1 CIAD/Mazatlán Unit for Aquaculture, AP 711, Mazatlán Sinaloa, Mexico 82000
2 CIBNOR, AP 128, La Paz, Baja California, Mexico 23090
3 Laboratory for Microbiology, K. L. Ledeganckstraat 35, Ghent University, Ghent 9000, Belgium
Correspondence
Bruno Gomez-Gil
bruno{at}victoria.ciad.mx
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ABSTRACT |
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INTRODUCTION |
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The taxonomy of Vibrio is in the process of revision due to the increasing data obtained with modern molecular biology techniques, where different genes are examined or where the whole genome is inspected. Special emphasis has been paid to the 16S rRNA, although other genes, such as those for 23S rRNA, 16S23S intergenic spacer region (IGS), or the gyrB gene, have been employed (Chun et al., 1999; Dorsch et al., 1992
; Venkateswaran et al., 1998
). Unfortunately, the 16S rRNA is unable to resolve closely related species (Nagpal et al., 1998
), such as the ones clustered in the Vibrio core group, namely V. alginolyticus, V. parahaemolyticus, V. harveyi, V. campbellii, V. natriegens and the newly described V. rotiferianus (Gomez-Gil et al., 2003
). The identification of vibrios isolated from the aquacultural environment has been imprecise and is labour-intensive, requiring many biochemical and/or physiological tests (Vandenberghe et al., 2003
).
Several highly powerful molecular tools, e.g. amplified fragment length polymorphism (AFLP, Rademaker et al., 2000; Gurtler & Mayall, 2001
) and repetitive extragenic palindromic elements polymerase chain reaction (REP-PCR) (Versalovic et al., 1991
), have become readily available for the identification of bacteria, including vibrios (Thompson et al., 2001a
; Sawabe et al., 2003
). Rep-PCR can also differentiate strains of the same species, e.g. Escherichia coli (Dombek et al., 2000
), Bradyrhizobium spp. (Vinuesa et al., 1998
), Bacillus subtilis (Versalovic et al., 1991
) and Vibrio cholerae (Rivera et al., 1995
). In this study, we report on the use of FAFLP, REP-PCR and IGS-PCR for the identification of V. harveyi-related species.
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METHODS |
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Genomic characterization.
DNA from each strain was extracted with the Promega Wizard Genomic DNA Purification kit (A1120). For DNADNA hybridization experiments, DNA from the selected strains was extracted following the technique of Pitcher et al. (1989). The DNA quality of both extractions was assessed by observations of an electrophoresed sample (1·0 % agarose, 1x TAE buffer, 1 h, 100 V). The same extracted DNA was employed in all genetic fingerprinting methods, except DNADNA hybridization, as explained above.
PCR reaction mix for REP-PCR contained 12·45 µl water, 1·25 µl dNTP mix (25 mM each), 2·5 µl DMSO, 5·0 µl 5x Gitschier buffer (Rademaker et al., 1998), 0·4 µl BSA (10 mg ml1), 1·0 µl of each primer (0·3 µg µl1), 0·4 µl Taq polymerase (5 U µl1, AmpliTaq; Applied Biosystems) and 1·0 µl DNA (50 ng µl1) for a final volume of 25 µl. For IGS amplification, the reaction mix contained 18·3 µl water, 2·5 µl dNTP mix (2 mM each), 2·5 µl 10x PCR buffer with 25 mM MgCl2 (Applied Biosystems), 0·26 µl of each primer (0·25 µg µl1), 0·15 µl of Taq polymerase (5 U µl1, AmpliTaq; Applied Biosystems) and 1·0 µl of DNA (50 ng µl1) for a final volume of 25 µl.
