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www.elsevier.nlrlocateraqua-online

Development of 16S rRNA targeted PCR methods

for the detection and differentiation of Vibrio

Õ

ulnificus in marine environments

Myoung Sug Kim, Hyun Do Jeong

)

Department of Aquatic Life Medicine, Pukyong National UniÕersity, 599-1 Dae Yeon Dong, Nam Ku, Pusan 608-737, South Korea

Received 12 April 2000; received in revised form 8 July 2000; accepted 8 July 2000

Abstract

A PCR method for the detection and differentiation of VibrioÕulnificus strains was developed

as an alternative to culture methods by using combined primers directed against the variable regions of 16S rRNA. Primers designed from two variable regions of the Vibrionaceae 16S rRNA Žcorresponding to nucleotide numbers 1006 to 1023 and 1278 to 1258 in Escherichia coli 16S

rRNA was found to be species-specific for V.Õulnificus by PCR. Additionally, tri-primer PCR of

16S rRNA was evaluated for the differentiation of V.Õulnificus strains. Although the third primer,

which was derived from the variable region, positions 454 to 473, cannot discriminate V.

Õulnificus from other bacteria, it was used to avoid the detection of type B 16S rRNA of this

organism in PCR. The resulting 825 bp fragment in the presence of the 273 bp fragment, which is specific to V.Õulnificus, in tri-primer PCR clearly differentiated type A 16S rRNA strains from

type B. Enumeration of V. Õulnificus in the samples of oyster and environmental samples was

Ž .

done by most probable numbers’ MPN method of five preenrichment tubes of alkaline peptone water supplemented with polymyxin B following up the confirmation of positive tubes by streaking the samples onto mCPC agar or by 16S rRNA gene amplification. Higher numbers of presumptive V.Õulnificus confirmed by selective media compared with those confirmed by PCR

method in MPN method suggested that there would be some bacteria that cannot be discriminated from V.Õulnificus on mCPC agar in environmental samples. In the biotyping of the V.Õulnificus

Ž .

isolates in oyster samples, the majority of the strains 92.5% belonged to biotype 1, and 7.5% of the strains belonged to biotype 2. However, strains of 16S rRNA of V.Õulnificus isolates in the

marine environment determined by tri-primer PCR appeared to be 35% type A and 65% type B.

)_{Corresponding author. Tel.:}_q_{82-51-620-6143; fax:}_q_{82-51-628-7430.}

Ž .

E-mail address: [email protected] H.D. Jeong .

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These results implied that the marine environment can serve as reservoir of both V. Õulnificus

Ž .

Keywords: V. Õulnificus; MPN most probable numbers ; Combined primers; PCR; Serotypes; 16S rRNA

types

1. Introduction

VibrioÕulnificus 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 ŽTacket 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

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 ŽWright 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

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

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 .

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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.

Õulnificus depending upon the types of 16S rRNA was also evaluated.

2. Materials and methods

2.1. Bacterial cultures and DNA extraction

Ž .

A total of 12 species of bacteria, 11 species of Gy bacteria including three V. Ž

Õulnificus strains, five Vibrio species, and Streptococcus sp. were used in this study. V.

Õulnificus CJVV04 from R & D Center of Cheiljedang, Korea; V.Õulnificus HUFP 5002,

V. alginolyticus HUFP 9701, V. parahemolyticus HUFP 9114 and V. cholerae POI 9001

. Ž .

from Dr. K. Muroga, Hiroshima University, Japan Table 1 . All of the Vibrio species

Ž .

were grown aerobically on brain heart infusion broth Difco, Detroit, MI, USA

Ž .

supplemented with 1% wrv NaCl at 258C for 18 h. Cultured cells were harvested by centrifugation at 8000=g for 10 min and lysed with 5.5% SDSr0.125 mgrml proteinase K solution. Bacterial nucleic acids were extracted by a phenol–chloroform–

Ž . Ž .

isoamyl alcohol 25:24:1 vrvrv mixture and chloroform–isoamyl alcohol 24:1 vrv mixture. The nucleic acids were precipitated by adding the two volumes of ethanol in the presence of 0.3-M sodium acetate.

2.2. Primers’ design

Ž . Ž .

Two senses Vib 1, Vib 2 and one antisense Vib 3R primers were constructed for

Ž .

