23 Salmonellosis

Salmonellosis remains the second most common form of bacterial food-poisoning in the UK and in most of the developed economies. Although the number of isolations per annum has declined since 2000, over 10, 000 laboratory-confirmed cases are recognized each year in England and Wales, and over 150, 000 in Europe. Most of infections are associated with contaminated food, particularly of poultry origin, but also may originate from cattle and pigs, and to a lesser extent, sheep. The most common serovars from cases of human infection is Enteritidis, followed by Typhimurium. Contact with pets, particularly reptiles and amphibians is becoming an increasing problem and infections can be severe, particularly in children. Accurate and reproducible methods of identification and subtyping are crucial for meaningful epidemiological investigations, and traditional phenotypic methods of typing are now being supplemented by DNA-based methods such as pulsed-field gel electrophoresis (PFGE), variable number of tandem repeats analysis, and multilocus sequence typing. The use of such methods in combination with phenotypic methods has been invaluable for outbreak control at the international level. The occurrence of resistance to antimicrobial drugs is an increasing problem, particularly in relation to the development of resistance to antimicrobials regarded as ‘critically-important’ for last resort therapy in humans. Control measures such as vaccination of poultry flocks appear to have had a substantial impact on the number of infections with Salmonella Enteritidis. Nevertheless good hygiene practices in both catering establishments and the home remain essential for the control of infections at the local level.

Salmonella infection in humans can be manifest as non-typhoidal enterocolitis, non-typhoidal focal disease, or typhoid (enteric) fever, with symptoms ranging from enteric fever (typhoid and paratyphoid) through to septicaemia, mild to moderate gastroenteritis, or be asymptomatic. From a clinical perspective, the serovars of salmonella are usually considered as two broad groups. Firstly, those which are generally human-host restricted and responsible for the systemic invasive disease known as enteric fever, which include S. Typhi and S. Paratyphi A, B and C. All other serovars are usually referred to as non-typhoidal salmonella (NTS) and tend to cause a less severe is self-limiting gastroenteritis. A few NTS serovars may be associated with an ability to cause invasive human infections. Enteric fever typically results from infection with Salmonella Typhi or S. Paratyphi A, B or C. In general such organisms are highly host specific and are only carried by humans or other primates. Such infections are common in developing countries and for the most part result from contamination of water systems by human faecal material. The incidence of typhoid salmonellosis has markedly increased in many countries worldwide. In 2000 it was estimated that 21.6 million cases worldwide of typhoid fever caused 216, 500 deaths (Crump et al. 2004). The incidence of typhoid fever in south-central Asia, Southeast Asia, and, possibly, southern Africa was particularly high with >100 cases per 100, 000 population per year. Infections with typhoidal salmonella bacteria are also becoming common in developed countries in travellers returning from endemic areas in south east Asia, the Indian sub-continent and South and Central America.

The most common form of salmonellosis in both developed and developing countries is self-limiting gastro-enterititis. Symptoms include headache, muscle aches, diarrhoea, vomiting, abdominal cramping, chills, fever, nausea and dehydration. Symptoms usually appear 6 to 72 hours after ingestion of the bacteria, but can be longer if fewer organisms are ingested. Non-typhoidal salmonellosis is seldom fatal but the disease can be severe in young children and in elderly or immunocompromised patients. Non-typhoidal salmonellosis causes significant morbidity and mortality in terms of both human and animal disease, and its socioeconomic consequences are felt worldwide. Although not as severe as typhoid, infections with non-typhoidal salmonella are a serious cause of food-poisoning related disease in many developed countries, with between 196, 000 and 160, 000 cases reported each year in the period 2002–2006 in the 24 European Union (EU) countries (EFSA 2007b), and between 16, 000 and 10, 000 infections in England and Wales each year in the period 2001–2008 (Health Protection Agency (HPA) 2009). The most common serovars during this period have been Enteritidis and Typhimurium.

Salmonellosis in animals is normally associated with symptoms such as scouring, which can be particularly debilitating in calves, and also abortion. In the UK the predominant serovars in cattle are Typhimurium and Dublin, and in pigs, Typhimurium and Derby (Veterinary Laboratories Agency 2008). The symptoms resulting from infections with these serovars are often severe, resulting in substantive losses to producers. Other serovars, such as Enteritidis, Infantis, Virchow and Hadar are particularly common in poultry. These serotypes may not cause overt disease in their hosts and in contrast to infections with Typhimurium and Dublin in cattle, and Typhimurium in pigs, may not be a cause of serious economic loss to producers.

Non-typhoidal salmonellosis is traditionally regarded as a zoonoses. The transmission of organisms to humans usually occurs via consumption of contaminated foods. The most common sources of salmonella organisms associated with food poisoning are poultry and poultry products such as eggs, beef, and pork meat. Improperly prepared fruits, vegetables, dairy products, and shellfish have also been implicated as sources of Salmonella. Substantive international outbreaks have also been associated with products such as chocolate, peanuts and powdered infant formula preparations. Human-to-human and animal-to-human transmissions can also occur and amphibian and reptile exposures are associated with approximately 74 000 Salmonella infections annually in the US.

