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Salmonella: Salmonellosis
Salmonella: Salmonellosis C Lo¨fstro¨m and T Hansen, Technical University of Denmark, Søborg, Denmark S Maurischat and B Malorny, Federal Institute for Risk Assessment, Berlin, Germany ã 2016 Elsevier Ltd. All rights reserved.
Introduction
Physiology
Salmonella remains one of the most important zoonotic pathogenic bacteria and is the causative agents of salmonellosis. The aim of this article is to give an overview of Salmonella and salmonellosis, starting by describing the characteristics of the microorganism Salmonella, including biochemical properties, physiology, classification, and nomenclature. Thereafter, the epidemiology of the organism is introduced, including the routes of transmission. Finally, the disease salmonellosis, the virulence mechanisms, and the prevalence in different types of food are described.
Characteristics of the Organism Biochemical Properties Salmonella are rod-shaped motile Gram-negative bacteria with flagella distributed uniformly over the cell surface (Figure 1). Nonflagellated variants and nonmotile strains containing dysfunctional flagella have been reported. Salmonellae are facultative anaerobic and nonspore-forming, able to utilize a wide variety of nutrients (chemoorganotrophic). They use both respiratory and fermentative metabolic pathways. Generally, Salmonellae are oxidase-negative and catalase-negative and grow on citrate as sole carbon source. Most strains produce hydrogen sulfide, decarboxylate lysine, and ornithine and do not utilize urea. Many of these traits are used when designing methods for identification, for example, selective xylose lysine deoxycholate agar plates used in the ISO 6579 standard method.
Classification and Nomenclature The Salmonella genus is a member of the family Enterobacteriaceae and contains two species, Salmonella enterica and Salmonella bongori, each consisting of multiple serotypes. S. enterica is subdivided into six subspecies: S. enterica ssp. enterica (I), S. enterica ssp. salamae (II), S. enterica ssp. arizonae (IIIa), S. enterica ssp. diarizonae (IIIb), S. enterica ssp. houtenae (IV), and S. enterica ssp. indica (VI). S. bongori was formerly classified as subspecies V. S. enterica ssp. enterica is mainly associated with humans and warm-blooded animals, whereas the other subspecies are found in cold-blooded animals and the environment. This classification scheme has historically undergone a number of rearrangements over time, as a result of the methodological improvements to reveal the true relationship between bacteria in this group. The subspecies of Salmonella are further separated into serovars according to the White–Kauffmann–Le Minor scheme. The separation of serovars is based on antigenic polymorphisms of lipopolysaccharide (O antigens), flagellar proteins (H antigens), and capsular polysaccharides (Vi antigens). Currently, about 2600 serovars of Salmonella have been identified, where most of the serotypes belong to S. enterica (Figure 2).
Encyclopedia of Food and Health
Salmonella can be found in natural environments, such as water, soil, and plants, but it does not seem to grow significantly there. However, since Salmonella are known to be quite resistant toward different environmental factors, it can survive for weeks in water and several years in soil, as long as the conditions are favorable. It has also been reported to survive for extended time periods in foods stored frozen or at room temperatures. The optimal growth temperature for Salmonella is 37 C, but the organism can grow in a quite broad range (7–45 C). In extreme cases, Salmonella growth in food has been observed at 2–4 C. Salmonella can grow at pH 4.5–9.5, with an optimum pH for growth at 6.5–7.5. The water activity (aw) needs to be 0.93 to support the growth of Salmonella in food and it is generally inhibited by the presence of 3–4% NaCl. Salmonella has a great ability to adapt to various more or less unfavorable environmental conditions, such as low pH, high temperature, and low aw. This makes these bacteria able to outcompete other microorganisms in, for example, different food production chains possibly leading to problems with cross contamination and biofilm formation in food production environments.
Epidemiology Salmonella is found worldwide and is an important zoonotic pathogen. It is estimated that Salmonella is responsible for 93.8 million cases of human gastroenteritis globally each year, and of these, 80.3 million cases are estimated to be foodborne. In the EU, 95 548 confirmed cases of human Salmonella infections were reported to the European Food Safety Authority in 2011. However, cases of salmonellosis are considered to be underreported as many of the sick individuals do not seek medical attention. Approximately 50 serovars (all in subspecies enterica) account for 99% of all clinical isolates from humans and warm-blooded animals. The two serotypes, Salmonella ser. Enteritidis and Salmonella ser. Typhimurium, are the most frequently reported serovars, together accounting for almost 70% of all human Salmonella infections in Europe. Human Salmonella ser. Enteritidis cases are mostly associated with the consumption of contaminated eggs and poultry meat, whereas Salmonella ser. Typhimurium cases most commonly are linked with the consumption of contaminated pork, beef, and poultry meat. The routes of Salmonella transmission to humans include the environment, contact with animals, and human-to-human contact (Figure 3). In industrialized countries, the main source of Salmonella infections is contaminated animal-derived food products, notably fresh meat and eggs as identified, for example, using source attribution models based on outbreak data. In developing countries, a large proportion of the human cases are from contaminated vegetables, water, and human-to-human
http://dx.doi.org/10.1016/B978-0-12-384947-2.00607-3
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transmission. Recently, there has been an increase in the reported outbreaks of salmonellosis caused by contamination of fruits and vegetables possibly related to the greater worldwide trade with fresh produce.
Salmonellosis Symptoms and Infectious Dose
1 mm Figure 1 A light microscope picture of Salmonella. ãDr. Jochen Reetz, Federal Institute for Risk Assessment (BfR), Berlin, Germany.