V. Õulnificus 16S rRNA sequence Table 2 . These are based upon a gene alignment

Table 1

Bacterial strains used in this study

Organism Source and strain Origin

V.Õulnificus CJVVO4 Human

HUFP5002 Hiroshima University

ATCC27562

V. alginolyticus HUFP9701 Hiroshima University

V. cholerae POI9001 Human

V. fluÕialis Isolate in lab. Sea water

V. mimicus Isolate in lab. Sea water

V. parahaemolyticus HUFP9114 Hiroshima University

Aeromonas hydrophila ATCC7966

Edwardsiella tarda EDK-2 Eel

E. coli HB101 Commercial

Streptococcus sp. Isolate in lab. Flounder a

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Table 2

Primers used in PCR

Primer Target gene Sequence Direction Position

X X

Cyt1 cytolysin 5 -ACAAAGACGGCCGCAAAGTGG-3 sense 1277–1297

X X

Cyt2 cytolysin 5 -AGCCCGCAGAGCCGTAAACC-3 antisense 1684–1665

X X

Vib 1 16S rDNA 5 -GTGGTAGTGTTAATAGCACT-3 sense 454–473

X X

Vib 2 16S rDNA 5 -TCTAGCGGAGACGCTGGA-3 sense 1006–1023

X X

Vib 3R 16S rDNA 5 -GCTCACTTTCGCAAGTTGGCC-3 antisense 1278–1258

Sequence positions for Vib 1, Vib 2, and Vib 3R are given according to the E. coli 16S rDNA numbering system.

using MACAW Version 2.0.5, National Center for Biotechnology Information, Na-.

tional Institutes of Health, USA program for 16S rDNA sequences of 16 Vibrio species

ŽÕ_{ulnificus ATCC 27562,} Õulnificus C7184, alginolyticus, anguillarum, campbelli,

carchariae, cholerae, diazotropicus, fluÕialis, parahaemolyticus, proteolyticus,

mimi-cus, ordalii, hollisae, penaeicida, furnissii and damsela re-classified as

Photobac-. Ž

terium damselae recently retrieved from the Entrez database accession numbers;

X56582, X76334, X56576, X71819, X56575, AF134581, X74696, X56577, X76335, X56580, X56579, X74713, X74718, X56583, AJ249719, X74704 and X74700,

respec-.

tively . Three primers were designed: Vib 1, Vib 2, and Vib 3R, which were comple-mentary to regions of 16S rRNA of V.Õulnificus ATCC 27562; base positions 454 to

473, 1006 to 1023, and 1278 to 1258 in Escherichia coli 16S rRNA Kita-Tsukamoto et

. Ž .

al., 1993 . Cyt1 and Cyt2 primers Table 2 were derived from the nucleotide sequence Ž

of the cytotoxin–hemolysin structural gene base positions 1277 to 1297 and 1684 to

. Ž .

1665, respectively cloned by Morris et al. 1987 and used for the detection of V.

Õulnificus by PCR gene amplification. All primers were synthesized with an automated

Ž .

DNA synthesizer Bioneer, Taejon, Korea by the phosphoramidite method. PCR amplification was carried out in a 50 ml reaction mixture containing the extracted

Ž .

bacterial nucleic acids 100 ng of the isolated total nucleic acids , 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl , 0.001% w2 rv gelatin, 0.5% Tween-20, 200mM each

dNTP, 1mM each primer, 1.25 U AmpliTaq DNA polymerase Perkin-Elmer, Norwalk,

. Ž .

CT, USA with an Perkin-Elmer 2400 thermal cycler Perkin-Elmer . Amplification consisted of 35 cycles at 948C for 30 s, 558C for 30 s, and 728C for 30 s in 0.2 ml thin-walled tubes. The results of amplification were analyzed by 2% agarose gel electrophoresis. The PCR products were purified by agarose gel electrophoresis using a

Ž .

Prep-A-Gene DNA Purification systems Bio-Rad and sequenced using Big dye termi-Ž

nator cycle DNA sequencing kit ABI PRISM, PE Applied Biosystems, Foster City, CA, .

USA and an automatic sequencer. RT-PCR amplification was performed for the isolated total nucleic acids from V. Õulnificus using the EZ-rTth DNA polymerase ŽPerkin-Elmer . The cDNAs to the 16S rRNA were synthesized in a reaction mixture.

