A Rapid and Simple PCR Analysis Indicates There Are Two Subgroups of Vibrio vulnificus Which Correlate with Clinical or Environmental Isolation

Abstract

Of all concerns which face the shellfish industry, none are currently as critical as the presence of V. vulnificus in oysters. In fact, 95% of all deaths resulting from seafood consumption in the United States are caused by this bacterium (25). Fatality rates of over 50%, with median incubation times to onset of symptoms ranging from as little as 7 hr to several days, have been reported (24). In Japan, a recent study reported a fatality rate of over 68% (15). Between 1989 and 2000, 274 cases involving oyster ingestion occurred in the United States, of which 142 (52%) were fatal. Most (ϳ95%) cases occur in males, since females are protected from the V. vulnificus endotoxin by estrogen (19). V. vulnificus is part of the normal bacterial flora of estuarine waters and occurs in high numbers in molluscan shellfish around the world (25,26). It has been isolated from coastal waters (35) and fish (29) of Japan, and clinical cases are also common in that country (18,28). Fujisawa et al. (10) determined that 29% of the necrotizing fasciitis cases diagnosed at one hospital were due to this pathogen. V. vulnificus infections and its isolation from waters and seafood are also common in Korea (6,30,36), China (3,7,38), Hong Kong (1, 5), and Taiwan (13). Despite this, a recent study by Osaka et al. (28) revealed that of 235 emergency room physicians in Japan, less than 16% had a basic knowledge of V. vulnificus infection.Most ( Abstract: Vibrio vulnificus is an estuarine bacterium which is the causative agent of both food-borne disease and wound infection. Although V. vulnificus is commonly found in molluscan shellfish at high numbers, the incidence of disease is relatively low, leading to the hypothesis that not all strains of V. vulnificus are equally virulent. Unfortunately, there is currently no easy test to identify virulent strains of this species. We have previously identified a 200 bp randomly amplified polymorphic DNA (RAPD) PCR amplicon associated with clinical isolates. DNA sequence data from this locus in six clinical and four environmental isolates showed that the strains could be divided into two groups, which we termed C-type (correlates with clinical origin) and E-type (correlates with environmental origin). We designed PCR primers that could distinguish between the two groups, and typed 55 randomly selected strains. We found that 90% of the C-type strains were clinical isolates, while 93% of environmental isolates were classified as E-type. The region directly downstream of this locus contained a heptanucleotide sequence repeated various times depending on the strain. Using a PCR-based assay to detect the repeat number present in a given strain, we found a statistically significant correlation with the C/E type classification and the number of repeats. The data reported here are consistent with the existence of two genotypes of V. vulnificus, with the C-type being a strong indicator of potential virulence.

pilF polymorphism-based real-time PCR to distinguish Vibrio vulnificus strains of human health relevance

“…Differences in the sequence of the small subunit 16S rRNA gene, as correlating with either clinical (pathogenic) and environmental (non-pathogenic) origin have been utilised previously (Aznar et al, 1994;Nilsson et al, 2003;Vickery et al, 2007). V. vulnificus biotype 1 strains have also been classified into two genotypes based on a virulence-correlated gene, vcg (Rosche et al, 2005). This genotype has been correlated with human infection for 90% of isolates from human cases having the vcgC sequence type and 87% of environmental strains having the vcgE variant (Rosche et al, 2005).…”

“…V. vulnificus biotype 1 strains have also been classified into two genotypes based on a virulence-correlated gene, vcg (Rosche et al, 2005). This genotype has been correlated with human infection for 90% of isolates from human cases having the vcgC sequence type and 87% of environmental strains having the vcgE variant (Rosche et al, 2005). Both 16S and vcg polymorphism can be used as a predictive assay to distinguish pathogenic potential in biotype 1 V. vulnificus strains, but have failed to adequately identify biotype 2 and biotype 3 strains, which are also potentially pathogenic to humans (Sanjuan et al, 2009;Roig et al, 2010).…”

Occurrence of clinical genotype Vibrio vulnificus in clam samples in Mangalore, Southwest coast of India

“…vcg Gene typing PCR typing was carried out targeting a 277 bp fragment of the virulence correlated gene using the two primer pairs P1 (5 0 -AGCTGCCGATAGCGATCT-3 0 ), and P3 (5 0 -CGCTTAGGATGATCGGTG-3 0 ) for vcgC type strains and P2 (5 0 -CTCAATTGACAATGATCT-3 0 ) and P3 for vcgE strains as described by Rosche et al (2005). PCR was carried out in a 30 ll reaction mixture as described above with an annealing temperature of 55°C for 1 min.…”

“…Recent independent genotyping studies based on DNA sequence polymorphism at individual genetic loci such as the 16S rRNA (Nilsson et al 2003) and virulence-correlated gene (vcg) (Rosche et al 2005) have also demonstrated that V. vulnificus populations could be significantly grouped as either the clinical or environmental genotype. An analysis of the 16S rRNA gene sequences of V. vulnificus isolated from the environmental and clinical origin is seen to differ by a total of 17 out of 1556 bases, with most of the polymorphism being centered around the first 500 base pairs of the forward strand (Van de Peer et al 1996).…”

“…Similarly, by a simple PCR method it has been established that DNA polymorphism at the virulence correlated gene (vcg) locus could differentiate V. vulnificus into two main genotypes vcgC and vcgE with the two alleles predominantly being associated with clinical or environmental source of isolation (Rosche et al 2005(Rosche et al , 2010.…”