Restriction Endonuclease Based Methods Pulsed Field Gel Electrophoresis

PFGE has become a widely used method in molecular subtyping. It has been used extensively in the analysis of isolates associated with outbreaks such as E. coli O157 and Salmonella. PFGE was selected as the method of choice for PulseNet, the national system used for monitoring and tracking foodborne pathogens and outbreaks in the United States (Swaminathan, Barrett, Hunter, Tauxe, & The CDC PulseNet Task Force, 2001). This database is linked to the Centers for Disease Control. A range of PFGE protocols for pathogens such as Salmonella, Listeria, Shigella, Campylobacter, Vibrio, Yersinia, and E. coli O157:H7 are available through PulseNet (http://www.cdc.gov/pulsenet/protocols.htm).

PFGE involves the use of restriction enzymes to digest a pathogen's total DNA into fragments. Bacterial cells of interest are suspended in an agarose plug with an optimum number of cells (108) required to ensure quality fingerprints. Cells are lysed in the agarose plugs with proteinase K, sarcosine, and detergents, leaving the DNA intact. Digestion products, detergents, and other contaminants are removed from the plugs by a series of washing steps. DNA in plugs is then digested with restriction enzymes that are considered to be ''rare cutting''. That is, they will cut the DNA into fragments ranging from 10 to 800 kb in size. Typical enzymes used in PFGE include Xbal (Foley et al., 2004; Liebana, Garcia-Migura, Breslin, Davies, & Woodward, 2001), AscI, Apal (Graves et al., 2005), Sall, or Smal (Suzuki, Ishihara, Saito, Ishikawa, & Yokochi, 1994; Wassenaar, Geilhausen, & Newell, 1998). There are a considerable number of restriction enzymes available for PFGE, and the reader is advised to choose enzymes based on the strain type and information desired. Restriction fragments are separated by gel electrophoresis in a specialized electrophoresis rig, as the fragments of DNA to be separated in this case are usually larger than those associated with other methods. PFGE depends on periodic inversion of the electric field in order that the strands of DNA trapped in the agarose reorient facilitating their movement through the gel. Most PFGE rigs use an array of electrodes arranged in a hexagonal formation, which generate electric fields at 120° to each other. Depending on size, DNA fragments move through the gel in a uniform manner, with the smaller fragments moving at a significantly faster rate than the larger ones resulting in a banding pattern (Carle, Frank, & Olson, 1986; Chu, Vollrath, & Davis, 1986).

PFGE is run in a specially designed rig with gel electrophoresis taking 18-24 h per run. Following electrophoresis, the DNA in the gel is stained with ethidium bromide to visualize the fingerprint. Images of the banding patterns are usually acquired electronically and imported into molecular analysis software such as BioNumerics® to generate cluster or dendrogram analyses useful in determining strain relatedness. Analysis software allows comparison of banding patterns and is useful in epidemiological investigations (see Fig. 18.2). One drawback of PFGE is that changes can occur in the banding patterns of strains during outbreaks as a

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Bacterial Cell

Embed cells in agarose plug

Generate fingerprint profile of fragments

Lysis, proteolytic cleavage of cells in the plug and washing

Section plug

Restriction enzyme digestion of DNA in plug

Post gel analysis using software

Generate fingerprint profile of fragments

Post gel analysis using software

E. coli from poultry

Load samples into agarose gel and subject to electrophoresis in alternating polarities

Fig. 18.2 Pulsed field gel electrophoresis result of repeated subculturing or on passage of a pathogen through the intestine of the host (Hanninen, Hakkinen, & Rautelin, 1999; Iguchi et al., 2002; On, 1998; Steinbruckner, Ruberg, & Kist, 2001). Also, Kudva et al. (2002) found that PFGE patterns of E. coli O157 strains differed as a result of insertions and deletions in their O islands. PFGE has, however, proven valuable in outbreak investigations when used as a means to link pathogen to host, sources, and vehicles. However, PFGE may not be as powerful in some situations where the diversity of the strains is great. In such cases, there may be too many banding patterns or profiles to form conclusions. Li et al. (2007) used PFGE to subtype 138 E. coli recovered from processed bison carcasses and found 96 distinct banding patterns among the isolates tested. When further analysis investigated PFGE patterns in relation to the antimicrobial resistance of the strains, no correlation was found between resistance phenotype and genotype, with the 23 strains tested exhibiting 22 distinct patterns. PFGE has greater discriminatory index when used in application for distinct outbreaks or more specific pathogen types as opposed to generic E. coli. In contrast, similar application of PFGE for Salmonella isolates recovered from a poultry slaughter line (Nde, Sherwood, Doetkott, & Logue, 2006) was useful in demonstrating the movement of strains through the line and the effect of defeathering (Nde, McEvoy, Sherwood, & Logue, 2007) on carcass contamination. PFGE also has been reported to provide better discrimination in the molecular analysis of S. Typhimurium than MLST (Fakhr, Nolan, & Logue, 2005). Others have found that PFGE is useful for strain discrimination in outbreak situations or in surveillance associated with Campy-lobacter and Salmonella (Bender et al., 2001; Fitzgerald et al., 2001; Gilpin et al., 2006; Suzuki et al., 1994). However, Hedberg et al. (2001) considered PFGE unsuitable for routine typing of strains. Foley et al. (2004) compared PFGE with MLST and REP PCR for E. coli O157 analysis and found that PFGE provided the greatest discrimination. PFGE has been used in investigations of Listeria (Graves et al., 2005; Sanders et al., 2003) and Campylobacter (Wang & Taylor, 1990; Wassenaar et al., 1998). Ronner, Borch, and Kaijser (2005) used PFGE to trace/link human campylobacteriosis in Sweden with isolates from Thailand. Of interest was the finding of similar profiles in isolates from both countries demonstrating the global distribution of some Campylobacter strains. No relationship was found between PFGE profiles and antimicrobial sensitivities. Suzuki et al. (1994) investigated PFGE as a method to discriminate C. jejuni from sporadic infections with isolates from outbreaks. They found differences in cleavage patterns among isolates from sporadic outbreaks, which were more heterogeneous in nature than those from outbreaks which showed the same unique restriction patterns.

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