Deciphering the ecoevolutionary recipe of milk microbiomes.

In a recent article, Keady et al. analyzed mammalian milk microbiomes across 47 species and found their assembly to be largely determined by stochastic (i.e., random) processes. In many ways, host-associated microbiomes are not random, but random events may have an underappreciated role in microbiome assembly, persistence, and ecology.

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.

Functional and Compositional Changes in the Fecal Microbiome of a Shorebird during Migratory Stopover.

Shorebirds migrate long distances twice annually, which requires intense physiological and morphological adaptations, including the ability to rapidly gain weight via fat deposition at stopover locations. The role of the microbiome in weight gain in avian hosts is unresolved, but there is substantial evidence to support the hypothesis that the microbiome is involved with host weight from mammalian microbiome literature. Here, we collected 100 fecal samples of Ruddy Turnstones to investigate microbiome composition and function during stopover weight gain in Delaware Bay, USA. Using 16S rRNA sequencing on 90 of these samples and metatranscriptomic sequencing on 22, we show that taxonomic composition of the microbiome shifts during weight gain, as do functional aspects of the metatranscriptome. We identified 10 genes that are associated with weight class, and polyunsaturated fatty acid biosynthesis in the microbiota is significantly increasing as birds gain weight. Our results support that the microbiome is a dynamic feature of host biology that interacts with both the host and the environment and may be involved in the rapid weight gain of shorebirds. IMPORTANCE Many animals migrate long distances annually, and these journeys require intense physiological and morphological adaptations. One such adaptation in shorebirds is the ability to rapidly gain weight at stopover locations in the middle of their migrations. The role of the microbiome in weight gain in birds is unresolved but is likely to play a role. Here, we collected 100 fecal samples from Ruddy Turnstones to investigate microbiome composition (who is there) and function (what they are doing) during stopover weight gain in Delaware Bay, USA. Using multiple molecular methods, we show that both taxonomic composition and function of the microbiome shifts during weight gain. We identified 10 genes that are associated with weight class, and polyunsaturated fatty acid biosynthesis in the microbiota is significantly increasing as birds gain weight. Our results support that the microbiome is a dynamic feature of host biology that interacts with both the host and the environment and may be involved in the rapid weight gain of shorebirds.

Multiomics Characterization of the Canada Goose Fecal Microbiome Reveals Selective Efficacy of Simulated Metagenomes.

16S rRNA amplicon sequences are predominantly used to identify the taxonomic composition of a microbiome, but they can also be used to generate simulated metagenomes to circumvent costly empirical shotgun sequencing. The effectiveness of using "simulated metagenomes" (shotgun metagenomes simulated from 16S rRNA amplicons using a database of full genomes closely related to the amplicons) in nonmodel systems is poorly known. We sought to determine the accuracy of simulated metagenomes in a nonmodel organism, the Canada goose (Branta canadensis), by comparing metagenomes and metatranscriptomes to simulated metagenomes derived from 16S amplicon sequencing. We found significant differences between the metagenomes, metatranscriptomes, and simulated metagenomes when comparing enzymes, KEGG orthologies (KO), and metabolic pathways. The simulated metagenomes accurately identified the majority (>70%) of the total enzymes, KOs, and pathways. The simulated metagenomes accurately identified the majority of the short-chain fatty acid metabolic pathways crucial to folivores. When narrowed in scope to specific genes of interest, the simulated metagenomes overestimated the number of antimicrobial resistance genes and underestimated the number of genes related to the breakdown of plant matter. Our results suggest that simulated metagenomes should not be used in lieu of empirical sequencing when studying the functional potential of a nonmodel organism's microbiome. Regarding the function of the Canada goose microbiome, we found unexpected amounts of fermentation pathways, and we found that a few taxa are responsible for large portions of the functional potential of the microbiome. IMPORTANCE The taxonomic composition of a microbiome is predominately identified using amplicon sequencing of 16S rRNA genes, but as a single marker, it cannot identify functions (genes). Metagenome and metatranscriptome sequencing can determine microbiome function but can be cost prohibitive. Therefore, computational methods have been developed to generate simulated metagenomes derived from 16S rRNA sequences and databases of full-length genomes. Simulated metagenomes can be an effective alternative to empirical sequencing, but accuracy depends on the genomic database used and whether the database contains organisms closely related to the 16S sequences. These tools are effective in well-studied systems, but the accuracy of these predictions in a nonmodel system is less known. Using a nonmodel bird species, we characterized the function of the microbiome and compared the accuracy of 16S-derived simulated metagenomes to sequenced metagenomes. We found that the simulated metagenomes reflect most but not all functions of empirical metagenome sequencing.

