Microbiology Letters

RESEARCH LETTER Analysis of16S^23S rRNA gene internal transcribed spacer of Vibrio anguillarum and Vibrio ordalii strains isolated from ¢sh Jorge Fernandez´ & Ruben Avendano-Herrera˜

Laboratorio de Veterquımica,´ Camino a Melipilla, Cerrillos, Santiago, Chile Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021

Correspondence: Ruben Avendano-Herrera,˜ Abstract Laboratorio de Veterquımica,´ Camino a Melipilla 5641, Cerrillos, Santiago, Chile. The 16S-23S rRNA intergenic spacer (ITS) of Vibrio anguillarum and Vibrio ordalii Tel.: 156 2 384 4109; fax: 156 2 384 4021; were PCR amplified and cloned with TA vector pCR2.1. PCR amplification e-mail: [email protected] obtained five products ranging from 917 to 437 bp. Three clones were obtained and analysed from all fragments with the exception of 437 bp. These products Present address: Ruben Avendano-Herrera,˜ were designated ITS-1, ITS-2, ITS-3 and ITS-4. ITS-1 contained genes for Universidad Andres´ Bello, Facultad de tRNAGlu(TTC), tRNALys(TTT), tRNAAla(TGC) and tRNAVal(TAC), while ITS-2 was Ciencias Basicas,´ Departamento de Ciencias almost the same as the ITS-1 sequence, but without tRNAVal(TAC). ITS-3 contained Basicas.´ Repu´ blica 217, Santiago, Chile. tRNAAla(TGC) and tRNAIle (GAT) and ITS-4, tRNAAla (GGC) or tRNAGlu(TTC). The

Received 30 January 2009; accepted 24 July number of copies of the ribosomal operon (rrn)inV. ordalii chromosome ranged 2009. from at least six to seven and V. anguillarum had at least seven rrn. The sequences Final version published online 1 September ITS-1, ITS-2 and ITS-3 showed a high similarity among the V. anguillarum and V. 2009. ordalii sequences (97.2% to 100%). Little variation was found for ITS-4, which does not seem to be sufficient to distinguish these two closely related species. Based DOI:10.1111/j.1574-6968.2009.01755.x on the findings, we confirm a close genetic relationship among V. anguillarum and V. ordalii and that they may be descended from a common ancestor in the Editor: Reggie Lo Vibrionaceae linage.

Keywords 16S–23S rRNA internal transcribed spacer; Vibrio anguillarum; Vibrio ordalii; tRNA.

rated by standard identification tests (Schiewe et al., 1981). Introduction In fact, the two species differ in important biochemical and Vibriosis due to Vibrio anguillarum is one of the most physiological traits, such as arginine dihydrolase, b-galacto- serious bacterial diseases, causing septicaemia and death in sidase, Voges–Proskauer reaction and ranges of temperature. both wild and cultured fish throughout the world (see Several serotypes are known to occur among V. anguillarum reviews Actis et al., 1999; Austin & Austin, 1999; Toranzo isolates, but only serotypes O1 and O2 have been associated et al., 2005). Vibrio ordalii, the former biotype 2 of V. with mortalities in salmonids (Toranzo et al., 1997). How- anguillarum, also causes vibriosis with comparable gross ever, previous studies from our group demonstrated that the pathological symptoms (Toranzo & Barja, 1993). This Chilean V. anguillarum isolates are antigenically homoge- microorganism is responsible for severe economic losses in neous, and that they could be classified as serotype O3 the culture of several salmonid species in the coastal waters (Silva-Rubio et al., 2008b). In contrast to V. anguillarum, all of America’s Pacific Northwest, Japan, Australia and New V. ordalii strains, including the Chilean isolates, are serolo- Zealand (Harrel et al., 1976; Ransom et al., 1984; Toranzo gically homogeneous (Silva-Rubio et al., 2008a) and share a et al., 1997). Recently, it has been reported from high common O-antigen with V. anguillarum O2 strains mortalities in populations of Atlantic salmon, Pacific sal- (Mutharia et al., 1993).

