Complex Microbial Communities Research Articles

Microbiota-derived genotoxin tilimycin generates colonic stem cell mutations

The DNA-alkylating metabolite tilimycin is a microbial genotoxin. Intestinal accumulation of tilimycin in individuals carrying til+ Klebsiella spp. causes apoptotic erosion of the epithelium and colitis. Renewal of the intestinal lining and response to injury requires the activities of stem cells located at the base of intestinal crypts. This study interrogates the consequences of tilimycin-induced DNA damage to cycling stem cells. We charted the spatial distribution and luminal quantities of til metabolites in Klebsiella-colonized mice in the context of a complex microbial community. Loss of marker gene G6pd function indicates genetic aberrations in colorectal stem cells that became stabilized in monoclonal mutant crypts. Mice colonized with tilimycin-producing Klebsiella displayed both higher frequencies of somatic mutation and more mutations per affected individual than animals carrying a non-producing mutant. Our findings imply that genotoxic til+ Klebsiella may drive somatic genetic change in the colon and increase disease susceptibility in human hosts.

The Gut Commensal Escherichia coli Aggravates High-Fat-Diet-Induced Obesity and Insulin Resistance in Mice.

Changes in the gut microbiota have been linked to metabolic endotoxemia as a contributing mechanism in the development of obesity and type 2 diabetes. Although identifying specific microbial taxa associated with obesity and type 2 diabetes remains difficult, certain bacteria may play an important role in initiating metabolic inflammation during disease development. The enrichment of the family Enterobacteriaceae, largely represented by Escherichia coli, induced by a high-fat diet (HFD) has been correlated with impaired glucose homeostasis; however, whether the enrichment of Enterobacteriaceae in a complex gut microbial community in response to an HFD contributes to metabolic disease has not been established. To investigate whether the expansion of Enterobacteriaceae amplifies HFD-induced metabolic disease, a tractable mouse model with the presence or absence of a commensal E. coli strain was established. With an HFD treatment, but not a standard-chow diet, the presence of E. coli significantly increased body weight and adiposity and induced impaired glucose tolerance. In addition, E. coli colonization led to increased inflammation in liver and adipose and intestinal tissue under an HFD regimen. With a modest effect on gut microbial composition, E. coli colonization resulted in significant changes in the predicted functional potential of microbial communities. The results demonstrated the role of commensal E. coli in glucose homeostasis and energy metabolism in response to an HFD, indicating contributions of commensal bacteria to the pathogenesis of obesity and type 2 diabetes. The findings of this research identified a targetable subset of the microbiota in the treatment of people with metabolic inflammation. IMPORTANCE Although identifying specific microbial taxa associated with obesity and type 2 diabetes remains difficult, certain bacteria may play an important role in initiating metabolic inflammation during disease development. Here, we used a mouse model distinguishable by the presence or absence of a commensal Escherichia coli strain in combination with a high-fat diet challenge to investigate the impact of E. coli on host metabolic outcomes. This is the first study to show that the addition of a single bacterial species to an animal already colonized with a complex microbial community can increase severity of metabolic outcomes. This study is of interest to a wide group of researchers because it provides compelling evidence to target the gut microbiota for therapeutic purposes by which personalized medicines can be made for treating metabolic inflammation. The study also provides an explanation for variability in studies investigating host metabolic outcomes and immune response to diet interventions.

Comparison of long- and short-read metagenomic assembly for low-abundance species and resistance genes.

Recent technological and computational advances have made metagenomic assembly a viable approach to achieving high-resolution views of complex microbial communities. In previous benchmarking, short-read (SR) metagenomic assemblers had the highest accuracy, long-read (LR) assemblers generated the most contiguous sequences and hybrid (HY) assemblers balanced length and accuracy. However, no assessments have specifically compared the performance of these assemblers on low-abundance species, which include clinically relevant organisms in the gut. We generated semi-synthetic LR and SR datasets by spiking small and increasing amounts of Escherichia coli isolate reads into fecal metagenomes and, using different assemblers, examined E. coli contigs and the presence of antibiotic resistance genes (ARGs). For ARG assembly, although SR assemblers recovered more ARGs with high accuracy, even at low coverages, LR assemblies allowed for the placement of ARGs within longer, E. coli-specific contigs, thus pinpointing their taxonomic origin. HY assemblies identified resistance genes with high accuracy and had lower contiguity than LR assemblies. Each assembler type's strengths were maintained even when our isolate was spiked in with a competing strain, which fragmented and reduced the accuracy of all assemblies. For strain characterization and determining gene context, LR assembly is optimal, while for base-accurate gene identification, SR assemblers outperform other options. HY assembly offers contiguity and base accuracy, but requires generating data on multiple platforms, and may suffer high misassembly rates when strain diversity exists. Our results highlight the trade-offs associated with each approach for recovering low-abundance taxa, and that the optimal approach is goal-dependent.

