03_vibrio with corrections fr Dr. Cariño Local Vibrio Isolates 23 ABSTRACT Science Diliman (January-June 2005) 17:1, 23-30 Local Vibrio Isolates Exhibit Molecular Characteristics Distinct from Reference V. harveyi and V. campbellii Strains Hanna H. Cortado1, Boris B. San Luis, Leobert dela Peña2, Rosario G. Monsalud3, and Cynthia T. Hedreyda1* 1National Institute of Molecular Biology and Biotechnology College of Science, University of the Philippines 1101 Diliman, Quezon City, Philippines 2Southeast Asian Fisheries Development Center (SEAFDEC) Tigbauan, Iloilo City 3National Institute of Molecular Biology and Biotechnology University of the Philippines Los Baños College, 4031 Laguna, Philippines Tel. No.: (632)920-5301 local 7048; Fax No.: (632)927-7516 E-mail: hedreyda@laguna.net Date received: April 14, 2003; Date accepted: May 17, 2005 Six Vibrio isolates identified biochemically as Vibrio campbellii from Southeast Asian Fisheries Development Center (SEAFDEC) in Tigbauan, Iloilo, were characterized by 16 rDNA sequence, total protein profile, and DNA profile analyses. Genomic DNA from the isolates were subjected to PCR using four sets of primers targeting gene fragments of hemolysin and toxR based on sequences from reference Vibrio harveyi (IFO15634), V. campbellii (IFO1563), and local isolates identified as V. harveyi. Total protein profile could not distinguish the isolates from one another and from the reference V. harveyi (IFO15634) and V. campbellii (IFO15631). Analysis of 16s rDNA sequences revealed high degree of sequence similarity (96% - 99%) of the six local isolates with other Vibrio species including V. campbellii and V. parahaemolyticus, indicating that this analysis will not be useful in resolving their identity. All six isolates exhibited characteristic reference V. harveyi PCR profile when a primer set designed to amplify a 308-bp fragment of hemolysin gene in that species was used. However, no amplicons were generated for these isolates using primers that amplify toxR gene fragments in V. harveyi. This suggests that the six isolates were not bonafide V. harveyi strains. The isolates also did not exhibit V. campbellii characteristics since the primer designed to target the toxR gene in V. campbellii could not amplify DNA from any of the six isolates, suggesting that they were not bonafide V. campbellii strains either. The toxR gene from the six isolates could be amplified using a primer based on toxR gene sequences from a SEAFDEC isolate previously identified as V. harveyi (PN-9801). These data suggest that the six isolates previously identified as V. campbellii as well as PN-9801 may be classified in one group separate from bonafide reference V. harveyi and reference V. campbellii strains, based on the identical results in the molecular analyses performed in this study. Keywords: Vibrio, toxR, hemolysin, protein profile, PCR *Corresponding author Cortado et al. 24 INTRODUCTION Penaeid shrimp farming, especially of the giant tiger prawn, Penaus monodon dominates crustacean production not only in the Philippines but also in the entire Southeast Asian region. In fact in 1995, a production of 0.48 million metric tons of black tiger prawn was recorded from Southeast Asia (Subasinghe et al., 1997). In the Philippines, production from tiger prawn totaled 36,798 metric tons, accounting for 20% of total yield in 1998 (Philippine Fisheries Profile– BFAR, 1998). One major concern in Philippine shrimp farming industry however, is the emergence of diseases, which results to significant reduction in production. Diseases in cultured shrimps, which have been causing great production loss in Philippine aquaculture industry, are attributed mainly to bacterial pathogens V. harveyi and V. campbellii. These pathogens were shown to be among the most dominant species associated with mortalities in black tiger shrimp in the Philippines (de la Peña et al., 2001). Biochemical tests were used to identify and classify the pathogens after heavy mortalities due to luminescent vibriosis have been observed among pond-cultured shrimps. In the same study (de la Pena et al., 2001), it was also demonstrated that several strains, including the six isolates used in this study, were pathogenic when injected intramuscularly to shrimps. However, identity of the local