S. enterica is a common agent of foodborne infections and the human infections can range from enteric fever (typhoid fever) to nontyphoidal Salmonella, which normally cause gastroenteritis. A few Salmonella can cause infection, especially for the more susceptible part of the population (e.g., young, elderly, and immunocompromised), but the infective dose is generally considered to be >104 CFU g1. Low infectious doses have been reported especially for food with a high fat content, including cocoa butter in chocolate and animal fat in meat or with food or ingredients with low aw values. Enteric fever is associated with serotypes Typhi and Paratyphi A, B, and C, and symptoms include diarrhea, prolonged and spiking fever, abdominal pain, headache, and prostration appearing after an incubation period of 7–28 days. After the initial acute phase of the disease, an asymptomatic chronic carrier state is common. Under certain conditions, it is possible for Salmonella to cause invasive (systemic) disease, entering the
Figure 2 General overview of the current classification of Salmonella enterica. Reprinted from Achtman et al. (2012). Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog 8(6), p. e1002776.
Salmonella: Salmonellosis
703
Effluent slurry sewage Wildlife reservoirs
Environmental pollution Streams/pastures
Products eaten by man
Animal importation
Farm livestock
Man
Human imports Man Products eaten by animals Imported animal protein
Meat/bone meal dried poultry waste etc.
Food
Pet animals
Offal
Figure 3 Routes of transmission for Salmonella. Reprinted with permission from the World Health Organization (1983). Guidelines on prevention and control of salmonellosis, A. H. Hinton (ed.), Geneva, p. 22. Available online: http://apps.who.int/iris/bitstream/10665/66402/1/VPH_83.42_%28p1p66%29.pdf (Date: July 2014).
blood stream and spreading throughout the body. The nature and severity of Salmonella infections depend on several factors such as the infecting Salmonella serotype, virulence of the strain, infection dose, and the host. Treatments of enteric fever include the use of supportive therapy and/or antibiotics to avoid systemic infections. However, strains resistant toward several antibiotics have been increasingly found, limiting the usefulness of antibiotic treatment. Salmonellosis with nontyphoidal Salmonella strains has a shorter incubation period of 8–72 h after ingestion of the pathogen. Gastrointestinal infections are primarily caused by serovars such as Enteritidis and Typhimurium. Usually, Salmonella colonize the intestine by attaching to the epithelial cells using the fimbria, followed by invasion of the intestinal mucosa and multiplication in the gut-associated lymphoid tissue. Symptoms include nonbloody diarrhea and abdominal pain and the illness is usually self-limiting. Usually, only supportive therapy is used to treat nontyphoidal salmonellosis since the use of antibiotics has been found to prolong the carrier state. Nontyphoidal salmonellosis can also sometimes result in systemic infections and chronic conditions such as aseptic reactive arthritis.
Pathogenicity and Virulence Factors Salmonella enterica possesses a large set of virulence factors enabling it to withstand the host defense mechanisms, invade the gut epithelial layer, and persist in a localized area during gastrointestinal infections or even spread systemically in the
case of typhoid fever. Because bacterial growth and the expression of those factors often lead to immune activation, it has to be well regulated depending on the invasion level by regulatory proteins or two-component-systems (TCSs). One of the first lines of defense is the antimicrobial condition of the stomach. To survive especially at low pH, Salmonella uses a complex acid tolerance response (ATR) that consists of more than 50 acid shock proteins. The ATR is regulated depending on the growth phase by either rpoS-encoded sS (organic acids) or PhoPQ TCS (inorganic acids) during exponential growth and the OmpR/EnvZ TCS in the stationary phase, which is therefore also essential for persistence in the host cell phagosome. Nevertheless, only 1% of the bacteria reach the gut and adhere to the epithelium by the fimbriae, nonfimbrial adhesins like SiiE or BapA, and autotransporter proteins like ShdA, MisL, or SadA. The repertoire of fimbriae is strain-specific and consists of more than 20 different loci, which might be important for tissue and cell tropism. Type 1 fimbriae (Fim) bind to enterocytes, long polar fimbriae (Lpf) bind to microfold (M) cells, whereas the plasmid-encoded fimbriae (Pef) and thin aggregative or curli fimbriae (Tafi) adhere to epithelial cells in vitro. For the subsequent invasion of epithelial cells, macrophages, and dendritic cells, as well as for the intracellular persistence, Salmonella possesses two type III secretion systems (T3SSs). They are encoded on two Salmonella pathogenicity islands (SPI-1 and SPI-2), which are the most studied SPIs out of 21 known to date. Most of them are found in the majority of subspecies I Salmonella strains but a few are
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Salmonella: Salmonellosis
serovar-specific, like SPI-14 for Salmonella ser. Typhimurium or SPI-7, SPI-15, SPI-17, and SPI-18 for Salmonella ser. Typhi. Salmonella uses its SPI-1 T3SS to secrete at least six effector proteins into the host cell cytosol that are important for the invasion process: Salmonella outer proteins SopB, SopE, and SopE2 induce by activating Rho GTPases Cdc42, Rac1, and RhoG an actin rearrangement, while Salmonella invasion proteins SipA and SipC stabilize the newly formed actin filaments. This leads to a membrane ruffling followed by an uptake of the bacteria into macropinosomes. Fission of the membranes is supported by SopD. Especially SopB, SopE, and SopE2 activity has, besides its role in the invasion process, other important consequences. SopE/E2 contribute to inflammation by inducing MAPK pathways leading to IL8 expression in epithelial cells and inducing IL1-b secretion and pyroptotic effects in macrophages by enhancing caspase-1 activation. SopB is directly involved in promoting diarrhea by elevating intracellular Ca2þ level and inducing a chloride and water efflux. Another SPI-1 effector, SipA, has also been