Environmental stress stimulates microbial activities as indicated by cyclopropane fatty acid enhancement

Soil microbial responses to environmental stress remain a critical question in microbial ecology. The content of cyclopropane fatty acid (CFA) in cytomembrane has been widely used to evaluate environmental stress on microorganisms. Here, we used CFA to investigate the ecological suitability of microbial communities and found a stimulating impact of CFA on microbial activities during wetland reclamation in Sanjiang Plain, Northeastern China. The seasonality of environmental stress resulted in the fluctuation of CFA content in the soil, which suppressed microbial activities due to nutrient loss upon wetland reclamation. After land conversion, the aggravation of temperature stress to microbes increased the CFA content by 5 % (autumn) to 163 % (winter), which led to the suppression of microbial activities by 7 %–47 %. By contrast, the warmer soil temperature and permeability decreased the CFA content by 3 % to 41 % and consequently aggravated the microbial reduction by 15 %–72 % in spring and summer. Complex microbial communities of 1300 CFA-produced species were identified using a sequencing approach, suggesting that soil nutrients dominated the differentiation in these microbial community structures. Further analysis with structural equation modeling highlighted the important function of CFA content to environmental stress and the stimulating influence of CFA induced by environmental stress on microbial activities. Our study shows the biological mechanisms of seasonal CFA content for microbial adaption to environmental stress under wetland reclamation. It advances our knowledge of microbial physiology affecting soil element cycling caused by anthropogenic activities.

Reduction in the Residues of Penthiopyrad in Processed Edible Vegetables by Various Soaking Treatments and Health Hazard Evaluation in China

Tomato and cucumber are two vital edible vegetables that usually appear in people's daily diet. Penthiopyrad is a new type of amide chiral fungicide, which is often used for disease control of vegetables (including tomato and cucumber) due to its wide bactericidal spectrum, low toxicity, good penetration, and strong internal absorption. Extensive application of penthiopyrad may have caused potential pollution in the ecosystem. Different processing methods can remove pesticide residues from vegetables and protect human health. In this study, the penthiopyrad removal efficiency of soaking and peeling from tomatoes and cucumbers was evaluated under different conditions. Among different soaking methods, heated water soaking and water soaking with additives (NaCl, acetic acid, and surfactant) presented a more effective reduction ability than other treatments. Due to the specific physicochemical properties of tomatoes and cucumbers, the ultrasound enhances the removal rate of soaking for tomato samples and inhibits it for cucumber samples. Peeling can remove approximately 90% of penthiopyrad from contaminated tomato and cucumber samples. Enantioselectivity was found only during tomato sauce storage, which may be related to the complex microbial community. Health risk assessment data suggests that tomatoes and cucumbers are safer for consumers after soaking and peeling. The results may provide consumers with some useful information to choose better household processing methods to remove penthiopyrad residues from tomatoes, cucumbers, and other edible vegetables.

Competitive Reactions during Ethanol Chain Elongation Were Temporarily Suppressed by Increasing Hydrogen Partial Pressure through Methanogenesis Inhibition.

Organic waste streams can be converted into high-value platform chemicals such as medium-chain carboxylic acids (MCCAs) using mixed microbial communities via chain elongation. However, the heterogeneity of waste streams and the use of complex microbial communities can lead to undesirable reactions, thus decreasing process efficiency. We explored suppressing excessive ethanol oxidation to acetate (EEO) by increasing the hydrogen partial pressure (PH2) through hydrogenotrophic methanogenesis inhibition by periodically adding 2-bromoethanesulfonate (2-BES) to an MCCA-producing bioreactor to reach 10 mM of 2-BES upon addition. The bioreactor was fed with pretreated food waste and brewery waste containing high concentrations of short-chain carboxylic acids and ethanol, respectively. While 2-BES addition initially reduced EEO, some methanogens (Methanobrevibacter spp.) persisted and resistant populations were selected over time. Besides changing the methanogenic community structure, adding 2-BES also changed the bacterial community structure due to its impact on PH2. While we demonstrated that PH2 could be manipulated using 2-BES to control EEO, methods that do not require the addition of a chemical inhibitor should be explored to maintain optimum PH2 for long-term suppression of EEO.