isolates should be confirmed because biochemical tests were not sufficient to distinguish the local isolates from each other or from reference strain V. harveyi and V. campbellii. The use of 16S rDNA sequences may not be suitable for the purpose, since these sequences in closely related species such as V. harveyi and V. campbellii have high homologies (about 96% to 99%). In this study, some molecular approaches were performed to characterize and confirm the identity of the six previously mentioned local isolates, identified as V. campbellii. The study was conducted with the following objectives: (1) To compare total protein profiles of the six SEAFDEC local isolates with those of reference V. campbelli and V. harveyi strains. (2) Evaluate if indeed 16s rDNA sequences will not be able to resolve their identity. (3) To compare PCR profiles of the six local isolates with those of reference V. harveyi and reference V. campbellii strains using four sets of primers namely Vhhemo which targets hemolysin gene fragments of V. harveyi, VhtoxR and VctoxR which amplifies toxR gene fragments of reference V. harveyi and V. campbellii, respectively, and PNtoxR designed to amplify toxR gene fragments of local isolates previously identified as V. harveyi. MATERIALS AND METHODS Bacterial isolates used The six local pathogenic Vibrio isolates, which this study aims to characterize, were obtained from Southeast Asian Fisheries Development Center (SEAFDEC) in Tigbauan, Iloilo (Table 1). This research also utilized two reference strains: V. harveyi (IFO 15634) and V. campbellii (IFO 15631), both from the Institute of Fermentation Osaka (IFO) Japan. Another local isolate (PN-9801) identified as V. harveyi based on biochemical tests was also used in this study. Vibrio cultures were continuously maintained at 30°C incubation in Nutrient agar media containing 1.5% NaCl. DNA extraction Genomic DNA from all Vibrio isolates used was extracted using the Nucleospin DNA extraction kit commercially available from Clontech Laboratories, Inc. (Palo Alto, CA, USA). Vc 1 Vc 2 Vc 3 Vc 4 Vc 5 Vc 6 CRW-9807 PIZ-9824 NRW-9805 BR-9807 BRW-9804 CRWP-9805 V. campbellii V. campbellii V. campbellii V. campbellii V. campbellii V. campbellii Designation ID number Biochemical identification (dela Peña et al., 2001) Table 1. Local isolates used in this study. Local Vibrio Isolates 25 16s rDNA sequence analysis The 16s rDNA fragments of the six isolates were amplified using specific primers targeting this gene. The PCR products were gel-purified and sequenced. Sequence homology of the isolates was then compared with known 16s rDNA sequences of various species deposited in the database using Blast sequence homology search, to determine the degree of relatedness of the isolates with the reference strains. Extraction of total proteins Total proteins were extracted from the six local Vibrio isolates, two reference strains: V. harveyi and V. campbelli and a local isolate (PN-9801) identified as V. harveyi. Cells were collected from a 10 ml overnight bacterial culture in liquid broth by centrifugation at 10,000 x g for 2 minutes at room temperature. The resulting pellet was resuspended in sterile distilled deionized water after which 200 mL of 2X Treatment buffer was added. The treatment buffer consist of 0.125M Tris-Cl, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.2M dithiothreitol (DTT) and 0.02% bromphenol blue. The cells in treatment buffer were boiled for 10 minutes and then centrifuged at 10,000 x g for 5 minutes at room temperature to pellet the debris and insoluble materials. The supernatant containing total protein extracts were transferred to fresh 1.5-ml tubes. The concentrations of the extracted proteins were determined by spectrophotometry at a wavelength of 280 nm (Ausubel, 1997). Bovine serum albumin (BSA) was used as protein standard. Sample concentration was standardized using 1X TB. SDS-polyacrylamide gel electrophoresis (PAGE) of total proteins from the isolates Total protein extracts of each isolate were run in a 7.5% acrylamide gel using the discontinuous Laemmli SDS- PAGE method. This was done in a Hoefer SE400 Sturdier Vertical Unit [Amersham] setup with 1.5 mm gel spacers. For each sample, 100 mg protein extract was loaded onto each well. Along with the samples, a