MICROBIOLOGY LETTERS MICROBIOLOGY mon and rainbow trout cultured in the South of Chile In the last two decades, bacterial phylogeny has been (Colquhoun et al., 2004; Silva-Rubio et al., 2008a, b). enriched with chronometers, for example rRNA genes (5S, These two pathogens are Gram-negative motile rods with 16S and 23S), to reconstruct bacterial phylogenies but also fermentative and respiratory metabolisms, but can be sepa- to be used as taxonomic markers for identiﬁcation

(Thompson et al., 2004). However, the two species of Vibrio DNA was maintained at 20 1C until they were used for show a high degree of similarity of the overall DNA base PCR reactions. One microlitre of each DNA solution was sequence, as revealed by chromosomal DNA–DNA pairing used in the respective amplification reaction. (Schiewe et al., 1981). In addition, DNA comparison of 5S rRNA gene sequences revealed a close relationship between PCR amplification of ITS V. anguillarum and V. ordalii, differing only in seven of 120 bp (MacDonell & Colwell, 1984; Pillidge & Colwell, Amplification of the spacer regions between the 16S and 23S 1988; Ito et al., 1995). Similarly, V. anguillarum is indis- rRNA genetic loci were carried out for both V. anguillarum tinguishable from V. ordalii on the basis of the 16S rRNA and V. ordalii strains using the universal primer pair gene sequence (Wiik et al., 1995). Therefore, there is a great designed by Jensen et al. (1993) and synthesized by Sigma- Genosys: G1 (50-GAA GTC GTA ACA AGG-30) and L1 (50- interest in finding genetic variation between both micro- Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021 CAA GGC ATC CAC CGT-30). The amplifications were organisms, which may be useful for the rapid detection of V. TM anguillarum and V. ordalii. obtained using the commercial kit Ready-To-Go PCR Numerous authors have used the spacer region between beads (Amersham Pharmacia Biotech), which included all the 16S and 23S rRNA genes (internal transcribed spacer, the reagents needed for the PCR reactions (buffer, nucleo- ITS) as targets on the identification of microorganisms at tides and Taq DNA polymerase), with the exception of the the species level. These genomic regions show a high degree specific primers and DNA template. One microlitre of each of variability between species, both in their base length and DNA solution, 25 mmol of each primer and water to their sequence (Jensen et al., 1993; Gurtler¨ & Stanisich, complete a volume of 25 mL were used in the amplification 1996). In fact, the 16S–23S rRNA gene sequences have reaction. All samples were subjected to 30 cycles of amplifi- already been used with marine Vibrio species (Chun et al., cation in a T Gradient thermocycler (Biometra) with the 1 1999; Maeda et al., 2000), as well as for other fish pathogens following programme: after heating to 95 C for 5 min, 1 1 1 (Casanova et al., 2001; Hassan et al., 2003; Osorio et al., 95 C for 1 min, 58 C for 1 min and 72 C for 1 min, with a 1 2005). In this study, the ITS nucleotide sequence of strains final extension step at 72 C for 6 min. In all cases, negative belonging to V. anguillarum and V. ordalii were analysed. controls, consisting of the same reaction mixture but with sterile distilled water instead of template DNA, were in- Materials and methods cluded in each batch of PCR reaction. The reproducibility of results was assessed by repetition of the amplifications in at least two independent PCR assays. Bacteria and growth conditions

The V. ordalii strains used in this work, Au2 and Au10, were Analysis of the PCR products isolated in Chile from clinically infected Atlantic salmon (Silva-Rubio et al., 2008a). In the case of V. anguillarum, two All the PCR products were separated by horizontal electro- strains isolated from Atlantic salmon coded as PF2 and PF8 phoresis on a 2% (w/v) agarose gel in 1 Tris-acetate-EDTA and belonging to serotype O3 of this fish pathogen (Silva- buffer, visualized with ethidium bromide (Bio-Rad Labora- Rubio et al., 2008b) were used. The reference strains of V. tories Inc.) and photographed under UV light. A 100-bp ordalii ATCC 33509T and V. anguillarum ATCC 19264 extended DNA ladder (Bioron) was used as a molecular (serotype O2) from the American Type Culture Collection mass marker. Each run was performed in duplicate and the were used for comparative purposes. All strains were routi- data analysis was performed using the GELCOMPAR II software nely cultivated on/in tryptone soya agar or broth supple- package (Applied Maths). mented with 1% (w/v) sodium chloride (TSA-1 or TSB-1, respectively) and incubated at 20 1C for 48–72 h. Stock Cloning and sequencing of the ITS cultures were maintained frozen at 70 1C in Criobille DNA fragments amplified using PCR were cloned into TA tubes (AES Laboratories Pvt Ltd, France). vector pCR2.1 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Nucleotide sequences of ITS DNA extraction inserts were determined using the ABI PRISM Kit reaction Chromosomal DNA was extracted using InstaGene Purifica- with AmpliTaq DNA polymerase on an ABI PRISM 310 tion Matrix (Bio-Rad Laboratories Inc.) according to the sequencer (Applied Biosystems, Foster) according to the manufacturer’s protocols. The concentration and quality of manufacturer’s recommendations. The sequence of the four each DNA sample was examined spectrophotometrically at Vibrio strains were aligned and compared with sequences of 260 nm and adjusted to a concentration of 20–30 ng mL1. V. anguillarum (AB255712 and AY227665) and V. ordalii All experiments were carried out with DNA obtained in two (AB255727) obtained from database Ribosomal Database different extractions for each bacterial strain. The extracted Project-II (RDP-II) and BLASTN (GenBank, NCBI, Bethesda).

