Unravelling the collateral damage of antibiotics on gut bacteria.

Antibiotics are used to fight pathogens but also target commensal bacteria, disturbing the composition of gut microbiota and causing dysbiosis and disease1. Despite this well-known collateral damage, the activity spectrum of different antibiotic classes on gut bacteria remains poorly characterized. Here we characterize further 144 antibiotics from a previous screen of more than 1,000 drugs on 38 representative human gut microbiome species2. Antibiotic classes exhibited distinct inhibition spectra, including generation dependence for quinolones and phylogeny independence for ß-lactams. Macrolides and tetracyclines, both prototypic bacteriostatic protein synthesis inhibitors, inhibited nearly all commensals tested but also killed several species. Killed bacteria were more readily eliminated from in vitro communities than those inhibited. This species-specific killing activity challenges the long-standing distinction between bactericidal and bacteriostatic antibiotic classes and provides a possible explanation for the strong effect of macrolides on animal3-5 and human6,7 gut microbiomes. To mitigate this collateral damage of macrolides and tetracyclines, we screened for drugs that specifically antagonized the antibiotic activity against abundant Bacteroides species but not against relevant pathogens. Such antidotes selectively protected Bacteroides species from erythromycin treatment in human-stool-derived communities and gnotobiotic mice. These findings illluminate the activity spectra of antibiotics in commensal bacteria and suggest strategies to circumvent their adverse effects on the gut microbiota.

Bacteriophages targeting protective commensals impair resistance against Salmonella Typhimurium infection in gnotobiotic mice.

Gut microbial communities protect the host against a variety of major human gastrointestinal pathogens. Bacteriophages (phages) are ubiquitous in nature and frequently ingested via food and drinking water. Moreover, they are an attractive tool for microbiome engineering due to the lack of known serious adverse effects on the host. However, the functional role of phages within the gastrointestinal microbiome remain poorly understood. Here, we investigated the effects of microbiota-directed phages on infection with the human enteric pathogen Salmonella enterica serovar Typhimurium (S. Tm), using a gnotobiotic mouse model (OMM14) for colonization resistance (CR). We show, that phage cocktails targeting Escherichia coli and Enterococcus faecalis acted in a strain-specific manner. They transiently reduced the population density of their respective target before establishing coexistence for up to 9 days. Infection susceptibility to S. Tm was markedly increased at an early time point after challenge with both phage cocktails. Surprisingly, OMM14 mice were also susceptible 7 days after a single phage inoculation, when the targeted bacterial populations were back to pre-phage administration density. Concluding, our work shows that phages that dynamically modulate the density of protective members of the gut microbiota can provide opportunities for invasion of bacterial pathogens, in particular at early time points after phage application. This suggests, that phages targeting protective members of the microbiota may increase the risk for Salmonella infection.

Metabolic reconstitution of germ-free mice by a gnotobiotic microbiota varies over the circadian cycle.

The capacity of the intestinal microbiota to degrade otherwise indigestible diet components is known to greatly improve the recovery of energy from food. This has led to the hypothesis that increased digestive efficiency may underlie the contribution of the microbiota to obesity. OligoMM12-colonized gnotobiotic mice have a consistently higher fat mass than germ-free (GF) or fully colonized counterparts. We therefore investigated their food intake, digestion efficiency, energy expenditure, and respiratory quotient using a novel isolator-housed metabolic cage system, which allows long-term measurements without contamination risk. This demonstrated that microbiota-released calories are perfectly balanced by decreased food intake in fully colonized versus gnotobiotic OligoMM12 and GF mice fed a standard chow diet, i.e., microbiota-released calories can in fact be well integrated into appetite control. We also observed no significant difference in energy expenditure after normalization by lean mass between the different microbiota groups, suggesting that cumulative small differences in energy balance, or altered energy storage, must underlie fat accumulation in OligoMM12 mice. Consistent with altered energy storage, major differences were observed in the type of respiratory substrates used in metabolism over the circadian cycle: In GF mice, the respiratory exchange ratio (RER) was consistently lower than that of fully colonized mice at all times of day, indicative of more reliance on fat and less on glucose metabolism. Intriguingly, the RER of OligoMM12-colonized gnotobiotic mice phenocopied fully colonized mice during the dark (active/eating) phase but phenocopied GF mice during the light (fasting/resting) phase. Further, OligoMM12-colonized mice showed a GF-like drop in liver glycogen storage during the light phase and both liver and plasma metabolomes of OligoMM12 mice clustered closely with GF mice. This implies the existence of microbiota functions that are required to maintain normal host metabolism during the resting/fasting phase of circadian cycle and which are absent in the OligoMM12 consortium.