Primers for REP-PCR were REP1R (5'-III ICG ICG ICA TCI GGC-3') and REP2 (5'-ICG ICT TAT CIG GCC TAC-3'). Inosine (I) contains the purine base hypoxanthine, capable of forming WatsonCrick base pairs with A, G, C or T (Versalovic et al., 1991). BOX-PCR employs the primer BOXA1R (5'-CTA CGG CAA GGC GAC GCT GAC G-3'), and (GTG)5-PCR the primer 5'-GTG GTG GTG GTG GTG-3' (Versalovic et al., 1994
). The amplification protocol for BOX-PCR was 95 °C for 2 min, followed by 35 cycles of 94 °C for 3 min, 92 °C for 30 s, 50 °C for 1 min and 65 °C for 8 min with a final extension of 65 °C for 8 min. The amplification protocol for REP- and (GTG)5-PCR was 95 °C for 2 min, followed by 35 cycles of 94 °C for 3 min, 92 °C for 30 s, 40 °C for 1 min and 65 °C for 8 min with a final extension of 65 °C for 8 min. Primers for the IGS-PCR 16S23S were V16S-1492F (5'-AAG TCG TAA CAA GGT ACG GCT-3') and V23S-68R (5'-GCC TCA TCT ACG CTT ATC GC-3'). The amplification protocol was 94 °C for 2 min followed by 35 cycles of 94 °C for 1 min, 70 °C for 1 min and 72 °C for 1 min with a final extension of 72 °C for 5 min.
The amplification products were resolved in a 1·5 % agarose gel in TAE buffer: 5 µl of a gel loading dye was mixed with the 25 µl of the reaction, and 10 µl of the mixture was loaded in the gel; 5 µl of a PCR molecular mass marker (Smartladder; Eurogenetec) was added every five lanes. The gel was electrophoresed at 48 °C for 15 h at 55 V. The gel was stained in an ethidium bromide solution with 1x TAE buffer for 20 min, destained for 5 min and photographed with a digital system. The resulting images were processed with Bionumerics 2.5 software (Applied Maths).
FAFLP (fluorescent amplified fragment length polymorphism) patterns were generated and analysed as described previously (Thompson et al., 2001a). Briefly, 1 µg high-molecular-mass DNA was digested with TaqI and HindIII followed by ligation of restriction half-site specific adapters to all restriction fragments with T4 ligase. Pre-selective PCR amplification was done with H00-ABI primer (5'-GAC TGC GTA CCA GCT T-3') and T00-ABI primer (5'-CGA TGA GTC CTG ACC GA-3'), and the selective PCR amplification with H01-6FAM primer (5'-GAC TGC GTA CCA GCT TTA-3') and T03-ABI (5'-CGA TGA GTC CTG ACC GAG-3').
DNADNA hybridization of representative strains of each cluster was done following the methodology described by Willems et al. (2001) under stringent conditions (39 °C) and data obtained from previous works (Gomez-Gil et al., 2003
; Thompson et al., 2001a
); in total, values for 13 strains were employed.
Numerical analyses.
Similarity among band patterns was calculated with the Dice similarity coefficient and dendrograms were constructed with the Ward algorithm. A band position tolerance of 0·50·7 % was allowed to compensate for misalignments of homologous bands due to technical imperfections (Thompson et al., 2001a). Correlation analysis of DNA homologies and similarity values from all the REP-PCR and IGS-PCR experiments were done with the Pearson product-moment coefficient. Data were analysed for normality using SigmaStat for Windows version 2.03 (SPSS). Fifty values were analysed for all tests, except for IGS-PCR, where only 39 values could be obtained.
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RESULTS |
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FAFLP analysis produced the highest number of bands per strain compared to the other analyses (mean 102·4, max. 147, min. 72, SD 13·41, n=49). Lower numbers were obtained, in descending order, with (GTG)5-PCR (mean 24·1, max. 31, min. 17, SD 2·93, n=49), BOX-PCR (mean 11·0, max. 17, min. 6, SD 2·92, n=48), REP-PCR (mean 14·8, max. 27, min. 5, SD 4·48, n=49) and IGS-PCR (mean 6·5, max. 9, min. 5, SD 1·04, n=44). REP-PCR produced the highest band sizes (mean 2384 bp, max. 9710, min. 153, SD 1806, n=49), followed by (GTG)5-PCR (mean 1414 bp, max. 4560, min. 187, SD 837, n=49), BOX-PCR (mean 1109 bp, max. 5130, min. 191, SD 786, n=48) and IGS-PCR (mean 611 bp, max. 894, min. 351, SD 161, n=44), and the lowest size of bands with FAFLP (215 bp, max. 534, min. 50, SD 120, n=49). No bands could be obtained for strain LMG 16828 with REP- and GTG5-PCR, nor for LMG 16835 with FAFLP. Strains LMG 21457 and CAIM 113 did not produce any bands with BOX-PCR. V. rotiferianus strains (LMG 21456 to LMG 21460T) were not tested in IGS-PCR, and no bands were obtained for strain LMG 16835.
