Host-associated Microbial Communities Research Articles

Antagonistic effects of actin-specific toxins on Salmonella Typhimurium invasion into mammalian cells.

Competition between bacterial species is a major factor shaping microbial communities. In this work, we explored the hypothesis that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by any of the four tested actin-specific toxins: Vibrio cholerae MARTX actin crosslinking and Rho GTPase inactivation domains (ACD and RID, respectively), TccC3 from Photorhabdus luminescens , and Salmonella's own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays confirmed by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. Together, the results suggest bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria.

Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria.

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise Sia for growth and/or cell surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-Sia forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

Host population crashes disrupt the diversity of associated marine microbiomes.

Host-associated microbial communities are shaped by myriad factors ranging from host conditions, environmental conditions and other microbes. Disentangling the ecological impact of each of these factors can be particularly difficult as many variables are correlated. Here, we leveraged earthquake-induced changes in host population structure to assess the influence of population crashes on marine microbial ecosystems. A large (7.8 magnitude) earthquake in New Zealand in 2016 led to widespread coastal uplift of up to ~6 m, sufficient to locally extirpate some intertidal southern bull kelp populations. These uplifted populations are slowly recovering, but remain at much lower densities than at nearby, less-uplifted sites. By comparing the microbial communities of the hosts from disturbed and relatively undisturbed populations using 16S rRNA gene amplicon sequencing, we observed that disturbed host populations supported higher functional, taxonomic and phylogenetic microbial beta diversity than non-disturbed host populations. Our findings shed light on microbiome ecological assembly processes, particularly highlighting that large-scale disturbances that affect host populations can dramatically influence microbiome structure. We suggest that disturbance-induced changes in host density limit the dispersal opportunities of microbes, with host community connectivity declining with the density of host populations.

Something in the water: aquatic microbial communities influence the larval amphibian gut microbiota, neurodevelopment and behaviour.

Microorganisms colonize the gastrointestinal tract of animals and establish symbiotic host-associated microbial communities that influence vertebrate physiology. More specifically, these gut microbial communities influence neurodevelopment through the microbiota-gut-brain (MGB) axis. We tested the hypothesis that larval amphibian neurodevelopment is affected by the aquatic microbial community present in their housing water. Newly hatched Northern Leopard Frog (Lithobates pipiens) tadpoles were raised in pond water that was unmanipulated (natural) or autoclaved. Tadpoles raised in autoclaved pond water had a gut microbiota with reduced bacterial diversity and altered community composition, had decreased behavioural responses to sensory stimuli, were larger in overall body mass, had relatively heavier brains and had altered brain shape when compared with tadpoles raised in natural pond water. Further, the diversity and composition of the gut microbiota were associated with tadpole behavioural responses and brain measurements. Our results suggest that aquatic microbial communities shape tadpole behaviour and brain development, providing strong support for the occurrence of the MGB axis in amphibians. Lastly, the dramatic role played by aquatic microbial communities on vertebrate neurodevelopment and behaviour should be considered in future wildlife conservation efforts.

Comparison of exoskeleton microbial communities of co-occurring native and invasive crayfish species

Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

18S rDNA gene metabarcoding of microeukaryotes and epi-endophytes in the holobiome of seven species of large brown algae.

Brown algae (Phaeophyceae) are habitat-forming species in coastal ecosystems and include kelp forests and seaweed beds that support a wide diversity of marine life. Host-associated microbial communities are an integral part of phaeophyte biology, and whereas the bacterial microbial partners have received considerable attention, the microbial eukaryotes associated with brown algae have hardly been studied. Here, we used broadly targeted "pan-eukaryotic" primers (metabarcoding) to investigate brown algal-associated eukaryotes (the eukaryome). Using this approach, we aimed to investigate the eukaryome of seven large brown algae that are important and common species in coastal ecosystems. We also aimed to assess whether these macroalgae harbor novel eukaryotic diversity and to ascribe putative functional roles to the host-associated eukaryome based on taxonomic affiliation and phylogenetic placement. We detected a significant diversity of microeukaryotic and algal lineages associated with the brown algal species investigated. The operational taxonomic units (OTUs) were taxonomically assigned to 10 of the eukaryotic major supergroups, including taxonomic groups known to be associated with seaweeds as epibionts, endobionts, parasites, and commensals. Additionally, we revealed previously unrecorded sequence types, including novel phaeophyte OTUs, particularly in the Fucus spp. samples, that may represent fucoid genomic variants, sequencing artifacts, or undescribed epi-/endophytes. Our results provide baseline data and technical insights that will be useful for more comprehensive seaweed eukaryome studies investigating the evidently lineage-rich and functionally diverse symbionts of brown algae.

