These results implied that the marine environment can serve as reservoir of both V. Õulnificus biotypes 1 and 2, and strains of 16S rRNA type B were more frequent than strains of type A in
that environment. q 2001 Elsevier Science B.V. All rights reserved.
Ž .
Keywords: V. Õulnificus; MPN most probable numbers ; Combined primers; PCR; Serotypes; 16S rRNA types
1. Introduction
Vibrio Õulnificus is a bacterium that is indigenous to coastal and estuarine waters and has been identified as the etiologic agent of certain serious, often fatal, human infections
Ž .
Tacket et al., 1984; Morris and Black, 1985 . The procedures for the isolation and identification of V. Õulnificus from marine environment have been improved during the
last decade with the development of a selective and differential cellobiose–polymyxin Ž
. B–colistin modified CPC agar in combination with selective preenrichment in alkaline
Ž peptone water supplemented with polymyxin B Massad and Oliver, 1987; Oliver et al.,
. 1992; Hoi et al., 1998 . After 24 to 48 h of incubation on CPC agar plates, V. Õulnificus
produces yellow colonies surrounded by a yellow zone due to cellobiose fermentation Ž
and are easily distinguishable from other bacteria Oliver et al., 1983; Massad and .
Oliver, 1987 . However, the final identification of V. Õulnificus usually relies upon the Ž
. biochemical tests andror immunoassays Tamplin et al., 1991 . The cost and labor
involved in these types of assessments can be prohibitive for many laboratories. It has been reported that DNA probes or PCR amplification of specific genes is
superior to the API 20E system for the identification of V. Õulnificus. V. Õulnificus Ž
. elaborates a cytotoxin–hemolysin Gray and Kreger, 1985; Morris et al., 1987 , and
DNA hybridization has shown that the gene for this protein is unique to this species Ž
. Wright et al., 1985; Morris et al., 1987 . The structural gene for the cytotoxin–hemo-
lysin has been sequenced, and oligonucleotide probes and PCR primers have been Ž
synthesized for DNA hybridization and gene amplification, respectively Wright et al., .
1985; Morris et al., 1987; Hill et al., 1991; Coleman et al., 1996 . Recently, many reports have also been published on the 16S rRNA sequences of
bacteria and the phylogenetic relationships deduced from analysis of these sequences Ž
. Collins et al., 1991; Dorsch et al., 1992; Kita-Tsukamoto et al., 1993 . Most of the
results indicate that phylogenetic relationships based on 16S rRNA sequences support the distinction of species among eubacteria, archaeobacteria, and eucaryotes. Because of
this feature, the need for rapid diagnostic methods to identify aquatic environment and animal-borne pathogens makes the variable 16S rRNA regions attractive targets for
Ž .
synthetic oligonucleotide probes and PCR primers Wayne et al., 1987 . Thus, many laboratories have employed 16S rRNA-targeted hybridization and PCR amplification for
Ž the identification and detection of several marine bacteria Heidelberg et al., 1993;
. Osorio et al., 1999 . Moreover, because they are essential constituents of all living
organisms, 16S rRNA genes are present in high copy numbers as essential and therefore, there is no problem with gene loss, which is a possibility for the cytotoxin–hemolysin
Ž .
gene Brauns et al., 1991; Aznar et al., 1994 .
Ž .
In the present study, a most probable number MPN
procedure was used to enumerate V. Õulnificus with the determination and comparison of the number of
presumptive positive tubes using selective media and PCR 16S rRNA gene amplifica- tion. The tri-primer PCR method for the differentiation of the confirmed isolates of V.
Õ ulnificus depending upon the types of 16S rRNA was also evaluated.
2. Materials and methods
