Characterizing the diversity of active bacteria in soil by comprehensive stable isotope probing of DNA and RNA with (H2O)-O-18

Current limitations in culture-based methods have lead to a reliance on culture- independent approaches, based principally on the comparative analysis of primary semantides such as ribosomal gene sequences. DNA can be remarkably stable in some environments, so its presence does not indicate live bacteria, but extracted ribosomal RNA (rRNA) has previously been viewed as an indicator of active cells. Stable isotope probing (SIP) involves the incorporation of heavy isotopes into newly synthesized nucleic acids, and can be used to separate newly synthesized from existing DNA or rRNA. H2 18O is currently the only potential universal bacterial substrate suitable for SIP of entire bacterial communities. The aim of our work was to compare soil bacterial community composition as revealed by total versus SIP-labeled DNA and rRNA. Soil was supplemented with H2 18O and after 38 days the DNA and RNA were co-extracted. Heavy nucleic acids were separated out by CsCl and CsTFA density centrifugation. The 16S rRNA gene pools were characterized by DGGE and pyrosequencing, and the sequence results analyzed using mothur. The majority of DNA (~60%) and RNA (~75%) from the microcosms incubated with H2 18O were labeled by the isotope. The analysis indicated that total and active members of the same type of nucleic acid represented similar community structures, which suggested that most dominant OTUs in the total nucleic acid extracts contained active members. It also supported that H2 18O was an effective universal label for SIP for both DNA and RNA. DNA and RNA-derived diversity was dissimilar. RNA from this soil more comprehensively recovered bacterial richness than DNA because the most abundant OTUs were less numerous in RNA than DNAderived community data, and dominant OTU pools didn’t mask rare OTUs as much in RNA.SD00H296-081HG from the South Dakota Agricultural Experiment Station to V. S. B. E. A. R. was supported by a fellowship from the NASA South Dakota Space Grant Consortium. We acknowledge use of the SDSU-Functional Genomics Core Facility, supported by NSF/EPSCoR Grant No. 0091948, the South Dakota 2010 Drought Initiative, and the South Dakota Agricultural Experiment Station.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2045-8827hb201

The Effects of Unfermented and Fermented Cow and Sheep Milk on the Gut Microbiota

A variety of fermented foods have been linked to improved human health, but their impacts on the gut microbiome have not been well characterized. Dairy products are one of the most popular fermented foods and are commonly consumed worldwide. One area we currently lack data on is how the process of fermentation changes the gut microbiota upon digestion. What is even less well characterized are the possible differences between cow and other mammals’ milks. Our aim was to compare the impact of unfermented skim milk and fermented skim milk products (milk/yogurt) originating from two species (cow/sheep) on the gut microbiome using a rat model. Male Sprague-Dawley rats were fed a dairy-free diet supplemented with one of four treatment dairy drinks (cow milk, cow yogurt, sheep milk, sheep yogurt) for 2 weeks. The viable starter culture bacteria in the yogurts were depleted in this study to reduce their potential influence on gut bacterial communities. At the end of the study, cecal samples were collected and the bacterial community profiles determined via 16S rRNA high-throughput sequencing. Fermentation status drove the composition of the bacterial communities to a greater extent than their animal origin. While overall community alpha diversity did not change among treatment groups, the abundance of a number of taxa differed. The cow milk supplemented treatment group was distinct, with a higher intragroup variability and a distinctive taxonomic composition. Collinsella aerofaciens was of particularly high abundance (9%) for this group. Taxa such as Firmicutes and Lactobacillus were found in higher abundance in communities of rats fed with milk, while Proteobacteria, Bacteroidetes, and Parabacteroides were higher in yogurt fed rats. Collinsella was also found to be of higher abundance in both milk (vs. yogurt) and cows (vs. sheep). This research provides new insight into the effects of unfermented vs. fermented milk (yogurt) and animal origin on gut microbial composition in a healthy host. A number of differences in taxonomic abundance between treatment groups were observed. Most were associated with the effects of fermentation, but others the origin species, or in the case of cow milk, unique to the treatment group. Future studies focusing on understanding microbial metabolism and interactions, should help unravel what drives these differences

