Examination of clinical and environmental Vibrio parahaemolyticus isolates by multi-locus sequence typing (MLST) and multiple-locus variable-number tandem-repeat analysis (MLVA).

Markus Fischer,Narjol Gonzalez-Escalona,Jessica L. Jones,Catharina H. M. LÃ¼deke

doi:10.3389/fmicb.2015.00564

Abstract

Vibrio parahaemolyticus is a leading cause of seafood-borne infections in the US. This organism has a high genetic diversity that complicates identification of strain relatedness and epidemiological investigations. However, sequence-based analysis methods are promising tools for these identifications. In this study, Multi-Locus Sequence Typing (MLST) and Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) was performed on 58 V. parahaemolyticus isolates (28 of oyster and 30 of clinical origin), to identify differences in phylogeny. The results obtained by both methods were compared to Pulsed-Field Gel Electrophoresis (PFGE) patterns determined in a previous study. Forty-one unique sequence types (STs) were identified by MLST among the 58 isolates. Almost half of the isolates (22) belonged to a new ST and added to the MLST database. A ST could not be generated for 5 (8.6%) isolates, primarily due to an untypable recA locus. Analysis with eBURST did not identify any clonal complex among the strains analyzed and revealed 37 singeltons with 4 of them forming 2 groups (1 of them SLV, and the other a DLV). An established MLVA assay, targeting 12 total genes through three separate 4-plex PCRs, was successfully adapted to high resolution melt (HRM) analysis with faster and easier experimental setup; resulting in 58 unique melt curve patterns. HRM-MLVA was capable of differentiating isolates within the same PFGE cluster and having the same ST. Conclusively, combining the three methods PFGE, MLST, and HRM-MLVA, for the phylogenetic analysis of V. parahaemolyticus resulted in a high resolution subtyping scheme for V. parahaemolyticus. This scheme will be useful as a phylogenetic research tool and as an improved method for outbreak investigations for V. parahaemolyticus.

Highlights

Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) was applied with the hypothesis that it will further discriminate isolates with identical sequence types (STs)
With 22 novel STs, this study substantially contributed to the diversity in the Multi-Locus Sequence Typing (MLST) database
The seven gene MLST protocol reported in a previous study was employed in this study (Gonzalez-Escalona et al, 2008)

Summary

Introduction

MLVA was applied with the hypothesis that it will further discriminate isolates with identical STs. In order to achieve this objective, an HRM-MLVA protocol for rapid and simple characterization of V. parahaemolyticus isolates was developed. In order to achieve this objective, an HRM-MLVA protocol for rapid and simple characterization of V. parahaemolyticus isolates was developed This is the first study, to the authors’ knowledge, to use the combined approach of the subtyping methods MLST, HRMMLVA, and PFGE for differentiation of V. parahaemolyticus isolates. Vibrio parahaemolyticus can cause acute gastroenteritis associated with consumption of raw or undercooked seafood (Nishibuchi and DePaola, 2005). This bacterium represents the most common cause for seafood-associated infections in the United States (Iwamoto et al, 2010).

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