M., Zhi, S. & Meng, J.

Yang, H.H., Gong, J., Zhang, J., Wang, M.L., Yang, J., Wu, G.Z., Quan, W.L., Gong, H.M.,

*Infection*, Vol.138, No.12, (December 2010), pp. 1765-1774, ISSN 1469-4409 Zhuang, R.Y., Beuchat, L.R., & Angulo, F.J. (1995). Fate of *Salmonella montevideo* on and in

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Foodborne salmonellosis is still today a serious public health issue: very common in poor developing countries, due to the bad general hygiene conditions, it is also largely widespread in developed countries. In the latter, 95% of recorded clinical cases are foodborne (Liu *et al*., 2011). According to EFSA epidemiological data (2011), in the European Union (EU) *Salmonella* is the second cause of foodborne disease after *Campylobacter* and it is still first in many EU States, such as Italy. Unlike *Campylobacter*, *Salmonella* often cause very large *multistate outbreaks* of food infection; this proves the greater resistance of this pathogen in the external environment and in food. In developed countries the main source of salmonellosis is still today food of animal origin, particularly meat (fresh and processed) and shell eggs. Also fresh fruits and vegetables can convey the bacteria to humans, as well as undrinkable water. *Salmonella* is quite resistant to adverse conditions and this allows them to persist in the environment and spread along the food chain, from the animals to the food of animal origin, or to plants that are fertilized with animal manure. Two species are currently registered into the genus *Salmonella*: *S. enterica* and *S. bongori*. The former is better adapted than the latter to live in the intestine of man and warm-blooded animals, whereas *S. bongori* travels in the external environment and is detectable in the intestinal contents of warm-blooded animals, so it is rare for it to be found in food for human consumption. The dangers for human health mainly arise from food contaminated with *Salmonella enterica*, which is often present in the intestines of livestock and pets, without causing any infection to the animals ("healthy carrier" condition). Humans can be healthy carriers of *S. enterica* in the intestine too. This may be a potential hazard to food hygiene, if the healthy carriers are the people involved in producing and handling the food. Usually a healthy carrier eliminates *Salmonella* in their faeces for several months after the episode of gastroenteritis through which they became carrier. In the case of *Salmonella* ser. Typhi, however, it has been demonstrated that humans can be asymptomatic carriers of the bacterium for decades (Weill, 2009). The genus *Salmonella* has more than 2,500 serotypes, and over 1,600 of these are within the *enterica* species, but not all serotypes have the same affinity for human and/or animals and they are not all found in the food that humans consume. Some serotypes (Typhy, Paratyphi A and C, some clones of Paratyphi B and Sendai) travel almost exclusively among men, and express their pathogenicity only when they infect a human being. Few serotypes travel exclusively among animals and do not infect humans, if not seldom (e.g. Abortusovis in sheep and Gallinarum-Pullorum in poultry). On the contrary, approximately 150 serotypes travel more or less constantly between the animal reservoir, the environment, food and man, starting from *Salmonella* ser. Typhimurium. Some serotypes, however, have a particular preference for some animal species: Enteritidis, Hadar, Heidelberg, Saintpaul,

Food as Cause of Human Salmonellosis 49

present some specific conditions. One of these is certainly the "minimal infective dose", i.e. the lowest charge that *Salmonella* must reach in the food for it to become dangerous to human health. Generally, it is accepted that *Salmonella* becomes truly dangerous for humans when it reaches in a food a charge of at least 104 cfu/g. However, it should be reminded that the bibliography reports some foodborne salmonellosis outbreaks caused by foods that contained less than 100 and sometimes less than 10 cfu of bacteria per gram of product. Fatty foods, such as cheeses, butter and chocolate, better protect the bacteria from the digestive enzymes in the stomach. In addition, the low water activity of these foods keeps the salmonellae in a latent phase, and this means that they do not proliferate in the food substrate, but can survive for very long time (Jansson *et al*., 2011; Finstad *et al*., 2011). The infective charge in one episode of salmonellosis which occurred in Canada and was caused by chocolate was estimated as low as 0,005 cfu/g (Komitopoulou & Penaloza, 2009). It is important to underline that the foods contaminated with Salmonella do not usually show any modification in their sensory characteristics even though the pathogens within have reached very high levels, concretely