Clustering of the strains with the FAFLP and REP-PCR methods produced similar groups (Figs 1 and 2). Type and reference strains of the three species analysed were clustered in different groups when a cut-off level of 45 % similarity was applied for the case of FAFLP (Thompson et al., 2001a
). In this case, 33 strains were assigned to V. campbellii, 11 to V. harveyi, and five to V. rotiferianus (Fig. 1
). The V. campbellii group is very diverse and the FAFLP cluster cutoff value for this species is less than 10 % similarity. V. harveyi and V. rotiferianus present tighter clusters well delimited at or above the 45 % cutoff FAFLP value.
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Eight strains were assigned to V. campbellii by DNADNA hybridization (Table 2); values above 70 % similarity were obtained between the type strain LMG 11216T and CAIM 372 (79·2 %), R-14899 (78·0 %), CAIM 128 (71·3 %), CAIM 333 (74·0 %), CAIM 415 (81·7 %), LMG 20369 (76·1 %), CAIM 113 (81·8 %) and LMG 16835 (77·6 %). Strains LMG 7890 and LMG 19643 were identified as V. harveyi because the DNA similarity value with the type strain LMG 4044T was 97·9 and 79·6 % respectively, and that between the two reference strains was 82·0 % (Thompson et al., 2001a
; Pedersen et al., 1998
). Values below the 70 % proposed by Wayne et al. (1987)
to delimit a species were obtained between the three type strains of the species analysed: 69·0 between V. campbellii LMG 11216T and V. harveyi LMG 4044T, 65·0 % between V. campbellii LMG 11216T and V. rotiferianus LMG 21460T, and 66·0 % between V. harveyi LMG 4044T and V. rotiferianus LMG 21460T (Gomez-Gil et al., 2003
).
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No clear phenotypic differences were found between the strains of V. harveyi and V. campbellii analysed in this study; at most, only a majority of strains had positive or negative reaction to eight tests (Table 4). None of the tests suggested by Holt et al. (1994)
to differentiate the two species (mentioned above) were useful because overlapping results were observed.
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DISCUSSION |
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It was suggested that a high number of fragments (bands) included in an analysis yields a higher correlation (Rademaker et al., 2000). In this study, although FAFLP had the highest number of bands (mean 102·4) and the highest correlation coefficient with DNADNA hybridization (0·761), no correlation could be established with the number of bands and hybridization values when all the methodologies were statistically analysed (Spearman correlation coefficient=0·083, P>0·05, n=4). It has been recommended that at least a minimum of 815 bands per sample (or lane) must be used for a rigorous comparative analysis (Versalovic et al., 1994
). (GTG)5-PCR has been used very rarely, and never, to our knowledge, correlated with DNADNA hybridization. Using this method, Gevers et al. (2001)
obtained a reliable identification of Lactobacillus species with a mean of 16·5 bands; they also found that combining data from BOX-, REP- and (GTG)5-PCR did not significantly enhance the discriminatory power compared to the increase in the amount of work needed. On the other hand, Nick et al. (1999)
preferred a combined dendrogram because a maximized specificity of the patterns was obtained. (GTG)5-PCR has also been useful to identify and describe new species of bacteria such as Vibrio coralliitycus (Ben Haim et al., 2003
), and Virgibacillus spp. (Heyrman et al., 2003
).
In our study, analysis of the IGS-PCR results produced an unrealistic dendrogram, which could be explained, in part, because of the low number of bands observed and the region analysed, which is only a small part of the genome, as compared to the other methods used. Apparently, the DNA sequence of these intergenic spacers can provide valuable information for the identification of many vibrios (Lee et al., 2002), although no specific sequences for V. campbellii could be found in the IGS types studied. Unfortunately, in the study of Lee et al. (2002)
V. harveyi was not analysed, and therefore it is not known if differences in the DNA sequences are useful to differentiate these very closely related species.
