Sequence Determination of rRNA Genes of Pathogenic Vibrio Species and Whole-Cell Identification of Vibrio vulnificus with rRNA-Targeted Oligonucleotide Probes

Abstract

A comparative analysis of seven new 16s rRNA gene sequences of pathogenic I/ibrio species with previously published vibrio sequences confirmed that Vibrio vulnificus represents a group that is not closely related to the core organisms of the genus Vibrio. In addition, we found that K vulnijicus, Listonella (Vibrio) anguillarum and I/ibrio diazotrophicus branch off separately from the core group. A comparison of the 16s rRNA gene sequences of V. vulniJicus strains belonging to biotypes 1 and 2 revealed that the sequences of all but four biotype 1 strains were identical to each other but slightly different (17 bases) from the sequences of the rest of the V. vulnificus strains investigated. In addition, the sequences of variable regions of the 23s rRNA genes of V'Zbrio fluvialis, VibrioJiLrnissii, Rbrio haweyi, Vibrio cholerae, and E vulnificus C7184 and TW1 were determined, aligned, and compared with all available bacterial 23s rRNA sequences in order to search for specific target sites. As a result, four oligonucleotide probes specific for V. vulniJicus were synthesized, and the specificities of these probes were evaluated by dot blot hybridization to membrane-bound RNAs from 21 V. vulnificus strains, 13 strains belonging to other fibrio species, 61 strains belonging to species that are members of the alpha, beta, and gamma subclasses of the Proteobacteria, and 3 eucaryotic microorganisms. Two probes hybridized with all of the V. vulnificus strains tested, and the other two probes distinguished V. vulnificus biotype 1 strains from all other organisms. In situ identification of V. vulnificus by using tetramethylrhodamine-or fluorescein-labelled oligonucleotides is now possible.The first description of the taxon that is designated Vibrio vulnificus was published by Baumann et al. (5); in a taxonomic study of marine bacteria, these authors reported that a group designated C2 could be clearly distinguished phenotypically. Subsequently, in 1976 Reichelt et al. (25) performed DNA-DNA hybridization studies whose results showed that this taxon was a separate species, which was then named Beneckea vulnifica. The present name, I/. vulnificus, was revived by Farmer in 1980 (13) to describe a group of lactose-positive Vibrio strains which were phenotypically and genetically distinct from other Vibrio species (31). These organisms are common inhabitants of estuarine and marine waters, and they can be isolated under a wide range of salinity and temperature conditions from oysters, clams, mussels, and fish, as well as from sediment and plankton. Recent studies of the incidence of I/. vulnificus in the United States have shown that it is present along the Gulf Coast, the Atlantic Coast, and the West Coast, and several cases of shellfish-associated T! vulnifcus infections have been reported (23). The disease syndromes caused by V. viilnijicus include septicemia and gastroenteritis following ingestion of shellfish and wound infections derived from contact with shellfish or the marine environment. In immunodepressed individuals the...

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

“…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).…”

“…Several studies have utilised different molecular markers in biotype 1 V. vulnificus as a proxy for potential human virulence, with varying degrees of success. 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).…”

Vibrio inhibens sp. nov., a novel bacterium with inhibitory activity against Vibrio species

“…6 Vibrio species are common inhabitants of aquatic environments, and they can be isolated under a wide range of salinity and temperature conditions from oysters, clams, mussels, and fish, as well as from sediment and plankton. 7,8 Some species have been reported to cause infections in humans and aquatic animals, 9,10 whereas a small number of other species have been used as probiotics in aquaculture. 11,12 In the present study, a Gram-negative bacterium (BFLP-10 T ), with inhibitory activity against Vibrio species, was isolated from faeces of wild long-snouted seahorses (H. guttulatus) captured in northwest Spain.…”

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

“…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). A restriction fragment analysis of the polymorphic region has further shown to generate two profiles 16S rRNA typeA or 16S rRNA typeB which significantly correlated respectively to its environmental or clinical origin (Aznar et al 1994;Kim and Jeong 2001;Nilsson et al 2003).…”