title: Molecular characterization of the serotype-associated plasmids of Salmonella enterica
Creators: name: Browning, Lynda M.
Publisher: name: University of Glasgow
Year: 1996

description: The clinical importance of Salmonella has been known for more than a century. The control of salmonellosis requires detailed understanding of both pathogenicity and epidemiology. Certain plasmids are involved in the virulence of the salmonellae and their analysis often contributes to epidemiological investigation. Molecular characterization of the serotype associated plasmids of the salmonellae was undertaken. A predefined strategy of restriction endonuclease fragmentation pattern (REFP) analysis revealed plasmids previously defined as "serotype specific" were present in different serotypes. Plasmids indistinguishable from of molecular variants of established serotype associated plasmids (SAP's) were detected in other serotypes of serogroup Dl. The results showed that related or identical plasmids were present in both strains which varied only slightly in their H antigens e.g. Enteritidis (gm), Moscow (gq) and Blegdam (gmq) as well as a strain of Antarctica which possessed the H antigens gz63,. In addition to plasmid similarity within a serogroup, plasmids were identified in strains of Wangata which although a member of serogroup D are outwith the g-complex of flagellar antigens (H = Z4Z23). Unexpectedly, these plasmids were closely related to Typhimurium which belongs to serogroup B. The incompatibility of the plasmids was tested with a cointegrate plasmid pOG669 (a cointegrate of pOG660, the Typhimurium plasmid and pOG670, an IncX R-plasmid) and confirmation of incompatibility to the Typhimiuium component of this plasmid was shown by introduction and compatibility with pOG670. Plasmid incompatibility analysis of these plasmids revealed all the SAP's, Except Dublin, were incompatible with Typhimurium and confirmed a family of related plasmids common to but not restricted in their distribution to individual serotypes. Co-resident plasmids of intermediate size (30 - 40 kb) were observed relatively frequently in certain serotypes of GpD1- notably Dublin, Enteritidis, Moscow, Blegdam and Antarctica. With the exception of Antarctica these plasmids exhibited IncX properties - and although the possibility of dual incompatibility was not investigated, these properties, by inference were impossible as it would have resulted in incompatibility to pOG669 also. Restriction endonuclease fragmentation pattern analysis of the serotype associated plasmids of the salmonellae revealed a high degree of relatedness between plasmids of Typhimurium, Wangata, Gallinarum and Pullorum and a low degree of REFP similarity with the plasmids of Dublin and Abortusovis and the other SAP's. The presence of a plasinid thought to be an evolutionary intermediate in the development of Typhimurium and Enteritidis has been suggested. This study demonstrated the presence of plasmid in Dublin which showed more REFP similarity to the plasmid of Gallinarum than to Dublin itself and may be an intermediate in the development of the Dublin plasmid. This was strengthened by the incompatibility analysis of the plasmids. All the SAP's except Dublin were incompatible with the Typhimurium plasmid only; the plasmid of Dublin exhibited dual incompatibility properties with both pOG660 and IncX. The intermediate Dublin plasmid pOG683 showed incompatibility to the Typhimurium plasmid only. The presence of other co-resident plasmids in this serotype which exhibit IncX properties as well as the identification of large cointegrate plasmids which were unstable, suggests that the SAP of Dublin has arisen via a cointegration event with an IncX plasmid. Molecular variation within serotypes was observed at a higher incidence in host adapted serotypes (23%-Dublin, 47%-Pullorum) than those of broad host range (5% for both Enteritidis and Typhimurium). This was contradictory to the hypothesis that the narrow range of ecological conditions encountered by these serotypes would reduce the possibility of genetic diversity. Chromosomal analysis of these serotypes has previously shown that they were relatively stable and consisted of a single world-wide clone and minor sub-clones. The location of restriction sites for PstI and SmaI were determined for the plasmid of Typhimurium and fragment similarity toother SAP's in relation to existing maps suggested. A 2.3 kb PstI fragment was demonstrated to be present in the plasmids of Typhimurium, Wangata, Gallinarum, Pullorum, Bovismorbificans, Dublin and the Dublin variant pOG683. Smaller fragments of Abortusovis and Choleraesuis hybridized and indicated partial sequence homology. No homology was detected in the plasmid of Enteritidis. Not only do these results confirm a family of related plasmids within the salmonellae, they indicate much more of the plasmid is conserved. These analyses suggest molecular divergence of the plasmids from a common ancestor (Typhimurium) has arisen by loss of DNA. The population genetics of the SAP's of the salmonellae parallel the findings of chromosomal analysis in as much as they demonstrate the presence of a common worldwide clone. However, they also demonstrate that the rate of evolution of the plasmid is much higher than previously thought.