Ruminococcoides bili gen. nov., sp. nov., a bile-resistant bacterium from human bile with autolytic behavior.

A strictly anaerobic, resistant starch-degrading, bile-tolerant, autolytic strain, IPLA60002T, belonging to the family Ruminococcaceae, was isolated from a human bile sample of a liver donor without hepatobiliary disease. Cells were Gram-stain-positive cocci, and 16S rRNA gene and whole genome analyses showed that Ruminococcus bromii was the phylogenetically closest related species to the novel strain IPLA60002T, though with average nucleotide identity values below 90 %. Biochemically, the new isolate has metabolic features similar to those described previously for gut R. bromii strains, including the ability to degrade a range of different starches. The new isolate, however, produces lactate and shows distinct resistance to the presence of bile salts. Additionally, the novel bile isolate displays an autolytic phenotype after growing in different media. Strain IPLA60002T is phylogenetically distinct from other species within the genus Ruminococcus. Therefore, we propose on the basis of phylogenetic, genomic and metabolic data that the novel IPLA60002T strain isolated from human bile be given the name Ruminococcoides bili gen. nov., sp. nov., within the new proposed genus Ruminococcoides and the family Ruminococcaceae. Strain IPLA60002T (=DSM 110008T=LMG 31505T) is proposed as the type strain of Ruminococcoides bili.

Ruminococcus bovis sp. nov., a novel species of amylolytic Ruminococcus isolated from the rumen of a dairy cow.

This study describes JE7A12T (=ATCC TSD-225T=NCTC 14479T), an isolate from the ruminal content of a dairy cow. Phenotypic and genotypic traits of the isolate were explored. JE7A12T was found to be a strictly anaerobic, catalase-negative, oxidase-negative, coccoid bacterium that grows in chains. The API 50 CH carbon source assay detected fermentation of d-glucose, d-fructose, d-galactose, glycogen and starch. HPLC showed acetate to be the major fermentation product as a result of carbohydrate fermentation. Phylogenetic analysis of JE7A12T based on 16S rRNA nucleotide sequence and amino acid sequences from the whole genome indicated a divergent lineage from the closest neighbours in the genus Ruminococcus. The results of 16S rRNA sequence comparison, whole genome average nucleotide identity (ANI) and DNA G+C content data indicate that JE7A12T represents a novel species which we propose the name Ruminococcus bovis with JE7A12T as the type strain.

Association of fibre degradation with ruminal dissolved hydrogen in growing beef bulls fed with two types of forages.

The present study investigated the association between fibre degradation and the concentration of dissolved molecular hydrogen (H2) in the rumen. Napier grass (NG) silage and corn stover (CS) silage were compared as forages with contrasting structures and degradation patterns. In the first experiment, CS silage had greater 48-h DM, neutral-detergent fibre (NDF) and acid-detergent fibre degradation, and total gas and methane (CH4) volumes, and lower 48-h H2 volume than NG silage in 48-h in vitro incubations. In the second experiment, twenty-four growing beef bulls were fed diets including 55 % (DM basis) NG or CS silages. Bulls fed the CS diet had greater DM intake (DMI), average daily gain, total-tract digestibility of OM and NDF, ruminal dissolved methane (dCH4) concentration and gene copies of protozoa, methanogens, Ruminococcus albus and R. flavefaciens, and had lower ruminal dH2 concentration, and molar proportions of valerate and isovalerate, in comparison with those fed the NG diet. There was a negative correlation between dH2 concentration and NDF digestibility in bulls fed the CS diet, and a lack of relationship between dH2 concentration and NDF digestibility with the NG diet. In summary, the fibre of CS silage was more easily degraded by rumen microorganisms than that of NG silage. Increased dCH4 concentration with the CS diet presumably led to the decreased ruminal dH2 concentration, which may be helpful for fibre degradation and growth of fibrolytic micro-organisms in the rumen.

Description of Mediterraneibacter massiliensis, gen. nov., sp. nov., a new genus isolated from the gut microbiota of an obese patient and reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus and Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., Mediterraneibacter lactaris comb. nov., Mediterraneibacter torques comb. nov., Mediterraneibacter gnavus comb. nov. and Mediterraneibacter glycyrrhizinilyticus comb. nov.

