Abstract
Vibrio fluvialis has been reported to cause sporadic infections and outbreaks of diarrhoea in humans (Huq et al., 1980; Lesmana et al., 2002; Lai et al., 2006) and has also been isolated from marine and estuarine environments (Seidler et al., 1980; Lee et al., 1981; Lockwood et al., 1982). However, the public health significance of this pathogen has not been studied in detail due to the lack of simple and reliable diagnostic tests. Although the bacterium is known to produce several potent toxins, their role in pathogenesis is not well established (Lockwood et al., 1982; Huq et al., 1985; Kothary et al., 2003; Chakraborty et al., 2005). Information regarding virulence genes, and standard genetic markers for the identification of this organism, are not fully exploited. Despite the use of an array of biochemical tests including commercial identification systems, proper identification of V. fluvialis still remains a problem due to its phenotypic similarity with Aeromonas species (Seidler et al., 1980).
toxR is an ancestral gene of the family Vibrionaceae which encodes a transcriptional activation domain (TAD), a transmembrane domain (TMD) and a periplasmic domain (PD) (Osorio & Klose, 2000). Among vibrios, there is a high level of homology within the TAD of the ToxR proteins and relatively conserved homology in the TMD and PD (Osorio & Klose, 2000). Interestingly, there is essentially no homology within the region between TAD and TMD. This region connects the TAD to the cytoplasmic membrane and it was therefore named the membrane tether region. toxR-based species-specific identification has been developed for Vibrio parahaemolyticus (Kim et al., 1999) and Vibrio hollisae (Vuddhakul et al., 2000). Here, we report the development of a PCR-based assay for the specific identification of V. fluvialis exploiting the sequence divergence within the membrane tether region of the toxR gene.
Forty non-cholera Vibrio strains isolated from hospitalized patients with acute diarrhoea at the Infectious Diseases Hospital or B. C. Roy Memorial Hospital for Children, Kolkata, India, between 1998 and 2001 were included in this study. These strains were screened for Vibrio cholerae by biochemical and serological tests and by species-specific ompW PCR (Nandi et al., 2000). The oxidase-positive and ompW-negative strains were further characterized using the API 20E identification system (bioMérieux). Salt tolerance was determined by growing the strains at 37 °C on nutrient agar (NA; Difco) plates containing 0 and 7 % NaCl. For identification of the strains, 16S rDNA sequencing (Microseq 500, 16S rDNA Bacterial Sequencing kit; Applied Biosystems) was performed following the manufacturer's instructions using an automated DNA sequencer (ABI Prism 310; Applied Biosystems).
To develop a PCR-based method for species-specific identification, primers targeted to the toxR gene of V. fluvialis were first tested with the strains that were previously identified as V. fluvialis by 16S rDNA sequencing (Table 1). The forward (5'-GACCAGGGCTTTGAGGTGGACGAC-3') and reverse (5'-AGGATACGGCACTTGAGTAAGACTC-3') primers were designed from the TAD and the membrane tether region of the toxR gene of V. fluvialis, respectively (accession no. AF170885; Osorio & Klose, 2000). The membrane tether region is highly variable, but unique for each Vibrio species. For PCR assay, the strains were grown in Luria broth (LB; Difco) containing 1 % NaCl at 37 °C with shaking at 200 r.p.m. overnight. For halophilic vibrios, the LB medium was supplemented with 3 % NaCl. Purified genomic DNA from these strains was used as template in the PCR assay. Amplification was carried out in a thermal cycler (Applied Biosystems) with a standard PCR reaction mixture that contained 50 ng DNA as template, 2.5 µl 10x PCR buffer with 15 mM MgCl2, 0.20 µl (1 U) Taq Polymerase (Takara Shuzo), 2.0 µl 2.5 mM (each) deoxynucleoside triphosphate, 2.5 µl of each primer (10 pmol µl1) and sterile distilled water to make the volume to 25 µl. The amplification conditions were initial denaturation at 94 °C for 5 min, followed by 30 cycles consisting of denaturation at 94 °C for 40 s, annealing at 65 °C for 40 s and extension at 72 °C for 1 min. To detect the sensitivity, the VF-toxR PCR was performed with different concentrations of chromosomal DNA ranging from 10 to 60 ng. PCR products were electrophoresed through 2 % agarose gel to resolve 217 bp amplicons and visualized under UV light in a gel documentation system (Gel-Doc 2000; Bio-Rad) after ethidium bromide staining.