Ribosomal RNA Gene Clone Research Articles

New insights into the structure and function of the prokaryotic communities colonizing plastic debris collected in King George Island (Antarctica): Preliminary observations from two plastic fragments

In Antarctic regions, the composition and metabolic activity of microbial assemblages associated with plastic debris (“plastisphere”) are almost unknown. A macroplastic item from land (MaL, 30 cm) and a mesoplastic from the sea (MeS, 4 mm) were collected in Maxwell Bay (King George Island, South Shetland) and analyzed by Fourier transform infrared spectroscopy in attenuated total reflectance geometry (FTIR-ATR), which confirmed a polystyrene foam and a composite high-density polyethylene composition for MaL and MeS, respectively. The structure and function of the two plastic-associated prokaryotic communities were studied by complementary 16S ribosomal RNA gene clone libraries, total bacterioplankton and culturable heterotrophic bacterial counts, enzymatic activities of the whole community and enzymatic profiles of bacterial isolates. Results showed that Gamma- and Betaproteobacteria (31% and 28%, respectively) dominated in MeS, while Beta- and Alphaproteobacteria (21% and 13%, respectively) in MaL. Sequences related to oil degrading bacteria (Alcanivorax,Marinobacter) confirmed the known anthropogenic pressure in King George Island. This investigation on plastic-associated prokaryotic structure and function represents the first attempt to characterize the ecological role of plastisphere in this Antarctic region and provides the necessary background for future research on the significance of polymer type, surface characteristics and environmental conditions in shaping the plastisphere.

Molecular characterization of microbial communities and quantification of Mycobacterium immunogenum in metal removal fluids and their associated biofilms.

A number of human health effects have been associated with exposure to metal removal fluids (MRFs). Multiple lines of research suggest that a newly identified organism, Mycobacterium immunogenum (MI), appears to have an etiologic role in hypersensitivity pneumonitis (HP) in case of MRFs exposed workers. However, our knowledge of this organism, other possible causative agents (e.g., Pseudomonads), and the microbial ecology of MRFs in general, is limited. In this study, culture-based methods and small subunit ribosomal RNA gene clone library approach were used to characterize microbial communities in MRF bulk fluid and associated biofilm samples collected from fluid systems in an automobile engine plant. PCR amplification data using universal primers indicate that all samples had bacterial and fungal contaminated. Five among 15 samples formed colonies on the Mycobacteria agar 7H9 suggesting the likely presence of Mycobacteria in these five samples. This observation was confirmed with PCR amplification of 16S rRNA gene fragment using Mycobacteria specific primers. Two additional samples, Biofilm-1 and Biofilm-3, were positive in PCR amplification for Mycobacteria, yet no colonies formed on the 7H9 cultivation agar plates. Real-time PCR was used to quantify the abundance of M. immunogenum in these samples, and the data showed that the copies of M. immunogenum 16S rRNA gene in the samples ranges from 4.33 × 10(4) copy/ml to 4.61 × 10(7) copy/ml. Clone library analysis revealed that Paecilomyces sp. and Acremonium sp. and Acremonium-like were dominant fungi in MRF samples. Various bacterial species from the major phylum of proteobacteria were found and Pseudomonas is the dominant bacterial genus in these samples. Mycobacteria (more specifically MI) were found in all biofilm samples, including biofilms collected from inside the MRF systems and from adjacent environmental surfaces, suggesting that biofilms may play an important role in microbial ecology in MRFs. Biofilms may provide a shield or sheltered microenvironment for the growth and/or colonization of Mycobacteria in MRFs.