According to the latest "European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks" (EFSA, 2011), in 2009 in the 27 EU Member States, the health authorities in charge have reported a total of 108,614 confirmed cases of human salmonellosis, with a prevalence of 23.7 cases/100,000 population. If we compare these levels with their equivalents reported from 2005 onwards, we discover that in the 2005/2009 period the cases of human salmonellosis have considerably dropped, estimated at -13%. In comparison, cases of campylobacteriosis have increased by +12%. In particular, between 2008 and 2009 there was a sharp decline in clinical cases of human salmonellosis caused by *Salmonella* ser. Enteritidis. All this indicates that the efforts made by health authorities and policies of individual EU states are obtaining positive and effective results. Furthermore, if we analyze the data regarding the spread of *Salmonella* among farm animals, we can find out that the importance of *Salmonella* as a cause of human foodborne disease is decreasing, also thanks to the decline in the spread of bacteria among livestock, starting with fowl. The decline in cases of foodborne salmonellosis among human beings does not tend to be consistent or regular in all 27 EU Members. The variations in the epidemiological pattern can be noticeable from one State to another. 10 states recorded a significant decline in cases; for 14 other states (including Italy) the epidemiological situation of human salmonellosis in food has remained essentially stable over the past five years, while Malta reported a sharp rise in cases (+24% compared to 2008), in contrast with the rest of the EU countries. Scandinavian and Central European countries are among the member states with the highest prevalence of human cases of foodborne salmonellosis while prevalence of salmonellosis among the population reported by the states bordering the Mediterranean are well below the previous. Epidemiologists interpret this as a sign of the single EU members' health authorities' increased awareness about the health of the populations under their responsibility. This increased attention to identify and report cases of foodborne salmonellosis explains the higher prevalence of human cases of salmonellosis in some northern European countries compared with the levels observed in Southern European countries. In most EU states food salmonellosis is a disease that patients contract "in their own country". Only Sweden, Finland, Denmark and the UK count a number of cases imported from abroad because they were contracted by people when they were out of the country. It should, however, be pointed out that some of the EU countries were not able

harmful to human health (Lindhardt *et al*., 2009).

**1.3 Epidemiology of foodborne human salmonellosis in the EU** 

Virchow, Senftenberg, Infantis and Kottbus find their main distribution channel in chickens, turkeys and ducks; Dublin and Bovismorbificans mainly infect cattle, while the Derby, Brandenburg and Panama serotypes frequently circulate among pigs (Weill, 2009). From the intestinal contents of livestock, the salmonellae can contaminate fresh meat, raw milk and egg shells. If the necessary hygienic precautions are not taken in the early stages of the production line (slaughter, milking, egg collecting), there is a risk that the salmonellae may then spread along each of their production chain, even polluting products such as cured meats, dairy products and egg-based dishes if they were made using raw milk or unpasteurized eggs. Moreover, through the faeces of animals and man, salmonellae can contaminate farmland, surface water flow and vegetables if they are fertilized with animal manure or dung that is not properly fermented. Vegetables, therefore, can be a source of disease to humans just like fresh meat, milk, shell eggs and by-products. Besides in animals, *Salmonella* can adhere well to the work surfaces, and from there spread to other foodstuffs by cross-contamination (Møretrø *et al*., 2011). The examples are numerous and blatant: in the U.S. a major *Salmonella* ser. Enteritidis outbreak occurred and was associated with the consumption of industrial ice cream premix which was transported in tanks that had been used for carrying unpasteurized liquid eggs and were not properly sanitized (Hennessy *et al*., 1996). An outbreak of salmonellosis due to *S*. Ealing caused by dehydrated powdered milk was traced back to the inadequate sanitization of production equipment (Rowe *et al*., 1987). The thorough cleaning of work surfaces, both in food manufacturing facilities and in domestic kitchens, is therefore one of the main strategies for the prevention of foodborne salmonellosis (Møretrø et al., 2011). Generally, forms of gastroenteritis caused by non-typhoid *Salmonella* are moderately serious diseases with a quick recovery and without the need to resort to specific therapies. Although in some cases – when young children, elderly, or immunocompromised subjects are affected – salmonellosis may also lead to the patient's death (Pathan *et al*., 2010). The severity of *Salmonella* infections can also be aggravated by the fact that in recent years more and more *Salmonella* strains have been spreading and they are resistant to one or more of the antibiotics which are widely used in human medicine, such as fluoroquinolones and third generation cephalosporins. In addition to the Typhimurium serotype, *Salmonella* strains which are multiresistant to many antibiotics have also been detected in the Agona, Anatum, Choleraesuis, Derby, Dublin, Heidelberg, Kentucky, Newport, Pullorum, Schwarzengrund, Senftenberg, and Uganda serotypes (Yan *et al*., 2010). In most cases, human infection manifests itself through diarrhoea, persistent fever and abdominal cramps which appear 12 to 72 hours after the infection. The disease is selflimiting and clears up by itself within 4-7 days, but it has rather significant side effects: it takes months for the patient to regain proper bowel function and they can remain healthy carriers for months. In addition, chronic complications may occur such as widespread polyarthritis (Reiter's syndrome), ocular and urinary disorders, and even occasional cases of endocarditis and appendicitis. All these diseases are hard to treat even with antibiotics (Castillo *et al*., 2011).

