Diversity of Microbial Communities in Production and Injection Waters of Algerian Oilfields Revealed by 16S rRNA Gene Amplicon 454 Pyrosequencing

The microorganisms inhabiting many petroleum reservoirs are multi-extremophiles capable of surviving in environments with high temperature, pressure and salinity. Their activity influences oil quality and they are an important reservoir of enzymes of industrial interest. To study these microbial assemblages and to assess any modifications that may be caused by industrial practices, the bacterial and archaeal communities in waters from four Algerian oilfields were described and compared. Three different types of samples were analyzed: production waters from flooded wells, production waters from non-flooded wells and injection waters used for flooding (water-bearing formations). Microbial communities of production and injection waters appeared to be significantly different. From a quantitative point of view, injection waters harbored roughly ten times more microbial cells than production waters. Bacteria dominated in injection waters, while Archaea dominated in production waters. Statistical analysis based on the relative abundance and bacterial community composition (BCC) revealed significant differences between production and injection waters at both OTUs_0.03 and phylum level. However, no significant difference was found between production waters from flooded and non-flooded wells, suggesting that most of the microorganisms introduced by the injection waters were unable to survive in the production waters. Furthermore, a Venn diagram generated to compare the BCC of production and injection waters of one flooded well revealed only 4% of shared bacterial OTUs. Phylogenetic analysis of bacterial sequences indicated that Alpha-, Beta- and Gammaproteobacteria were the main classes in most of the water samples. Archaeal sequences were only obtained from production wells and each well had a unique archaeal community composition, mainly belonging to Methanobacteria, Methanomicrobia, Thermoprotei and Halobacteria classes. Many of the bacterial genera retrieved had already been reported as degraders of complex organic molecules and pollutants. Nevertheless, a large number of unclassified bacterial and archaeal sequences were found in the analyzed samples, indicating that subsurface waters in oilfields could harbor new and still-non-described microbial species.     

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.     

Dynamics of Cathode-Associated Microbial Communities and Metabolite Profiles in a Glycerol-Fed Bioelectrochemical System

Electrical current can be used to supply reducing power to microbial metabolism. This phenomenon is typically studied in pure cultures with added redox mediators to transfer charge. Here, we investigate the development of a current-fed mixed microbial community fermenting glycerol at the cathode of a bioelectrochemical system in the absence of added mediators and identify correlations between microbial diversity and the respective product outcomes. Within 1 week of inoculation, a Citrobacter population represented 95 to 99% of the community and the metabolite profiles were dominated by 1,3-propanediol and ethanol. Over time, the Citrobacter population decreased in abundance while that of a Pectinatus population and the formation of propionate increased. After 6 weeks, several Clostridium populations and the production of valerate increased, which suggests that chain elongation was being performed. Current supply was stopped after 9 weeks and was associated with a decrease in glycerol degradation and alcohol formation. This decrease was reversed by resuming current supply; however, when hydrogen gas was bubbled through the reactor during open-circuit operation (open-circuit potential) as an alternative source of reducing power, glycerol degradation and metabolite production were unaffected. Cyclic voltammetry revealed that the community appeared to catalyze the hydrogen evolution reaction, leading to a +400-mV shift in its onset potential. Our results clearly demonstrate that current supply can alter fermentation profiles; however, further work is needed to determine the mechanisms behind this effect. In addition, operational conditions must be refined to gain greater control over community composition and metabolic outcomes.     

Shedding Light on the Microbial Community of the Macropod Foregut Using 454-Amplicon Pyrosequencing

