Vibrio (V.) parahaemolyticus is naturally present in coastal waters worldwide [1–4]. It is associated with self-limiting gastroenteritis due to the ingestion of contaminated raw or undercooked seafood [5, 6]. In 1996 the pandemic O3:K6 serotype emerged in Asia and was identified as the predominant cause of numerous outbreaks throughout the world [7–10]. In recent years, other serotypes, esp. serovariants of O3:K6, were associated with severe outbreaks .
To distinguish between different lineages of V. parahaemolyticus various techniques have been used so far (e.g. serotyping, PFGE, rep-PCR), most promising multilocus sequence typing (MLST). In MLST analysis the genotypic relatedness of bacterial strains is analyzed basing on the sequences of internal fragments of usually 6 to 8 housekeeping genes [11, 12]. Due to the nucleotide sequence based typing the comparison of results obtained by others and exchange via public databases is possible and allows continuously increasing understanding of the molecular epidemiology and evolution of the typed bacteria [12–14].
The population of V. parahaemolyticus is characterized by a high degree of genotypic diversity that diversifies in the first step via recombination and is thus called a semi-clonal population [13, 15]. In its habitat the marine and estuarine environment V. parahaemolyticus encounters changing environmental conditions . Better adapted or faster adapting clones arise from the background of the diverse and highly recombinogenic bacterial population leading to the “pandemic” model of clonal expansion . In MLST analyses such adapted clones are termed clonal complexes (CC) and are characterized by strains of allelic profiles or Sequence Types that differ in at most one allele. The pandemic clone of V. parahaemolyticus, consisting of O3:K6 strains and its serovariants, shares the same genetic properties (trh
+, GS-PCR+) and forms the distinct cluster of clonal complex 3 (CC3) founded by Sequence Type 3 (ST3). On the contrary the converse argument is not true as CC3 is also formed by non-pathogenic strains . Since ST and serotype are not linked, a diverse set of serotypes constitutes ST3 (largely caused by serotype switching via recombination) [9, 13, 17–20].
The overall genotypic diversities differ depending on the pathogenicity of strains: Pandemic strains show a high uniformity, whereas non-pandemic strains are highly diverse, leading to the observation that an analyzed geographically restricted subpopulation was genetically as diverse as the entire worldwide pubMLST database [21–24]. In contrast, environmental tdh
V. parahaemolyticus are as diverse as the non-pathogenic populations . Diversity also depends on water temperature, with a less diverse cold water adapted population replaced by more diverse strains when temperature rises . The environmental populations are characterized by a fast evolution observable in the rapid turnover of predominant strains [25, 26]. But some clones and strain groups can persist for years in a specific habitat, creating an endemic population . With the application of MLST a high degree of genetic similarity between environmental and pandemic or non-pandemic infectious isolates as well as the mentioned environmental clade of CC3 isolates was shown, emphasizing the potential threat even of environmental strains to human health . A clustering of strains in regard to specific properties, like sampling time, habitat or origin is desired to establish a relationship between these properties and the genotype (in the case of MLST the ST) of a strain. However, in the case of V. parahaemolyticus this was not possible in general [13, 19, 25]. Theethakaew et al. were able to identify distinct clusters of strains sampled either from farmed prawns or clinical cases . Due to the high genetic diversity especially of environmental strains, the identification of related strains can lack reliability; therefore clustering of strains on the basis of their amino acid sequence was applied to V. parahaemolyticus[24, 28].
Even though some studies already used MLST analysis to characterize V. parahaemolyticus strain sets, they were restricted to specific geographical areas (e.g. U.S. coast, Thailand and Peru) [23, 24, 27, 29], focused exclusively on pandemic or non-pandemic pathogenic isolates [17, 21, 22, 25, 26, 29] or were based on a limited strain number. Whether strains, originating from a specific habitat but of different geographic regions, possess similar properties concerning their genetic diversity has not been investigated yet.
Thus, the goal of this study was to investigate four different V. parahaemolyticus strain sets, each of distinct geographical origin (a cold water population originating from the German North Sea and the Baltic Sea, two prawn associated strain sets originating from Sri Lanka and Ecuador and additionally seafood isolates from German retail) by using MLST analysis, in order to define sequence polymorphism of the strains, investigate genetic polymorphisms and relationships among strains of the different regions and to analyze the probable evolutionary relationships among the strains. Therefore differences in the relationship of isolates in regard to sequence type, clonal complex and peptide sequence type affiliation were considered. To analyze peptide based differences a peptide-based MLST scheme was implemented into the pubMLST database. To obtain a more global overview previously available MLST data of isolates from other countries and continents were included.