Avian gut microbiomes taking flight.

Birds harbor complex gut bacterial communities that may sustain their ecologies and facilitate their biological roles, distribution, and diversity. Research on gut microbiomes in wild birds is surging and it is clear that they are diverse and important - but strongly influenced by a series of environmental factors. To continue expanding our understanding of how the internal ecosystems of birds work in their natural settings, we believe the most pressing needs involve studies on the functional and evolutionary aspects of these symbioses. Here we summarize the state of the field and provide a roadmap for future studies on aspects that are pivotal to understanding the biology of avian gut microbiomes, emphasizing prospects for integrating gut microbiome work in avian conservation and host health monitoring.

Playing by their own rules: the vertebrate archaeome.

In a recent paper, Youngblut et al. used comparative methods to study the archaeal component of the vertebrate microbiome. This study showed that the vertebrate archaeome contains abundant variation, differs from the vertebrate bacteriome and lays a foundation for future comparative studies.

Sex Differences in Gut Microbial Development of Preterm Infant Twins in Early Life: A Longitudinal Analysis.

Infant gut microbiota plays a vital role in immune response, mediates neurobehavioral development and health maintenance. Studies of twins' gut microbiota found that gut microbiota composition and diversity tend to be mature and stable with increasing postnatal age (PNA). Preterm infant gut microbiome shifts dramatically when they were staying in the neonatal intensive care unit (NICU). Compositions and shifting characteristics of gut microbiota among neonatal preterm twins and triplets during their early life are still unknown, which impedes a better understanding of the mechanism underpinning neurobehavioral development and precise intervention/health of preterm neonates. This longitudinal cohort study used a twins/triplets design to investigate the interaction of genetic (e.g., male vs. female) and environmental factors influencing the development of the gut microbiome in early life. We included 39 preterm infants, 12 were Female twins/triplets (Female T/T) including 3 twins pairs and 2 triplets, 12 were male twins (Male T) including 6 twins pairs, and 15 were mixed-sex twins/triplets (Mix T/T) including 6 twins pairs and 1 triplet (8 females and 7 males) during the first four weeks of NICU stay. Weekly gut microbiota patterns between females and males were compared by linear discriminant analysis (LDA) effect size (LEfSe). Metagenomics function of gut microbiota was predicted by using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Weekly function (KEGG pathways) differences between females and males were detected by using Statistical Analysis of Metagenomic Profiles (STAMP). Results found that female pairs and male pairs were significantly different in gut microbiome diversity, compositions, and predicted metabolic profiles, importantly, females and males were also significantly dissimilar within their co-twin/triplet pairs of the mixed-sex group, infants of co-twins/triplets shared more similar features than un-related infants from different twins' pair. Future research developing personalized interventions for vulnerable high-risk infants should consider sex, and the interaction of sex and environmental factors.

Red Fluorescence of European Hedgehog (Erinaceus europaeus) Spines Results from Free-Base Porphyrins of Potential Microbial Origin.

Bioluminescence has been recognized as an important means for inter- and intra-species communication. A growing number of reports of red fluorescence occurring in keratinaceous materials have become available. The fluorophore(s) in these cases were shown to be, or suspected to be, free base porphyrins. The red fluorescence found in the downs of bustards was associated with inter-species signaling in mate selection. First reported in 1925, we confirm that spines of the European hedgehog (Erinaceus europaeus) when irradiated with UV (365-395 nm) light display red fluorescence localized in the light-colored sections of their proximal ends. Using reflectance fluorescence spectroscopy, we confirmed that the fluorophores responsible for the emission are free-base porphyrins, as suspected in the original report. Base-induced degradation of the spine matrix and subsequent HPLC, UV-vis, and ESI+ mass spectrometry analysis revealed the presence of a mixture of coproporphyrin III and uroporphyrin III as predominant porphyrins and a minor fraction of protoporphyrin IX. Investigation of the spine microbiome uncovered the abundant presence of bacteria known to secrete and/or interconvert porphyrins and that are not present on the non-fluorescing quills of the North American porcupine (Erethizon dorsatum). Given this circumstantial evidence, we propose the porphyrins could originate from commensal bacteria. Furthermore, we hypothesize that the fluorescence may be incidental and of no biological function for the hedgehog.

