**4.1.1 Serotyping**

352 Salmonella – A Dangerous Foodborne Pathogen

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

**Tests Substrate Reaction (-) Results (+) Results** *Salmonella* 

dihydrolase yellow red/orange -

decarboxylase yellow red/orange +

decarboxylase yellow red/orange +

hydrolysis yellow red/orange -

blue-green/

blue -

deposit <sup>+</sup>

(in 2 min) -

(in 10 min) -

diffusion -

black

blue-green yellow +

blue-green yellow +

blue-green yellow -

blue-green yellow +

blue-green yellow +

blue-green yellow -

blue-green yellow +

blue-green yellow -

blue-green yellow +

pale to green/yellow

production colorless/gray black

production yellow red

production colorless pink/red

of black

**ONPG** ONPG betagalactosidase colorless yellow -

**TDA** tryptophan deaminase yellow brown-red -

Table 1. Biochemical reactions involved in API 20E (bioMérieux, Inc., France) test kits and

*spp.*

suspicious colonies were identified with biochemical test (as mentioned above).

Utilization

H2S

Urea

Acetoin

**GLU** glucose fermentation/oxidation blue/

**MAN** mannitol fermentation/oxidation blue/

**INO** inositol fermentation/oxidation blue/

**SOR** sorbitol fermentation/oxidation blue/

**RHA** rhamnose fermentation/oxidation blue/

**SAC** sucrose fermentation/oxidation blue/

**MEL** melibiose fermentation/oxidation blue/

**AMY** amygdalin fermentation/oxidation blue/

**ARA** arabinose fermentation/oxidation blue/

gelatin Gelatinase no diffusion

**ADH** arginine Arginine

**LDC** lysine Lysine

**ODC** ornithine Ornithine

**CIT** citrate Citrate

**IND** tryptophan Indole

**H2S** Na

**URE** urea

**VP** Na-

**GEL** charcoal

thiosulfate

pyruvate

typical *Salmonella* reactions.

Serotyping is the initial step for routine diagnostics of *Salmonella* strains and performed with commercially available omni-, poly- and monovalent antisera. Up to date, over 2500 serotypes of *Salmonella* has been identified and classified in the Kaufmann-White scheme. This scheme differentiates between O (=somatic) antigens of the cell surface, H1 and H2 (=flagellar) antigens of the phase 1 or phase 2, respectively (Selander et al., 1996) and the Vi (=capsular) antigens which, however, may only be present in very few serotype, such as *Typhi*, *Paratyphi C* or *Dublin*.

Each *Salmonella* serogroup has a group specific O-antigen. Within each O-group, different serovars are distinguished by the combination of O- and H-antigens that are present. Each serotype has a specific antigenic formula where the O-antigens are indicated by Arabic numbers, the H1-antigens by lower case letters and the H2- antigens again by Arabic numbers. In these formulas, underlined antigens may only be expressed once the culture is lysogenised by the corresponding converting phage whereas letters or numbers in brackets indicate antigens which may be present or absent without relation to phage conversion (Le Minor, 1984).

For most of the isolates assigned to *S. enterica* and the subspecies I, antigenic formula corresponds to a serotype name. In contrast, serotypes identified after 1996 in the subspecies *salamae*, *houtenae* and *indica* and in the subspecies *bongori* are designated only by antigenic formula (Brenner et al., 2000).


Table 2. Examples for the antigenic formulas of *Salmonella enterica* subsp. *enterica* serotypes according to Kaufmann-White scheme (Poppoff and Le Minor, 2001).

The detection of the presence of *Salmonella* O- and H- antigens were tested by slide agglutination with the commercially available antisera. One loop of appropriate antisera was dropped onto a cleaned glass slide. One loop of overnight culture grown on agar was dispersed in the drop to obtain a homogeneous and turbid suspension. The slide was rocked gently for 30 s and clumping was monitored by a magnifying glass. The scheme to obtain the serotype was given in **Figure 2.**

Laboratory Typing Methods for Diagnostic of

to any recognized phage types).

**4.1.2 Phage typing** 

Hald et al., 2007)

difficulties of a *Salmonella* strain.

through the bottom of the plate (Ward et al., 1987).

barely visible turbidity.