The evidence provided in this study and others strongly supports the use of AFLP (or FAFLP) and (GTG)5-PCR as alternative or supportive methods to DNA hybridization to delineate a species.
V. harveyi and V. campbellii are genetically related species with a DNADNA similarity value of 69 % (Gomez-Gil et al., 2003) and a 16S rRNA similarity higher than 97 %. The ability of V. harveyi to produce ornithine decarboxylase is a key phenotypic feature to separate it from V. campbellii (Alsina & Blanch, 1994
). Based on this and other characters many strains were identified as V. campbellii, although they shared characters with V. harveyi and thus were named V. campbellii-like (Hameed et al., 1996
). The results obtained in this study reinforce the findings of Gauger & Gomez-Chiarri (2002)
and of Thompson (2003)
that phenotypic characters are not useful to differentiate these species. None of the characters could clearly discriminate the two species (Table 4
). Therefore it is recommended that molecular fingerprinting methods be used to identify these species, such as rep-PCR [preferably (GTG)5] or FAFLP.
Sequencing of the 16S rRNA gene of some of the strains employed in this study (Gauger & Gomez-Chiarri, 2002), namely LMG 4044T, LMG 780, LMG 11216T, R-14905 and LMG 16828, and others, produced a dendrogram that supports the results of this study. A similar result was obtained by Pedersen et al. (1998)
with some of the strains used here; all the strains identified as V. harveyi here clustered in the AFLP cluster 1 of Pedersen's work, while all the V. campbellii clustered in the AFLP groups 4 and 5. Thompson et al. (2001a)
also analysed some of the strains used in this study, and found a similar arrangement, where V. harveyi strains formed a tight cluster (A36) and the V. campbellii strains were divided in two (A14 and A37); the delineation thresholds used by these authors were 50 % and 45 %, respectively, useful for V. harveyi strains but not for the more genetically diverse V. campbellii strains. For V. campbellii strains, a 10 % similarity threshold in the FAFLP dendrogram had to be used to include all the strains that were identified as this species based on the DNA hybridization studies (Fig. 1
). It was suggested that identification with FAFLP should be made when a similarity pattern of 6070 % was adopted (Thompson, 2003
), which corresponds to a DNA hybridization value of 70 %; clearly, this threshold can be used for the majority of the Vibrio species. The subdivision of the V. campbellii strains into many clusters could be explained by the great heterogeneity of this species.
Of particular interest was strain LMG 20977 (=AK2), which was identified as V. shilonii by Kushmaro et al. (2001) due to being phenotypically identical to the type strain AK1T (=LMG 19703T). Later, Thompson et al. (2001b)
corrected the identification to V. mediterranei but considering only the type strain and not LMG 20977. In the present study, strain LMG 20977 was grouped, with all methods except IGS-PCR, in the V. harveyi cluster. The FAFLP similarity between LMG 20977 and the V. harveyi type strain (LMG 4044T) was 66·33 % and with the V. campbellii type strain (LMG 11216T) only 49·08 %. It is not clear if strain LMG 20977 is pathogenic for corals (Kushmaro et al., 2001
), but if so, then V. harveyi should also be considered as pathogenic for corals, and many other marine organisms.
The present study highlights the inadequacy of dichotomous keys (Alsina & Blanch, 1994) for the identification of V. harveyi-related species. Presumptive V. harveyi isolates associated with diseased organisms (see Soto-Rodríguez et al., 2003
) analysed in this study turned out to be V. campbellii on the basis of both molecular fingerprinting and DNADNA hybridization, suggesting that V. campbellii may be an important pathogenic species of aquatic organisms (Gauger & Gomez-Chiarri, 2002
; Soto-Rodríguez et al., 2003
).
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ACKNOWLEDGEMENTS |
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Received 30 September 2003;
revised 13 February 2004;
accepted 20 February 2004.
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