Characterization of fungal community and dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine by means of multiple culture-independent methods

The objective of this study was to investigate the fungal community and dynamics during the traditional fermentation of Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine using culture-independent approaches based on nested PCR-denaturing gradient gel electrophoresis (PCR-DGGE), 18S ribosomal RNA (rRNA) gene clone libraries and species-specific quantitative PCR (qPCR). Both of nested PCR-DGGE and 18S rRNA gene clone libraries revealed that the predominant microorganisms varied in different brewing phases. The relative proportions of some fungal species (such as Monascus purpureus, Rhizopus oryzae, Pichia guilliermondii and Saccharomycopsis fibuligera) detected at the early brewing stage decreased as the fermentation progressed, while Saccharomyces cerevisiae became the dominant species at the later stage. The present study revealed that PCR-DGGE fingerprint and 18S rRNA gene clone libraries presented the similar results. Nevertheless, the differences of the fungal species detected by the two different molecular biological methods demonstrated that the combined approach of nested PCR-DGGE and 18S rRNA gene clone libraries would give a more comprehensive profile of the fungal dynamics than either alone. Finally, species-specific qPCR was also performed to quantify some dominant fungal species detected by nested PCR-DGGE and 18S rRNA gene clone libraries. Results showed that R. oryzae increased sharply and dominated during the early brewing period, but decreased continuously from day 10 to the end of the traditional brewing. S. fibuligera and P. guilliermondii were the dominant yeast species at early stage, while S. cerevisiae increased sharply during the early phase and became the primary species at the end of the traditional brewing process. This study provides the first detailed evaluation of fungal community and dynamics in Wuyi Hong Qu glutinous rice wine traditional brewing process using multiplex culture-independent methods, which would show great value in scientifically understanding of the brewing mechanism and be useful for the selection of the suitable and beneficial fungal strains to improve the wine quality.

Rhizosphere bacterial community of Typha angustifolia L. and water quality in a river wetland supplied with reclaimed water.

Wetland plant rhizosphere microorganisms play a significant role in the purification of ecological restoration of reclaimed water replenishment wetlands. In this study, water quality discriminant analysis indicated the wetland had a distinctive role in the purification of total nitrogen (TN), total phosphorus (TP), and nitrate (NO3 (-)) from reclaimed water, of which removal rates were 42.15, 47.34, and 28.56 % respectively. All the sequences of 16S ribosomal RNA (16S rRNA) gene clone library were affiliated with Proteobacteria (74.50 %), Bacteroidetes (6.54 %), Gemmatimonadetes (5.88 %), Chloroflexi (4.25 %), Chlorobi (2.94 %), Nitrospira (2.61 %), Acidobacteria (2.29 %), and Actinobacteria (0.98 %). Assessment of water quality purification and rhizosphere bacterial properties revealed that the major biogeochemical reactions were nitrogen, phosphorus, carbon, and sulfur cycles (33.70, 15.40, 14.40, and 4.90 %, respectively). In addition, approximately 5.90 and 4.60 % of the clones are closely related with the minor biogeochemical degradations of antibiotics and halogenated hydrocarbons, which were the typical characteristics of reclaimed water wetland different from freshwater wetlands. The finding of water quality discriminant is consistent with that of bacterial community, but the latter was a more powerful method than the former which reveals possible implications of wetland plant purification on the reclaimed water.

Comparison of soil bacterial communities in a natural hardwood forest and coniferous plantations in perhumid subtropical low mountains.

BackgroundThe bacterial community of forest soils is influenced by environmental disturbance and/or meteorological temperature and precipitation. In this study, we investigated three bacterial communities in soils of a natural hardwood forest and two plantations of conifer, Calocedrus formosana and Cryptomeria japonica, in a perhumid, low mountain area. By comparison with our previous studies with similar temperature and/or precipitation, we aimed to elucidate how disturbance influences the bacterial community in forest soils and whether bacterial communities in similar forest types differ under different climate conditions.ResultsAnalysis of 16S ribosomal RNA gene clone libraries revealed that Acidobacteria and Proteobacteria were the most abundant phyla in the three forest soil communities, with similar relative abundance of various bacterial groups. However, UniFrac analysis based on phylogenetic information revealed differences of bacterial communities between natural hardwood forest and coniferous plantation soils. The diversities of bacterial communities of the replanted Calocedrus and Cryptomeria forests were higher than that in natural hardwood forest. The bacterial diversity of these three forest soil were all higher than those in the same forest types at other locations with less precipitation or with lower temperature. In addition, the distribution of some of the most abundant operational taxonomic units in the three communities differed from other forest soils, including those related to Acidobacteria, α-, β- and γ-Proteobacteria.ConclusionsReforestation could increase the bacterial diversity. Therefore, soil bacterial communities could be shaped by the forestry management practices and climate differences in warm and humid conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0050-x) contains supplementary material, which is available to authorized users.