An anaerobic isolate, strain AT7T, was cultivated from a stool sample of a morbidly obese French woman using a microbial culturomics approach. The 16S rRNA gene sequence analysis showed that strain AT7T exhibited 96% nucleotide sequence similarity with Ruminococcus torques strain JCM 6553T (= ATCC 27756T = VPI B2-51T), currently the closest related species with a validly published name. The strain was observed to be a Gram-stain positive, non-motile, asporogenous and coccobacillary-shaped bacterium. It was found to be catalase positive and oxidase negative. Its major fatty acids were identified as C16:0 (54%) and C18:1n9 (30%). The draft genome of strain AT7T is 3,069,882 bp long with 42.4% G+C content. 2925 genes were predicted, including 2867 protein-coding genes and 58 RNAs. Based on phenotypic, biochemical, phylogenetic and genomic evidence, we propose the creation of the new genus Mediterraneibacter and species, Mediterraneibacter massiliensis, that contains strain AT7T (= CSUR P2086T = DSM 100837T), and the reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus, Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., with type strain Eg2T (= KCTC 5757T = JCM15917T), Mediterraneibacter lactaris comb. nov., with type strain ATCC 29176T (= VPI X6-29T), Mediterraneibacter torques comb. nov., with type strain ATCC 27756T (= VPI B2-51T), Mediterraneibacter gnavus comb. nov., with type strain ATCC 29149T (= VPI C7-9T) and Mediterraneibacter glycyrrhizinilyticus comb. nov., with type strain ZM35T (= JCM 13368T = DSM 17593T), respectively.

Metataxonomic Analysis of Individuals at BMI Extremes and Monozygotic Twins Discordant for BMI.

OBJECTIVE: The human gut microbiota has been demonstrated to be associated with a number of host phenotypes, including obesity and a number of obesity-associated phenotypes. This study is aimed at further understanding and describing the relationship between the gut microbiota and obesity-associated measurements obtained from human participants. SUBJECTS/METHODS: Here, we utilize genetically informative study designs, including a four-corners design (extremes of genetic risk for BMI and of observed BMI; N = 50) and the BMI monozygotic (MZ) discordant twin pair design (N = 30), in order to help delineate the role of host genetics and the gut microbiota in the development of obesity. RESULTS: Our results highlight a negative association between BMI and alpha diversity of the gut microbiota. The low genetic risk/high BMI group of individuals had a lower gut microbiota alpha diversity when compared to the other three groups. Although the difference in alpha diversity between the lean and heavy groups of the BMI-discordant MZ twin design did not achieve significance, this difference was observed to be in the expected direction, with the heavier participants having a lower average alpha diversity. We have also identified nine OTUs observed to be associated with either a leaner or heavier phenotype, with enrichment for OTUs classified to the Ruminococcaceae and Oxalobacteraceae taxonomic families. CONCLUSION: Our study presents evidence of a relationship between BMI and alpha diversity of the gut microbiota. In addition to these findings, a number of OTUs were found to be significantly associated with host BMI. These findings may highlight separate subtypes of obesity, one driven by genetic factors, the other more heavily influenced by environmental factors.

Western diet feeding influences gut microbiota profiles in apoE knockout mice.

BACKGROUND: Gut microbiota plays an important role in many metabolic diseases such as diabetes and atherosclerosis. Apolipoprotein E (apoE) knock-out (KO) mice are frequently used for the study of hyperlipidemia and atherosclerosis. However, it is unknown whether apoE KO mice have altered gut microbiota when challenged with a Western diet. METHODS: In the current study, we assessed the gut microbiota profiling of apoE KO mice and compared with wild-type mice fed either a normal chow or Western diet for 12 weeks using 16S pyrosequencing. RESULTS: On a western diet, the gut microbiota diversity was significantly decreased in apoE KO mice compared with wild type (WT) mice. Firmicutes and Erysipelotrichaceae were significantly increased in WT mice but Erysipelotrichaceae was unchanged in apoE KO mice on a Western diet. The weighted UniFrac principal coordinate analysis exhibited clear separation between WT and apoE KO mice on the first vector (58.6%) with significant changes of two dominant phyla (Bacteroidetes and Firmicutes) and seven dominant families (Porphyromonadaceae, Lachnospiraceae, Ruminococcaceae, Desulfovibrionaceae, Helicobacteraceae, Erysipelotrichaceae and Veillonellaceae). Lachnospiraceae was significantly enriched in apoE KO mice on a Western diet. In addition, Lachnospiraceae and Ruminococcaceae were positively correlated with relative atherosclerosis lesion size in apoE KO. CONCLUSIONS: Collectively, our study showed that there are marked changes in the gut microbiota of apoE KO mice, particularly challenged with a Western diet and these alterations may be possibly associated with atherosclerosis.