The nucleotide sequences obtained in this study were M1234567M analysed with BIOEDIT program and alignments were performed with CLUSTALX program (Thompson et al., 1997). The phylogenetic analysis was performed with PHYLIP program 1500 917 (Felsenstein, 1997). The evolutive distances were estimated 805 637 with DNADIST NEIGHBOR (neighbour-joining method). The 600 526 reproducibility patterns were performed with SEQBOOT 437 300 (bootstrap = 100) and the phylogenetic tree was constructed with CONSENSE PHYLIP software. The detection and identiﬁca- tion of tRNA genes was performed using the program

TRNASCAN SE SEARCH SERVER Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021 - (http://lowelab.ucsc.edu/tRNAs Fig. 1. Electrophoresis with a 1.5% agarose gel of PCR-amplified can-SE/) (Lowe & Eddy, 1997). 16S–23S rRNA gene ITS regions of Vibrio anguillarum and Vibrio ordalii. Lanes: M, molecular weight marker (100-bp ladder); 1, negative control Nucleotide sequence accession numbers (no DNA); 2, ATCC 19264; 3, PF2; 4, PF8; 5, ATCC 33509T; 6, Au2 and 7, Au10. Numbers on the left indicate the position of molecular size The 16S–23S rRNA gene ITS sequences from this study have marker in bp. Numbers on the right indicate the size of the amplified been deposited in the GenBank database under accession products in bp. numbers EU570962–EU570973 and FJ666329–FJ666336. analysed, in strains V. anguillarum PF2, PF8 and ATCC Determination of rrn operon copy number by 19264, the ITS sequenced were 820, 725, 598 and 510 bp, pulsed-field gel electrophoresis (PFGE) analysis while for V. ordalii Au2 and Au10, they were 820, 725, 609 DNA samples were prepared according to Bag et al. (1999). and 487 bp. With respect to the type V. ordalii strain, sizes of Briefly, agarose-embedded DNA was digested at 37 1C with 820, 725, 598 and 476 bp were found. The identification of 20 U of I-CeuI endonuclease according to the manufac- four kinds of ITS suggested that the genomes of these turer’s instructions. PFGE was performed on a CHEF DR III microorganisms have at least four rrn operons. However, system (Bio-Rad Laboratories Inc.) with the pulse time bands with double intensity were evidenced, which could interpolated between 10 and 50 s for 24 h at 10 V cm1 at comprise more than one distinct rrn-containing DNA frag- 4 1C. DNA of Salmonella braenderup cleaved by XbaI was ment as demonstrated below. used as a molecular size marker. Following electrophoresis, On the other hand, the nucleotide sequences of each the gels were stained with ethidium bromide (0.5 mgmL1) clone containing the 437-bp product was different and none for 30 min, destained in distilled water for 1 h, and visua- of them could be aligned with sequencing described in the lized with a UV transilluminator. All gels were also scanned public database. It is important to denote that for sequen- and the images were captured using a Gel Doc-2000 gel cing this less-abundant c. 437-bp PCR fragment higher DNA documentation system (Bio-Rad Laboratories Inc.). concentration and more rounds of agarose gel were needed to carry out the cloning procedures. Examination of the sequences showed the presence of different clones, indicat- Results and discussion ing that the 437-bp PCR product exhibits polymorphisms. The primers L1 and G1 have complementary sequences of Further studies will be developed in order to separate clones the 16S–23S rRNA genes and amplified the spacer region from the 437-bp fragment and to establish whether they located between them. PCR with these primers yielded a were from different ITS. nearly identical product pattern for the V. anguillarum and When the copy of rrn operon was studied by PFGE using V. ordalii strains containing five products of about 917, 805, the restriction enzyme I-CeuI, six and seven fragments were 637, 526 and 437 bp (Fig. 1). It can be seen that the 637-bp detected in the ethidium bromide-stained gel, indicating product is at least a doublet. that there are at least seven rrn operons in all strains To compare structures between the differently sized ITS of included in the study, except in the V. ordalii type strain that the Vibrio species, the five PCR fragments of all V. anguillar- had at least six. It is important to denote that both bacterial um and V. ordalii strains were cloned into the TA vector species showed different electrophoresis patterns (Fig. 2). pCR2.1 and maintained in Escherichia coli. The recombinant Because I-CeuI cleaves only rrn genes and because the plasmids were sequenced separately to reveal all possible number and locations of these genes are highly conserved amplicons. From the products of 917, 805, 637 and 526 bp, in the genomes among various strains within species (Liu & three clones were obtained and analysed from each frag- Sanderson, 1995; Bag et al., 1999; Valcheva et al., 2007), we ment. These products were designated as ITS-1, ITS-2, ITS-3 also used this technique, demonstrating that it is also a and ITS-4. Taking together the results for the strains valuable tool for the elucidation of the numbers of rrn