Salmonella Strains, the "Old" Organism That Continued Challenges 355

Individual isolates of many *Salmonella* serotypes vary in their susceptibility to lysis by different bacteriophages and this has led to a typing scheme based on reactivity to a panel of bacteriophage. Therefore, a *Salmonella* strain is subjected to a specified set of typing phages and the lytic pattern obtained commonly allows the assignment to a specific phage type. The strains exhibiting a lytic pattern that does not correspond to a known phage type are classified as RDNC (= Reacting with the typing phage, but lytic pattern Did Not Correspond

Phage typing is mostly performed for serotypes such as *S. Typhimurium*, *S. Enteritidis*, *S. Typhi* or *S. Paratyphi*, although phage typing systems are also available for a number of additional serotypes, including *S. Virchow*. Phage typing has led to the discrimination of over 200 *S. typhimurium* phage types (Threlfall & Frost, 1990) and, together with antimicrobial susceptibility analyses, led to detection of several large-scale, international epidemics including the dissemination of a multi-drug resistant clone of *S. typhimurium*  DT104, (definitive phage type, DT, 104) (Threlfall, 2000). In Denmark, phage typing as described by the World Health Organization (WHO) Collaborative Centre for phage typing of Salmonella (Health Protection Agency (HPA), Colindale, United Kingdom) has been applied for surveillance of S. Enteritidis and S. Typhimurium in humans, food and food production animals. Phage typing has proven to be an important tool for strain characterisation and the results obtained have been used since the mid-90s in surveillance, source attribution and outbreak investigations (Baggesen & Wegener, 1994;

In general, phage typing is only performed by the National Reference Centers, since only these institutions have access to the defined sets of typing phages. The interpretation of the results requires considerable experience (Riley, 2004). Although, phage typing in *Salmonella* epidemiology has been used since the 1950s, the stability of phage types can be limited by phage type conversion (Rabsch et al., 2002), even during an outbreak (Mmolawa et al., 2002). This is due to the acquisition of a temperate phage or a plasmid. Besides, host-controlled phage defence mechanisms such as restriction/modification systems and phage adsorption inhibition are also responsible for the phage typing

By means of a sterile inoculation loop, the test culture was inoculated into a test tube containing 4 mL double strength nutrient broth with a special care for heavy inoculum to give visible turbidity for *S. Enteritidis* and a very light inoculum for *S. Typhimurium* to give a

The culture was incubated by shaking at 200 rpm at 37°C for 1-1.5 h for *S. Enteritidis* and for *S. Typhimurium* 1.5 h without agitation to obtain a very light growth in early log phase. After incubation, it was flooded over the surface of double strength nutrient agar using a flooding pipette and the excess of culture was removed. As soon as the surface of agar dried, the appropriate typing phages at routine test dilutions were applied to the dried surface by a multipoint inoculation loop. When the phage spots dried, the agar plate was incubated at 37°C for 18 h. At the end of the incubation, the agar plate was read using a magnifying glass

Phage susceptibilities were evaluated by means of the plaque number, size and transparency. The pattern was compared with known phage type patterns in the database and defined. If the culture did not react with any of the typing phages, it was defined as non-typable (NT); and if the culture reacted with the typing phages, but gave a different

Serotyping is easy to perform and standardized antisera are commercially available. However, it only allows the assignment of *Salmonella* strains to a specific serotype, and no further differentiation between strains of the same serotype is achieved.

Fig. 2. Serotyping analysis scheme for *Salmonella*

During the 1980's, a tremendous increase in *S. enteritidis* was identified, particularly in the Northeastern U.S. (Rodrigue et al.,1990). Studies linked *S. enteritidis* to contaminated shell eggs or foods that contained eggs (Mishu et al., 1994). During 1987-1997, five serotypes accounted for 66% of all clinical infections in which a *Salmonella* isolate was identified to the serotype level. *S. typhimurium* accounted for 24% of these isolates, *S. enteritidis* (22%), *S. heidelberg* (9%), *S. newport* (5%) and *S. hadar* (4%) followed (Olsen et al., 2001). When clinical outbreaks were distinguished from sporadic infections, *S. enteritidis* was implicated in 55% of *Salmonella* cases associated with a clinical outbreak (Olsen et al., 2001).

In Tunisia, from 1994 to 2004, 16.214 *Salmonella* isolates were reported to the national Centre of Enteropathogenic bacteria at Pasteur Institute, Tunis, Tunisia. (Ridha et al., 2007). The largest proportion of *Salmonella* isolates was from human origin (n=6815) followed by isolates from food (n=5539). During the surveillance period, the top five reported *Salmonella*  serotypes were: *Enteritidis*, *Anatum*, *Corvallis*, *Braenderup*, and *Livingstone.* These five serotypes accounted for 3479 strains of all *Salmonella* isolates from food. (Ridha et al., 2007). Finally, *Salmonella* isolates reported from environmental origin cam in last position (n=1611) after isolates from animal origin (n=2249) (Ridha et al., 2007).

Serological analysis usually remains the first step in an epidemiological investigation of *Salmonella* and may be sufficient for epidemiological investigations associated with uncommon serotypes (Threlfall & Frost, 1990). However, smaller labs often do not have access to the pools of serum required for this analysis and may need to rely on other techniques to analyze isolates. The multiplex PCR, an easier molecular method, has been developed to differenciate between the most common serotypes of *Salmonella enterica* subsp. *enterica* (Imen et al. 2010).