Phylogenetic diversity of microorganisms in subseafloor crustal fluids from Holes 1025C and 1026B along the Juan de Fuca Ridge flank.

To expand investigations into the phylogenetic diversity of microorganisms inhabiting the subseafloor biosphere, basalt-hosted crustal fluids were sampled from Circulation Obviation Retrofit Kits (CORKs) affixed to Holes 1025C and 1026B along the Juan de Fuca Ridge (JdFR) flank using a clean fluid pumping system. These boreholes penetrate the crustal aquifer of young ocean crust (1.24 and 3.51 million years old, respectively), but differ with respect to borehole depth and temperature at the sediment-basement interface (147 m and 39°C vs. 295 m and 64°C, respectively). Cloning and sequencing of PCR-amplified small subunit ribosomal RNA genes revealed that fluids retrieved from Hole 1025C were dominated by relatives of the genus Desulfobulbus of the Deltaproteobacteria (56% of clones) and Candidatus Desulforudis of the Firmicutes (17%). Fluids sampled from Hole 1026B also contained plausible deep subseafloor inhabitants amongst the most abundant clone lineages; however, both geochemical analysis and microbial community structure reveal the borehole to be compromised by bottom seawater intrusion. Regardless, this study provides independent support for previous observations seeking to identify phylogenetic groups of microorganisms common to the deep ocean crustal biosphere, and extends previous observations by identifying additional lineages that may be prevalent in this unique environment.

Impact of protists on a hydrocarbon-degrading bacterial community from deep-sea Gulf of Mexico sediments: A microcosm study

In spite of significant advancements towards understanding the dynamics of petroleum hydrocarbon degrading microbial consortia, the impacts (direct or indirect via grazing activities) of bacterivorous protists remain largely unknown. Microcosm experiments were used to examine whether protistan grazing affects the petroleum hydrocarbon degradation capacity of a deep-sea sediment microbial community from an active Gulf of Mexico cold seep. Differences in n-alkane content between native sediment microcosms and those treated with inhibitors of eukaryotes were assessed by comprehensive two-dimensional gas chromatography following 30–90 day incubations and analysis of shifts in microbial community composition using small subunit ribosomal RNA gene clone libraries. More biodegradation was observed in microcosms supplemented with eukaryotic inhibitors. SSU rRNA gene clone libraries from oil-amended treatments revealed an increase in the number of proteobacterial clones (particularly γ-proteobacteria) after spiking sediments with diesel oil. Bacterial community composition shifted, and degradation rates increased, in treatments where protists were inhibited, suggesting protists affect the hydrocarbon degrading capacity of microbial communities in sediments collected at this Gulf of Mexico site.

Comparative Analysis of the Methanogen Diversity in Horse and Pony by UsingmcrAGene and Archaeal 16S rRNA Gene Clone Libraries

Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing the α-subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. The mcrA clone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P < 0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both the mcrA and the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds of Equus caballus.

A Comparison of the Bacterial Diversity of Two Shallow Freshwater Lakes in China

The objective of the present study was to determine the bacterial diversity present in the water samples taken from two shallow freshwater lakes in China. The water samples were collected from the Lake Nanhu, a hyper-eutrophic lake, and Lake Niushan, a mesotrophic lake. To determine the bacterial diversity, a 16S ribosomal RNA gene clone library from each lake, using the bacterial PCR primers 27F and 1492R, was prepared. A total of 119 near-full-length sequences were analyzed and 44 Operational Taxonomic Units (OTUs) were identified for Lake Nanhu. Seven different phyla were identified in which 42 OTUs were classified. Most of the OTUs contained sequences belonging to the phylum Proteobacteria (40.9 %), followed by Actinobacteria (15.9 %), Cyanobacteria (11.4 %), Verrucomicrobia (11.4 %), Plantomycetes (6.8 %), Bacteroidetes (4.5 %) and Chloroflexi (4.5 %). A phylum could not be assigned for clones within 2 OTUs (4.5 %). A total of 90 partial sequences were analyzed and 50 OTUs were identified for Lake Niushan. Most of the OTUs contained sequences belonging to the phyla Proteobacteria (40.0 %), followed by Bacteroidetes (16.0 %), Cyanobacteria (12.0 %), Plantomycetes (8.0 %), Verrucomicrobia, (6.0 %), Actinobacteria (4.0 %), Firmicutes (4.0 %) and Candidate Division OP10 (2.0 %). A phylum could not be assigned for clones within 4 OTUs (8.0 %).