Contribution of bacterial and host factors to pathogen "blooming" in a gnotobiotic mouse model for Salmonella enterica serovar Typhimurium-induced enterocolitis.

Inflammation has a pronounced impact on the intestinal ecosystem by driving an expansion of facultative anaerobic bacteria at the cost of obligate anaerobic microbiota. This pathogen "blooming" is also a hallmark of enteric Salmonella enterica serovar Typhimurium (S. Tm) infection. Here, we analyzed the contribution of bacterial and host factors to S. Tm "blooming" in a gnotobiotic mouse model for S. Tm-induced enterocolitis. Mice colonized with the Oligo-Mouse-Microbiota (OMM12), a minimal bacterial community, develop fulminant colitis by day 4 after oral infection with wild-type S. Tm but not with an avirulent mutant. Inflammation leads to a pronounced reduction in overall intestinal bacterial loads, distinct microbial community shifts, and pathogen blooming (relative abundance >50%). S. Tm mutants attenuated in inducing gut inflammation generally elicit less pronounced microbiota shifts and reduction in total bacterial loads. In contrast, S. Tm mutants in nitrate respiration, salmochelin production, and ethanolamine utilization induced strong inflammation and S. Tm "blooming." Therefore, individual Salmonella-specific inflammation-fitness factors seem to be of minor importance for competition against this minimal microbiota in the inflamed gut. Finally, we show that antibody-mediated neutrophil depletion normalized gut microbiota loads but not intestinal inflammation or microbiota shifts. This suggests that neutrophils equally reduce pathogen and commensal bacterial loads in the inflamed gut.

CCL17 Promotes Colitis-Associated Tumorigenesis Dependent on the Microbiota.

Colorectal cancer is one of the most common cancers and a major cause of mortality. Proinflammatory and antitumor immune responses play critical roles in colitis-associated colon cancer. CCL17, a chemokine of the C-C family and ligand for CCR4, is expressed by intestinal dendritic cells in the steady state and is upregulated during colitis in mouse models and inflammatory bowel disease patients. In this study, we investigated the expression pattern and functional relevance of CCL17 for colitis-associated colon tumor development using CCL17-enhanced GFP-knockin mice. CCL17 was highly expressed by dendritic cells but also upregulated in macrophages and intermediary monocytes in colon tumors induced by exposure to azoxymethane and dextran sodium sulfate. Despite a similar degree of inflammation in the colon, CCL17-deficient mice developed fewer tumors than did CCL17-competent mice. This protective effect was abrogated by cohousing, indicating a dependency on the microbiota. Changes in microbiota diversity and composition were detected in separately housed CCL17-deficient mice, and these mice were more susceptible to azoxymethane-induced early apoptosis in the colon affecting tumor initiation. Immune cell infiltration in colitis-induced colon tumors was not affected by the lack of CCL17. Taken together, our results indicate that CCL17 promotes colitis-associated tumorigenesis by influencing the composition of the intestinal microbiome and reducing apoptosis during tumor initiation.

Establishing causality in Salmonella-microbiota-host interaction: The use of gnotobiotic mouse models and synthetic microbial communities.

