Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium which is a major cause of gastroenteritis worldwide. In the present study, the genetic relationships and population structure of isolates originating from clinical and seafood production sources in Thailand were investigated by multilocus sequence typing (MLST). Nucleotide sequence variation of virulence-related genes including haemolysin and TTSS1 genes among Thai and worldwide isolates was also analyzed. The outer membrane proteome of V. parahaemolyticus isolate RIMD2210633 was predicted using bioinformatic approaches, and the outer membrane proteomes of eight isolates from different sources and representing different MLST sequence type characterized using proteomics.
The 101 Thai V. parahaemolyticus isolates examined were recovered from clinical samples (n=15), healthy human carriers (n=18), various fresh seafood (n=18), frozen shrimps (n=16), fresh-farmed shrimp tissue (n=18) and shrimp-farm water (n=16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples, farmed shrimp and water samples represented five distinct clusters. The majority of clinical isolates were resolved into two genetic clusters and none of these isolates were found to share sequence types (STs) with strains isolated from human carriers, seafood, or water. Similarly, STs representing human carrier isolates differed from those of clinical, seafood and water isolates. The limited genetic diversity of the clinical isolates suggested non-random selection for pathogenic strains, but the absence of such strains in local seafood raises questions about the likely source of infection. Extensive serotypic diversity occurred among isolates representing the same STs and recovered from the same source at the same time point. Furthermore, evidence of interspecies horizontal gene transfer and intragenic recombination was observed at the recA locus in a large proportion of isolates; this has a substantial effect on the apparent phylogenetic relationships of the isolates. Notably, the majority of these recombinational exchanges occurred among clinical and carrier isolates, suggesting that the human intestinal tract is serving as a reservoir that is driving evolutionary change and leading to the emergence of new, potentially pathogenic strains. MLST was also applied to study genetic relationships between V. parahaemolyticus isolates from Thailand (n=101) and those from European countries (n=9). With the exception of the pandemic ST3 which was resolved from two isolates from Thai human carriers, two clinical isolates from England and a clinical isolate from Norway, none of the other European isolates examined in this study shared the same ST with the Thai isolates.
This study demonstrated that Thai human carrier isolates are capable of harbouring virulence-related genes including the haemolysin-encoding genes tdhA, tdhS, trh1 and trh2, and the TTSS1-related genes vcrD1, vscC2 and VP1680, that are present in clinical isolates. In particular, the Thai human carrier isolate VP132 shared identical TTSS1-related gene fragments with the pandemic V. parahaemolyticus serotype O3:K6 (RIMD2210633) and related strains (AQ3810, AQ4037, Peru466, AN5034 and K5030) of worldwide distribution.
A total of 117 outer membrane proteins (OMPs) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633. A total 73 OMPs proteins were identified from eight V. parahaemolyticus isolates recovered from clinical samples, human carriers, oyster, shrimp tissue and water in Thailand. Of the 117 predicted OMPS, 32 were identified in eight strains by proteomic analysis. OmpU, a non-specific porin protein, represents the most abundantly expressed protein in all eight isolates. OMPs involved in TTSSs (YscW, YscJ, YscC, PopN and VscC2) and iron uptake (IrgA, putative 83 Da decaheme outer membrane cytochrome C, PvuA1, PvuA2, LutA, FhuE, HutA and putative-regulated protein B) were predicted from the genome of V. parahaemolyticus isolate RIMD2210633, but were not recovered from any of the eight Thai isolates. The absence of TTSS and iron uptake related OMPs in the eight representative strains that were grown under in vitro conditions may suggest an important requirement for in vivo growth conditions to induce expression of important virulence factor-related OMPs in V. parahaemolyticus. There was no clear association between OMP profile and the source of isolation, ST or serotype. However, a high degree of variation of OMP profiles was observed in isolates from different sources as well as in the isolates representing the same ST.
This study demonstrated the usefulness of a multidisciplinary approach that includes MLST, virulence-related gene DNA sequence analysis, bioinformatic prediction and gel-based proteomic analyses for the study of molecular evolutionary relationships and the epidemiology of V. parahaemolyticus isolates from clinical and seafood production sources. The outcomes of this study highlight the role of human carriers as a reservoir of potentially pathogenic V. parahaemolyticus and this should be considered as one of the possible contamination sources in the surveillance of seafood safety