Genomic properties of Marine Group A bacteria indicate a role in the marine sulfur cycle

Marine Group A (MGA) is a deeply branching and uncultivated phylum of bacteria. Although their functional roles remain elusive, MGA subgroups are particularly abundant and diverse in oxygen minimum zones and permanent or seasonally stratified anoxic basins, suggesting metabolic adaptation to oxygen-deficiency. Here, we expand a previous survey of MGA diversity in O2-deficient waters of the Northeast subarctic Pacific Ocean (NESAP) to include Saanich Inlet (SI), an anoxic fjord with seasonal O2 gradients and periodic sulfide accumulation. Phylogenetic analysis of small subunit ribosomal RNA (16S rRNA) gene clone libraries recovered five previously described MGA subgroups and defined three novel subgroups (SHBH1141, SHBH391, and SHAN400) in SI. To discern the functional properties of MGA residing along gradients of O2 in the NESAP and SI, we identified and sequenced to completion 14 fosmids harboring MGA-associated 16S RNA genes from a collection of 46 fosmid libraries sourced from NESAP and SI waters. Comparative analysis of these fosmids, in addition to four publicly available MGA-associated large-insert DNA fragments from Hawaii Ocean Time-series and Monterey Bay, revealed widespread genomic differentiation proximal to the ribosomal RNA operon that did not consistently reflect subgroup partitioning patterns observed in 16S rRNA gene clone libraries. Predicted protein-coding genes associated with adaptation to O2-deficiency and sulfur-based energy metabolism were detected on multiple fosmids, including polysulfide reductase (psrABC), implicated in dissimilatory polysulfide reduction to hydrogen sulfide and dissimilatory sulfur oxidation. These results posit a potential role for specific MGA subgroups in the marine sulfur cycle.

Bacterial community dynamics during the traditional brewing of Wuyi Hong Qu glutinous rice wine as determined by culture-independent methods

Wuyi Hong Qu (black-skin-red-koji) glutinous rice wine, as one of the most typical representatives of Hong Qu glutinous rice wine in Fujian province of China, is brewed under non-sterile and uncontrolled fermentation condition based on empirical knowledge, causing uncontrollability of fermentation process and instability of the final quality. The objective of this study was to investigate the bacterial dynamics during the traditional fermentation of Wuyi Hong Qu glutinous rice wine using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and 16S ribosomal RNA (rRNA) gene clone libraries analysis.The DGGE profile indicated that the dominant bacterial species in the traditional wine fermentation starters were Pediococcus pentosaceus, Pediococcus acidilactici and Bacillus sp. (including Bacillus aryabhattai or Bacillus megaterium and Bacillus amyloliquefaciens). Bacterial dynamic obtained from the PCR-DGGE revealed the presence of Bacillus sp. and LAB (including Lactobacillus plantarum group, Lactobacillus brevis, P. acidilactici and P. pentosaceus) during the traditional fermentation process, but they varied in different brewing phases. The relative proportions of some bacterial species (such as Bacillus sp., P. acidilactici, L. brevis and P. pentosaceus) detected at early fermentation stage decreased as the fermentation progressed. While L. plantarum group was consistently detected with high light band intensity throughout the fermentation process. 16S rRNA gene clone libraries revealed that the two different molecular biological methods gave similar results, but clone library analysis was more representative of the bacterial community to some extent. For example, Leuconostoc mesenteroides was detected by 16S rRNA gene clone library but not discovered by bacterial DGGE profile throughout the whole fermentation process. Therefore, the combined approach of nested PCR-DGGE and 16S rRNA gene clone libraries would give a more comprehensive profile of the bacterial dynamics than either alone. Finally, species-specific multiplex PCR was also performed to confirm the L. plantarum group. Result showed that only L. plantarum can be detected from the total bacterial DNA extracted from samples of different fermentation phases.This is the first report to reveal the dynamics of bacterial species involved in Wuyi Hong Qu glutinous rice wine brewing process using culture-independent methods. It might be useful to control wine production systems and improve wine quality.

