RESUMO
The human gut microbiota is a complex community of prokaryotic and eukaryotic microbes and viral particles that is increasingly associated with many aspects of host physiology and health. However, the classical microbiology approach of axenic culture cannot provide a complete picture of the complex interactions between microbes and their hosts in vivo. As such, recently there has been much interest in the culture of gut microbial ecosystems in the laboratory as a strategy to better understand their compositions and functions. In this review, we discuss the model platforms and methods available in the contemporary microbiology laboratory to study human gut microbiomes, as well as current knowledge surrounding the isolation of human gut microbes for the potential construction of defined communities for use in model systems.
Assuntos
Microbioma Gastrointestinal , Microbiota , HumanosRESUMO
MOTIVATION: Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species. RESULTS: isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections. AVAILABILITY AND IMPLEMENTATION: isolateR is available at: https://github.com/bdaisley/isolateR.
Assuntos
Bactérias , Software , Bactérias/genética , Bactérias/classificação , Análise de Sequência de DNA/métodos , Humanos , Archaea/genética , Fungos/genética , Biblioteca GênicaRESUMO
OBJECTIVES: We set out to identify and characterize prophages within genomes of published Fusobacterium strains, and to develop qPCR-based methods to characterize intra- and extra-cellular induction of prophage replication in a variety of environmental contexts. METHODS: Various in silico tools were used to predict prophage presence across 105 Fusobacterium spp. Genomes. Using the example of the model pathogen, Fusobacterium nucleatum subsp. animalis strain 7-1, qPCR was used with DNase I treatment to determine induction of its 3 predicted prophages ɸFunu1, ɸFunu2, and ɸFunu3, across several conditions. RESULTS: 116 predicted prophage sequences were found and analyzed. An emerging association between the phylogenetic history of a Fusobacterium prophage and that of its host was detected, as was the presence of genes encoding putative host fitness factors (e.g. ADP-ribosyltransferases) in distinct subclusters of prophage genomes. For strain 7-1, a pattern of expression for ɸFunu1, ɸFunu2, and ɸFunu3 was established indicating that ɸFunu1 and ɸFunu2 are capable of spontaneous induction. I Salt and mitomycin C exposure were able to promote induction of ɸFunu2. A range of other biologically relevant stressors, including exposure to pH, mucin and human cytokines showed no or minimal induction of these same prophages. ɸFunu3 induction was not detected under tested conditions. CONCLUSION: The heterogeneity of Fusobacterium strains is matched by their prophages. While the role of Fusobacterium prophages in host pathogenicity remains unclear, this work provides the first overview of clustered prophage distribution among this enigmatic genus and describes an effective assay for quantifying mixed samples of prophages that cannot be detected by plaque assay.
Assuntos
Fusobacterium , Prófagos , Humanos , Prófagos/genética , FilogeniaRESUMO
Many species, including humans, host communities of symbiotic microbes. There is a vast literature on the ways these microbiomes affect hosts, but here we argue for an increased focus on how hosts affect their microbiomes. Hosts exert control over their symbionts through diverse mechanisms, including immunity, barrier function, physiological homeostasis, and transit. These mechanisms enable hosts to shape the ecology and evolution of microbiomes and generate natural selection for microbial traits that benefit the host. Our microbiomes result from a perpetual tension between host control and symbiont evolution, and we can leverage the host's evolved abilities to regulate the microbiota to prevent and treat disease. The study of host control will be central to our ability to both understand and manipulate microbiotas for better health.
Assuntos
Evolução Biológica , Interações entre Hospedeiro e Microrganismos , Microbiota , Seleção Genética , Simbiose , Animais , Humanos , Homeostase , Interações entre Hospedeiro e Microrganismos/genética , Microbiota/genéticaRESUMO
Microbiomes feature complex interactions between diverse bacteria and bacteriophages. Synthetic microbiomes offer a powerful way to study these interactions; however, a major challenge is obtaining a representative bacteriophage population during the bacterial isolation process. We demonstrate that colony isolation reliably excludes virulent viruses from sample sources with low virion-to-bacteria ratios such as feces, creating "virulent virus-free" controls. When the virulent dsDNA virome is reintroduced to a 73-strain synthetic gut microbiome in a bioreactor model of the human colon, virulent viruses target susceptible strains without significantly altering community structure or metabolism. In addition, we detected signals of prophage induction that associate with virulent predation. Overall, our findings indicate that dilution-based isolation methods generate synthetic gut microbiomes that are heavily depleted, if not devoid, of virulent viruses and that such viruses, if reintroduced, have a targeted effect on community assembly, metabolism, and prophage replication.
Assuntos
Bactérias , Bacteriófagos , Fezes , Microbioma Gastrointestinal , Bacteriófagos/genética , Bacteriófagos/fisiologia , Humanos , Fezes/microbiologia , Fezes/virologia , Bactérias/virologia , Bactérias/genética , Prófagos/genética , Prófagos/fisiologia , Viroma , Reatores Biológicos/microbiologia , Reatores Biológicos/virologia , Colo/microbiologia , Colo/virologia , Microbiota , VirulênciaRESUMO
The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles.
Assuntos
Microbioma Gastrointestinal , Microbiota , Humanos , Reprodutibilidade dos Testes , Reatores Biológicos , ColoRESUMO
The study of bacteriophage communities reproducing in the gastrointestinal tract is limited by the quality of model systems supporting experimental manipulation in vitro. Traditionally, studies aiming to experimentally address phage-bacteria dynamics have utilized gnotobiotic mice inoculated with defined bacterial communities. While mouse models simulate complex interactions between microbes and their host, they also forestall the study of phage-bacteria dynamics in isolation of host factors. Here, we established a method for manipulating phage-bacteria dynamics using an in vitro chemostat bioreactor model of the distal human gut. We create defined communities representing a subset of bacteria in the feces of two human individuals, cultivated these communities in chemostat bioreactors, developed methods to purify the autochthonous viromes associated with each cultured community, and trialed a system for transmitting live or heat-killed viruses between chemostat bioreactors to decipher outcomes of virus-mediated perturbation. We found that allochthonous viromes were detectable via metagenomic sequencing against the autochthonous virome background and that shifts in bacterial community diversity and composition were detectable in relation to time posttreatment. These microbiome composition changes spanned multiple phyla, including Bacteroidetes, Firmicutes, and Actinobacteria. We also found that compositional changes occurred when using live viruses regardless of whether intrasubject or intersubject viruses were used as the perturbation agents. Our results supported the use of chemostat bioreactors as a platform for studying complex bacteria-phage dynamics in vitro. IMPORTANCE Bacteriophages are relatively ubiquitous in the environment and are highly abundant in the human microbiome. Phages can be commonly transmitted between close contacts, but the impact that such transmissions may have on their bacteria counterparts in our microbiomes is unknown. We developed a chemostat cultivation system to simulate individual-specific features of human distal gut microbiota that can be used to transmit phages between ecosystems and measure their impacts on the microbiota. We used this system to transfer phage communities between chemostats that represented different human subjects. We found that there were significant effects on overall microbiota diversity and changes in the relative abundances of Bacteroidetes, Firmicutes, and Actinobacteria, when intersubject perturbations were performed, compared to intrasubject perturbations. These changes were observed when perturbations were performed using live phages, but not when heat-killed phages were used, and they support the use of chemostat systems for studying complex human bacteria-phage dynamics.