Characterization of the Nero Siciliano Pig Fecal Microbiota after a Liquid Whey-Supplemented Diet.

The utilization of dairy by-products as animal feed, especially in swine production, is a strategy to provide functional ingredients to improve gut health. This study explored the potential effect of a liquid whey-supplemented diet on the fecal microbiota of eleven pigs belonging to the Nero Siciliano breed. Five pigs were assigned to the control group and fed with a standard formulation feed, whereas six pigs were assigned to the experimental group and fed with the same feed supplemented with liquid whey. Fecal samples were collected from each individual before the experimental diet (T0), and one (T1) and two (T2) months after the beginning of the co-feed supplementation. Taxonomic analysis, based on the V3-V4 region of the bacterial 16S rRNA, showed that pig feces were populated by a complex microbial community with a remarkable abundance of Firmicutes, Bacteroidetes, and Spirochaetes phyla and Prevotella, Lactobacillus, Clostridium, and Treponema genera. Alpha and beta diversity values suggested that the experimental diet did not significantly affect the overall fecal microbiota diversity. However, analysis of abundance at different time points revealed significant variation in several bacterial genera, suggesting that the experimental diet potentially affected some genera of the microbial community.

The media composition as a crucial element in high-throughput metabolic network reconstruction.

In recent years, metagenome-assembled genomes (MAGs) have provided glimpses into the intra- and interspecies genetic diversity and interactions that form the bases of complex microbial communities. High-throughput reconstruction of genome-scale metabolic networks (GEMs) from MAGs is a promising avenue to disentangle the myriad trophic interactions stabilizing these communities. However, high-throughput reconstruction of GEMs relies on accurate gap filling of metabolic pathways using automated algorithms. Here, we systematically explore how the composition of the media (specification of the available nutrients and metabolites) during gap filling influences the resulting GEMs concerning predicted auxotrophies for fully sequenced model organisms and environmental isolates. We expand this analysis by using 106 MAGs from the same species with differing quality. We find that although the completeness of MAGs influences the fraction of gap-filled reactions, the composition of the media plays the dominant role in the accurate prediction of auxotrophies that form the basis of myriad community interactions. We propose that constraining the media composition for gap filling through both experimental approaches and computational approaches will increase the reliability of high-throughput reconstruction of genome-scale metabolic models from MAGs and paves the way for culture independent prediction of trophic interactions in complex microbial communities.

Effects of Euterpe oleracea Mart. extract on Candida spp. biofilms.

Candida spp. biofilms are complex microbial communities that have been associated with increasing resistance to clinically available antifungal drugs. Hence, novel pharmacological approaches with ability to inhibit biofilm formation have been investigated. The aim was to analyze in vitro antifungal activity of Euterpe oleracea Mart. (açaí berry) extract on biofilm strains of Candida albicans, C. parapsilosis, and C. tropicalis that were formed on abiotic surfaces. Biofilms of C. albicans, C. parapsilosis, and C. tropicalis were grown in vitro. They were then treated with E. oleracea Mart. extract at different concentrations (7.8, 15.6, 31.2, 62.5, 125, 250, 500, and 1000μg/mL) for evaluation of both biofilm removal and anti-biofilm activity. All Candida species analyzed formed biofilms on abiotic surfaces. Yet, increased biofilm formation was displayed for C. tropicalis in comparison with the other two species. E. oleracea Mart. extract was shown to inhibit biofilm formation at all concentrations used when compared to no treatment (p < 0.05). In the current study, the extract of E. oleracea Mart. demonstrated antifungal activity against Candida albicans, C. parapsilosis, and C. tropicalis biofilms, regardless of the dose utilized. These results are important to evaluate a natural product as antifungal for Candida species.

