rRNA Gene PCR Amplicons Research Articles

Detection of pathogenic bacteria in ticks from Isiolo and Kwale counties of Kenya using metagenomics.

Ticks are arachnid ectoparasites that rank second only to mosquitoes in the transmission of human diseases including bacteria responsible for anaplasmosis, ehrlichiosis, spotted fevers, and Lyme disease among other febrile illnesses. Due to the paucity of data on bacteria transmitted by ticks in Kenya, this study undertook a bacterial metagenomic-based characterization of ticks collected from Isiolo, a semi-arid pastoralist County in Eastern Kenya, and Kwale, a coastal County with a monsoon climate in the southern Kenyan border with Tanzania. A total of 2,918 ticks belonging to 3 genera and 10 species were pooled and screened in this study. Tick identification was confirmed through the sequencing of the Cytochrome C Oxidase Subunit 1 (COI) gene. Bacterial 16S rRNA gene PCR amplicons obtained from the above samples were sequenced using the MinION (Oxford Nanopore Technologies) platform. The resulting reads were demultiplexed in Porechop, followed by trimming and filtering in Trimmomatic before clustering using Qiime2-VSearch. A SILVA database pretrained naïve Bayes classifier was used to classify the Operational Taxonomic Units (OTUs) taxonomically. The bacteria of clinical interest detected in pooled tick assays were as follows: Rickettsia spp. 59.43% of pools, Coxiella burnetii 37.88%, Proteus mirabilis 5.08%, Cutibacterium acnes 6.08%, and Corynebacterium ulcerans 2.43%. These bacteria are responsible for spotted fevers, query fever (Q-fever), urinary tract infections, skin and soft tissue infections, eye infections, and diphtheria-like infections in humans, respectively. P. mirabilis, C. acnes, and C. ulcerans were detected only in Isiolo. Additionally, COI sequences allowed for the identification of Rickettsia and Coxiella species to strain levels in some of the pools. Diversity analysis revealed that the tick genera had high levels of Alpha diversity but the differences between the microbiomes of the three tick genera studied were not significant. The detection of C. acnes, commonly associated with human skin flora suggests that the ticks may have contact with humans potentially exposing them to bacterial infections. The findings in this study highlight the need for further investigation into the viability of these bacteria and the competency of ticks to transmit them. Clinicians in these high-risk areas also need to be appraised for them to include Rickettsial diseases and Q-fever as part of their differential diagnosis.

Gut microbes differ in postmenopausal women responding to prunes to maintain hip bone mineral density.

Foods high in phenolics such as prunes have been shown to exert protective effects on bone mineral density (BMD), but only certain individuals experience these benefits. This post-hoc analysis of a 12-month randomized controlled trial aimed to identify the relationship among the gut microbiome, immune responses, and bone protective effects of prunes on postmenopausal women. Subjects who consumed 50-100 g prunes daily were divided into responders (n = 20) and non-responders (n = 32) based on percent change in total hip bone mineral density (BMD, ≥1% or ≤-1% change, respectively). DXA scans were used to determine body composition and BMD. Immune markers were measured using immunoassays and flow cytometry. Targeted phenolic metabolites were analyzed using ultra performance liquid chromatography-tandem mass spectrometry. The fecal microbiota was characterized through 16S rRNA gene PCR amplicon sequencing. After 12 months of prune consumption, anti-inflammatory markers showed responders had significantly lower levels of IL-1β and TNF-α. QIIME2 sequence analysis showed that microbiomes of responders and non-responders differed in alpha (Shannon and Faith PD, Kruskal-Wallis p < 0.05) and beta diversity (unweighted Unifrac, PERMANOVA p < 0.04) metrics both before and after prune treatment. Furthermore, responders had a higher abundance of bacterial families Oscillospiraceae and Lachnospiraceae (ANCOM-BC p < 0.05). These findings provide evidence that postmenopausal women with initial low BMD can benefit from prunes if they host certain gut microbes. These insights can guide precision nutrition strategies to improve BMD tailored to diet and microbiome composition.