Salmonellae are Gram-negative motile bacilli that conform to the definition of the Enterobacteriaceae. The genus Salmonella was named after Daniel E. Salmon, an American veterinarian who first isolated Salmonella choleraesuis from pigs with hog cholera in 1884. Most strains are motile. Apart from a few exceptions, they produce acid and gas from glucose and mannitol, and usually also from sorbitol; they ferment sucrose or adonitol rarely, and rarely form indole. They do not hydrolyse urea or deaminate phenylalanine, usually form H₂S on triple sugar iron agar and use citrate as sole carbon source. They form lysine and ornithine decarboxylases. Salmonellae grow over a wide temperature range from 7 to 48°C, at pH 4–8 and at water activities above 0.93. Under special conditions, they may proliferate at <4°C and withstand extremes of pH <4. Over 2,500 serovars have been identified, which are closely related to each other by somatic and flagella antigens. Additionally most strains show diphasic variation of the flagella antigens (Threlfall 2005).

The Salmonella genus is now considered to comprise two species: S. enterica and S. bongori. There are 6 subspecies of S. enterica, the most important of which is S. enterica subsp. enterica (subspecies I) which includes the typhoid and paratyphoid bacilli and most of the other serotypes responsible for widespread disease in mammals (Table 23.1). Members of the other five subspecies (II–VI) are in the main parasites of cold-blooded animals or are found in the natural environment.

The terminology introduced by early workers accorded specific rank to each antigenically distinguishable salmonella type. The species names given were generally descriptive of the disease or the host with which the serotype was associated, and were sometimes incorrect. Thereafter, the convention was established that each new type should be named after the place in which it was

first isolated. Whereas the first published table contained some 20 serotypes, each considered to be a species, the current number is 2,579 (Popoff et al. 2004; Bale et al. 2007), an increase of 82 since 1984 (Table 20.1).

Salmonella organisms are no longer accorded specific status because more modern taxonomic techniques suggested that all serotypes of Salmonella probably belonged to one DNA hybridization group within which the seven subgroups are identified (Le Minor et al. 1982). DNA hybridization studies suggested that DNA subgroup V (S. enterica subsp. bongori) had evolved significantly from the other six subspecies (Le Minor et al. 1986). These observations were supported by multilocus enzyme electrophoresis (MLEE) studies. The species S. bongori was therefore proposed and agreed (Reeves et al. 1989) and subsequently confirmed by MLST http://pubmlst.org/databases.shtml.

In the latest classification only serovars of subsp. enterica are still named; serovars of other subspecies of S. enterica and of S. bongori are no longer named but designated by their antigenic formulae. Designations such as Salmonella ser. Typhimurium, Salmonella Typhimurium or even simply Typhimurium are convenient for use in clinical situations and indicate that the named serotype is a member of subsp. enterica. Subtypes of serotypes recognized by phage typing or biotyping should not be named as if they were serotypes. Thus, the names of salmonellae (e.g. ‘S. Java’) distinguishable from established serotypes by biotype characters has been deleted from the updated version of the Kauffmann–White scheme as published by Popoff et al. (2004). Nevertheless it is important to realise that for some serovars, particularly S. Paratyphi B, which caused both enteric fever and more typical gastrointestinal disease, biotype classification may be important. Thus when infections with S. Paratyphi B biotype variant Java are recognized then the initial designation of S. Paratyphi B is sometimes supplemented by the use of the ‘biotype Java’ designation for epidemiological investigations.

The antigens used to define the serological types of salmonellae include the O antigens—heat-stable polysaccharides that form part of the cell wall lipopolysaccharide (LPS), the H antigens—heat-labile proteins of the flagella that in salmonellae have the almost unique character of diphasic variation and surface polysaccharides that inhibit the agglutinability of the organisms by homologous O antisera, of which the Vi antigen of Typhi and Dublin are two of the most important examples.

Serotyping, based on antigenic variation in the ‘O’ and ‘H’ antigen structure and encapsulated in the Kauffmann-White scheme, is the basic method for the primary subdivision of salmonella organisms after preliminary speciation. The method, which has been in use since the 1930s, is internationally accepted and although several DNA- and array-based methods are being developed (Fields et al. 2002), serotyping still remains the internationallyrecognized method for the preliminary identification of subtypes within species.

Due to the common occurrence of infections caused by Salmonella, and due to the variety of sources of the infection in particular regarding NTS, it is in most cases not sufficient to simply perform a successful serotyping procedure in order to gain insights into the epidemiology and roots of the infections. Therefore, numerous subtyping/fingerprinting methods have been developed to dissect further the origin of salmonella isolates. Such methods may broadly be considered as phenotypic, genotypic and sequence-based. Different techniques are useful in different circumstances, depending on the reason for the investigations. In practice a combination of methods is often used.

The principal phenotypic subtyping methods used are phage typing and antimicrobial susceptibility determination.

The underlying principle of phage typing is the host specificity of bacteriophages and on this basis several phage-typing schemes have been developed for serotypes of clinical or epidemiological importance (Threlfall et al. 1990).