Ixodes scapularis microbiome correlates with life stage, not the presence of human pathogens, in ticks submitted for diagnostic testing.

Ticks are globally distributed arthropods and a public health concern due to the many human pathogens they carry and transmit, including the causative agent of Lyme disease, Borrelia burgdorferi. As tick species' ranges increase, so do the number of reported tick related illnesses. The microbiome is a critical part of understanding arthropod biology, and the microbiome of pathogen vectors may provide critical insight into disease transmission and management. Yet we lack a comprehensive understanding of the microbiome of wild ticks, including what effect the presence of multiple tick-borne pathogens (TBPs) has on the microbiome. In this study we chose samples based on life stage (adult or nymph) and which TBPs were present. We used DNA from previously extracted Ixodes scapularis ticks that tested positive for zero, one, two or three common TBPs (B. burgdorferi, B. miyamotoi, Anaplasma phagocytophilum, Babesia microti). We produced 16S rRNA amplicon data for the whole tick microbiome and compared samples across TBPs status, single vs multiple coinfections, and life stages. Focusing on samples with a single TBP, we found no significant differences in microbiome diversity in ticks that were infected with B. burgdorferi and ticks with no TBPs. When comparing multiple TBPs, we found no significant difference in both alpha and beta diversity between ticks with a single TBP and ticks with multiple TBPs. Removal of TBPs from the microbiome did not alter alpha or beta diversity results. Life stage significantly correlated to variation in beta diversity and nymphs had higher alpha diversity than adult ticks. Rickettsia, a common tick endosymbiont, was the most abundant genus. This study confirms that the wild tick microbiome is highly influenced by life stage and much less by the presence of human pathogenic bacteria.

Spatial heterogeneity of the shorebird gastrointestinal microbiome.

The gastrointestinal tract (GIT) consists of connected structures that vary in function and physiology, and different GIT sections potentially provide different habitats for microorganisms. Birds possess unique GIT structures, including the oesophagus, proventriculus, gizzard, small intestine, caeca and large intestine. To understand birds as hosts of microbial ecosystems, we characterized the microbial communities in six sections of the GIT of two shorebird species, the Dunlin and Semipalmated Sandpiper, identified potential host species effects on the GIT microbiome and used microbial source tracking to determine microbial origin throughout the GIT. The upper three GIT sections had higher alpha diversity and genus richness compared to the lower sections, and microbial communities in the upper GIT showed no clustering. The proventriculus and gizzard microbiomes primarily originated from upstream sections, while the majority of the large intestine microbiome originated from the caeca. The heterogeneity of the GIT sections shown in our study urges caution in equating data from faeces or a single GIT component to the entire GIT microbiome but confirms that ecologically similar species may share many attributes in GIT microbiomes.

Context Is Key: Comparative Biology Illuminates the Vertebrate Microbiome.

Microbes affect vertebrates on timescales from daily to evolutionary, and the cumulative effect of these interactions is immense. However, how microbiomes compare across (host) species is poorly understood, as most studies focus on relatively few species. A recent mBio article by S. J. Song, J. G. Sanders, F. Delsuc, J. Metcalf, et al. (mBio 11:e02901-19, 2019, https://doi.org/10.1128/mBio.02901-19) expands our collective understanding of the vertebrate microbiome by analyzing ∼900 species. They demonstrate that patterns within mammals contrast with those within birds. Their results suggest many hypotheses about the role of host ecology and evolution on microbiome variation. Bats, the only volant mammals, appear to contradict many of the general mammal microbiome trends, in some ways resembling birds. What role has powered flight, and the evolution thereof, played in microbiome structure and function? Comparative methods, mechanistic hypotheses, and theory will elucidate this exciting question (and others) that we can ask using Song, Sanders et al.'s data and results.

Evolutionary signal in the gut microbiomes of 74 bird species from Equatorial Guinea.

