scholarly journals Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
William H. DePas ◽  
Ruth Starwalt-Lee ◽  
Lindsey Van Sambeek ◽  
Sripriya Ravindra Kumar ◽  
Viviana Gradinaru ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Growth Rate ◽  
Microbial Communities ◽  
Spatial Organization ◽  
Single Cells ◽  
Host Cells ◽  
Individual Species ◽  
Complex Environments ◽  
Host Interactions

ABSTRACT Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus , Pseudomonas aeruginosa , Streptococcus sp., and Achromobacter xylosoxidans , we demonstrate a spectrum of aggregation states: from mostly single cells ( A. xylosoxidans ), to medium-sized clusters ( S. aureus ), to a mixture of single cells and large aggregates ( P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum. IMPORTANCE The advent of metagenomic and metatranscriptomic analyses has improved our understanding of microbial communities by empowering us to identify bacteria, calculate their abundance, and profile gene expression patterns in complex environments. We are still technologically limited, however, in regards to the many questions that bulk measurements cannot answer, specifically in assessing the spatial organization of microbe-microbe and microbe-host interactions. Here, we demonstrate the power of an enhanced optical clearing method, MiPACT, to survey important aspects of bacterial physiology (aggregation, host interactions, and growth rate), in situ , with preserved spatial information when coupled to rRNA detection by HCR. Our application of MiPACT-HCR to cystic fibrosis patient sputum revealed species-specific aggregation patterns, yet slow growth characterized the vast majority of bacterial cells regardless of their cell type. More broadly, MiPACT, coupled with fluorescent labeling, promises to advance the direct study of microbial communities in diverse environments, including microbial habitats within mammalian systems.

scholarly journals Highly Multiplexed Spatial Mapping of Microbial Communities

2019 ◽  
Author(s):  
Hao Shi ◽  
Warren Zipfel ◽  
Ilana Brito ◽  
Iwijn De Vlaminck
Keyword(s):  
Microbial Communities ◽  
Spatial Organization ◽  
Single Cells ◽  
Cost Effective ◽  
Probe Design ◽  
E Coli ◽  
Rich Diversity ◽  
Effective Technology ◽  
Phylogenetic Resolution

ABSTRACTMapping the complex biogeography of microbial communities in situ with high taxonomic and spatial resolution poses a major challenge because of the high density and rich diversity of species in environmental microbiomes and the limitations of optical imaging technology. Here, we introduce High Phylogenetic Resolution microbiome mapping by Fluorescence In-Situ Hybridization (HiPR-FISH), a versatile and cost-effective technology that uses binary encoding and spectral imaging and machine learning based decoding to create micron-scale maps of the locations and identities of hundreds of microbial species in complex communities. We demonstrate the ability of 10-bit HiPR-FISH to distinguish 1023 E. coli strains, each fluorescently labeled with a unique binary barcode. HiPR-FISH, in conjunction with custom algorithms for automated probe design and segmentation of single-cells in the native context of tissues, reveals the intricate spatial architectures formed by bacteria in the human oral plaque microbiome and disruption of spatial networks in the mouse gut microbiome in response to antibiotic treatment. HiPR-FISH provides a framework for analyzing the spatial organization of microbial communities in tissues and the environment at single cell resolution.

scholarly journals A metaproteomics method to determine carbon sources and assimilation pathways of species in microbial communities

2018 ◽  
Author(s):  
Manuel Kleiner ◽  
Xiaoli Dong ◽  
Tjorven Hinzke ◽  
Juliane Wippler ◽  
Erin Thorson ◽  
...  
Keyword(s):  
Microbial Community ◽  
Microbial Communities ◽  
Carbon Isotope ◽  
Isotope Ratio ◽  
Single Cells ◽  
Stable Carbon Isotope ◽  
Carbon Sources ◽  
Food Sources ◽  
Individual Species ◽  
Stable Carbon

AbstractMeasurements of the carbon stable isotope ratio (δ13C) are widely used in biology to address major questions regarding food sources and metabolic pathways used by organisms. Measurement of these so called stable carbon isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals have led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide limited taxonomic resolution, such as with the use of lipid biomarkers, or are limited in throughput, such as NanoSIMS imaging of single cells.Here we present “direct Protein-SIF” and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ13C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we show that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria-animal symbiosis showed that direct Protein-SIF confirms previous physiological hypotheses and can provide unexpected new insights into the symbionts’ metabolism. This confirms the usefulness of this new approach to accurately determine δ13C values for different species in microbial community samples.SignificanceTo understand the roles that microorganisms play in diverse environments such as the open ocean and the human intestinal tract, we need an understanding of their metabolism and physiology. A variety of methods such as metagenomics and metaproteomics exist to assess the metabolism of environmental microorganisms based on gene content and gene expression. These methods often only provide indirect evidence for which substrates are used by a microorganism in a community. The direct Protein-SIF method that we developed allows linking microbial species in communities to the environmental carbon sources they consume by determining their stable carbon isotope signature. Direct Protein-SIF also allows assessing which carbon fixation pathway is used by autotrophic microorganisms that directly assimilate CO2.