shown to contribute to fluid efflux. The SPI-2 T3SS and its effectors have been shown to promote intracellular survival although some effectors of both T3SSs are acting at the same time, which presumes a complex coordination of both SPI-1 and SPI-2 effector genes. HilA encoded by SPI-1 is known as master regulator of the SPI-1 effector expression, whereas three different TCSs SpiR/SsrB, EnvZ/OmpR, and PhoPQ are regulators of the SPI-2 virulence genes. The cross talk of the SPI-1/SPI-2 regulatory network is mediated by HilD, which controls both HilA expression and SsrAB expression. The main function of the 20 different SPI-2 effectors known to date is to facilitate intracellular replication by redirecting intracellular vesicular trafficking and maturation of the Salmonella-containing vacuole (SCV). The main effectors in that process are SifA, SseG, and SseF, which provoke the motion of the SCV in a juxtanuclear position, and SopD2, SifA, and PipB2, which are required for Salmonella-induced filament (Sif) formation by interacting with kinesin and the microtubule network. The function of Sifs for pathogenesis is still not completely understood but a Sif-forming ability has been shown to be necessary for full virulence in mice. In addition to the SPIs, a lot of virulence factors are encoded on mobile elements like prophages and plasmids. Certain Salmonella serovars harbor a serovar-specific, 50–100 kb large, low-copy-number plasmid. Part of that plasmid is the 8 kb Salmonella plasmid virulence (spv) operon. Signals resulting in spv transcription are typically conditions of the SCV interior like iron or nutrient depletion, elevated temperatures, a low pH or the stationary growth phase. The operon is regulated by SpvR and the chromosomal rpoS-encoded sS. This plasmid seems to be an important component for intracellular persistence and growth, cytotoxicity, and the systemic spread although such a plasmid is missing in prominent serovars causing typhoid or paratyphoid like Salmonella Typhi and Paratyphi A. In addition to spv, some serovars harbor specific virulence genes on their plasmids like rck that influences the serum resistance of Salmonella ser. Typhimurium and Enteritidis. The fimbrial gene pef is encoded on Salmonella ser. Typhimurium, Enteritidis, and Choleraesuis but not on Salmonella ser. Dublin or Gallinarum plasmids. Recently, a putative virulence plasmid called pR(ST98) has been described in Salmonella ser. Typhi, which supports drug
resistance and cytotoxicity to macrophages. Even antimicrobial resistance is often transmitted by the so-called R plasmids. For example, the multidrug resistance (MDR) phenotype of Salmonella ser. Typhi is associated with an (Inc)HI1 plasmid. In addition to those factors, Salmonella ser. Typhi harbors some exclusive virulence factors, which might explain its strong virulence in humans. A so-called typhoid toxin (CdtB/PltA/PltB) similar to the pertussis toxin has been identified, which is expressed intracellularly and causes para- and autocrinely cytotoxic effects. Target specificity is accomplished by specific carbohydrate moieties on surface glycoproteins. Another important factor is the expression of a virulence (Vi) capsular polysaccharide encoded by the viaB locus in a few Salmonella serovars. Depending on osmolarity, the expression of the Vi antigen is regulated by TviA and the EnvZ/OmpR TCS. Due to a regulatory network with RcsB, the induction of tviA and suppression of flagellin as well as T3SS genes are coregulated and support evasion from immune recognition. Altogether, Salmonella evolved a complex network of virulence genes and regulators enabling it to successfully adapt to different environmental conditions and evade or rather manipulate the host immune defense mechanisms in a serovar-specific and host species-specific manner to form its own niche.
Occurrence in Food Persistence in the Food Chain Environment The adaptation and persistence of Salmonella in food production chains have been suggested to be partly a result of bacterial attachment and surface colonization. Colonization of Salmonella on surfaces in contact with food provides a reservoir of bacteria that can increase the risk of contamination in the production line. Salmonella present in biofilm are generally more resistant toward environmental stresses, for example, disinfections, desiccation, organics acids, and heat. Besides attaching to the materials used in the production line, Salmonella can also attach to surfaces of the food product or be localized inside the food item for products such as vegetables and fruits.
Salmonella in Meat and Eggs Salmonella can enter at any stage of the food chain, from the livestock feed, at the slaughterhouse or packing plant; in manufacturing, processing, and retailing of food; and through catering and cooking at home. Colonized pigs constitute the main reservoir for carcass contamination in slaughterhouses and many of the carcasses harbor Salmonella on the skin or in the rectum. However, the slaughter line is an open process with many possibilities for cross contamination. Carcasses can throughout the slaughter line be contaminated from tools and equipment used for the slaughtering, and since the equipment can be very difficult to clean and sterilize, the Salmonella has the possibility to colonize and form biofilms. Eggs and egg products are another important source of Salmonella and salmonellosis. Salmonella are usually located on the shell surface but can also be found inside the egg making sanitation
Salmonella: Salmonellosis
difficult. Outbreaks associated with the consumption of eggs or egg products are common. Recent data on monitoring of the Salmonella prevalence in the United States found the following percent positive rate of Salmonella per product class for the calendar year 2012: young chicken (4.3%), market hog (1.3%), cow/bull (0.0%), steer/ heifer (1.1%), ground beef (1.9%), ground chicken (28%), ground turkey (11%), and turkey (2.2%). Data from the European Union report the following % positive rates: broiler meat and products thereof (5.9%), pig meat and products thereof (0.7%), and eggs and egg products (0.1%). Note that these data are difficult to compare as the sampling systems and analysis methods differ between different countries.