broad-range protein ladder from Biorad served as molecular weight marker. Proteins run in the gels were stained using the Vorum Silver Staining protocol (Mortz et al., 2001). PCR-based characterization of Vibrio isolates Four sets of primers namely Vhhemo, VhtoxR, VctoxR and PNtoxR, which were designed in earlier studies to target specific regions of the hemolysin and toxR genes of reference V. harveyi and V. campbellii strains as well as two local Vibrio isolates, were used in this study. Vhhemo, VhtoxR and PNtoxR primers were designed by Conejero (2002) for the specific detection of different strains of V. harveyi. The VctoxR primer set was designed to amplify a fragment of toxR from V. campbellii (Sobrepeña et al., 2002). Sequences of each primer set used appear in Table 2. PCR using hemolysin primer Vhhemo DNA extracted from the isolates and reference strains, V. harveyi and V. campbellii were used as templates for PCR with primers originally designed for a hemolysin gene fragment. The Vhhemo primer set targets a unique 308-bp region in the hemolysin gene of the reference V. harveyi (IFO 15634) and a local Vibrio harveyi strain (PN-9801). DNA samples were subjected to PCR in Perkin Elmer Gene Amp System (Perkin Elmer Corporation, Singapore). Conditions for PCR with Vhhemo primers were as follows: 30 cycles at 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min with an initial denaturation at 94°C for 5 minutes and a final extension step at 72°C for 7 minutes. Vhhemo Vhtoxr Vctoxr PNtoxr Table 2. PCR primer sequences used in the study (Conejero & Hedreyda, 2003). Forward Reverse Forward Reverse Forward Reverse Forward Reverse TCA GTGCCT CTC AAG TAA GA GCT TGA TAA CAC TTT GCG GT TTC TGA AGC AGC ACT CAC TCG ACT GGT GAA GAC TCA TTC TGA AGC AGC ACT CAC TTC TGA AGC AGC ACT CAC AGC AGC TGC TCC AGT TGA CTG CTC AAT TGA TGG CAG Primer Sequence 5’ – 3’ Cortado et al. 26 PCR using VhtoxR primer VhtoxR primers were used to amplify genes from the six local isolates and from reference strains, V. harveyi and V. campbellii. The primer was based on partial toxR gene sequence of a reference V. harveyi strain (IFO 15634). Conditions for PCR with VhtoxR primers were as follows: 30 cycles at 94°C for 1 min, 63°C for 1 min, and 72°C for 1 min with an initial denaturation at 94°C for 5 minutes and a final extension step at 72°C for 7 minutes. PCR using the VctoxR primer VctoxR primer set was previously designed to amplify a 230-bp fragment of the toxR from V. campbellii. This primer set was used in PCR using DNA templates from the six isolates and from reference strains, V. campbellii and V. harveyi. PCR of the samples with VctoxR was accomplished using the following conditions: 30 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min with an initial denaturation at 94°C for 5 min and a final extension step at 72°C for 7 min. PCR using the PNtoxR primers PNtoxR primers were based on partial toxR sequence of a local Vibrio isolate (PN-9801) previously identified as V. harveyi. The primer set was designed to target the 226-bp fragment of toxR from local Vibrio isolates. DNA from the six local isolates, reference V. campbellii (IFO-15631) and a local isolate (PN-9801) were used as template in the following run: 30 cycles at 92°C for 45 secs, 63°C for 1 min, and 72°C for 1.5 min with an initial denaturation at 94°C for 5 min and a final extension step at 72°C for 7 min. The PCR mix without the DNA template was used as a negative control for all runs. The components for each 20-mL reaction mix are as follows: 13.9 mL sterile distilled water, 2 mL buffer, 0.6 mL MgCl2, 0.4 mL dNTPs, 1 mL forward and 1 mL reverse 10mM primers, 0.1 mL Taq polymerase, and 1 mL template. PCR products along with a 50-bp or 100-bp molecular weight marker were run in 1.5% agarose gels, stained with ethidium bromide and visualized under ultraviolet light. RESULTS AND DISCUSSION The six Vibrio isolates used in this study (CRW-9807, PIZ-9824, NRW-9805, BR-9807, BRW-9804, CRWP- 9805) were previously identified by traditional biochemical tests as V. campbellii (de la Peña et al., 2001). However, variable responses to biochemical tests are often observed in Vibrio species. These tests are also seldom adequate to distinguish between Vibrio species especially closely related ones. In a species