Quantitative qPCR Analysis of Ruminal Microorganisms in Beef Cattle Grazing in Pastures in the Rainy Season and Supplemented with Different Protein Levels.

We tested the hypothesis that supplementation with three protein levels improves fermentation parameters without changing the rumen microbial population of grazing beef cattle in the rainy season. Four rumen-cannulated Nellore bulls (432 ± 21 kg of body weight) were used in a 4 × 4 Latin square design with four supplements and four experimental periods of 21 days each. The treatments were mineral supplement (ad libitum) and supplements with low, medium (MPS), and high protein supplement (HPS), supplying 106, 408, and 601 g/day of CP, respectively. The abundance of each target taxon was calculated as a fraction of the total 16S rRNA gene copies in the samples, using taxon-specific and domain bacteria primers. Supplemented animals showed lower (P < 0.05) proportions of Ruminococcus flavefaciens and greater (P < 0.05) proportions of Ruminococcus albus and Butyrivibrio fibrisolvens than animals that received only the mineral supplement. The HPS supplement resulted in higher (P < 0.05) proportions of Fibrobacter succinogenes, R. flavefaciens, and B. fibrisolvens and lower (P < 0.05) proportions of R. albus than the MPS supplement. Based on our results, high protein supplementation improves the ruminal conditions and facilitates the growth of cellulolytic bacteria in the rumen of bulls on pastures during the rainy season.

Direct identification of Ruminococcus gnavus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a positive anaerobic blood culture bottle.

We report a case of bloodstream infection with the anaerobic bacterium Ruminococcus gnavus (R. gnavus), associated with intestinal perforation in a patient undergoing chemotherapy for multiple myeloma and cancer of the sigmoid colon. Gram staining of positive anaerobic blood cultures revealed both diplococci and short chains of gram-positive cocci. MALDI-TOF MS done directly on the blood culture bottle identified the bacterium as R. gnavus, and 16S rRNA gene sequencing confirmed the identification.

A bloodstream infection by Ruminococcus gnavus in a patient with a gall bladder perforation.

Ruminococcus gnavus is frequently found among human gut microbiome. However, human bloodstream infections by R. gnavus have been reported only three times. Clinical details were lacking for one case; the other two cases with concurrent bacteremia in patients with diverticulitis. We report a case of R. gnavus bloodstream infection in a patient with a gall bladder perforation suggesting its association with damage to the gastrointestinal tract. R. gnavus was misidentified using biochemical test but 16S rRNA sequencing and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were useful for correct identification. With the advancement of identification method in clinical laboratory, more frequent identification of R. gnavus from clinical specimens is expected.

Isolation and whole genome sequencing of a Ruminococcus-like bacterium, associated with irritable bowel syndrome.

In our previous studies on the intestinal microbiota in irritable bowel syndrome (IBS), we identified a bacterial phylotype with higher abundance in patients suffering from diarrhea than in healthy controls. In the present work, we have isolated in pure culture strain RT94, belonging to this phylotype, determined its whole genome sequence and performed an extensive genomic analysis and phenotypical testing. This revealed strain RT94 to be a strict anaerobe apparently belonging to a novel species with only 94% similarity in the 16S rRNA gene sequence to the closest relatives Ruminococcus torques and Ruminococcus lactaris. The G + C content of strain RT94 is 45.2 mol% and the major long-chain cellular fatty acids are C16:0, C18:0 and C14:0. The isolate is metabolically versatile but not a mucus or cellulose utilizer. It produces acetate, ethanol, succinate, lactate and formate, but very little butyrate, as end products of glucose metabolism. The mechanisms underlying the association of strain RT94 with diarrhea-type IBS are discussed.

Analysis of the rumen bacterial diversity of goats during shift from forage to concentrate diet.