The first phage typing scheme based on the principle of phage adaptation was that developed for the differentiation of Typhi (Craigie et al. 1938); in this scheme progressive adaptations were made of Vi phage II, which is specific for the Vi (capsular) antigen of Typhi. Phage typing schemes for other serotypes depend to a limited extent on phage adaptability and, for the most part, are based on patterns of lysis produced by serologically distinct phages isolated from a variety of sources. Other serotypes for which published phage typing schemes are in routine use in the UK include: Paratyphi B, Typhimurium Hadar, Enteritidis and Virchow. More than 80 phage types are now recognized in the Enteritidis scheme. For the Typhimurium scheme, 232 phage types were designated and a further 50 types have been subsequently recognized. Phage typing schemes for Enteritidis and Typhimurium use in different countries of the EU have now been rationalized as part of an international collaborative venture for salmonella surveillance (Fisher et al. 1994; Fisher, 1999). Thus, while apparently ‘old-fashioned,’ phage typing still represents a fairly robust and discriminating subtyping approach and is internationallyaccepted. More recently attempts have been made to develop DNA-based phage typing for Typhimurium, utilizing phage-type specific markers revealed by fluorescent amplified fragment length polymorphism fingerprinting) of isolates of Australian origin (Hu et al. 2002). It is possible that this method may be used for the development of a microarray-based molecular phage-typing scheme for this and other serotypes.

Determination of the pattern of sensitivity to a defined panel of antimicrobial drugs (antimicrobials) can be used for subdivision within serovar or phage type. Although the antimicrobial susceptibility pattern cannot be used as definitive, such patterns can be used to define clones, such as the internationally distributed clone of S. Typhimurium definitive phage type (DT) 104 with resistance to ampicillin (A), chloramphenicol (C), streptomycin (S), sulphonamides (Su) and tetracyclines (T) (= S. Typhimurium DT 104 ACSSuT) (Threlfall 2000), or as the clone is referred to in North America, penta-resistant DT104. Nevertheless it should be realized that although antimicrobial susceptibility patterns may provide useful information at individual and population level, the same phenotypic resistance pattern may be due to different genetic mechanisms. Additionally resistance may change very rapidly, often in response to selection pressure impose by antimicrobial usage.

Molecular biological tools have been developed for subtyping serovars of Salmonella for many years. Genetic typing methods can be broadly divided into extrachromosomal analysis (plasmid profiling, restriction digest of plasmids) and chromosomal analysis (ribotyping, IS200 typing; macrorestriction of genomic DNA visualized by PFGE typing). Plasmid profiling has been used for many years to subtype salmonellae in outbreak investigations. While technically fairly convenient, plain comparison of plasmid profiles can be misleading as plasmids of the same size may be quite different when subjected to restriction enzyme digest analysis. The use of PFGE has become the ‘gold standard’ for international comparison of salmonella isolates. One reason for this is the considerable progress in standardization of methods. International networks, such as PulseNet (Swaminathaan et al. 2006), and Enter-net (Fisher 1999), have ensured reproducibility and comparability of typing data between different laboratories. Furthermore, the typing data can be stored in silico as reference material, or for further comparison.

Typing schemes based on variation in particular DNA sequences have the advantage of being digital in nature. This means that the same results should be achieved wherever the test is performed and any comparison of results is simple, quantitative and absolute in nature. Sequence-based typing schemes can also be considered as classification schemes and so genetic and evolutionary inferences can be made. Current DNA- sequence-based typing methods include the detection of DNA repeats and single nucleotide polymorphisms (SNPs). Variable Number of Tandem Repeat variation (VNTR) does not penetrate into the actual DNA sequence but produces data on the copy number of short repetitive sequences of individual isolates, by determining the size of the PCR product (Lindstedt 2005). To date, VNTR is able to differentiate reliably between isolates of Typhimurium, and VNTR for the differentiation of Enteritidis is underway. MLST, which is targeted at comparing sequence diversity at multiple, conserved housekeeping genes (Maiden et al. 1998), has been applied to seven housekeeping genes in a collection of 26 isolates of S. Typhi collected from diverse geographical locations (Kidgell 2003). The results confirmed earlier findings from MLEE typing and conclusively demonstrated the existence of four sequence types of S. Typhi from distinct geographical areas as well as providing an estimate of the age of the organism (ca. 50, 000 years old) (Kidgell et al. 2002). Because the genes chosen for MLST are selectively neutral, not under selective pressure, the variation detected represents the true phylogenetic relationships between strains. At present the main promise for MLST is as a replacement for serotyping. Kidgell et al. demonstrated that MLST can form the basis of an identification scheme for S. Typhi but for sub-typing, more SNPs from around the genome are needed (Holt et al. 2008) Typing using SNP-based methods are under development for S. Typhi (Baker et al. 2008)and for S. Paratyphi A (Holt et al. 2009). At present these methods are expensive and so the detection of SNPs remains the preserve of the research institutions for evolutionary studies and of well-funded reference laboratories for molecular epidemiology.

