Microbial community structure of a pilot-scale thermophilic anaerobic digester treating poultry litter

The microbial community structure of a stable pilot-scale thermophilic continuous stirred tank reactor digester stabilized on poultry litter was investigated. This 40-m³ digester produced biogas with 57 % methane, and chemical oxygen demand removal of 54 %. Bacterial and archaeal diversity were examined using both cloning and pyrosequencing that targeted 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes, constituting 93 % of the clones and 76 % of the pyrotags. Of the Firmicutes, class Clostridia (52 % pyrotags) was most abundant followed by class Bacilli (13 % pyrotags). The bacterial libraries identified 94 operational taxonomic units (OTUs) and pyrosequencing identified 577 OTUs at the 97 % minimum similarity level. Fifteen OTUs were dominant (≥2 % abundance), and nine of these were novel unclassified Firmicutes. Several of the dominant OTUs could not be classified more specifically than Clostridiales, but were most similar to plant biomass degraders, including Clostridium thermocellum. Of the rare pyrotag OTUs (<0.5 % abundance), 75 % were Firmicutes. The dominant methanogen was Methanothermobacter which has hydrogenotrophic metabolism, and accounted for >99 % of the archaeal clones. Based on the primary methanogen, as well as digester chemistry (high VA and ammonia levels), we propose that bacterial acetate oxidation is the primary pathway in this digester for the control of acetate levels.     

Assessment of bacterial diversity in agricultural by-product compost by sequencing of cultivated isolates and amplified rDNA restriction analysis

An investigation of bacterial diversity in compost was performed using molecular chronometer in order to reveal its phylogeny. Thirty-three bacterial isolates isolated from compost were analyzed by 16S rRNA gene sequencing which revealed phylogenetic lineage of class Bacilli, γ, β-Proteobacteria, and Actinobacteria. Among these lineages, isolates belonging to class Bacilli consisted of species from genera Staphylococcus, Bacillus, Terribacillus, and Lysinibacillus. From phylum Actinobacteria: Microbacterium barkeri and Kocuria sp. were identified. Other bacterial groups had phylogenetic linkage with genera Comamonas and Acidovorax (class β-Proteobacteria); Serratia, Klebsiella, and Enterobacter (class γ-Proteobacteria). Similar isolates were analyzed through ARDRA. Amplified product of 16S rRNA gene from each isolates was subjected to cleavage by enzymes HpaII, HinfI, and MspI in separate reaction tubes. HpaII generated 2–6 bands ranging from 90–688 bp, HinfI generated 2–5 bands of 71–1,038 bp, and MspI 2–7 bands of 69–793 bp. The restriction patterns from HpaII, HinfI, and MspI were normalized separately and combined by means of pattern recognition software “Diversity Database.” HpaII had highest discrimination index (0.72) than HinfI (0.68) and MspI (0.65), and the combination of all three showed discrimination index (0.69). Numerical analysis of ARDRA patterns demonstrated sufficient phylogenetic information for characterizing bacterial diversity. Phylogenetic relationship obtained among isolates through ARDRA was compared with 16S rRNA gene sequence and ARDRA results showed sufficiently similar 16S rRNA gene sequence analysis, but not an overlapping. It has been observed that ARDRA technique facilitates the identification of bacteria in less than 36 h as compared to traditional 16S rRNA gene sequencing.     

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.     

Phylogenetic analysis of 16S rRNA gene sequences reveals distal gut bacterial diversity in wild wolves (Canis lupus)

The aim of this study was to describe the microbial communities in the distal gut of wild wolves (Canis lupus). Fecal samples were collected from three healthy unrelated adult wolves captured at the nearby of Dalai Lake Nature Reserve in Inner Mongolia of China. The diversity of fecal bacteria was investigated by constructing PCR-amplified 16S rRNA gene clone libraries using the universal bacterial primers 27 F and 1493 R. A total of 307 non-chimeric near-full-length 16S rRNA gene sequences were analyzed and 65 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified. Seventeen OTUs (26%) showed less than 98% sequence similarity to 16S rRNA gene sequences were reported previously. Five different bacterial phyla were identified, with the majority of OTUs being classified within the phylum Firmicutes (60%), followed by Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%) and Actinobacteria (4.6%). The majority of clones fell within the order Clostridiales (53.8% of OTUs). It was predominantly affiliated with five families: Lachnospiraceae was the most diverse bacterial family in this order, followed by Ruminococcaceae, Clostridiaceae, Peptococcaceae and Peptostreptococcaceae.     