High-grain feeding used in the animal production is known to affect the host rumen bacterial community, but our understanding of consequent changes in goats is limited. This study was therefore aimed to evaluate bacterial population dynamics during 20 days adaptation of 4 ruminally cannulated goats to the high-grain diet (grain: hay - ratio of 40:60). The dietary transition of goats from the forage to the high-grain-diet resulted in the significant decrease of rumen fluid pH, which was however still higher than value established for acute or subacute ruminal acidosis was not diagnosed in studied animals. DGGE analysis demonstrated distinct ruminal microbial populations in hay-fed and grain-fed animals, but the substantial animal-to-animal variation were detected. Quantitative PCR showed for grain-fed animals significantly higher number of bacteria belonging to Clostridium leptum group at 10 days after the incorporation of corn into the diet and significantly lower concentration of bacteria belonging to Actinobacteria phylum at the day 20 after dietary change. Taxonomic distribution analysed by NGS at day 20 revealed the similar prevalence of the phyla Firmicutes and Bacteroidetes in all goats, significantly higher presence of the unclassified genus of groups of Bacteroidales and Ruminococcaceae in grain-fed animals and significantly higher presence the genus Prevotella and Butyrivibrio in the forage-fed animals. The three different culture-independent methods used in this study show that high proportion of concentrate in goat diet does not induce any serious disturbance of their rumen ecosystem and indicate the good adaptive response of caprine ruminal bacteria to incorporation of corn into the diet.

Modulatory effects of condensed tannin fractions of different molecular weights from a Leucaena leucocephala hybrid on the bovine rumen bacterial community in vitro.

BACKGROUND: Condensed tannin (CT) fractions of different molecular weights (MWs) may affect rumen microbial metabolism by altering bacterial diversity. In this study the effects of unfractionated CTs (F0) and five CT fractions (F1-F5) of different MWs (F1, 1265.8 Da; F2, 1028.6 Da; F3, 652.2 Da; F4, 562.2 Da; F5, 469.6 Da) from Leucaena leucocephala hybrid-Rendang (LLR) on the structure and diversity of the rumen bacterial community were investigated in vitro. RESULTS: Real-time polymerase chain reaction assay showed that the total bacterial population was not significantly (P > 0.05) different among the dietary treatments. Inclusion of higher-MW CT fractions F1 and F2 significantly (P < 0.05) increased the Fibrobacter succinogenes population compared with F0 and CT fractions F3-F5. Although inclusion of F0 and CT fractions (F1-F5) significantly (P < 0.05) decreased the Ruminococcus flavefaciens population, there was no effect on the Ruminococcus albus population when compared with the control (without CTs). High-throughput sequencing of the V3 region of 16S rRNA showed that the relative abundance of genera Prevotella and unclassified Clostridiales was significantly (P < 0.05) decreased, corresponding with increasing MW of CT fractions, whereas cellulolytic bacteria of the genus Fibrobacter were significantly (P < 0.05) increased. Inclusion of higher-MW CT fractions F1 and/or F2 decreased the relative abundance of minor genera such as Ruminococcus, Streptococcus, Clostridium XIVa and Anaeroplasma but increased the relative abundance of Acinetobacter, Treponema, Selenomonas, Succiniclasticum and unclassified Spirochaetales compared with the control and lower-MW CT fractions. CONCLUSION: This study indicates that CT fractions of different MWs may play an important role in altering the structure and diversity of the rumen bacterial community in vitro, and the impact was more pronounced for CT fractions with higher MW. © 2016 Society of Chemical Industry.

Reclassification of Ruminococcus obeum as Blautia obeum comb. nov.

During our previous studies we reclassified Clostridium coccoides and a number of misclassified ruminococci into a novel genus Blautia within the family Lachnospiraceae. However, the Rules of the Bacteriological Code currently require that the types of all species and subspecies with new names (including new combinations) be deposited in two different collections in two different countries. The type strain of Ruminococcus obeum was, at that period in time, only deposited in the American Type Culture Collection (ATCC) and a second independent deposit, as required by the Code, was not available. Consequently, the transfer of this species to the genus Blautia could not be made, because the resulting species name would not conform to the Rules governing the valid publication of species names and deposit of type material (Rules 27 and 30) and consequently would not be considered to be validly published. This resulted in a nomenclatural and taxonomic anomaly with R. obeum being phylogenetically placed among members of the genus Blautia with 16S rRNA gene sequence similarities of between 91.8 and 96.6 %. In order to rectify this unsatisfactory situation, through our discussions with the ATCC, the deposit of strain R. obeum ATCC 29174(T) to the DSMZ as strain number DSM 25238(T) was completed. Hence, the transfer of R. obeum to the genus Blautia as Blautia obeum comb. nov. is now proposed. The type strain is ATCC 29174(T) ( = DSM 25238(T) = KCTC 15206(T)).