scholarly journals Time-series genome-centric analysis unveils bacterial response to operational disturbance in activated sludge

2019 ◽  
Author(s):  
María Victoria Pérez ◽  
Leandro D. Guerrero ◽  
Esteban Orellana ◽  
Eva L. Figuerola ◽  
Leonardo Erijman
Keyword(s):  
Growth Rate ◽  
Time Series ◽  
Wastewater Treatment ◽  
Activated Sludge ◽  
Microbial Communities ◽  
Wastewater Treatment Plant ◽  
Treatment Plant ◽  
Operating Conditions ◽  
The Face

ABSTRACTUnderstanding ecosystem response to disturbances and identifying the most critical traits for the maintenance of ecosystem functioning are important goals for microbial community ecology. In this study, we used 16S rRNA amplicon sequencing and metagenomics to investigate the assembly of bacterial populations in a full-scale municipal activated sludge wastewater treatment plant over a period of three years, including a period of nine month of disturbance, characterized by short-term plant shutdowns. Following the reconstruction of 173 metagenome-assembled genomes, we assessed the functional potential, the number of rRNA gene operons and thein situgrowth rate of microorganisms present throughout the time series. Operational disturbances caused a significant decrease in bacteria with a single copy of the ribosomal RNA (rrn) operon. Despite only moderate differences in resource availability, replication rates were distributed uniformly throughout time, with no differences between disturbed and stable periods. We suggest that the length of the growth lag phase, rather than the growth rate, as the primary driver of selection under disturbed conditions. Thus, the system could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions.IMPORTANCEIn this work we investigated the response of microbial communities to disturbances in a full-scale activated sludge wastewater treatment plant over a time-scale that included periods of stability and disturbance. We performed a genome-wide analysis, which allowed us the direct estimation of specific cellular traits, including the rRNA operon copy number and the in situ growth rate of bacteria. This work builds upon recent efforts to incorporate growth efficiency for the understanding of the physiological and ecological processes shaping microbial communities in nature. We found evidence that would suggest that activated sludge could maintain its function in the face of disturbance by recruiting bacteria with the capacity to rapidly resume growth under unsteady operating conditions. This paper provides relevant insights into wastewater treatment process, and may also reveal a key role for growth traits in the adaptive response of bacteria to unsteady environmental conditions.

scholarly journals Predicting global dynamics of spatial microbial communities from local interaction rules

2020 ◽  
Author(s):  
Simon van Vliet ◽  
Christoph Hauert ◽  
Martin Ackermann ◽  
Alma Dal Co
Keyword(s):  
Growth Rate ◽  
Microbial Communities ◽  
Molecular Mechanisms ◽  
Single Cells ◽  
Cell Types ◽  
Cell Interactions ◽  
Community Level ◽  
Global Dynamics ◽  
Mathematical Framework ◽  
Cell Cell

AbstractInteractions between cells drive biological processes across all of life, from microbes in the environment to cells in multicellular organisms. Interactions often arise in spatially structured settings, where cells mostly interact with their neighbors. A central question is how the properties of biological systems emerge from local interactions. This question is very relevant in the context of microbial communities, such as biofilms, where cells live close by in space and are connected via a dense network of biochemical interactions. To understand and control the functioning of these communities, it is essential to uncover how community-level properties, such as the community composition, spatial arrangement, and growth rate, arise from these interactions. Here, we develop a mathematical framework that can predict community-level properties from the molecular mechanisms underlying the cell-cell interactions for systems consisting of two cell types. Our predictions match quantitative measurements from an experimental cross-feeding community. For these cross-feeding communities, the community growth rate is reduced when cells interact only with few neighbors; as a result, some communities can co-exist in a well-mixed system, but not in a spatial one. In general, our framework shows that key molecular parameters underlying the cell-cell interactions (e.g. the uptake and leakage rates of molecules) determine community level properties. Our framework can be extended to a variety of systems of two interacting cell types, within and beyond the microbial world, and contributes to our understanding of how biological functions arise from interactions between single cells.

scholarly journals Spatial charting of single cell transcriptomes in tissues

2021 ◽  
Author(s):  
Nicholas Navin ◽  
Runmin Wei ◽  
Siyuan He ◽  
Shanshan Bai ◽  
Emi Sei ◽  
...  
Keyword(s):  
Single Cell ◽  
Spatial Organization ◽  
Spatial Information ◽  
Ductal Carcinoma ◽  
Single Cells ◽  
Cell Types ◽  
Spatial Structures ◽  
Tissue Sections ◽  
Normal Mouse Brain