Characterization of the methanogen community in a household anaerobic digester fed with swine manure in China

Household anaerobic digesters have been installed across rural China for biogas production, but information on methanogen community structure in these small biogas units is sparsely available. By creating clone libraries for 16S rRNA and methyl coenzyme M reductase alpha subunit (mcrA) genes, we investigated the methanogenic consortia in a household biogas digester treating swine manure. Operational taxonomic units (OTUs) were defined by comparative sequence analysis, seven OTUs were identified in the 16S rRNA gene library, and ten OTUs were identified in the mcrA gene library. Both libraries were dominated by clones highly related to the type strain Methanocorpusculum labreanum Z, 64.0 % for 16S rRNA gene clones and 64.3 % for mcrA gene clones. Additionally, gas chromatography assays showed that formic acid was 84.54 % of the total volatile fatty acids and methane was 57.20 % of the biogas composition. Our results may help further isolation and characterization of methanogenic starter strains for industrial biogas production.     

Genotypic and Phenotypic Diversity of Cyanobacteria in Biological Soil Crusts of the Succulent Karoo and Nama Karoo of Southern Africa

Biological soil crusts (BSCs) are communities of cryptogamic organisms, occurring in arid and semiarid regions all over the world. Based on both morphological identification and genetic analyses, we established a first cyanobacterial inventory using the biphasic approach for BSCs within two major biomes of southern Africa. The samples were collected at two different sites in the Succulent Karoo and one in the Nama Karoo. After cultivation and morphological identification, the 16S rRNA gene was sequenced from the cyanobacterial cultures. From the soil samples, the DNA was extracted, and the 16S rRNA gene sequenced. All the sequences of the clone libraries from soil and cultures were compared with those of the public databases. Forty-five different species were morphologically identified in the samples of the Succulent Karoo (observatories of Soebatsfontein and Goedehoop). Based on the genetic analyses, 60 operational taxonomic units (OTUs) were identified for the Succulent Karoo and 43 for the Nama Karoo (based on 95 % sequence similarity). The cloned sequences corresponded well with the morphologically described taxa in cultures and sequences in the public databases. Besides known species of typical crust-forming cyanobacterial genera (Microcoleus, Phormidium, Tolypothrix and Scytonema), we found sequences of so far undescribed species of the genera Leptolyngbya, Pseudanabaena, Phormidium, Oscillatoria, Schizothrix and Microcoleus. Most OTUs were restricted to distinct sites. Grazed soils showed lower taxa numbers than undisturbed soils, implying the presence of early successional crust types and reduced soil surface protection. Our combined approach of morphological identification and genetic analyses allowed both a taxa inventory and the analysis of species occurring under specific habitat conditions.     

Impact of crop species on bacterial community structure during anaerobic co-digestion of crops and cow manure

The bacterial communities in three continuously stirred tank reactors co-digesting cow manure with grass silage, oat straw, and sugar beet tops, respectively, were investigated by 16S rRNA gene-based fingerprints and clone libraries. The analyses revealed both clearly distinct and similar phylotypes in the bacterial communities between the reactors. The major groups represented in the three reactors were Clostridia, unclassified Bacteria, and Bacteroidetes. Phylotypes affiliated with Bacilli or Deltaproteobacteria were unique to the sugar beet and straw reactor, respectively. Unclassified Bacteria dominated in sugar beet reactor while in the straw and grass reactor Clostridia was the dominant group. An increase in organic loading rate from 2 to 3 kg volatile solids m^?3 d^?1 resulted in larger changes in the bacterial community in the straw compared to grass reactor. The study shed more light on the evolution of bacterial community during anaerobic co-digestion of different crops and manure to methane.     

Marine Actinobacteria from the Gulf of California: diversity, abundance and secondary metabolite biosynthetic potential

The Gulf of California is a coastal marine ecosystem characterized as having abundant biological resources and a high level of endemism. In this work we report the isolation and characterization of Actinobacteria from different sites in the western Gulf of California. We collected 126 sediment samples and isolated on average 3.1–38.3 Actinobacterial strains from each sample. Phylogenetic analysis of 136 strains identified them as members of the genera Actinomadura, Micromonospora, Nocardiopsis, Nonomuraea, Saccharomonospora, Salinispora, Streptomyces and Verrucosispora. These strains were grouped into 26–56 operational taxonomic units (OTUs) based on 16S rRNA gene sequence identities of 98–100 %. At 98 % sequence identity, three OTUs appear to represent new taxa while nine (35 %) have only been reported from marine environments. Sixty-three strains required seawater for growth. These fell into two OTUs at the 98 % identity level and include one that failed to produce aerial hyphae and was only distantly related (≤95.5 % 16S identity) to any previously cultured Streptomyces sp. Phylogenetic analyses of ketosynthase domains associated with polyketide synthase genes revealed sequences that ranged from 55 to 99 % nucleotide identity to experimentally characterized biosynthetic pathways suggesting that some may be associated with the production of new secondary metabolites. These results indicate that marine sediments from the Gulf of California harbor diverse Actinobacterial taxa with the potential to produce new secondary metabolites.     