Formate oxidation in the intestinal mucus layer enhances fitness of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium induces intestinal inflammation to create a niche that fosters the outgrowth of the pathogen over the gut microbiota. Under inflammatory conditions, Salmonella utilizes terminal electron acceptors generated as byproducts of intestinal inflammation to generate cellular energy through respiration. However, the electron donating reactions in these electron transport chains are poorly understood. Here, we investigated how formate utilization through the respiratory formate dehydrogenase-N (FdnGHI) and formate dehydrogenase-O (FdoGHI) contribute to gut colonization of Salmonella. Both enzymes fulfilled redundant roles in enhancing fitness in a mouse model of Salmonella-induced colitis, and coupled to tetrathionate, nitrate, and oxygen respiration. The formic acid utilized by Salmonella during infection was generated by its own pyruvate-formate lyase as well as the gut microbiota. Transcription of formate dehydrogenases and pyruvate-formate lyase was significantly higher in bacteria residing in the mucus layer compared to the lumen. Furthermore, formate utilization conferred a more pronounced fitness advantage in the mucus, indicating that formate production and degradation occurred predominantly in the mucus layer. Our results provide new insights into how Salmonella adapts its energy metabolism to the local microenvironment in the gut. IMPORTANCE Bacterial pathogens must not only evade immune responses but also adapt their metabolism to successfully colonize their host. The microenvironments encountered by enteric pathogens differ based on anatomical location, such as small versus large intestine, spatial stratification by host factors, such as mucus layer and antimicrobial peptides, and distinct commensal microbial communities that inhabit these microenvironments. Our understanding of how Salmonella populations adapt its metabolism to different environments in the gut is incomplete. In the current study, we discovered that Salmonella utilizes formate as an electron donor to support respiration, and that formate oxidation predominantly occurs in the mucus layer. Our experiments suggest that spatially distinct Salmonella populations in the mucus layer and the lumen differ in their energy metabolism. Our findings enhance our understanding of the spatial nature of microbial metabolism and may have implications for other enteric pathogens as well as commensal host-associated microbial communities.

Connect the dots: sketching out microbiome interactions through networking approaches.

Microbiome networking analysis has emerged as a powerful tool for studying the complex interactions among microorganisms in various ecological niches, including the human body and several environments. This analysis has been used extensively in both human and environmental studies, revealing key taxa and functional units peculiar to the ecosystem considered. In particular, it has been mainly used to investigate the effects of environmental stressors, such as pollution, climate change or therapies, on host-associated microbial communities and ecosystem function. In this review, we discuss the latest advances in microbiome networking analysis, including methods for constructing and analyzing microbiome networks, and provide a case study on how to use these tools. These analyses typically involve constructing a network that represents interactions among microbial taxa or functional units, such as genes or metabolic pathways. Such networks can be based on a variety of data sources, including 16S rRNA sequencing, metagenomic sequencing, and metabolomics data. Once constructed, these networks can be analyzed to identify key nodes or modules important for the stability and function of the microbiome. By providing insights into essential ecological features of microbial communities, microbiome networking analysis has the potential to transform our understanding of the microbial world and its impact on human health and the environment.

Skin bacterial community differences among three species of co-occurring Ranid frogs.

Skin microbial communities are an essential part of host health and can play a role in mitigating disease. Host and environmental factors can shape and alter these microbial communities and, therefore, we need to understand to what extent these factors influence microbial communities and how this can impact disease dynamics. Microbial communities have been studied in amphibian systems due to skin microbial communities providing some resistance to the amphibian chytrid fungus, Batrachochytrium dendrobatidis. However, we are only starting to understand how host and environmental factors shape these communities for amphibians. In this study, we examined whether amphibian skin bacterial communities differ among host species, host infection status, host developmental stage, and host habitat. We collected skin swabs from tadpoles and adults of three Ranid frog species (Lithobates spp.) at the Mianus River Gorge Preserve in Bedford, New York, USA, and used 16S rRNA gene amplicon sequencing to determine bacterial community composition. Our analysis suggests amphibian skin bacterial communities change across host developmental stages, as has been documented previously. Additionally, we found that skin bacterial communities differed among Ranid species, with skin communities on the host species captured in streams or bogs differing from the communities of the species captured on land. Thus, habitat use of different species may drive differences in host-associated microbial communities for closely-related host species.

