Genetic and serological identification of three Vibrio parahaemolyticus strains as candidates for novel provisional O serotypes

Highlights

• Three novel provisional O serotypes of V. parahaemolyticus were identified.

• Intra-species recombination might lead to the formation of novel serotypes.

• A PCR assay was developed for the identification of these novel serotypes.

Abstract

Vibrio parahaemolyticus is a Gram-negative, halophilic Vibrio that naturally inhabits marine and estuarine environments worldwide and has recently been recognized as one of the most important foodborne pathogens. To date, 13 O serotypes and 71 K serotypes of V. parahaemolyticus have been identified. However, untypeable V. parahaemolyticus strains are frequently found during routine detection, indicating that other forms of serotypes exist and suggesting the necessity for extension of the antigenic scheme. In this work, through the genetic analysis of the O serotype genetic determinants (OGDs) and the production of antisera and serological tests, we identified three novel O serotypes of V. parahaemolyticus. Further analyses showed that recombination and gene-set deletions/insertions within OGDs may play key roles in the generation of V. parahaemolyticus O serotype diversity. A PCR method was developed for the identification of these novel O serotypes, and specificity and sensitivity were evaluated. A double-blind test including 283 clinical isolates was performed, giving perfect correlation with the agglutination test results. Generally, our study expanded the O-antigenic scheme of V. parahaemolyticus from 13 to 16 and provided a tool with the potential for the detection and identification of V. parahaemolyticus strains (especially untypeable strains) isolated from both the clinic and the environment.

Introduction

Vibrio parahaemolyticus (V. parahaemolyticus) was first identified as a cause of foodborne illness in Japan in 1950 after an outbreak of food poisoning that sickened 272 patients and killed 20 individuals (Fujino et al., 1953). The bacterium has spread widely throughout the world, especially in Asian countries, which account for approximately 90% of global aquaculture production (Okuda et al., 1997, Wong et al., 2000), and has been recognized as one of the most important foodborne pathogens and the leading causal agent of human acute gastroenteritis primarily due to the consumption of raw, undercooked or mishandled seafood and marine products (Chowdhury et al., 2000, DePaola et al., 2003).

V. parahaemolyticus is divided into 13 O serotypes based on differences in its heat-stable somatic antigen and 71 K serotypes based on its capsular antigen (Ishibashi et al., 2000). Many unique serotypes have been identified as pandemic clones, although O3:K6, O4:K68 and O1:K25 have been recognized as the predominant groups responsible for most outbreaks since 1996 and are generally considered more virulent than other serotypes (Ansaruzzaman et al., 2005, Broberg et al., 2011, Martinez-Urtaza et al., 2005, Velazquez-Roman et al., 2014). Despite recent developments in molecular typing methods, serotyping based on polysaccharide antigens remains the ‘gold standard’ for pathogen detection, epidemiological surveillance and tracing (Wang et al., 2010). However, untypeable strains are frequently found during routine detection (Gavilan et al., 2013, Gil et al., 2007, Hashii et al., 2000, Jones et al., 2012). During the routine screening of 165 clinical V. parahaemolyticus isolates in Shanghai Municipal Center for Disease Control and Prevention, China, from 2012 to 2014 using an agglutination test with specific antisera, we obtained five strains that showed a negative result with any specific antisera against the well-known O1–O13 serotypes. These findings suggest the existence of V. parahaemolyticus clones with additional K or O serotypes. Thus, the diversity of surface polysaccharide forms of V. parahaemolyticus may be still under construction, and further extension of the antigenic scheme for this pathogen may be necessary.

Lipopolysaccharide (LPS) is an important component of the outer membrane of Gram-negative bacteria and usually consists of three distinct regions: lipid A, core oligosaccharide (core OS), and O antigen (Samuel et al., 2004). The variability of the O antigen provides the primary basis for serotyping schemes for many Gram-negative bacteria (Liu et al., 2014). However, the LPS of V. parahaemolyticus consists of only lipid A and core OS, with the latter recognized as the determinant of the O serotypes (Han and Chai, 1992, Iguchi et al., 1995). The locus of O serotype genetic determinants (OGDs) has been defined between dgkA and gmhD genes, which encode a diacylglycerol kinase and an epimerase, respectively, and different sugar synthesis genes and sugar transferase genes in the OGD region exhibit high sequence variability and specificity for each serotype.

In this study, through the genetic analysis of OGDs and the production of antisera and serological tests, three novel O serotypes of V. parahaemolyticus were identified, thereby increasing the number of O serotype forms to 16, which is important for the epidemiological surveillance and tracing of this vital foodborne pathogen. A probable genetic basis for the diffusion and formation of the V. parahaemolyticus O serotypes was discussed. Moreover, a PCR-based serotyping method was developed, and the specificity and sensitivity of this assay were evaluated.