Molecular diversity of Bacteroides spp. in human fecal microbiota as determined by group-specific 16S rRNA gene clone library analysis

Bacteroides spp. represent a prominent bacterial group in human intestinal microbiota with roles in symbiosis and pathogenicity; however, the detailed composition of this group in human feces has yet to be comprehensively characterized. In this study, the molecular diversity of Bacteroides spp. in human fecal microbiota was analyzed from a seven-member, four-generation Chinese family using Bacteroides spp. group-specific 16S rRNA gene clone library analysis. A total of 549 partial 16S rRNA sequences amplified by Bacteroides spp.-specific primers were classified into 52 operational taxonomic units (OTUs) with a 99% sequence identity cut-off. Twenty-three OTUs, representing 83% of all clones, were related to 11 validly described Bacteroides species, dominated by Bacteroides coprocola, B. uniformis, and B. vulgatus. Most of the OTUs did not correspond to known species and represented hitherto uncharacterized bacteria. Relative to 16S rRNA gene universal libraries, the diversity of Bacteroides spp. detected by the group-specific libraries was much higher than previously described. Remarkable inter-individual differences were also observed in the composition of Bacteroides spp. in this family cohort. The comprehensive observation of molecular diversity of Bacteroides spp. provides new insights into potential contributions of various species in this group to human health and disease.     

Rapid identification of nitrogen-fixing and legume-nodulating Burkholderia species based on PCR 16S rRNA species-specific oligonucleotides

Several novel N₂-fixing Burkholderia species associated with plants, including legume-nodulating species, have recently been discovered. Presently, considerable interest exists in studying the diazotrophic Burkholderia species, both for their ecology and their great potential for agro-biotechnological applications. However, the available methods used in the identification of these Burkholderia species are time-consuming and expensive. In this study, PCR species-specific primers based on the 16S rRNA gene were designed, which allowed rapid, easy, and correct identification of most known N₂-fixing Burkholderia. With this approach, type and reference strains of Burkholderia kururiensis, B. unamae, B. xenovorans, B. tropica, and B. silvatlantica, as well as the legume-nodulating B. phymatum, B. tuberum, B. mimosarum, and B. nodosa, were unambiguously identified. In addition, the PCR species-specific primers allowed the diversity of the diazotrophic Burkholderia associated with field-grown tomato and sorghum plants to be determined. B. tropica and B. xenovorans were the predominant species found in association with tomato, but the occurrence of B. tropica with sorghum plants was practically exclusive. The efficiency of the species-specific primers was validated with the detection of B. tropica and B. xenovorans from DNA directly recovered from tomato rhizosphere soil samples. Additionally, using PCR species-specific primers, all of the legume-nodulating Burkholderia were correctly identified, even from single nodules collected from inoculated common bean plants. These primers could contribute to rapid identification of the diazotrophic and nodulating Burkholderia species associated with important crop plants and legumes, as well as revealing their environmental distribution.     

Quantitative real-time PCR (qPCR) detection chemistries affect enumeration of the Dehalococcoides 16S rRNA gene in groundwater

Quantitative real-time PCR (qPCR) commonly uses the fluorogenic 5′ nuclease (TaqMan) and SYBR Green I (SG) detection chemistries to enumerate biomarker genes. Dehalococcoides (Dhc) are keystone bacteria for the detoxification of chlorinated ethenes, and the Dhc 16S ribosomal RNA (rRNA) gene serves as a biomarker for monitoring reductive dechlorination in contaminated aquifers. qPCR enumeration of Dhc biomarker genes using the TaqMan or SG approach with the same primer set yielded linear calibration curves over a seven orders of magnitude range with similar amplification efficiencies. The TaqMan assay discriminates specific from nonspecific amplification observed at low template concentrations with the SG assay, and had a 10-fold lower limit of detection of ~3 copies per assay. When applied to Dhc pure cultures and Dhc-containing consortia, both detection methods enumerated Dhc biomarker genes with differences not exceeding 3-fold. Greater variability was observed with groundwater samples, and the SG chemistry produced false-positive results or yielded up to 6-fold higher biomarker gene abundances compared to the TaqMan method. In most cases, the apparent error associated with SG detection resulted from quantification of nonspecific amplification products and was more pronounced with groundwater samples that had low biomarker concentrations or contained PCR inhibitors. Correction of the apparent error using post-amplification melting curve analysis produced 2 to 21-fold lower abundance estimates; however, gel electrophoretic analysis of amplicons demonstrated that melting curve analysis was insufficient to recognize all nonspecific amplification. Upon exclusion of nonspecific amplification products identified by combined melting curve and electrophoretic amplicon analyses, the SG method produced false-negative results compared to the TaqMan method. To achieve sensitive and accurate quantification of Dhc biomarker genes in environmental samples (e.g., groundwater) and avoid erroneous conclusions, the analysis should rely on TaqMan detection chemistry, unless additional analyses validate the results obtained with the SG approach.     