Unraveling the microbial succession during the natural fermentation of grass carp and their correlation with volatile flavor formation

Complex microbial communities contribute significantly to the flavor formation of traditional fermented fish products. However, the relationship between microorganisms and flavor formation in traditional fermented grass carp products is still unclear. In this study, the diversity and succession of microbial communities and the variation of volatile compounds during natural fermentation of grass carp were analyzed using high-throughput sequencing of 16S rRNA and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), respectively. The core functional microorganism and key volatile compounds were identified, and their potential relationship was revealed using a correlation network model analysis. The microbial community analysis result showed that the microbial diversity during natural fermentation of grass carp decreased markedly with increasing fermentation time, and Lactiplantibacillus, Staphylococcus, and Enterobacter were the dominant genera in naturally fermented grass carp. HS-SPME-GC-MS analysis result showed that 45 volatile compounds were identified from fermented samples, among which 13 compounds (e.g., hexanal, heptanal, nonanal, decanal, 3-octanone, 3-methyl-1-butanol, 1-hexanol, 1-heptanol, 1-octen-3-ol, 1-octanol, ethyl acetate, 3-methyl-1-butanol acetate, and 2-methoxy-4-vinylphenol) were identified as the key volatile compounds. Additionally, the correlation network model analysis result revealed that Lactiplantibacillus showed significantly positive correlations with most of the key volatile compounds, making an important contribution to the formation of volatile flavor in naturally fermented grass carp. This study may lead to an understanding of the role of core functional microorganisms in the formation of volatile flavor during the natural fermentation of grass carp and provide some theoretical guidance for the industrial production of high-quality fermented grass carp products.

Symbiont-screener: A reference-free tool to separate host sequences from symbionts for error-prone long reads

Metagenomic sequencing facilitates large-scale constitutional analysis and functional characterization of complex microbial communities without cultivation. Recent advances in long-read sequencing techniques utilize long-range information to simplify repeat-aware metagenomic assembly puzzles and complex genome binning tasks. However, it remains methodologically challenging to remove host-derived DNA sequences from the microbial community at the read resolution due to high sequencing error rates and the absence of reference genomes. We here present Symbiont-Screener (https://github.com/BGI-Qingdao/Symbiont-Screener), a reference-free approach to identifying high-confidence host’s long reads from symbionts and contaminants and overcoming the low sequencing accuracy according to a trio-based screening model. The remaining host’s sequences are then automatically grouped by unsupervised clustering. When applied to both simulated and real long-read datasets, it maintains higher precision and recall rates of identifying the host’s raw reads compared to other tools and hence promises the high-quality reconstruction of the host genome and associated metagenomes. Furthermore, we leveraged both PacBio HiFi and nanopore long reads to separate the host’s sequences on a real host-microbe system, an algal-bacterial sample, and retrieved an obvious improvement of host assembly in terms of assembly contiguity, completeness, and purity. More importantly, the residual symbiotic microbiomes illustrate improved genomic profiling and assemblies after the screening, which elucidates a solid basis of data for downstream bioinformatic analyses, thus providing a novel perspective on symbiotic research.

Studying Fungal-Bacterial Relationships in the Human Gut Using an In Vitro Model (TIM-2)

The complex microbial community found in the human gut consist of members of multiple kingdoms, among which are bacteria and fungi. Microbiome research mainly focuses on the bacterial part of the microbiota, thereby neglecting interactions that can take place between bacteria and fungi. With the rise of sequencing techniques, the possibilities to study cross-kingdom relationships has expanded. In this study, fungal-bacterial relationships were investigated using the complex, dynamic computer-controlled in vitro model of the colon (TIM-2). Interactions were investigated by disruption of either the bacterial or fungal community by the addition of antibiotics or antifungals to TIM-2, respectively, compared to a control without antimicrobials. The microbial community was analyzed with the use of next generation sequencing of the ITS2 region and the 16S rRNA. Moreover, the production of SCFAs was followed during the interventions. Correlations between fungi and bacteria were calculated to investigate possible cross-kingdom interactions. The experiments showed that no significant differences in alpha-diversity were observed between the treatments with antibiotics and fungicide. For beta-diversity, it could be observed that samples treated with antibiotics clustered together, whereas the samples from the other treatments were more different. Taxonomic classification was done for both bacteria and fungi, but no big shifts were observed after treatments. At the level of individual genera, bacterial genus Akkermansia was shown to be increased after fungicide treatment. SCFAs levels were lowered in samples treated with antifungals. Spearman correlations suggested that cross-kingdom interactions are present in the human gut, and that fungi and bacteria can influence each other. Further research is required to gain more insights in these interactions and their molecular nature and to determine the clinical relevance.

