DNA Fingerprinting ofSalmonella entericasubsp.entericaSerovar Typhimurium with Emphasis on Phage Type DT104 Based on Variable Number of Tandem Repeat Loci

Abstract

Seventy-eight human and environmental strains of Salmonella enterica subsp. enterica serovar Typhimurium, as well as 18 isolates of other Salmonella serovars and 6 isolates of Escherichia coli, were subjected to a novel variable number of tandem repeats (VNTR)-based fingerprinting method that showed high discrimination and reproducibility for typing serovar Typhimurium isolates. The method is based on capillary separation of PCR products from fluorescence-labeled VNTR in the serovar Typhimurium genome. The serovar Typhimurium isolates displayed 54 VNTR patterns, and the VNTR assay correctly identified strains from a well-characterized outbreak. Among 37 serovar Typhimurium phage type DT104 isolates, 28 distinct VNTR patterns were found. This VNTR-based method is fast and suitable for complete automation. Our VNTR-based method was capable of high discrimination within the homogeneous serovar Typhimurium DT104 phage type and can be used to trace outbreaks and to monitor DT104 as well as other phage types. The VNTR assay was compared to XbaI pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, integron-cassette profiles and gene PCR of intI1, qacE⌬1, sulI1, and floR. The VNTR assay showed greatly improved resolution compared to all other tested methods in this study.The class of repetitive DNA named variable number of tandem repeats (VNTR) is, in general, a source of genetic polymorphism in humans. VNTR have been extensively studied in humans and can consist of several hundreds to several thousands of base pairs of DNA in head-to-tail repetition of short sequence motifs of about 10 to 100 bp (20). The spontaneous mutation rate to new alleles is sufficiently high to be measured in human pedigrees, and VNTR can possess several alleles (3, 19) and be implicated in human disease (28). VNTR have gained interests in prokaryotes when complete bacterial genomes were sequenced. Several bacterial strains have multiple VNTR in their genome, and in some instances, they have been adopted for typing purposes. A VNTR-based assay has recently been developed for typing the pathogen Bacillus anthracis using eight VNTR loci. Characterization of 426 B. anthracis isolates with this method gave 89 distinct genotypes (22). Polymorphic VNTR regions which can be used for typing purposes have been identified and tested in Yersinia pestis (1, 23), Francisella tularensis (21), Mycobacterium tuberculosis (10-12, 37, 40, 41), Xylella fastidiosa (6), Haemophilus influenzae (43,44,49), and Bacillus anthracis (2,8,18,22,38). The VNTR-based typing approach is promising in all of these strains. A database of tandem repeats in several completely sequenced genomes is also available (26). VNTR appear to contain a high level of polymorphism, which gives a high discriminatory capacity.Salmonella enterica serovar Typhimurium is the only salmonella serovar that causes recurrent domestic infections in Norway. The method of choice for typing serovar Typhimurium as a means of source identification, outbreak investigation, and phy...

Use of multilocus variable-number tandem repeat analysis in molecular subtyping of Salmonella enterica serovar Typhi isolates

“…The primer screening process selected 34 TRs that were easily amplified by PCR and had only one amplicon in the Y.-Y. Tien and others (Liu et al, 2003); STTR5 (Sty44) (Lindstedt et al, 2003), Sal02 (Sty37), Sal06 (Sty39), Sal20 (Sty40) and Sal10 (Sty43) (Ramisse et al, 2004); and TR4500 (Sty20) and TR4699 (Sty25) (Octavia & Lan, 2009). Two novel VNTRs, Sty2 and Sty3, were discovered in the present study.…”

“…Based on the analyses of a small set of highly variable loci, MLVA is sufficient to replace PFGE in resolving closely (Liu et al, 2003); STTR5 (Lindstedt et al, 2003); Sal02, Sal06 and Sal20 (Ramisse et al, 2004); TR4500 and TR4699 (Octavia & Lan, 2009). DThe range of repeat units for allele 173 of Sty20, which has a 7 bp deletion at the 59-flanking region immediately adjacent to the repeats, is reported instead of that for the fragment size (bp).…”

“…Multilocus variable-number tandem repeat analysis (MLVA) is a well-known typing method that has been successfully developed for many bacterial species or for serovars within a species (Liang et al, 2007;Liao et al, 2006;Lindstedt et al, 2003;Marsh et al, 2006;Noller et al, 2003). Based on the analysis of multilocus variable-number tandem repeats (VNTRs), MLVA can be an extremely powerful method for distinguishing between closely related isolates in the investigation of disease outbreaks (Liang et al, 2007;Noller et al, 2003;Torpdahl et al, 2007).…”

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

“…Multilocus variable-number tandem-repeat analysis (MLVA) has been developed for various Salmonella serovars (Cho et al, 2007;Lindstedt et al, 2004;Liu et al, 2003;Witonski et al, 2006). It has allowed a high level of resolution of variant strains of the relatively homogeneous S. Typhimurium definitive type 104 (Lindstedt et al, 2003).…”

Caracterización por electroforesis de campo pulsado de aislamientos de Salmonella Typhimurium recuperados en el programa de vigilancia de enfermedad diarreica aguda en Colombia, 1997-2004

“…como la determinación de la susceptibilidad antimicrobiana (3,7,10) y la fagotipificación (11), las cuales permiten establecer relaciones epidemiológicas. Las técnicas de subtipificación molecular como la electroforesis en gel de campo pulsado (PFGE), considerada la prueba de oro para la diferenciación y el agrupamiento de aislamientos de Salmonella spp.…”