Salmonella enterica is a highly diverse species with more than 2600 serovars. The ability to distinguish serovars within the species is important for surveillance. With the uptake of whole-genome sequencing technology, serovar determination by traditional serotyping is being to be replaced by inference of serotypes from genome sequences. In this study, we aimed to identify new serovar-specific gene markers for the common Salmonella serovars and to validate the genomic signatures for accurate detection of five most common serovars in Australia.
A total of 2258 publicly available Salmonella genomes were selected to include serovars with at least 5 genomes available. The genomes were annotated using PROKKA. Pan-genome and core-genome were analyzed by Roary using an 80% sequence identity threshold. The genes specific to each serovar were identified from the pan-genome’s accessory genes using an in-house python script with a cutoff of 20% false negatives and 10% false positives. Multiple Cross Displacement Amplification (MCDA) was employed to develop highly sensitive assay to detect the common serovars.
Comparative genomics of 2258 Salmonella accessory genomes identified a minimum of 131 serovar-specific gene markers to allow in silico typing of the 106 serovars with 95.1% accuracy. To evaluate the usefulness of gene markers for assay development, we selected seven genes which were specific to the five most common Salmonella serovars in Australia: Typhimurium, Enteritidis, Virchow, Saintpaul, and Infantis. Seven MCDA assays targeting these seven genes for rapid identification of the five Salmonella serovars were developed and evaluated. They demonstrated a sensitivity of 50 fg/ul (10 copies) on the pure DNA and were specific to the target serovars. The assays were also rapid with a result detectable within 8 minutes.
A new in silico serotyping method based on genomic data of the common Salmonella serovars is described. The gene markers identified in this study have also been used to develop a rapid, accurate and sensitive serotyping MCDA assay which opens the way for culture independent serotyping directly from clinical samples.