Salmonella in Fresh Produce Recently, there have been an increasing number of reported cases and outbreaks due to Salmonella in fruits and vegetables, for example, herbs, melons, and tomatoes, and products derived from fruits and vegetables, for example, peanut butter. Moreover, Salmonella has the ability to attach and survive in or on fresh produce and might contaminate through cultivation practices, handling, or processing. A large number of outbreaks have been caused by the use of contaminated water sources used to irrigate and wash produce crops. Fresh produce is mostly consumed without a heating step that would kill Salmonella, and as Salmonella also can be found inside the crops, rinsing is sometimes ineffective.
Other Sources Salmonella can also be found in a variety of other food items, such as milk, chocolate, and fish, and outbreaks due to these vehicles have been reported. Other routes of transmission include contact with animals and person-to-person transmission (Figure 3).
Conclusions Salmonella and salmonellosis (both enteric fever and nontyphoidal salmonellosis) remain significant problems worldwide, leading to increased public health concerns and economic burden on society. Salmonella adapts easily to various environmental conditions, giving it a competitive advantage toward other microorganisms, and enables survival in many different food products and food production chains. The molecular mechanisms for salmonellosis are complex, combining several different systems. This also contributes to the success of Salmonella to survive and proliferate in its hosts. Important vehicles contributing to the spread of Salmonella are different types of food, in particular meat, eggs, and fresh produce.
Acknowledgments The preparation of this article has been supported by a grant from the Ministry of Food, Agriculture and Fisheries of
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Denmark, the GUDP project UltraSal (grant number 340511-0349).
See also: Biofilms; Codex Alimentarius Commission: Role in International Food Standards Setting; Diarrheal Diseases; Eggs: Composition and Health Effects; Emerging Foodborne Enteric Bacterial Pathogens; Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects; Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection; Foodborne Pathogens; HACCP and ISO22000: Risk Assessment in Conjunction with Other Food Safety Tools Such as FMEA, Ishikawa Diagrams and Pareto; Laboratory Management: Microbiological Safety; Listeria: Listeriosis; Listeria: Properties and Occurrences; Meat: Eating Quality and Preservation; Milk Powder; Milk: Processing of Milk; Pasteurization: Principles and Applications; Pork Meat Quality, Production and Processing on; Poultry: Processing; Preservation of Foods; Risk Assessment of Foods and Chemicals in Foods; Salmonella: Detection; Salmonella: Properties and Occurrence; Shigella; Soy Beans: Processing; Staphylococcus: Food Poisoning; Staphylococcus: Occurrence and Properties; Vibrio: Types, Properties, and Determination; Yersinia enterocolitica: Properties and Occurrence; Yersinia enterocolitica: Detection and Treatment; Zoonoses.
Further Reading Salmonella in domestic animals. Barrow PA and Methner U (eds.) (2013) Wallingford, UK: CABI Publishing. Salmonella. Da Silva N, et al. (ed.) (2013) Microbiological examination methods of food and water, pp. 217–248. Leiden, The Netherlands: CRC Press/Balkema. EFSA and ECDC (2014) The European union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA Journal 12(2): 3547. http://dx.doi.org/10.2903/j.efsa.2014.3547. Fabrega A and Vila J (2013) Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clinical Microbiology Reviews 26(2): 308–341. Grimont AD and Weill FX (2007) Antigenic formulae of the Salmonella serovars, 9th ed. Paris: WHO collaborating centre for research on Salmonella, Institut Pasteur. Haeusler GM and Curtis N (2013) Non-typhoidal Salmonella in children: microbiology, epidemiology and treatment. Advances in Experimental Medicine and Biology 764: 13–26. International Organisation for Standardization (2002) ISO 6579:2002 Microbiology of food and animal feeding stuffs – horizontal method for the detection of Salmonella spp. Geneva, Switzerland: International Organisation for Standardization. Josefsen MH, Lo¨fstro¨m C, Olsen KEP, Mølbak K, and Hoorfar J (2011) Salmonella. In: Liu D (ed.) Molecular detection of human bacterial pathogens, pp. 1023–1035. Boca Raton, FL: CRC Press. Kothari A, Pruthi A, and Chugh TD (2008) The burden of enteric fever. Journal of Infection in Developing Countries 2(4): 253–259. Lee MB and Greig JD (2013) A review of nosocomial Salmonella outbreaks: infection control interventions found effective. Public Health 127(3): 199–206. Levin RE (2009) Salmonella. In: Rapid detection and characterization of foodborne pathogens by molecular techniques, pp. 79–138. Boca Raton, FL: CRC Press. Montville TJ, Matthews KR and Kniel KE (2012) Food microbiology an introduction, 3rd ed. Washington, DC: ASM Press. Foodborne infections and intoxications. Morris JG and Potter M (eds.) (2013) London, UK: Elsevier Science. Popoff MY and Le Minor LE (2005) Salmonella. In: Garrity GM, et al. (ed.) Bergey´s manual of systematic bacteriology. New York: Springer. Salmonella: from genome to function. Porwollik S (ed.) (2011) Poole, UK: Caister Academic Press.