Characterization of a novel Jerseyvirus phage T102 and its inhibition effect on biofilms of multidrug-resistant Salmonella

Biofilm, as a complex microbial community, is a serious and major safety concern in the food industry. Interestingly, some phages could effectively disrupt biofilms. This study characterized a novel isolated Salmonella bacteriophage T102, and its ability to control and remove biofilm produced by multidrug-resistant Salmonella. Phage T102 exhibited a broad host range within the Salmonella genus, especially drug-resistant Salmonella. The genome of phage T102 was comprised of 41,941 bp with 49.7% G + C composition, and with no genes associated with antibiotic resistance or virulence factors. The structural protein profile of phage T102 was subjected to SDS-PAGE and UPLC-MS/MS analysis, among them, 34 peptides were consistent with the hypothetical protein sequences annotated in the genome of T102. The biofilm inhibition assay revealed that phage T102 inhibited the formation of 6 h biofilms by two multidrug-resistant S. Typhimurium strains by 43.17 and 32.42%, respectively. 24 h biofilms formed by S. Typhimurium decreased by 54.94 and 53.67%, respectively, after 2 h of exposure to phage T102. Microscopic observation confirmed the inhibition effect of phage T102 on biofilm formation on spiked lettuce. Overall, our results support new research into the application of bacteriophage for biofilm reduction.

Gut Microbiome Proteomics in Food Allergies.

Food allergies (FA) have dramatically increased in recent years, particularly in developed countries. It is currently well-established that food tolerance requires the strict maintenance of a specific microbial consortium in the gastrointestinal (GI) tract microbiome as alterations in the gut microbiota can lead to dysbiosis, causing inflammation and pathogenic intestinal conditions that result in the development of FA. Although there is currently not enough knowledge to fully understand how the interactions between gut microbiota, host responses and the environment cause food allergies, recent advances in '-omics' technologies (i.e., proteomics, genomics, metabolomics) and in approaches involving systems biology suggest future headways that would finally allow the scientific understanding of the relationship between gut microbiome and FA. This review summarizes the current knowledge in the field of FA and insights into the future advances that will be achieved by applying proteomic techniques to study the GI tract microbiome in the field of FA and their medical treatment. Metaproteomics, a proteomics experimental approach of great interest in the study of GI tract microbiota, aims to analyze and identify all the proteins in complex environmental microbial communities; with shotgun proteomics, which uses liquid chromatography (LC) for separation and tandem mass spectrometry (MS/MS) for analysis, as it is the most promising technique in this field.

Mid-Infrared Photothermal-Fluorescence In Situ Hybridization for Functional Analysis and Genetic Identification of Single Cells.

Simultaneous identification and metabolic analysis of microbes with single-cell resolution and high throughput are necessary to answer the question of "who eats what, when, and where" in complex microbial communities. Here, we present a mid-infrared photothermal-fluorescence in situ hybridization (MIP-FISH) platform that enables direct bridging of genotype and phenotype. Through multiple improvements of MIP imaging, the sensitive detection of isotopically labeled compounds incorporated into proteins of individual bacterial cells became possible, while simultaneous detection of FISH labeling with rRNA-targeted probes enabled the identification of the analyzed cells. In proof-of-concept experiments, we showed that the clear spectral red shift in the protein amide I region due to incorporation of 13C atoms originating from 13C-labeled glucose can be exploited by MIP-FISH to discriminate and identify 13C-labeled bacterial cells within a complex human gut microbiome sample. The presented methods open new opportunities for single-cell structure-function analyses for microbiology.

Diversity and structure of bacterial and archaeal communities associated with the vulnerable sponge Halichondria cebimarensis.

Sponges are essential components of the marine benthos and well known for their complex and abundant associated microbial communities. There are five endemic species of the genus Halichondria on the Brazilian coast and H. cebimarensis is one of the least studied. This sponge has a very limited geographic distribution and is classified as vulnerable. In order to understand the bacterial and archaeal communities associated with this sponge, samples of H. cebimarensis were collected from the southwestern Atlantic coast (Brazil, São Paulo state). Choanosome samples were separated and processed to be (i) inoculated in three different culture media; (ii) investigated by transmission electron microscopy; (iii) submitted to 16S rRNA metabarcoding. Forty isolates were obtained and 12 were identified as belonging to the Bacilli class. The culture-dependent approaches allowed us to access unique members of the microbial community. Our analyses revealed that this animal belongs to the Low Microbial Abundance group of sponges. Culture-independent approaches showed that the H. cebimarensis microbiome is dominated by the heterotrophic Gammaproteobacteria AqS2 ("Ca. Amphirhobacter heronislandensis"). This is the first study to reveal details of the microbiome of this vulnerable sponge and is an important step in understanding how this sponge functions, its biotechnological potential and a contribution to conservation efforts.