Microbial life associated with low‐temperature alteration of ultramafic rocks in the Leka ophiolite complex

Water-rock interactions in ultramafic lithosphere generate reduced chemical species such as hydrogen that can fuel subsurface microbial communities. Sampling of this environment is expensive and technically demanding. However, highly accessible, uplifted oceanic lithospheres emplaced onto continental margins (ophiolites) are potential model systems for studies of the subsurface biosphere in ultramafic rocks. Here, we describe a microbiological investigation of partially serpentinized dunite from the Leka ophiolite (Norway). We analysed samples of mineral coatings on subsurface fracture surfaces from different depths (10-160 cm) and groundwater from a 50-m-deep borehole that penetrates several major fracture zones in the rock. The samples are suggested to represent subsurface habitats ranging from highly anaerobic to aerobic conditions. Water from a surface pond was analysed for comparison. To explore the microbial diversity and to make assessments about potential metabolisms, the samples were analysed by microscopy, construction of small subunit ribosomal RNA gene clone libraries, culturing and quantitative-PCR. Different microbial communities were observed in the groundwater, the fracture-coating material and the surface water, indicating that distinct microbial ecosystems exist in the rock. Close relatives of hydrogen-oxidizing Hydrogenophaga dominated (30% of the bacterial clones) in the oxic groundwater, indicating that microbial communities in ultramafic rocks at Leka could partially be driven by H2 produced by low-temperature water-rock reactions. Heterotrophic organisms, including close relatives of hydrocarbon degraders possibly feeding on products from Fischer-Tropsch-type reactions, dominated in the fracture-coating material. Putative hydrogen-, ammonia-, manganese- and iron-oxidizers were also detected in fracture coatings and the groundwater. The microbial communities reflect the existence of different subsurface redox conditions generated by differences in fracture size and distribution, and mixing of fluids. The particularly dense microbial communities in the shallow fracture coatings seem to be fuelled by both photosynthesis and oxidation of reduced chemical species produced by water-rock reactions.

Ruminal fermentation and microbial ecology of buffaloes and cattle fed the same diet

Although buffaloes and cattle are ruminants, their digestive capabilities and rumen microbial compositions are considered to be different. The purpose of this study was to compare the rumen microbial ecology of crossbred water buffaloes and cattle that were fed the same diet. Cattle exhibited a higher fermentation rate than buffaloes. Methane production and methanogen density were lower in buffaloes. Phylogenetic analysis of Fibrobacter succinogenes-specific 16S ribosomal RNA gene clone library showed that the diversity of groups within a species was significantly different (P < 0.05) between buffalo and cattle and most of the clones were affiliated with group 2 of the species. Population densities of F.succinogenes, Ruminococcus albus and R. flavefaciens were higher until 6 h post-feeding in cattle; however, buffaloes exhibited different traits. The population of anaerobic fungi decreased at 3 h in cattle compared to buffaloes and was similar at 0 h and 6 h. The diversity profiles of bacteria and fungi were similar in the two species. The present study showed that the profiles of the fermentation process, microbial population and diversity were similar in crossbred water buffaloes and crossbred cattle.

Comparison of wintertime eukaryotic community from sea ice and open water in the Baltic Sea, based on sequencing of the 18S rRNA gene

The Baltic Sea is one of the world’s largest brackish water basins and is traditionally considered to be species poor. Here, we assessed the diversity of the nano-sized eukaryotic microbial wintertime community, using molecular ecological methods based on sequencing of small-subunit ribosomal RNA gene clone libraries. The results demonstrate that a rich community of small eukaryotes inhabits the Baltic Sea ice and water during winter. The community was dominated by alveolates and stramenopiles. Ciliates and cercozoans were the richest groups present, while in contrast to previous studies, diatoms showed a lower richness than expected. Furthermore, fungi and parasitic Syndiniales were present both in the water and in the sea ice. Some of the organisms in the sea-ice community were active, based on the RNA data, but a number of organisms were inactive or remnants from the freezing process. The results demonstrate that the sea-ice communities in the Baltic Sea are highly diverse and that water and ice of different ages include different protistan assemblages. Our study emphasizes the potential loss in biodiversity through diminishing ice cover as a result of climate change.

