Intergenic Transcribed Spacer Research Articles

Detection and Genetic Diversity of Bartonella vinsonii Subsp. berkhoffii Strains Isolated from Dogs in Ankara, Turkey

Bartonella vinsonii subsp. berkhoffii has been identified as an important pathogen in dogs and is an emerging pathogen in people with zoonotic potential. This study aimed to isolate Bartonella spp. in 250 blood samples collected from dogs in the province of Ankara, Turkey, between October 2006 and March 2007. The typing of the 23 isolates was carried out by PCR for citrate synthase (gltA) and the 16S-23S intergenic transcribed spacer (ITS). The prevalence of bacteremia was 9.2% in 250 samples. Among 170 shelter dogs, 21 (12.4%) were bacteremic for B. vinsonii subsp. berkhoffii, while the B. vinsonii subsp. berkhoffii bacteremia rate was 5% (2/40) for the stray dogs. B. vinsonii subsp. berkhoffii was not isolated from the pet dogs. The prevalence of bacteremia was found to be 25.5% (13/51) in shelter dogs aged less than one year old. All of the isolates were identified as B.vinsonii subsp. berkhoffii genotype III. In some isolates, MseI digest for gltA was found to be different from the American and European strains due to a single nucleotide change.

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Multiple scales of diversification within natural populations of archaea in hydrothermal chimney biofilms

Corroborative data collected from 16S rRNA clone libraries, intergenic transcribed spacer (ITS) region clone libraries, and 16S rRNA hypervariable region tag pyrosequencing demonstrate microdiversity within single-species archaeal biofilms of the Lost City Hydrothermal Field. Both 16S rRNA clone libraries and pyrosequencing of the V6 hypervariable region show that Lost City Methanosarcinales (LCMS) biofilms are dominated by a single sequence, but the pyrosequencing data set also reveals the presence of an additional 1654 rare sequences. Clone libraries constructed with DNA spanning the V6 hypervariable region and ITS show that multiple ITS sequences are associated with the same dominant V6 sequence. Furthermore, ITS variability differed among three chimney samples, and the sample with the highest ITS diversity also contained the highest V6 diversity as measured by clone libraries as well as tag pyrosequencing. These results indicate that the extensive microdiversity detected in V6 tag sequences is an underestimate of genetic diversity within the archaeal biofilms.

Genotypic diversity of lactic acid bacteria isolated from African traditional alkaline-fermented foods

To identify and compare lactic acid bacteria (LAB) isolated from alkaline fermentations of cassava (Manihot esculenta Crantz) leaves, roselle (Hibiscus sabdariffa) and African locust bean (Parkia biglobosa) seeds for production of, respectively, Ntoba Mbodi, Bikalga and Soumbala. A total of 121 LAB were isolated, identified and compared by phenotyping and genotyping using PCR amplification of 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The results revealed a diversity of genera, species and subspecies of LAB in African alkaline fermentations. The isolates were characterized as nonmotile (in most cases) Gram-positive rods, cocci or coccobacilli, catalase and oxidase negative. ITS-PCR allowed typing mainly at species level, with differentiation of a few bacteria at subspecies level. Rep-PCR permitted typing at subspecies level and revealed significant genotypic differences between the same species of bacteria from different raw materials. DNA sequencing combined with use of API 50CHL and API 20Strep systems allowed identification of bacteria as Weissella confusa, Weissella cibaria, Lactobacillus plantarum, Pediococcus pentosaceus, Enterococcus casseliflavus, Enterococcus faecium, Enterococcus faecalis, Enterococcus avium and Enterococcus hirae from Ntoba Mbodi; Ent. faecium, Ent. hirae and Pediococcus acidilactici from Bikalga and Soumbala. LAB found in African alkaline-fermented foods belong to a range of genera, species and subspecies of bacteria and vary considerably according to raw material. Our study confirms that LAB survive in alkaline fermentations, a first crucial stage in determining their significance and possible value as probiotic bacteria.