characterization study of de la Peña and co-workers (2001) for example, only the ability to decarboxylate ornithine differentiates V. harveyi from V. campbellii. Similar characteristics were exhibited by both strains in all other reactions tested. So to confirm whether the six local SEAFDEC isolates are strains of bonafide V. campbellii, molecular tests were performed. A molecular approach offers the speed and ease of method without compromising accuracy of identification. The isolates can be characterized based on their 16s rDNA sequences as well as protein and DNA profiles using powerful molecular tools such as SDS-PAGE and gene- targeted PCR for discrimination between various strains. Characterization using total protein profile The isolates were first characterized based on the protein profiles generated after resolution of total soluble protein in an SDS-PAGE. Using protein profile analysis, relationships between bacteria can be determined based on the presence of unique and shared bands. This was demonstrated by Que (2002) to deduce genetic relatedness in several Vibrio reference strains where several protein bands were found to be characteristic of each species. Isolates were therefore expected to exhibit distinct protein profiles that could identify them with any of the reference strains used based on the presence of similar protein bands. Surprisingly, similar total protein profiles were generated by the six isolates (Fig.1), the two reference strains, V. harveyi (IFO 15634) and V. campbellii (IFO 15634) and a local isolate identified as V. harveyi (PN- 9801). All were characterized by the presence of two heavily stained bands near the 45 kDa marker and four lightly stained high molecular weight bands between the 97.4 and 66 kDa markers. Compared to the rest, Local Vibrio Isolates 27 one slight difference though, can be observed with the profile of V. harveyi, which is the presence of a distinct band just below the 31 kDa marker. These observed similarities in total protein profiles indicate the close relatedness of the six local isolates with V. harveyi and V. campbellii reference strains and with a locally isolated V. harveyi strain. Protein profile analysis was not able to sufficiently distinguish the isolates from each other nor identify them with any of the two reference strains used. Analysis of 16s rDNA sequence Another way to elucidate genetic relatedness among bacteria is the use of 16s rDNA sequence analysis. Dorsch and co-researchers (1992) have shown the close relationship of V. alginolyticus, V. campbellii, V. harveyi, V. proteolyticus, V. parahaemolyticus, and V. natriegens by analyzing conserved and variable sequences in this gene. An available database containing known 16s rDNA sequences of various bacterial species allow alignment of new sequences with those already recorded in the database. The use of Blast sequence homology search to analyze newly obtained 16s rDNA sequences of the six local isolates [GenBank Accession Nos. DQ017901 (CRW-9807), DQ017902 (PIZ-9824), DQ017903 (NRW-9805), DQ017904 (BR-9807), DQ017905 (BRW-9804), and DQ017906 (CRWP- 9805)] revealed high degree of sequence similarity (96%-99%) in 16s rDNA of the six local isolates with other Vibrio species including V. campbellii and V. parahaemolyticus (Table 3). The high interspecies homology observed in 16s rDNA was also confirmed by phylogenetic analysis using the MEGA v2.1 software (Fig. 2). These observations suggest the need for other molecular typing methods that target specific gene sequences in Vibrio species for more accurate classification. PCR targeting hemolysin gene fragments The hemolysin gene, found to be present in several Vibrio species codes for an important virulence factor in bacteria. Pathogenic activity of hemolysins includes erythrocyte disruption in various species and toxicity for cells and small experimental animals (Nishibuchi & Kaper, 1995). To detect the presence of hemolysin gene and to test if primers designed based on V. harveyi would work with the six local isolates, Vhhemo primers were used in PCR. These primers were shown to generate a characteristic 308-bp amplicon from V. harveyi reference strains as well as from a variant local isolate (PN-9801) identified as V. harveyi (Conejero & Hedreyda, 2004). Amplification of the 308-bp hemolysin gene fragment using DNA templates from the five of six local isolates was observed (Fig. 3), indicating that a gene homologue is present in the five local isolates and the sequences in the primer annealing M 1 2 3 4 5 6 7 8 9 200 kDa 97.4 66.2 45.0 31.0 Fig. 1. Total protein profile of the Vibrio isolates. Lanes 1-6: CRW-9807, PIZ-9824, NRW-9805, BR-9807, BRW-9804, CRWP-9805; lanes 7 and 8 are reference strains V. harveyi (IFO15634) and V. campbellii (IFO15631), respectively; lane 9 is a local isolate (PN-9801) identified as V. harveyi. M is a broad-range protein ladder from Biorad. Table 3. 