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Concept: Biotechnology


Dispersal of microbes between humans and the built environment can occur through direct contact with surfaces or through airborne release; the latter mechanism remains poorly understood. Humans emit upwards of 10(6) biological particles per hour, and have long been known to transmit pathogens to other individuals and to indoor surfaces. However it has not previously been demonstrated that humans emit a detectible microbial cloud into surrounding indoor air, nor whether such clouds are sufficiently differentiated to allow the identification of individual occupants. We used high-throughput sequencing of 16S rRNA genes to characterize the airborne bacterial contribution of a single person sitting in a sanitized custom experimental climate chamber. We compared that to air sampled in an adjacent, identical, unoccupied chamber, as well as to supply and exhaust air sources. Additionally, we assessed microbial communities in settled particles surrounding each occupant, to investigate the potential long-term fate of airborne microbial emissions. Most occupants could be clearly detected by their airborne bacterial emissions, as well as their contribution to settled particles, within 1.5-4 h. Bacterial clouds from the occupants were statistically distinct, allowing the identification of some individual occupants. Our results confirm that an occupied space is microbially distinct from an unoccupied one, and demonstrate for the first time that individuals release their own personalized microbial cloud.

Concepts: Archaea, Bacteria, Organism, Microbiology, Ribosomal RNA, Biotechnology, 16S ribosomal RNA, Microorganism


Microbial communities are ubiquitous in both natural and artificial environments. However, microbial diversity is usually reduced under strong selection pressures, such as those present in habitats rich in recalcitrant or toxic compounds displaying antimicrobial properties. Caffeine is a natural alkaloid present in coffee, tea and soft drinks with well-known antibacterial properties. Here we present the first systematic analysis of coffee machine-associated bacteria. We sampled the coffee waste reservoir of ten different Nespresso machines and conducted a dynamic monitoring of the colonization process in a new machine. Our results reveal the existence of a varied bacterial community in all the machines sampled, and a rapid colonisation process of the coffee leach. The community developed from a pioneering pool of enterobacteria and other opportunistic taxa to a mature but still highly variable microbiome rich in coffee-adapted bacteria. The bacterial communities described here, for the first time, are potential drivers of biotechnologically relevant processes including decaffeination and bioremediation.

Concepts: Natural selection, Bacteria, Microbiology, Biotechnology, Coffee, Caffeine, Tea, Microorganism


Wine grapes present a unique biogeography model, wherein microbial biodiversity patterns across viticultural zones not only answer questions of dispersal and community maintenance, they are also an inherent component of the quality, consumer acceptance, and economic appreciation of a culturally important food product. On their journey from the vineyard to the wine bottle, grapes are transformed to wine through microbial activity, with indisputable consequences for wine quality parameters. Wine grapes harbor a wide range of microbes originating from the surrounding environment, many of which are recognized for their role in grapevine health and wine quality. However, determinants of regional wine characteristics have not been identified, but are frequently assumed to stem from viticultural or geological factors alone. This study used a high-throughput, short-amplicon sequencing approach to demonstrate that regional, site-specific, and grape-variety factors shape the fungal and bacterial consortia inhabiting wine-grape surfaces. Furthermore, these microbial assemblages are correlated to specific climatic features, suggesting a link between vineyard environmental conditions and microbial inhabitation patterns. Taken together, these factors shape the unique microbial inputs to regional wine fermentations, posing the existence of nonrandom “microbial terroir” as a determining factor in regional variation among wine grapes.

Concepts: Bacteria, Biotechnology, Vitis vinifera, Wine, Grape, Vitis, Viticulture, Terroir


This paper describes a method of generating three-dimensional (3D) cell-laden microstructures by applying the principle of origami folding technique and cell traction force (CTF). We harness the CTF as a biological driving force to fold the microstructures. Cells stretch and adhere across multiple microplates. Upon detaching the microplates from a substrate, CTF causes the plates to lift and fold according to a prescribed pattern. This self-folding technique using cells is highly biocompatible and does not involve special material requirements for the microplates and hinges to induce folding. We successfully produced various 3D cell-laden microstructures by just changing the geometry of the patterned 2D plates. We also achieved mass-production of the 3D cell-laden microstructures without causing damage to the cells. We believe that our methods will be useful for biotechnology applications that require analysis of cells in 3D configurations and for self-assembly of cell-based micro-medical devices.

Concepts: DNA, Gene, Causality, Enzyme, Organism, Cell biology, Biotechnology, Cell wall


Understanding of soil processes is essential for addressing the global issues of food security, disease transmission and climate change. However, techniques for observing soil biology are lacking. We present a heterogeneous, porous, transparent substrate for in situ 3D imaging of living plants and root-associated microorganisms using particles of the transparent polymer, Nafion, and a solution with matching optical properties. Minerals and fluorescent dyes were adsorbed onto the Nafion particles for nutrient supply and imaging of pore size and geometry. Plant growth in transparent soil was similar to that in soil. We imaged colonization of lettuce roots by the human bacterial pathogen Escherichia coli O157:H7 showing micro-colony development. Micro-colonies may contribute to bacterial survival in soil. Transparent soil has applications in root biology, crop genetics and soil microbiology.