Colonization resistance (CR), the ability to block infections by potentially harmful microbes, is a fundamental function of host-associated microbial communities and highly conserved between animals and humans. Environmental factors such as antibiotics and diet can disturb microbial community composition and thereby predispose to opportunistic infections. The most prominent is Clostridioides difficile, the causative agent of diarrhea and pseudomembranous colitis. In addition, the risk to succumb to infections with genuine human enteric pathogens like nontyphoidal Salmonella (NTS) is also increased by a low-diverse, diet or antibiotic-disrupted microbiota. Despite extensive microbial community profiling efforts, only a limited set of microorganisms have been causally linked with protection against enteric pathogens. Furthermore, it remains a challenge to predict colonization resistance from complex microbiome signatures due to context-dependent action of microorganisms. In the past decade, the study of NTS infection has led to the description of several fundamental principles of microbiota-host-pathogen interaction. In this review, I will give an overview on the current state of knowledge in this field and outline experimental approaches to gain functional insight to the role of specific microbes, functions and metabolites in Salmonella-microbiota-host interaction. In particular, I will highlight the value of mouse infection models, which, in combination with culture collections, synthetic communities and gnotobiotic models have become essential tools to screen for protective members of the microbiota and establishing causal relationship and mechanisms in infection research.

Monitoring and contamination incidence of gnotobiotic experiments performed in microisolator cages.

With the increased interest in the microbiome research, gnotobiotic animals and techniques emerged again as valuable tools to investigate functional effects of host-microbe and microbe-microbe interactions. The increased demand for gnotobiotic experiments has resulted in the greater need for housing systems for short-term maintenance of gnotobiotic animals. During the last six years, the gnotobiotic facility of the Hannover Medical School has worked intensively with different housing systems for gnotobiotic animals. Here, we report our experience in handling, contamination incidence, and monitoring strategies that we apply for controlling gnotobiotic experiments. From our experience, the risk of introducing contaminants to animals housed in microisolator cages is higher than in isolators. However, with strict operating protocols, the contamination rate in these systems can be minimized. In addition to spore-forming bacteria and fungi from the environment, spore-forming bacteria from defined bacterial communities used in experiments represent the major risk for contamination of gnotobiotic experiments performed in microisolator cages. The presence/absence of contaminants in germ-free animals can be easily monitored by preparation of wet mounts and Gram staining of fecal samples. Contaminants in animals colonized with specific microorganisms need to be tracked with methods such as next-generation sequencing. However, when using PCR-based methods it is important to consider that relatively small amounts of bacterial DNA detected likely originates from food, bedding, or reagents and is not to be interpreted as true contamination.

Genomic convergence between Akkermansia muciniphila in different mammalian hosts.

BACKGROUND: Akkermansia muciniphila is a member of the human gut microbiota where it resides in the mucus layer and uses mucin as the sole carbon, nitrogen and energy source. A. muciniphila is the only representative of the Verrucomicrobia phylum in the human gut. However, A. muciniphila 16S rRNA gene sequences have also been found in the intestines of many vertebrates. RESULTS: We detected A. muciniphila-like bacteria in the intestines of animals belonging to 15 out of 16 mammalian orders. In addition, other species belonging to the Verrucomicrobia phylum were detected in fecal samples. We isolated 10 new A. muciniphila strains from the feces of chimpanzee, siamang, mouse, pig, reindeer, horse and elephant. The physiology and genome of these strains were highly similar in comparison to the type strain A. muciniphila MucT. Overall, the genomes of the new strains showed high average nucleotide identity (93.9 to 99.7%). In these genomes, we detected considerable conservation of at least 75 of the 78 mucin degradation genes that were previously detected in the genome of the type strain MucT. CONCLUSIONS: The low genomic divergence observed in the new strains may indicate that A. muciniphila favors mucosal colonization independent of the differences in hosts. In addition, the conserved mucus degradation capability points towards a similar beneficial role of the new strains in regulating host metabolic health.

The human symbiont Mucispirillum schaedleri: causality in health and disease.

Trillions of bacteria inhabit the mammalian gastrointestinal tract. In the majority of hosts, these symbionts contribute largely to beneficial functions promoting microbe-host homeostasis. However, an increasing number of human diseases is associated with altered microbiota composition and enrichment of certain bacterial species. A well-known example of this is Mucispirillum schaedleri, which has been associated with inflammatory conditions in the intestine. Mucispirillum spp. belong to the phylum Deferribacteres and are prevalent but low abundant members of the rodent, pig and human microbiota. Recently, M. schaedleri was causally linked to the development of Crohn's disease-like colitis in immunodeficient mice. While this study certifies a considerable pathogenic potential, the same organism can also promote health in the immunocompetent host: M. schaedleri protects from Salmonella enterica serovar Typhimurium (S. Tm)-induced colitis by interfering with the expression of the pathogen´s invasion machinery. In this review, we summarize the current knowledge on the mammalian gut symbiont M. schaedleri and its role in intestinal homeostasis and discuss open questions and perspectives for future research.