16S rDNA sequence analysis. CRW-9807 PIZ-9824 NRW-9805 BR-9807 BRW-9804 CRWP-9805 98% V. campbellii, Vibrio sp, V. parahaemolyticus 96% V. campbellii, Vibrio sp, V. parahaemolyticus 99% V. campbellii, Vibrio sp, V. parahaemolyticus 99% V. campbellii, Vibrio sp, V. parahaemolyticus 99% V. parahaemolyticus, 98% V. campbellii 99% V. campbellii, V. parahaemolyticus Isolate ID number 16S rDNA sequence similarity Cortado et al. 28 sites were significantly homologous to that of the V. harveyi reference strain. At 53°C annealing temperature, five local isolates were able to produce distinct 308-bp amplicons with the Vc2 and Vc4 (PIZ- 9824 and BR-9807, respectively) both exhibiting an additional band of about 600-bp in their profiles which could not be explained at this point. One isolate, Vc6 (CRWP-9805), gave only a 600-bp product instead of the expected 308-bp fragment. The absence of the 308-bp band in the profile of reference strain V. campbellii indicates that hemolysin gene sequence variation in the primer annealing sites are present in V. campbellii. Data also suggest that hemolysin gene sequences of the six local isolates being studied maybe exhibiting greater homology with reference strain V. harveyi compared to the hemolysin sequences of V. campbellii. This adds to the confusion in species confirmation of these local isolates. Although they were previously identified by biochemical means as V. campbellii, these results show that they are not exhibiting bonafide V. campbellii characteristics. PCR targeting toxR gene fragments VhtoxR primers The toxR gene is considered a main regulator for coordinating expression of various virulence genes in 100 54 67 100 0.04 0.020.03 92 61 36 61 0.01 0.00 Vc5 BRW-9804 V. campbellii V. harveyi V. parahaemolyticus V. natriegens V. alginolyticus Vc6 CRWP-9805 Vc1 CRW-9807 Vc3 NRW-9805 Vc2 PIZ-9824 Vc4 BR-9807 V. anguillarum V. fluvialis V. vulnificus V. mimicus V. cholerae M 1 2 3 4 5 6 7 8 9 600 bp 308 bp100 bp Fig. 3. PCR profile of the six Vibrio isolates using Vhhemo primers. Lanes 1-6 contains the local isolates: CRW-9807, PIZ-9824, NRW-9805, BR-9807, BRW-9804, CRWP-9805; lanes 7 and 8 are products from the reference strains V. harveyi (IFO 15634) and V. campbellii (IFO 15631), respectively; lane 9 is the negative control (no template). M is the 100-bp molecular weight marker. Fig. 2. Unrooted tree constructed by the neighbor-joining method based on the 16s rRNA gene. Percent bootstrap values from 1,000 replicates generated are given at the branching points. Bars indicate genetic distance. Local Vibrio Isolates 29 bacteria. It is also hypothesized to be part of the conserved genes in the ancestral chromosome of members of the family Vibrionaceae (Osorio et al., 2000). Sequencing of the toxR gene revealed the presence of variable sequences that can be utilized for the identification of specific species of Vibrio. A toxR homologue from a reference strain V. harveyi (IFO 15634) was demonstrated (Conejero, 2002) to be useful for designing primers (VhtoxR) specific for V. harveyi reference strains. Based on this homologue another primer PNtoxR was designed for the detection of toxR fragments in locally derived Vibrio isolates pathogenic to P. monodon. Another set of primers (VctoxR) targeting a toxR gene fragment in V. campbellii was designed (Sobrepeña et al., 2002) based on the partial toxR sequence of a reference V. campbellii strain (IFO 15631). All the above-mentioned primers were used to test which primer will result in amplification of the toxR gene fragment and consequently to test if indeed they will exhibit the characteristic V. campbellii results, that is producing amplicons only when using