Concepts: Genetics, Bacteria, Organism, Microbiology, Escherichia coli, Biotechnology, Escherichia coli O157:H7, Root


Whole genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples this could further reduce diagnostic time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatics tools for analysis of the sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria and by directly sequencing on pellets from the urine. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples, but only in pure culture from 17. WGS improved the identification of the cultivated bacteria and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibility. Complete agreement was observed between species identification, multi-locus-sequence typing and phylogenetic relationship for the Escherichia coli and Enterococcus faecalis isolates when comparing the results of WGS of cultured isolates and directly from the urine samples. Sequencing directly from the urine enabled bacterial identification in polymicrobic samples. Additional putative pathogenic strains were observed in some culture negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic time. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem a publicly available bioinformatics tool was developed in this study.

Concepts: DNA, Bacteria, Molecular biology, Microbiology, Urinary tract infection, Urine, Escherichia coli, Biotechnology


The characterization of microbial community structure via 16S rRNA gene profiling has been greatly advanced in recent years by the introduction of amplicon pyrosequencing. The possibility of barcoding gives the opportunity to massively screen multiple samples from environmental or clinical sources for community details. However, an on-going debate questions the reproducibility and semi-quantitative rigour of pyrotag sequencing, similar to the early days of community fingerprinting. In this study we demonstrate the reproducibility of bacterial 454 pyrotag sequencing over biological and technical replicates of aquifer sediment bacterial communities. Moreover, we explore the potential of recovering specific template ratios via quantitatively defined template spiking to environmental DNA. We sequenced pyrotag libraries of triplicate sediment samples taken in annual sampling campaigns at a tar oil contaminated aquifer in Düsseldorf, Germany. The abundance of dominating lineages was highly reproducible with a maximal standard deviation of ~4% read abundance across biological, and ~2% across technical replicates. Our workflow also allows for the linking of read abundances within defined assembled pyrotag contigs to that of specific ‘in vivo’ fingerprinting signatures. Thus we demonstrate that both terminal restriction fragment length polymorphism (T-RFLP) analysis and pyrotag sequencing are capable of recovering highly comparable community structure. Overall diversity was roughly double in amplicon sequencing. Pyrotag libraries were also capable of linearly recovering increasing ratios (up to 20%) of 16S rRNA gene amendments from a pure culture of Aliivibrio fisheri spiked to sediment DNA. Our study demonstrates that 454 pyrotag sequencing is a robust and reproducible method, capable of reliably recovering template abundances and overall community structure within natural microbial communities.

Concepts: DNA, Scientific method, Molecular biology, Microbiology, Ribosomal RNA, Biotechnology, 16S ribosomal RNA, Restriction fragment length polymorphism


Advances in genome sequencing have progressed at a rapid pace, with increased throughput accompanied by plunging costs. But these advances go far beyond faster and cheaper. High-throughput sequencing technologies are now routinely being applied to a wide range of important topics in biology and medicine, often allowing researchers to address important biological questions that were not possible before. In this review, we discuss these innovative new approaches-including ever finer analyses of transcriptome dynamics, genome structure and genomic variation-and provide an overview of the new insights into complex biological systems catalyzed by these technologies. We also assess the impact of genotyping, genome sequencing and personal omics profiling on medical applications, including diagnosis and disease monitoring. Finally, we review recent developments in single-cell sequencing, and conclude with a discussion of possible future advances and obstacles for sequencing in biology and health.

Concepts: Medicine, Genetics, Biology, Biotechnology


Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic.

Concepts: DNA, Archaea, Bacteria, Microbiology, Ribosomal RNA, Biotechnology, 16S ribosomal RNA, Pathogenic bacteria


Massively parallel high throughput sequencing technologies allow us to interrogate the microbial composition of biological samples at unprecedented resolution. The typical approach is to perform high-throughout sequencing of 16S rRNA genes, which are then taxonomically classified based on similarity to known sequences in existing databases. Current technologies cause a predicament though, because although they enable deep coverage of samples, they are limited in the length of sequence they can produce. As a result, high-throughout studies of microbial communities often do not sequence the entire 16S rRNA gene. The challenge is to obtain reliable representation of bacterial communities through taxonomic classification of short 16S rRNA gene sequences. In this study we explored properties of different study designs and developed specific recommendations for effective use of short-read sequencing technologies for the purpose of interrogating bacterial communities, with a focus on classification using naïve Bayesian classifiers. To assess precision and coverage of each design, we used a collection of ∼8,500 manually curated 16S rRNA gene sequences from cultured bacteria and a set of over one million bacterial 16S rRNA gene sequences retrieved from environmental samples, respectively. We also tested different configurations of taxonomic classification approaches using short read sequencing data, and provide recommendations for optimal choice of the relevant parameters. We conclude that with a judicious selection of the sequenced region and the corresponding choice of a suitable training set for taxonomic classification, it is possible to explore bacterial communities at great depth using current technologies, with only a minimal loss of taxonomic resolution.

Concepts: DNA, Archaea, Bacteria, Ribosomal RNA, Biotechnology, Taxonomy, 16S ribosomal RNA, Sequence