How the microbiome interacts with hosts across evolutionary time is poorly understood. Data sets including many host species are required to conduct comparative analyses. Here, we analyzed 142 intestinal microbiome samples from 92 birds belonging to 74 species from Equatorial Guinea, using the 16S rRNA gene. Using four definitions for microbial taxonomic units (97%OTU, 99%OTU, 99%OTU with singletons removed, ASV), we conducted alpha and beta diversity analyses. We found that raw abundances and diversity varied between the data sets but relative patterns were largely consistent across data sets. Host taxonomy, diet and locality were significantly associated with microbiomes, at generally similar levels using three distance metrics. Phylogenetic comparative methods assessed the evolutionary relationship between the microbiome as a trait of a host species and the underlying bird phylogeny. Using multiple ways of defining "microbiome traits", we found that a neutral Brownian motion model did not explain variation in microbiomes. Instead, we found a White Noise model (indicating little phylogenetic signal), was most likely. There was some support for the Ornstein-Uhlenbeck model (that invokes selection), but the level of support was similar to that of a White Noise simulation, further supporting the White Noise model as the best explanation for the evolution of the microbiome as a trait of avian hosts. Our study demonstrated that both environment and evolution play a role in the gut microbiome and the relationship does not follow a neutral model; these biological results are qualitatively robust to analytical choices.

No evidence for phylosymbiosis in western chipmunk species.

Phylosymbiosis refers to a congruent pattern between the similarity of microbiomes of different species and the branching pattern of the host phylogeny. Phylosymbiosis has been detected in a variety of vertebrate and invertebrate hosts, but has only been assessed in geographically isolated populations. We tested for phylosymbiosis in eight (sub)species of western chipmunks with overlapping ranges and ecological niches; we used a nuclear (Acrosin) and a mitochondrial (CYTB) phylogenetic marker because there are many instances of mitochondrial introgression in chipmunks. We predicted that similarity among microbiomes increases with: (1) increasing host mitochondrial relatedness, (2) increasing host nuclear genome relatedness and (3) decreasing geographic distance among hosts. We did not find statistical evidence supporting phylosymbiosis in western chipmunks. Furthermore, in contrast to studies of other mammalian microbiomes, similarity of chipmunk microbiomes is not predominantly determined by host species. Sampling site explained most variation in microbiome composition, indicating an important role of local environment in shaping microbiomes. Fecal microbiomes of chipmunks were dominated by Bacteroidetes (72.2%), followed by Firmicutes (24.5%), which is one of the highest abundances of Bacteroidetes detected in wild mammals. Future work will need to elucidate the effects of habitat, ecology and host genomics on chipmunk microbiomes.

Correction: Longitudinal microbiome profiling reveals impermanence of probiotic bacteria in domestic pigeons.

[This corrects the article DOI: 10.1371/journal.pone.0217804.].

Microbiomes, Community Ecology, and the Comparative Method.

Microbiomes contain many levels of biological information, and integrating across the levels creates a holistic understanding of host-microbiome interactions. In my research on the evolution and ecology of avian microbiomes, I use two complementary frameworks: the microbiome as a community and the microbiome as a trait of the host. We draw on classic ecological and evolutionary theory and modern statistical models to advance our understanding in each of these frameworks and then integrate what we have learned into a better understanding of host-associated microbiomes, host evolution, and microbial biodiversity. Ecological theories that bear on processes such as community assembly and metacommunities are well suited for application to microbiomes. Phylogenetic comparative methods can quantify the fit of evolutionary models and detect correlations between traits and correlations between traits and the rate of evolution; these methods allow the inference of evolutionary process from contemporary patterns.

Longitudinal microbiome profiling reveals impermanence of probiotic bacteria in domestic pigeons.