Twenty macropods from five locations in Queensland, Australia, grazing on a variety of native pastures were surveyed and the bacterial community of the foregut was examined using 454-amplicon pyrosequencing. Specifically, the V3/V4 region of 16S rRNA gene was examined. A total of 5040 OTUs were identified in the data set (post filtering). Thirty-two OTUs were identified as ‘shared’ OTUS (i.e. present in all samples) belonging to either Firmicutes or Bacteroidetes (Clostridiales/Bacteroidales). These phyla predominated the general microbial community in all macropods. Genera represented within the shared OTUs included: unclassified Ruminococcaceae, unclassified Lachnospiraceae, unclassified Clostridiales, Peptococcus sp. Coprococcus spp., Streptococcus spp., Blautia sp., Ruminoccocus sp., Eubacterium sp., Dorea sp., Oscillospira sp. and Butyrivibrio sp. The composition of the bacterial community of the foregut samples of each the host species (Macropus rufus, Macropus giganteus and Macropus robustus) was significantly different allowing differentiation between the host species based on alpha and beta diversity measures. Specifically, eleven dominant OTUs that separated the three host species were identified and classified as: unclassified Ruminococcaceae, unclassified Bacteroidales, Prevotella spp. and a Syntrophococcus sucromutans. Putative reductive acetogens and fibrolytic bacteria were also identified in samples. Future work will investigate the presence and role of fibrolytics and acetogens in these ecosystems. Ideally, the isolation and characterization of these organisms will be used for enhanced feed efficiency in cattle, methane mitigation and potentially for other industries such as the biofuel industry.     

454高通量技术比较两种不同臭豆腐卤水中微生物多样性

利用454高通量技术对两种不同臭豆腐卤水中微生物不同分类水平的多样性进行了评估和统计分析。经过与SILVA数据库对比,发现了7个门、10个纲、17个目、26个科、24个菌属。经分析发现两个样品的微生物不同分类学差异显著,主要集中在属水平上,分布都非常不均一。在样品A中厌氧球菌属和卟啉单胞菌属所占的比例最高,分别为40.22%、37.54%,成为优势菌属。两个样品之间相同的微生物属只有6个,比例差别极大。在样品B中氨基杆菌属的比例很大,为86.94%,占绝对优势。说明卤水原料来源不同对微生物的差异显著,同时发现臭豆腐卤水的发酵过程只与少数主要菌种有关。     

The Effect of Long-Term Continuous Cropping of Black Pepper on Soil Bacterial Communities as Determined by 454 Pyrosequencing

In the present study, 3 replanted black pepper orchards with continuously cropping histories for 10, 21, and 55 years in tropical China, were selected for investigating the effect of monoculture on soil physiochemical properties, enzyme activities, bacterial abundance, and bacterial community structures. Results showed long-term continuous cropping led to a significant decline in soil pH, organic matter contents, enzymatic activities, and resulted in a decrease in soil bacterial abundance. 454 pyrosequencing analysis of 16S rRNA genes revealed that the Acidobacteria and Proteobacteria were the main phyla in the replanted black pepper orchard soils, comprising up to 73.82% of the total sequences; the relative abundances of Bacteroidetes and Firmicutes phyla decreased with long-term continuous cropping; and at genus level, the Pseudomonas abundance significantly depleted after 21 years continuous cropping. In addition, bacterial diversity significantly decreased after 55 years black pepper continuous cropping; obvious variations for community structures across the 3 time-scale replanted black pepper orchards were observed, suggesting monoculture duration was the major determinant for bacterial community structure. Overall, continuous cropping during black pepper cultivation led to a significant decline in soil pH, organic matter contents, enzymatic activities, resulted a decrease in soil bacterial abundance, and altered soil microbial community membership and structure, which in turn resulted in black pepper poor growth in the continuous cropping system.     

Strain Identification and Quantitative Analysis in Microbial Communities

Microbiology has long studied the ways in which subtle genetic differences between closely related microbial strains can have profound impacts on their phenotypes and those of their surrounding environments and communities. Despite the growth in high-throughput microbial community profiling, however, such strain-level differences remain challenging to detect. Once detected, few quantitative approaches have been well-validated for associating strain variants from microbial communities with phenotypes of interest, such as medication usage, treatment efficacy, host environment, or health. First, the term “strain” itself is not used consistently when defining a highly-resolved taxonomic or genomic unit from within a microbial community. Second, computational methods for identifying such strains directly from shotgun metagenomics are difficult, with several possible reference- and assembly-based approaches available, each with different sensitivity/specificity tradeoffs. Finally, statistical challenges exist in using any of the resulting strain profiles for downstream analyses, which can include strain tracking, phylogenetic analysis, or genetic association studies. We provide an in depth discussion of recently available computational tools that can be applied for this task, as well as statistical models and gaps in performing and interpreting any of these three main types of studies using strain-resolved shotgun metagenomic profiling of microbial communities.     