Introduction
Physiology
Salmonella remains one of the most important zoonotic pathogenic bacteria and is the causative agents of salmonellosis. The aim of this article is to give an overview of Salmonella and salmonellosis, starting by describing the characteristics of the microorganism Salmonella, including biochemical properties, physiology, classification, and nomenclature. Thereafter, the epidemiology of the organism is introduced, including the routes of transmission. Finally, the disease salmonellosis, the virulence mechanisms, and the prevalence in different types of food are described.
Characteristics of the Organism Biochemical Properties Salmonella are rod-shaped motile Gram-negative bacteria with flagella distributed uniformly over the cell surface (Figure 1). Nonflagellated variants and nonmotile strains containing dysfunctional flagella have been reported. Salmonellae are facultative anaerobic and nonspore-forming, able to utilize a wide variety of nutrients (chemoorganotrophic). They use both respiratory and fermentative metabolic pathways. Generally, Salmonellae are oxidase-negative and catalase-negative and grow on citrate as sole carbon source. Most strains produce hydrogen sulfide, decarboxylate lysine, and ornithine and do not utilize urea. Many of these traits are used when designing methods for identification, for example, selective xylose lysine deoxycholate agar plates used in the ISO 6579 standard method.
Classification and Nomenclature The Salmonella genus is a member of the family Enterobacteriaceae and contains two species, Salmonella enterica and Salmonella bongori, each consisting of multiple serotypes. S. enterica is subdivided into six subspecies: S. enterica ssp. enterica (I), S. enterica ssp. salamae (II), S. enterica ssp. arizonae (IIIa), S. enterica ssp. diarizonae (IIIb), S. enterica ssp. houtenae (IV), and S. enterica ssp. indica (VI). S. bongori was formerly classified as subspecies V. S. enterica ssp. enterica is mainly associated with humans and warm-blooded animals, whereas the other subspecies are found in cold-blooded animals and the environment. This classification scheme has historically undergone a number of rearrangements over time, as a result of the methodological improvements to reveal the true relationship between bacteria in this group. The subspecies of Salmonella are further separated into serovars according to the White–Kauffmann–Le Minor scheme. The separation of serovars is based on antigenic polymorphisms of lipopolysaccharide (O antigens), flagellar proteins (H antigens), and capsular polysaccharides (Vi antigens). Currently, about 2600 serovars of Salmonella have been identified, where most of the serotypes belong to S. enterica (Figure 2).
Encyclopedia of Food and Health
Salmonella can be found in natural environments, such as water, soil, and plants, but it does not seem to grow significantly there. However, since Salmonella are known to be quite resistant toward different environmental factors, it can survive for weeks in water and several years in soil, as long as the conditions are favorable. It has also been reported to survive for extended time periods in foods stored frozen or at room temperatures. The optimal growth temperature for Salmonella is 37 C, but the organism can grow in a quite broad range (7–45 C). In extreme cases, Salmonella growth in food has been observed at 2–4 C. Salmonella can grow at pH 4.5–9.5, with an optimum pH for growth at 6.5–7.5. The water activity (aw) needs to be 0.93 to support the growth of Salmonella in food and it is generally inhibited by the presence of 3–4% NaCl. Salmonella has a great ability to adapt to various more or less unfavorable environmental conditions, such as low pH, high temperature, and low aw. This makes these bacteria able to outcompete other microorganisms in, for example, different food production chains possibly leading to problems with cross contamination and biofilm formation in food production environments.
Epidemiology Salmonella is found worldwide and is an important zoonotic pathogen. It is estimated that Salmonella is responsible for 93.8 million cases of human gastroenteritis globally each year, and of these, 80.3 million cases are estimated to be foodborne. In the EU, 95 548 confirmed cases of human Salmonella infections were reported to the European Food Safety Authority in 2011. However, cases of salmonellosis are considered to be underreported as many of the sick individuals do not seek medical attention. Approximately 50 serovars (all in subspecies enterica) account for 99% of all clinical isolates from humans and warm-blooded animals. The two serotypes, Salmonella ser. Enteritidis and Salmonella ser. Typhimurium, are the most frequently reported serovars, together accounting for almost 70% of all human Salmonella infections in Europe. Human Salmonella ser. Enteritidis cases are mostly associated with the consumption of contaminated eggs and poultry meat, whereas Salmonella ser. Typhimurium cases most commonly are linked with the consumption of contaminated pork, beef, and poultry meat. The routes of Salmonella transmission to humans include the environment, contact with animals, and human-to-human contact (Figure 3). In industrialized countries, the main source of Salmonella infections is contaminated animal-derived food products, notably fresh meat and eggs as identified, for example, using source attribution models based on outbreak data. In developing countries, a large proportion of the human cases are from contaminated vegetables, water, and human-to-human
http://dx.doi.org/10.1016/B978-0-12-384947-2.00607-3
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transmission. Recently, there has been an increase in the reported outbreaks of salmonellosis caused by contamination of fruits and vegetables possibly related to the greater worldwide trade with fresh produce.
Salmonellosis Symptoms and Infectious Dose
1 mm Figure 1 A light microscope picture of Salmonella. ãDr. Jochen Reetz, Federal Institute for Risk Assessment (BfR), Berlin, Germany.
