**3.1 Food samples**

350 Salmonella – A Dangerous Foodborne Pathogen

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

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

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 epidemiologically-

*Salmonella* are Gram negative, short plump shaped rods, nonsporeforming, noncapsulated, aerobic and facultatively anaerobic organisms and classified under the family

*Salmonella* nomenclature has changed many times and still is not stable. The genus *Salmonella* was previously differentiated into two species: *Salmonella enterica* and *Salmonella bongori*. However, a new species, *Salmonella subterranea* was identified and validated (Shelobolina et al., 2004; Validation List No: 102, 2005). Among them, the species *Salmonella enterica (S. enterica)* is further divided into the six subspecies *S. enterica* subsp. *enterica* (I), *S. enterica* subsp. *salamae* (II), *S. enterica* subsp. *arizonae* (IIIa), *S. enterica* subsp. *diarizonae* (IIIb), *S. enterica* subsp. *houtenae* (IV), and *S. enterica* subsp. *indica* (VI). Formerly, *S. bongori* was the

Fermentation of selected substances, such as dulcitol, malonate, sorbitol, d-tartrate, galacturonate, mucate, salicine, ONPG, and lactose, as well as production of enzymes such as gelatinase, -glutamyl-transferase or -glucuronidase, but also lysis by phage O1 allow a

Furthermore, the genus composed of over 2500 serotypes differentiated according to three different types of surface antigens discussed bellow in more detail. 99% of these serotypes belong to *S. enterica* and nearly 60% of them are in *S. enterica* subsp. *enterica*. The average DNA sequence similarity between *Salmonella* serotypes is 96-99% (Edwards et al., 2002).

A standard technique was used to isolate *Salmonella* strains in many laboratories. The

certain *Salmonella* using molecular methods.

**2. General properties of the genus** *Salmonella*

subspecies V, but later considered as a separate species (Fluit, 2005).

differentiation between the different species and subspecies (Le Minor 1984).

discrimination is often needed.

Enterobacteriaceae (Freeman, 1985).

**3. Bacterial isolations** 

technique is explained bellow.

linked isolates.

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

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

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

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

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