Survey of Microbial Diversity in Flood Areas during Thailand 2011 Flood Crisis Using High-Throughput Tagged Amplicon Pyrosequencing

The Thailand flood crisis in 2011 was one of the largest recorded floods in modern history, causing enormous damage to the economy and ecological habitats of the country. In this study, bacterial and fungal diversity in sediments and waters collected from ten flood areas in Bangkok and its suburbs, covering residential and agricultural areas, were analyzed using high-throughput 454 pyrosequencing of 16S rRNA gene and internal transcribed spacer sequences. Analysis of microbial community showed differences in taxa distribution in water and sediment with variations in the diversity of saprophytic microbes and sulfate/nitrate reducers among sampling locations, suggesting differences in microbial activity in the habitats. Overall, Proteobacteria represented a major bacterial group in waters, while this group co-existed with Firmicutes, Bacteroidetes, and Actinobacteria in sediments. Anaeromyxobacter, Steroidobacter, and Geobacter were the dominant bacterial genera in sediments, while Sulfuricurvum, Thiovirga, and Hydrogenophaga predominated in waters. For fungi in sediments, Ascomycota, Glomeromycota, and Basidiomycota, particularly in genera Philipsia, Rozella, and Acaulospora, were most frequently detected. Chytridiomycota and Ascomycota were the major fungal phyla, and Rhizophlyctis and Mortierella were the most frequently detected fungal genera in water. Diversity of sulfate-reducing bacteria, related to odor problems, was further investigated using analysis of the dsrB gene which indicated the presence of sulfate-reducing bacteria of families Desulfobacteraceae, Desulfobulbaceae, Syntrobacteraceae, and Desulfoarculaceae in the flood sediments. The work provides an insight into the diversity and function of microbes related to biological processes in flood areas.     

Bacterial Communities in the Gut and Reproductive Organs of Bactrocera minax (Diptera: Tephritidae) Based on 454 Pyrosequencing

The citrus fruit fly Bactrocera minax is associated with diverse bacterial communities. We used a 454 pyrosequencing technology to study in depth the microbial communities associated with gut and reproductive organs of Bactrocera minax. Our dataset consisted of 100,749 reads with an average length of 400 bp. The saturated rarefaction curves and species richness indices indicate that the sampling was comprehensive. We found highly diverse bacterial communities, with individual sample containing approximately 361 microbial operational taxonomic units (OTUs). A total of 17 bacterial phyla were obtained from the flies. A phylogenetic analysis of 16S rDNA revealed that Proteobacteria was dominant in all samples (75%–95%). Actinobacteria and Firmicutes were also commonly found in the total clones. Klebsiella, Citrobacter, Enterobacter, and Serratia were the major genera. However, bacterial diversity (Chao1, Shannon and Simpson indices) and community structure (PCA analysis) varied across samples. Female ovary has the most diverse bacteria, followed by male testis, and the bacteria diversity of reproductive organs is richer than that of the gut. The observed variation can be caused by sex and tissue, possibly to meet the host''s physiological demands.     

Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009–0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.     

AmpliconDuo: A Split-Sample Filtering Protocol for High-Throughput Amplicon Sequencing of Microbial Communities

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.     