Quantitative metaproteomics reveals composition and metabolism characteristics of microbial communities in Chinese liquor fermentation starters.

Daqu, the Chinese liquor fermentation starter, contains complex microbial communities that are important for the yield, quality, and unique flavor of produced liquor. However, the composition and metabolism of microbial communities in the different types of high-temperature Daqu (i.e., white, yellow, and black Daqu) have not been well understood. Herein, we used quantitative metaproteomics based on data-independent acquisition (DIA) mass spectrometry to analyze a total of 90 samples of white, yellow, and black Daqu collected in spring, summer, and autumn, revealing the taxonomic and metabolic profiles of different types of Daqu across seasons. Taxonomic composition differences were explored across types of Daqu and seasons, where the under-fermented white Daqu showed the higher microbial diversity and seasonal stability. It was demonstrated that yellow Daqu had higher abundance of saccharifying enzymes for raw material degradation. In addition, considerable seasonal variation of microbial protein abundance was discovered in the over-fermented black Daqu, suggesting elevated carbohydrate and amino acid metabolism in autumn black Daqu. We expect that this study will facilitate the understanding of the key microbes and their metabolism in the traditional fermentation process of Chinese liquor production.

MetaBinner: a high-performance and stand-alone ensemble binning method to recover individual genomes from complex microbial communities

Binning aims to recover microbial genomes from metagenomic data. For complex metagenomic communities, the available binning methods are far from satisfactory, which usually do not fully use different types of features and important biological knowledge. We developed a novel ensemble binner, MetaBinner, which generates component results with multiple types of features by k-means and uses single-copy gene information for initialization. It then employs a two-stage ensemble strategy based on single-copy genes to integrate the component results efficiently and effectively. Extensive experimental results on three large-scale simulated datasets and one real-world dataset demonstrate that MetaBinner outperforms the state-of-the-art binners significantly.

A pilot study to disentangle the infant gut microbiota composition and identification of bacteria correlates with high fat mass.

Background: At birth, the human intestine is colonized by a complex community of microorganisms known as gut microbiota. These complex microbial communities that inhabit the gut microbiota are thought to play a key role in maintaining host physiological homeostasis. For this reason, correct colonization of the gastrointestinal tract in the early stages of life could be fundamental for human health. Furthermore, alterations of the infant microbiota are correlated with the development of human inflammatory diseases and disorders. In this context, the possible relationships between intestinal microbiota and body composition during infancy are of great interest. Methods: In this study, we have performed a pilot study based on 16S rRNA gene profiling and metagenomic approaches on repeatedly measured data on time involving a cohort of 41 Italian newborns, which is aimed to investigate the possible correlation between body fat mass percentage (FM%) and the infant gut microbiota composition. Results and conclusion: The taxonomical analysis of the stool microbiota of each infant included in the cohort allowed the identification of a specific correlation between intestinal bacteria, such as Bifidobacterium and Veillonella, and the increase in FM%. Moreover, the analysis of the infant microbiome's metabolic capabilities suggested that the intestinal microbiome functionally impacts the human host and its possible influence on host physiology.

Targeted Enrichment of Low-Abundance and Uncharacterized Taxon Members in Complex Microbial Community with Primer-Free FISH ProbesDesigned from Next Generation Sequencing Dataset.

Methods to obtain high-quality assembled genomic information of rare and unclassified member species in complex microbial communities remain a high priority in microbial ecology. Additionally, the supplementation of three-dimensional spatial information that highlights the morphology and spatial interaction would provide additional insights to its ecological role in the community. Fluorescent in-situ hybridization (FISH) coupling with fluorescence-activated cell sorting (FACS) is a powerful tool that enables the detection, visualization, and separation of low-abundance microbial members in samples containing complex microbial compositions. Here, we have described the workflow from designing the appropriate FISH probes from metagenomics or metatranscriptomics datasets to the preparation and treatment of samples to be used in FISH-FACS procedures.