**4.2.4 Insertion sequence (IS) typing**

358 Salmonella – A Dangerous Foodborne Pathogen

Despite that PFGE is usually considered as the method of choice to determine the molecular relatedness among *Salmonella* strains; this method is relatively slow, often taking three days to complete, and requires the presence of expensive specialized equipment, high quality chemicals, and a considerable experience in the preparation of the DNA-containing agarose slices. Moreover, single genetic events, such as point mutations, integration, deletion or recombination events, can result in differences in the

The Fingerprinting of rRNA coding sequences, termed ribotyping, describes the

Multiple copies of the rRNA operon are present within the *Salmonella* chromosome (Mendoza & Landeras, 1999). The rRNA genes themselves are quite homologous among these copies and between isolates, but the intervening sequences vary in length and

Ribotyping begins with separating endonuclease-digested chromosomal DNA on agarose gels, DNA then is transferred to a membrane and fragments are hybridized to a probe that recognizes 16S and 23S rRNA. Analysis of multiple restriction endonucleases can improve

Ribotype analysis is clearly able to subtype some of the isolates that fall within some common serotypes and phage types (Landeras et al., 1996). Lin et al. (Lin et al., 1996) detected 7 different ribotypes among 17 *S. enteritidis* PT 8 isolates when chromosomal DNA was digested with SphI. Using rRNA gene restriction patterns to investigate the relatedness of S. Enteritidis strains isolated in São Paulo, from 1975 to 1995; Fernandes et al. showed that ribotyping is a genomic profiling method that is reproducible and suitable for tracing the spread of S. Enteritidis. They found that the restriction endonuclease SphI discriminated best between subtypes of this serotype. Dambaugh et al. presented evidence suggesting that the ribotyping of Salmonella using the restriction enzyme PvuII increased the incidence of discreet ribotype patterns for the most common Salmonella serovars. This study evaluates the potential of PvuII to generate serotype-specific DNA fingerprints. However, studies have identified isolates that belong to different phage types yet demonstrate identical ribotypes (Fontana et al., 2002). Therefore, ribotyping is considered not suitable for local epidemiological studies or surveillance studies in a

Comparisons of ribotyping with PFGE have been somewhat unpredictable and often depend on the enzymes used for digestion as well as the nature of the population being tested. Several studies have found PFGE to be more discriminating than ribotype analysis (Fontana et al., 2002) while others have found the two procedures equivalent (Navarro et al., 1996) or ribotype analysis superior (Liebana et al., 2001). Ribotype analysis using two restriction enzymes, Pst I -SphI or HindIII - EcoRV, can improve discrimination (Liebana et al., 2001). Particular care must be taken when analyzing chromosomal patterns of *S. typhi.* The rapid genomic reassortment that occurs in *S. typhi* can affect ribotype analysis

Though most laboratories continue to perform ribotyping manually, machinery has been developed to perform this entire procedure in an automated fashion. Data is stored

hybridization of restriction-digested DNA fragments with probes specific for rDNA.

the discriminatory powers of ribotyping (Millemann et al., 1995).

fragment patterns (Herschleb et al., 2007).

**4.2.3 Ribotyping** 

nucleotide composition.

restricted region (Riley, 2004).

(Ng et al., 1999).

IS200 is a mobile element found in a variety of eubacterial genera, such as *Salmonella*, *Escherichia*, *Shigella*, *Vibrio, Enterococcus, Clostridium, Helicobacter,* and *Actinobacillus*. IS200 elements are very small (707-711 bp) and contain a single gene. Unlike typical mobile elements, IS200 transposes rarely. A consequence of IS200 self-restraint is that the number and distribution of IS200 elements remain fairly constant in natural populations of bacteria. This stability makes IS200 a suitable molecular marker for epidemiological and ecological studies, especially when the number of IS200 copies is high. IS200 typing, has been used to evaluate the molecular relationships between *Salmonella* isolates. In *Salmonella enterica*, IS200 fingerprinting is extensively used for strain discrimination. It is a 708 bp insertion sequence that is present in multiple copies within the *Salmonella* chromosome (Lam & Roth, 1983). Hybridization of digested chromosomal DNA with an IS200 probe has been useful in describing the clonal heritage of *Salmonella* from various serotypes, but has not been as discriminating as phage typing itself for *S. enteritidis*, *S. typhi* and others (Threlfall et al., 1994). For certain phage types of *S. typhimurium*, such as the multidrug resistant DT204c and 193 types common in the U.K., IS200 typing can result in strain discrimination and in some studies has been superior to PFGE and ribotyping (Jeoffreys et al., 2001). More frequently, PFGE has performed better than IS200 typing (Amavisit et al., 2001).