Phylogenetic comparative approach reveals evolutionary conservatism, ancestral composition, and integration of vertebrate gut microbiota.

How host-associated microbial communities evolve as their hosts diversify remains equivocal: How conserved is their composition? What was the composition of ancestral microbiota? Do microbial taxa covary in abundance over millions of years? Multivariate phylogenetic models of trait evolution are key to answering similar questions for complex host phenotypes, yet they are not directly applicable to relative abundances, which usually characterize microbiota. Here, we extend these models in this context, thereby providing a powerful approach for estimating phylosymbiosis (the extent to which closely related host species harbor similar microbiota), ancestral microbiota composition, and integration (evolutionary covariations in bacterial abundances). We apply our model to the gut microbiota of mammals and birds. We find significant phylosymbiosis that is not entirely explained by diet and geographic location, indicating that other evolutionary-conserved traits shape microbiota composition. We identify main shifts in microbiota composition during the evolution of the two groups and infer an ancestral mammalian microbiota consistent with an insectivorous diet. We also find remarkably consistent evolutionary covariations among bacterial orders in mammals and birds. Surprisingly, despite the substantial variability of present-day gut microbiota, some aspects of their composition are conserved over millions of years of host evolutionary history.

Ectotherm heat tolerance and the microbiome: current understanding, future directions and potential applications.

Climate change and increasing global temperatures are a leading threat to ectothermic animals worldwide. Ectotherm persistence under climate change will depend on a combination of host and environmental factors; recently it has become clear that host-associated microbial communities contribute significantly to the response of ectotherms to environmental warming. However, several unanswered questions about these relationships remain before accurate predictions can be made regarding the microbiome's influence on host ecology and evolution under climate warming. In this Commentary, we provide a brief background of what is currently known about the influence of the microbiome on heat tolerance in both invertebrate and vertebrate ectothermic animals, and the mechanisms behind these effects. We then outline what we feel are important priorities for future work in the field, and how these goals could be accomplished. We specifically highlight a need for more diversity in study systems, especially through increasing representation of vertebrate hosts and hosts across a variety of life-history traits and habitats, as well as a greater understanding of how these relationships manifest in field settings. Lastly, we discuss the implications of microbiome-mediated heat tolerance for animal conservation under climate change and the possibility of 'bioaugmentation' approaches to bolster host heat tolerance in vulnerable populations.

Pulsed antibiotic treatments of gnotobiotic mice manifest in complex bacterial community dynamics and resistance effects

Bacteria can evolve to withstand a wide range of antibiotics (ABs) by using various resistance mechanisms. How ABs affect the ecology of the gut microbiome is still poorly understood. We investigated strain-specific responses and evolution during repeated AB perturbations by three clinically relevant ABs, using gnotobiotic mice colonized with a synthetic bacterial community (oligo-mouse-microbiota). Over 80days, we observed resilience effects at the strain and community levels, and we found that they were correlated with modulations of the estimated growth rate and levels of prophage induction as determined from metagenomics data. Moreover, we tracked mutational changes in the bacterial populations, and this uncovered clonal expansion and contraction of haplotypes and selection of putative AB resistance-conferring SNPs. We functionally verified these mutations via reisolation of clones with increased minimum inhibitory concentration (MIC) of ciprofloxacin and tetracycline from evolved communities. This demonstrates that host-associated microbial communities employ various mechanisms to respond to selective pressures that maintain community stability.

Body Size Poorly Predicts Host-Associated Microbial Diversity in Wild Birds.

The composition and diversity of avian microbiota are shaped by many factors, including host ecologies and environmental variables. In this study, we examine microbial diversity across 214 bird species sampled in Malawi at five major body sites: blood, buccal cavity, gizzard, intestinal tract, and cloaca. Microbial community dissimilarity differed significantly across body sites. Ecological theory predicts that as area increases, so does diversity. We tested the hypothesis that avian microbiota diversity is correlated with body size, used as a proxy for area, using comparative phylogenetic methods. Using Pagel's lambda, we found that few microbial diversity metrics had significant phylogenetic signals. Phylogenetic generalized least squares identified a significant but weak negative correlation between host size and microbial diversity of the blood and a similarly significant but weakly positive correlation between the cloacal microbiota and host size among birds within the order Passeriformes. Phylosymbiosis, or a congruent branching pattern between host phylogeny and their associated microbiota similarity, was tested and found to be weak or not significant in four of the body sites with sufficient sample size (blood, buccal, cloaca, and intestines). Taken together, these results suggest that the avian microbiome is highly variable, with microbiota diversity demonstrating few clear associations with bird size. Finally, the blood microbiota have a unique relationship with host size. IMPORTANCE All animals coexist and interact with microorganisms, including bacteria, archaea, microscopic eukaryotes, and viruses. These microorganisms can have an enormous influence on the biology and health of macro-organisms. However, the general rules that govern these host-associated microbial communities are poorly described, especially in wild animals. In this paper, we investigate the microbial communities of over 200 species of birds from Malawi and characterize five body site bacterial microbiota in depth. Because the evolutionary relationships of the host underlie the relationship between any host-associated microbiota relationships, we use phylogenetic comparative methods to account for this relationship. We find that the size of a host (the bird) and the diversity and composition of the microbiota are largely uncorrelated. We also find that the general pattern of similarity between host phylogeny and microbiota similarity is weak. Together, we see that bird microbiota are not strongly tied to host size or evolutionary history.

