DNA fingerprinting of Salmonella enterica subsp. enterica serovar 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, qacEDelta1, sulI1, and floR. The VNTR assay showed greatly improved resolution compared to all other tested methods in this study.

FIG. 1.

Electropherogram from pooled capillary electrophoresis run of FAM-labeled products from the STTR5, STTR6, STTR3, STTR7, and STTR2 VNTR loci of the 1011095 DT104 isolate (upper panel) and the 10111243 DT104 isolate (lower panel). Vertical scale is relative fluorescence. Horizontal scale is size in base pairs. The STTR5 VNTR is 1 repeat unit longer (+6 bp) in 10111243 than in 1011095, and the STTR6 VNTR is 1 repeat unit shorter (−6 bp) in 10111243 than in 1011095.

FIG. 2.

Example of PCR amplification of integrons using primers directed to the integron variable region (gene cassette region). Lane A, 100-bp ladder; lane B, the integron profile of serovar Typhimurium DT104 isolate 10111243; lane C, the integron profile of serovar Typhimurium DT104 isolate 1805/96; lane D, the integron profile of serovar Typhimurium DT104 isolate 10402; lane E, the integron profile of serovar Typhimurium DT104 isolate 11327; lane F, a control PCR with a strain known to harbor a 1,600-bp integron; lanes G and H, 100-bp ladder. Bands: 1, 1,000 bp; 2, 1,200 bp; 3, 1,600 bp; P, 200-bp PCR artifact caused by primer mispairing.

FIG. 3.

Dendrogram of all the VNTR-typed serovar Typhimurium strains. Abbreviations: nd, phage typing or PFGE not done; nn, origin of strain not certain.

FIG. 4.

Dendrogram of the VNTR-typed serovar Typhimurium DT104 strains only. nn, origin of strain not certain.

FIG. 5.

XbaI PFGE gel with the following profiles: lane A, λ size marker; lane B, A profile; lane C, Aa profile from 3544/00; lane D, Aa profile from 3919/00; lane E, B profile; lane F, F profile; lane G, D profile from 3019/98; lane H, E profile; lane I, C profile; lane J, H profile; lane K, D profile from 1176/96; lane L, λ size marker.

FIG. 6.

AFLP electropherograms of two serovar Typhimurium DT104 isolates. The two strains have identical AFLP patterns, and this pattern is shared by all the DT104 isolates in this study.

FIG. 7.

AFLP electropherograms of two serovar Typhimurium isolates, one DT104 (upper panel) and one not DT104 but multiresistant to antibiotics (lower panel). Some variations between the two patterns can be seen. The variation is marked with filled peaks and arrows in the lower panel.

FIG. 8.

Comparison of the serovar Typhimurium strains with isolates of other Salmonella serovars and isolates of E. coli. Abbreviations: nd, phagetyping not done; nn, origin of strain not certain; nc, year of isolation not certain.