The availability of a large amount of genomic sequence from different strains of Salmonella in combination with microarray technology opens yet another perspective for typing of salmonellae. Microarrays containing all known sequences from S. enterica serotypes have already been prepared and used to interrogate the gene content of different serotypes. Although not currently suitable for diagnostic laboratories these methods may find utility in reference labs for defining some aspects of genetic diversity such as antimicrobial resistance. Perhaps in the era of high-throughput nucleic acid re-sequencing whole genome analysis will be the ultimate typing method—this is, however, not yet possible.

The advantages and disadvantages of the various phenotypic, genotypic and sequence-based typing methods for the differentiation of Salmonella for outbreak tracing have recently been reviewed by Cook et al. (2006).

The ability of salmonella organisms to invade, survive and replicate within eukaryotic cells is essential for successful infection. S. enterica usually enters the host by the oral route. Although the infectious dose varies among Salmonella strains, a large inoculum is thought to be necessary to overcome stomach acidity and to compete with normal intestinal flora. Large inocula are also associated with higher rates of illness and shorter incubation periods. In general, about 10⁶ bacterial cells are needed to cause infection. Low gastric acidity, which is common in elderly persons and among individuals who use antacids, can decrease the infective dose to 10³ cells, while prior vaccination can increase the number to 10⁹ cells.

In contrast to other invasive enteric bacteria such as Yersinia spp. and enteropathogenic Escherichia coli, salmonellae require neither motility nor fimbriae to adhere to and invade eukaryotic cells. The induction of de novo protein synthesis is necessary for the invasion and is regulated by the microenvironment (low oxygen), growth phase, or the epithelial cell surface. Only viable, metabolically-active salmonellas can adhere to host cell surfaces. Adherence is followed almost immediately by internalization into the host cells.

After ingestion, infection with salmonellae is characterized by attachment of the bacteria by fimbriae or pili to cells lining the intestinal lumen. Salmonellae selectively attach to specialized epithelial cells (M cells) of the Peyer patches. The bacteria are then internalized by receptor-mediated endocytosis and transported within phagosomes to the lamina propria, where they are released, thereby effecting a massive efflux of electrolytes and water into the intestinal lumen. Once there, salmonellae induce an influx of macrophages (typhoidal strains) or neutrophils (non-typhoidal strains).

Salmonellae possess several virulence-promoting factors which contribute to the disease process. These virulence factors are generally conserved between species, as are the mechanisms by which salmonella bacteria interact with the host cell. Such virulence factors are complex and encoded both on the organism’s chromosome and on large (34–120 kd) plasmids. Ten Salmonella pathogenicity islands have been identified that amongst other properties contributing to virulence, mediate uptake of the bacteria into epithelial cells (type III secretion system [TTSS]), non-phagocytic cell invasion (Salmonella pathogenicity-island 1 [SPI-1]), and survival and replication within macrophages (Salmonella pathogenicity-island 2 [SPI-2], phoP/phoQ). Other putative virulence factors include outer membrane proteins, secreted proteins, Vi antigen encoding genes, virulence markers located in prophages (e.g. sopE1, encoded by phage SopEphi), and genes belonging to various fimbrial clusters (Hensel 2004).

The Col V plasmid and related plasmids of the FI plasmid incompatibility group have been found to possess genes coding for the production of the siderophore aerobactin, a hydroxamate iron transport compound which enhances growth of host organisms in iron-deplete media such as blood. Genes from the Salmonella Pathogenicity Islands (SPIs) tend to be highly conserved throughout serovars although some genes may be deleted in some strains. Other putative virulence factors are those associated with genes coding for antimicrobial drug resistance (AMR), such as Salmonella Genomic Island 1 (SGI1) (Boyd at al. 2001), although as yet an association between the presence of SGI1 and enhanced virulence has not been conclusively established.

Salmonella strains belonging to a range of serovars possess plasmids of high relative molecular mass (M_I) which can be regarded as ‘serovar-specific’. Serovars which harbour such plasmids include Enteritidis, Typhimurium, Dublin, Gallinarum, Pullorum, Abortusovis, Cholerae-suis, Paratyphi C, Blegdam, Rostock, Newport, and Moscow. In contrast such plasmids have not been identified in epidemiologically-important serovars such as Hadar, Infantis or Virchow, and there are phage type differences in serovars such as Typhimurium. Such plasmids range in M_I from about 45 kb in Choleraesuis to 93 kb in Typhimurium. Although for the most part serovar-specific, there are some exceptions. The closely-related serovars Rostock and Dublin harbour an identical virulence plasmid, and different virulence plasmids are found in Enteritidis and Pullorum. Such plasmids have been shown to be involved in the virulence of their host organisms for mice and chickens. Genes on the virulence region include orfE, spvD, spvC, spvB, spvA, spvR, vagC and vagD (Gulig et al. 1993). Six spv genes within a common 8 kb SalI–XhoI fragment have now been sequenced, namely spvR, spvA, spvB, spvC, spvD and spvE. The spvE gene encodes a 13 kDa protein, the spvD gene a 25 kDa protein and the spvC gene a 28 kDa protein, which is located both in the outer membrane and the cytoplasm. The latter protein has a demonstrable role in virulence, as it has been shown that transposon insertion mutants in the spvC gene of SSP result in a significant drop in the number of Typhimurium isolated from the spleen of orally infected mice. The spvB gene encodes a 65 kDa protein of unknown function which is located both in the outer membrane and the cytoplasm, and the spvA gene a 28 kDa protein also of unknown function.