Phylogeny and taxonomy of a diverse collection of Bradyrhizobium strains based on multilocus sequence analysis of the 16S rRNA gene, ITS region and glnII, recA, atpD and dnaK genes

The genus Bradyrhizobium encompasses a variety of bacteria that can live in symbiotic and endophytic associations with legumes and non-legumes, and are characterized by physiological and symbiotic versatility and broad geographical distribution. However, despite indications of great genetic variability within the genus, only eight species have been described, mainly because of the highly conserved nature of the 16S rRNA gene. In this study, 169 strains isolated from 43 different legumes were analysed by rep-PCR with the BOX primer, by sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic transcribed spacer (ITS) and by multilocus sequence analysis (MLSA) of four housekeeping genes, glnII, recA, atpD and dnaK. Considering a cut-off at a level of 70 % similarity, 80 rep-PCR profiles were distinguished, which, together with type strains, were clustered at a very low level of similarity (24 %). In both single and concatenated analyses of the 16S rRNA gene and ITS sequences, two large groups were formed, with bootstrap support of 99 % in the concatenated analysis. The first group included the type and/or reference strains of Bradyrhizobium japonicum, B. betae, B. liaoningense, B. canariense and B. yuanmingense and B. japonicum USDA 110, and the second group included strains related to Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T) and B. jicamae PAC68(T). Similar results were obtained with MLSA of glnII, recA, atpD and dnaK. Greatest variability was observed when the atpD gene was amplified, and five strains related to B. elkanii revealed a level of variability never reported before. Another important observation was that a group composed of strains USDA 110, SEMIA 5080 and SEMIA 6059, all isolated from soybean, clustered in all six trees with high bootstrap support and were quite distinct from the clusters that included B. japonicum USDA 6(T). The results confirm that MLSA is a rapid and reliable way of providing information on phylogenetic relationships and of identifying rhizobial strains potentially representative of novel species.

Genotyping and Antifungal Drug Susceptibility of the Pathogenic Yeast Trichosporon asahii Isolated from Thai Patients

Trichosporonosis due to Trichosporon asahii is a life-threatening infection with a very poor prognosis. We analyzed the genotype of intergenic transcribed spacer (IGS) region 1 of the rRNA gene and determined the drug susceptibility of 101 T. asahii isolates obtained from Thai patients to collect basic information on trichosporonosis in Thailand. Of the five genotypes in the IGS region identified in this study, types 1 and 3 were predominant in Thailand. The distribution in Thailand differs from that in other countries, suggesting that there is a geographic substructure among T. asahii clinical isolates. Voriconazole appeared to be the most active drug.

Genetic characterization of Anisakis typica and Anisakis physeteris from marine mammals and fish from the Atlantic Ocean off Brazil

Until now, Anisakis typica has been the sole anisakid identified by means of genetic markers from the Atlantic Ocean off Brazil. In this study we developed, and applied to larvae and adults, an A. typica-specific molecular identification protocol based on the complete intergenic transcribed spacer (ITS) region. Anisakids from the dolphin Sotalia guianensis and from the frigate tuna, Auxis thazard, caught off the coast of Brazil, were processed using two DNA extraction methods. Molecular diagnosis identified A. typica in 13 of 15 samples. Complete ITS analysis showed that the remaining two isolates were in fact A. physeteris. Maximum parsimony analysis of complete ITS region (915 bp) confirmed these results. Our study verified the dominance of A. typica infecting hosts from the Brazilian coast and showed, for the first time, the presence of A. physeteris in the intermediate/paratenic host A. thazard in this region.