(a) MW 1 2 (b) MW 3 4

2316 2176 2316 2176 1135 1135 Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021

669 669 622 600 452 452 445 422

Fig. 2. PFGE patterns obtained for Vibrio ordalii (a) and Vibrio anguillarum (b) strains after chromosomal DNA digestion with I-CeuI. Lanes: 78.2 68 78.2 1, ATCC 33509T; 2, Au10; 3, ATCC 19264; 4, 40.5 33 PF2. DNA of Salmonella braenderup cleaved by 32 30 30.5 28 XbaI was used as the molecular size marker. 28.8 20 Numbers on the right indicate the position of the molecular size marker in kb.

operons in these Vibrio species. Among the Vibrionaceae When the nucleotide sequences were aligned and the 30 species, it could be noted that Vibrio parahaemolyticus end of the 16S rRNA gene and the 50 end of the 23S rRNA contains nine rrn operons (Bag et al., 1999; Maeda et al., gene were compared, the ITS sequence alignments of the six 2000) and there are eight rrn operons in Vibrio cholerae Vibrio strains revealed a high difference between two species according to the genomic sequence (Nandi et al., 1997; attributable to deletions and insertions. However, ITS-1 and Heidelberg et al., 2000). ITS-2 sequences are highly conserved within the V. ordalii The results of the sequence analyses are summarized in and V. anguillarum species, with identity levels ranging from Fig. 3. Partial sequences of ITS-1 and ITS-2 from both Vibrio 99% to 100%. Instead, the sequences of the ITS-4 of V. species were identical, with sizes of 820 and 725 nucleotides anguillarum and V. ordalii strains possessed two ‘conserved (i.e. 917- and 805-bp amplicons, respectively). In ITS-2, a sequence blocks’, which were identical to each other on the deletion of 96 bases at nucleotides 290–386 was detected. On 81 positions immediately following the 16S rRNA gene- the other hand, Chilean V. anguillarum strains and both coding sequences and the last 144 positions immediately reference strains ATCC 43306 and ATCC 43307 also shared preceding the 23S rRNA gene-coding sequences. Also, these similar sizes of 598 and 510 nucleotides (i.e. 637- and 526- two species presented two conserved blocks in the ITS-3 bp amplicons, respectively). sequence, being also identical to each other in the ﬁrst 304 The sequences of ITS-3 from all V. ordalii isolates and last 123 nucleotides. A unique exception was the V. consisted of 609 nucleotides, while the V. ordalii type strain ordalii type strain that contained, in the beginning of both had a shorter version and differed from the Chilean isolates ITS fragments, a nucleotide substitution of thymine by by 11 nucleotides. In the case of ITS-4, a similar difference adenine at position 31 bp in the conserved block. These was observed among Chilean V. ordalii strains and type observations agree with the very little sequence variation strain. The lengths were 487 and 476 bp, respectively. The found for the ﬁrst 50 and last 150 nucleotides for ITS in DNA size of these ITS sequences are similar to those Vibrio strains (Lee et al., 2002). described for the ITS region of other species of the Vibrio- In V. anguillarum, the difference between ITS-3 and ITS-4 naceae family, ranging from 277 to 803 bp (Chun et al., 1999; sequences is attributed to a 129-bp insertion at nucleotide Maeda et al., 2000; Osorio et al., 2005). positions 87–215 in ITS-4. Another fragment in the same