Impact of elevated atmospheric CO2 on the wheat rhizomicrobiome under the additional influence of warming, drought, and nitrogen fertilization

The rhizomicrobiome is an important component of agricultural soil that may face vulnerability due to global change. Here, we focused on its response in a 2-year cropland free air carbon dioxide enrichment (FACE) experiment on winter wheat in combination with warming (+3 °C) and drought. To characterize bacterial abundance and prokaryotic diversity, 16S rRNA gene PCR amplicons obtained from soil DNA were analysed. Elevated CO2 (e[CO2]) increased under conventional water supply bacterial abundance, along with higher above-ground plant biomass, but had no tangible effect on the overall diversity and community composition. In contrast, drought reduced bacterial abundance under e[CO2] despite higher plant biomass, while it did not affect bacterial abundance at ambient CO2. Warming and N-fertilizer supplies increased bacterial abundance and community composition, but these responses were not modified by CO2. Compared to the wheat growth stage and variation in N-supply, the effect of CO2 on the overall community composition was minor, explaining 2.2% of its variation. While 7 taxa, representing only 0.3% of relative abundance and most of them affiliated with Acidobacteria, changed with the CO2 level, many more responded to growth stages and nitrogen supply. Fewer growth stage and N supply-responsive taxa were detected with e[CO2]. Our results provide strong evidence that wheat plant responses to e[CO2] transfer to the rhizomicrobiome affecting the abundance composition of their constituting microbes. Additional drought conditions can strongly change the direction of these responses and should thus be considered for predicting implications of global change for the cropland soil microbiomes.

Gut flora alterations due to lipopolysaccharide derived from Porphyromonas gingivalis.

Gut dysbiosis induces 'leaky gut,' a condition associated with diabetes, NASH, and various auto-immune diseases. Porphyromonas gingivalis is a periodontopathic bacterium which causes periodontal tissue breakdown, and often enters the systemic blood flow. Oral administration of P. gingivalis induced gut dysbiosis in mice model, but no systemic administration of P. gingivalis has been reported thus far. In the present study, we investigated the effect of P. gingivalis-derived lipopolysaccharide (Pg-LPS) on the intestinal flora of our established mouse model. Eight-week-old C57BL/6J mice were intraperitoneally administered Pg-LPS. Three months later, DNA was extracted from stool, and RNA from the small and large intestines. After euthanizing the mice, pathological sections of the intestinal tract were prepared and stained with hematoxylin and eosin (H&E). Tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 expression levels were evaluated using quantitative PCR. 16S rRNA gene PCR amplicon analysis data were acquired using NGS. Microbial diversity and composition were analyzed using Quantitative Insights into Microbial Ecology 2. Furthermore, alterations in microbial function were performed by PICRUSt2. No significant inflammatory changes were observed in the H&E. No significant differences in the mRNA levels of IL-1β, IL-6, and TNF-α were observed between the groups. Pg-LPS administration decreased the abundance of Allobacterium in the gut. A predictive metagenomic analysis by PICRUSt2 and STAMP showed that 47 pathways increased and 17 pathways decreased after Pg-LPS administration. Systemic application of periodontal pathogens may cause changes in the intestinal flora which may affect the physiological functions of the intestinal tract.

Gullies and Moraines Are Islands of Biodiversity in an Arid, Mountain Landscape, Asgard Range, Antarctica.

Cold, dry, and nutrient-poor, the McMurdo Dry Valleys of Antarctica are among the most extreme terrestrial environments on Earth. Numerous studies have described microbial communities of low elevation soils and streams below glaciers, while less is known about microbial communities in higher elevation soils above glaciers. We characterized microbial life in four landscape features (habitats) of a mountain in Taylor Valley. These habitats varied significantly in soil moisture and include moist soils of a (1) lateral glacial moraine, (2) gully that terminates at the moraine, and very dry soils on (3) a southeastern slope and (4) dry sites near the gully. Using rRNA gene PCR amplicon sequencing of Bacteria and Archaea (16S SSU) and eukaryotes (18S SSU), we found that all habitat types harbored significantly different bacterial and eukaryotic communities and that these differences were most apparent when comparing habitats that had macroscopically visible soil crusts (gully and moraine) to habitats with no visible crusts (near gully and slope). These differences were driven by a relative predominance of Actinobacteria and a Colpodella sp. in non-crust habitats, and by phototrophic bacteria and eukaryotes (e.g., a moss) and predators (e.g., tardigrades) in habitats with biological soil crusts (gully and moraine). The gully and moraine also had significantly higher 16S and 18S ESV richness than the other two habitat types. We further found that many of the phototrophic bacteria and eukaryotes of the gully and moraine share high sequence identity with phototrophs from moist and wet areas elsewhere in the Dry Valleys and other cold desert ecosystems. These include a Moss (Bryum sp.), several algae (e.g., a Chlorococcum sp.) and cyanobacteria (e.g., Nostoc and Phormidium spp.). Overall, the results reported here broaden the diversity of habitat types that have been studied in the Dry Valleys of Antarctica and suggest future avenues of research to more definitively understand the biogeography and factors controlling microbial diversity in this unique ecosystem.