Investigating the relationship between raw milk bacterial composition, as described by intergenic transcribed spacer-PCR fingerprinting, and pasture altitude

To assess the bacterial biodiversity level in bovine raw milk used to produce Fontina, a Protected Designation of Origin cheese manufactured at high-altitude pastures and in valleys of Valle d'Aosta region (North-western Italian Alps) without any starters. To study the relation between microbial composition and pasture altitude, in order to distinguish high-altitude milk against valley and lowland milk. The microflora from milks sampled at different alpine pasture, valley and lowland farms were fingerprinted by PCR of the 16S-23S intergenic transcribed spacers (ITS-PCR). The resulting band patterns were analysed by generalized multivariate statistical techniques to handle discrete (band presence-absence) and continuous (altitude) information. The fingerprints featured numerous bands and marked variability indicating complex, differentiated bacterial communities. Alpine pasture milks were distinguished from lowland ones by cluster analysis, while this technique less clearly discriminated alpine pasture and valley samples. Generalized principal component analysis and clustering-after-ordination enabled a more effective distinction of alpine pasture, valley and lowland samples. Alpine raw milks for Fontina production contain highly diverse bacterial communities, the composition of which is related to the altitude of the pasture where milk was produced. This research may provide analytical support to the important issue represented by the authentication of the geographical origin of alpine milk productions.

Sequestering from host and characterization of sequence of a ribosomal RNA operon ( rrn) from “ Candidatus Liberibacter asiaticus”

A 5005 bp rrn DNA sequence of “ Candidatus Liberibacter asiaticus” was obtained by PCR using primers conserved to Rhizobiaceae of alpha-proteobacteria. The rrn locus consisted of a 16S rRNA gene ( rrs), an intergenic transcribed spacer (ITS) containing two genes of tRNA Ile and tRNA Ala, a 23S rRNA gene ( rrl), an ITS without tRNA gene and a 5S rRNA gene ( rrf). Like rrs, rrl and rrf were also related (89–90%) to those of the members of Rhizobiaceae. Interestingly, the genes of tRNA Ile and tRNA Ala were more related to those of non- Rhizobiaceae alpha-proteobacteria. The non-tRNA gene regions of the 16S-23S ITS and 23S-5S rRNA ITS did not share significant similarity to any known non-Liberibacter DNA sequences, and could be used for specific and efficient detection of “ Ca. L. asiaticus”.

Diversity of auxin‐producing bacteria associated to Pseudomonas savastanoi ‐induced olive knots

Forty three strains were isolated from knots induced by Pseudomonas savastanoi in different olive cultivars. All the selected bacteria were shown to produce variable amounts of the plant growth hormone indole-3-acetic acid (IAA). Amplification of the intergenic transcribed spacers (ITS) between 16S and 23S rDNA genes, allowed the clustering of the isolates into seven distinct groups. All isolates from ITS group 1 were positive to the Pseudomonas savastanoi pv. savastanoi specific iaa L gene as shown by PCR. Partial sequencing of 16S rDNA gene confirmed the identity of these isolates to Pseudomonas savastanoi strains and allowed to tentatively assign the other isolates from the remaining ITS groups to Pantoea oleae/agglomerans, Burkholderia cepacia, Pseudomonas putida, Stenotrophomonas maltophilia and Hafnia alvei. Identification of endophytic knot-derived isolates revealed association of various saprophytic and putative human pathogenic bacteria with P. savastanoi pv. savastanoi in knot environment of olive infected trees.

DNA fingerprinting of Paenibacillus popilliae and Paenibacillus lentimorbus using PCR-amplified 16S–23S rDNA intergenic transcribed spacer (ITS) regions

