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Keywords:

  • R72H polymerase chain reaction;
  • DNA–DNA hybridization;
  • Biochemical identification;
  • Vibrio parahaemolyticus;
  • Vibrio alginolyticus

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

We compared the efficiencies of biochemical methods and polymerase chain reaction (PCR) for the identification of Vibrio parahaemolyticus strains. The 122 isolates studied, identified by biochemical tests as V. parahaemolyticus or Vibrio alginolyticus, were tested by R72H PCR assay. The results obtained with the two methods were consistent for 90% of the strains studied. PCR amplification of the R72H fragment generated two unique amplicons, 387 bp and 320 bp in length. For 11% of the strains from seawater, the results of biochemical identification did not correlate with PCR results. DNA–DNA hybridization experiments provided evidence that some strains identified as V. alginolyticus in biochemical tests should be considered members of the V. parahaemolyticus species. We therefore suggest that biochemical tests are not accurate enough for the identification of V. parahaemolyticus isolates and we demonstrate that amplification of the R72H fragment, whether the amplicon is 320 bp or 387 bp long, is a powerful tool for the reliable identification of V. parahaemolyticus.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Vibrio parahaemolyticus occurs naturally in aquatic environments. Some bacteria of this species are toxigenic and may cause acute gastroenteritis, following the consumption of contaminated foods, mostly raw or undercooked fish and shellfish [1,2]. The identification by biochemical means of V. parahaemolyticus from clinical sources is straightforward. In contrast, the phenotypic variability of isolates from the environment and from food makes it difficult to distinguish accurately between V. parahaemolyticus and other members of this genus, particularly Vibrio alginolyticus, by means of biochemical tests. V. alginolyticus is isolated from coastal waters and sediments all over the world. In addition to being pathogenic for man, this bacterium is one of the most important pathogens in aquaculture, causing tremendous damage in shellfish and crustaceans[3]. V. parahaemolyticus and V. alginolyticus isolates are very closely related[4]. V. alginolyticus was prior designated as a biotype 2 of V. parahaemolyticus, but the Subcommittee on the taxonomy of Vibrios of the International Committee on systematic Bacteriology recognized it to be a distinct species, now universally known as V. alginolyticus[5,6]. DNA–DNA re-association studies revealed that V. alginolyticus shares only 60–70% homology with V. parahaemolyticus[7].

Public health concerns, heightened by an outbreak of V. parahaemolyticus gastroenteritis in the USA (Texas, New York and Washington)[8] and in Asian countries[9], have emphasized the importance of developing molecular methods for the differentiation and identification of these two bacterial species. Polymerase chain reaction (PCR) was found to be a rapid and highly specific method for detecting V. parahaemolyticus in environmental water samples, clinical samples and various food products. Venkateswaran et al.[10] proposed the use of the gyrB gene, encoding the B subunit of DNA gyrase, for the detection of V. parahaemolyticus and closely related V. alginolyticus strains. PCR primers amplifying only the gyrB fragment of V. parahaemolyticus were designed and used to detect this pathogen specifically in shrimp. However, Kim et al.[11] raised doubts concerning the usefulness of this identification system, reporting that the amplicon used to identify V. parahaemolyticus was also present in V. alginolyticus strains. The toxR gene was initially described as the regulatory gene of the cholera toxin operon and of other virulence determinants of V. cholerae; it was subsequently found in V. parahaemolyticus[12]. Two toxR gene-targeted PCR protocols were established for the specific detection of V. parahaemolyticus, but non-specific amplicons were generated by some strains of V. vulnificus and V. alginolyticus[11]. Khan et al.[13] recently characterized V. parahaemolyticus O3:K6 strains from an outbreak of gastroenteritis; they used enterobacterial repetitive intergenic consensus sequences (ERIC-PCR) and selected primers that specifically amplified a nucleotide fragment from outbreak isolates. However, this PCR method can only be used to detect V. parahaemolyticus isolates belonging to serovar O3:K6. The results suggest that these probes were not able to differentiate V. parahaemolyticus from related species. For identification, it is essential to use a nucleotide sequence that is specific and well conserved in the species. Two possible nucleotide sequences with such properties have been identified. Firstly, Bej et al.[14] suggested that PCR amplification of the tl gene, which encodes a thermolabile hemolysin, was V. parahaemolyticus-specific and could be used as a species-specific marker. Secondly, Lee et al. [15,16] described the R72H DNA sequence, which is highly conserved in V. parahaemolyticus strains and can be used for species-specific detection, although its function is unknown. Analysis of the sequences flanking the V. parahaemolyticus R72H fragment showed that this fragment was located after an rRNA operon and was composed of a non-coding region and a phosphatidylserine synthetase gene.

