Human microbiome composition is closely tied to health, but how the host manages its microbial inhabitants remains unclear. One important, but understudied, factor is the natural host environment: mucus, which contains gel-forming glycoproteins (mucins) that display hundreds of glycan structures with potential regulatory function. Leveraging a tractable culture-based system to study how mucins influence oral microbial communities, we found that mucin glycans enable the coexistence of diverse microbes, while resisting disease-associated compositional shifts. Mucins from tissues with unique glycosylation differentially tuned microbial composition, as did isolated mucin glycan libraries, uncovering the importance of specific glycan patterns in microbiome modulation. We found that mucins shape microbial communities in several ways: serving as nutrients to support metabolic diversity, organizing spatial structure through reduced aggregation, and possibly limiting antagonism between competing taxa. Overall, this work identifies mucin glycans as a natural host mechanism and potential therapeutic intervention to maintain healthy microbial communities.
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Abstract Background Elucidating the spatial structure of host-associated microbial communities is essential for understanding taxon-taxon interactions within the microbiota and between microbiota and host. Macroalgae are colonized by complex microbial communities, suggesting intimate symbioses that likely play key roles in both macroalgal and bacterial biology, yet little is known about the spatial organization of microbes associated with macroalgae. Canopy-forming kelp are ecologically significant, fixing teragrams of carbon per year in coastal kelp forest ecosystems. We characterized the micron-scale spatial organization of bacterial communities on blades of the kelp
Nereocystis luetkeana using fluorescence in situ hybridization and spectral imaging with a probe set combining phylum-, class-, and genus-level probes to localize and identify > 90% of the microbial community.Results We show that kelp blades host a dense microbial biofilm composed of disparate microbial taxa in close contact with one another. The biofilm is spatially differentiated, with clustered cells of the dominant symbiont
Granulosicoccus sp. (Gammaproteobacteria ) close to the kelp surface and filamentousBacteroidetes andAlphaproteobacteria relatively more abundant near the biofilm-seawater interface. A community rich inBacteroidetes colonized the interior of kelp tissues. Microbial cell density increased markedly along the length of the kelp blade, from sparse microbial colonization of newly produced tissues at the meristematic base of the blade to an abundant microbial biofilm on older tissues at the blade tip. Kelp from a declining population hosted fewer microbial cells compared to kelp from a stable population.Conclusions Imaging revealed close association, at micrometer scales, of different microbial taxa with one another and with the host. This spatial organization creates the conditions necessary for metabolic exchange among microbes and between host and microbiota, such as provisioning of organic carbon to the microbiota and impacts of microbial nitrogen metabolisms on host kelp. The biofilm coating the surface of the kelp blade is well-positioned to mediate interactions between the host and surrounding organisms and to modulate the chemistry of the surrounding water column. The high density of microbial cells on kelp blades (105–107cells/cm2), combined with the immense surface area of kelp forests, indicates that biogeochemical functions of the kelp microbiome may play an important role in coastal ecosystems.
