Rapid in situ detection of virulent Vibrio vulnificus strains in raw oyster matrices using real‐time PCR

Abstract

Vibrio vulnificus is a Gram-negative bacterial pathogen responsible for the vast majority of bacterially mediated fatalities from the consumption of raw or undercooked seafood in the USA. Vibrio vulnificus-associated septicaemia can occur rapidly (< 24 h); however, methods for the isolation and confirmation of V. vulnificus from seafood samples typically require several days. A real-time PCR assay was developed for V. vulnificus biotype 1 that provides a rapid means of identifying a gene fragment (vcgC) previously indicated as a strong predictor of potential virulence. PCR probe specificity was confirmed by amplification of 17 clinical V. vulnificus strains and by the lack of amplification with seven non-pathogenic V. vulnificus isolates and a wide range of closely related bacteria. Oyster and seawater samples were amended with a range of environmentally realistic concentrations of C-genotype V. vulnificus cells, which were quantitatively and unambiguously identified according to biotype. Of some significance, we utilized a sample processing and nucleic acid extraction procedure that allowed identification of pathogenic strains of V. vulnificus from oyster matrices without prior enrichment or culturing of strains. This outlined approach allowed the detection of as little as 50 cfu of V. vulnificus in less than 5 h, which compares favourably with culture-based approaches. The results indicate the applicability of this approach for monitoring purposes or as a potential diagnostic tool in clinical settings.

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

“…The species specificity of the real-time PCR assay was evaluated by testing pathogenic and non-pathogenic V. vulnificus strains (deemed pathogenic to humans based on previous human serum reactivity analysis), as well as a range of closely and distantly related bacteria. To compare pilF with established pathogenicity analysis we analysed strains using vcgC realtime PCR, an assay previously shown to successfully identify pathogenic biotype 1 V. vulnificus isolates (Baker-Austin et al, 2010b). In agreement with previous work (Roig et al, 2010), the results here demonstrated that the pilF was capable of correctly identifying the vast majority of human serum-resistant strains, irrespective of biotype (Table 2).…”

“…No amplification of closely related vibrio or non-vibrio strains were observed, indicating the specificity of the pilF realtime PCR assay ( Table 2). The pilF assay thus contrasts with other targets used to identify pathogenic strains of V. vulnificus, such as 16S rRNA (Aznar et al, 1994;Nilsson et al, 2003;Vickery et al, 2007) and vcgC polymorphisms (Rosche et al, 2005;Warner and Oliver, 2008;Baker-Austin et al, 2010b), which have shown limited usefulness in detecting biotype 2 and biotype 3 V. vulnificus strains potentially dangerous to human health (Roig et al, 2010;Sanjuan et al, 2009). Both biotype 2 (serovar E and serovar I) and biotype 3 V. vulnificus strains represent important human pathogens (Amaro and Biosca, 1996;Bisharat et al, 1999), and the ability to identify potentially virulent strains rapidly is of paramount importance.…”

“…In this regard, we believe that pilF is a useful and reliable additional target for epidemiological and diagnostic tests alongside other reported assays for virulence testing in V. vulnificus, such as 16S rRNA (Aznar et al, 1994;Nilsson et al, 2003;Vickery et al, 2007), capsular polysaccharide genes (Han et al, 2009), and vcgC and vcgE analysis (Rosche et al, 2005;Baker-Austin et al, 2010b;Warner and Oliver, 2008).…”

“…The digestive glands were subsequently pooled together, and then finely chopped using a sterile razor blade. Homogenates were then prepared by treating the chopped digestive glands with 100 mg/ml Proteinase K solution (30 U/mg; Promega) as previously described (Jothikumar et al, 2005;Baker-Austin et al, 2010b). Homogenates were stored at 4 C prior to testing.…”

The Biology of Vibrio vulnificus

“…vulnificus and V. vulnificus infections have been reported throughout Europe, Scandinavia, South America, the Far East, South Pacific, as well as on all coasts of the United States (15). Along with oysters and other molluscan bivalves and estuarine/coastal waters, V. vulnificus has been reported in fish (82,83,84), sediments (85,86), and plankton (73). The presence of this pathogen appears to be spreading to areas where it was not previously reported, and this is likely due to global warming (see below).…”