Systematic analysis of drug combinations against Gram-positive bacteria.

Drug combinations can expand options for antibacterial therapies but have not been systematically tested in Gram-positive species. We profiled ~8,000 combinations of 65 antibacterial drugs against the model species Bacillus subtilis and two prominent pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Thereby, we recapitulated previously known drug interactions, but also identified ten times more novel interactions in the pathogen S. aureus, including 150 synergies. We showed that two synergies were equally effective against multidrug-resistant S. aureus clinical isolates in vitro and in vivo. Interactions were largely species-specific and synergies were distinct from those of Gram-negative species, owing to cell surface and drug uptake differences. We also tested 2,728 combinations of 44 commonly prescribed non-antibiotic drugs with 62 drugs with antibacterial activity against S. aureus and identified numerous antagonisms that might compromise the efficacy of antimicrobial therapies. We identified even more synergies and showed that the anti-aggregant ticagrelor synergized with cationic antibiotics by modifying the surface charge of S. aureus. All data can be browsed in an interactive interface ( https://apps.embl.de/combact/ ).

proGenomes3: approaching one million accurately and consistently annotated high-quality prokaryotic genomes.

The interpretation of genomic, transcriptomic and other microbial 'omics data is highly dependent on the availability of well-annotated genomes. As the number of publicly available microbial genomes continues to increase exponentially, the need for quality control and consistent annotation is becoming critical. We present proGenomes3, a database of 907 388 high-quality genomes containing 4 billion genes that passed stringent criteria and have been consistently annotated using multiple functional and taxonomic databases including mobile genetic elements and biosynthetic gene clusters. proGenomes3 encompasses 41 171 species-level clusters, defined based on universal single copy marker genes, for which pan-genomes and contextual habitat annotations are provided. The database is available at http://progenomes.embl.de/.

Cultivation-independent genomes greatly expand taxonomic-profiling capabilities of mOTUs across various environments.

BACKGROUND: Taxonomic profiling is a fundamental task in microbiome research that aims to detect and quantify the relative abundance of microorganisms in biological samples. Available methods using shotgun metagenomic data generally depend on the deposition of sequenced and taxonomically annotated genomes, usually from cultures of isolated strains, in reference databases (reference genomes). However, the majority of microorganisms have not been cultured yet. Thus, a substantial fraction of microbial community members remains unaccounted for during taxonomic profiling, particularly in samples from underexplored environments. To address this issue, we developed the mOTU profiler, a tool that enables reference genome-independent species-level profiling of metagenomes. As such, it supports the identification and quantification of both "known" and "unknown" species based on a set of select marker genes. RESULTS: We present mOTUs3, a command line tool that enables the profiling of metagenomes for >33,000 species-level operational taxonomic units. To achieve this, we leveraged the reconstruction of >600,000 draft genomes, most of which are metagenome-assembled genomes (MAGs), from diverse microbiomes, including soil, freshwater systems, and the gastrointestinal tract of ruminants and other animals, which we found to be underrepresented by reference genomes. Overall, two thirds of all species-level taxa lacked a reference genome. The cumulative relative abundance of these newly included taxa was low in well-studied microbiomes, such as the human body sites (6-11%). By contrast, they accounted for substantial proportions (ocean, freshwater, soil: 43-63%) or even the majority (pig, fish, cattle: 60-80%) of the relative abundance across diverse non-human-associated microbiomes. Using community-developed benchmarks and datasets, we found mOTUs3 to be more accurate than other methods and to be more congruent with 16S rRNA gene-based methods for taxonomic profiling. Furthermore, we demonstrate that mOTUs3 increases the resolution of well-known microbial groups into species-level taxa and helps identify new differentially abundant taxa in comparative metagenomic studies. CONCLUSIONS: We developed mOTUs3 to enable accurate species-level profiling of metagenomes. Compared to other methods, it provides a more comprehensive view of prokaryotic community diversity, in particular for currently underexplored microbiomes. To facilitate comparative analyses by the research community, it is released with >11,000 precomputed profiles for publicly available metagenomes and is freely available at: https://github.com/motu-tool/mOTUs . Video Abstract.

Biosynthetic potential of the global ocean microbiome.

Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters ('Candidatus Eudoremicrobiaceae') that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments.

Critical Assessment of Metagenome Interpretation: the second round of challenges.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses.

Calorie restriction improves metabolic state independently of gut microbiome composition: a randomized dietary intervention trial.