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ABSTRACT The European common cuttlefish, Sepia officinalis , is used extensively in biological and biomedical research, yet its microbiome remains poorly characterized. We analyzed the microbiota of the digestive tract, gills, and skin in mariculture-raised S. officinalis using a combination of 16S rRNA amplicon sequencing, quantitative PCR (qPCR), and fluorescence spectral imaging. Sequencing revealed a highly simplified microbiota consisting largely of two single bacterial amplicon sequence variants (ASVs) of Vibrionaceae and Piscirickettsiaceae . The esophagus was dominated by a single ASV of the genus Vibrio . Imaging revealed bacteria in the family Vibrionaceae distributed in a discrete layer that lines the esophagus. This Vibrio was also the primary ASV found in the microbiota of the stomach, cecum, and intestine, but occurred at lower abundance, as determined by qPCR, and was found only scattered in the lumen rather than in a discrete layer via imaging analysis. Treatment of animals with the commonly used antibiotic enrofloxacin led to a nearly 80% reduction of the dominant Vibrio ASV in the esophagus but did not significantly alter the relative abundance of bacteria overall between treated versus control animals. Data from the gills were dominated by a single ASV in the family Piscirickettsiaceae , which imaging visualized as small clusters of cells. We conclude that bacteria belonging to the Gammaproteobacteria are the major symbionts of the cuttlefish Sepia officinalis cultured from eggs in captivity and that the esophagus and gills are major colonization sites. IMPORTANCE Microbes can play critical roles in the physiology of their animal hosts, as evidenced in cephalopods by the role of Vibrio ( Aliivibrio ) fischeri in the light organ of the bobtail squid and the role of Alpha - and Gammaproteobacteria in the reproductive system and egg defense in a variety of cephalopods. We sampled the cuttlefish microbiome throughout the digestive tract, gills, and skin and found dense colonization of an unexpected site, the esophagus, by a microbe of the genus Vibrio , as well as colonization of gills by Piscirickettsiaceae . This finding expands the range of organisms and body sites known to be associated with Vibrio and is of potential significance for understanding host-symbiont associations, as well as for understanding and maintaining the health of cephalopods in mariculture.more » « less
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Abstract Motivation Spectral unmixing methods attempt to determine the concentrations of different fluorophores present at each pixel location in an image by analyzing a set of measured emission spectra. Unmixing algorithms have shown great promise for applications where samples contain many fluorescent labels; however, existing methods perform poorly when confronted with autofluorescence-contaminated images.
Results We propose an unmixing algorithm designed to separate fluorophores with overlapping emission spectra from contamination by autofluorescence and background fluorescence. First, we formally define a generalization of the linear mixing model, called the affine mixture model (AMM), that specifically accounts for background fluorescence. Second, we use the AMM to derive an affine nonnegative matrix factorization method for estimating fluorophore endmember spectra from reference images. Lastly, we propose a semi-blind sparse affine spectral unmixing (SSASU) algorithm that uses knowledge of the estimated endmembers to learn the autofluorescence and background fluorescence spectra on a per-image basis. When unmixing real-world spectral images contaminated by autofluorescence, SSASU greatly improved proportion indeterminacy as compared to existing methods for a given relative reconstruction error.
Availability and implementation The source code used for this paper was written in Julia and is available with the test data at https://github.com/brossetti/ssasu.
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Abstract Plastic marine debris (PMD) affects spatial scales of life from microbes to whales. However, understanding interactions between plastic and microbes in the “Plastisphere”—the thin layer of life on the surface of PMD—has been technology‐limited. Research into microbe–microbe and microbe–substrate interactions requires knowledge of community phylogenetic composition but also tools to visualize spatial distributions of intact microbial biofilm communities. We developed a CLASI‐FISH (combinatorial labelling and spectral imaging – fluorescence
in situ hybridization) method using confocal microscopy to study Plastisphere communities. We created a probe set consisting of three existing phylogenetic probes (targeting all Bacteria,Alpha ‐, andGammaproteobacteria ) and four newly designed probes (targetingBacteroidetes ,Vibrionaceae ,Rhodobacteraceae andAlteromonadaceae ) labelled with a total of seven fluorophores and validated this probe set using pure cultures. Our nested probe set strategy increases confidence in taxonomic identification because targets are confirmed with two or more probes, reducing false positives. We simultaneously identified and visualized these taxa and their spatial distribution within the microbial biofilms on polyethylene samples in colonization time series experiments in coastal environments from three different biogeographical regions. Comparing the relative abundance of 16S rRNA gene amplicon sequencing data with cell‐count abundance data retrieved from the microscope images of the same samples showed a good agreement in bacterial composition. Microbial communities were heterogeneous, with direct spatial relationships between bacteria, cyanobacteria and eukaryotes such as diatoms but also micro‐metazoa. Our research provides a valuable resource to investigate biofilm development, succession and associations between specific microscopic taxa at micrometre scales.