Although essential for the virulence of the host organism in certain strains of mice the role of these genes in the virulence process in humans and other animal species is not clear. Serovar-specific virulence plasmids have been shown to be necessary for intracellular survival of salmonellas in mouse phagocytes and it is possible that they may contribute to the invasive process in humans and food animals such as cattle after the initial infection and cellular attachment process.

The severity of illness in individuals with salmonellosis is determined not only by the virulence factors of the infecting strain but also by host properties. Such properties include corticosteroid use, malignancy, diabetes, HIV infection, prior antimicrobial therapy, and immunosuppressive therapy. In developing countries sickle cell disease, malaria, schistosomiasis, bartonellosis, and pernicious anaemia have been mentioned as other co-morbidities that predispose to salmonellosis.

Since the early 1980s the epidemiology of Salmonella infection in humans in the UK and most European countries has been dominated by two serovars, namely Enteritidis and Typhimurium (Fig. 23.1).

Enteritidis is commonly among the top three serovars from cases of human infection in many developed countries world wide. In the UK, for example, reported isolations increased 16-fold in the period 1981–94 whilst in the EU there were estimated to be 180, 000 Enteritidis infections in the years 2004–2006 (EFSA 2007b). The main reservoir of S. Enteritidis is poultry, with poultry meat and contaminated eggs being important vehicles of human infection. The unprecedented epidemic of Enteritidis in the USA, UK and many several other countries in the late 1980s and early 1990s was caused by a strain of phage type (PT) 4 which spread in poultry flocks, selected by environmental pressures associated with poultry husbandry. An unusual feature of the epidemic was the transovarian spread of the strain in the avian host.

Since 1997 in the UK there has been a dramatic decline in incidence (Fig. 23.1). The reasons for this decline are multi-factorial. Several codes of practice for the control of salmonellas in chickens have been in operation in the UK since 1993. There have also been many improvements in the poultry industry in infection control and hygiene at breeding sites, and vaccination against S. Enteritidis started in breeder flocks in the UK in 1994 and in layer flocks in 1998 (Davies et al. 2003). Nevertheless, Enteritidis still remains the most common serotype from humans in the UK and since 2002 there have been a substantial number of outbreaks of infection caused by phage types other than PT 4 (O’Brien et al. 2004). In 2002–03 national surveillance databases at the Health Protection Agency (HPA) identified an increase in the incidence of infection due to a number of specific strains of Salmonella. Cases were widely distributed. Interlocking national and local epidemiological investigations were conducted to determine the source of these strains. These investigations demonstrated associations between infection and the consumption of foods prepared in the commercial catering and retail sectors and containing raw shell egg. The findings generated a further series of multidisciplinary food and environmental investigations coordinated by the HPA Centre for Infections and involving a number of Health Protection Units, local authorities and laboratories throughout England. Eggs imported from Spain used by caterers were implicated in local outbreaks and microbiological investigations showed that Spanish eggs were contaminated at high rates with strains of Salmonella. The nationally collated data from local outbreak investigations identified key interventions in catering practices.

In 2004 a Standing Multi-agency National Outbreak Control Team (comprised of the HPA, Food Standards Agency (FSA), Department of Environment, Food and Rural Affairs (Defra), Health Protection [Scotland] and National Public Health Service for Wales) was convened to coordinate the development and implementation of local and national control measures. Guidance documents were produced for the catering, healthcare, retail, and wholesale sectors and discussions were held with the Spanish Government Agencies and egg producers. These actions have resulted in a sharp decline in the importation of eggs from Spain and a concomitant fall in the incidence of the outbreak strains of Salmonella.

Salmonella Typhimurium is one of the top three serovars isolated world wide over many decades from humans, domestic and wild animals, foodstuffs, and the environment, with cattle and poultry important sources of infection for humans (Palmer et al. 1986). Over the last two decades the key organism within serovar Typhimurium has been the MDR clone of S. Typhimurium DT 104 (=MDR DT 104) (see above).

Strains of MR DT104 were identified in cattle in the United Kingdom in the late 1980s. The strain was subsequently transmitted to humans through the food chain, but also became common in poultry (particularly turkeys), pigs and sheep. Throughout the 1990s, MDR DT104 spread to other parts of the world, particularly in Europe and North America where molecular epidemiological studies have contributed to the consensus that MDR DT104 represents a globally disseminated clone. Although isolations of MDR DT104 have, in general been decreasing since 1997 (Fig. 23.2), this phage type continues to be of clinical concern not only due to its rapid dissemination, but also because it has been associated with increased morbidity and mortality and because of its ability to readily acquire additional resistance to other clinically important antimicrobials such as the fluoroquinolones, trimethoprim, aminoglycosides, and cephalosporins.