In this study, the biochemical identification of V. parahaemolyticus and V. alginolyticus strains was checked by PCR amplification of the R72H sequence and the tl gene. We assessed the correlation between the results obtained for biochemical detection, and molecular detection of the R72H fragment. If these two methods gave contradictory results, then we used DNA–DNA hybridization to identify the species.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Bacterial strains and biochemical identification

A total of 108 strains from seawater sampled at a fish farming plant and from aquarium facilities on the northwestern coast of France were included in this study. These strains, which were considered to belong to the species V. parahaemolyticus (72 isolates) or V. alginolyticus (36 isolates), were kindly donated by Dr. J. Lesne (Laboratoire d'Etude et de Recherche en Environnement et Santé, Ecole Nationale de la Santé Publique, Rennes, France). Clinical strains and strains isolated from food were also included in this study. The origin, number and source of each strain tested are listed in Table 1.

Table 1.  Correlation between biochemical identification and amplification of the R72H sequence specific to V. parahaemolyticus
  1. aCIP: Collection de l'Institut Pasteur; CNRVC: Collection du Centre National de Référence des Vibrions et du Choléra, Institut Pasteur; LERES: Collection du Laboratoire d'Etudes et de Recherches en Environnement et Santé, Rennes.

Biochemical identificationSourcea (accession number)Number of strainsOriginAmplification of R72H sequence by PCR, positive/total
V. parahaemolyticusCIP (75.2T)1Human1/1
 CIP (78.26)1Human1/1
 CIP (70.63)1Human1/1
 CNRVC4Human4/4
 CIP (73.30)1Seafood1/1
 CIP (71.1)1Seafood1/1
 CNRVC1Seafood1/1
 LERES72Seawater63/72
V. alginolyticusCIP (73.29)1Human0/1
 CIP (71.3)1Human0/1
 CIP (71.4)1Human0/1
 CIP (103336T)1Seafood0/1
 LERES36Seawater3/36

Bacteria were grown overnight on marine agar (MA; Difco Laboratories, Detroit, MA, USA) at 30°C and colonies isolated from pure culture were tested for Gram-staining and cytochrome oxidase. We used growth assays in salt-free peptone water and in the presence of different concentrations of NaCl ranging from 1% to 10% NaCl. Api 20E diagnostic strips (BioMerieux, Marnes-la-Coquette, France) were used to study the biochemical properties of the strains. The activities of arginine dihydrolase, lysine and ornithine decarboxylases were determined in ADH-ODC-LDC broth (Bio-Rad S.A., Yvry sur Seine, France). We added NaCl to the inoculum diluent and the culture medium, to a final concentration of 1%. Incubation was performed at 30°C for 24 h, the optimal temperature for these environmental bacteria. Identification was based on analytical profile index (fifth edition, BioMerieux, Marcy l'Etoile, France).

