Polymerase Chain Reaction-amplified 16S rRNA Research Articles

Microbial Community Structure in Soilless Substrates Used for Nursery Crops

Soilless substrates are widely used for plant cultivation. However, little is known about how soilless substrate components, plant growth, or their interactions impact microbial communities in soilless media. The objectives of this study were to analyze microbial communities in typical pine bark substrates used for nursery crop production and determine the impacts of substituting peat with a compost substrate, and planting, on microbial community dynamics over a production cycle. Three soilless substrate mixtures were compared. The substrate mixes consisted of 80:20:0, 80:10:10, and 80:0:20 (volume:volume:volume) ratios of pine bark:peatmoss:leaf compost, respectively. One set of each treatment was planted with a single birch (Betula nigra ‘Cully’) liner and another set was not planted. The treatments (n = 3) were maintained in a nursery production setting, and samples were taken after 0, 1, 2, 3, and 4 months. Bacterial and fungal communities were characterized by sequencing polymerase chain reaction-amplified 16s rRNA genes and internal transcribed spacer regions. Initially, the two substrate mixtures that contained compost had more phyla than the substrate mixture that only contained peat and bark. After 1 month, microbial communities in all treatments contained similar phyla, but at different relative abundances based on the amount of compost they contained. Over time, Nitrosomonadaceae and Acetobacteraceae were the most abundant bacterial families in substrate mixes containing 10% and 20% compost, but they were absent from treatments without compost. The communities were dynamic and changed the most over the first 2 months. Microbial communities and their dynamics were similar between planted and unplanted treatments. Planting had less of an effect on microbial communities than compost amendment. Among the fungal communities, differences were observed based on both compost amendment and plant presence. Ascomycota and Basidiomycota were the most abundant fungal phyla and resembled those originally in the peat and compost, respectively. These findings could be used to understand the importance and dynamics of specific microbial communities present in substrate components and how they develop during greenhouse production.

Saliva Microbiome Changes in Patients With Periodontitis With and Without Chronic Obstructive Pulmonary Disease.

Objective: The oral microbiota plays a key part in the initial colonization by pathogens and the chronic inflammatory reaction of the host. We measured variations in the salivary microbiota and evaluated their potential associations with periodontitis and chronic obstructive pulmonary disease (COPD).Methods: We investigated the salivary microbiota of patients with COPD and periodontitis (n = 21) compared with that in patients with periodontitis alone (n = 36) and with healthy controls (HCs; n = 14), using pyrosequencing of polymerase chain reaction-amplified 16s rRNA genes.Results: Bacterial richness and diversity were significantly higher in patients suffering from COPD, and the bacterial family Lachnospiraceae was observed frequently only among patients with COPD and periodontitis. Veillonella, Rothia, Actinomyces, and Fusobacterium were the core bacterial genera that showed significant differences among patients with coincident COPD and periodontitis, patients with periodontitis alone, and HCs (p < 0.05). Veillonella, Rothia, and Actinomyces were observed much more frequently in patients with COPD and periodontitis, compared with that in HCs. All tested populations were divided into subgroups based on sex, smoking, or periodontitis index. In the subgroup with a bleeding index >2, Rothia was significantly different in periodontitis with and without COPD groups compared with HCs. In the subgroup with a plaque index >2.5, Rothia and Veillonella showed significant differences in periodontitis with and without COPD groups compared with HCs.Conclusion: Variations in salivary microbiota may be associated with COPD and periodontitis.

Coexistence of sulfate reducers with the other oil bacterial groups in Diyarbakır oil fields