A Possible Aquatic Origin of the Thiaminase TenA of the Human Gut Symbiont Bacteroides thetaiotaomicron

TenA thiamin-degrading enzymes are commonly found in prokaryotes, plants, fungi and algae and are involved in the thiamin salvage pathway. The gut symbiont Bacteroides thetaiotaomicron (Bt) produces a TenA protein (BtTenA) which is packaged into its extracellular vesicles. An alignment of BtTenA protein sequence with proteins from different databases using the basic local alignment search tool (BLAST) and the generation of a phylogenetic tree revealed that BtTenA is related to TenA-like proteins not only found in a small number of intestinal bacterial species but also in some aquatic bacteria, aquatic invertebrates, and freshwater fish. This is, to our knowledge, the first report describing the presence of TenA-encoding genes in the genome of members of the animal kingdom. By searching metagenomic databases of diverse host-associated microbial communities, we found that BtTenA homologues were mostly represented in biofilms present on the surface of macroalgae found in Australian coral reefs. We also confirmed the ability of a recombinant BtTenA to degrade thiamin. Our study shows that BttenA-like genes which encode a novel sub-class of TenA proteins are sparingly distributed across two kingdoms of life, a feature of accessory genes known for their ability to spread between species through horizontal gene transfer.

Co-infections mask pathogen-specific associations with the gut microbiota in wild voles.

Research Highlight: Brila, I., Lavirinienko, A., Tukalenko, E., Kallio, E. R., Mappes, T. & Watts, P. C. (2022). Idiosyncratic effects of coinfection on the association between systemic pathogens and the gut microbiota of a wild rodent, the bank vole (Myodes glareolus). Journal of Animal Ecology, https://besjournals.onlinelibrary.wiley.com/doi/10.1111/1365-2656.13869. Interactions between pathogens and host-associated microbial communities can influence host fitness, disease progression and pathogen emergence. The vast majority of studies characterize interactions between single pathogens and bacterial commensals, yet co-infections with multiple pathogens are the norm in nature. In their paper on pathogen-microbiome interactions, Brila et al. (2022) examine how co-infections with four systemic pathogens associate with the gut microbiota in wild bank voles. Building on a series of tests, the authors show that excluding co-infection information from statistical models masks pathogen-specific patterns and confounds interpretations. This paper advances on previous studies by generating surveillance data on a phylogenetically diverse suite of vole pathogens to address the question as to whether pathogens exhibit unique or universal associations with gut commensals. They report that even bacterial pathogens with similar transmission ecology have divergent associations with gut microbes, and highlight that a mechanistic understanding of host-pathogen interactions is necessary for decoding the diverse consequences for gut microbial communities.

Effects of anthropogenic stress on hosts and their microbiomes: Treated wastewater alters performance and gut microbiome of a key detritivore (Asellus aquaticus).