2.2DNA purification and PCR assay

Genomic DNA was isolated from bacterial strains, as described by Ausubel[17], by a mini-prep method with the following minor modifications. Bacteria were grown overnight on MA at 30°C. Two or three colonies were directly resuspended in 567 μl of TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0). The efficiency of extraction was assessed by electrophoresis of DNA samples in a 1% (w/v) agarose gel. Oligonucleotide primers specific for the R72H fragment[15] and the tl gene[14] were used for PCR, under the conditions specified by the authors, to confirm the correct identification of strains as V. parahaemolyticus or V. alginolyticus by biochemical means. PCR-amplified DNA (10 μl) was subjected to electrophoresis in a 2% (w/v) agarose gel (Gibco BRL, Cergy Pontoise, France). The gels were run in Tris–borate–EDTA buffer (Gibco BRL, Cergy Pontoise, France) at 80 V for 1 h, with a 100-bp DNA ladder (Amersham Pharmacia Biotech, Les Ulis, France) as molecular mass markers. The gels were then immersed in TBE buffer containing 0.5 μg ml−1 ethidium bromide. DNA fragments were visualized by UV transillumination.

2.3Restriction analysis for confirmation of PCR products

The nucleotide sequence of the 387-bp R72H fragment from strain 93 deposited in GenBank (accession numbers L30116 and Y13093) was analyzed. The Hae III restriction enzyme (Boehringer Mannheim, France), which cleaves the R72H amplicon into two bands (235 and 152 bp) easily distinguishable by electrophoresis in a 2% agarose gel, was used for the analysis. Five 320-bp amplicons and five 387-bp amplicons, from clinical, seafood or seawater isolates, were digested with Hae III.

2.4DNA sequencing

Primers based on the flanking sequences of the R72H DNA fragment were used to amplify and to sequence the DNA of these amplicons, on an ABI PRISM 373 automated DNA sequencer (Genome Express, Meylan, France). V. parahaemolyticus type strain (CIP 75.2T), which generated an amplicon of the expected size (387 bp), and one strain (L56) from seawater generating a 320-bp amplicon were used for sequencing.

2.5DNA–DNA hybridization

DNA was prepared by a procedure adapted from that described by Ausubel[17]. Absorbance ratios (A280, A260 and A230) were used to assess the purity of each DNA preparation. Genomic DNA was then sheared by sonication, to give a range of DNA fragments of various sizes, up to about 500 bp. DNA probes from V. parahaemolyticus (CIP 75.2T) and V. alginolyticus (CIP 103336T) type strains were prepared by random priming, using the Megaprime kit (Amersham Pharmacia Biotech, UK). Total genomic DNA was labeled with both [3H]dCTP and [3H]dGTP (NEN Life Science Products, Inc., Boston, MA, USA), each to a final level of approximately 20 μCi μg−1 of DNA (8 μg DNA was used for each experiment). The labeled DNA was then treated for 90 min at 50°C with S1 nuclease. DNA duplexes were produced by incubating 15–20 μl of denatured 3H-labeled DNA from V. parahaemolyticus or V. alginolyticus type strains (CIP 75.2T and CIP 103336T, respectively) with 75 μg of denatured, unlabeled DNA from seawater isolates. The DNA of each type strain served as a positive control in homologous experiments. Calf thymus DNA (Sigma, D-8661) was used as the negative control. Tm values were estimated according to the G+C content reported for V. parahaemolyticus and V. alginolyticus by Baumann[18] and hybridization was carried out for 16 h, at a temperature fixed at 10°C below Tm, under stringent conditions. DNA duplexes were incubated with S1 nuclease for 1 h at 50°C and the percentage re-association was calculated from the radioactivity of the hybrids with respect to the homologous controls, with subtraction of the non-specific background radiation resulting from non-homologous hybridization[19]. We used 70% as the cutoff point for considering organisms to be members of the same species.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Biochemical identification and confirmation by PCR assay

In Api 20E system, the ability to ferment sucrose is the only biochemical characteristic that can be used to differentiate between V. parahaemolyticus and V. alginolyticus. According to this observation, 82 of the 122 strains unable to ferment sucrose were identified as V. parahaemolyticus, and the remaining 40 strains able to ferment sucrose were identified as V. alginolyticus (data not shown). However, exceptions were described; most V. parahaemolyticus strains are unable to ferment sucrose, but 11–25% of V. alginolyticus strains are also reported to be unable to ferment sucrose[20]. Therefore, the use of this biochemical test could be inadequate and might therefore result in the misidentification of these strains.

