Molecular characterization of endemic salmonella infections ... - Evira

2.5.2.2 Plasmid fingerprinting and identification of plasmid-mediated virulence genes
Discrimination between plasmids of similar molecular mass, or determination of the degree of
molecular relatedness between plasmids of different size, may be achieved by digesting the
plasmids with a restriction endonuclease and comparing the number and size of the restriction
fragments generated (Maslow et al 1993; Threlfall et al 1994). The same restriction pattern should
be seen in identical plasmids (Grimont et al 2000), but the results may be extremely difficult to
interpret if a strain carries multiple plasmids (Threlfall et al 1994). However, a study by Rankin et al
(1995) demonstrated the importance of plasmid fingerprinting, as plasmid analysis, on its own, can
easily lead to the assumption that plasmids of the same molecular weight are the same plasmids.
Their work showed plasmids of the same molecular weight could be unrelated whereas plasmids
of different molecular weight could be closely related. In a study by Browning et al (1995),
considerable plasmid diversity and evolutionary divergence was detected in the serotype-specific
plasmid of Salmonella Dublin. Baggesen et al (1992) observed that plasmid profiling followed by
restriction enzyme analysis was useful as it showed variations in the serotype-specific plasmid and
also the presence of co-migrating plasmids of the same size as the serotype-specific plasmid.
Plasmids carried by certain serotypes (serotype-specific plasmids) possess a highly conserved
common area which carries genes responsible for the virulence of the serotype to certain strains of
mice (Williamson et al 1988). A DNA probe specific for these salmonella plasmid virulence (spv)
genes makes rapid screening of a large number of strains possible. In epidemiological
investigations, the presence or absence of serotype-specific plasmids can be useful (Threlfall et al
1994). At least 12 serotypes contain virulence plasmids. The size of the plasmids range from 54 to
98 kb, and phage type differences were found in plasmid carriage within a serotype (Lax et al
1995).
2.5.2.3 Ribotyping
Restriction fragment length polymorphism (RFLP) associated with the ribosomal operon and
detected by Southern blot analysis is called ribotyping (Stull et al 1988). Initially ribotyping,
detecting variations in the copy number and location of 16S and 23S rRNA (rrn) loci, was used as
a taxonomic tool (Grimont and Grimont 1986). The complete nucleotide sequence of a 16S
ribosomal RNA gene obtained from E. coli was determined by Brosius et al and published in 1978.
Stull et al (1988) tested the ability of E. coli rRNA to hybridize with heterologous DNA purified from
genetically diverse gram-negative bacteria. These authors found cross-hybridization to occur
between the E. coli rRNA probe and unrelated species. Therefore a probe derived from the E. coli
ribosomal operon can be used widely (Maslow et al 1993).
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