Ž .

containing 10 mM Tris–HCl, 50 mM Bicine, 115 mM potassium acetate, 8% wrv

Ž .

glycerol, pH 8.2, 2.5 mM Mn OAc , 12 mM of each Vib 2 and Vib 3R primer, 300mM each dNTP, 1 Urml RNase inhibitor and 2.5 U EZ-rTth DNA polymerase at 608C for 45 min followed PCR amplification on the same tube containing cDNA produced using

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the 30 cycles of denaturation at 958C for 30 s, annealing at 658C for 30 s and extension at 728C for 30 s.

2.3. Collection and treatment of samples

Coastal sea water, sediment, and cultured oyster samples were obtained at the Hadong area, located at the southern coast of Korea, in late August, 1999. The temperature of the water was 228C and the salinity, as determined from the refractive index, was 32 ppt. To enumerate the V. Õulnificus, samples were analyzed by using a five-tube MPN method with alkaline peptone water preenrichment supplemented with

Ž .

polymyxin B APWP followed by the identification of presumptive V. Õulnificus positive tubes using mCPC agar plating or PCR gene amplification with specific primers

Ž .

against V. Õulnificus. Top-layer sediment approximately 500 g and oysters obtained

Ž .

from local farmers 10 oysters, average body weight 15 g were transported in a sealed Ž sterile plastic bag to the laboratory and homogenized with an equal volume of PBS pH

. Ž .

7.5 in a stainless steel commercial blender Sam Sung at high speed for 60 s. Water Ž

samples were collected along with oyster samples. Homogenized samples 0.01, 0.1, and

. Ž .

1 g were inoculated into 10 ml of APWP five tubes for each weight of sample and incubated at 378C for 24 h. For the determination of the positive tubes of V.Õulnificus by the MPN method, samples showing turbidity were analyzed by PCR gene amplifica-tion with the primers Vib 2 and Vib 3R or by the appearance of yellow colonies after streaking onto mCPC agar plates with incubation at 408C for 24 h. MPN tables were

Ž used to estimate the number of V. Õulnificus cells originally present in a sample US

. Food and Drug Administration, 1984 .

2.4. Identification and characterization of V.Õulnificus isolates

From the colonies on mCPC agar plates of different samples, 111 V.Õulnificus-like yellow colonies were picked randomly and used to isolate total nucleic acid. PCR gene

Ž .

amplification was performed with two primers Vib 2 and Vib 3R to confirm that the isolated colonies were V. Õulnificus. All verified isolates as V. Õulnificus were further

Ž .

analyzed to determine the biotypes by indole production in tryptone broth Difco supplemented with Kovacs indole reagent and the 16S rRNA types were determined by

Ž .

PCR gene amplification using tri-primers Vib 1,Vib 2, and Vib 3R .

3. Results

3.1. DeÕelopment of the PCR method

Amplification of genomic DNA isolated from three different strains of V.Õulnificus with primers Vib 2 and Vib 3R resulted in a product with the predicted length of 273 bp, while no products were obtained from those of other species of bacteria including six

Ž .

different species of Vibrionaceae Fig. 1 . For verification, the PCR product derived from the amplification of the total nucleic acid of V.Õulnificus, CJVVO4 was sequenced

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Ž .

Fig. 1. Agarose 2% gel electrophoresis analysis of the PCR products from nucleic acids of bacteria with V.

Õulnificus specific primers, Vib 2 and Vib 3R. Lane 1: V.Õulnificus CJVVO4; Lane 2: V.Õulnificus HUFP

5002; Lane 3: V.Õulnificus ATCC 27562; Lane 4: V. alginolyticus HUFP 9701; Lane 5: V. cholerae POI

9001; Lane 6: V. fluÕialis; Lane 7: V. mimicus; Lane 8: V. parahaemolyticus HUFP 9114; Lane 9: A. hydrophila ATCC7966; Lane 10: E. coli HB101; Lane 11: Edw. tarda EDK-2; Lane 12: Streptococcus sp.;

Lane M: 100 bp DNA ladder.

and showed no differences compared with the same target region in the 16S rRNA gene

Ž .

of V.Õulnificus biotype 1 strain ATCC 27562 data not shown . The PCR amplification of a 10-fold serial dilution of V.Õulnificus DNA indicated that at least 5 pg of nucleic