The inconstant gut microbiota of Drosophila species revealed by 16S rRNA gene analysis

The gut microorganisms in some animals are reported to include a core microbiota of consistently associated bacteria that is ecologically distinctive and may have coevolved with the host. The core microbiota is promoted by positive interactions among bacteria, favoring shared persistence; its retention over evolutionary timescales is evident as congruence between host phylogeny and bacterial community composition. This study applied multiple analyses to investigate variation in the composition of gut microbiota in drosophilid flies. First, the prevalence of five previously described gut bacteria (Acetobacter and Lactobacillus species) in individual flies of 21 strains (10 Drosophila species) were determined. Most bacteria were not present in all individuals of most strains, and bacterial species pairs co-occurred in individual flies less frequently than predicted by chance, contrary to expectations of a core microbiota. A complementary pyrosequencing analysis of 16S rRNA gene amplicons from the gut microbiota of 11 Drosophila species identified 209 bacterial operational taxonomic units (OTUs), with near-saturating sampling of sequences, but none of the OTUs was common to all host species. Furthermore, in both of two independent sets of Drosophila species, the gut bacterial community composition was not congruent with host phylogeny. The final analysis identified no common OTUs across three wild and four laboratory samples of D. melanogaster. Our results yielded no consistent evidence for a core microbiota in Drosophila. We conclude that the taxonomic composition of gut microbiota varies widely within and among Drosophila populations and species. This is reminiscent of the patterns of bacterial composition in guts of some other animals, including humans.     

Comparison of bacteroides-prevotella 16S rRNA genetic markers for fecal samples from different animal species

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.     

Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils

The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups.     

Isolation of endophytic bacteria from arboreal species of the Amazon and identification by sequencing of the 16S rRNA encoding gene

Endophytic bacteria from three arboreal species native to the Amazon (Carapa guianenses, Ceiba pentandra, and Swietenia macrophylla), were isolated and identified, through partial sequencing of the 16S rRNA encoding gene. From these, 16 isolates were obtained, although, when compared to sequences deposited in GenBank, only seven had produced identifiable fragments. Bacillus, Pantoea and two non-culturable samples were identified. Results obtained through sequence analysis revealed low genetic diversity across the isolates, even when analyzing different species and plant structures. This is the first report concerning the isolation and identification of endophytic bacteria in these plant species.     

Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Evaluation of selenite bioremoval from liquid culture by Enterococcus species

The genus Enterococcus belong to the genera of bacteria that produce lactic acid and can confer health benefits to living organisms. Selenium (Se) is an essential micronutrient for humans and animals. Thirty-six Enterococcus species isolated from dairy products were screened for Se(IV) sorption capacity for use as a probiotics in animal nutrition. Several isolates grew luxuriantly and significantly removed Se(IV) from Se(IV) amended medium. Two isolates, LAB 14 and LAB 18, identified by 16S rRNA gene sequence analysis as Enterococcus faecalis (98% nucleotide sequence similarity) and Enterococcus faecium (97% nucleotide sequence similarity), respectively, were selected for further studies. The two isolates grew optimally and removed selenium at initial pH 7.0. Optimum removal of Se(IV) from the medium was recorded at 25 °C. Time course studies showed that after 8 h of incubation LAB 14 and LAB 18 cultures displayed the highest biomass production and Se(IV) bioremoval and most selenite in culture depleted in 24 h. At initial concentrations of 10 mg L⁻¹ and 60 mg L⁻¹, E. faecium (LAB 18) removed 9.91 mg L⁻¹ and 59.70 mg L⁻¹, respectively after 24 h. Similar Se(IV) bioremoval capacity was recorded with E. faecalis (LAB 14). Substantial amount of Se was detected in biomass of E. faecium (0.4599 mg g⁻¹ of dry weight) and E. faecalis (0.4759 mg g⁻¹ of dry weight). The significant uptake and transformation of Se(IV) by the Enterococcus species observed in this study suggest that they can be used to deliver dietary Se through feed augmentation with Se(IV)-enriched Enterococcus biomass.     