Identification of Antibiotics That Diminish Disease in a Murine Model of Enterohemorrhagic Escherichia coli Infection.

Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic-uremic syndrome (HUS) and are the most common cause of renal failure in children in high-income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided, as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium ϕstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearance of the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics might be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development.

EDEN: evolutionary dynamics within environments.

SUMMARY: Metagenomics revolutionized the field of microbial ecology, giving access to Gb-sized datasets of microbial communities under natural conditions. This enables fine-grained analyses of the functions of community members, studies of their association with phenotypes and environments, as well as of their microevolution and adaptation to changing environmental conditions. However, phylogenetic methods for studying adaptation and evolutionary dynamics are not able to cope with big data. EDEN is the first software for the rapid detection of protein families and regions under positive selection, as well as their associated biological processes, from meta- and pangenome data. It provides an interactive result visualization for detailed comparative analyses. AVAILABILITY AND IMPLEMENTATION: EDEN is available as a Docker installation under the GPL 3.0 license, allowing its use on common operating systems, at http://www.github.com/hzi-bifo/eden. CONTACT: alice.mchardy@helmholtz-hzi.de. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Intestinal Dysbiosis, Barrier Dysfunction, and Bacterial Translocation Account for CKD-Related Systemic Inflammation.

CKD associates with systemic inflammation, but the underlying cause is unknown. Here, we investigated the involvement of intestinal microbiota. We report that collagen type 4 α3-deficient mice with Alport syndrome-related progressive CKD displayed systemic inflammation, including increased plasma levels of pentraxin-2 and activated antigen-presenting cells, CD4 and CD8 T cells, and Th17- or IFNγ-producing T cells in the spleen as well as regulatory T cell suppression. CKD-related systemic inflammation in these mice associated with intestinal dysbiosis of proteobacterial blooms, translocation of living bacteria across the intestinal barrier into the liver, and increased serum levels of bacterial endotoxin. Uremia did not affect secretory IgA release into the ileum lumen or mucosal leukocyte subsets. To test for causation between dysbiosis and systemic inflammation in CKD, we eradicated facultative anaerobic microbiota with antibiotics. This eradication prevented bacterial translocation, significantly reduced serum endotoxin levels, and fully reversed all markers of systemic inflammation to the level of nonuremic controls. Therefore, we conclude that uremia associates with intestinal dysbiosis, intestinal barrier dysfunction, and bacterial translocation, which trigger the state of persistent systemic inflammation in CKD. Uremic dysbiosis and intestinal barrier dysfunction may be novel therapeutic targets for intervention to suppress CKD-related systemic inflammation and its consequences.

Microbiota Dysbiosis Controls the Neuroinflammatory Response after Stroke.

UNLABELLED: Acute brain ischemia induces a local neuroinflammatory reaction and alters peripheral immune homeostasis at the same time. Recent evidence has suggested a key role of the gut microbiota in autoimmune diseases by modulating immune homeostasis. Therefore, we investigated the mechanistic link among acute brain ischemia, microbiota alterations, and the immune response after brain injury. Using two distinct models of acute middle cerebral artery occlusion, we show by next-generation sequencing that large stroke lesions cause gut microbiota dysbiosis, which in turn affects stroke outcome via immune-mediated mechanisms. Reduced species diversity and bacterial overgrowth of bacteroidetes were identified as hallmarks of poststroke dysbiosis, which was associated with intestinal barrier dysfunction and reduced intestinal motility as determined by in vivo intestinal bolus tracking. Recolonizing germ-free mice with dysbiotic poststroke microbiota exacerbates lesion volume and functional deficits after experimental stroke compared with the recolonization with a normal control microbiota. In addition, recolonization of mice with a dysbiotic microbiome induces a proinflammatory T-cell polarization in the intestinal immune compartment and in the ischemic brain. Using in vivo cell-tracking studies, we demonstrate the migration of intestinal lymphocytes to the ischemic brain. Therapeutic transplantation of fecal microbiota normalizes brain lesion-induced dysbiosis and improves stroke outcome. These results support a novel mechanism in which the gut microbiome is a target of stroke-induced systemic alterations and an effector with substantial impact on stroke outcome. SIGNIFICANCE STATEMENT: We have identified a bidirectional communication along the brain-gut microbiota-immune axis and show that the gut microbiota is a central regulator of immune homeostasis. Acute brain lesions induced dysbiosis of the microbiome and, in turn, changes in the gut microbiota affected neuroinflammatory and functional outcome after brain injury. The microbiota impact on immunity and stroke outcome was transmissible by microbiota transplantation. Our findings support an emerging concept in which the gut microbiota is a key regulator in priming the neuroinflammatory response to brain injury. These findings highlight the key role of microbiota as a potential therapeutic target to protect brain function after injury.