The existence of sulfate-reducing bacteria (SRB) is a major concern in oil industry due to the detrimental effects of SRB in oil technology. SRB are co-habited with diverse microbial populations in oil fields. The presence of other bacterial groups in oil fields may alter SRB activity in different ways. Therefore, understanding this coexistence may provide insights into problems induced by SRB activity and possible solutions to these problems. To investigate this aspect, not only the presence and abundance of SRB but also bacterial population that coexists with SRB in sulfate-reducing enrichment cultures obtained from the Diyarbakır oil fields in southeast of Turkey was determined by using cultivation- and molecular-based approaches. The most probable number technique (MPN) was used to determine the number of sulfidogenic bacteria in the enrichments. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction-amplified 16S rRNA gene fragments was performed to examine the bacterial diversity of the enrichments. The results demonstrated that the number of sulfidogenic bacteria in the enrichments was low (<103 cells/mL). The DGGE analysis indicated that community members belonging to the Firmicutes were more abundant than those of other phyla. Members belonging to SRB mainly consisted of the genera Desulfosporosinus, Desulfovibrio, Thermodesulfovibrio, and Desulfotomaculum. Fermentative bacteria, acetogens, nitrate reducers, and sulfur reducers were also detected in the enrichments. The results of this study not only provide information regarding the diversity of the cultivable portion of the bacterial community that coexists with cultivable SRB, but they also offer insights into the interactions of bacteria in complex microbial communities that inhabit natural environments.

Soluble Corn Fiber Increases Calcium Absorption Associated with Shifts in the Gut Microbiome: A Randomized Dose-Response Trial in Free-Living Pubertal Females

Background: Soluble corn fiber (SCF; 12 g fiber/d) is shown to increase calcium absorption efficiency, associated with shifts in the gut microbiota in adolescent males and females who participated in a controlled feeding study.Objective: We evaluated the dose response of 0, 10, and 20 g fiber/d delivered by PROMITOR SCF 85 (85% fiber) on calcium absorption, biochemical bone properties, and the fecal microbiome in free-living adolescents.Methods: Healthy adolescent females (n = 28; aged 11–14 y) randomly assigned into a 3-phase, double-blind, crossover study consumed SCF for 4 wk at each dose (0, 10, and 20 g fiber/d from SCF) alongside their habitual diet and were followed by 3-d clinical visits and 3-wk washout periods. Stable isotope (44Ca and 43Ca) enrichment in pooled urine was measured by inductively coupled plasma mass spectrometry. Fecal microbial community composition was assessed by high-throughput sequencing (Illumina) of polymerase chain reaction–amplified 16S rRNA genes. Mixed model ANOVA and Friedman analysis were used to determine effects of SCF on calcium absorption and to compare mean microbial proportions, respectively.Results: Calcium absorption increased significantly with 10 (13.3% ± 5.3%; P = 0.042) and 20 g fiber/d (12.9% ± 3.6%; P = 0.026) from SCF relative to control. Significant differences in fecal microbial community diversity were found after consuming SCF (operational taxonomic unit measures of 601.4 ± 83.5, 634.5 ± 83.8, and 649.6 ± 75.5 for 0, 10, and 20 g fiber/d, respectively; P < 0.05). Proportions of the genus Parabacteroides significantly increased with SCF dose (1.1% ± 0.8%, 2.1% ± 1.6%, and 3.0% ± 2.0% for 0, 10, and 20 g fiber/d from SCF, respectively; P < 0.05). Increases in calcium absorption positively correlated with increases in Clostridium (r = 0.44, P = 0.023) and unclassified Clostridiaceae (r = 0.40, P = 0.040).Conclusions: SCF, a nondigestible carbohydrate, increased calcium absorption in free-living adolescent females. Two groups of bacteria may be involved, one directly fermenting SCF and the second fermenting SCF metabolites further, thereby promoting increased calcium absorption. This trial was registered at clinicaltrials.gov as NCT01660503.

Microbial Diversity of Ammonia Oxidizing Bacteria through Waste Water Genomics

Autotrophic ammonia-oxidizing bacteria are a critical factor of the microbial community in industrial wastewater treatment systems. We evaluated the diversity and community composition of β-proteobacterial ammonia-oxidizing bacteria in two full-scale treatment reactors - a sand filter and a biological aerated filter - receive an identical wastewater. Polymerase chain reaction of the 16S rRNA gene fragments of ammonia-oxidizing bacteria-selective primers was merged with denaturing gradient gel electrophoresis to allow the comparative analysis of the dominant ammonia-oxidizing bacteria populations. The phylogenetic affinities of the dominant ammonia-oxidizing bacteria were verified by cloning and sequencing of polymerase chain reaction-amplified 16S rRNA gene fragments. Denaturing gradient gel electrophoresis profiles were evaluated using a probability-based similarity index. An exploitation of a probabilistic index of similarity permitted us to consider the differences and similarities observed in ammonia-oxidizing bacteria community structure in different samples were statistically significant or could be accounted for random matching of bands in denaturing gradient gel electrophoresis profiles that would propose random colonization of the reactors at different ammonia-oxidizing bacteria. All Possibly-like sequences recognized, grouped within the Nitrosomonas genus. A greater diversity of ammonia-oxidizing bacteria were detected in trickling filters than the BAF on all samples analyzed were initiate to be dominated by ammonia oxidizing bacteria most closely linked to Nitrosococcus mobilis. Numerical investigation of the denaturing gradient gel electrophoresis profiles indicated that the ammonia-oxidizing bacteria community in depth profiles from the filter beds was selected in a non-random manner.