Identification of Mediterranean Diplodus spp. and Dentex dentex (Sparidae) by means of DNA Inter-Simple Sequence Repeat (ISSR) markers

The aim of this paper was to test the effectiveness of Inter-Simple Sequence Repeat (ISSR) technique in detecting specific molecular markers to genetically discriminate some species of teleost fish. As model species, we used four Mediterranean sea bream species (i.e. Diplodus annularis, D. puntazzo, D. sargus sargus, and D. vulgaris), and the common Mediterranean dentex Dentex dentex. Furthermore, the occurrence in our samples of two partially albino sparids (suspected, on a morphological basis, to probably belong to D. sargus sargus and to D. dentex, respectively) allowed ISSRs to be tested on doubtful specimens. Eight ISSR primers produced 95 out of 97 scorable polymorphic loci with a large number (63) of species-private bands. Maximum parsimony analysis, ΔK statistics, and assignment test were consistent in separating the five species and solving the taxonomic position of specimens of dubious attribution. The results obtained evidence the usefulness of ISSR markers in specimen and species identification, particularly when the morphological traits of fish make the classification problematic.     

Chemotaxonomy of Pseudowintera: Sesquiterpene dialdehyde variants are species markers

Two sesquiterpene dialdehydes, the 1β-E-coumaroyl-5α-hydroxypolygodial plus the known 1β-E-cinnamoyl-5α-hydroxypolygodial, were isolated from the recently described species Pseudowintera insperata. This discovery is a further example of the rare sesquiterpene dialdehyde coumarate/cinnamate combination being found exclusively in the family Winteraceae. Another sesquiterpene dialdehyde, isopaxidal, with the rare rearranged drimane skeleton, was isolated from Pseudowintera axillaris. The sesquiterpene dialdehyde contents of leaves of 25 individual plants of the four Pseudowintera species, all endemic to New Zealand, were measured by HPLC. P. insperata individuals all had high levels (3.0-6.9% of leaf dry wt.) of the coumarate, P. axillaris had high levels (2.2-6.9%) of paxidal, and Pseudowintera colorata from different areas of New Zealand contained varying levels of polygodial (1.4-2.9%) and 9-deoxymuzigadial (0-2.9%). Therefore the sesquiterpene dialdehydes are good species markers.     

Species delimitation in the European species of Clavulina (Cantharellales, Basidiomycota) inferred from phylogenetic analyses of ITS region and morphological data

The identification of the conventionally accepted species of Clavulina (Cantharellales, Basidiomycota) in Europe (Clavulina amethystina, Clavulina cinerea, Clavulina cristata, and Clavulina rugosa) is often difficult and many specimens are not straightforwardly assignable to any of those four species, which is why some authors have questioned their identity. In order to assess the status of those species, a morphological examination was combined with the molecular analysis of the ITS region. The same six major clades were obtained in the Bayesian and parsimony phylogenetic analyses, and all six clades were well-supported at least by one of the analyses. Morphological characters, such as the overall branching pattern, the presence and intensity of grey colour, the cristation of the apices, and basidiospore size and shape were to various extents correlated with the phylogenetic signal obtained from the ITS region. The congruence between the molecular analyses and morphology, rather than geographical origin, suggests the existence of several species that can be delimited using a combined phylogenetic and morphological species recognition. The analyses revealed that C. cristata and C. rugosa are well-delimited species. In contrast, more than one taxa could be subsumed under the names C. amethystina and C. cinerea, the taxonomical complexity of which is discussed. The ITS region is proved to be adequate to separate phylogenetic species of Clavulina.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR

A real time quantitative PCR combined with propidium monoazide (PMA) treatment of samples was implemented to quantify live C. jejuni, C. coli and C. lari on broiler chicken carcasses at selected processing steps in the slaughterhouse. The samples were enumerated by culture for comparison. The Campylobacter counts determined with the PMA-qPCR and the culture method were not concordant. We conclude that the qPCR combined with PMA treatment of the samples did not fully reduce the signal from dead cells.     

Generic allocation of Indian and Sri Lankan Philautus (Anura: Rhacophoridae) inferred from 12S and 16S rRNA genes

The generic allocation of Indian and Sri Lankan Philautus needs further examination. In this study, a comprehensive understanding of the phylogeny of Indian and Sri Lankan Philautus is obtained based on 12S and 16S rRNA genes. All phylogenetic analyses indicate that Indian-Sri Lankan Philautus, Philautus menglaensis, Philautus longchuanensis, and Philautus gryllus form a well supported clade, separate from Philautus of Sunda Islands that form another well supported clade representing true Philautus. This result supports the designation of the genus Pseudophilautus to accommodate the Indian and Sri Lankan species. Pseudophilautus consists of two major lineages, one comprises the majority of Indian species, Chinese species, and Southeast Asian species, and one comprises all Sri Lankan species and a few Indian species. Pseudophilautus may have originated in South Asia and dispersed into Southeast Asia and China. Based on the results, we further suggest that Philautus cf. gryllus (MNHN1997.5460) belongs to the genus Kurixalus.     