Ž .

acid was required to yield a visible fragment on agarose gel electrophoresis Fig. 2 . The

Fig. 2. Detection limit of the total nucleic acids isolated from V.Õulnificus CJVVO4 by PCR and RT-PCR

Ž . Ž .

with V.Õulnificus specific primers. A Product of PCR gene amplification with primers Cyt1 and Cyt2; B Ž .

product of PCR gene amplification with primers Vib 2 and Vib 3R; C product of RT-PCR gene amplification with primers Vib 2 and Vib 3R; Lane 1 through 4; 50 pg, 5 pg, 500 fg, 50 fg of V.Õulnificus CJVVO4 nucleic

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sensitivity was close to that of PCR with Cyt1 and Cyt2 primers. With RT-PCR amplification to increase the sensitivity, it was found that 500 fg of the nucleic acids, which was 10-fold lower than the threshold sensitivity of PCR amplification, was

Ž .

sufficient to yield a visible fragment by agarose gel electrophoresis Fig. 2 .

3.2. Determination of rRNA types of V.Õulnificus

V.Õulnificus could be separated into the following two groups corresponding to the

two different 16S rRNA sequences: type A, 16S rRNA corresponding to V.Õulnificus ATCC 27562; and type B, 16S rRNA corresponding to V. Õulnificus C7184 or TW1 Ž_{Aznar et al., 1994 . The complete 16S rRNA sequences of type A strains were slightly}.

Ž . Ž

different 17 bases from the corresponding sequences of the type B strains Aznar et al., .

1994 . Primers Vib 2 and Vib 3R used in the experiment above were targeted to the variable regions of 16S rDNA of Vibrio species and were specific for both rRNA types of V.Õulnificus. Another PCR sense primer Vib 1 is complementary to position 454 to 473 in 16 rDNA of V. Õulnificus ATCC 27562 known to be rRNA type A strain. Although it is not complementary to the target region of the 16S rRNA type B strain of

V.Õulnificus, it can hybridize to the 16S rDNA of other species of bacteria depending

upon the phylogenetic relatedness of the 16S rRNA. Upon PCR amplification with

Ž .

Fig. 3. Agarose 2% gel electrophoresis of amplicons generated by tri-primers PCR with the total nucleic acid isolated from V.Õulnificus strains of different 16S rRNA types and other eubacteria. Lane 1: V.Õulnificus

Ž . Ž .

ATCC 27562 type A 16S rRNA ; Lane 2: V.Õulnificus CJVVO4 type B 16S rRNA ; Lane 3: mixture of the Ž

isolated nucleic acids from five different strains of Vibrionaceae V. alginolyticus HUFP 9701, V. cholerae

POI 9001, V. fluÕialis, V. mimicus, V. parahaemolyticus HUFP 9114 ; Lane 4: mixture of three different

Ž . Ž .

strains of Gy bacteria A. hydrophila ATCC 7966, E. tarda EDK-2, E. coli HB101 ; Lane M: 100 bp

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Ž .

tri-primers Vib 1, Vib 2, and Vib 3R , the presence of two amplified products, the 273 and 825 bp fragments, discriminated the nucleic acid of rRNA type A V.Õulnificus from

Ž that of rRNA type B V.Õulnificus, which produced a single fragment 273 bp long Fig.

3 . The sequence of the 825 bp fragment was exactly the same as that of V.Õulnificus

Ž . Ž

C7184 data not shown , which is reported to be a type B 16S rRNA strain Aznar et al., .

1994 .

Tri-primer PCR amplification using nucleic acid isolated from other strains of bacteria as a template yielded no fragments or only a single fragment 825 bp long.

3.3. Characteristics of V.Õulnificus occurred in oyster and enÕironmental samples

Õulnificus. The other one was PCR analysis with the primers Vib 2 and Vib 3R against

the total nucleic acids isolated from the bacteria of the cultured tube samples. In a comparison of these two methods, the number of V.Õulnificus counted when samples from oyster, coastal mud, and sea water was analyzed by PCR appeared to be 49%, 13%, and 12% of those counted with subculturing on mCPC agar plate, respectively ŽTable 3 ..

