Molecular Typing of Salmonella enterica Serovar Typhi Isolates from Various Countries in Asia by a Multiplex PCR Assay on Variable-Number Tandem Repeats

Abstract

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Clonal Expansion and Microevolution of Quinolone-Resistant Salmonella enterica Serotype Typhi in Vietnam from 1996 to 2004

“…Another method, multiple-locus variable-number tandem-repeat analysis, which was successfully used to subtype S. enterica serotype Typhimurium DT104 clone (19), has been developed for serotype Typhi. Two schemes have been published (22,32). They use three to six polymorphic loci.…”

Distribution and characterization of staphylococcal interspersed repeat units (SIRUs) and potential use for strain differentiation

“…These loci have a characteristic structure consisting of a variable number of near-identical repeated DNA sequences arranged consecutively. A general term for these repeats is variable-number tandem repeats (VNTRs), and true VNTRs have now been reported in Bacillus anthracis, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella enterica and Escherichia coli O157 (Keim et al, 2000;Liu et al, 2003;Noller et al, 2003;Onteniente et al, 2003;Pourcel et al, 2003). With the exception of M. tuberculosis, these studies have been confined to comparisons of molecular strain differentiation techniques.…”

Reverse Transcription-Multiplex PCR Assay for Simultaneous Detection of Escherichia coli O157:H7, Vibrio cholerae O1, and Salmonella Typhi

“…MPCR methods for detecting several genes of these bacteria have been reported previously for E. coli O157:H7 (9,10,13,16,(21)(22)(23)(24), Salmonella spp (13,21,(25)(26)(27), and V. cholerae (21,28,29 ). However, none of the reported MPCR methods simultaneously detected all three viable pathogens present in the same sample.…”