Diversity and Community Composition of Methanogenic Archaea in the Rumen of Scottish Upland Sheep Assessed by Different Methods

Ruminal archaeomes of two mature sheep grazing in the Scottish uplands were analysed by different sequencing and analysis methods in order to compare the apparent archaeal communities. All methods revealed that the majority of methanogens belonged to the Methanobacteriales order containing the Methanobrevibacter, Methanosphaera and Methanobacteria genera. Sanger sequenced 1.3 kb 16S rRNA gene amplicons identified the main species of Methanobrevibacter present to be a SGMT Clade member Mbb. millerae (≥91% of OTUs); Methanosphaera comprised the remainder of the OTUs. The primers did not amplify ruminal Thermoplasmatales-related 16S rRNA genes. Illumina sequenced V6–V8 16S rRNA gene amplicons identified similar Methanobrevibacter spp. and Methanosphaera clades and also identified the Thermoplasmatales-related order as 13% of total archaea. Unusually, both methods concluded that Mbb. ruminantium and relatives from the same clade (RO) were almost absent. Sequences mapping to rumen 16S rRNA and mcrA gene references were extracted from Illumina metagenome data. Mapping of the metagenome data to16S rRNA gene references produced taxonomic identification to Order level including 2–3% Thermoplasmatales, but was unable to discriminate to species level. Mapping of the metagenome data to mcrA gene references resolved 69% to unclassified Methanobacteriales. Only 30% of sequences were assigned to species level clades: of the sequences assigned to Methanobrevibacter, most mapped to SGMT (16%) and RO (10%) clades. The Sanger 16S amplicon and Illumina metagenome mcrA analyses showed similar species richness (Chao1 Index 19–35), while Illumina metagenome and amplicon 16S rRNA analysis gave lower richness estimates (10–18). The values of the Shannon Index were low in all methods, indicating low richness and uneven species distribution. Thus, although much information may be extracted from the other methods, Illumina amplicon sequencing of the V6–V8 16S rRNA gene would be the method of choice for studying rumen archaeal communities.     

Diversity of 16S rRNA genes in metagenomic community of the freshwater sponge Lubomirskia baicalensis

Phylogenetic analysis of the nucleotide sequences of 16S rRNA genes in the metagenomic community of Lubomirskia baicalensis has revealed taxonomic diversity of bacteria associated with the endemic freshwater sponge. Fifty-four operational taxonomic units (OTUs) belonging to six bacterial phyla (Actinobacteria, Proteobacteria (class ??-Proteobacteria and ??-Proteobacteria) Verrucomicrobia, Bacteroidetes, Cyanobacteria, and Nitrospira) have been identified. Actinobacteria, whose representatives are known as antibiotic producers, is the dominant phylum of the community (37%, 20 OTUs). All sequences detected shared the maximal homology with unculturable microorganisms from freshwater habitats. The wide diversity of bacteria closely coexisting with the Baikal sponge indicate the complex ecological relationships in the community formed under the unique conditions of Lake Baikal.     

Comparison of the Biolog OmniLog Identification System and 16S ribosomal RNA gene sequencing for accuracy in identification of atypical bacteria of clinical origin

The Biolog OmniLog Identification System (Biolog) and the 16S ribosomal RNA (rRNA) gene sequencing methods were compared to conventional microbiological methods and evaluated for accuracy of bacterial identification. These methods were evaluated using 159 clinical isolates. Each isolate was initially identified by conventional biochemical tests and morphological characteristics and subsequently placed into one of seven categories: aerobic Actinomycetes, Bacillus, Coryneforms, fastidious Gram-negative rods (GNR), non-fermenting GNR, miscellaneous Gram-positive rods (GPR), and Vibrio/Aeromonas. After comparison to the conventional identification, the Biolog system and 16S rRNA gene sequence identifications were classified as follows: a) correct to the genus and species levels; b) correct to the genus level only; or c) neither (unacceptable) identification. Overall, 16S rRNA gene sequencing had the highest percent accuracy with 90.6% correct identifications, while the Biolog system identified 68.3% of the isolates correctly. For each category, 16S rRNA gene sequencing had a substantially higher percent accuracy compared to the conventional methods. It was determined that the Biolog system is deficient when identifying organisms in the fastidious GNR category (20.0%). The observed data suggest that 16S rRNA gene sequencing provides a more accurate identification of atypical bacteria than the Biolog system.     

