Comparative Phenotypic, Molecular, and Virulence Characterization of Vibrio parahaemolyticus O3:K6 Isolates

Abstract

Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.Vibrio parahaemolyticus is reported as an agent of foodborne illness in the United States (13) and around the globe. Human infection with this pathogen is associated most frequently with the consumption of seafood, primarily raw or improperly cooked shellfish (4). Consumption of sufficiently high numbers of organisms of virulent V. parahaemolyticus strains can cause gastroenteritis, septicemia, and even death. Since 1996, an increased incidence of gastroenteritis in many parts of the world (7, 19) has been associated with V. parahaemolyticus serotype O3:K6. The recent predominance of the O3:K6 serotype as a causative agent of multiple gastroenteritis outbreaks is noteworthy, because, in general, V. parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. The association of this "new" O3:K6 serotype with large-scale food-borne disease outbreaks suggests this organism may have an unusual capacity to be transmitted by foods and/or to cause human infection.The current standard method for definitive identification of V. parahaemolyticus O3:K6 requires serotyping (9). This method is expensive, tedious, and time-consuming. Because O3:K6 isolates recovered since 1996 have been shown to be genetically distinct from previously isolated O3:K6 strains (3, 17, 19),...

Characterization of Pathogenic Vibrio parahaemolyticus Isolates from Clinical Sources in Spain and Comparison with Asian and North American Pandemic Isolates

“…Isolates from Asia and the United States belonging to serotypes O3:K6, O1:KUT, and O4:K68 showed a high degree of clonality; this is consistent with previous studies (3,7,8,29,33) and supports the hypothesis of a related origin of these three new serotypes (22). However, a combination of NotI and SfiI PFGE showed slight differences between the restriction patterns from isolates belonging to the different serotypes.…”

Global Dissemination of Vibrio parahaemolyticus Serotype O3:K6 and Its Serovariants

“…Starting with AP-PCR (17,18,57,69), a variety of molecular techniques, including ribotyping and pulsed-field gel electrophoresis (18,25,101,105), revealed that O3:K6 isolates from widely different geographic areas were genetically similar to each other and distinct from O3:K6 isolates obtained before 1995 and from non-O3:K6 serotypes. The O3:K6 isolates appearing after 1995 carried the tdh but not the trh gene, did not produce urease, and were defined by a positive group-specific PCR (GS-PCR) based on the mismatched nucleotides at seven base positions of the toxRS gene sequences and ORF8 from the f237 phage.…”

On the origin and function of an insertion element VPaI-1 specific to post-1995 pandemic Vibrio parahaemolyticus strains

“…On the other hand, the synonymous divergence between two Vibrios at other loci is similar to each other, although face values appear different due to large standard errors because of the small number of synonymous sites of each gene (Table 4). Swarming related gene In V. parahaemolyticus, it is well known that there are two types of cells, translucent (TR) and opaque (OR), of which the former has a swarming ability (Jaques and McCarter, 2006) and that the swarming ability is one of characteristics for pandemic strains (Yeung et al, 2002). Although recently three regulators of the swarming ability have been reported, results suggest the presence of additional factors of the swarming regulation in V. parahaemolyticus (Jaques and McCarter, 2006).…”