Fingerprinting of freshly separated and cultured cyanobionts from different Azolla species using morphological and molecular markers

Differentiation of freshly separated (uncultured) and cultured cyanobionts of Azolla species was carried out employing morphological markers and profiles generated using SDS-PAGE and PCR–RFLP of 16S rRNA. The cell dimensions of vegetative cells, heterocysts and heterocyst frequency (25–28%) of the freshly separated cyanobionts were distinctly higher than that those recorded for the cultured cyanobionts (7–10%). The SDS-PAGE profiles of whole cell proteins of cultured cyanobionts (comprising 28–30 bands) and those of freshly separated cyanobionts (consisting of 6–10 bands) exhibited distinct differences and unique bands. AluI was able to discriminate freshly separated cyanobionts from cultured cyanobionts of same species of Azolla. The profiles of cyanobionts (freshly separated and cultured) of A. rubra (RU6503) generated using the restriction enzyme AluI were distinctly different from other cyanobionts and unique bands were observed in both cyanobionts. The cultured cyanobionts from A. microphylla (MI4018), A. filiculoides (FI1001) and A. pinnata (PP7001) showed the presence of a distinct band of 450, 622 and 307 bp, respectively. Three common bands of 500, 400 and 275 bp were recorded in the AluI restriction profiles of all the freshly separated cyanobionts. 16S rRNA PCR–RFLP analyses confirmed the existence of primary and secondary cyanobionts in the leaf cavities of different Azolla species. These techniques can be utilized for discriminating between freshly separated and cultured cyanobionts of Azolla and provide reliable fingerprints for these strains.     

A new species of ferret-badger, Genus Melogale, from Vietnam

Ferret-badgers, genus Melogale, are distributed in the Indochinese region, Java, Bali and NE-Borneo. There are currently four species described each having very similar phenotypes. In March 2005, a living ferret-badger of a different phenotype was confiscated by rangers from Cuc Phuong National Park, Vietnam. This individual died and the carcass was not preserved. In January 2006, a newly deceased individual of the same phenotype was found at the Endangered Primate Rescue Center, Cuc Phuong National Park. Due to several different characteristics these individuals vary greatly from the current species. Thus, we describe an additional species, M. cucphuongensis sp. nov. from northern Vietnam, which occurs sympatrically with M. moschata and M. personata, but differs from both species clearly in skull morphology and other features.Based on a 423 bp-long fragment of the mitochondrial cytochrome b gene, M. cucphuongensis sp. nov. is a member of the genus Melogale and represents a sister lineage to a clade consisting of M. personata and M. moschata.     

Evaluation of real-time PCR targeting hbb gene for Borrelia species identification

Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 °C), B. burgdorferi sensu stricto (Tm 68.18 °C), Borreliaspielmanii (Tm 59.45 °C) and Borreliavalaisiana (Tm 59.62 °C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 °C, while subtype Mlg4 was characterized by Tm 56.87 °C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 °C while two tick isolates shared Tm 61.77 °C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.     

Hybridizations and genetic relationships among Lindernia species (Scrophulariaceae): L. procumbens and two subspecies of L. dubia

Lindernia procumbens and L. dubia are common annual weeds in flooded rice fields of Japan. Two subspecies of L. dubia, subsp. major and subsp. dubia, are usually recognized in Japan but they are both regarded as synonyms of L. dubia elsewhere. In a cluster analysis based on AFLP, most L. dubia subsp. major formed a separate cluster from L. dubia subsp. dubia although 11% of haplotypes classified using AFLP were not coincident with classification using the shape of leaf bases, which is the commonly used identification trait. Artificial F₁ plants between L. procumbens and L. dubia subsp. major, and between L. procumbens and L. dubia subsp. dubia did not produce seed. Forty percent of capsules produced by F₁ plants from these two subspecies were slimmer and 80% pollen were sterile in slimmer capsules. However, seed number of most F₁ capsules was not different from that of self-fertilized plants, suggesting that there was no complete reproductive isolation between the subspecies. Natural hybridization of these subspecies may have occurred but we are not aware of it because F₁ plants are rare and F₂ plants are indistinguishable from these subspecies.