Microbial Community Composition in Crude Oils and Asphalts from the Kurdistan Region of Iraq

To identify hydrocarbon-degrading microorganisms contributing to the formation of heavy oil we investigated the microbial community composition in different types of crude oils from oil-production facilities and in crude oil and asphalt from different natural seeps from the Kurdistan Region of Iraq (KRI). Crude oils from five out of six production facilities did not contain microorganisms detectable by 16S rRNA gene PCR amplicon sequencing likely reflecting a low microbial abundance in these samples. Crude oil and asphalt from the natural seeps hosted diverse microbial communities. The same phylotypes of uncultivated Deferribacteres and Thermodesulfobacteraceae were predominant community members across crude oils and asphalts from separate geographical locations. Soils surrounding seeps did not contain these phylotypes suggesting that they originate from the subsurface and although they seem commonly detected in hydrocarbon-rich environments their role in hydrocarbon-degradation is unknown. GC-MS analyses showed that mainly aromatic hydrocarbons were present in the crude oil and asphalt and that they were undergoing biodegradation - likely with sulfate and nitrate as terminal oxidants. In agreement, only bssA gene, but not assA gene-carrying microorganisms were detectable in the analyzed sampled. Overall our study identified several abundant uncultivated taxa with likely roles in transformation of nitrate, sulfate and hydrocarbons.

The Vaginal Microbiome as a Tool to Predict rASRM Stage of Disease in Endometriosis: a Pilot Study.

Endometriosis remains a challenge to understand and to diagnose. This is an observational cross-sectional pilot study to characterize the gut and vaginal microbiome profiles among endometriosis patients and control subjects without the disease and to explore their potential use as a less-invasive diagnostic tool for endometriosis. Overall, 59 women were included, n = 35 with endometriosis and n = 24 controls. Rectal and vaginal samples were collected in two different periods of the menstrual cycle from all subjects. Gut and vaginal microbiomes from patients with different rASRM (revised American Society for Reproductive Medicine) endometriosis stages and controls were analyzed. Illumina sequencing libraries were constructed using a two-step 16S rRNA gene PCR amplicon approach. Correlations of 16S rRNA gene amplicon data with clinical metadata were conducted using a random forest-based machine-learning classification analysis. Distribution of vaginal CSTs (community state types) significantly differed between follicular and menstrual phases of the menstrual cycle (p = 0.021, Fisher's exact test). Vaginal and rectal microbiome profiles and their association to severity of endometriosis (according to rASRM stages) were evaluated. Classification models built with machine-learning methods on the microbiota composition during follicular and menstrual phases of the cycle were built, and it was possible to accurately predict rASRM stages 1-2 verses rASRM stages 3-4 endometriosis. The feature contributing the most to this prediction was an OTU (operational taxonomic unit) from the genus Anaerococcus. Gut and vaginal microbiomes of women with endometriosis have been investigated. Our findings suggest for the first time that vaginal microbiome may predict stage of disease when endometriosis is present.

Diets with and without edible cricket support a similar level of diversity in the gut microbiome of dogs.

The gut microbiome plays an important role in the health of dogs. Both beneficial microbes and overall diversity can be modulated by diet. Fermentable sources of fiber in particular often increase the abundance of beneficial microbes. Banded crickets (Gryllodes sigillatus) contain the fermentable polysaccharides chitin and chitosan. In addition, crickets are an environmentally sustainable protein source. Considering crickets as a potential source of both novel protein and novel fiber for dogs, four diets ranging from 0% to 24% cricket content were fed to determine their effects on healthy dogs’ (n = 32) gut microbiomes. Fecal samples were collected serially at 0, 14, and 29 days, and processed using high-throughput sequencing of 16S rRNA gene PCR amplicons. Microbiomes were generally very similar across all diets at both the phylum and genus level, and alpha and beta diversities did not differ between the various diets at 29 days. A total of 12 ASVs (amplicon sequence variants) from nine genera significantly changed in abundance following the addition of cricket, often in a dose-response fashion with increasing amounts of cricket. A net increase was observed in Catenibacterium, Lachnospiraceae [Ruminococcus], and Faecalitalea, whereas Bacteroides, Faecalibacterium, Lachnospiracaeae NK4A136 group and others decreased in abundance. Similar changes in Catenibacterium and Bacteroides have been associated with gut health benefits in other studies. However, the total magnitude of all changes was small and only a few specific taxa changed in abundance. Overall, we found that diets containing cricket supported the same level of gut microbiome diversity as a standard healthy balanced diet. These results support crickets as a potential healthy, novel food ingredient for dogs.