Comparison of 16S rRNA sequencing with biochemical testing for species-level identification of clinical isolates of Neisseria spp.

We aimed to compare accuracy of genus and species level identification of Neisseria spp. using biochemical testing and 16S rRNA sequence analysis. These methods were evaluated using 85 Neisseria spp. clinical isolates initially identified to the genus level by conventional biochemical tests and API NH system (Bio-Mérieux^®). In 34 % (29/85), more than one possibility was given by 16S rRNA sequence analysis. In 6 % (5/85), one of the possibilities offered by 16S rRNA gene sequencing, agreed with the result given by biochemical testing. In 4 % (3/85), the same species was given by both methods. 16S rRNA gene sequencing results did not correlate well with biochemical tests.     

Bacterial and archaeal communities in the acid pit lake sediments of a chalcopyrite mine

Bacterial and archaeal community structures and diversity of three different sedimentary environments (BH1A, BH2A and BH3A) in the acid pit lake of a chalcopyrite mine at Touro (Spain) were determined by 16S rRNA gene PCR-DGGE and sequencing of clone libraries. DGGE of bacterial and archaeal amplicons showed that the sediments harbor different communities. Bacterial 16S rRNA gene sequences were assigned to Acidobacteria, Actinobacteria, Cyanobacteria, Planctomycetes, Proteobacteria, Chloroflexi and uncultured bacteria, after clustering into 42 operational taxonomic units (OTUs). OTU 2 represented approximately 37, 42 and 37 % of all sequences from sediments BH1A, BH2A and BH3A, respectively, and was phylogenetically related to uncultured Chloroflexi. Remaining OTUs were phylogenetically related to heterotrophic bacteria, including representatives of Ferrithrix and Acidobacterium genera. Archaeal 16S rRNA gene sequences were clustered into 54 OTUs. Most of the sequences from the BH1A sediment were assigned to Euryarchaeota, whereas those from BH2A sediment were assigned to Crenarchaeota. The majority of the sequences from BH3A sediment were assigned to unclassified Archaea, and showed similarities to uncultured and unclassified environmental clones. No sequences related to Acidithiobacillus and Leptospirillum, commonly associated with acid mine drainage, were detected in this study.     

Identification of medically important yeasts by sequence analysis of the internal transcribed spacer and D1/D2 region of the large ribosomal subunit

BackgroundThe prevalence of opportunistic yeast infections has increased in recent decades as the result of an increasing immunocompromised patient population.AimsTo evaluate ribosomal RNA (rRNA) gene sequence to identify medically important yeast species, to investigate the performance of both the rRNA gene internal transcribed spacer (ITS) and D1/D2 region in identifying clinically relevant yeasts, and to compare these results with those of a standard phenotypic method.MethodsBoth regions from 50 yeast strains, comprising 45 clinical isolates and 5 reference strains, were amplified using PCR and then sequenced. The sequences were compared to reference data available from the GenBank database of the National Center for Biotechnology Information using the BLASTn tool.ResultsUsing ID32C, 88% (44/50) of all strains were identified accurately at the species level, although 6% were misidentified; two Candida eremophila isolates were identified as Candida glabrata and Candida tropicalis, and one Saprochaete clavata isolate was identified as Saprochaete capitata. Two of the four isolates identified by phenotypic methods as Trichosporon asahii were defined so by analyzing the ITS region, but the remaining two were not distinguishable from closely related species. Based on the D1/D2 region, these four isolates had 100% sequence identity with T. asahii, Trichosporon japonicum, and Trichosporon asteroides. The isolate identified as Trichosporon inkin using ID32C could not be distinguished from Trichosporon ovoides by analyzing the ITS and D1/D2 regions.ConclusionsIdentifying medically important yeasts by sequencing the ITS and D1/D2 region is a rapid and reliable alternative to conventional identification methods. For a diagnostic algorithm, we suggest a two-step procedure integrating conventional methods (e.g. microscopic morphology on corn meal agar with Tween® 80 and API ID32C®) and sequence analysis of the ITS and D1/D2 region.     

