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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 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 185 (Thompson et al., 2004). However, the two species of Vibrio DNA was maintained at À 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 Â 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 À 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 mLÀ1. 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).
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