Microbial diversity in deep-sea sediment from the cobalt-rich crust deposit region in the Pacific Ocean

Cobalt-rich crusts are important metallic mineral resources with great economic potential, usually distributed on seamounts located in the Pacific Ocean. Microorganisms are believed to play a role in the formation of crusts as well as in metal cycling. To explore the microbial diversity related to cobalt-rich crusts, 16S ribosomal RNA gene clone libraries were constructed from three consecutive sediment layers. In total, 417 bacterial clones were obtained from three bacterial clone libraries, representing 17 distinct phylogenetic groups. Proteobacteria dominated in the bacterial communities, followed by Acidobacteria and Planctomycetes. Compared with high bacterial diversity, archaea showed a remarkably low diversity, with all 137 clones belonging to marine archaeal group I except one novel euryarchaeotal clone. The microbial communities were potentially involved in sulfur, nitrogen and metal cycling in the area of cobalt-rich crusts. Sulfur oxidation and metal oxidation were potentially major sources of energy for this ecosystem. This is the first reported investigation of microbial diversity in sediments associated with cobalt-rich crusts, and it casts fresh light on the microbial ecology of these important ecosystems.

Bile Acid Is a Host Factor That Regulates the Composition of the Cecal Microbiota in Rats

Alterations in the gastrointestinal microbiota have been associated with metabolic diseases. However, little is known about host factors that induce changes in gastrointestinal bacterial populations. We investigated the role of bile acids in this process because of their strong antimicrobial activities, specifically the effects of cholic acid administration on the composition of the gut microbiota in a rat model. Rats were fed diets supplemented with different concentrations of cholic acid for 10 days. We used 16S ribosomal RNA gene clone library sequencing and fluorescence in situ hybridization to characterize the composition of the cecal microbiota of the different diet groups. Bile acids in feces, organic acids in cecal contents, and some blood parameters were also analyzed. Administration of cholic acid induced phylum-level alterations in the composition of the gut microbiota; Firmicutes predominated at the expense of Bacteroidetes. Cholic acid feeding simplified the composition of the microbiota, with outgrowth of several bacteria in the classes Clostridia and Erysipelotrichi. Externally administered cholic acid was efficiently transformed into deoxycholic acid by a bacterial 7α-dehydroxylation reaction. Serum levels of adiponectin decreased significantly in rats given the cholic acid diet. Cholic acid regulates the composition of gut microbiota in rats, inducing similar changes to those induced by high-fat diets. These findings improve our understanding of the relationship between metabolic diseases and the composition of the gastrointestinal microbiota.

Characterization of Bacterial Community Diversity in Chronic Rhinosinusitis Infections Using Novel Culture‐Independent Techniques

Objective: Chronic rhinosinusitis (CRS) is a common condition involving a microbial component. Only a small fraction of the total range of bacterial species present in CRS is detected through conventional culture-dependent techniques. We aimed to characterize the bacterial diversity in CRS samples using the novel molecular technique TRFLP. Method: Through 16S ribosomal RNA (rRNA) gene clone sequencing and Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis, the bacteria present in 70 clinical samples from 43 CRS patients undergoing endoscopic sinus surgery were characterized. Results: Bacterial TRFLP profiles were generated for 70 from 73 samples, and a total of 48 separate bands were detected. Species belonging to 34 genera were identified as present by clone sequence analysis. Of the species detected, those within the genera Pseudomonas, Citrobacter, Haemophilus, Propionibacterium, Staphylococcus, and Streptococcus were found to be numerically dominant, with Pseudomonas aeruginosa being the most frequently detected species. Conclusion: This study has validated the use of the culture-independent molecular technique TRFLP in sinonasal samples. Preliminary characterization of the microbial diversity in CRS suggests a complex range of common and novel bacterial species within the upper airway in CRS, providing further evidence for the polymicrobial etiology of this disease.