S. enterica is a common agent of foodborne infections and the human infections can range from enteric fever (typhoid fever) to nontyphoidal Salmonella, which normally cause gastroenteritis. A few Salmonella can cause infection, especially for the more susceptible part of the population (e.g., young, elderly, and immunocompromised), but the infective dose is generally considered to be >104 CFU g1. Low infectious doses have been reported especially for food with a high fat content, including cocoa butter in chocolate and animal fat in meat or with food or ingredients with low aw values. Enteric fever is associated with serotypes Typhi and Paratyphi A, B, and C, and symptoms include diarrhea, prolonged and spiking fever, abdominal pain, headache, and prostration appearing after an incubation period of 7–28 days. After the initial acute phase of the disease, an asymptomatic chronic carrier state is common. Under certain conditions, it is possible for Salmonella to cause invasive (systemic) disease, entering the
Figure 2 General overview of the current classification of Salmonella enterica. Reprinted from Achtman et al. (2012). Multilocus sequence typing as a replacement for serotyping in Salmonella enterica. PLoS Pathog 8(6), p. e1002776.
Salmonella: Salmonellosis
703
Effluent slurry sewage Wildlife reservoirs
Environmental pollution Streams/pastures
Products eaten by man
Animal importation
Farm livestock
Man
Human imports Man Products eaten by animals Imported animal protein
Meat/bone meal dried poultry waste etc.
Food
Pet animals
Offal
Figure 3 Routes of transmission for Salmonella. Reprinted with permission from the World Health Organization (1983). Guidelines on prevention and control of salmonellosis, A. H. Hinton (ed.), Geneva, p. 22. Available online: http://apps.who.int/iris/bitstream/10665/66402/1/VPH_83.42_%28p1p66%29.pdf (Date: July 2014).
blood stream and spreading throughout the body. The nature and severity of Salmonella infections depend on several factors such as the infecting Salmonella serotype, virulence of the strain, infection dose, and the host. Treatments of enteric fever include the use of supportive therapy and/or antibiotics to avoid systemic infections. However, strains resistant toward several antibiotics have been increasingly found, limiting the usefulness of antibiotic treatment. Salmonellosis with nontyphoidal Salmonella strains has a shorter incubation period of 8–72 h after ingestion of the pathogen. Gastrointestinal infections are primarily caused by serovars such as Enteritidis and Typhimurium. Usually, Salmonella colonize the intestine by attaching to the epithelial cells using the fimbria, followed by invasion of the intestinal mucosa and multiplication in the gut-associated lymphoid tissue. Symptoms include nonbloody diarrhea and abdominal pain and the illness is usually self-limiting. Usually, only supportive therapy is used to treat nontyphoidal salmonellosis since the use of antibiotics has been found to prolong the carrier state. Nontyphoidal salmonellosis can also sometimes result in systemic infections and chronic conditions such as aseptic reactive arthritis.
Pathogenicity and Virulence Factors Salmonella enterica possesses a large set of virulence factors enabling it to withstand the host defense mechanisms, invade the gut epithelial layer, and persist in a localized area during gastrointestinal infections or even spread systemically in the
case of typhoid fever. Because bacterial growth and the expression of those factors often lead to immune activation, it has to be well regulated depending on the invasion level by regulatory proteins or two-component-systems (TCSs). One of the first lines of defense is the antimicrobial condition of the stomach. To survive especially at low pH, Salmonella uses a complex acid tolerance response (ATR) that consists of more than 50 acid shock proteins. The ATR is regulated depending on the growth phase by either rpoS-encoded sS (organic acids) or PhoPQ TCS (inorganic acids) during exponential growth and the OmpR/EnvZ TCS in the stationary phase, which is therefore also essential for persistence in the host cell phagosome. Nevertheless, only 1% of the bacteria reach the gut and adhere to the epithelium by the fimbriae, nonfimbrial adhesins like SiiE or BapA, and autotransporter proteins like ShdA, MisL, or SadA. The repertoire of fimbriae is strain-specific and consists of more than 20 different loci, which might be important for tissue and cell tropism. Type 1 fimbriae (Fim) bind to enterocytes, long polar fimbriae (Lpf) bind to microfold (M) cells, whereas the plasmid-encoded fimbriae (Pef) and thin aggregative or curli fimbriae (Tafi) adhere to epithelial cells in vitro. For the subsequent invasion of epithelial cells, macrophages, and dendritic cells, as well as for the intracellular persistence, Salmonella possesses two type III secretion systems (T3SSs). They are encoded on two Salmonella pathogenicity islands (SPI-1 and SPI-2), which are the most studied SPIs out of 21 known to date. Most of them are found in the majority of subspecies I Salmonella strains but a few are
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Salmonella: Salmonellosis
serovar-specific, like SPI-14 for Salmonella ser. Typhimurium or SPI-7, SPI-15, SPI-17, and SPI-18 for Salmonella ser. Typhi. Salmonella uses its SPI-1 T3SS to secrete at least six effector proteins into the host cell cytosol that are important for the invasion process: Salmonella outer proteins SopB, SopE, and SopE2 induce by activating Rho GTPases Cdc42, Rac1, and RhoG an actin rearrangement, while Salmonella invasion proteins SipA and SipC stabilize the newly formed actin filaments. This leads to a membrane ruffling followed by an uptake of the bacteria into macropinosomes. Fission of the membranes is supported by SopD. Especially SopB, SopE, and SopE2 activity has, besides its role in the invasion process, other important consequences. SopE/E2 contribute to inflammation by inducing MAPK pathways leading to IL8 expression in epithelial cells and inducing IL1-b secretion and pyroptotic effects in macrophages by enhancing caspase-1 activation. SopB is directly involved in promoting diarrhea by elevating intracellular Ca2þ level and inducing a chloride and water efflux. Another SPI-1 