#### **1.2 The infective dose "issue"**

According to the regulations currently in force in the European Union, it is the manufacturer's responsibility to ensure the hygiene of their production processes on a daily basis, seeing to prevent any possible *hazard* that may contaminate food and be harmful to human health. The system used by food manufacturers to control processing hygiene in their facilities is the wellknown HACCP system. In view of the fundamental principles of HACCP, if *Salmonella* contaminates a food, this is a *Hazard* because its presence could potentially cause harm to human health. It is, however, a hypothetical danger, as, for it to become real, the food has to

Virchow, Senftenberg, Infantis and Kottbus find their main distribution channel in chickens, turkeys and ducks; Dublin and Bovismorbificans mainly infect cattle, while the Derby, Brandenburg and Panama serotypes frequently circulate among pigs (Weill, 2009). From the intestinal contents of livestock, the salmonellae can contaminate fresh meat, raw milk and egg shells. If the necessary hygienic precautions are not taken in the early stages of the production line (slaughter, milking, egg collecting), there is a risk that the salmonellae may then spread along each of their production chain, even polluting products such as cured meats, dairy products and egg-based dishes if they were made using raw milk or unpasteurized eggs. Moreover, through the faeces of animals and man, salmonellae can contaminate farmland, surface water flow and vegetables if they are fertilized with animal manure or dung that is not properly fermented. Vegetables, therefore, can be a source of disease to humans just like fresh meat, milk, shell eggs and by-products. Besides in animals, *Salmonella* can adhere well to the work surfaces, and from there spread to other foodstuffs by cross-contamination (Møretrø *et al*., 2011). The examples are numerous and blatant: in the U.S. a major *Salmonella* ser. Enteritidis outbreak occurred and was associated with the consumption of industrial ice cream premix which was transported in tanks that had been used for carrying unpasteurized liquid eggs and were not properly sanitized (Hennessy *et al*., 1996). An outbreak of salmonellosis due to *S*. Ealing caused by dehydrated powdered milk was traced back to the inadequate sanitization of production equipment (Rowe *et al*., 1987). The thorough cleaning of work surfaces, both in food manufacturing facilities and in domestic kitchens, is therefore one of the main strategies for the prevention of foodborne salmonellosis (Møretrø et al., 2011). Generally, forms of gastroenteritis caused by non-typhoid *Salmonella* are moderately serious diseases with a quick recovery and without the need to resort to specific therapies. Although in some cases – when young children, elderly, or immunocompromised subjects are affected – salmonellosis may also lead to the patient's death (Pathan *et al*., 2010). The severity of *Salmonella* infections can also be aggravated by the fact that in recent years more and more *Salmonella* strains have been spreading and they are resistant to one or more of the antibiotics which are widely used in human medicine, such as fluoroquinolones and third generation cephalosporins. In addition to the Typhimurium serotype, *Salmonella* strains which are multiresistant to many antibiotics have also been detected in the Agona, Anatum, Choleraesuis, Derby, Dublin, Heidelberg, Kentucky, Newport, Pullorum, Schwarzengrund, Senftenberg, and Uganda serotypes (Yan *et al*., 2010). In most cases, human infection manifests itself through diarrhoea, persistent fever and abdominal cramps which appear 12 to 72 hours after the infection. The disease is selflimiting and clears up by itself within 4-7 days, but it has rather significant side effects: it takes months for the patient to regain proper bowel function and they can remain healthy carriers for months. In addition, chronic complications may occur such as widespread polyarthritis (Reiter's syndrome), ocular and urinary disorders, and even occasional cases of endocarditis and appendicitis. All these diseases are hard to treat even with antibiotics (Castillo *et al*., 2011).