Failure to identify correctly the milky disease bacteria, Paenibacillus popilliae and Paenibacillus lentimorbus, has resulted in published research errors and commercial production problems. A DNA fingerprinting procedure, using PCR amplification of the 16S–23S rDNA intergenic transcribed spacer (ITS) regions, has been shown to easily and accurately identify isolates of milky disease bacteria. Using 34 P. popilliae and 15 P. lentimorbus strains, PCR amplification of different ITS regions produced three DNA fingerprints. For P. lentimorbus phylogenic group 2 strains and for all P. popilliae strains tested, electrophoresis of amplified DNA produced a migratory pattern (i.e., ITS-PCR fingerprint) exhibiting three DNA bands. P. lentimorbus group 1 strains also produced this ITS-PCR fingerprint. However, the fingerprint was phase-shifted toward larger DNA sizes. Alignment of the respective P. popilliae and P. lentimorbus group 1 ITS DNA sequences showed extensive homology, except for a 108 bp insert in all P. lentimorbus ITS regions. This insert occurred at the same location relative to the 23S rDNA and accounted for the phase-shift difference in P. lentimorbus group 1 DNA fingerprints. At present, there is no explanation for this 108 bp insert. The third ITS-PCR fingerprint, produced by P. lentimorbus group 3 strains, exhibited approximately eight DNA bands. Comparison of the three fingerprints of milky disease bacteria to the ITS-PCR fingerprints of other Paenibacillus species demonstrated uniqueness. ITS-PCR fingerprinting successfully identified eight unknown isolates as milky disease bacteria. Therefore, this procedure can serve as a standard protocol to identify P. popilliae and P. lentimorbus.

Use of the 16S–23S ribosomal genes spacer region for the molecular typing of sphingomonads

The ability of sphingomonads in drinking water to cause community- and hospital-acquired opportunistic infections has raised the need to establish reproducible identification assays. In this study, a total of 129 isolates recovered from drinking water with yellow- to orange-pigmented colonies were distributed among 10 biotypes on the basis of colony morphology. Polymorphisms, based on the amplification and restriction digestion of the intergenic transcribed spacer (ITS) region within the 10 assigned biotypes and 18 ATCC reference strains, were used to investigate the ability of this approach to differentiate closely related sphingomonads. ITS size, which ranged between 400 and 1100 bp, did not vary enough among the different genera. However, 16 distinct banding patterns within the ATCC reference strains and 9 within the 10 biotypes were obtained through ITS restriction digestion, and the majority of the tested biotypes produced patterns similar to those generated by the ATCC strains. To our knowledge, this study is not only the first comprehensive record of the size of the ITS region in sphingomonads, it is also the first study that describes the use of ITS restriction digestion to subtype those isolates.

Population of Aphanizomenon from the Gulf of Gdańsk (Southern Baltic Sea): differences in phenotypic and genotypic characteristics

In this study, one of the major bloom-forming cyanobacteria in the Gulf of Gdansk (Southern Baltic Sea), Aphanizomenon Morren ex Bornet et Flahaut has been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology as well as ultrastructure. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). We have found the Aphanizomenon population from the Gulf of Gdansk to be significantly different in ultrastructure, morphology from freshwater A. flos-aquae and according to the traditional approach; it could be assigned to different taxonomic units. However, genetic relationship with regard to sequences of the 16S rRNA gene, showed an high overall sequence identity (97.5–99%) to freshwater isolates. Similarly, ITS sequence identity among populations from the Baltic Sea and different freshwater isolates was as high as 90.3–97.7% suggesting one and the same species.

Exploration of methods used to describe bacterial communities in silage of maize (Zea mays) cultivars

Different techniques to assess bacterial community structure and diversity were evaluated in silages prepared with four different maize cultivars, three conventional and one transgenic (cv. Tundra, event Bt-176). Plants were cultivated in the greenhouse and harvested after 30 days of growth. Silage samples were collected at successive times during fermentation and analyzed for bacterial counts and by various DNA-based fingerprinting techniques. Bacterial counts were similar between cultivars for the total culturable bacteria, sporeforming, and mesophilic and thermophilic lactic acid bacteria (LAB). Further analysis of the species composition of 388 LAB strains by intergenic transcribed spacer (ITS) PCR followed by sequencing of 16S rRNA gene did not reveal differences between cultivars. In contrast, molecular fingerprinting methods targeting whole bacterial communities, such as automated ribosomal intergenic spacers analysis (ARISA) and 16S rRNA gene length heterogeneity-PCR (LH-PCR), indicated that different maize silage batches or cultivars hosted different bacterial communities. Thus, ARISA and LH-PCR fingerprinting techniques offer a fast and sensitive method to compare bacterial communities, and to detect differences in silage bacterial communities.