Unraveling the diversity of sedimentary sulfate-reducing prokaryotes (SRP) across Tibetan saline lakes using epicPCR

Sulfate reduction is an important biogeochemical process in the ecosphere; however, the major taxa of sulfate reducers have not been fully identified. Here, we used epicPCR (Emulsion, Paired Isolation, and Concatenation PCR) technology to identify the phylogeny of sulfate-reducing prokaryotes (SRP) in sediments from Tibetan Plateau saline lakes. A total of 12,519 OTUs and 883 SRP-OTUs were detected in ten lakes by sequencing of 16S rRNA gene PCR amplicons and epicPCR products of fused 16S rRNA plus dsrB gene, respectively, with Proteobacteria, Firmicutes, and Bacteroidetes being the dominant phyla in both datasets. The 120 highly abundant SRP-OTUs (> 1% in at least one sample) were affiliated with 17 described phyla, only 7 of which are widely recognized as SRP phyla. The majority of OTUs from both the whole microbial communities and the SRPs were not detected in more than one specific lake, suggesting high levels of endemism. The α-diversity of the entire microbial community and SRP sub-community showed significant positive correlations. The pH value and mean water temperature of the month prior to sampling were the environmental determinants for the whole microbial community, while the mean water temperature and total nitrogen were the major environmental drivers for the SRP sub-community. This study revealed there are still many undocumented SRP in Tibetan saline lakes, many of which could be endemic and adapted to specific environmental conditions.

Prebiotic supplementation over a cold season and during antibiotic treatment specifically modulates the gut microbiota composition of 3-6 year-old children.

Supplementing kindergarten children during a cold season with a prebiotic inulin-type fructans product with shorter and longer fructan chains has been shown to reduce febrile episodes requiring medical attention and to lower the incidence of sinusitis. These beneficial effects may be connected to the specific modulation of children's gut microbiota. By applying quantitative and qualitative microbiota analysis this study aimed at characterising the gut microbiota composition and at exploring effects of prebiotic intervention on the gut microbiota during a 24-weeks intervention and during antibiotic treatment in healthy children. The study was a randomised, placebo-controlled trial with 258 healthy children aged 3 to 6 years consuming 6 g/day prebiotic inulin-type fructans or maltodextrin. During the course of the study, faecal samples were collected and subject to targeted qPCR analysis and phylogenetic profiling by multiplexed high throughput sequencing of the prokaryotic 16S rRNA gene PCR amplicons. The microbiota composition of the cohort could be clustered into three distinct constellations (enterotypes). Prebiotic intake resulted in a selective modulation of the gut microbiota composition. Relative abundance of Bifidobacterium was significantly higher in the prebiotic group (n=104) compared to control group (n=105) and this effect was found for all three enterotypes. Antibiotic administration decreased the relative abundance of Bifidobacterium in both groups. Nonetheless, children of the prebiotic group receiving antibiotic treatment displayed significantly higher levels of Bifidobacterium than children receiving the placebo control. Prebiotic supplementation induced specific changes in the gut microbiota composition of children aged 3 to 6 years. Moreover, it attenuated antibiotic-induced disturbances in the gut microbiota composition as shown by higher relative abundance of bifidobacteria at the end of the antibiotic treatment in the prebiotic group. With the previously reported benefits on immune function, the study contributes to the evidence on the immune-modulating effects of prebiotics through gut microbiota modifications. The study was registered as NCT03241355 ( https://clinicaltrials.gov/show/NCT03241355 ).

Community composition of arbuscular mycorrhizal fungi associated with native plants growing in a petroleum-polluted soil of the Amazon region of Ecuador.