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections, but do not have single cell genomic resolution. Here, we developed a computational approach called SChart, that combines these two datasets to achieve single cell spatial mapping of cell types, cell states and continuous phenotypes. We applied SChart to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues to validate our approach. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied SChart to identify subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data shows that SChart can accurately map single cells in diverse tissue types to resolve their spatial organization into cellular neighborhoods and tissue structures.

scholarly journals Visualization of loop extrusion by DNA nanoscale tracing in single human cells

2021 ◽  
Author(s):  
KS Beckwith ◽  
Ø Ødegård-Fougner ◽  
NR Morero ◽  
C Barton ◽  
F Schueder ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Spatial Organization ◽  
Driving Forces ◽  
Single Cells ◽  
Human Cells ◽  
Random Coil ◽  
Loop Formation ◽  
Chromosomal Dna ◽  
3D Genome

SummaryThe spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensates and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random structures such as chromatin compartments and topologically associating domains2,3. However, if the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we present LoopTrace, a FISH workflow for high-resolution reconstruction of 3D genome architecture without DNA denaturation. Classical fluorescence in situ hybridization approaches can link chromatin architecture to DNA sequence but disrupt chromatin structure at the critical nanoscale of individual loops. Our method conserves chromatin structure and can resolve the 3D-fold of chromosomal DNA with better than 5-kb-resolution in single human cells. Our results show that the chromatin fiber behaves as a random coil that can be further structured in a manner consistent with loop formation, explaining the emergence of topologically associated domain-like features in cell population averages. Mining a large amount of single-cell data computationally, we reveal chromatin folding intermediates consistent with progressive loop extrusion and stabilized loops, highlighting the power of our method to visualize the nanoscale features of genome organization in situ.

Probing the spatial organization of nucleic acids within cells by nonisotopic in situ hybridization

1992 ◽  
Vol 50 (1) ◽  
pp. 10-11
Author(s):  
Jeanne Bentley Lawrence
Keyword(s):  
In Situ Hybridization ◽  
Nucleic Acids ◽  
Spatial Organization ◽  
Experimental Approach ◽  
Single Cells ◽  
Potential Applications ◽  
A Cell ◽  
Differential Gene ◽  
High Degree

In situ hybridization is a powerful experimental approach that directly couples molecular and cytological information in a visual context. Advances in hybridization procedures over recent years, coupled with previously described non-isotopic labelling methods developed in a number of laboratories, now provide a way to detect nucleic acids within cells with a high degree of resolution and sensitivity. Adaptations of this technology allow either DNA or RNA to be detected and visualized either with the light microscope, using fluorescence or colorimetric methods, or with the electron microscope using antibodies conjugated to gold or peroxidase. The potential applications of this technology are relevant to numerous areas of biomedical research and range from the more straightforward study of differential gene expression in single cells within a population to the precise localization of individual genes or RNAs within the cytoplasm or nucleus of a cell.

The polymicrobial nature of airway infections in cystic fibrosis: Cangene Gold Medal LectureThis article is based on a presentation by Dr. Christopher Sibley at the 60th Annual Meeting of the Canadian Society of Microbiologists in Hamilton, Ontario, on 15 June 2010. Dr. Sibley was the recipient of the 2010 Cangene Gold Medal as the Canadian Graduate Student Microbiologist of the Year, an annual award sponsored by Cangene Corporation intended to recognize excellence in graduate research.

2011 ◽  
Vol 57 (2) ◽  
pp. 69-77 ◽  
Author(s):  
Christopher D. Sibley ◽  
Michael G. Surette
Keyword(s):  
Cystic Fibrosis ◽  
Microbial Communities ◽  
Gold Medal ◽  
Emergent Properties ◽  
Host Interactions ◽  
Airway Infections ◽  
Future Challenges ◽  
Culture Independent ◽  
Airway Microbiome ◽  
Dynamic Communities

Microbial communities characterize the airways of cystic fibrosis (CF) patients. Members of these diverse and dynamic communities can be thought of as pathogens, benign commensals, or synergens — organisms not considered pathogens in the traditional sense but with the capacity to alter the pathogenesis of the community through microbe–microbe or polymicrobe–host interactions. Very few bacterial pathogens have been implicated as clinically relevant in CF; however, the CF airway microbiome can be a reservoir of previously unrecognized but clinically relevant organisms. A combination of culture-dependent and culture-independent approaches provides a more comprehensive perspective of CF microbiology than either approach alone. Here we review these concepts, highlight the future challenges for CF microbiology, and discuss the implications for the management of CF airway infections. We suggest that the success of treatment interventions for chronic CF lung disease will rely on the context of the microbes within microbial communities. The microbiology of CF airways may serve as a model to investigate the emergent properties of other clinically relevant microbial communities in the human body.

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