16S rRNA gene 23S rRNA gene pG1 pL1 Size ITS-1 820 50 5 27 72 22 73 34 73 29 5 72 118 240

ITS-2 795 50 27 72 22 73 72 118 240

ITS-3a 609 Fig. 3. Schematic representation of 16S–23S

31 50 5 4 74 47 73 43 26 240 16 rRNA gene ITS regions of Vibrio anguillarum and Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021 Vibrio ordalii. ITS-3a and ITS-4b, corresponding to ITS-3b 598 Chilean V. ordalii strains; ITS-3b, corresponding to 31 50 74 47 73 43 15 240 16 V. anguillarum strains and the V. ordalii type strain ATCC 33509T; ITS-4a, corresponding to ITS-4a 510 V. anguillarum strains and ITS-4c to V. ordalii type strain ATCC 33509T. Numbers on the left indicate 31 50 5 677427 240 16 the sequence lengths of the ITS. The type of tDNAs was identiﬁed using the program ITS-4b 487 TRNASCAN-SE SEARCH SERVER (http://lowelab.ucsc.edu/ 31 50 8 73 43 26 240 16 tRNAscan-SE/). Colours indicate discrete sequence blocks that are common between ITS-4c 476 different ITS. Numbers at the bottom indicate the 31 50 8 73 43 15 240 16 lengths of the discrete sequence blocks.

ITS-4 sequence showed relevant variability located at posi- 1995), Salmonella enterica (Perez´ et al., 1998), V. cholerae tion 218, presenting a deletion of 99 nucleotides. and Vibrio mimicus (Chun et al., 1999) and Photobacterium On the other hand, V. ordalii sequences revealed several damselae (Osorio et al., 2005), the same size and nucleotide highly variable regions in both fragments. In fact, the position might have occurred in a common ancestor of V. type strain and the V. ordalli isolates shared the 122-bp anguillarum and V. ordalii. deletions at nucleotide positions 89–210 in ITS-4. Impor- On the other hand, tDNAAla(GGC) was also found in the tant similarities were observed in the largest sequences of ITS-4 amplicon of the V. ordalii strains, with the same size Chilean V. ordalii isolates (Au2 and Au10), showing two and nucleotide position as ITS-3. The other sequence of the insertions located at position 328–333 and 344–348. Inter- tRNAGlu(TTC) gene was found in the ITS-4 V. anguillarum estingly, these insertions were also found in the ITS-4 strains. None of the tDNAs from V. anguillarum and V. sequence of all strains, but not in the V. ordalii type strain ordalii sequences have been described or deposited in a (Fig. 4). public database. The number of tRNA genes (tDNAs) coexisting in a The similarity values used in the phylogenetic compar- bacterial ITS region varies from zero to four (Osorio et al., ison among the V. anguillarum and V. ordalii sequences, 2005). Sequence analysis of the ITS-1 demonstrated that including the reference strains, were calculated from 596 bp V. anguillarum and V. ordalii strains examined in this of ITS-3 fragment, revealing a high level of similarity study contained four tDNAs coding for tRNAGlu(TTC), ranging from 97.2% to 99.8% (Fig. 5). This high percentage tRNALys(TTT), tRNAAla(TGC) and tRNAVal(TAC) (anticodons of sequence similarity between the two Vibrio species clearly are indicated in parentheses). The tDNAs in the ITS-2 are supports the taxonomic placement of V. ordalli (previously arranged similar to the tDNAs found in ITS-1, but without designated as V. anguillarum biotype 2), and as reported in tRNAVal(TAC). Analysis of the ITS-3 demonstrated that the V. phenotypic studies, they should not share species (Schiewe anguillarum and V. ordalii strains contained two tDNAs et al., 1981). In fact, the similarity levels containing either coding for tRNAAla(TGC) and tRNAIle(GAT). It is important to the ITS-1 or the ITS-2 among the V. ordalii and V. note that these tDNAs from V. anguillarum were identical to anguillarum isolates, including the reference strains, are the deposited sequences in GenBank to date (AY227665). relatively higher (99.0–100%), indicating that these regions Although the combinations of tRNAAla and tRNAIle have are conserved within the genus. been reported in several Gram-negative bacteria including In addition, between the V. ordalii and V. anguillarum Aeromonas hydrophila (East & Collins, 1993), Cythophaga strains the sequences of ITS-4 had the lowest similarities (Andresson´ & Fridjonsson,´ 1994), E. coli (Condon et al., (58.9%), while the similarity within V. anguillarum species