The free movement of people and foodstuffs between countries are effective ways of distributing disease-causing salmonella organisms internationally. To control international outbreaks there is a requirement for a mechanism whereby data and information on potential salmonella outbreaks can be disseminated rapidly to those who need to know. Within Europe this requirement was fulfilled until 2007 by the Enter-net dedicated surveillance network complimented by the Salm-gene molecular typing network (Fisher et al. 2005). Data on epidemiological and microbiological features on current cases, as well as background levels of infections were immediately available within the Enter-net databases. The Salm-gene network with its database of harmonized salmonella PFGE patterns from the participating European countries provided immediate, and electronically exchangeable, DNA fingerprints of outbreak strains. This prompt electronic dissemination of information regarding unusual events with international implications ensured that public health interventions could be implemented and cases of food-borne salmonella disease prevented. Examples of international salmonella outbreaks recognized by Enter-net from 2000–2007 are shown in Table 23.2.

In 2007 Enter-net was subsumed into the European Centre for Disease Prevention and Control (ECDC), and investigations of outbreaks have been coordinated by ECDC.

In the UK in 2009 tetracycline-resistant S. Typhimurium DT 191a associated with pet snakes have caused over 200 infections in humans. The source of the antimicrobial-resistant strain is thought to be imported frozen mice used as food for the reptiles (Anon. 2009; Hawker et al. 2010). Reptiles, including snakes and turtles, have also been associated with substantive outbreaks of salmonellosis in other European countries and the USA (Bertrand et al. 2008; Lee et al. 2008; Anon. 2007). In the USA there have also been reports of the transmission of strains of S. Typhimurium and S. Virchow from pets to humans (CDC. 2001; Sato et al. 2000; Swanson et al. 2007). In Australia, ornamental fish tanks have been identified as reservoirs for MDR S. Paratyphi variant Java (Levings et al. 2006). Although not strictly from pets, in Canada MDR  S. Newport associated with pet treats has caused infections in both humans and dogs (Pitout et al. 2003).

In the UK information on Salmonella in livestock is collated by the Veterinary Laboratories Agency. In 2007 the most common serovars in cattle were Dublin (59% of incidents) followed by Typhimurium (14%). In pigs it was Typhimurium (70% of incidents) followed by Derby (8%). In poultry it was Enteritidis (53%) followed by Typhimurium (13%) (VLA, 2008). The most common phage type in Typhimurium from cattle was DT104, and in pigs U288 (66% of incidents). In isolates of Enteritidis from poultry PT 4 predominated (47% of incidents). In the EU the most common serovar in both breeder flocks and layer flocks in 2006 was Enteritidis, but the most common serovar identified in broiler meat was Infantis. In pig meat the most common serovar in 2006 was Typhimurium, followed by Derby, and in cattle, Typhimurium predominated followed by Goldcoast (EFSA 2007b).

Control of salmonella is a multifactorial process. Reduction of the occurrence of specific types in their food animal host by measures such as immunization is obviously key in reducing the overall burden of infection, as has been demonstrated with the protracted S. Enteritidis outbreaks referred to above. Reduction in the use of antimicrobials in food-producing animals will result in a reduction in the occurrence of specific types exhibiting resistance to such antimicrobials, as exemplified by MR S. Typhimurium DT 104. Withdrawal of a contaminated product is also a method of control, but normally relates to products such as salads, salad vegetables and chocolate. Contamination of such products often involves organisms originating from a food production animal at some stage in the production process.

Good hygiene practices in restaurants and mass catering establishments will also contribute to salmonella control. Despite a plethora of advice and guidelines, outbreaks of infection still occur when basic hygiene practices are ignored, such as correct storage, defrosting and thorough cooking. Salmonella bacteria are killed when food is thoroughly cooked. This means cooking minced beef and similar products to at least 68°C and ensuring that all food is cooked properly. Once cooked, any food held in a buffet should be kept hotter than 60°C. Cross-contamination may be avoided by using different utensils, plates, cutting boards, and counter tops before and after cooking. Cooked food that stands at room temperature for a long time, especially poultry, is at risk. Infected persons should not be allowed to handle food or work in the kitchen before at least three negative samples have been submitted.

In the home environment basic hygiene procedures such as washing hands in hot soapy water after toileting and before handling foods are essential. Frozen foods should be thoroughly defrosted before cooking and refrigerator temperatures should be kept colder than 4°C. Raw milk and recipes involving raw or lightly-cooked eggs should be avoided. Because fruits and vegetables have now been identified as a source of salmonella, it is important that these food items be thoroughly washed in running water before they are eaten. Cutting boards for raw meat and poultry should not be used for cheese, raw vegetables and other foods that will not be cooked before being served.

Additionally infections caused by highly pathogenic salmonella bacteria are becoming increasingly associated with domestic pets, including snakes, terrapins and other reptiles as well as dogs and cats. It is essential that hand washing procedures such as outlined above are implemented after handling pets and in the case of young children, access to pets such as snakes and reptiles should be actively discouraged.

