Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States

Abstract

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.

Figure 1. REP-PCR patterns and representative dendrogram.

The electrophoresis banding patterns of 167 V. parahaemolyticus isolates assayed by REP-PCR is shown. BioNumerics analysis of patterns revealed 39 unique REP-PCR groups comprised of N isolates. The corresponding BioNumerics dendrogram illustrates the genetic relatedness between REP-PCR groups, which we grouped into three major clusters (I, II, III). Groups 27, 28 and 3 comprise cluster I while groups 11, 29 and 34 comprise cluster III and all remaining groups comprise cluster II. Electrophoresis banding patterns shown with scale indicating fragment size in base pairs (bp).

Figure 2. MLST majority consensus phylogeny.

A majority consensus phylogeny of 77 V. parahaemolyticus isolates based on 7 concatenated housekeeping loci (dnaE, gyrB, recA, dtdS, pntA, pryC and tnaA) and representing 3,682 total nucleotides was constructed using the Bayesian Markov chain Monte Carlo (MCMC) method as implemented in MrBayes v3.2. The 77 isolates included in this phylogeny were separated into three major clusters (I, II, III) and 12 distinct clades (1–12). Sequence typing (ST) designations for MLST analysis describe the 24 MLST sequence types comprising each of the 12 clades. Distinct clades clearly highlighted by alternating blue and gray shading. Nodes are labeled with posterior probabilities (0–1) while cladogram shading is indicative of branches with weak support (red) and strong support (black).

Figure 3. NeighborNet analysis.

SplitsTree v4 NeigborNet analysis of 77 V. parahaemolyticus isolates based on 7 concatenated housekeeping loci (dnaE, gyrB, recA, dtdS, pntA, pryC and tnaA) representing a total 3,682 nucleotides. Sequence typing (ST) designations for MLST analysis and phylogenetic clades (1–12) included for reference. Regions of the network showing extensive reticulation (e.g., clades 8 and 10), consistent with higher rates of recombination, contrast with the less reticulated nature of clade 12. Highlights in blue distinguish groups of isolates sharing ST and clade designations and function to facilitate comparison with Figure 2.