Arbuscular mycorrhizal fungi (AMF) are worldwide distributed plant symbionts. However, their occurrence in hydrocarbon‐polluted environments is less investigated, although specific communities may be present with possible interest for remediation strategies. Here, we investigated the AMF community composition associated with the roots of diverse plant species naturally recolonizing a weathered crude oil pond in the Amazon region of Ecuador. Next generation 454 GS‐Junior sequencing of an 800 bp LSU rRNA gene PCR amplicon was used. PCR amplicons were affiliated to a maximum‐likelihood phylogenetic tree computed from 1.5 kb AMF reference sequences. A high throughput phylogenetic annotation approach, using an evolutionary placement algorithm (EPA) allowed the characterization of sequences to the species level. Fifteen species were detected. Acaulospora species were identified as dominant colonizers, with 73% of relative read abundance, Archaeospora (19.6%) and several genera from the Glomeraceae (Rhizophagus, Glomus macrocarpum‐like, Sclerocystis, Dominikia and Kamienskia) were also detected. Although, a diverse community belonging to Glomeraceae was revealed, they represented <10% of the relative abundance in the Pond. Seventy five % of the species could not be identified, suggesting possible new species associated with roots of plants under highly hydrocarbon‐polluted conditions.

Beneficial changes in rumen bacterial community profile in sheep and dairy calves as a result of feeding the probiotic Bacillus amyloliquefaciens H57.

The probiotic Bacillus amyloliquefaciens H57 increased weight gain, increased nitrogen retention and increased feed intake in ruminants when administered to the diet. This study aims to develop a better understanding of this probiotic effect by analysing changes in the rumen prokaryotic community. Sequencing the 16S rRNA gene PCR amplicons of the rumen microbiome, revealed that ewes fed H57 had a significantly different rumen microbial community structure to Control sheep. In contrast, dairy calves showed no significant differences in rumen community structure between treatment groups. In both instances, H57 was below detection in the rumen community profile and was only present at low relative abundance as determined by qPCR. The altered rumen microbial community in sheep likely contributes to increased weight gain through more efficient digestion of plant material. As no change occurred in the rumen community of dairy calves it is suggested that increased weight gain may be due to changes in community function rather than structure. The low relative abundance of H57 as determined by qPCR, suggests that weight gain was not directly mediated by the probiotic, but rather by influencing animal behaviour (feed consumption) and/or altering the native rumen community structure or function. This study provides a novel look at the rumen prokaryotic community in both sheep and dairy calves when fed H57. These findings improve our understanding for the potential rumen community involvement in H57-enabled weight gain. The study reveals that the probiotic B. amyloliquefaciens H57 is capable of benefiting ruminants without colonizing the rumen, suggesting an indirect mechanism of action.

Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, ‘universal’ SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by ‘universal’ primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology.

Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate 13C-acetate in coastal marine sediment.

Stable isotope probing (SIP) of deoxyribonucleic acid (DNA) was used to identify microbes incorporating (13) C-labeled acetate in sulfate-reducing sediment from Aarhus Bay, Denmark. Sediment was incubated in medium containing 10 mM sulfate and different (13) C-acetate (10, 1, 0.1 mM) concentrations. The resultant changes in microbial community composition were monitored in total and SIP-fractionated DNA during long-term incubations. Chemical analyses demonstrated metabolic activity in all sediment slurries, with sulfate-reducing activity largely determined by initial acetate concentrations. Sequencing of 16S rRNA gene PCR amplicons showed that the incubations shifted the bacterial but not the archaeal community composition. After 3 months of incubation, only sediment slurries incubated with 10 mM (13) C-acetate showed detectable (13) C-DNA labeling. Based on 16S rRNA and dsrB gene PCR amplicon sequencing, the (13) C-labeled DNA pool was dominated by a single type of sulfate reducer representing a novel genus in the family Desulfobacteraceae. In addition, members of the uncultivated Crenarchaeotal group C3 were enriched in the (13) C-labeled DNA. Our results were reproducible across biological replicate experiments and provide new information about the identities of uncultured acetate-consuming bacteria and archaea in marine sediments.

