Microevolution of Pandemic Vibrio parahaemolyticus Assessed by the Number of Repeat Units in Short Sequence Tandem Repeat Regions

Katherine García,Manfred G Höfle,Romilio T Espejo,Jaime Martínez-Urtaza,Ronnie G Gavilán,Baochuan Lin

doi:10.1371/journal.pone.0030823

Abstract

The emergence of the pandemic strain Vibrio parahaemolyticus O3:K6 in 1996 caused a large increase of diarrhea outbreaks related to seafood consumption in Southeast Asia, and later worldwide. Isolates of this strain constitutes a clonal complex, and their effectual differentiation is possible by comparison of their variable number tandem repeats (VNTRs). The differentiation of the isolates by the differences in VNTRs will allow inferring the population dynamics and microevolution of this strain but this requires knowing the rate and mechanism of VNTRs' variation. Our study of mutants obtained after serial cultivation of clones showed that mutation rates of the six VNTRs examined are on the order of 10−4 mutant per generation and that difference increases by stepwise addition of single mutations. The single stepwise mutation (SSM) was deduced because mutants with 1, 2, 3, or more repeat unit deletions or insertions follow a geometric distribution. Plausible phylogenetic trees are obtained when, according to SSM, the genetic distance between clusters with different number of repeats is assessed by the absolute differences in repeats. Using this approach, mutants originated from different isolates of pandemic V. parahaemolyticus after serial cultivation are clustered with their parental isolates. Additionally, isolates of pandemic V. parahaemolyticus from Southeast Asia, Tokyo, and northern and southern Chile are clustered according their geographical origin. The deepest split in these four populations is observed between the Tokyo and southern Chile populations. We conclude that proper phylogenetic relations and successful tracing of pandemic V. parahaemolyticus requires measuring the differences between isolates by the absolute number of repeats in the VNTRs considered.

Highlights

Diarrhea associated with seafood consumption is caused primarily by pathogenic Vibrio parahaemolyticus
We show that the analysis of the relation between variants generated by stepwise mutation (SSM) show plausible results using the Minimum Spanning Trees (MST) method with the Manhattan category which builds the tree considering the sum of the differences in repeat units of any two mutants [24]
We compared the strains collected in southern Chile; northern Chile, where an outbreak caused by the pandemic strain was observed in 1997 [28]; and Southeast Asia to determine if Multiple locus variable analysis (MLVA) (Manhattan approach) allows us to distinguish between strains according to the geographic origin and to establish possible relationships among these populations

Summary

Introduction

Diarrhea associated with seafood consumption is caused primarily by pathogenic Vibrio parahaemolyticus. Cases of diarrhea related to seafood consumption increased worldwide with the emergence of pandemic strain O3:K6, which was originally observed in Southeast Asia [2]. Isolates in this group are commonly recognized by genetic markers, including the O3:K6 antigens, which determine the serovar; the presence of genes toxRS/new [3], orf8 [4], and tdh; and the absence of trh, which is found in some pathogenic strains. Multiple loci VNTR analysis of a number of representative pandemic V. parahaemolyticus strains from Asia, Peru, and Chile using seven polymorphic loci divided the populations into two genetically distinct groups [23]. When a set of distances is given between n samples, a minimum spanning tree is the tree that connects all samples in such a way that the summed distance of all branches of the tree is minimized

Materials and Methods

Results

Discussion