Furthermore, the proportion of V. Õulnificus isolates confirmed by PCR analysis among the presumptive colonies of V. Õulnificus on mCPC agar plates also showed similar to that of the compared result of MPN method described above and appeared to be 47%, 25%, and 15% with the colonies of the tube samples of oyster, coastal mud, and

Ž .

sea water, respectively Table 4 . For the classification of the isolated V. Õulnificus

Ž .

depending upon the biochemical characteristics biotype and the sequence of rRNA Ž_{rRNA type , a total of 40 V.}. Õulnificus isolates from oyster 35 , coastal mud 3 , andŽ . Ž .

Ž .

sea water 2 were tested for indole production and the presence of the 825 fragment on

Ž .

tri-primer PCR amplification Table 4 . The prevalence of V.Õulnificus biotype 2 in the samples of oyster, coastal mud, and sea water was 2 of 33, 1 of 2, and 0 of 2, respectively. However, the rRNA types of the isolated V.Õulnificus did not show such a

Table 3

Characteristics and confirmation of the identities of V.Õulnificus isolates

Ž .

Samples Total number of Number of V.Õulnificus cellsr100 ml or g

Ž .

bacteria g or ml identified by

mCPC plating PCR

a 3 3

Oyster ND 7.0=10 2.4=10

5 4 3

Coastal mud 2.1=10 1.6=10 2.1=10

2 2

Sea water 1.2=10 1.3=10 1.5=10

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Table 4

Comparison of the number of V.Õulnificus identified by PCR and mCPC agar plating in five-tube MPN

method

a b

Samples Number of colonies Biotypes of the 16S rRNA types of

verified isolates the verified isolates Examined on Verified by

c ₁ ₂ _A _B

mCPC PCR

Oyster 74 35 33 2 11 24

Coastal mud 12 3 2 1 2 1

Sea water 13 2 2 0 1 1

Total 111 40 37 3 14 26

Determined by indole test in tryptone broth. b

Determined by tri-primer PCR gene amplification. c

Examined yellow colonies on the mCPC agar plate were verified by PCR.

biased result as the biotype, and appeared to be 35% type A and 65% type B in the marine environmental samples.

4. Discussion

Several different PCR techniques with primers targeting 16S rRNA have also been Ž

used for the detection of Vibrio species Rehnstam et al., 1989; Toyama et al., 1994; .

Genmoto et al., 1996 . The primers designed as antisense and sense sequences from

Ž . Ž .

conserved region 786 to 805, E. coli numbering and variable region 455 to 478 were Ž

found to be species-specific for V. penaeicida after RT-PCR gene amplification Genmoto .

et al., 1996 . Despite many reports of the identification of species using primers targeted at 16S rRNA sequences by PCR amplification, however, it was still not known whether this assay method could be applied to the detection of V.Õulnificus from other marine

Vibrio species.

By comparative analysis of known 16S rRNA sequences of Vibrio species, we identified four variable regions, positions 70 to 110, 454 to 473, 1006 to 1023 and 1258 to 1278, that could be useful as target sequences to design candidate primers specific for the amplification of 16S rRNA genes of V.Õulnificus.

Unfortunately, none of these sequences in PCR using only the antisense primer for

Ž . Ž X

the conserved region of Gy bacteria 1351–1331 in E. coli numbering, 5

-ATTAC-X

TAGCGATTCCGTCTTC-3 , was completely specific to the different strains of V.

Õulnificus. The sequence of positions 74–110 was similar to those of V. diazotropicus

and V. fluÕialis. Moreover, the most variable known region, position 450–473, and

Ž .

position 1006–1023 4 out of 18 bases, sparsely both showed intrastrain variation depending upon the 16S rRNA types of V.Õulnificus. Because of this, we tried to do PCR gene amplification for the identification of V. Õulnificus with combined primers that could compensate for the similarity of one primer to the target region of the 16S rRNA gene of the phylogenetically related Vibrionaceae by the other primer. In an

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analysis of specificity with PCR amplification using the combination of different primers designed from four variable regions, the sequences used in this study for the

Ž . Ž .

sense 1006–1023 and antisense 1278–1258 primers were able to uniquely distinguish

Ž .

V. Õulnificus from other Gy or Vibrio species, even though sequences of the sense

and antisense primers compared with the target region of 16S rRNA of V. cholerae and

V. mimicus, and V. fluÕialis and V. ordalii, respectively, did not show enough

differ-Ž .

ences by themselves to distinguish these species from V.Õulnificus Fig. 1 . This result implied that the specificity problem resulting from the use of one primer in PCR amplification could be overcome by using two primers derived from different variable regions.

