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.

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

“…DAll primers designed by modifications based on AFLP fragments refer to Hu et al (2002), bacteriophage ST64B refers to Ross & Heuzenroeder (2005) and bacteriophage P22, Gifsy-1 and -2 refer to Mikasova et al (2005). Table 2.…”

“…Thirty-two sets of primers were designed, based on previously published primers and S. Typhimurium phage-typerelated AFLP fragment sequences. Of these, 14 sets were based on bacteriophage ST64B (Ross & Heuzenroeder, 2005), 12 on bacteriophages P22, Gifsy-1 and Gifsy-2 (Mikasova et al, 2005), and six on the AFLP fragments (Hu et al, 2002). All primers were designed or modified so that they were 18-28 bp in length, with melting temperatures between 59 and 71 uC and product sizes in the range 90-249 bp (Table 2).…”

“…A 30 ml multiplex PCR mixture containing 32 primer sets was prepared using the Qiagen Hotstar Taq PCR system as follows: 3.0 ml of 106 PCR buffer, 4.5 mM MgCl 2 , 0.2 mM dNTP, 0.5 mM each forward and reverse primer, 2 ml template DNA, 0.2 ml Qiagen Hotstar Taq polymerase (5 U ml 21 ) and water added to a total volume of 30 ml. A touchdown PCR was performed according to the protocol described previously (Ross & Heuzenroeder, 2005) with decreasing primer-annealing temperatures from 59 to 52 uC. One representative isolate from each of 46 phage types was also tested by single PCR using each primer set to validate the mPCR results.…”

“…Based on published sequence data, we designed or modified 32 primer sets targeting sequences from phages ST64B (Ross & Heuzenroeder, 2005), P22, Gifsy-1 and Gifsy-2 (Mikasova et al, 2005) and S. Typhimurium phage type-related AFLP fragments (Hu et al, 2002). They were designed to produce short PCR products with similar physical and biochemical characteristics to allow simultaneous amplification in a multiplex reaction.…”

“…A number of phage type-specific sequences have been identified in S. Typhimurium and its phages and used to distinguish phenotypically related definitive types of S. Typhimurium by PCR and sequencing (Hu et al, 2002;Lan et al, 2007;Mikasova et al, 2005;Ross & Heuzenroeder, 2005). As this involves multiple single PCRs and sequencing, it is time-consuming and unsuitable for routine identification and typing.…”