Multilocus variable-number tandem-repeat analysis for molecular typing of Shigella sonnei

Abstract

A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method was developed and evaluated for the subtyping of Shigella sonnei isolates. A total of 26 VNTR loci were identified by exploring the repeat sequence loci in the genomic sequences of S. sonnei strains Ss046 and 53G and by testing 536 isolates that had previously been characterized by pulsed-field gel electrophoresis (PFGE). The discriminatory power of MLVA (Simpson's index of diversity [D], 0.9524; 95% confidence interval [CI], 0.9373 to 0.9564) for the 536 isolates was significantly higher than that of PFGE (D, 0.8882; CI, 0.8667 to 0.9097). MLVA typing with the four and eight most variable loci had D values of 0.9468 and 0.9481, respectively, results approaching that of 26 loci. The usefulness of MLVA for outbreak investigation was evaluated using 151 isolates from 10 shigellosis outbreaks and 22 PFGE-indistinguishable isolates collected from nine epidemiologically unrelated events in five different countries. The evaluations indicated that MLVA was a powerful typing tool to distinguish isolates for outbreak investigation and that it exhibited a good discrimination of the 22 PFGE-indistinguishable isolates. Single-locus variants did occur during the outbreak; therefore, S. sonnei isolates with MLVA profiles differing at no more than a single locus should be considered part of the same outbreak. The present study suggests that MLVA has the potential to replace PFGE as a standard method of typing S. sonnei isolates for disease surveillance and outbreak investigation.

FIG. 1.

Minimum spanning tree of the MLVA genotypes for the S. sonnei strains collected from 10 shigellosis outbreaks. The clustering was constructed by a minimum spanning tree algorithm. Arabic numerals (1 to 10) and indicate the outbreaks from which the MLVA strains were collected. The circle size is proportional to the number of isolates belonging to the indicated MLVA genotype. MLVA types differing by zero or one VNTR locus are regarded as a group and are marked with gray. Differences in loci between the two MLVA types are numbered. The IST genotypes for the outbreak strains are indicated. MLVA codes are marked in the circles.

FIG. 2.

Minimum spanning tree of the MLVA genotypes for 22 Shigella sonnei isolates with indistinguishable PFGE patterns (J16N09.0015) collected from nine epidemiologically unrelated events (E1 to E9) in five different countries. Each event is noted with the country of infection, the year of occurrence, and the IST genotype. The circle size is proportional to the number of isolates belonging to the MLVA genotype. MLVA types differing by zero or one VNTR locus are regarded as a group and are colored gray. The differences in loci between two MLVA types are numbered. The MLVA codes are indicated in the circles.