BACKGROUND & AIMS-Non-alcoholic fatty liver disease affects up to 30% of the U.S. population, but the mechanisms underlying this condition are incompletely understood. We investigated how diet standardization and choline deficiency influence the composition of the microbial community in the human gastrointestinal (GI) tract and the development of fatty liver under conditions of choline deficiency.

We report the results of pyrosequencing of DNA collected from the activated sludge basin of a wastewater treatment plant in Charlotte, NC. Using the 454-FLX technology, we generated 378,601 sequences with an average read length of 250.4 bp. Running the 454 assembly algorithm over our sequences yielded very poor assembly, with only 0.3% of our sequences participating in assembly of significant contigs. Of the 117 contigs greater than 500 bp long that were assembled, the most common annotations were to transposases and hypothetical proteins. Comparing our sequences to known microbial genomes showed nonspecific recruitment, indicating that previously described taxa are only distantly related to the most abundant microbes in this treatment plant. A comparison of proteins generated by translating our sequence set to translations of other sequenced microbiomes shows a distinct metabolic profile for activated sludge with high counts for genes involved in metabolism of aromatic compounds and low counts for genes involved in photosynthesis. Taken together, these data document the substantial levels of microbial diversity within activated sludge and further establish the great utility of pyrosequencing for investigating diversity in complex ecosystems.Although largely invisible in the urban landscape when they are functioning well, wastewater treatment plants are integral to the municipal obligation to protect public health, aquatic ecosystems, and the quality of life. At the heart of wastewater treatment plants is a process whereby a dense microbial consortium is employed to remove organic and nutrient contaminants. The microbes used to treat wastewater are a crucial tool in environmental protection. The current use of molecular techniques that do not require the isolation and cultivation of microorganisms (1, 33), including 16S rRNA analysis (6,13,20) and fluorescent in situ hybridization (8), have greatly expanded our understanding of wastewater microbial communities. Researchers have identified many bacteria of importance to wastewater treatment, including the bacteria involved in biological phosphorus removal (5, 16, 29), nitrifiers (8, 19, 25), denitrifiers (3, 12, 17), and methanogens (18, 36). Molecular techniques have also improved our understanding of fundamental processes such as nitrification and denitrification, as well as plant upsets, such as foaming (9, 24), which can decrease treatment efficiency.In this paper, we apply recently developed pyrosequencing technology to probe the molecular diversity of the aerobic basin of a wastewater treatment plant in Charlotte, NC. In line with other studies of complex microbial communities (28, 32), we observed astounding levels of diversity. We found that substantial regions of the genomes of the most prevalent microbes in the wastewater treatment plant are poorly described by existing sequence databases. Our results demonstrate that despite recent technological advances that allow identification of microorganisms, the microbial population of wastewater treatment plan...

Even in the absence of weight gain, fructose rapidly causes liver damage that we suggest is secondary to endotoxemia and MT. HS relates to the duration of fructose consumption and total calories consumed. These data support fructose inducing both MT and ectopic fat deposition in primates.

When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6, and V6-V7 variable regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Pronounced differences between primer sets, however, occurred in the "rare biosphere" involving taxa that we observed in fewer than 11 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest-neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, we observed pronounced differences between these analysis schemes in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere, both the experimental conditions and analysis choices will have a profound impact on the observed results.For nearly 3 decades, investigations of the distribution of microbes in complex environments have focused on the use of rRNA genes (1,2,4,11,16,18,19,22,24). Because the full-length 16S rRNA sequence can be obtained with pairedend reads via traditional Sanger sequencing, until recently most studies of the 16S rRNA gene captured most or nearly most of the 16S sequence length. New pyrosequencing technologies, however, have recently been introduced that greatly reduce the per base cost of sequencing but with shorter read lengths than traditional Sanger sequencing (17). This new approach has proven powerful, yielding a previously unobtainable view of rare taxa (7,(12)(13)(14)25).The shorter reads produced by pyrosequencing require the choice of a particular region of the 16S rRNA gene to target for pyrosequencing as well as the choice of an algorithm to classify the taxonomy of the shorter reads. In their initial surveys of microbial diversity with pyrosequencing (12, 14, 25), Sogin and colleagues targeted the V6 variable region, in part because it is was small enough to be captured with the 100-bp reads of the pyrosequencing technology available at the time. Recently, the read length of 454 pyrosequencing machines has been increased to an average of ϳ250 bp. This allows for more flexibility in primer design and opens up the possibility of targeting regions of the 16S rRNA gene other than V6. In recent work, Huse et al. took advantage of this new capability to compare the classifications made for the human gut microbiome with the V6 and longer V3 regions (13). Plotti...

The evolution of pulmonary disease in cystic fibrosis (CF) usually begins when bacteria get trapped in mucus in the lungs and become established as a chronic infection. While most CF patients experience periods of stability, pulmonary exacerbations (PEs) can occur multiple times per year and result in permanent damage to the lungs. Little is known of the shift from a period of stability to a PE, but this shift is likely to be attributed to changes in the bacterial community. Here, we identified changes in the lung microbiota to determine if they reflect patient health, indicate the onset of exacerbations, or are related to antibiotic treatment. In contrast to most bacterial studies on CF, we collected weekly samples from an adult CF patient over a period of 3 years and performed quantitative PCR (qPCR) and Illumina sequencing on those samples. While many DNA-based studies have shown the CF microbiota to be relatively stable, we observed an increase in the total bacterial abundance over time (P < 0.001), while the number of different taxa (bacterial richness) and the number of different taxa and their abundances (diversity) significantly decreased over time (P < 0.03), which was likely due to repeated antibiotic exposure. Using genus-specific primers with qPCR, we observed an increase in the abundance of Burkholderia multivorans, a CF-associated pathogen, prior to the occurrence of a PE (P ‫؍‬ 0.006). Combining these DNA-based techniques with frequent sampling identified a potential initiator for exacerbations and described a response of the CF microbiota to time and antibiotic treatment not observed in previous CF microbiota studies. Bacterial infections with consequent progressive lung disease are the leading cause of death in persons with cystic fibrosis (CF), a disease that affects an estimated 30,000 people in the United States and 70,000 people worldwide (1). Prior to the past 2 decades, it was assumed that the CF lungs were colonized with only a few different bacteria, including Pseudomonas aeruginosa, Haemophilus influenzae, Staphylococcus aureus, and members of the Burkholderia cepacia complex (BCC) (2). It has been shown in CF patients that chronic infection with these CF-related bacteria (CFRB) is linked to an increase in mortality (3, 4).Pulmonary exacerbations (PEs), which may develop multiple times per year in CF patients, are often caused by a disturbance to a stable chronic bacterial infection (5, 6). The exact cause of a PE, often identified by an increase in pulmonary disease symptoms, remains uncertain but is commonly attributed to factors associated with established bacteria, and possibly to viruses or newly acquired bacterial strains (7,8). In 2004, Rogers et al. used terminal restriction fragment length polymorphism (TRFLP) analysis to target the bacterial 16S rRNA gene in order to analyze DNA extracted from sputum samples from CF patients. This method of analysis revealed a complexity that included 15 species not previously identified in the lungs. The study by Rogers et al. laid the founda...