The oral microbiota of patients with recurrent aphthous stomatitis

BackgroundSpecific pathogenic bacteria have been implicated in recurrent aphthous stomatitis (RAS), a chronic inflammatory condition characterised by ulcerations in the oral mucosa. However, the aetiology behind this condition still remains unclear.ObjectiveThe buccal microbiota of patients with RAS was compared to that of control subjects to investigate its potential role for this condition.DesignBuccal swabs were obtained from non-ulcerative areas of 60 patients, of whom 42 patients had lesions at the time of sampling, and 60 healthy age- and gender-matched controls. Bacterial DNA was extracted and analysed by Terminal-Restriction Fragment Length Polymorphism, using enzymatic digestion of the polymerase chain reaction-amplified 16S rRNA gene, yielding a series of peaks, each representing a bacterial taxon.ResultsTwo peaks, 60 and 489, were more prevalent in patients with RAS than controls. Conversely, peaks 58 and 490 were less common in patients than controls. When the patients were divided into subgroups, we found that the observed differences in peak-pattern were related to the presence of lesions during sampling.ConclusionsThe microbiota of the non-inflamed buccal mucosa differed between patients and controls. The differences were most pronounced in patients who presented with lesions during sampling, suggesting that a disturbance in the normal buccal microbiota triggers the presence of lesions or that presence of lesions alters the microbiota.

Denaturing gradient gel electrophoresis analysis of gut bacterial community for the Japanese earthworms

The gut bacterial community structure for Pheretima hilgendorfi and P. heteropoda (Family Megascolecidae), and Allolobophora japonica (Family Lumbricidae) collected from agricultural grasslands in Japan was analyzed by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S rRNA gene fragments (PCR-DGGE) and compared with those in the surrounding soils. Denaturing gradient gel electrophoresis (DGGE) profiles indicated that each earthworm species had their own specific bacterial communities, and multidimentional scaling analysis grouped the DGGE profiles into three groups: gut samples from P. hilgendorfi and P. heteropoda, gut samples from A. japonica and samples from the surrounding soils. Nine dominant bands were identified by their direct sequencing and cloning. Major three bands from P. hilgendorfi and P. heteropoda were closely related to Bacillus species belonging to the phylum Firmicutes. Major four and two bands from A. japonica were closely related to the phyla Proteobacteria and Bacteroidetes, respectively.

Nonrandom Assembly of Bacterial Populations in Activated Sludge Flocs

The aim of this work was to investigate the dynamics of assembly of bacterial populations in activated sludge flocs. We approached this question by following the development of active bacterial populations during floc development in four replicated lab-scale activated sludge reactors, in which solid retention time (SRT) was set at 4 days. The null hypothesis was that the similarities in community composition could be accounted for by the probability that the same organisms occur in more than one replicated reactor. Microscopic imaging showed that the size of flocs in reactors with biomass retention increased during the first few days until a steady-state size was reached. The diversity and community structure of the sludge in all reactors were analyzed during a period of up to ten SRT, using denaturing gradient gel electrophoresis (DGGE) of reverse-transcription polymerase chain reaction-amplified 16S rRNA. High rates of change in DGGE profiles from consecutive sampling points suggested a high level of dynamics in all reactors. This conclusion was confirmed by the application of the Raup and Crick probability-based similarity index (S(RC)) for the comparison of rRNA-based fingerprinting patterns, which indicated that bacterial communities within reactors were not significantly similar after three SRT (0.05 < S(RC) < 0.95) and became significantly dissimilar after five SRT (S(RC) < 0.05). More importantly, significant similarity between replicate reactors was observed at all times analyzed (S(RC) > 0.95). The fact that the patterns between replicates were more reproducible than expected by chance under highly dynamic conditions allowed us to reject the null hypothesis that activated sludge floc communities assemble randomly from the available source pool of bacteria. We suggest that communities progressively recruit from the available pool of bacterial species, each with particular ecological requirements that determine their time of emergence into the community.