effector, SipA, has also been shown to contribute to fluid efflux. The SPI-2 T3SS and its effectors have been shown to promote intracellular survival although some effectors of both T3SSs are acting at the same time, which presumes a complex coordination of both SPI-1 and SPI-2 effector genes. HilA encoded by SPI-1 is known as master regulator of the SPI-1 effector expression, whereas three different TCSs SpiR/SsrB, EnvZ/OmpR, and PhoPQ are regulators of the SPI-2 virulence genes. The cross talk of the SPI-1/SPI-2 regulatory network is mediated by HilD, which controls both HilA expression and SsrAB expression. The main function of the 20 different SPI-2 effectors known to date is to facilitate intracellular replication by redirecting intracellular vesicular trafficking and maturation of the Salmonella-containing vacuole (SCV). The main effectors in that process are SifA, SseG, and SseF, which provoke the motion of the SCV in a juxtanuclear position, and SopD2, SifA, and PipB2, which are required for Salmonella-induced filament (Sif) formation by interacting with kinesin and the microtubule network. The function of Sifs for pathogenesis is still not completely understood but a Sif-forming ability has been shown to be necessary for full virulence in mice. In addition to the SPIs, a lot of virulence factors are encoded on mobile elements like prophages and plasmids. Certain Salmonella serovars harbor a serovar-specific, 50–100 kb large, low-copy-number plasmid. Part of that plasmid is the 8 kb Salmonella plasmid virulence (spv) operon. Signals resulting in spv transcription are typically conditions of the SCV interior like iron or nutrient depletion, elevated temperatures, a low pH or the stationary growth phase. The operon is regulated by SpvR and the chromosomal rpoS-encoded sS. This plasmid seems to be an important component for intracellular persistence and growth, cytotoxicity, and the systemic spread although such a plasmid is missing in prominent serovars causing typhoid or paratyphoid like Salmonella Typhi and Paratyphi A. In addition to spv, some serovars harbor specific virulence genes on their plasmids like rck that influences the serum resistance of Salmonella ser. Typhimurium and Enteritidis. The fimbrial gene pef is encoded on Salmonella ser. Typhimurium, Enteritidis, and Choleraesuis but not on Salmonella ser. Dublin or Gallinarum plasmids. Recently, a putative virulence plasmid called pR(ST98) has been described in Salmonella ser. Typhi, which supports drug
resistance and cytotoxicity to macrophages. Even antimicrobial resistance is often transmitted by the so-called R plasmids. For example, the multidrug resistance (MDR) phenotype of Salmonella ser. Typhi is associated with an (Inc)HI1 plasmid. In addition to those factors, Salmonella ser. Typhi harbors some exclusive virulence factors, which might explain its strong virulence in humans. A so-called typhoid toxin (CdtB/PltA/PltB) similar to the pertussis toxin has been identified, which is expressed intracellularly and causes para- and autocrinely cytotoxic effects. Target specificity is accomplished by specific carbohydrate moieties on surface glycoproteins. Another important factor is the expression of a virulence (Vi) capsular polysaccharide encoded by the viaB locus in a few Salmonella serovars. Depending on osmolarity, the expression of the Vi antigen is regulated by TviA and the EnvZ/OmpR TCS. Due to a regulatory network with RcsB, the induction of tviA and suppression of flagellin as well as T3SS genes are coregulated and support evasion from immune recognition. Altogether, Salmonella evolved a complex network of virulence genes and regulators enabling it to successfully adapt to different environmental conditions and evade or rather manipulate the host immune defense mechanisms in a serovar-specific and host species-specific manner to form its own niche.
Occurrence in Food Persistence in the Food Chain Environment The adaptation and persistence of Salmonella in food production chains have been suggested to be partly a result of bacterial attachment and surface colonization. Colonization of Salmonella on surfaces in contact with food provides a reservoir of bacteria that can increase the risk of contamination in the production line. Salmonella present in biofilm are generally more resistant toward environmental stresses, for example, disinfections, desiccation, organics acids, and heat. Besides attaching to the materials used in the production line, Salmonella can also attach to surfaces of the food product or be localized inside the food item for products such as vegetables and fruits.
Salmonella in Meat and Eggs Salmonella can enter at any stage of the food chain, from the livestock feed, at the slaughterhouse or packing plant; in manufacturing, processing, and retailing of food; and through catering and cooking at home. Colonized pigs constitute the main reservoir for carcass contamination in slaughterhouses and many of the carcasses harbor Salmonella on the skin or in the rectum. However, the slaughter line is an open process with many possibilities for cross contamination. Carcasses can throughout the slaughter line be contaminated from tools and equipment used for the slaughtering, and since the equipment can be very difficult to clean and sterilize, the Salmonella has the possibility to colonize and form biofilms. Eggs and egg products are another important source of Salmonella and salmonellosis. Salmonella are usually located on the shell surface but can also be found inside the egg making sanitation
Salmonella: Salmonellosis
difficult. Outbreaks associated with the consumption of eggs or egg products are common. Recent data on monitoring of the Salmonella prevalence in the United States found the following percent positive rate of Salmonella per product class for the calendar year 2012: young chicken (4.3%), market hog (1.3%), cow/bull (0.0%), steer/ heifer (1.1%), ground beef (1.9%), ground chicken (28%), ground turkey (11%), and turkey (2.2%). Data from the European Union report the following % positive rates: broiler meat and products thereof (5.9%), pig meat and products thereof (0.7%), and eggs and egg products (0.1%). Note that these data are difficult to compare as the sampling systems and analysis methods differ between different countries.