Microbiota differences between commercial breeders impacts the post-stroke immune response.

Experimental reproducibility between laboratories is a major translational obstacle worldwide, particularly in studies investigating immunomodulatory therapies in relation to brain disease. In recent years increasing attention has been drawn towards the gut microbiota as a key factor in immune cell polarization. Moreover, manipulation of the gut microbiota has been found effective in a diverse range of brain disorders. Within this study we aimed to test the impact of microbiota differences between mice from different sources on the post-stroke neuroinflammatory response. With this rationale, we have investigated the correlation between microbiota differences and the immune response in mice from three commercial breeders with the same genetic background (C57BL/6). While overall bacterial load was comparable, we detected substantial differences in species diversity and microbiota composition on lower taxonomic levels. Specifically, we investigated segmented filamentous bacteria (SFB)-which have been shown to promote T cell polarization-and found that they were absent in mice from one breeder but abundant in others. Our experiments revealed a breeder specific correlation between SFB presence and the ratio of Treg to Th17 cells. Moreover, recolonization of SFB-negative mice with SFB resulted in a T cell shift which mimicked the ratios found in SFB-positive mice. We then investigated the response to a known experimental immunotherapeutic approach, CD28 superagonist (CD28SA), which has been previously shown to expand the Treg population. CD28SA treatment had differing effects between mice from different breeders and was found to be ineffective at inducing Treg expansion in SFB-free mice. These changes directly corresponded to stroke outcome as mice lacking SFB had significantly larger brain infarcts. This study demonstrates the major impact of microbiota differences on T cell polarization in mice during ischemic stroke conditions, and following immunomodulatory therapies.

Temperate phages promote colicin-dependent fitness of Salmonella enterica serovar Typhimurium.

Bacteria employ bacteriocins for interference competition in microbial ecosystems. Colicin Ib (ColIb), a pore-forming bacteriocin, confers a significant fitness benefit to Salmonella enterica serovar Typhimurium (S. Tm) in competition against commensal Escherichia coli in the gut. ColIb is released from S. Tm into the environment, where it kills susceptible competitors. However, colicin-specific release proteins, as they are known for other colicins, have not been identified in case of ColIb. Thus, its release mechanism has remained unclear. In the current study, we have established a new link between ColIb release and lysis activity of temperate, lambdoid phages. By the use of phage-cured S. Tm mutant strains, we show that the presence of temperate phages and their lysis genes is necessary and sufficient for release of active ColIb into the culture supernatant. Furthermore, phage-mediated lysis significantly enhanced S. Tm fitness in competition against a ColIb-susceptible competitor. Finally, transduction with the lambdoid phage 933W rescued the defect of E. coli strain MG1655 with respect to ColIb release. In conclusion, ColIb is released from bacteria in the course of phage lysis. Our data reveal a new mechanism for colicin release and point out a novel function of temperate phages in enhancing colicin-dependent bacterial fitness.

Inflammation fuels colicin Ib-dependent competition of Salmonella serovar Typhimurium and E. coli in enterobacterial blooms.