tRNA

Fig. 4. Aligned sequences of Vibrio anguillarum and Vibrio ordalii ITS-4. Nucleotide positions that are conserved relative to the sequence located at the top are indicated by dots. Gaps included to preserve the alignments are indicated by dashes. The tDNA sequences and their positions are indicated by dark grey boxes. Slashes indicate deletion sequences. Regions indicated by boxes show homology with sequences in strains.

V. parahaemolyticus (AY298805)

V. orientalis (gAB255728)

V. parahaemolyticus (AY298808)

V. harveyi (DQ095212)

V. alginolyticus (AB255709)

V. alginolyticus (AB255710)

69 V. ordalii ATCC-33509 (AB255727)

V. nereis (AB255725)

V. splendidus (AF413023) Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021

V. tubiashii (AB255736)

V. tubiashii (AB255737) 98 V. furnissii (DQ095208)

V. harveyi (AB255714)

V. vulnificus (DQ854745)

V. vulnificus (DQ462504)

V. gazogenes (AB255720)

V. fluvialis (AY245214)

V. mimicus (AF114748) 100 V. mimicus (AF114747)

V. cholerae (AF114741)

V. ichthyoenteri (AB255722)

V. proteolyticus (AF413017)

V. diazotrophicus (AB255718)

V. tapetis (AB255734)

V. aestuarianus (AF412994)

V. aestuarianus (AF412993)

V. anguillarum (AB255712)

V. anguillarum ATCC 19264

93 V. anguillarum ATCC-19264 (AY227665) Au10

Au2

PF8

PF2

V. ordalii ATCC 33509

V. logei (AB255723) 100 V. salmonicida (AF413020)

88 V. salmonicida (AB255731)

0.1

Fig. 5. Phylogenics of Vibrio anguillarum and Vibrio ordalii strains based on the 16S–23S rRNA gene ITS region sequence (258 bp) obtained using the neighbour-joining algorithm. Bars indicate the difference between sequences.