Unique aspects of fiber degradation by the ruminal ethanologen Ruminococcus albus 7 revealed by physiological and transcriptomic analysis.

BACKGROUND: Bacteria in the genus Ruminococcus are ubiquitous members of the mammalian gastrointestinal tract. In particular, they are important in ruminants where they digest a wide range of plant cell wall polysaccharides. For example, Ruminococcus albus 7 is a primary cellulose degrader that produces acetate usable by its bovine host. Moreover, it is one of the few organisms that ferments cellulose to form ethanol at mesophilic temperatures in vitro. The mechanism of cellulose degradation by R. albus 7 is not well-defined and is thought to involve pilin-like proteins, unique carbohydrate-binding domains, a glycocalyx, and cellulosomes. Here, we used a combination of comparative genomics, fermentation analyses, and transcriptomics to further clarify the cellulolytic and fermentative potential of R. albus 7. RESULTS: A comparison of the R. albus 7 genome sequence against the genome sequences of related bacteria that either encode or do not encode cellulosomes revealed that R. albus 7 does not encode for most canonical cellulosomal components. Fermentation analysis of R. albus 7 revealed the ability to produce ethanol and acetate on a wide range of fibrous substrates in vitro. Global transcriptomic analysis of R. albus 7 grown at identical dilution rates on cellulose and cellobiose in a chemostat showed that this bacterium, when growing on cellulose, utilizes a carbohydrate-degrading strategy that involves increased transcription of the rare carbohydrate-binding module (CBM) family 37 domain and the tryptophan biosynthetic operon. CONCLUSIONS: Our data suggest that R. albus 7 does not use canonical cellulosomal components to degrade cellulose, but rather up-regulates the expression of CBM37-containing enzymes and tryptophan biosynthesis. This study contributes to a revised model of carbohydrate degradation by this key member of the rumen ecosystem.

Ruminococcus gnavus: an unusual pathogen in septic arthritis.

Ruminococcus gnavus is an anaerobic Gram positive coccus that can be found in the gastrointestinal tract of animals and humans. We report a case of septic arthritis caused by R. gnavus that was identified by mass spectrometry and confirmed by 16S rRNA sequencing.

Cryptic diversity of cellulose-degrading gut bacteria in industrialized humans.

Humans, like all mammals, depend on the gut microbiome for digestion of cellulose, the main component of plant fiber. However, evidence for cellulose fermentation in the human gut is scarce. We have identified ruminococcal species in the gut microbiota of human populations that assemble functional multienzymatic cellulosome structures capable of degrading plant cell wall polysaccharides. One of these species, which is strongly associated with humans, likely originated in the ruminant gut and was subsequently transferred to the human gut, potentially during domestication where it underwent diversification and diet-related adaptation through the acquisition of genes from other gut microbes. Collectively, these species are abundant and widespread among ancient humans, hunter-gatherers, and rural populations but are rare in populations from industrialized societies thus indicating potential disappearance in response to the westernized lifestyle.

Ruminococcus champanellensis sp. nov., a cellulose-degrading bacterium from human gut microbiota.

A strictly anaerobic, cellulolytic strain, designated 18P13(T), was isolated from a human faecal sample. Cells were Gram-positive non-motile cocci. Strain 18P13(T) was able to degrade microcrystalline cellulose but the utilization of soluble sugars was restricted to cellobiose. Acetate and succinate were the major end products of cellulose and cellobiose fermentation. 16S rRNA gene sequence analysis revealed that the isolate belonged to the genus Ruminococcus of the family Ruminococcaceae. The closest phylogenetic relative was the ruminal cellulolytic strain Ruminococcus flavefaciens ATCC 19208(T) (<95% 16S rRNA gene sequence similarity). The DNA G+C content of strain 18P13(T) was 53.05±0.7 mol%. On the basis of phylogenetic analysis, and morphological and physiological data, strain 18P13(T) can be differentiated from other members of the genus Ruminococcus with validly published names. The name Ruminococcus champanellensis sp. nov. is proposed, with 18P13(T) (=DSM 18848(T)=JCM 17042(T)) as the type strain.

High-level dietary fibre up-regulates colonic fermentation and relative abundance of saccharolytic bacteria within the human faecal microbiota in vitro.