BACKGROUND: The gut microbiota has been suggested to play a significant role in the development of overweight and obesity. However, the effects of calorie restriction on gut microbiota of overweight and obese adults, especially over longer durations, are largely unexplored. METHODS: Here, we longitudinally analyzed the effects of intermittent calorie restriction (ICR) operationalized as the 5:2 diet versus continuous calorie restriction (CCR) on fecal microbiota of 147 overweight or obese adults in a 50-week parallel-arm randomized controlled trial, the HELENA Trial. The primary outcome of the trial was the differential effects of ICR versus CCR on gene expression in subcutaneous adipose tissue. Changes in the gut microbiome, which are the focus of this publication, were defined as exploratory endpoint of the trial. The trial comprised a 12-week intervention period, a 12-week maintenance period, and a final follow-up period of 26 weeks. RESULTS: Both diets resulted in ~5% weight loss. However, except for Lactobacillales being enriched after ICR, post-intervention microbiome composition did not significantly differ between groups. Overall weight loss was associated with significant metabolic improvements, but not with changes in the gut microbiome. Nonetheless, the abundance of the Dorea genus at baseline was moderately predictive of subsequent weight loss (AUROC of 0.74 for distinguishing the highest versus lowest weight loss quartiles). Despite the lack of consistent intervention effects on microbiome composition, significant study group-independent co-variation between gut bacterial families and metabolic biomarkers, anthropometric measures, and dietary composition was detectable. Our analysis in particular revealed associations between insulin sensitivity (HOMA-IR) and Akkermansiaceae, Christensenellaceae, and Tanerellaceae. It also suggests the possibility of a beneficial modulation of the latter two intestinal taxa by a diet high in vegetables and fiber, and low in processed meat. CONCLUSIONS: Overall, our results suggest that the gut microbiome remains stable and highly individual-specific under dietary calorie restriction. TRIAL REGISTRATION: The trial, including the present microbiome component, was prospectively registered at ClinicalTrials.gov NCT02449148 on May 20, 2015.

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.

mOTUs: Profiling Taxonomic Composition, Transcriptional Activity and Strain Populations of Microbial Communities.

The mOTU profiler, or mOTUs for short, is a software tool that enables the profiling of microbial communities in terms of their taxonomic composition, relative abundance of metabolically active members, and diversity of strain populations. To this end, it maintains a database of single-copy phylogenetic marker gene sequences, which are used as a reference to which short read metagenomic and metatranscriptomic reads are mapped for the identification and quantification of microbial taxa. Here, we describe the most common use cases of the mOTU profiler in two basic protocols. Additional supporting protocols provide information on its installation and in-depth guidance on adjusting its settings for increasing or decreasing the stringency with which taxa are detected and quantified, as well as for customizing the output file format. Guidelines for understanding the profiling results are provided, along with additional information on unique features, methodological details, and the development history of the tool. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Metagenomic and metatranscriptomic mOTU profiling Basic Protocol 2: Metagenomic SNV profiling Support Protocol 1: Installing mOTUs Support Protocol 2: Profiling pipeline-step by step Support Protocol 3: The mOTUs profiling routine using advanced parameters Support Protocol 4: Metagenomic SNV calling: advanced parameters.

Metabolic cooperation and spatiotemporal niche partitioning in a kefir microbial community.

Microbial communities often undergo intricate compositional changes yet also maintain stable coexistence of diverse species. The mechanisms underlying long-term coexistence remain unclear as system-wide studies have been largely limited to engineered communities, ex situ adapted cultures or synthetic assemblies. Here, we show how kefir, a natural milk-fermenting community of prokaryotes (predominantly lactic and acetic acid bacteria) and yeasts (family Saccharomycetaceae), realizes stable coexistence through spatiotemporal orchestration of species and metabolite dynamics. During milk fermentation, kefir grains (a polysaccharide matrix synthesized by kefir microorganisms) grow in mass but remain unchanged in composition. In contrast, the milk is colonized in a sequential manner in which early members open the niche for the followers by making available metabolites such as amino acids and lactate. Through metabolomics, transcriptomics and large-scale mapping of inter-species interactions, we show how microorganisms poorly suited for milk survive in-and even dominate-the community, through metabolic cooperation and uneven partitioning between grain and milk. Overall, our findings reveal how inter-species interactions partitioned in space and time lead to stable coexistence.

Changes in Plasma Short-Chain Fatty Acid Levels after Dietary Weight Loss Among Overweight and Obese Adults over 50 Weeks.