c 2009 Federation of European Microbiological Societies FEMS Microbiol Lett 299 (2009) 184–192 Published by Blackwell Publishing Ltd. All rights reserved 16S–23S rRNA genes of Vibrio species 191 was 100%. Besides, this difference in the ITS-4 sequence References data could be useful in separating V. ordalii from the closely Actis LA, Tolmasky ME & Crosa JH (1999) Vibriosis. Fish related V. anguillarum, the analyses are time consuming, Diseases and Disorders Vol. 3: Viral, Bacterial and Fungal require sophisticated equipment and are not adapted for Infections (Woo PTK & Bruno DW, eds), pp. 523–557. CAB field-based diagnostics. International, London. Also, each ITS sequence of V. anguillarum and V. ordalii Andresson´ OS & Fridjonsson´ OH (1994) The sequence of the was compared with the sequences AB255712, AY227665 and single 16S rRNA gene of the thermophilic eubacterium AB255727 as well as the ITS sequences from other Vibrio Rhodothermus marinus reveals a distant relationship to the species and related taxa available in the database RDP-II and group containing Flexibacter, Bacteroides, and Cytophaga BLASTN. The phylogenetic tree based on ITS sequences (Fig. 5) species. J Bacteriol 176: 6165–6169. showed consistent differences among all the V. ordalii Austin B & Austin DA (1999) Bacterial Fish Pathogens: Disease of Downloaded from https://academic.oup.com/femsle/article/299/2/184/647810 by guest on 30 September 2021 strains examined and the type strain (AB255727), while the Farmed and Wild Fish, 3rd edn. Springer-Verlag KG, Berlin. V. anguillarum ATCC 19264 and the two sequences deposited Bag PK, Nandi S, Bhadra RK, Ramamurthy T, Bhattacharya SK, (AB255712 and AY227665) were very similar, if not identical. Nishibuchi M, Hamabata T, Yamasaki S, Takeda Y & Nair B Interestingly, an alignment with previously reported ITS (1999) Clonal diversity among recently emerged strains of from V. ordalii AB255727 showed that the sequences of the Vibrio parahaemolyticus O3: K6 associated with pandemic Chilean V. ordalii isolates studied are completely different, spread. J Clin Microbiol 37: 2354–2357. with a similarity of 59.6%. It is therefore not clear that the Casanova A, Obreque J, Sandino AM & Jashes´ M (2001) tRNA 16S–23S rRNA gene sequence (accession number AB255727) genes were found in Piscirickettsia salmonis 16S–23S rDNA generated in the study of Mutsuyama et al. (2006) corre- spacer region (ITS). FEMS Microbiol Lett 197: 19–22. sponds to V. ordalii type strain, which was also included in Chun J, Huq A & Colwell RR (1999) Analysis of 16S–23S rRNA our study, with the exception that the sequence AB255727 intergenic spacer regions of Vibrio cholerae and Vibrio corresponds to the ITS polymorphic region, which was not mimicus. Appl Environ Microb 65: 2202–2208. completely examined in this study. Colquhoun DJ, Aase IL, Wallace C, Baklien & Gravningen K In addition, we analysed the same strain by other DNA- (2004) First description of Vibrio ordalii from Chile. Bull Eur based methods such as PFGE, random amplified poly- Assn Fish P 24: 185–188. morphic DNA, repetitive element-PCR and enterobacterial Condon C, Squires C & Squires CL (1995) Control of rRNA repetitive intergenic consensus-PCR, demonstrating that the transcription in Escherichia coli. Microbiol Rev 59: 623–645. V. ordalii included in this study is the type strain (Silva- East AK & Collins MD (1993) Molecular characterization of DNA Rubio et al., 2008a, b). encoding 23S rRNA and 16S–23S rRNA intergenic spacer On the other hand, the values for the interspecies regions of Aeromonas hydrophila. FEMS Microbiol Lett 106: comparison of the 16S–23S rRNA gene sequences were 129–133. Felsenstein J (1997) An alternating least squares approach to lower, with identities of 85.1% with the ITS of Vibrio inferring phylogenies from pairwise distances. Syst Biol 46: aestuarinus (GenBank accession numbers AF412993 and 101–111. AF412994). Important similarities were observed with other Gurtler¨ V & Stanisich VA (1996) New approaches to typing and ITS of Vibrio species such as Vibrio diazotrophicus (72.6%) identification of bacteria using the 16S–23S rDNA spacer and Vibrio tapetis (63.8%) (GenBank accession numbers region. Microbiology 142: 1255–1265. AB255718 and AB255734, respectively). Although we ex- Harrel LW, Novotny AJ, Schiewe MJ & Hodgins HO (1976) pected to find significant heterogeneity among Vibrio ITS Isolation and description of two vibrios pathogenic to Pacific sequences, it was surprising to find a high identity, because salmon in Puget Sound, Washington. Fish Bull 74: 447–449. previous analysis of the V. ordalii type strain examined here Hassan AA, Khan IU, Abdulmawjood A & Lammler¨ C (2003) had showed differences (55.5%). Inter- and intraspecies variations of the 16S–23S rDNA intergenic spacer region of various streptococcal species. Syst Appl Microbiol 26: 97–103. Acknowledgements Heidelberg JF, Eisen JA, Nelson WC et al. (2000) DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. The authors thank Andres´ Silva-Rubio for his technical Nature 406: 477–483. help. This work was supported by Grant IPC 019 from the Ito H, Uchida I, Sekizaki T & Terakado N (1995) A specific Program Bicentenario Ciencia y Tecnologıa,´ CONICYT, oligonucleotide probe based on 5S rRNA sequences for Chile. The authors also acknowledge the anonymous re- identification of Vibrio anguillarum and Vibrio ordalii. Vet viewers, who provided many useful suggestions that im- Microbiol 43: 167–171. proved this manuscript, and Dr C.R. Osorio (Universidad Jensen MA, Webster JA & Straus N (1993) Rapid identification of de Santiago de Compostela, Spain) for helpful comments. bacteria on the basis of polymerase chain reaction-amplified

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