Biochemical and genetic diversity of pectolytic enterobacteria causing soft rot disease of potatoes in New Zealand

The diversity of 89 pectolytic enterobacteria collected from potatoes in New Zealand was investigated by comparing their physiological, biochemical and genetic characteristics with those of type strains of Pectobacterium and strains previously identified as isolates of the genus Dickeya. Biochemical and physiological analyses demonstrated that the predominant pectolytic enterobacterium present in New Zealand is Pectobacterium carotovorum subspecies carotovorum. Isolates of Pectobacterium atrosepticum and strains closely related to Dickeya were also detected. Species and subspeciesspecific PCR confirmed the identity of the majority of the P. carotovorum subsp. carotovorum and P. atrosepticum strains. However, several atypical isolates classified by Biolog as P. carotovorum subsp. carotovorum failed to grow at 37C or to amplify the pectate lyase-encoding gene (pel gene) characteristic of this subspecies. Restriction fragment length polymorphism (RFLP) analyses of the 16S–23S intergenic transcribed spacer (ITS) also identified atypical isolates with unique ITS-RFLP profiles. Further characterisation of the atypical strains, by phylogenetic analysis of their 16S rRNA sequences, clustered them into four groups distinct from pectolytic enterobacteria previously found on potato. Several of the atypical strains proved highly virulent on both tubers and stems of potato, suggesting that a variety of novel species or subspecies are responsible for soft rot of potatoes in New Zealand, including Pectobacterium wasabiae.

Characterization of true‐branching cyanobacteria from geothermal sites and hot springs of Costa Rica

Costa Rica is at the centre of the Mesoamerican biodiversity hotspot. Little is known about cyanobacteria from this region so far. Here, four isolates of the order Stigonematales (section V) were characterized in a polyphasic approach. All strains were isolated from geothermal sites and hot springs of Costa Rica. However, one of them, identified as Westiellopsis sp. Ar73, did not grow at more than 40 degrees C. Based on its identical 16S rRNA to several previously isolated Westiellopsis sp. and Fischerella muscicola strains, a ubiquitous distribution throughout tropical and subtropical regions can be implied. In contrast, the isolates MV9, MV11 and RV14 grew well up to 50-55 degrees C. Based on morphologic, ultrastructural, molecular and physiologic data, MV9, MV11 and RV14 were identified to belong to the genus Fischerella. Two distinct intergenic transcribed spacer (ITS) types, with or without tRNA genes, were detected for all Stigonematales analysed here, indicating ITS polymorphism as a characteristic feature of heterocystous cyanobacteria. In phylogenetic trees, these Fischerella spp. formed a new and distinct clade within the wider lineage of thermophilic Fischerella (Mastigocladus cf. laminosus), which might represent a geographic lineage. Thus, geographic isolation may be an underestimated aspect of microbial evolution. The strains presented here are suitable as new models to study this group of cyanobacteria.

Development and field assessment of a quantitative PCR for the detection and enumeration of the noxious bloom‐former Anabaena planktonica

Anabaena planktonica is a harmful, bloom‐forming freshwater cyanobacterium, which has arrived recently in New Zealand. In the short time since its incursion (<10 yr), A. planktonica has spread rapidly throughout lakes in the North Island. To date, the identification and enumeration of A. planktonica has been undertaken using light microscopy. There is an urgent demand for a highly sensitive and specific quantitative detection method that can be combined with a high sample processing capability in order to increase sampling frequency. In this study, we sequenced 36 cyanobacterial 16S rRNA genes (partial), complete intergenic transcribed spacers (ITS), and 23S rRNA genes (partial) of fresh‐water cyanobacteria found in New Zealand. The sequences were used to develop an A. planktonica specific TaqMan QPCR assay targeting the long ITS1‐L and the 5′ terminus of the 23S rRNA gene. The QPCR method was linear (R2 = 0.999) over seven orders of magnitude with a lower end sensitivity of approximately five A. planktonica cells in the presence of exogenous DNA. The quantitative PCR (QPCR) method was used to assess the spatial distribution and seasonal population dynamics of A. planktonica from the Lower Karori Reservoir (Wellington, New Zealand) over a five‐month period. The QPCR results were compared directly to microscopic cell counts and found to correlate significantly (95% confidence level) under both bloom and non‐bloom conditions. The current QPCR assay will be an invaluable tool for routine monitoring programs and in research investigating environmental factors that regulate the population dynamics and the blooming of A. planktonica.