Pyrosequencing Reveals Diverse and Distinct Sponge-Specific Microbial Communities in Sponges from a Single Geographical Location in Irish Waters

Marine sponges are host to numerically vast and phylogenetically diverse bacterial communities, with 26 major phyla to date having been found in close association with sponge species worldwide. Analyses of these microbial communities have revealed many sponge-specific novel genera and species. These endosymbiotic microbes are believed to play significant roles in sponge physiology including the production of an array of bioactive secondary metabolites. Here, we report on the use of culture-based and culture-independent (pyrosequencing) techniques to elucidate the bacterial community profiles associated with the marine sponges Raspailia ramosa and Stelligera stuposa sampled from a single geographical location in Irish waters and with ambient seawater. To date, little is known about the microbial ecology of sponges of these genera. Culture isolation grossly underestimated sponge-associated bacterial diversity. Four bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria) were represented amongst ~200 isolates, compared with ten phyla found using pyrosequencing. Long average read lengths of ~430 bp (V1-V3 region of 16S rRNA gene) allowed for robust resolution of sequences to genus level. Bacterial OTUs (2,109 total), at 95% sequence similarity, from ten bacterial phyla were recovered from R. ramosa, 349 OTUs were identified in S. stuposa representing eight phyla, while 533 OTUs from six phyla were found in surrounding seawater. Bacterial communities differed significantly between sponge species and the seawater. Analysis of the data for sponge-specific taxa revealed that 2.8% of classified reads from the sponge R. ramosa can be defined as sponge-specific, while 26% of S. stuposa sequences represent sponge-specific bacteria. Novel sponge-specific clusters were identified, whereas the majority of previously reported sponge-specific clusters (e.g. Poribacteria) were absent from these sponge species. This deep and robust analysis provides further evidence that the microbial communities associated with marine sponge species are highly diverse and divergent from one another and appear to be host-selected through as yet unknown processes.     

Bacterial Diversity Analysis of Zhenjiang Yao Meat During Refrigerated and Vacuum-Packed Storage by 454 Pyrosequencing

Zhenjiang Yao meat is a traditional variety of cooked marinated and jellied pork food in China. It is usually stored refrigerated to prevent gelatine liquefaction. Our understanding of the bacterial populations found in Zhenjiang Yao meat is limited. This study was designed to explore both the bacterial diversity and the main bacterial flora of Zhenjiang Yao meat using pyrosequencing of tagged amplicons from the V3 and V9 regions of the 16S rRNA gene. A total of 53,363 bacterial sequences from five samples and an average 10,672 reads of each sample were acquired and used in the analysis of microbial diversity. The bacterial diversity was observed to increase weekly, and the main bacterial flora changed significantly under refrigerated vacuum-packaged storage. The predominant bacteria were Vibrio during the first 7 days of storage, whereas Shewanella, Lactobacillus, Lactococcus, Yersinia, and other Enterobacteriaceae members were the main groups after 15 days in refrigerated storage. The bacterial population changed significantly after 15 days, and the bacterial communities in the 30 days sample were significantly different from all other samples. In comparison with culture-dependant method, pyrosequencing provides a more comprehensive estimate of bacterial diversity and more close to the real bacterial composition in Zhenjiang Yao meat.     

Pyrosequencing Reveals the Microbial Communities in the Red Sea Sponge Carteriospongia foliascens and Their Impressive Shifts in Abnormal Tissues

Abnormality and disease in sponges have been widely reported, yet how sponge-associated microbes respond correspondingly remains inconclusive. Here, individuals of the sponge Carteriospongia foliascens under abnormal status were collected from the Rabigh Bay along the Red Sea coast. Microbial communities in both healthy and abnormal sponge tissues and adjacent seawater were compared to check the influences of these abnormalities on sponge-associated microbes. In healthy tissues, we revealed low microbial diversity with less than 100 operational taxonomic units (OTUs) per sample. Cyanobacteria, affiliated mainly with the sponge-specific species “Candidatus Synechococcus spongiarum,” were the dominant bacteria, followed by Bacteroidetes and Proteobacteria. Intraspecies dynamics of microbial communities in healthy tissues were observed among sponge individuals, and potential anoxygenic phototrophic bacteria were found. In comparison with healthy tissues and the adjacent seawater, abnormal tissues showed dramatic increase in microbial diversity and decrease in the abundance of sponge-specific microbial clusters. The dominated cyanobacterial species Candidatus Synechococcus spongiarum decreased and shifted to unspecific cyanobacterial clades. OTUs that showed high similarity to sequences derived from diseased corals, such as Leptolyngbya sp., were found to be abundant in abnormal tissues. Heterotrophic Planctomycetes were also specifically enriched in abnormal tissues. Overall, we revealed the microbial communities of the cyanobacteria-rich sponge, C. foliascens, and their impressive shifts under abnormality.     