the primers VctoxR designed based on V. campbellii sequences. PCR using DNA templates from the six local isolates with VhtoxR primers did not result in the amplification of the 390-bp toxR fragment that is produced in PCR using V. harveyi reference strain DNA template. Although the Vhhemo primer generated the unexpected 308-bp amplicons for the isolates previously identified as V. campbellii, these six isolates did not exhibit profiles characteristic of V. harveyi reference strain in PCR using the VhtoxR primers. This further complicates the analysis because the use of two different primer set in PCR resulted in conflicting result, one suggesting closer relatedness to V. harveyi and the other does not. VctoxR primers When the DNA from six local isolates were subjected to PCR using VctoxR primers, designed based on sequences of toxR gene fragments of reference V. campbellii,, no amplicons were observed either (data not shown). Interestingly, data reveals that the toxR gene fragment in the six local isolates may have significant sequence variation particularly at the primer annealing sites from those of reference strains V. campbellii. Therefore, the six isolates are not exhibiting bonafide V. campbellii characteristics. These findings point out to the possibility that the local isolates could be very close relatives of Vibrio harveyi or Vibrio campbellii but may later be classified as an entirely different group separate from these two species. PNtoxR primers Another set of primers (PNtoxR) based on partial sequences of the toxR gene isolated from a local isolate PN-9801, previously identified as V. harveyi, could generate a 226-bp band in PCR using DNA template from that local isolate (PN-9801). When DNA from the six isolates used in this study were subjected to PCR using PNtoxR primers, the 226-bp amplicon was also produced in all six samples (Fig. 4). No amplicon was produced in PCR using DNA templates from reference V. harveyi and V. campbellii. These data strongly support the hypothesis that the local isolates previously identified as V. harveyi and the local isolates used in this study previously identified as V. campbellii, could be belonging to the same classification. Preliminary total protein profile analysis of these local isolates revealed almost identical profiles. The hemolysin gene fragment from these isolates could not be amplified by three primers used to amplify hemolysin gene fragments from bonafide V. harveyi isolates but the primer set Vhhemo that was able to produce a 308-bp amplicon in reference strain V. M 1 2 3 4 5 6 7 8 9 850 bp 226 bp 350 bp Fig. 4. PCR profile of the six Vibrio isolates using PNtoxR primers. Lanes 1-6 contains the six local isolates: CRW-9807, PIZ-9824, NRW-9805, BR-9807, BRW-9804, CRWP-9805; lanes 7 and 8 are products from the reference strains V. campbellii (IFO 15631) and a local isolate PN-9801, respectively; lane 9 is the negative control (no template). M is the 50-bp molecular weight marker. Cortado et al. 30 harveyi as well as the all the local isolates in this study. DNA templates from 6 local isolates in this study, could not be amplified by the VhtoxR and the VctoxR primers that are used to amplify toxR gene fragments from V. harveyi and V campbellii, respectively. The molecular data from this study provide preliminary evidence for a proposed separate classification of a local Vibrio isolates from previous studies and from this present study, distinct and separate from bonafide V. harveyi and V. campbellii. where they are currently included based on biochemical tests. One approach to resolve this classification problem involves DNA sequence analysis of additional genes that are present in all Vibrios in order to provide additional data for grouping isolates together or discriminating members of different but closely related Vibrio species. Candidate gene markers that are being considered include the gyrase B gene, toxR gene, and the gene for hemolyisn (Conejero & Hedreyda, 2003; Conejero & Hedreyda, 2004). ACKNOWLEDGMENTS The authors would like to thank Mia Judith U. Conejero-Viray for the use of her toxR and hemolysin gene primers. REFERENCES Conejero, M., 2002. Identification of gene sequences for the specific detection of Vibrio harveyi. 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