The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of Enterobacteriaceae ("inflammation-inflicted blooms"). Here, we investigated competition of the human pathogenic Salmonella enterica serovar Typhimurium strain SL1344 (S. Tm) and commensal E. coli in inflammation-inflicted blooms. S. Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related Enterobacteriaceae. Production of ColIb conferred a competitive advantage to S. Tm over sensitive E. coli strains in the inflamed gut. In contrast, an avirulent S. Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (cib) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive E. coli, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both S. Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of S. Tm and commensal E. coli in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the Enterobacteriaceae family.

Granulocytes impose a tight bottleneck upon the gut luminal pathogen population during Salmonella typhimurium colitis.

Topological, chemical and immunological barriers are thought to limit infection by enteropathogenic bacteria. However, in many cases these barriers and their consequences for the infection process remain incompletely understood. Here, we employed a mouse model for Salmonella colitis and a mixed inoculum approach to identify barriers limiting the gut luminal pathogen population. Mice were infected via the oral route with wild type S. Typhimurium (S. Tm) and/or mixtures of phenotypically identical but differentially tagged S. Tm strains ("WITS", wild-type isogenic tagged strains), which can be individually tracked by quantitative real-time PCR. WITS dilution experiments identified a substantial loss in tag/genetic diversity within the gut luminal S. Tm population by days 2-4 post infection. The diversity-loss was not attributable to overgrowth by S. Tm mutants, but required inflammation, Gr-1+ cells (mainly neutrophilic granulocytes) and most likely NADPH-oxidase-mediated defense, but not iNOS. Mathematical modelling indicated that inflammation inflicts a bottleneck transiently restricting the gut luminal S. Tm population to approximately 6000 cells and plating experiments verified a transient, inflammation- and Gr-1+ cell-dependent dip in the gut luminal S. Tm population at day 2 post infection. We conclude that granulocytes, an important clinical hallmark of S. Tm-induced inflammation, impose a drastic bottleneck upon the pathogen population. This extends the current view of inflammation-fuelled gut-luminal Salmonella growth by establishing the host response in the intestinal lumen as a double-edged sword, fostering and diminishing colonization in a dynamic equilibrium. Our work identifies a potent immune defense against gut infection and reveals a potential Achilles' heel of the infection process which might be targeted for therapy.

The mouse gut microbiome revisited: From complex diversity to model ecosystems.

Laboratory mice are the most commonly used animal model in translational medical research. In recent years, the impact of the gut microbiota (i.e. communities of microorganisms in the intestine) on host physiology and the onset of diseases, including metabolic and neuronal disorders, cancers, gastrointestinal infections and chronic inflammation, became a focal point of interest. There is abundant evidence that mouse phenotypes in disease models vary greatly between animal facilities or commercial providers, and that this variation is associated with differences in the microbiota. Hence, there is a clear discrepancy between the widespread use of mouse models in research and the patchwork knowledge on the mouse gut microbiome. In the present manuscript, we summarize data pertaining to the diversity and functions of the mouse gut microbiota, review existing work on gnotobiotic mouse models, and discuss challenges and opportunities for current and future research in the field.

Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing.

The animal and human intestinal mucosa secretes an assortment of compounds to establish a physical barrier between the host tissue and intestinal contents, a separation that is vital for health. Some pathogenic microorganisms as well as members of the commensal intestinal microbiota have been shown to be able to break down these secreted compounds. Our understanding of host-compound degradation by the commensal microbiota has been limited to knowledge about simplified model systems because of the difficulty in studying the complex intestinal ecosystem in vivo. In this study, we introduce an approach that overcomes previous technical limitations and allows us to observe which microbial cells in the intestine use host-derived compounds. We added stable isotope-labeled threonine i.v. to mice and combined fluorescence in situ hybridization with high-resolution secondary ion mass spectrometry imaging to characterize utilization of host proteins by individual bacterial cells. We show that two bacterial species, Bacteroides acidifaciens and Akkermansia muciniphila, are important host-protein foragers in vivo. Using gnotobiotic mice we show that microbiota composition determines the magnitude and pattern of foraging by these organisms, demonstrating that a complex microbiota is necessary in order for this niche to be fully exploited. These results underscore the importance of in vivo studies of intestinal microbiota, and the approach presented in this study will be a powerful tool to address many other key questions in animal and human microbiome research.