Genotypic and phenotypic analysis of strains assigned to the widespread cyanobacterial morphospecies Phormidium autumnale (Oscillatoriales)

In this study, ten cyanobacterial strains assigned to the oscillatorian species Phormidium autumnale have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, and pigment content. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The strains were quite homogenous in their morphologic features. Their thylakoids showed a stacked or fascicular pattern. Some, but not all strains contained phycoerythrin. Only one strain (P. autumnale UTCC 476) deviated significantly in its phenotype by lacking a calyptra. In neighbour-joining and maximum Parsimony trees most 16S rRNA sequences were located on a single well-defined branch, which, however, also harboured sequences assigned to other cyanobacterial genera. Two strains (P. autumnale UTCC 476 and P. autumnale UTEX 1580) were found on distant branches. The presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. Our results reconfirm that the morphospecies P. autumnale and the Phormidium group in general are not phylogenetically coherent and require revision. However, as indicated by sequence similarities most of the strains assigned to P. autumnale except P. autumnale UTCC 476 and P. autumnale UTEX 1580 are phylogenetically related and might belong to a single genus.

Diversity and detection of Korean Erwinia pyrifoliae strains as determined by plasmid profiling, phylogenetic analysis and PCR

Twenty‐five strains of Erwinia pyrifoliae were investigated for their plasmid profiles and genetic relatedness. Four types of plasmid profile were observed for the first time, suggesting intraspecific plasmid profile diversity in E. pyrifoliae. Moreover, BOX‐PCR and phylogenetic analysis based on the 16S‐23S intergenic transcribed spacer (ITS) region showed genetic variations among E. pyrifoliae strains, although all strains were clustered in one group and separated from E. amylovora. On the other hand, ERIC‐PCR and phylogenetic analysis based on partial groEL gene sequences revealed close genetic relatedness among the strains. Amplification with EpSPF and EpSPR primers of a fragment of approximately 0·65 kb from the genomic DNA of all E. pyrifoliae strains, but not from E. amylovora strains, suggested that this primer set is useful for identification of this pathogen.

Identification of Bacillus spp. from Bikalga, fermented seeds of Hibiscus sabdariffa: phenotypic and genotypic characterization

To identify Bacillus spp. responsible of the fermentation of Hibiscus sabdariffa for production of Bikalga, an alkaline fermented food used as a condiment in Burkina Faso. Seventy bacteria were isolated from Bikalga produced in different regions of Burkina Faso and identified by phenotyping and genotyping using PCR amplification of the 16S-23S rDNA intergenic transcribed spacer (ITS-PCR), repetitive sequence-based PCR (rep-PCR) and DNA sequencing. The isolates were characterized as motile, rod-shaped, endospore forming, catalase positive, Gram-positive bacteria. ITS-PCR allowed typing mainly at species level. Rep-PCR was more discriminative and allowed a typing at ssp. level. The DNA sequencing combined with the Blast search program and fermentation profiles using API 50CHB system allowed an identification of the bacteria as Bacillus subtilis, B. licheniformis, B. cereus, B. pumilus, B. badius, Brevibacillus bortelensis, B. sphaericus and B. fusiformis. B. subtilis were the predominant bacterium (42) followed by B. licheniformis (16). Various species and ssp. of Bacillus are involved in fermentation of H. sabdariffa for production of Bikalga. Selection of starter cultures of Bacillus for controlled production of Bikalga, selection of probiotic bacteria.