In a recent study, oligonucleotide probes for dot blot hybridization targeted to the variable region of 23S rRNA were able to separate V.Õulnificus strains into two groups

corresponding to two different 16S rRNA sequences, type A and type B Aznar et al., .

1994 . In the present study, gene amplification with three primers, Vib 2 and Vib 3R, and Vib 1 primers designed to detect both types and only the A type of 16S rRNA gene of V.Õulnificus, respectively, resulted in the discrimination of V.Õulnificus based on the

Ž .

type of 16S rRNA Fig. 3 . These results suggested that PCR amplification with combined primers designed from different variable regions of the 16S rRNA sequences could be a useful method both for the development of species and gene type, types A and B, specific gene amplification techniques.

In addition to the specificity of primers, it also important to consider the sensitivity or detection limit of various assays of V. Õulnificus, since it is well known that V.

Ž .

Õulnificus is present in low numbers in the environment. In 1991, Brauns et al. 1991

reported that as little as 72 pg of DNA from culturable V. Õulnificus cells could be detected by PCR amplification with primers flanking a 340 bp fragment of the cytotoxin–hemolysin gene. However, more sensitive methods have been demonstrated with the use of the sequences of 16S rRNA for RT-PCR assays. In a comparative analysis of PCR and RT-PCR assays for the detection of V. penaeicida, RT-PCR targeted at both 16S rDNA and rRNA had 100-fold higher sensitivity, 10 fg of total

Ž .

nucleic acid, than PCR targeted at 16S rDNA only Aznar et al., 1994 . In the present study, about 5 and 0.5 pg of DNA from V.Õulnificus could be detected with the primers

Ž .

designed for PCR and RT-PCR assays, respectively Fig. 2 . Less increased sensitivity

Ž .

of RT-PCR, 10 times, compared to the study of Genmoto et al. 1996 may be derived from the used EZ-rTth polymerase, which contained both cDNA synthesis and DNA polymerization activity but is less stable than Taq polymerase at high temperatures in

Ž .

the thermocycler. In agreement with Hiney and Smith 1998 , who pointed out practical problems in the analysis of environmental samples, we found that the method was 10–100 times less sensitive for detecting DNA obtained from oysters artificially contaminated with V.Õulnificus than for detecting DNA obtained from bacteria enriched

Ž .

in media data not shown .

A previously described technique for enumerating V.Õulnificus in samples with MPN method requires preenrichment in alkaline peptone water followed by subculturing on

selective media for the identification of presumptive V.Õulnificus Massad and Oliver, .

1987 . However, differentiation and confirmation of the presumptive isolates requires several laborious, time-consuming follow-up steps. To avoid these problems, PCR

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Õulnificus detected by confirmation of the presumptive positive tubes by mCPC media

for the MPN method was approximately two times higher than those confirmed by PCR

Ž . Ž .

amplification Table 3 . With the oyster samples, it also was found that the ratio 2:1 of the V. Õulnificus isolates between the yellow colonies on mCPC media and confirmed isolates by PCR was similar to that enumerated on MPN with the determination of the

Ž .

presumptive positive tubes by two different methods, mCPC media and PCR Table 4 .

Ž .

These results differ from those of Hoi et al. 1998 who found that more than 95% of the presumptive isolates on mCPC agar were identified as V.Õulnificus when they were tested by PCR amplification with the cytolysin primers. Because of the geographic environment in that study, there would have been some marine bacteria that could grow up with the fermentation of cellobiose on mCPC media.

The characterization of V.Õulnificus strains has led to subdivision of the species into two biotypes: biotype 1 strains are pathogenic for humans and are indole positive, biotype 2 strains appear to be virulent for both humans and eels and the majority of

these strains are indole negative Amaro et al., 1995; Biosca et al., 1996; Hoi et al., .

1997 . Until recently, biotype 2 strains had been isolated only from diseased eels and a

Ž .

single clinical case Veenstra et al., 1992; Amaro and Biosca, 1996 .

Ž .