Next-Generation Pyrosequencing Analysis of Microbial Biofilm Communities on Granular Activated Carbon in Treatment of Oil Sands Process-Affected Water

The development of biodegradation treatment processes for oil sands process-affected water (OSPW) has been progressing in recent years with the promising potential of biofilm reactors. Previously, the granular activated carbon (GAC) biofilm process was successfully employed for treatment of a large variety of recalcitrant organic compounds in domestic and industrial wastewaters. In this study, GAC biofilm microbial development and degradation efficiency were investigated for OSPW treatment by monitoring the biofilm growth on the GAC surface in raw and ozonated OSPW in batch bioreactors. The GAC biofilm community was characterized using a next-generation 16S rRNA gene pyrosequencing technique that revealed that the phylum Proteobacteria was dominant in both OSPW and biofilms, with further in-depth analysis showing higher abundances of Alpha- and Gammaproteobacteria sequences. Interestingly, many known polyaromatic hydrocarbon degraders, namely, Burkholderiales, Pseudomonadales, Bdellovibrionales, and Sphingomonadales, were observed in the GAC biofilm. Ozonation decreased the microbial diversity in planktonic OSPW but increased the microbial diversity in the GAC biofilms. Quantitative real-time PCR revealed similar bacterial gene copy numbers (>10⁹ gene copies/g of GAC) for both raw and ozonated OSPW GAC biofilms. The observed rates of removal of naphthenic acids (NAs) over the 2-day experiments for the GAC biofilm treatments of raw and ozonated OSPW were 31% and 66%, respectively. Overall, a relatively low ozone dose (30 mg of O₃/liter utilized) combined with GAC biofilm treatment significantly increased NA removal rates. The treatment of OSPW in bioreactors using GAC biofilms is a promising technology for the reduction of recalcitrant OSPW organic compounds.     

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.     

Influence of DNA Extraction Method, 16S rRNA Targeted Hypervariable Regions,and Sample Origin on Microbial Diversity Detected by 454 Pyrosequencing in Marine Chemosynthetic Ecosystems

Next-generation sequencing (NGS) opens up exciting possibilities for improving our knowledge of environmental microbial diversity, allowing rapid and cost-effective identification of both cultivated and uncultivated microorganisms. However, library preparation, sequencing, and analysis of the results can provide inaccurate representations of the studied community compositions. Therefore, all these steps need to be taken into account carefully. Here we evaluated the effects of DNA extraction methods, targeted 16S rRNA hypervariable regions, and sample origins on the diverse microbes detected by 454 pyrosequencing in marine cold seep and hydrothermal vent sediments. To assign the reads with enough taxonomic precision, we built a database with about 2,500 sequences from Archaea and Bacteria from deep-sea marine sediments, affiliated according to reference publications in the field. Thanks to statistical and diversity analyses as well as inference of operational taxonomic unit (OTU) networks, we show that (i) while DNA extraction methods do not seem to affect the results for some samples, they can lead to dramatic changes for others; and (ii) the choice of amplification and sequencing primers also considerably affects the microbial community detected in the samples. Thereby, very different proportions of pyrosequencing reads were obtained for some microbial lineages, such as the archaeal ANME-1, ANME-2c, and MBG-D and deltaproteobacterial subgroups. This work clearly indicates that the results from sequencing-based analyses, such as pyrosequencing, should be interpreted very carefully. Therefore, the combination of NGS with complementary approaches, such as fluorescence in situ hybridization (FISH)/catalyzed reporter deposition (CARD)-FISH or quantitative PCR (Q-PCR), would be desirable to gain a more comprehensive picture of environmental microbial communities.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.