According to the regulations currently in force in the European Union, it is the manufacturer's responsibility to ensure the hygiene of their production processes on a daily basis, seeing to prevent any possible *hazard* that may contaminate food and be harmful to human health. The system used by food manufacturers to control processing hygiene in their facilities is the wellknown HACCP system. In view of the fundamental principles of HACCP, if *Salmonella* contaminates a food, this is a *Hazard* because its presence could potentially cause harm to human health. It is, however, a hypothetical danger, as, for it to become real, the food has to

**1.2 The infective dose "issue"** 

present some specific conditions. One of these is certainly the "minimal infective dose", i.e. the lowest charge that *Salmonella* must reach in the food for it to become dangerous to human health. Generally, it is accepted that *Salmonella* becomes truly dangerous for humans when it reaches in a food a charge of at least 104 cfu/g. However, it should be reminded that the bibliography reports some foodborne salmonellosis outbreaks caused by foods that contained less than 100 and sometimes less than 10 cfu of bacteria per gram of product. Fatty foods, such as cheeses, butter and chocolate, better protect the bacteria from the digestive enzymes in the stomach. In addition, the low water activity of these foods keeps the salmonellae in a latent phase, and this means that they do not proliferate in the food substrate, but can survive for very long time (Jansson *et al*., 2011; Finstad *et al*., 2011). The infective charge in one episode of salmonellosis which occurred in Canada and was caused by chocolate was estimated as low as 0,005 cfu/g (Komitopoulou & Penaloza, 2009). It is important to underline that the foods contaminated with Salmonella do not usually show any modification in their sensory characteristics even though the pathogens within have reached very high levels, concretely harmful to human health (Lindhardt *et al*., 2009).

#### **1.3 Epidemiology of foodborne human salmonellosis in the EU**

According to the latest "European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks" (EFSA, 2011), in 2009 in the 27 EU Member States, the health authorities in charge have reported a total of 108,614 confirmed cases of human salmonellosis, with a prevalence of 23.7 cases/100,000 population. If we compare these levels with their equivalents reported from 2005 onwards, we discover that in the 2005/2009 period the cases of human salmonellosis have considerably dropped, estimated at -13%. In comparison, cases of campylobacteriosis have increased by +12%. In particular, between 2008 and 2009 there was a sharp decline in clinical cases of human salmonellosis caused by *Salmonella* ser. Enteritidis. All this indicates that the efforts made by health authorities and policies of individual EU states are obtaining positive and effective results. Furthermore, if we analyze the data regarding the spread of *Salmonella* among farm animals, we can find out that the importance of *Salmonella* as a cause of human foodborne disease is decreasing, also thanks to the decline in the spread of bacteria among livestock, starting with fowl. The decline in cases of foodborne salmonellosis among human beings does not tend to be consistent or regular in all 27 EU Members. The variations in the epidemiological pattern can be noticeable from one State to another. 10 states recorded a significant decline in cases; for 14 other states (including Italy) the epidemiological situation of human salmonellosis in food has remained essentially stable over the past five years, while Malta reported a sharp rise in cases (+24% compared to 2008), in contrast with the rest of the EU countries. Scandinavian and Central European countries are among the member states with the highest prevalence of human cases of foodborne salmonellosis while prevalence of salmonellosis among the population reported by the states bordering the Mediterranean are well below the previous. Epidemiologists interpret this as a sign of the single EU members' health authorities' increased awareness about the health of the populations under their responsibility. This increased attention to identify and report cases of foodborne salmonellosis explains the higher prevalence of human cases of salmonellosis in some northern European countries compared with the levels observed in Southern European countries. In most EU states food salmonellosis is a disease that patients contract "in their own country". Only Sweden, Finland, Denmark and the UK count a number of cases imported from abroad because they were contracted by people when they were out of the country. It should, however, be pointed out that some of the EU countries were not able

