**1. Introduction**

372 Salmonella – A Dangerous Foodborne Pathogen

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*Salmonella* is the etiologic agent of Salmonellosis in humans causing severe illness in infants, the elderly, and immunocompromised patients (Cross et al. 1989; Tauxe 1991; Smith 1994; Baumler et al. 2000). Salmonellosis symptoms include watery diarrhea, abdominal pain, nausea, fever, headache and occasional constipation with hospitalization required in cases of severe infections. The genus currently contains two species, *Salmonella bongori and Salmonella enterica* (including six subspecies: *enterica* (I), *salamae* (II), *arizonae* (IIIa), *diarizonae* (IIIb), *houtenae* (IV), and *indica* (VI). However, there are more than 2,500 serovars of *Salmonella* based on the Kauffmann-White antigenic scheme for the classification of Salmonellae (Popoff et al. 1994). *Salmonella* is a gram-negative, non-spore forming rod and facultative anaerobe that can ferment glucose belonging to the family Enterobacteriaceae. Most strains are motile with peritrichous flagella and can reduce nitrate to nitrite (Grimont et al. 2000). The organism is mesophilic with optimum growth temperature in the range of 32 – 37°C but capable of growth within a wide temperature range of 6 – 46oC. *Salmonella* is ubiquitous in the environment originating from the gastrointestinal tracts of domesticated and wild animals and can be present without causing apparent illness. Most infections result from the ingestion of foods of animal origin contaminated with *Salmonella* species such as beef, chicken, turkey, pork, eggs, and milk (D'Aoust 1997; D'Aoust 2000; Olsen et al. 2000). Other vehicles, including non-animal foods such as fresh fruits and vegetables (Mahon et al. 1997), reptiles (Friedman et al. 1998), water (Angulo et al. 1997), and direct person-to-person transmission (Lyons et al. 1980), have also been implicated. However, certain serotypes of *Salmonella* such as *S.* Enteritidis, which can penetrate poultry reproductive organs resulting in the contamination of egg contents has been a prominent cause of human illness for several decades (Gantois et al. 2009). In addition to faecal contamination, cross-contamination of foods by *Salmonella* during food preparation can be an important source of foodborne illness.

Generally, detection methods are based on physiological and biochemical markers of the organism (Williams 1981). Cultural methods are based on nutrient acquisition, biochemical characteristics, and metabolic products unique to *Salmonella* spp. (Ricke et al. 1998). More rapid immunological and molecular screening methods of detection have been devised to detect cell surface markers and nucleic acids, respectively. This chapter will provide an overview of various culture based methods and rapid methods currently available for the detection of *Salmonella* in foods and food ingredients. We will focus our discussion on

*Salmonella* Detection Methods for Food and Food Ingredients 375

reaction, lysine decarboxylase, negative indole test, H2S production, and fermentation of dulcitol. Serological confirmation tests typically utilize polyvalent antisera for flagellar (H) and somatic (O) antigens. Isolates with a typical biochemical profile, which agglutinate with both H and O antisera are identified as *Salmonella* species. Where results are inconclusive, it may be necessary to perform additional biochemical tests. Positive isolates are often sent for further serotyping to identify the serovar using specific antisera as per the Kauffman-White (KW) typing scheme recognizing 46 O antigens, and 119 H antigens, thereby permitting the characterization of 2,541 serotypes (Shipp and Rowe 1980). Serotyping is a useful epidemiological tool in identifying circulating serotypes and to characterize outbreaks. The antigenic formulae of Le Minor and Popoff (2001) is a standard method for naming the serovars. However, serotyping is normally undertaken at reference laboratories and is rarely performed in routine food or clinical laboratories. Reference laboratories are also able to further type isolates using techniques such a phage typing (Anderson and Williams 1956; Callow 1959; Anderson 1964; Anderson et al. 1977), antibiotic susceptibility (Bauer et al. 1966), pulsed-field gel electrophoresis (PFGE), or other emerging genetic typing technologies such Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and

Although standard culture methods are excessively time-consuming, there is potential for further improvements, and thus many attempts have been made to maximize their efficiency by introducing new technologies, making reliability of detection more convenient, user friendly, as well as by reducing the costs of materials and labour (de Boer and Beumer 1999; Weenk, 1992). For example, biochemical confirmatory tests may be easily replaced by commercial identification kits such as the API 20E (BioMérieux) or other commercially available bacterial identification kits. The detection of sub-lethally damaged cells is of utmost importance as these may still pose a risk to human health and may lead to false negative results. Strategies for the recovery of injured bacteria are based on overlay methods such as tryptic soy agar (TSA) overlayed on XLD selective agar (Kang and Fung 2000) and other approaches also include the development of single enrichment broths where multiple step enrichments are usually required (Baylis et al. 2000). Other novel approaches include the addition of bacteriophages for the elimination of background microflora that may out-compete the target organism. For example, RapidChek® SELECTTM Salmonella (Strategic Diagnostics Inc.) employs a primary enrichment media supplemented with a bacteriophage cocktail as a selective agent, which reduces the level of background flora in high burden samples allowing *Salmonella* to grow with minimal competition. In addition, there is also the development of enrichment broths for the concurrent enrichment of pathogens thereby reducing laboratory workloads with respect to the preparation of sample homogenates since different enrichment broths would no longer be required, and multiple analyses could be performed from a single universal enrichment culture (Kim and Bhunia 2008). Amendments to media have also been performed such as the addition of novobiocin (Restaino et al. 1977; Devenish et al. 1986), and cycloheximide to decrease fungal overgrowth (Ricke et al. 1998). Lastly, and perhaps the most important advancement is the use of chromogenic or fluorogenic substrates in selective agars, permitting identification to be performed directly on the isolation plate, thereby expediting or eliminating the use of subculture media or additional biochemical tests as these media provide highly specific reactions, and help reduce the workload for unnecessary examination of suspect colonies arising from poor specificity of conventional agars (Manafi 1996; Manafi 2000). A number of selective chromogenic agar media

Multilocus sequence typing (MLST) (Kruy et al. 2011).

advances introduced for the improvement of conventional culture methods, the use of Polymerase Chain Reaction (PCR) technology, immunology-based methods, and bacteriophage based assays. Whenever possible, examples from the academic literature as well as from commercial applications will be considered. The importance of sample preparation will be examined throughout as it relates to its impact on sensitivity and turnaround time for detection. Specific *Salmonella* serovars will be named according to the nomenclature of Leminor and Popoff (2001), e.g. *Salmonella* Enteritidis or *S.* Enteritidis.