Gut microbial-derived short-chain fatty acids (SCFAs) may regulate energy homeostasis and exert anti-carcinogenic, immunomodulatory and anti-inflammatory effects. Smaller trials indicate that dietary weight loss may lead to decreased SCFA production, but findings have been inconclusive. SCFA concentrations were measured by HPLC-MS/MS in plasma samples of 150 overweight or obese adults in a trial initially designed to evaluate the metabolic effects of intermittent (ICR) versus continuous (CCR) calorie restriction (NCT02449148). For the present post hoc analyses, participants were classified by quartiles of weight loss, irrespective of the dietary intervention. Linear mixed models were used to analyze weight-loss-induced changes in SCFA concentrations after 12, 24 and 50 weeks. There were no differential changes in SCFA levels across the initial study arms (ICR versus CCR versus control) after 12 weeks, but acetate concentrations significantly decreased with overall weight loss (mean log-relative change of -0.7 ± 1.8 in the lowest quartile versus. -7.6 ± 2 in the highest, p = 0.026). Concentrations of propionate, butyrate and other SCFAs did not change throughout the study. Our results show that weight-loss, achieved through calorie restriction, may lead to smaller initial decreases in plasma acetate, while plasma SCFAs generally remain remarkably stable over time.

proGenomes2: an improved database for accurate and consistent habitat, taxonomic and functional annotations of prokaryotic genomes.

Microbiology depends on the availability of annotated microbial genomes for many applications. Comparative genomics approaches have been a major advance, but consistent and accurate annotations of genomes can be hard to obtain. In addition, newer concepts such as the pan-genome concept are still being implemented to help answer biological questions. Hence, we present proGenomes2, which provides 87 920 high-quality genomes in a user-friendly and interactive manner. Genome sequences and annotations can be retrieved individually or by taxonomic clade. Every genome in the database has been assigned to a species cluster and most genomes could be accurately assigned to one or multiple habitats. In addition, general functional annotations and specific annotations of antibiotic resistance genes and single nucleotide variants are provided. In short, proGenomes2 provides threefold more genomes, enhanced habitat annotations, updated taxonomic and functional annotation and improved linkage to the NCBI BioSample database. The database is available at http://progenomes.embl.de/.

Meta-analysis of fecal metagenomes reveals global microbial signatures that are specific for colorectal cancer.

Association studies have linked microbiome alterations with many human diseases. However, they have not always reported consistent results, thereby necessitating cross-study comparisons. Here, a meta-analysis of eight geographically and technically diverse fecal shotgun metagenomic studies of colorectal cancer (CRC, n = 768), which was controlled for several confounders, identified a core set of 29 species significantly enriched in CRC metagenomes (false discovery rate (FDR) < 1 × 10-5). CRC signatures derived from single studies maintained their accuracy in other studies. By training on multiple studies, we improved detection accuracy and disease specificity for CRC. Functional analysis of CRC metagenomes revealed enriched protein and mucin catabolism genes and depleted carbohydrate degradation genes. Moreover, we inferred elevated production of secondary bile acids from CRC metagenomes, suggesting a metabolic link between cancer-associated gut microbes and a fat- and meat-rich diet. Through extensive validations, this meta-analysis firmly establishes globally generalizable, predictive taxonomic and functional microbiome CRC signatures as a basis for future diagnostics.

Microbial abundance, activity and population genomic profiling with mOTUs2.

Metagenomic sequencing has greatly improved our ability to profile the composition of environmental and host-associated microbial communities. However, the dependency of most methods on reference genomes, which are currently unavailable for a substantial fraction of microbial species, introduces estimation biases. We present an updated and functionally extended tool based on universal (i.e., reference-independent), phylogenetic marker gene (MG)-based operational taxonomic units (mOTUs) enabling the profiling of >7700 microbial species. As more than 30% of them could not previously be quantified at this taxonomic resolution, relative abundance estimates based on mOTUs are more accurate compared to other methods. As a new feature, we show that mOTUs, which are based on essential housekeeping genes, are demonstrably well-suited for quantification of basal transcriptional activity of community members. Furthermore, single nucleotide variation profiles estimated using mOTUs reflect those from whole genomes, which allows for comparing microbial strain populations (e.g., across different human body sites).

Magnetosomes Extracted from Magnetospirillum gryphiswaldense as Theranostic Agents in an Experimental Model of Glioblastoma.

Magnetic fluid hyperthermia (MFH) with chemically synthesized nanoparticles is currently used in clinical trials as it destroys tumor cells with an extremely localized deposition of thermal energy. In this paper, we investigated an MFH protocol based on magnetic nanoparticles naturally produced by magnetotactic bacteria: magnetosomes. The efficacy of such protocol is tested in a xenograft model of glioblastoma. Mice receive a single intratumoral injection of magnetosomes, and they are exposed three times in a week to an alternating magnetic field with concurrent temperature measurements. MRI is used to visualize the nanoparticles and to monitor tumor size before and after the treatment. Statistically significant inhibition of the tumor growth is detected in subjects exposed to the alternating magnetic field compared to control groups. Moreover, thanks to magnetosomes high transversal relaxivity, their effective delivery to the tumor tissue is monitored by MRI. It is apparent that the efficacy of this protocol is limited by inhomogeneous delivery of magnetosomes to tumor tissue. These results suggest that naturally synthesized magnetosomes could be effectively considered as theranostic agent candidates for hyperthermia based on iron oxide nanoparticles.