A conserved arbuscular mycorrhizal fungal core-species community colonizes potato roots in the Andes

Plant-symbiotic arbuscular mycorrhizal fungi (AMF) are of high global ecological and economic importance, but describing environmental communities of AMF at the species level remains a challenge, despite the need to understand AMF-plant preferences and to apply AMF in sustainable agriculture. Here, the potato-associated AMF species community composition was assessed for three Andean countries along an altitudinal gradient and at different plant stages, by using 454 GS-FLX+ sequencing of a 760 bp LSU rRNA gene PCR amplicon. Two methods were compared: defining OTUs based on a simple sequence similarity threshold, or affiliating reference sequences to species based on a high throughput phylogenetic annotation approach using an evolutionary placement algorithm (EPA). The EPA-based approach was not only more precise, but also fundamental to robustly unveil the AMF species community composition. The principal advantage of this approach was also demonstrated by using artificially constructed datasets based on validated public database sequences. The affiliation of sequence reads to species using phylogenetic annotation revealed a surprisingly conserved AMF core-species community structure in Andean potatoes, regardless of different plant stages and environmental factors. In total, 41 species were detected and in some cases more than 25 species were found colonizing an individual root system. Acaulospora species were identified as dominant colonizers, co-occurring with Cetraspora nodosa and certain Claroideoglomus and Rhizophagus species in most potato root samples.

Conversion of activated-sludge reactors to microbial fuel cells for wastewater treatment coupled to electricity generation

Wastewater can be treated in microbial fuel cells (MFCs) with the aid of microbes that oxidize organic compounds using anodes as electron acceptors. Previous studies have suggested the utility of cassette-electrode (CE) MFCs for wastewater treatment, in which rice paddy-field soil was used as the inoculum. The present study attempted to convert an activated-sludge (AS) reactor to CE-MFC and use aerobic sludge in the tank as the source of microbes. We used laboratory-scale (1L in capacity) reactors that were initially operated in an AS mode to treat synthetic wastewater, containing starch, yeast extract, peptone, plant oil, and detergents. After the organics removal became stable, the aeration was terminated, and CEs were inserted to initiate an MFC-mode operation. It was demonstrated that the MFC-mode operation treated the wastewater at similar efficiencies to those observed in the AS-mode operation with COD-removal efficiencies of 75-80%, maximum power densities of 150-200mWm(-2) and Coulombic efficiencies of 20-30%. These values were similar to those of CE-MFC inoculated with the soil. Anode microbial communities were analyzed by pyrotag sequencing of 16S rRNA gene PCR amplicons. Comparative analyses revealed that anode communities enriched from the aerobic sludge were largely different from those from the soil, suggesting that similar reactor performances can be supported by different community structures. The study demonstrates that it is possible to construct wastewater-treatment MFCs by inserting CEs into water-treatment tanks.

Use of denaturing high-performance liquid chromatography (DHPLC) to characterize the bacterial and fungal airway microbiota of cystic fibrosis patients

The aim of this study was to evaluate the use of denaturing high-performance liquid chromatography (DHPLC) to characterize cystic fibrosis (CF) airway microbiota including both bacteria and fungi. DHPLC conditions were first optimized using a mixture of V6, V7 and V8 region 16S rRNA gene PCR amplicons from 18 bacterial species commonly found in CF patients. Then, the microbial diversity of 4 sputum samples from 4 CF patients was analyzed using cultural methods, cloning/sequencing (for bacteria only) and DHPLC peak fraction collection/sequencing. DHPLC analysis allowed identifying more bacterial and fungal species than the classical culture methods, including well-recognized pathogens such as Pseudomonas aeruginosa. Even if a lower number of bacterial Operational Taxonomic Units (OTUs) was identified by DHPLC, it allowed to find OTUs unidentified by cloning/sequencing. The combination of both techniques permitted to correlate the majority of DHPLC peaks to defined OTUs. Finally, although Aspergillus fumigatus detection using DHPLC can still be improved, this technique clearly allowed to identify a higher number of fungal species versus classical culture-based methods. To conclude, DHPLC provided meaningful additional data concerning pathogenic bacteria and fungi as well as fastidious microorganisms present within the CF respiratory tract. DHPLC can be considered as a complementary technique to culture-dependent analyses in routine microbiological laboratories.

