Extended phage locus typing of Salmonella enterica serovar Typhimurium, using multiplex PCR-based reverse line blot hybridization

Abstract

Salmonella enterica serovar Typhimurium (S. Typhimurium) is the commonest pathogen causing food-borne disease among humans and animals in Australia. A multiplex PCR-based reverse line blot (mPCR/RLB) system was developed to rapidly identify S. Typhimurium phage types and strains within them. The system comprised 32 biotin-labelled primer sets and 38 amino-labelled probes, based on sequences that were either phage-type-related or derived from temperate phages ST64B, P22, Gifsy-1 or Gifsy-2. The system was developed and evaluated using 168 S. Typhimurium isolates, representing 46 phage types. RLB patterns, based on a combination of positive hybridization and grading of signal intensities, validated by sequencing, differentiated S. Typhimurium isolates into 102 types. Some clusters contained isolates belonging to a single phage type while others contained isolates belonging to more than one. Most phage types exhibited at least two RLB profiles. The feasibility of this system was evaluated during investigations of three outbreaks, due to two different phage types. Within each outbreak, isolates showed identical RLB patterns, whereas sporadic isolates of corresponding phage types showed various patterns. The mPCR/RLB system was compared with multilocus variable-number tandem-repeat analysis (MLVA). The two methods demonstrated similar discriminatory abilities. Based on these preliminary results, the mPCR/RLB system is a promising tool for molecular identification of most common S. Typhimurium phage types. It could be used as an alternative to, or in conjunction with, MLVA for rapid strain typing during outbreaks.