Probiotics are bacterial species or assemblages that are applied to animals and plants with the intention of altering the microbiome in a beneficial way. Probiotics have been linked to positive health effects such as faster disease recovery times in humans and increased weight gain in poultry. Pigeon fanciers often feed their show pigeons probiotics with the intention of increasing flight performance. The objective of our study was to determine the effect of two different probiotics, alone and in combination, on the fecal microbiome of Birmingham Roller pigeons. We sequenced fecal samples from 20 pigeons divided into three probiotic treatments, including prior to, during, and after treatment. Pre-treatment and control group samples were dominated by Actinobacteria, Firmicutes, Proteobacteria, and Cyanobacteria. Administration of a probiotic pellet containing Enterococcus faecium and Lactobacillus acidophilus resulted in increase in average relative abundance of Lactobacillus spp. from 4.7 ± 2.0% to 93.0 ± 5.3%. No significant effects of Enterococcus spp. were detected. Probiotic-induced shifts in the microbiome composition were temporary and disappeared within 2 days of probiotic cessation. Administration of a probiotic powder in drinking water that contained Enterococcus faecium and three Lactobacillus species had minimal effect on the microbiome. We conclude that supplementing Birmingham roller pigeons with the probiotic pellets, but not the probiotic powder, temporarily changed the microbiome composition. A next step is to experimentally test the effect of these changes in microbiome composition on host health and physical performance.

Microbial Community Succession and Nutrient Cycling Responses following Perturbations of Experimental Saltwater Aquaria.

Although aquaria are common features of homes and other buildings, little is known about how environmental perturbations (i.e., tank cleaning, water changes, addition of habitat features) impact the diversity and succession of aquarium microbial communities. In this study, we sought to evaluate the hypotheses that newly established aquaria show clear microbial successional patterns over time and that common marine aquarium-conditioning practices, such as the addition of ocean-derived "live rocks" (defined as any "dead coral skeleton covered with crustose coralline algae" transferred into an aquarium from open ocean habitats) impact the diversity of microbial populations as well as nitrogen cycling in aquaria. We collected water chemistry data alongside water and sediment samples from two independent and newly established saltwater aquaria over a 3-month period. Microbial communities in samples were assessed by DNA extraction, amplification of the 16S rRNA gene, and Illumina MiSeq sequencing. Our results showed clear and replicable patterns of community succession in both aquaria, with the existence of multiple stable states for aquarium microbial assemblages. Notably, our results show that changes in aquarium microbial communities do not always correlate with water chemistry measurements and that operational taxonomic unit (OTU)-level patterns relevant to nitrogen cycling were not reported as statistically significant. Overall, our results demonstrate that aquarium perturbations have a substantial impact on microbial community profiles of aquarium water and sediment and that the addition of live rocks improves nutrient cycling by shifting aquarium communities toward a more typical saltwater assemblage of microbial taxa.IMPORTANCE Saltwater aquaria are living systems that support a complex biological community of fish, invertebrates, and microbes. The health and maintenance of saltwater tanks are pressing concerns for home hobbyists, zoos, and professionals in the aquarium trade; however, we do not yet understand the underlying microbial species interactions and community dynamics which contribute to tank setup and conditioning. This report provides a detailed view of ecological succession and changes in microbial community assemblages in two saltwater aquaria which were sampled over a 3-month period, from initial tank setup and conditioning with "live rocks" through subsequent tank cleanings and water replacement. Our results showed that microbial succession appeared to be consistent and replicable across both aquaria. However, changes in microbial communities did not always correlate with water chemistry measurements, and aquarium microbial communities appear to have shifted among multiple stable states without any obvious buildup of undesirable nitrogen compounds in the tank environment.

The Cloacal Microbiome of Five Wild Duck Species Varies by Species and Influenza A Virus Infection Status.

Waterfowl, especially ducks of the genus Anas, are natural reservoir species for influenza A virus (IAV). Duck populations contain nearly all the known diversity of IAVs, and the birds are asymptomatic to infection. Previous work established that IAV infection status is correlated with changes in the cloacal microbiome in juvenile mallards. Here, we analyze five Anas species to determine whether these duck species have similar IAV+ and IAV- cloacal microbiomes, or if the relationships among a host, influenza virus, and the microbiome are species specific. We assessed taxonomic composition of the microbiome, alpha diversity, and beta diversity and found very few patterns related to microbiome and infection status across species, while detecting strong differences within species. A host species-specific signal was stronger in IAV- ducks than IAV+ ducks, and the effect size of host species on the microbiome was three times higher in IAV- birds than IAV+ birds. The mallards and the northern shovelers, the species with highest sample sizes but also with differing feeding ecology, showed especially contrasting patterns in microbiome composition, alpha diversity, and beta diversity. Our results indicate that the microbiome may have a unique relationship with influenza virus infection at the species level.IMPORTANCE Waterfowl are natural reservoir species for influenza A virus (IAV). Thus, they maintain high levels of pathogen diversity, are asymptomatic to the infection, and also contribute to the risk of a global influenza pandemic. An individual's microbiome is a critical part in how a vertebrate manages pathogens and illness. Here, we describe the cloacal microbiome of 300 wild ducks, from five species (four with previously undescribed microbiomes), including both IAV-negative and IAV-positive individuals. We demonstrate that there is not one consistent "flu-like" microbiome or response to flu across species. Individual duck species appear to have unique relationships between their microbiomes and IAV, and IAV-negative birds have a stronger tie to host species than the IAV-positive birds. In a broad context, understanding the role of the microbiome in IAV reservoir species may have future implications for avian disease management.

