Adaptation of Multilocus Sequencing for Studying Variation within a Major Clone: Evolutionary Relationships of Salmonella Enterica Serovar Typhimurium

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ABSTRACT

Serovar Typhimurium of Salmonella enterica is a model organism for studies of pathogenesis that exhibits phage-type variation and variation in host range and virulence, but in a recent study showed no sequence variation in four genes, indicating the clonal nature of this serovar. We determined the relationships of 46 Typhimurium isolates of nine phage types using mutational changes detected either by matching AFLP (amplified fragment length polymorphism) fragments to computer-modeled LT2 AFLP fragments or by sequencing intergenic regions. Fifty-one polymorphic sites were detected, which gave a single phylogenetic tree. Comparison with genome sequences of five other serovars, Typhi, Paratyphi A, Gallinarum, Enteritidis, and Pullorum, enabled determination of the root of the tree. Only two parallel events were observed, giving high confidence in the tree branching order. The mutation-based tree provided a high level of consistency and a clear lineage for the Typhimurium isolates studied. This enabled us to show that for seven of the nine phage types used, the isolates studied have a single origin, but that two phage types clearly have more than one independent origin. We found that sequencing intergenic regions provides a good strategy for detection of mutational polymorphisms and study of phylogenetic relationships of closely related isolates and would be applicable to many other species.

SALMONELLA entericaserovar Typhimurium (denoted O Typhimurium in this article) is a common cause of salmonellosis among humans and domestic animals worldwide. Most Typhimurium strains have a broad host range, causing diseases in multiple species, including humans, farm livestock, domestic fowl, rodents, and birds, whereas some Typhimurium variants have a very narrow host range, such as pigeon-adapted variants (BAUMLER et al. 1998; RABSCH et al. 2002).

The Anderson phage-typing scheme (ANDERSON et al. 1977) is commonly used in epidemiological surveillance of Typhimurium infections and provides a means of following the rise and fall of different forms and geographical distribution of Typhimurium strains. A few phage types tend to dominate within a geographical region for a significant period of time. For example, multidrug-resistant DTl 04 has become a widespread pathogen in humans and animals in Europe and the United States since 1994 (Low et al. 1997; GLYNN et al. 1998), while remaining rare in Australia. DT9 and DTl 35 are dominant in Typhimurium infections in Australia (FOWLING 1997a,b), but DT9 is rare in Europe and the United States. The availability of a phage-typing scheme for Typhimurium, and the wealth of data generated using it, make Typhimurium an excellent model for studying variation within a major bacterial clone.

We previously reported (Hu et al. 2002) the use of amplified fragment length polymorphism (AFLP; Vos et al. 1995) to explore the relationships of nine phage types of Typhimurium. The dendrogram based on presence or absence of AFLP fragments showed good correlation with phage type, grouping most isolates of the same phage type together, demonstrating potential for development of a PCR- or microarray-based typing scheme.

However, the variation was mostly due to gain or loss of mobile DNA elements, which overwhelmed mutational variation in the AFLP tree (Hu et al. 2002). Mutational changes, which accumulate over time, would better reflect phylogenetic relationships of Typhimurium isolates than polymorphisms relating to mobile elements, such as phages and plasmids. In this article, we focus on mutational differences to reveal the phylogenetic relationships of 46 isolates of nine phage types used in our previous study (Hu et al. 2002).

Because the isolates in this study are within a major clone and closely related, mutational changes are expected to be rare, and a recent study (FAKHR et al. 2005) confirmed this with no variation among 85 Typhimurium isolates in a four-gene multilocus sequence typing (MLST) study, although MLST gave good discriminatory power for S. …