In the present study, however, 7.5% three strains of the 40 V. Õulnificus isolates

Ž .

appeared to be indole negative biotype 2 strains Table 4 . These results are different

Ž .

from those of Hoi et al. 1998 who found these strains in the sea water of Denmark at a frequency of 0.4%. The difference may be attributable to differences in the isolation

Ž .

procedures, geographical characteristics, biological samples wild fishrcultured oyster and number of analyzed isolates. However, our findings and other studies indicate that the marine environment should be regarded as a reservoir and possible vehicle of transmission for V.Õulnificus biotype 2.

Ž .

The assignment of V.Õulnificus strains to two biotypes biotypes 1 and 2 defined by biochemical properties does not reflect the natural relationships or genetic heterogeneity of the strains. The relationship of the strains that could be deduced from the 16S rRNA sequences of these organisms would supply us important information related to ecology and degree of heterogeneity of these organisms in marine environment. However, determined rRNA types of 40 isolates of V.Õulnificus by tri-primers PCR showed that the distribution of types A and B of these organisms in the coastal area was not biased

Ž .

as found in biotypes 1 and 2, and appeared to be 35% and 65%, respectively Table 4 . It suggested that the differences of rRNA type might not affect the viability or be the determining factor of the dominant strains of these organisms in marine environment. Comparison with other studies in different areas has to be done in the near future. Acknowledgements

Ž .

This work 1998-023-H00016 was supported by the Korea Research Foundation Grant of 1998. We wish to thank Dr. Jack L. Komisar, WRAIR-Washington, DC, for reviewing the manuscript.

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Ž .

tri-primers Vib 1, Vib 2, and Vib 3R , the presence of two amplified products, the 273 and 825 bp fragments, discriminated the nucleic acid of rRNA type A V.Õulnificus from

that of rRNA type B V.Õulnificus, which produced a single fragment 273 bp long Fig.

3 . The sequence of the 825 bp fragment was exactly the same as that of V.Õulnificus

Ž . Ž

C7184 data not shown , which is reported to be a type B 16S rRNA strain Aznar et al.,

1994 .

Tri-primer PCR amplification using nucleic acid isolated from other strains of bacteria as a template yielded no fragments or only a single fragment 825 bp long. 3.3. Characteristics of V.Õulnificus occurred in oyster and enÕironmental samples

For counting of V. Õulnfificus by using a five-tube MPN method, two different methods were employed to determine the presence of V. Õulnificus in the tubes with turbidity. One was the subculturing of the tube samples preenriched in APWP followed by observation of the yellow colonies that appeared on mCPC agar as presumptive V. Õulnificus. The other one was PCR analysis with the primers Vib 2 and Vib 3R against the total nucleic acids isolated from the bacteria of the cultured tube samples. In a comparison of these two methods, the number of V.Õulnificus counted when samples from oyster, coastal mud, and sea water was analyzed by PCR appeared to be 49%, 13%, and 12% of those counted with subculturing on mCPC agar plate, respectively

ŽTable 3 ..

Ž .

sea water, respectively Table 4 . For the classification of the isolated V. Õulnificus

Ž .

depending upon the biochemical characteristics biotype and the sequence of rRNA

Ž_{rRNA type , a total of 40 V.}. Õulnificus isolates from oyster 35 , coastal mud 3 , andŽ . Ž . Ž .

sea water 2 were tested for indole production and the presence of the 825 fragment on

Ž .

Table 3

Characteristics and confirmation of the identities of V.Õulnificus isolates

Ž .

Samples Total number of Number of V.Õulnificus cellsr100 ml or g

Ž .

bacteria g or ml identified by

mCPC plating PCR

a 3 3

Oyster ND 7.0=10 2.4=10

5 4 3

Coastal mud 2.1=10 1.6=10 2.1=10

2 2

Sea water 1.2=10 1.3=10 1.5=10

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Table 4

Comparison of the number of V.Õulnificus identified by PCR and mCPC agar plating in five-tube MPN

method

a b

Samples Number of colonies Biotypes of the 16S rRNA types of verified isolates the verified isolates Examined on Verified by

c ₁ ₂ _A _B

mCPC PCR

Oyster 74 35 33 2 11 24

Coastal mud 12 3 2 1 2 1

Sea water 13 2 2 0 1 1

Total 111 40 37 3 14 26

Determined by indole test in tryptone broth.

Determined by tri-primer PCR gene amplification.