Food as Cause of Human Salmonellosis 51

serovar 4, [5], 12:i- has been observed in many European countries (Hopkins *et al*., 2010). It is resistant to ampicillin, streptomycin, sulphonamides and tetracycline in food-borne infections, in pigs and pork. The results indicate that genetically related strains of serovar 4, [5], 12:i:- of the DT193 and DT120 phage types with resistance to ampicillin, streptomycin, sulphonamides and tetracycline have emerged in many European countries and that pigs are the likely reservoir of the infection. A survey by the European Food Safety Authority has established the prevalence of *Salmonella* in pigs for slaughter in the EU-25 plus Norway (EFSA, 2008). This survey, as well as discovering that one pig every ten is affected, also identified the prevalent serotypes in infected pigs (*S*. Typhimurium and *S*. Derby), the same

Cattle are often colonized by S. Dublin and S. Typhimurium, with infections that vary in duration and clinical manifestation (Graziani *et al*., 2005). Cattle are particularly susceptible to infection by *Salmonella* in the first weeks of life (Cantoni & Ripamonti, 1998). S. Dublin can stay in the host for a long time, in some cases all its life and often causes serious bouts of illness (Graziani *et al*., 2005). As healthy carriers, they can pass S. Dublin and S. Typhimurium in their faeces, and those can remain viable in the outside for at least six

In the meat-processing industry, eggs and poultry meat are the main groups of raw materials which usually carry *Salmonella* (D'Aoust & Maurer, 2007) and in many States they overshadow other sources such as pork, beef and mutton as a means of infection (WHO, 1988). To conclude, we can say that the biological cycle of *Salmonella* spp. is complex (see Table 1) and involves animals, environment and food (D'Aoust & Maurer, 2007), and that animals

act as the most important reservoirs for its conservation (Graziani et al., 2005).

Table 1. *Salmonella* life cycle and transmission to humans (adapted from WHO, 1988).

ones as in the cases of human infection.

months (Cantoni & Ripamonti, 1998).

to ascertain and report to the EFSA the proportion of "national" cases of salmonellosis and those "acquired" from abroad. We would like to recall that in 2005 the EU issued the 2073/2005 (EC) Regulation which identified the *food safety criteria* for some of the major food groups most at risk of transmitting diseases to man. *Salmonella* was adopted as a parameter for the safety of fresh meat and products derived from it, raw milk and dairy products made with it, edible bivalve molluscs, as well as for pre-cut fruits and vegetables. In accordance with the EU provisions, *Salmonella* must be absent from 25 or 10 grams of examined sample of these foods in order for them to be destined for human consumption. In the EU which foodstuffs did not comply with this criterion and exceeded it? In 2009, as in 2008, the highest percentage of non-compliance was found in food derived from fresh meat, and particularly from minced meat and meat preparations containing chicken or turkey (8.7% of the total non-complying foods). Secondly, in order of prevalence, are bivalve molluscs and echinoderms, which are often traditionally consumed raw or hardly cooked (3.4% of all samples). Much less at risk are currently liquid eggs which go through a pasteurization process before entering the food manufacturing industry. Some concern arises from the fact that there are rather large percentages of non-compliance even among meat preparations for raw human consumption (the samples tested positive for *Salmonella* during official tests ranged from 1,2% to 1,7 % of the total tested samples).