Although numbers are dropping, salmonellosis remains the second most common form of bacterial food-poisoning in the UK and in most European countries. The majority of infections are associated with contaminated food, particularly of poultry origin but also from originating from cattle and pigs, and to a lesser extent, sheep. The occurrence of resistance to ‘critically important’ antimicrobial drugs is increasing. Control measures such as vaccination of poultry flocks appear to have had a substantial impact on the number of infections with Salmonella Enteritidis. Nevertheless good hygiene practices in both catering establishments and the home remain essential for the control of infections at the local level and should be actively encouraged by health practitioners at all times.

Antimicrobial drug resistance in Salmonella has increased worldwide leading to treatment failures in human and animal infectious diseases. Serious concerns about such resistance have been growing for a number of years and have been raised at both national and international levels. For Salmonella, of particular concern are quinolone resistance, resistance to third- and fourth-generation cephalosporins (WHO 2007), and the appearance and spread of strains exhibiting multiple resistance as a result of the acquisition of the Salmonella Genomic Island 1 (Boyd et al. 2001; Amar et al. 2008) (see above).

Two fundamental types of quinolone resistance in Salmonella have been identified, namely chromosomally-mediated quinolone resistance and plasmid-mediated quinolone resistance (PMQR). Chromosomal resistance to quinolones, arises spontaneously under antimicrobial pressure due to point mutations that result in: (i) amino acid substitutions within the topoisomerase II (DNA gyrase) and IV subunits gyrA, gyrB, parC or parE, (ii) decreased expression of outer membrane porins or alteration of LPS, or (iii) over expression of multi-drug efflux pumps. Mutations in the gyrA, gyrB, parC, or parE genes in regions that form the fluoroquinolone binding site (termed the Quinolone Resistance-Determining Region, QRDR) change the topoisomerase structure in a way that fluoroquinolones (FQs) are unable to bind to these target sites. Single mutations affect firstly only older quinolones such as nalidixic acid in their inhibitory action. The MIC for nalidixic acid is in the range of 64–128 mg/l, whereas the MICs for FQs are generally in the range of 0.25–1.0 mg/l. This level of resistance is generally regarded as ‘epidemiological’. Additional mutations are required to decrease the susceptibility to later and more recently-introduced FQs such as ciprofloxacin. These additional mutations result in the development of ‘clinical resistance’, with MICs of greater than 2 mg/l.

Plasmid-mediated quinolone resistance (PMQR) is mediated by genes (qnr) encoding proteins that protect DNA gyrase from inhibition by ciprofloxacin. One such gene, qnrA confers resistance to nalidixic acid (MIC; 8–16 mg/l) and epidemiological resistance to FQs (ciprofloxacin MIC: 0.25–1.0 mg/l). The basal level of quinolone resistance provided by qnr genes is low and strains can appear susceptible to quinolones according to the USA Clinical Laboratory Standards Institute (CLSI) guidelines. Their clinical importance lies in increasing the MIC of quinolone-resistant strains of Salmonella to levels that are clinically-relevant.

In the UK fluoroquinolones were licensed for veterinary use in 1993. Subsequent studies of the occurrence of resistance to quinolones in S. Typhimurium DT104 showed a temporal increase of quinolone-resistant isolates of MDR S. Typhimurium DT104 from humans, cattle, poultry, and pigs (Threlfall et al. 1999).

Over the five-year period 2000–2004, there has been an overall increase in cases of human infection in the EU by strains of S. Enteritidis exhibiting resistance to nalidixic acid and epidemiological resistance to ciprofloxacin, with the occurrence of resistance to both antimicrobials increasing from 10% to 26%. Over this period resistance remained constant at approximately 6% in S. Typhimurium; the highest incidence of resistance was seen in S. Virchow, with 68% of isolates resistant to nalidixic acid in 2002 (Meakins et al. 2008). From 2005–2006 to nalidixic acid/ciprofloxacin increased in both Enteritidis (135 to 15%) and Typhimurium (75 to 8%) (Table 23.3).

Ciprofloxacin resistance was commonly found in isolates of S. Enteritidis from broiler meat and hens from countries in southern Europe but also from certain countries in northern Europe. With the exception of certain new member states, such resistance was relatively uncommon in isolates of S. Typhimurium from pork, pigs and cattle. With the exception of one northern European country, there was a high incidence of quinolone resistance in Salmonella from turkeys (EFSA 2007b).

Foods have been implicated in several major national and international outbreaks of S. Typhimurium exhibiting epidemiological resistance to ciprofloxacin. Eggs contaminated with nalidixic acid-resistant S. Enteritidis have been linked to numerous outbreaks of salmonellosis in several European countries since 2000, although it has not been possible to precisely ascertain how many infections have been associated with contaminated eggs. Isolates with PMQR have been reported in several countries, but such strains were mostly associated with travel to countries outside of the EU (Hopkins et al. 2008). Of clinical concern is that the acquisition of plasmid-mediated quinolone resistance can raise the FQ MIC to clinical levels.