Salmonella in Fresh Produce Recently, there have been an increasing number of reported cases and outbreaks due to Salmonella in fruits and vegetables, for example, herbs, melons, and tomatoes, and products derived from fruits and vegetables, for example, peanut butter. Moreover, Salmonella has the ability to attach and survive in or on fresh produce and might contaminate through cultivation practices, handling, or processing. A large number of outbreaks have been caused by the use of contaminated water sources used to irrigate and wash produce crops. Fresh produce is mostly consumed without a heating step that would kill Salmonella, and as Salmonella also can be found inside the crops, rinsing is sometimes ineffective.
Other Sources Salmonella can also be found in a variety of other food items, such as milk, chocolate, and fish, and outbreaks due to these vehicles have been reported. Other routes of transmission include contact with animals and person-to-person transmission (Figure 3).
Conclusions Salmonella and salmonellosis (both enteric fever and nontyphoidal salmonellosis) remain significant problems worldwide, leading to increased public health concerns and economic burden on society. Salmonella adapts easily to various environmental conditions, giving it a competitive advantage toward other microorganisms, and enables survival in many different food products and food production chains. The molecular mechanisms for salmonellosis are complex, combining several different systems. This also contributes to the success of Salmonella to survive and proliferate in its hosts. Important vehicles contributing to the spread of Salmonella are different types of food, in particular meat, eggs, and fresh produce.
Acknowledgments The preparation of this article has been supported by a grant from the Ministry of Food, Agriculture and Fisheries of
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Denmark, the GUDP project UltraSal (grant number 340511-0349).
See also: Biofilms; Codex Alimentarius Commission: Role in International Food Standards Setting; Diarrheal Diseases; Eggs: Composition and Health Effects; Emerging Foodborne Enteric Bacterial Pathogens; Escherichia coli and Other Enterobacteriaceae: Food Poisoning and Health Effects; Escherichia coli and Other Enterobacteriaceae: Occurrence and Detection; Foodborne Pathogens; HACCP and ISO22000: Risk Assessment in Conjunction with Other Food Safety Tools Such as FMEA, Ishikawa Diagrams and Pareto; Laboratory Management: Microbiological Safety; Listeria: Listeriosis; Listeria: Properties and Occurrences; Meat: Eating Quality and Preservation; Milk Powder; Milk: Processing of Milk; Pasteurization: Principles and Applications; Pork Meat Quality, Production and Processing on; Poultry: Processing; Preservation of Foods; Risk Assessment of Foods and Chemicals in Foods; Salmonella: Detection; Salmonella: Properties and Occurrence; Shigella; Soy Beans: Processing; Staphylococcus: Food Poisoning; Staphylococcus: Occurrence and Properties; Vibrio: Types, Properties, and Determination; Yersinia enterocolitica: Properties and Occurrence; Yersinia enterocolitica: Detection and Treatment; Zoonoses.
Further Reading Salmonella in domestic animals. Barrow PA and Methner U (eds.) (2013) Wallingford, UK: CABI Publishing. Salmonella. Da Silva N, et al. (ed.) (2013) Microbiological examination methods of food and water, pp. 217–248. Leiden, The Netherlands: CRC Press/Balkema. EFSA and ECDC (2014) The European union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2012. EFSA Journal 12(2): 3547. http://dx.doi.org/10.2903/j.efsa.2014.3547. Fabrega A and Vila J (2013) Salmonella enterica serovar Typhimurium skills to succeed in the host: virulence and regulation. Clinical Microbiology Reviews 26(2): 308–341. Grimont AD and Weill FX (2007) Antigenic formulae of the Salmonella serovars, 9th ed. Paris: WHO collaborating centre for research on Salmonella, Institut Pasteur. Haeusler GM and Curtis N (2013) Non-typhoidal Salmonella in children: microbiology, epidemiology and treatment. Advances in Experimental Medicine and Biology 764: 13–26. International Organisation for Standardization (2002) ISO 6579:2002 Microbiology of food and animal feeding stuffs – horizontal method for the detection of Salmonella spp. Geneva, Switzerland: International Organisation for Standardization. Josefsen MH, Lo¨fstro¨m C, Olsen KEP, Mølbak K, and Hoorfar J (2011) Salmonella. In: Liu D (ed.) Molecular detection of human bacterial pathogens, pp. 1023–1035. Boca Raton, FL: CRC Press. Kothari A, Pruthi A, and Chugh TD (2008) The burden of enteric fever. Journal of Infection in Developing Countries 2(4): 253–259. Lee MB and Greig JD (2013) A review of nosocomial Salmonella outbreaks: infection control interventions found effective. Public Health 127(3): 199–206. Levin RE (2009) Salmonella. In: Rapid detection and characterization of foodborne pathogens by molecular techniques, pp. 79–138. Boca Raton, FL: CRC Press. Montville TJ, Matthews KR and Kniel KE (2012) Food microbiology an introduction, 3rd ed. Washington, DC: ASM Press. Foodborne infections and intoxications. Morris JG and Potter M (eds.) (2013) London, UK: Elsevier Science. Popoff MY and Le Minor LE (2005) Salmonella. In: Garrity GM, et al. (ed.) Bergey´s manual of systematic bacteriology. New York: Springer. Salmonella: from genome to function. Porwollik S (ed.) (2011) Poole, UK: Caister Academic Press.