**1. Introduction**

348 Salmonella – A Dangerous Foodborne Pathogen

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*Salmonella* are enteric gram negative organisms that are widely dispersed in nature. These organisms can reside as common commensals in the gastrointestinal tracts of animals and man or cause disease states that range from self-limited diarrhea to bacteremia with enteric fever or invasion of vascular structures, bone or other localized sites (Hook, 1990).

Organisms can be highly host adapted, where they infect only a limited number of species, or can be much more ubiquitous. The most significant human host-adapted organism is *S. typhi*, the cause of typhoid fever. Man remains the only known reservoir for these isolates. Similarly, *S. pullorum* and *S. gallinarum* are poultry associated organisms that are so hostadapted that even upon transmission to man they usually remain non-pathogenic (Ziprin & Hume, 2001). More frequently, animal host-adapted organisms can be transmitted to man causing symptomatic disease. *S. choleraesuis* is normally a porcine organism though it can cause gastroenteritis and enteric fever, when transmitted to man (especially in children). Other organisms, such as *S. typhimurium*, have a broad host range and these serotypes are responsible for the majority of human infections.

Thus, *Salmonella* strains, the well and "old" pathogens, continued threat to public health. In fact, despite that, the incidence of salmonellosis has decreased substantially especially in developed country, recent events and several articles illustrate continued challenges in *Salmonella* control. The first challenge in *Salmonella* control is the widespread distribution of food; in fact contaminated food produced in one country may cause illness far away demonstrating the importance of robust control programmes. Likewise, this organism cause substantial economic loss resulting from mortality, morbidity, poor growth of infected animals, poultry and human beings; hazardous of transmitting food poisoning with gastroenteritis to human and so represents a serous problem for the food industry (Khan et al., 2007).

The second challenge is traceability, in fact, the complexity of the food supply chains and/ or the lack of identifying markers on foods can make it extremely difficult to trace back to their origin.

Laboratory Typing Methods for Diagnostic of

**3.1 Food samples** 

**3.2 Stool sample** 

Salmonella Strains, the "Old" Organism That Continued Challenges 351

Samples were analysed according to French Norm for *Salmonella spp.* NFV 08-052/97. From each sample, 25 g was pre-enriched in 275 ml buffered peptone water (Oxoid, Dardilly Cedex, France) at 37°C for 24h. Afterwards, 0.1 ml of the pre-enrichment sample was incubated in 9.9 ml of buffered Rappaport-Vassiliadis medium (Oxoid, Dardilly Cedex, France) and 2 ml in 20 ml of buffered selenite cystine medium for another 24 h at 42 °C and 37 °C, respectively. The enrichment samples were then applied onto Hecktoen and

Suspicious colonies were identified by Gram staining performed according to the conventional method and also with biochemical test (oxydase reaction). Both Gram-negative and oxidase-negative isolates were further tested. Biochemical tests other than oxidase test

The plastic strips holding twenty mini-test tubes were inoculated with the saline suspensions of the cultures according to manufacturer's directions. This process also rehydrated the desiccated medium in each tube. A few tubes were completely filled (CIT, VP and GEL), and some tubes were overlaid with mineral oil such that anaerobic reactions

After incubation in a humidity chamber for 18-24 hours at 37°C, the colour reactions were read (some with the aid of added reagents as supplied by the kit). The data were analysed by the manufacturer's software and positive results with ≥89% probabilities were confirmed as *Salmonella*. The list of the biochemical tests performed by API 20E test kit and typical

Each stool sample was streaked onto Hecktoen agar and pre-enriched in selenite broth at 37 °C for 24 h. The pre-enrichment sample was streaked onto Hecktoen agar, and after incubation at 37 °C for another 24 h, the suspicious colonies were identified with

From each sample, 100 ml was pre-enriched in 100 ml double concentrated buffered peptone water (Oxoid, Dardilly Cedex, France) at 37 °C for 24 h. Afterwards, 0.1 ml of the preenrichment sample was incubated in 9.9 ml of buffered Rappaport-Vassiliadis medium (Oxoid, Dardilly Cedex, France) and 1 ml in 9 ml of buffered selenite cystine medium for another 24 h at 37 °C; The enrichment samples were then applied onto Hecktoen and

Kampelmacher agar. Both selective media were incubated during 24 h at 37 °C.

were done by using API 20E test kit (bioMérieux, Inc., France).

could be carried out (ADH, LDC, ODC, H2S, URE) **(Figure 1)**.

Fig. 1. Typical *Salmonella* reaction of API 20E test kit.

reactions exhibited by *Salmonella spp*. are given in **Table 1**.

biochemical test (as mentioned above).

**3.3 Environmental water samples** 

The third is antimicrobial resistance; in fact, over the last decade, strains of *Salmonella enterica* with multiples drug resistance have been distributed widely in many countries.

The fourth is capacity building to enhance outbreak detection through routinely subtyping certain *Salmonella* using molecular methods.

To contain this organism, it is essential to maintain continued vigilance, including rapid identification of similar strains and the immediate sharing of information within the public health community. Many nations have established extensive surveillance systems to track *Salmonella* infections and disrupt epidemic spread. Most of these surveillance projects rely on traditional serotype and phage type analyses to identify trends and potential outbreaks. Many clinical outbreaks cluster among a few serotypes so further discrimination is often needed.

Molecular epidemiological techniques have been used to enhance surveillance and discriminate outbreak strains within these common serotypes. The institution of these techniques has led to enhanced detection of outbreaks worldwide. In this chapter, we review the theoretical and practical basis of laboratory typing method for diagnostic of *salmonella* strains with emphasis on molecular methods which would contribute to the monitoring of human and animal *Salmonella* infections. Overall, traditional serotype surveillance in association with one or several molecular typing techniques, appears to provide the most reproducible and comparable discrimination of epidemiologicallylinked isolates.