Evolutionary Biology Needs Wild Microbiomes.

The microbiome is a vital component to the evolution of a host and much of what we know about the microbiome derives from studies on humans and captive animals. But captivity alters the microbiome and mammals have unique biological adaptations that affect their microbiomes (e.g., milk). Birds represent over 30% of known tetrapod diversity and possess their own suite of adaptations relevant to the microbiome. In a previous study, we showed that 59 species of birds displayed immense variation in their microbiomes and host (bird) taxonomy and ecology were most correlated with the gut microbiome. In this Frontiers Focused Review, I put those results in a broader context by discussing how collecting and analyzing wild microbiomes contributes to the main goals of evolutionary biology and the specific ways that birds are unique microbial hosts. Finally, I outline some of the methodological considerations for adding microbiome sampling to the research of wild animals and urge researchers to do so. To truly understand the evolution of a host, we need to understand the millions of microorganisms that inhabit it as well: evolutionary biology needs wild microbiomes.

Community-Level Differences in the Microbiome of Healthy Wild Mallards and Those Infected by Influenza A Viruses.

Waterfowl, especially ducks and geese, are primary reservoirs for influenza A viruses (IAVs) that evolve and emerge as important pathogens in domestic animals and humans. In contrast to humans, where IAVs infect the respiratory tract and cause significant morbidity and mortality, IAVs infect the gastrointestinal tract of waterfowl and cause little or no pathology and are spread by fecal-oral transmission. For this reason, we examined whether IAV infection is associated with differences in the cloacal microbiome of mallards (Anas platyrhyncos), an important host of IAVs in North America and Eurasia. We characterized bacterial community composition by sequencing the V4 region of 16S rRNA genes. IAV-positive mallards had lower species diversity, richness, and evenness than IAV-negative mallards. Operational taxonomic unit (OTU) cooccurrence patterns were also distinct depending on infection status. Network analysis showed that IAV-positive mallards had fewer significant cooccurring OTUs and exhibited fewer coassociation patterns among those OTUs than IAV-negative mallards. These results suggest that healthy mallards have a more robust and complex cloacal microbiome. By combining analytical approaches, we identified 41 bacterial OTUs, primarily representatives of Streptococcus spp., Veillonella dispar, and Rothia mucilaginosa, contributing to the observed differences. This study found that IAV-infected wild mallards exhibited strong differences in microbiome composition relative to noninfected mallards and identified a concise set of putative biomarker OTUs. Using Random Forest, a supervised machine learning method, we verified that these 41 bacterial OTUs are highly predictive of infection status. IMPORTANCE Seasonal influenza causes 3 to 5 million severe illnesses and 250,000 to 500,000 human deaths each year. While pandemic influenza viruses emerge only periodically, they can be devastating-for example, the 1918 H1N1 pandemic virus killed more than 20 million people. IAVs infect the respiratory tract and cause significant morbidity and mortality in humans. In contrast, IAVs infect the gastrointestinal tract of waterfowl, producing little pathology. Recent studies indicated that viruses can alter the microbiome at the respiratory and gastrointestinal mucosa, but there are no reports of how the microbiota of the natural host of influenza is affected by infection. Here we find that the mallard microbiome is altered during IAV infection. Our results suggest that detailed examination of humans and animals infected with IAVs may reveal individualized microbiome profiles that correspond to health and disease. Moreover, future studies should explore whether the altered microbiome facilitates maintenance and transmission of IAVs in waterfowl populations.