Analysis of bacterial community shifts in the gastrointestinal tract of pigs fed diets supplemented with β-glucan from Laminaria digitata, Laminaria hyperborea and Saccharomyces cerevisiae

This study was designed to evaluate the effects of algal and yeast β-glucans on the porcine gastrointestinal microbiota, specifically the community of Lactobacillus, Bifidobacterium and coliforms. A total of 48 pigs were fed four diets over a 28-day period to determine the effect that each had on these communities. The control diet consisted of wheat and soya bean meal. The remaining three diets contained wheat and soya bean meal supplemented with β-glucan at 250 g/tonne from Laminaria digitata, Laminaria hyperborea or Saccharomyces cerevisiae. Faecal samples were collected from animals before feeding each diet and after the feeding period. The animals were slaughtered the following day and samples were collected from the stomach, ileum, caecum, proximal colon and distal colon. Alterations in Lactobacillus in the gastrointestinal tract (GIT) were analysed using denaturing gradient gel electrophoresis (DGGE) profiles generated by group-specific 16S rRNA gene PCR amplicons. Plate count analysis was also performed to quantify total coliforms. DGGE profiles indicated that all β-glucan diets provoked the emergence of a richer community of Lactobacillus. The richest community of lactobacilli emerged after feeding L. digitata (LD β-glucan). Plate count analysis revealed that the L. hyperborea (LH β-glucan) diet had a statistically significant effect on the coliform counts in the proximal colon in comparison with the control diet. β-glucan from L. digitata and S. cerevisiae also generally reduced coliforms but to a lesser extent. Nevertheless, the β-glucan diets did not significantly reduce levels of Lactobacillus or Bifidobacterium. DGGE analysis of GIT samples indicated that the three β-glucan diets generally promoted the establishment of a more varied range of Lactobacillus species in the caecum, proximal and distal colon. The LH β-glucan had the most profound reducing effect on coliform counts when compared with the control diet and diets supplemented with L. digitata and S. cerevisiae β-glucans.

Groundtruthing Next-Gen Sequencing for Microbial Ecology–Biases and Errors in Community Structure Estimates from PCR Amplicon Pyrosequencing

Analysis of microbial communities by high-throughput pyrosequencing of SSU rRNA gene PCR amplicons has transformed microbial ecology research and led to the observation that many communities contain a diverse assortment of rare taxa–a phenomenon termed the Rare Biosphere. Multiple studies have investigated the effect of pyrosequencing read quality on operational taxonomic unit (OTU) richness for contrived communities, yet there is limited information on the fidelity of community structure estimates obtained through this approach. Given that PCR biases are widely recognized, and further unknown biases may arise from the sequencing process itself, a priori assumptions about the neutrality of the data generation process are at best unvalidated. Furthermore, post-sequencing quality control algorithms have not been explicitly evaluated for the accuracy of recovered representative sequences and its impact on downstream analyses, reducing useful discussion on pyrosequencing reads to their diversity and abundances. Here we report on community structures and sequences recovered for in vitro-simulated communities consisting of twenty 16S rRNA gene clones tiered at known proportions. PCR amplicon libraries of the V3–V4 and V6 hypervariable regions from the in vitro-simulated communities were sequenced using the Roche 454 GS FLX Titanium platform. Commonly used quality control protocols resulted in the formation of OTUs with >1% abundance composed entirely of erroneous sequences, while over-aggressive clustering approaches obfuscated real, expected OTUs. The pyrosequencing process itself did not appear to impose significant biases on overall community structure estimates, although the detection limit for rare taxa may be affected by PCR amplicon size and quality control approach employed. Meanwhile, PCR biases associated with the initial amplicon generation may impose greater distortions in the observed community structure.

Prevalence of a Novel Division-Level Bacterial Lineage in Lake Dhanmondi, Dhaka, Bangladesh, as Revealed by Deep Sequencing of 16S rRNA Gene Amplicons

A culture-independent study of the bacterial diversity in Lake Dhanmondi, located in the central region of Dhaka city, Bangladesh, was carried out using deep sequence analysis of 16S rRNA gene PCR amplicons. The results revealed the presence of a group of bacteria, termed LD11, phylogenetically unrelated to any previously cultivated bacteria at the phylum level. LD11 sequences comprised about 1.7% of the total sequence reads after quality assessment. LD11 appears to constitute a novel division with a deep evolutionary lineage apparently branching between the Chloroflexi and Thermi-Deinococci phyla. Sequence similarity with molecular data from freshwater environments indicates that LD11 represents a widespread and novel clade of freshwater bacteria for which no cultivated representatives are yet available.