Antibiotic Treatment Drives the Diversification of the Human Gut Resistome

Despite the documented antibiotic-induced disruption of the gut microbiota, the impact of antibiotic intake on strain-level dynamics, evolution of resistance genes, and factors influencing resistance dissemination potential remains poorly understood. To address this gap we analyzed public metagenomic datasets from 24 antibiotic treated subjects and controls, combined with an in-depth prospective functional study with two subjects investigating the bacterial community dynamics based on cultivation-dependent and independent methods. We observed that short-term antibiotic treatment shifted and diversified the resistome composition, increased the average copy number of antibiotic resistance genes, and altered the dominant strain genotypes in an individual-specific manner. More than 30% of the resistance genes underwent strong differentiation at the single nucleotide level during antibiotic treatment. We found that the increased potential for horizontal gene transfer, due to antibiotic administration, was ∼3-fold stronger in the differentiated resistance genes than the non-differentiated ones. This study highlights how antibiotic treatment has individualized impacts on the resistome and strain level composition, and drives the adaptive evolution of the gut microbiota

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

GC‐Clamp Primer Batches Yield 16S rRNA Amplicon Pools with Variable GC Clamps, Affecting Denaturing Gradient Gel Electrophoresis Profiles

Fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pools have become a popular tool for comparisons between microbial communities. The GC‐clamp portion of primers for DGGE amplicon preparation provides a key component in resolving fragments of similar size but different sequence. We hypothesized that repeat syntheses of identical 40‐base GC‐clamp primers lead to different DGGE profiles. Three repeat syntheses of the same GC‐clamp primer and two different GC‐clamp primers directed at the V3–5 region of the 16S rRNA gene were compared. Genomic DNA of two separate soil bacterial communities and three bacterial species was amplified and resolved by DGGE. The DGGE profiles obtained with repeat‐synthesized primers differed among each other as much as with alternate primers, for both soil DNA and pure single species. The GC‐clamp portion of members of amplicon pools varied among each other, deviating from the design sequence, and was the likely cause for multiple bands derived from a single 16S rRNA gene sequence. We recommend procuring an oligonucleotide batch large enough to conduct an entire project. This should help to avoid any DGGE profile variations due to performance differences between repeat syntheses of GC‐clamp oligonucleotide primers

The Gut Microbiome Is Altered in Postmenopausal Women With Osteoporosis and Osteopenia

ABSTRACT Osteoporosis and its precursor osteopenia are common metabolic bone diseases in postmenopausal women. A growing body of evidence suggests that the gut microbiota is involved in the regulation of bone metabolism; however, there are few studies examining how gut microbiomes in osteoporosis and osteopenia may differ from those in healthy individuals. The aim of this study was to characterize the diversity, composition, and functional gene potential of the gut microbiota of healthy, osteopenic, and osteoporotic women. Body composition, bone density, and fecal metagenomes were analyzed in 86 postmenopausal women. The women were classified as healthy, osteopenic, or osteoporotic based on T‐scores. The taxonomic and functional gene compositions of the microbiome were analyzed using shotgun metagenomic sequencing. Both osteoporotic and osteopenic taxonomic compositions were found to be significantly different from healthy participants. Linear discriminant‐analysis effect‐size analyses identified that healthy participants had more unclassified Clostridia and methanogenic archaea (Methanobacteriaceae) than in both osteoporotic and osteopenic participants. Bacteroides was found to be more abundant in osteoporosis and osteopenia groups. Some KEGG pathways, including carbohydrate metabolism, biosynthesis of secondary metabolites, and cyanoamino acid metabolism, were found to be more abundant in both osteoporosis and osteopenia. These results show that osteoporosis and osteopenia alter the gut microbiome of postmenopausal women and identify potential microbial taxonomic and functional pathways that may be involved in this disease. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research

Transcriptional interactions suggest niche segregation among microorganisms in the human gut

The human gastrointestinal (GI) tract is the habitat for hundreds of microbial species, of which many cannot be cultivated readily, presumably because of the dependencies between species(1). Studies of microbial co-occurrence in the gut have indicated community substructures that may reflect functional and metabolic interactions between cohabiting species(2,3). To move beyond species co-occurrence networks, we systematically identified transcriptional interactions between pairs of coexisting gut microbes using metagenomics and microarray-based metatranscriptomics data from 233 stool samples from Europeans. In 102 significantly interacting species pairs, the transcriptional changes led to a reduced expression of orthologous functions between the coexisting species. Specific species-species transcriptional interactions were enriched for functions important for H-2 and CO2 homeostasis, butyrate biosynthesis, ATP-binding cassette (ABC) transporters, flagella assembly and bacterial chemotaxis, as well as for the metabolism of carbohydrates, amino acids and cofactors. The analysis gives the first insight into the microbial community-wide transcriptional interactions, and suggests that the regulation of gene expression plays an important role in species adaptation to coexistence and that niche segregation takes place at the transcriptional level