We amplified the R72H fragment to confirm the biochemical identification of the 122 strains studied, which were isolated from clinical, seafood or seawater sources (Table 1). The results for biochemical identification and PCR amplification of the R72H fragment were correlated for the 10 clinical strains tested: an amplicon was detected only for strains identified biochemically as V. parahaemolyticus. In contrast, only 66 of 75 (88%) strains from seafood and seawater showed a correlation between biochemical profile and amplification of the R72H fragment by PCR. For the other nine strains, biochemically identified as V. parahaemolyticus, no PCR product was generated. Conversely, three of the 37 strains biochemically identified as V. alginolyticus on the basis of sucrose fermentation generated an R72H amplicon. If the R72H fragment was amplified, then one of two PCR products, approximately 320 or 387 bp in length, was obtained, for clinical strains and strains from seafood and seawater, regardless of their origin. The 387-bp amplicon was described in a previous report[15]. The difference in the size of the fragment seems to be independent of the source of the strain. The smaller amplicon (320 bp) was found in 30% (23/76) of the V. parahaemolyticus strains studied here. We also used primers previously reported to be specific to V. parahaemolyticus for amplification of the tl gene[14]. Nine strains, biochemically identified as V. parahaemolyticus and giving positive results in the R72H PCR assay, or biochemically identified as V. alginolyticus and showing no amplification of the R72H fragment, were chosen at random. Unfortunately, amplicons were obtained with both V. parahaemolyticus and V. alginolyticus strains, showing that these two species display sequence similarities in the region binding to the oligonucleotide primers. Thus, primers targeting the tl gene cannot be used to differentiate V. parahaemolyticus from V. alginolyticus.

3.2Restriction pattern and sequence analysis of generated amplicons

We checked that the PCR products corresponded to specific amplification of the R72H fragment by restriction endonuclease digestion of the amplicon. Endonuclease digestion of the 387-bp amplicon gave the pattern predicted from the GenBank sequence: the sizes of the two restriction fragments were consistent with the restriction map we established, except for type strain (CIP 75.2T), indicating probable rearrangements in the nucleotide sequence of this strain. As expected, smaller restriction fragments (approximate sizes 220 and 100 bp) were generated by digestion of the 320-bp amplicons (data not shown).

We compared the DNA sequences of the 320-bp and 387-bp amplicons. The nucleotide sequences were aligned and compared with the R72H nucleotide sequence of strain 93 deposited in GenBank (Fig. 1). The 387-bp R72H sequences of strain 93 and the V. parahaemolyticus type strain were closely related, and displayed 99% sequence identity. However, analysis of the type strain sequence showed that the Hae III restriction site was lost due to nucleotide substitutions in the type strain, accounting for the lack of restriction fragments after Hae III digestion. The 320-bp amplicon of the strain (L56) from seawater was more divergent, displaying only 81% sequence identity due to the loss of a small fragment or DNA rearrangement, accounting for the smaller size of this PCR product. However, the amplicons of these two strains were very similar to the amplicon of strain 93, suggesting that the presence of either a 320-bp or a 387-bp amplicon should be considered to be indicative of the amplification of a V. parahaemolyticus species-specific sequence.

image

Figure 1. R72H nucleotide sequence of reference strain 93[15] compared with 387-bp (V. parahaemolyticus type strain 75.2T) and 320-bp (V. parahaemolyticus strain L56 from seawater) amplification product sequences. *, deleted nucleotides; –, identical nucleotides; underlining indicates the relative positions of primers. The Hae III restriction site is shown in a box.

