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

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.

Keywords: HRM; MLST; MLVA; Vibrio parahaemolyticus; phylogenetics.

Figure 1

MLST minimum evolution tree of the 58 V. parahaemolyticus isolates. The tree was built with Mega six software using concatenated sequences. The scale represents the evolutionary distance and the branches show bootstrap values above 50%.

Figure 2

Combined dendrogram of MLVA melting curves of the three multiplex PCRs built with BioNumerics software version 6.6. using Pearson correlation and the unweighted pair group method using arithmetic averages (UPGMA). Isolates originated from oysters starting with “FDA,” isolates from clinical origin labeled with “CDC.” The PFGE pattern designations are as previously reported (Ludeke et al., 2014).

Figure 3

Dendrogram of MLVA melting curves of the three multiplex PCRs for the isolates carrying ST3 and ST36 built with BioNumerics software version 6.6. using Pearson correlation and the unweighted pair group method using arithmetic averages (UPGMA). The PFGE cluster designations are as previously reported (Ludeke et al., 2014).