Human activity is a major driver of ecological and evolutionary change in wild populations and can have diverse effects on eukaryotic organisms as well as on environmental and host-associated microbial communities. Although host-microbiome interactions can be a major determinant of host fitness, few studies consider the joint responses of hosts and their microbiomes to anthropogenic changes. In freshwater ecosystems, wastewater is a widespread anthropogenic stressor that represents a multifarious environmental perturbation. Here, we experimentally tested the impact of treated wastewater on a keystone host (the freshwater isopod Asellus aquaticus) and its gut microbiome. We used a semi-natural flume experiment, in combination with 16S rRNA amplicon sequencing, to assess how different concentrations (0%, 30%, and 80%) of nonfiltered wastewater (i.e. with chemical toxicants, nutrients, organic particles, and microbes) versus ultrafiltered wastewater (i.e. only dissolved pollutants and nutrients) affected host survival, growth, and food consumption as well as mid- and hindgut bacterial community composition and diversity. Our results show that while host survival was not affected by the treatments, host growth increased and host feeding rate decreased with nonfiltered wastewater - potentially indicating that A. aquaticus fed on organic matter and microbes available in nonfiltered wastewater. Furthermore, even though the midgut microbiome (diversity and composition) was not affected by any of our treatments, nonfiltered wastewater influenced bacterial composition (but not diversity) in the hindgut. Ultrafiltered wastewater, on the other hand, affected both community composition and bacterial diversity in the hindgut, an effect that in our system differed between sexes. While the functional consequences of microbiome changes and their sex specificity are yet to be tested, our results indicate that different components of multifactorial stressors (i.e. different constituents of wastewater) can affect hosts and their microbiome in distinct (even opposing) manners and have a substantial impact on eco-evolutionary responses to anthropogenic stressors.

Interrogating the role of the milk microbiome in mastitis in the multi-omics era.

There is growing interest in a functional understanding of milk-associated microbiota as there is ample evidence that host-associated microbial communities play an active role in host health and phenotype. Mastitis, characterized by painful inflammation of the mammary gland, is prevalent among lactating humans and agricultural animals and is associated with significant clinical and economic consequences. The etiology of mastitis is complex and polymicrobial and correlative studies have indicated alterations in milk microbial community composition. Recent evidence is beginning to suggest that a causal relationship may exist between the milk microbiota and host phenotype in mastitis. Multi-omic approaches can be leveraged to gain a mechanistic, molecular level understanding of how the milk microbiome might modulate host physiology, thereby informing strategies to prevent and ameliorate mastitis. In this paper, we review existing studies that have utilized omics approaches to investigate the role of the milk microbiome in mastitis. We also summarize the strengths and challenges associated with the different omics techniques including metagenomics, metatranscriptomics, metaproteomics, metabolomics and lipidomics and provide perspective on the integration of multiple omics technologies for a better functional understanding of the milk microbiome.

Impact of Nesting Mode, Diet, and Taxonomy in Structuring the Associated Microbial Communities of Amazonian Ants

Studies of ant biodiversity are important to understand their group better, as well as to extend our knowledge on the evolution of their associated organisms. Host-associated microbial communities, and particularly bacterial communities, are shaped by different host factors such as habitat, diet, and phylogeny. Here, we studied the structures of bacterial and microbial eukaryote communities associated with Amazonian ants collected from two habitats: the rainforest and the city. We collected 38 ant species covering a large taxonomic range, and we used 16S rRNA and 18S rRNA amplicon sequencing to study the impact of the host’s ecological and phylogenetic factors on their microbial communities. Our results show that (1) habitat does not structure ant microbial communities, (2) ant diet and nesting mode impact bacterial communities, while only nesting mode structures microbial eukaryote communities, and (3) microbial diversity is not correlated with host phylogeny, although several ant genera have conserved bacterial communities. As we continue to uncover the diversity and function of insect-associated microbes, this work explores how host ecology and evolutionary history shape ant microbial communities.

Beyond the theory: From holobiont concept to microbiome engineering.

Holobiont research has increasingly moved from descriptive studies to sophisticated field- and laboratory-based manipulations; however, the extent to which changes in the holobiont persist remains largely unknown. In this Burning Question, we ask whether the underlying principles of the holobiont concept, whereby an externally applied evolutionary pressure can lead to a beneficial change in host-associated microbial community composition, could be used to facilitate microbiome engineering and thereby addition of a new ecosystem service that persists across generations. The answer to this question has potential implications for diverse fields including symbiosis, conservation and biotechnology.

Integrated multi-omics analyses of microbial communities: a review of the current state and future directions.

Integrated multi-omics analyses of microbiomes have become increasingly common in recent years as the emerging omics technologies provide an unprecedented opportunity to better understand the structural and functional properties of microbial communities. Consequently, there is a growing need for and interest in the concepts, approaches, considerations, and available tools for investigating diverse environmental and host-associated microbial communities in an integrative manner. In this review, we first provide a general overview of each omics analysis type, including a brief history, typical workflow, primary applications, strengths, and limitations. Then, we inform on both experimental design and bioinformatics analysis considerations in integrated multi-omics analyses, elaborate on the current approaches and commonly used tools, and highlight the current challenges. Finally, we discuss the expected key advances, emerging trends, potential implications on various fields from human health to biotechnology, and future directions.