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3.3Confirmation of identification of V. parahaemolyticus by DNA–DNA hybridization

DNA–DNA hybridization, the standard reference method, regarded as the final arbiter for bacterial species classification[21], was used to study isolates for which the results obtained with biochemical methods and by PCR were not consistent. DNA–DNA hybridization experiments (Table 2) showed that the strains 1 and 2 biochemically identified as V. parahaemolyticus and confirmed to be V. parahaemolyticus by amplification of a 320-bp R72H fragment on PCR were closely related to the V. parahaemolyticus type strain, and displayed 76–92% DNA sequence identity to this strain. Strains 3–5 identified as V. alginolyticus on the basis of biochemical tests but from which a 387-bp fragment was amplified were assigned to the V. parahaemolyticus species, to which they displayed 71–86% sequence identity. These results suggest that R72H fragment amplification is more reliable than biochemical identification for identification of the V. parahaemolyticus species. Positive results in sucrose utilization tests have not previously been reported for V. parahaemolyticus strains, probably because only the classical biochemical identification method was used. In contrast, low levels of re-association (3/4 53%) were observed between the V. parahaemolyticus type strain and the nine strains 6–14 biochemically identified as V. parahaemolyticus but presenting no R72H amplicon. We studied seven of these nine strains further by DNA–DNA hybridization, using DNA from the V. alginolyticus type strain as a probe (Table 2). Three of these seven strains showed at least 73% sequence identity to the probe, confirming that these strains belonged to the V. alginolyticus species, despite their inability to ferment sucrose, which resulted in their biochemical identification as V. parahaemolyticus. The remaining four strains displayed less than 64% DNA sequence identity to the V. alginolyticus type strain. These strains probably belong to other species of the genus Vibrio.

Table 2.  Results of DNA–DNA hybridization
  1. aND, not determined.

StrainBiochemical identificationR72H amplification (length of amplicon)Re-association (%) with 3H-labeled DNA from
   V. parahaemolyticusV. alginolyticus
   (CIP 75.2T)(CIP 103336T)
Type strains
V. parahaemolyticus (CIP 75.2T)V. parahaemolyticusPositive (387 bp)10043
V. alginolyticus (CIP 103336T)V. alginolyticusNegative42100
Isolates from seawater
1V. parahaemolyticusPositive (320 bp)9253
2V. parahaemolyticusPositive (320 bp)7642
3V. alginolyticusPositive (387 bp)7149
4V. alginolyticusPositive (387 bp)7640
5V. alginolyticusPositive (387 bp)8656
6V. parahaemolyticusNegative5375
7V. parahaemolyticusNegative3979
8V. parahaemolyticusNegative4173
9V. parahaemolyticusNegative3864
10V. parahaemolyticusNegative4350
11V. parahaemolyticusNegative4361
12V. parahaemolyticusNegative3841
13V. parahaemolyticusNegative41NDa
14V. parahaemolyticusNegative38NDa

In conclusion, we attempted to confirm the identification of V. parahaemolyticus isolates on the basis of cultural and biochemical characters by amplification of the R72H sequence. The results obtained with the two methods were correlated in all clinical strains and in 88% of strains from seafood and seawater. PCR amplification of the R72H DNA fragment generated two unique amplicons, 387 and 320 bp in size. Restriction and sequence analysis confirmed that both PCR products were the result of amplification of the R72H sequence. All strains displaying amplification of the R72H fragment were shown by DNA–DNA hybridization to belong to the species V. parahaemolyticus, regardless of the results of biochemical identification (V. parahaemolyticus or V. alginolyticus). Finally, the results of DNA–DNA hybridization demonstrated that positive amplification of the R72H sequence can be used to assign isolates to the V. parahaemolyticus species in a reliable manner. We conclude that amplification of the R72H sequence is specific to the V. parahaemolyticus species and provides a specific means of identifying V. parahaemolyticus strains among environmental and food isolates, avoiding the misidentification that may occur in conventional studies based principally on biochemical characters.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

The authors thank Marie-Laure Quilici and Bruno Dassy for critical review of this manuscript and helpful comments.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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