Characterization of Bacterial Community Diversity in Chronic Rhinosinusitis Infections Using Novel Culture-independent Techniques

Chronic rhinosinusitis (CRS) with or without polyps is a common chronic upper airway condition of multifactorial origin. Fundamental to effective treatment of any infection is the ability to accurately characterize the underlying cause. Many studies have shown that only a small fraction of the total range of bacterial species present in CRS is detected through conventional culture-dependent techniques. Consequently, culture data are often unrepresentative of the true diversity of the microbial community within the sample. These drawbacks, along with the length of time required to complete the analysis, strongly support the development of alternative means of assessing which bacterial species are present. As such, molecular microbiological approaches that assess the content of clinical samples in a culture-independent manner could significantly enhance the range and quality of data obtained routinely from such samples. We aimed to characterize the bacterial diversity present in tissue and mucus samples taken from the CRS setting using molecular nonculture-dependent techniques. Through 16S ribosomal RNA (rRNA) gene clone sequencing and terminal restriction fragment length polymorphism (T-RFLP) analysis, the bacteria present in 70 clinical samples from 43 CRS patients undergoing endoscopic sinus surgery were characterized. Bacterial T-RFLP profiles were generated for 70 of 73 samples and a total of 48 separate bands were detected. Species belonging to 34 genera were identified as present by clone sequence analysis. Of the species detected, those within the genera Pseudomonas, Citrobacter, Haemophilus, Propionibacterium, Staphylococcus, and Streptococcus were found numerically dominant, with Pseudomonas aeruginosa the most frequently detected species. This study has validated the use of the culture-independent technique T-RFLP in sinonasal samples. Preliminary characterization of the microbial diversity in CRS suggests a complex range of common and novel bacterial species within the upper airway in CRS, providing further evidence for the polymicrobial etiology of CRS.

Analysis of bacterial community structure in Saba-Narezushi (Narezushi of Mackerel) by 16S rRNA gene clone library

Narezushi, a derivation of sushi, is a traditional Japanese food made by fermenting salted fish meat and cooked rice together. In this study, the microbial diversity of saba-narezushi (narezushi of mackerel, Scomber japonicus) was analyzed by the 16S ribosomal RNA gene clone library method. Chemical composition was also analyzed to compare with different kinds of narezushi. The chemical composition of the narezushi was similar to those obtained from samma-narezushi. Ninety-four clones were randomly selected and DNA sequences of cloned fragments (approx. 890bp) were analyzed. The DNA sequences obtained were phylogenetically analyzed. The expected operational taxonomy units (OTUs) by Chao1 estimates and Shannon-Wiener index (H') at 97% identity threshold were 48 and 1.822, respectively. The sequence similarity of the cloned fragment was equal to or higher than 98% of the sequence of cultivated bacterial species in the public database. Most of the clones (85%) belonged to lactic acid bacteria (LAB). Lactobacillus curvatus was the most abundant species followed by Lactococcus piscium and Leuconostoc gasicomitatum, suggesting that these bacteria play important roles in the fermentation of saba-narezushi.

Molecular profiling of bacterial species in the geese cecum

The purpose of this study was to analyse the microbial diversity in the caecum of geese using a 16S ribosomal RNA gene (rRNA) clone library approach. A total of 160 clones and 124 clones were sequenced and phylogenetically analysed from the contents and mucosa of the caecum of Yang Zhou geese, respectively. The result indicated that there was a rich variety of bacteria in the caecum contents. Forty-six operational taxonomic units (OTUs) based on a 98% similarity criterion were classified in the contents of goose caecum, as compared to 29 OTUs based on a 97% similarity criterion in the mucosa of goose caecum. The sequences were assigned to 7 and 5 groups in the contents and mucosa of goose caecum, respectively. Contents of goose caecum were dominantly occupied by Clostridia-related species (58.7%) with other abundant sequences being related to Bacteroidetes (26.9%) and Erysipelotrichi (11.2%). Gammaproteobacteria (59.6%) and Clostridia (20.1%) were predominant in the mucosa of goose caecum.