Bacterial Communities Associated with the Digestive Tract of the Predatory Ground Beetle, Poecilus chalcites, and Their Modification by Laboratory Rearing and Antibiotic Treatment

Ground beetles such as Poecilus chalcites (Coleoptera: Carabidae) are beneficial insects in agricultural systems where they contribute to the control of insect and weed pests. We assessed the complexity of bacterial communities occurring in the digestive tracts of field-collected P. chalcites using terminal restriction fragment length polymorphism analyses of polymerase chain reaction-amplified 16S rRNA genes. Bacterial identification was performed by the construction of 16S rRNA gene clone libraries and sequence analysis. Intestinal bacteria in field-collected beetles were then compared to those from groups of beetles that were reared in the lab on an artificial diet with and without antibiotics. Direct cell counts estimated 1.5x10(8) bacteria per milliliter of gut. The digestive tract of field-collected P. chalcites produced an average of 4.8 terminal restriction fragments (tRF) for each beetle. The most abundant clones were affiliated with the genus Lactobacillus, followed by the taxa Enterobacteriaceae, Clostridia, and Bacteriodetes. The majority of the sequences recovered were closely related to those reported from other insect gastrointestinal tracts. Lab-reared beetles produced fewer tRF, an average of 3.1 per beetle, and a reduced number of taxa with a higher number of clones from the family Enterobacteriaceae compared to the field-collected beetles. Antibiotic treatment significantly (p<0.05) reduced the number of tRF per beetle and selected for a less diverse set of bacterial taxa. We conclude that the digestive tract of P. chalcites is colonized by a simple community of bacteria that possess autochthonous characteristics. Laboratory-reared beetles harbored the most common bacteria found in field-collected beetles, and these bacterial communities may be manipulated in the laboratory with the addition of antibiotics to the diet to allow study of functional roles.

Selective colonization of insoluble substrates by human faecal bacteria

Insoluble plant polysaccharides and endogenous mucin are important energy sources for human colonic microorganisms. The object of this study was to determine whether or not specific communities colonize these substrates. Using faecal samples from four individuals as inocula for an anaerobic in vitro continuous flow system, the colonization of wheat bran, high amylose starch and porcine gastric mucin was examined. Recovered substrates were extensively washed and the remaining tightly attached bacterial communities were identified using polymerase chain reaction-amplified 16S rRNA gene sequences and fluorescent in situ hybridization. The substrate had a major influence on the species of attached bacteria detected. Sequences retrieved from bran were dominated by clostridial cluster XIVa bacteria, including uncultured relatives of Clostridium hathewayi, Eubacterium rectale and Roseburia species. Bacteroides species were also detected. The most abundant sequences recovered from starch were related to the cultured species Ruminococcus bromii, Bifidobacterium adolescentis, Bifidobacterium breve and E. rectale. The most commonly recovered sequences from mucin were from Bifidobacterium bifidum and uncultured bacteria related to Ruminococcus lactaris. This study suggests that a specific subset of bacteria is likely to be the primary colonizers of particular insoluble colonic substrates. For a given substrate, however, the primary colonizing species may vary between host individuals.

