Identification of Vibrio anguillarum in fish by using partial 16S rRNA sequences and a specific 16S rRNA oligonucleotide probe.

16S rRNA from seven different Vibrio anguillarum strains was partially sequenced and compared. From this sequence information we could design a 25-base-long oligonucleotide and use it as a specific probe for identification of V. anguillarum. This was determined by RNA-DNA colony hybridization and slot-blot hybridization. Strong, specific hybridization to the probe was observed for all V. anguillarum strains tested. Furthermore, no cross-hybridization could be seen against five other bacterial species. The detection limit was 5 x 10(3) bacteria per ml. It was even possible to detect V. anguillarum, by slot-blot hybridization, directly in a homogenized kidney from a fish that had died of vibriosis. The partial sequence information revealed small but significant differences between strains of the same species. These sequence differences are sufficiently significant to allow serotyping on the RNA level. Comparing strains of different serotypes revealed a 10-base and an 11-base difference in V. anguillarum serotypes O8 and O9, respectively, in a 122-base partial sequence.     

Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA.

Partial 16S rDNA from Vibrio collection type strains and recent isolates of Vibrio-related strains were sequenced and compared with previously published sequences. A 24-base DNA oligonucleotide (VaV3) was designed and used as a specific probe for detection and identification of Vibrio anguillarum. Its specificity was tested against collection type strains and environmental isolates and no cross-reaction was found. The probe detected 8 of the 10 V. anguillarum serovars. It was applied to screen different Vibrio-related strains isolated from marine hatcheries and fish farms. The detection limit in DNA-DNA slot blot hybridization was 150 pg.     

Taxonomic studies of predatory bdellovibrios based on 16S rRNA analysis, ribotyping and the hit locus and characterization of isolates from the gut of animals

The aim of our study was to obtain data for the molecular characterization of bdellovibrio bacteria, which were recently split into the genus Bdellovibrio and the newly designated genus Bacteriovorax. We determined the 16S rDNA sequences of five reference strains and performed a phylogenetic analysis including published 16S rRNA sequences of bdellovibrios. A comparison of the secondary structure showed significant differences in two regions of the 16S rRNAs of the species Bdellovibrio bacteriovorus, Bacteriovorax starrii, and Bacteriovorax stolpii. In addition, ribotyping techniques gave specific hybridization patterns and revealed that two rRNA operons are present in the investigated strains. A hybridization probe derived from the genetic locus hit, associated with the host independent (HI) phenotype of B. bacteriovorus, was found to be specific for this species. Sequence comparison of the hit locus revealed few base pair changes between host independent (HI) and host dependent (HD) strains. Ribotyping and hybridization experiments using the hit probe were applied to characterize bdellovibrio strains isolated from the gut of animals and humans and one isolate from sewage.     

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

Genetic variability of the heme uptake system among different strains of the fish pathogen Vibrio anguillarum: identification of a new heme receptor

The ability to utilize heme compounds as iron sources was investigated in Vibrio anguillarum strains belonging to serotypes O1 to O10. All strains, regardless of their serotype or isolation origin could utilize hemin and hemoglobin as sole iron sources. Similarly, all of the isolates could bind hemin and Congo red, and this binding was mediated by cell envelope proteins. PCR and Southern hybridization were used to assay the occurrence of heme transport genes huvABCD, which have been previously described in serotype O1. Of 23 strains studied, two serotype O3 isolates proved negative for all huvABCD genes, whereas nine strains included in serotypes O2, O3, O4, O6, O7, and O10 tested negative for the outer membrane heme receptor gene huvA. A gene coding for a novel outer membrane heme receptor was cloned and characterized in a V. anguillarum serotype O3 strain lacking huvA. The new heme receptor, named HuvS, showed significant similarity to other outer membrane heme receptors described in Vibrionaceae, but little homology (39%) to HuvA. This heme receptor was present in 9 out of 11 of the V. anguillarum strains that tested negative for HuvA. Furthermore, complementation experiments demonstrated that HuvS could substitute for the HuvA function in Escherichia coli and V. anguillarum mutants. The huvS and huvA sequences alignment, as well as the analysis of their respective upstream and downstream DNA sequences, suggest that horizontal transfer and recombination might be responsible for generating this genetic diversity.     

Ribotypes and plasmid contents of Vibrio anguillarum strains in relation to serovar.

Eighty-six strains of the 10 major agglutination types of Vibrio anguillarum (serovars O1 to O10) and 6 nontypeable strains of V. anguillarum have been characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by plasmid profile analysis. Forty-four different ribotypes were observed with the restriction enzyme HindIII. Ribotype similarity was compared by using the Dice coefficient (Sd), and three significantly different levels of homogeneity within the V. anguillarum serovars were observed (serovars O1, O3A, O7, and O9, Sds of > 90%; serovars O2B, O4, and O10, Sds of 80 to 90%; serovars O2A, O3B, O5, and O8, Sds between 46 and 70%). None of the ribotype patterns of V. anguillarum strains were observed among 20 other Vibrio strains typed for comparison. By cluster analysis, the V. anguillarum strains were divided into a main cluster containing 83 strains, while all strains of serovar O3B, one strain (each) of serovars O2A, O5, and O8, and a nontypeable strain were separated from this cluster by at least 15% difference in similarity coefficients. Plasmids were demonstrated in only six strains other than serovar O1. In serovar O1, a 67- to 70-kilobase-pair (kb) plasmid molecule was present in 17 of 19 strains tested; of the two remaining strains, one strain harbored two plasmids (45 and 6.5 kb) and one strain had no plasmids.     