Examined yellow colonies on the mCPC agar plate were verified by PCR.

biased result as the biotype, and appeared to be 35% type A and 65% type B in the marine environmental samples.

4. Discussion

Several different PCR techniques with primers targeting 16S rRNA have also been

used for the detection of Vibrio species Rehnstam et al., 1989; Toyama et al., 1994;

Genmoto et al., 1996 . The primers designed as antisense and sense sequences from

Ž . Ž .

conserved region 786 to 805, E. coli numbering and variable region 455 to 478 were

found to be species-specific for V. penaeicida after RT-PCR gene amplification Genmoto

Unfortunately, none of these sequences in PCR using only the antisense primer for

Ž . Ž X

the conserved region of Gy bacteria 1351–1331 in E. coli numbering, 5 -ATTAC-X

TAGCGATTCCGTCTTC-3 , was completely specific to the different strains of V. Õulnificus. The sequence of positions 74–110 was similar to those of V. diazotropicus and V. fluÕialis. Moreover, the most variable known region, position 450–473, and

Ž .

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analysis of specificity with PCR amplification using the combination of different primers designed from four variable regions, the sequences used in this study for the

Ž . Ž .

sense 1006–1023 and antisense 1278–1258 primers were able to uniquely distinguish

Ž .

V. Õulnificus from other Gy or Vibrio species, even though sequences of the sense and antisense primers compared with the target region of 16S rRNA of V. cholerae and V. mimicus, and V. fluÕialis and V. ordalii, respectively, did not show enough

differ-Ž .

In a recent study, oligonucleotide probes for dot blot hybridization targeted to the variable region of 23S rRNA were able to separate V.Õulnificus strains into two groups

corresponding to two different 16S rRNA sequences, type A and type B Aznar et al.,

Ž .

In addition to the specificity of primers, it also important to consider the sensitivity or detection limit of various assays of V. Õulnificus, since it is well known that V.

Ž .

Õulnificus is present in low numbers in the environment. In 1991, Brauns et al. 1991 reported that as little as 72 pg of DNA from culturable V. Õulnificus cells could be detected by PCR amplification with primers flanking a 340 bp fragment of the cytotoxin–hemolysin gene. However, more sensitive methods have been demonstrated with the use of the sequences of 16S rRNA for RT-PCR assays. In a comparative analysis of PCR and RT-PCR assays for the detection of V. penaeicida, RT-PCR targeted at both 16S rDNA and rRNA had 100-fold higher sensitivity, 10 fg of total

Ž .

nucleic acid, than PCR targeted at 16S rDNA only Aznar et al., 1994 . In the present study, about 5 and 0.5 pg of DNA from V.Õulnificus could be detected with the primers

Ž .

designed for PCR and RT-PCR assays, respectively Fig. 2 . Less increased sensitivity

Ž .

in media data not shown .

A previously described technique for enumerating V.Õulnificus in samples with MPN method requires preenrichment in alkaline peptone water followed by subculturing on

selective media for the identification of presumptive V.Õulnificus Massad and Oliver,

1987 . However, differentiation and confirmation of the presumptive isolates requires several laborious, time-consuming follow-up steps. To avoid these problems, PCR

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amplification with primers Vib 2 and Vib 3R specific to V.Õulnificus was applied to the total nucleic acid isolated from bacteria cultured in the preenrichment tubes for the determination of presumptive positive tubes by the MPN method. The number of V. Õulnificus detected by confirmation of the presumptive positive tubes by mCPC media for the MPN method was approximately two times higher than those confirmed by PCR

Ž . Ž .

Ž .

presumptive positive tubes by two different methods, mCPC media and PCR Table 4 .

Ž .

these strains are indole negative Amaro et al., 1995; Biosca et al., 1996; Hoi et al.,

1997 . Until recently, biotype 2 strains had been isolated only from diseased eels and a

Ž .

single clinical case Veenstra et al., 1992; Amaro and Biosca, 1996 .

Ž .

In the present study, however, 7.5% three strains of the 40 V. Õulnificus isolates

Ž .

appeared to be indole negative biotype 2 strains Table 4 . These results are different

Ž .

from those of Hoi et al. 1998 who found these strains in the sea water of Denmark at a frequency of 0.4%. The difference may be attributable to differences in the isolation

Ž .

Acknowledgements