Anaerobic redox cycling of iron by freshwater sediment microorganisms

The potential for microbially mediated anaerobic redox cycling of iron (Fe) was examined in a first-generation enrichment culture of freshwater wetland sediment microorganisms. Most probable number enumerations revealed the presence of significant populations of Fe(III)-reducing (approximately 10(8) cells ml(-1)) and Fe(II)-oxidizing, nitrate-reducing organisms (approximately 10(5) cells ml(-1)) in the freshwater sediment used to inoculate the enrichment cultures. Nitrate reduction commenced immediately following inoculation of acetate-containing (approximately 1 mM) medium with a small quantity (1% v/v) of wetland sediment, and resulted in the transient accumulation of NO(2)(-) and production of a mixture of gaseous end-products (N(2)O and N(2)) and NH(4)(+). Fe(III) oxide (high surface area goethite) reduction took place after NO(3)(-) was depleted and continued until all the acetate was utilized. Addition of NO(3)(-) after Fe(III) reduction ceased resulted in the immediate oxidation of Fe(II) coupled to reduction of NO(3)(-) to NH(4)(+). No significant NO(2)(-) accumulation was observed during nitrate-dependent Fe(II) oxidation. No Fe(II) oxidation occurred in pasteurized controls. Microbial community structure in the enrichment was monitored by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified 16S rDNA and reverse transcription polymerase chain reaction-amplified 16S rRNA, as well as by construction of 16S rDNA clone libraries for four different time points during the experiment. Strong similarities in dominant members of the microbial community were observed in the Fe(III) reduction and nitrate-dependent Fe(II) oxidation phases of the experiment, specifically the common presence of organisms closely related (>or= 95% sequence similarity) to the genera Geobacter and Dechloromonas. These results indicate that the wetland sediments contained organisms such as Geobacter sp. which are capable of both dissimilatory Fe(III) reduction and oxidation of Fe(II) with reduction of NO(3)(-) to NH(4)(+). Our findings suggest that microbially catalysed nitrate-dependent Fe(II) oxidation has the potential to contribute to a dynamic anaerobic Fe redox cycle in freshwater sediments.

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood‐feeding cockroach

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.

Use of Polymerase Chain Reaction-amplified 16S rRNA Gene Sequences to Identify Pink-pigmented Bacteria Found in a Potable Water Treatment System.

Two strains of pink-pigmented, chlorine-resistant bacteria, GK101 and GK118, previously isolated from pink-colored microbial biomass occurring in a potable water treatment system were studied phylogenetically by using 16S rRNA gene sequence information. 16S ribosomal DNA (rDNA) fragments of approximately 1.5kbp were amplified by the polymerase chain reaction (PCR) from crude cell lysates of the isolates and sequenced directly by a combined method consisting of linear PCR sequencing and automated fluorescence detection. Sequence comparisons and a phylogenetic tree analysis showed that the pink-pigmented isolates were closely related to Methylobacterium extorquens. Smaller fragments of 16S rDNA were also amplified from the bulk DNA extracted from the pink biomass, with a pair of PCR primers specific to the methylotrophs with the serine pathway or to proteobacteria of the alpha subclass. The PCR product was cloned by using the T-A cloning technique and sequenced as noted above. The results of this molecular approach suggested that bacteria phylogenetically the same as strain GK101 predominated in the pink-pigmented population in situ and that some other bacteria of the genus Methylobacterium might also be involved in formation of the pink-colored aggregates.

Identification of mycobacteria from animals by restriction enzyme analysis and direct DNA cycle sequencing of polymerase chain reaction-amplified 16S rRNA gene sequences

Two methods, based on analysis of the polymerase chain reaction-amplified 16S rRNA gene by restriction enzyme analysis (REA) or direct cycle sequencing, were developed for rapid identification of mycobacteria isolated from animals and were compared to traditional phenotypic typing. BACTEC 7H12 cultures of the specimens were examined for "cording," and specific polymerase chain reaction amplification was performed to identify the presence of tubercle complex mycobacteria. Combined results of separate REAs with HhaI, MspI, MboI, and ThaI differentiated 12 of 15 mycobacterial species tested. HhaI, MspI, and ThaI restriction enzyme profiles differentiated Actinobacillus species from mycobacterial species. Mycobacterium bovis could not be differentiated from M. bovis BCG or Mycobacterium tuberculosis. Similarly, Mycobacterium avium and Mycobacterium paratuberculosis could not be distinguished from each other by REA but were differentiated by cycle sequencing. Compared with traditional typing, both methods allowed rapid and more accurate identification of acid-fast organisms recovered from 21 specimens of bovine and badger origin. Two groups of isolates were not typed definitively by either molecular method. One group of four isolates may constitute a new species phylogenetically very closely related to Mycobacterium simiae. The remaining unidentified isolates (three badger and one bovine) had identical restriction enzyme profiles and shared 100% nucleotide identify over the sequenced signature region. This nucleotide sequence most closely resembled the data base sequence of Mycobacterium senegalense.