A cluster of atypical Yersinia strains with a distinctive 16S rRNA signature

Thirty-eight bacterial isolates from raw milk samples in Queensland, Australia were identified as members of the genus Yersinia on the basis of biochemical profile, ability to hybridize with a genus-specific DNA probe, comparative 16S rDNA sequence analysis, and the presence of characteristic 16S rDNA signature nucleotides which occur in all Yersinia spp. Twenty-five of these isolates reacted with typing sera (O:22 or O:58) of Y. enterocolitica; the remainder were non-typable. None of the isolates displayed any of the phenotypic or genetic virulence-associated characteristics of Y. enterocolitica. Comparative 16S rDNA sequence analysis revealed that members of this group appear to represent a new sub-line within the genus Yersinia, most closely related to Y. frederiksenii hybridization group 2 (unnamed genomospecies 2). This finding was confirmed by DNA hybridization studies which indicated that the strains belonged to the unnamed genomospecies, Yersinia frederiksenii genomospecies 2, which is biochemically indistinguishable from Y. frederiksenii (Y. frederiksenii genomospecies 1). A 23-nucleotide 16S rDNA signature stretch which characterised these strains was identified.     

Characterization of Vibrio strains isolated from turbot (Scophthalmus maximus) culture by phenotypic analysis,ribotyping and 16S rRNA gene sequence comparison

AIM: The aim of the present study was to clarify the taxonomic status of Vibrio strains isolated from an aquaculture system and to compare the results of the identifications made by phenotypic and molecular methods. METHODS AND RESULTS: Fifty-one Vibrio strains isolated from a turbot (Scophthalmus maximus) aquaculture system were characterized by ribotyping and 16S rRNA gene sequencing. The strains had been identified phenotypically in a previous numerical taxonomy analysis as Vibrio anguillarum, V. mediterranei, V. splendidus, V. aestuarianus, V. ordalii, V. fischeri and V. scophthalmi. Cluster analysis of ribotype patterns showed that the strains were separated into two main groups: V. splendidus-V. lentus and V. scophthalmi groups. The use of 16S rRNA gene sequence allowed differentiation among V. splendidus biovar I and V. lentus strains. CONCLUSIONS: The molecular methods identified strains of V. splendidus biovar I, V. lentus and V. scophthalmi, showing discrepancies with phenotypic characterization. SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular methods, as 16S rRNA gene sequence analysis, are necessary for the identification of phenotypically close species to avoid mis-identifications. Interestingly, this is the first report of V. lentus strains associated to turbot culture.     

Comparative Serology of the Marine Fish Pathogen Vibrio anguillarum

The different serotyping systems, based on thermostable O antigens, reported for Vibrio anguillarum and V. ordalii were compared by quantitative agglutination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent silver staining or Western blotting (immunoblotting) of purified lipopolysaccharide (LPS), using polyclonal rabbit antisera. The results demonstrate that 16 different serotypes within V. anguillarum (designated O1 to O16) can be distinguished. Each of these serotypes is characterized by a distinct polysaccharide banding pattern, as revealed by silver-stained gels of purified LPS. The comparative analysis allowed a complete alignment of the different serotypes for the first three serovars: O1, O2, and O3. Moreover, immunoblotting showed that strains belonging to each of these serotypes had the same LPS banding pattern independent of the origin of the strain. Serotype O2 contains different subtypes, O2a and O2b. While no differences were apparent between these subgroups in silver-stained gels, they could be separated by quantitative agglutination (titer determination) or immunoblotting. V. ordalii, the former biotype II of V. anguillarum, strongly reacts with anti-V. anguillarum O2a antiserum. Strains of the two species can be separated on the basis of different LPS profiles in the high-molecular-weight region of silver-stained gels of purified LPS. The silver-stained LPS profiles of the different serotypes of V. anguillarum that have been established are provided for further comparison in the future.     

Identification of Campylobacter species by southern hybridization of genomic DNA using an oligonucleotide probe for 16S rRNA genes

Nine strains of four species of Campylobacter (C. jejuni, C. fetus, C. coli, C. laridis) were studied by genomic Southern hybridization. Restriction digests of chromosomal DNA prepared by treatment with either Eco RV or Rsa I were probed with an oligonucleotide specific for Campylobacter 16S ribosomal RNA genes. Six distinct hybridization patterns were obtained, each indicating the presence of 2–4 copies of the 16S rRNA gene are encoded in Campylobacter DNA. Differences in the hybridization patterns were observed not only between members of two species, but also between individual strains of the same species. Of the four C. jejuni strains tested, two different hybridization patterns were evident. Similar results were observed with different strains of C. coli and C. laridis. The relative simplicity of the patterns obtained, combined with the apparent diversity among individual strains, suggests that DNA-fingerprinting with the 16S rRNA gene probe could be a potentially useful identification method in epidemiological studies of Campylobacter infection.     