Comparative analysis of bacterial diversity in the rhizosphere of tomato by culture-dependent and -independent approaches

The microbiome in the rhizosphere–the region surrounding plant roots–plays a key role in plant growth and health, enhancing nutrient availability and protecting plants from biotic and abiotic stresses. To assess bacterial diversity in the tomato rhizosphere, we performed two contrasting approaches: culture-dependent and -independent. In the culture-dependent approach, two culture media (Reasoner’s 2A agar and soil extract agar) were supplemented with 12 antibiotics for isolating diverse bacteria from the tomato rhizosphere by inhibiting predominant bacteria. A total of 689 bacterial isolates were clustered into 164 operational taxonomic units (OTUs) at 97% sequence similarity, and these were found to belong to five bacterial phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes). Of these, 122 OTUs were retrieved from the antibiotic-containing media, and 80 OTUs were recovered by one specific antibiotic-containing medium. In the culture-independent approach, we conducted Illumina MiSeq amplicon sequencing of the 16S rRNA gene and obtained 19,215 high-quality sequences, which clustered into 478 OTUs belonging to 16 phyla. Among the total OTUs from the MiSeq dataset, 22% were recovered in the culture collection, whereas 41% of OTUs in the culture collection were not captured by MiSeq sequencing. These results showed that antibiotics were effective in isolating various taxa that were not readily isolated on antibiotic-free media, and that both contrasting approaches provided complementary information to characterize bacterial diversity in the tomato rhizosphere.     

Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data

Background

The intra- and inter-species genetic diversity of bacteria and the absence of ‘reference’, or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia.

Methods

A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM) of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization.

Results

The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52%) corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as ‘centroids’ in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578.

Conclusion

The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra-species variability.     

Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.     

Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing

Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1 393 579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health- and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.     

Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea

The bacterial communities in the guts of the adults and larvae of the Asian honey bee Apis cerana and the European honey bee Apis mellifera were surveyed by pyrosequencing the 16S rRNA genes. Most of the gut bacterial 16S rRNA gene sequences were highly similar to the known honey bee-specific ones and affiliated with Pasteurellaceae or lactic acid bacteria (LAB). The numbers of operational taxonomic units (OTUs, defined at 97% similarity) were lower in the larval guts (6 or 9) than in the adult guts (18 or 20), and the frequencies of Pasteurellaceae-related OTUs were higher in the larval guts while those of LAB-related OTUs in the adult guts. The frequencies of Lactococcus, Bartonella, Spiroplasma, Enterobacteriaceae, and Flavobacteriaceae-related OTUs were much higher in A. cerana guts while Bifidobacterium and Lachnospiraceae-related OTUs were more abundant in A. mellfera guts. The bacterial community structures in the midguts and hindguts of the adult honey bees were not different for A. cerana, but significantly different for A. mellifera. The above results substantiated the previous observation that honey bee guts are dominated by several specific bacterial groups, and also showed that the relative abundances of OTUs could be markedly changed depending on the developmental stage, the location within the gut, and the honey bee species. The possibility of using the gut bacterial community as an indicator of honey bee health was discussed.     

Intestinal Microbiota and Species Diversity of Campylobacter and Helicobacter spp. in Migrating Shorebirds in Delaware Bay

Using 16S rRNA gene sequencing analysis, we examined the bacterial diversity and the presence of opportunistic bacterial pathogens (i.e., Campylobacter and Helicobacter) in red knot (Calidris canutus; n = 40), ruddy turnstone (Arenaria interpres; n = 35), and semipalmated sandpiper (Calidris pusilla; n = 22) fecal samples collected during a migratory stopover in Delaware Bay. Additionally, we studied the occurrence of Campylobacter spp., enterococci, and waterfowl fecal source markers using quantitative PCR (qPCR) assays. Of 3,889 16S rRNA clone sequences analyzed, the bacterial community was mostly composed of Bacilli (63.5%), Fusobacteria (12.7%), Epsilonproteobacteria (6.5%), and Clostridia (5.8%). When epsilonproteobacterium-specific 23S rRNA gene clone libraries (i.e., 1,414 sequences) were analyzed, the sequences were identified as Campylobacter (82.3%) or Helicobacter (17.7%) spp. Specifically, 38.4%, 10.1%, and 26.0% of clone sequences were identified as C. lari (>99% sequence identity) in ruddy turnstone, red knot, and semipalmated sandpiper clone libraries, respectively. Other pathogenic species of Campylobacter, such as C. jejuni and C. coli, were not detected in excreta of any of the three bird species. Most Helicobacter-like sequences identified were closely related to H. pametensis (>99% sequence identity) and H. anseris (92% sequence identity). qPCR results showed that the occurrence and abundance of Campylobacter spp. was relatively high compared to those of fecal indicator bacteria, such as Enterococcus spp., E. faecalis, and Catellicoccus marimammalium. Overall, the results provide insights into the complexity of the shorebird gut microbial community and suggest that these migratory birds are important reservoirs of pathogenic Campylobacter species.