BACKGROUND: Health authorities around the world advise citizens to increase their intake of foods rich in dietary fibre because of its inverse association with chronic disease. However, a few studies have measured the impact of increasing mixed dietary fibres directly on the composition of the human gut microbiota. AIMS OF THE STUDY: We studied the impact of high-level mixed dietary fibre intake on the human faecal microbiota using an in vitro three-stage colonic model. METHODS: The colonic model was maintained on three levels of fibre, a basal level of dietary fibre, typical of a Western-style diet, a threefold increased level and back to normal level. Bacterial profiles and short chain fatty acids concentrations were measured. RESULTS: High-level dietary fibre treatment significantly stimulated the growth of Bifidobacterium, Lactobacillus-Enterococcus group, and Ruminococcus group (p < 0.05) and significantly increased clostridial cluster XIVa and Faecalibacterium prausnitzii in vessel 1 mimicking the proximal colon (p < 0.05). Total short chain fatty acids concentrations increased significantly upon increased fibre fermentation, with acetate and butyrate increasing significantly in vessel 1 only (p < 0.05). Bacterial species richness changed upon increased fibre supplementation. The microbial community and fermentation output returned to initial levels once supplementation with high fibre ceased. CONCLUSIONS: This study shows that high-level mixed dietary fibre intake can up-regulate both colonic fermentation and the relative abundance of saccharolytic bacteria within the human colonic microbiota. Considering the important role of short chain fatty acids in regulating human energy metabolism, this study has implications for the health-promoting potential of foods rich in dietary fibres.

Oligosaccharides in 4 different milk groups, Bifidobacteria, and Ruminococcus obeum.

OBJECTIVES: The aim of this study was to identify a link between the total amount of breast milk oligosaccharides and faecal microbiota composition of newborns at the end of the first month of life, with special attention paid to bifidobacteria, and establish the role, if any, of the different oligosaccharides in determining the gut microbiota composition. SUBJECTS AND METHODS: Milk oligosaccharide groups were identified by high-performance anion exchange chromatography analysis. DPCRNA from newborns' faecal samples at 30 days of life was isolated and processed by polymerase chain reaction analyses that allow the identification of 6 species of bifidobacteria (adolescentis, bifidum, breve, catenulatum, longum, infantis) and Ruminococcus spp; denaturing gradient gel electrophoresis analysis was also performed. RESULTS: No substantial differences in bifidobacteria species composition within milk groups 1, 2, and 3 were observed; however, infants fed with group 4 milk show a microbiota characterised by a greater frequency of Bifidobacteria adolescentis and the absence of Bifidobacteria catenulatum. For the first time, a high percentage of the Ruminococcus genus in infants fed with all milk groups was found. CONCLUSIONS: Our data show that milk groups 1, 2, and 3, containing an amount of oligosaccharides ranging within 10 to 15 g/L, share a substantially identical composition of the intestinal microbiota in breast-fed infants, despite quali-quantitative difference in oligosaccharides content. Newborns taking milk with only 5 g/L of oligosaccharides (group 4) harbour a different intestinal microbiota.

Integrating taxonomic, functional, and strain-level profiling of diverse microbial communities with bioBakery 3.

Culture-independent analyses of microbial communities have progressed dramatically in the last decade, particularly due to advances in methods for biological profiling via shotgun metagenomics. Opportunities for improvement continue to accelerate, with greater access to multi-omics, microbial reference genomes, and strain-level diversity. To leverage these, we present bioBakery 3, a set of integrated, improved methods for taxonomic, strain-level, functional, and phylogenetic profiling of metagenomes newly developed to build on the largest set of reference sequences now available. Compared to current alternatives, MetaPhlAn 3 increases the accuracy of taxonomic profiling, and HUMAnN 3 improves that of functional potential and activity. These methods detected novel disease-microbiome links in applications to CRC (1262 metagenomes) and IBD (1635 metagenomes and 817 metatranscriptomes). Strain-level profiling of an additional 4077 metagenomes with StrainPhlAn 3 and PanPhlAn 3 unraveled the phylogenetic and functional structure of the common gut microbe Ruminococcus bromii, previously described by only 15 isolate genomes. With open-source implementations and cloud-deployable reproducible workflows, the bioBakery 3 platform can help researchers deepen the resolution, scale, and accuracy of multi-omic profiling for microbial community studies.