Discrimination within phenotypically closely related definitive types of Salmonella enterica serovar typhimurium by the multiple amplification of phage locus typing technique

Abstract

Multilocus sequence typing (MLST) is a relatively new high-resolution typing system employed for epidemiological studies of bacteria, including Salmonella. Discrimination based on MLST of housekeeping genes may be problematical, due to the high identity of gene sequences of closely related Salmonella species. The presence of genomic sequences derived from stable temperate phages in Salmonella offers an alternative for MLST of Salmonella. We have used MLST of prophage loci in Salmonella enterica serovar Typhimurium to discriminate closely related isolates of serovar Typhimurium. We have compared these results to MLST of five housekeeping genes, as well as pulsed-field gel electrophoresis (PFGE). The presence or absence of prophage loci in the 73 serovar Typhimurium isolates tested, as well as allelic variation as detected by sequencing, provided greater discrimination between isolates than either MLST of housekeeping genes or PFGE. Amplification of prophage loci alone separated serovar Typhimurium isolates into 27 groups comprising multiple isolates or individual strains. Sequencing of isolates found within the clusters separated isolates even further. By contrast, PFGE could only divide the 73 isolates into five distinct groups. MLST using housekeeping genes did not provide any significant separation of isolates in comparison to amplification or MLST of prophage loci. The results demonstrate that the amplification and sequencing of prophage loci provides a high-resolution, objective method for the discrimination of closely related isolates of serovar Typhimurium. It is proposed that multiple amplification of phage locus typing may provide sufficient discrimination for epidemiological purposes without recourse to MLST.

FIG. 1.

Dendrogram of PCR profiles for separation of serovar Typhimurium isolates based on MAPLT. The dendrogram was generated by Dice coefficient with clustering by UPGMA, based on the presence or absence of amplified product. A total of six clusters of isolates with identical PCR profiles were generated, as well as a total of 21 isolates with unique profiles. Boldface numbers in the dendrogram refer to cluster numbers. Cluster 1 comprises the eight non-DT126 isolates, and cluster 5 comprises the four DT126 var isolates. Abbreviations: N.S.W., New South Wales; N.T., Northern Territory; Qld., Queensland; S.A., South Australia; Vic., Victoria.

FIG. 2.

Separation of serovar Typhimurium isolates based on PFGE. Dendrogram generated by Dice coefficient with clustering by UPGMA. Five different profiles were generated. Profiles 2 and 3 had >90% similarity, suggesting isolates with these profiles are genetically closely related. Serovar Typhimurium DT126 isolates were either profile 4 or 5; the remaining serovar Typhimurium isolates were found in all five profiles. Marker sizes are in kilobases × 10³.

FIG. 3.

Sequence types (ST) within the immC region of phage ST64B. The base position is the position within the sequenced region; hence, position 1 is position 28047 in GenBank accession number AY055382, which includes ST1. Eight different sequence types including ST1 were detected in the 73 serovar Typhimurium isolates. Where there was no nucleotide difference to ST1, this is indicated by a −. The filled boxes indicate regions covered by primers BIM1 to BIM4.