Resistance to third- and fourth-generation cephalosporins in Salmonella is primarily caused by production of extended-spectrum β-lactamases (ESBLs) and/or AmpC enzymes. Both classes of enzymes confer resistance to extended-spectrum cephalosporins and to other β-lactam antimicrobials, with substrate specificity depending on the mechanism and sequential mutations involved. In particular, in different salmonella serovars ESBLs and/or AmpC enzymes have often been identified on plasmids. These plasmid-mediated resistances have frequently been found together with resistance determinants for e.g. aminoglycosides, chloramphenicol and florfenicol, sulphonamides, tetracyclines and/or trimethoprim, leading to efficient spread via co-selection. In addition, the down regulation of porins in some resistant isolates may also contribute to a decreased activity of antimicrobials that use the same entry pathway, such as FQs.

Extended-spectrum cephalosporins (ESBLs), along with fluoroquinolones, have been classified by WHO as ‘critically-important’ antimicrobials (WHO 2007). ESBLs with the ability to hydrolyse and confer resistance to cefotaxime (= CTX-M) are generally regarded as having the most impact for public health.

Strains of Salmonella enterica with CTX-M enzymes were first reported in isolates of S. Typhimurium made in 1998 from cases of human infection in Greece, Hungary and Latvia. In 2000 cases of human infection in Spain in 1997 and 1998 with S. Virchow possessing CTX-M-were reported and in two retrospective studies of CTX-M in salmonella infections in the UK, cases of CTX-M-producing S. Virchow in the UK in 1997 and 1999 were identified which were associated with travel to Spain and in which the causative strain possessed CTX-M-9. Subsequently CTX-M enzymes have been identified in cases of human infection in a further 14 European countries, from food animals—poultry in five countries, and in seafood in one country outwith Europe, since 2001 salmonella organisms with CTX-M enzymes have been reported in 21 countries in five continents. The great majority of organisms have been from cases of human infection, although infections associated with imported poultry meat have been reported in Japan CTX-M-producing serovars with an international distribution within Europe included Virchow, Typhimurium, Enteritidis, and Concord. With the exception of S. Concord the organisms were not clonal in respect of their CTX-M types, with four CTX-M types being identified in S. Virchow, six in S. Typhimurium and three in S. Enteritidis. In contrast to the plethora of CTX-M-producing serovars from cases of human infection, the only serovars from food production animals in which CTX-M enzymes have been positively identified are S. Virchow and S. Java. As such S. Virchow with CTX-M types 2, 9, and 32 have been isolated from poultry and S. Java with CTX-M types 1, 2 and 9 also from poultry. Of particular note has been the rapid spread of CTX-M- producing S. Java in poultry in the Netherlands, with the percentage of such isolates increasing from 0% in 1999 to 20% in 2005, with a slight decline in isolations in 2006 (MARAN 2007) In almost all serovars and strains CTX-M genes have been plasmid-mediated. Other than in Europe the occurrence of salmonella strains possessing CTX-M-inactivating enzymes is low. Although such strains have emerged, and in certain cases have caused serious infections, in countries outwith Europe most CTX-M possessing salmonella strains seem to be confined to cases of human infection and, with the possible exception of China, there is no evidence of either epidemic spread or a food-animal reservoir.

SGI1 is a chromosomally-located gene cluster originally identified in the UK in MDR S. Typhimurium DT104 in the early 1990s (Threlfall et al. 1993). MDR DT104 was subsequently detected in the US in 1985 and its prevalence increased in the 1990s worldwide (Threlfall 2000). DT104 is zoonotic in origin and causes numerous infections in humans. One of the characteristics of DT104 is that it is phenotypically resistant to ampicillin (A), chloramphenicol/florfenicol (C), streptomycin (S), sulphonamides (Su), and tetracyclines (T). DT104 is also associated with enhanced ability to cause disease (virulence) SGI1 was first described in 2000 as a 43 kb chromosomal region in a Canadian isolate of DT104 and was sequenced in 2001 (Boyd et al. 2001). SGI1 has recently been detected in other, epidemic salmonella serovars as well as the pandemic DT104, but which has also recently been detected in other sub-species (or serovars) of S. enterica (e,g., Agona, Albany, Newport, Java) (Doublet et al. 2004), indicating that there may be a relationship between potentially enhanced virulence and MDR and the presence of this gene cluster. SGI1 seems to spread horizontally so it poses a public health risk to the future treatment of salmonella infections. Although horizontal transfer between salmonella serovars may be the mechanism by which SGI1 spreads, a biological reservoir in other organisms cannot be excluded.

The use of antimicrobials in humans and animals is widely regarded as a major driving force in the emergence and spread of both antimicrobial resistance (AMR) and antimicrobial-resistant bacteria. The question of whether antimicrobial resistance in NTS associated with food animals is a human public health problem in developed countries has been a contentious issue since the late 1960s. In 1969 the Joint Committee on the Use of Antibiotics in Animal Husbandry and Veterinary Medicine (the Swann Committee) recommended that certain therapeutic antibiotics, at that time widely used in food animals without prescription, should be available only on prescription (Anon. 1969). The committee also recommended that therapeutic antimicrobials should not be used for the purpose of growth promotion in food-producing animals. The practice of using antimicrobials for growth promotion in food production animals has subsequently been forbidden in the EU since 2006, but such strictures have not been adopted worldwide, for example in the USA.