Horizontal transfer of segments of the 16S rRNA genes between species of the Streptococcus anginosus group

The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes.     

Characterization of Vibrio anguillarum and closely related species isolated from farmed fish in Norway.

A total of 264 bacterial strains tentatively or definitely classified as Vibrio anguillarum were examined. The strains were isolated from diseased or healthy Norwegian fish after routine autopsy. With the exception of five isolates from wild saithe (Pollachius virens), the strains originated from nine different species of farmed fish. The bacteria were subjected to morphological, physiological, and biochemical studies, numerical taxonomical analyses, serotyping by slide agglutination and enzyme-linked immunosorbent assay, DNA-plasmid profiling, and in vitro antimicrobial drug susceptibility testing. The results of the microbiological studies were correlated to anamnestic information. The bacterial strains were identified as V. anguillarum serovar O1 (n = 132), serovar O2 (n = 89), serovar O4 (n = 2), serovar O8 (n = 1), and not typeable (n = 1) as well as Vibrio splendidus biovar I (n = 36) and biovar II (n = 1), Vibrio tubiashii (n = 1), and Vibrio fischerii (n = 1). V. anguillarum serovar O1 or O2 was isolated in 176 out of 179 cases of clinical vibriosis in Atlantic salmon (Salmo salar). V. anguillarum serovar O1 was the only serovar isolated from salmonid fish species other than Atlantic salmon, while V. anguillarum serovar O2 was isolated from all marine fish suffering from vibriosis. A 48-Mda plasmid was isolated from all V. anguillarum serovar O1 isolates examined. Serovar O2 isolates did not harbor any plasmids. Resistance against commonly used antibiotic compounds was not demonstrated among V. anguillarum isolates. Neither V. splendidus biovar I nor other V. anguillarum-related species appeared to be of clinical importance among salmonid fish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intragenomic diversity of the V1 regions of 16S rRNA genes in high-alkaline protease-producing Bacillus clausii spp

Alkaliphilic Bacillus sp. strain KSM-K16, which produces high-alkaline M-protease, was characterized phenotypically, biochemically and genetically. This strain was identified as Bacillus clausii based on the results of taxonomic studies, including sequencing of the 16S rRNA gene and DNA-DNA hybridization. Seven rRNA operons in the genome were identified by pulsed-field gel electrophoresis. Sequencing of cloned 16S rRNA genes revealed two distinct types of variable region V1. Moreover, some cloned 16S rRNA genes in some of the reference strains of B. clausii had a V1 region of yet another type. The B. clausii strains could clearly be divided into at least two subgroups based on the frequencies of the types of cloned V1 sequence. Bacillus sp. strain KSM-K16 was found to be in a different phylogenetic position from other high-alkaline protease-producing strains of B. clausii.     

Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers

Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.     

Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes

Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03—species, 0.05—genus, 0.10—family) based on full-length 16S rRNA gene sequences. However, partial sequences (700 bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700 bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases.     

Ribotyping and plasmid profiling of Vibrio anguillarum serovar O2 and Vibrio ordalii

One hundred and twenty-nine strains of Vibrio anguillarum serovar O2 and 14 strains of Vibrio ordalii were ribotyped and examined for plasmid contents. A total of 35 different ribotypes were detected. The V. anguillarum serovar O2 strains were divided into 32 different ribotypes. The V. ordalii strains showed three different ribotypes, clearly distinct from those of the V. anguillarum strains.
Ribotypes were separated into seven clusters, of which one comprised the V. ordalii strains. Clustering of the strains indicated a genetic difference between North European and South European V. anguillarum O2 strains. Sero-subgroups O2a and O2b shared ribotypes; however, three of the clusters did not include O2a strains.
All V. ordalii strains had a plasmid of 32 kb. This plasmid was not detected in any of the V. anguillarum strains. Seventeen different plasmid profiles with 17 different sized plasmids were detected among the V. anguillarum strains. Most of the plasmids were small (< 6 kb) and found in several strains. Except for one South European strain, plasmids were detected only in the North European strains of V. anguillarum O2.     

Benchmarking of 16S rRNA gene databases using known strain sequences

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at various taxonomic levels using various methods and databases.     

Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA

Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the construction of partially complementary 'side'-labeled probes, based on secondary structure models of the rRNA sequences. The specificity and routine applicability of the FISH-based method for yeast identification were tested by analyzing different wine isolates. Investigation of the prevalence of Dekkera/Brettanomyces yeasts in the German viticultural regions Wonnegau, Nierstein and Bingen (Rhinehesse, Rhineland-Palatinate) resulted in the isolation of 37 D. bruxellensis strains from 291 wine samples.