Co-cultivation is a powerful approach to produce a robust functionally designed synthetic consortium as a live biotherapeutic product (LBP).

The success of fecal microbiota transplants (FMT) has provided the necessary proof-of-concept for microbiome therapeutics. Yet, feces-based therapies have many associated risks and uncertainties, and hence defined microbial consortia that modify the microbiome in a targeted manner have emerged as a promising safer alternative to FMT. The development of such live biotherapeutic products has important challenges, including the selection of appropriate strains and the controlled production of the consortia at scale. Here, we report on an ecology- and biotechnology-based approach to microbial consortium construction that overcomes these issues. We selected nine strains that form a consortium to emulate the central metabolic pathways of carbohydrate fermentation in the healthy human gut microbiota. Continuous co-culturing of the bacteria produces a stable and reproducible consortium whose growth and metabolic activity are distinct from an equivalent mix of individually cultured strains. Further, we showed that our function-based consortium is as effective as FMT in counteracting dysbiosis in a dextran sodium sulfate mouse model of acute colitis, while an equivalent mix of strains failed to match FMT. Finally, we showed robustness and general applicability of our approach by designing and producing additional stable consortia of controlled composition. We propose that combining a bottom-up functional design with continuous co-cultivation is a powerful strategy to produce robust functionally designed synthetic consortia for therapeutic use.

Gut microbiota composition as a candidate risk factor for dimethyl fumarate-induced lymphopenia in multiple sclerosis.

Mounting evidence points towards a pivotal role of gut microbiota in multiple sclerosis (MS) pathophysiology. Yet, whether disease-modifying treatments alter microbiota composition and whether microbiota shape treatment response and side-effects remain unclear. In this prospective observational pilot study, we assessed the effect of dimethyl fumarate (DMF) on gut microbiota and on host/microbial metabolomics in a cohort of 20 MS patients. Combining state-of-the-art microbial sequencing, metabolome mass spectrometry, and computational analysis, we identified longitudinal changes in gut microbiota composition under DMF-treatment and an increase in citric acid cycle metabolites. Notably, DMF-induced lymphopenia, a clinically relevant safety concern, was correlated with distinct baseline microbiome signatures in MS patients. We identified gastrointestinal microbiota as a key therapeutic target for metabolic properties of DMF. By characterizing gut microbial composition as a candidate risk factor for DMF-induced lymphopenia, we provide novel insights into the role of microbiota in mediating clinical side-effects.

Combination of Whole Genome Sequencing and Metagenomics for Microbiological Diagnostics.

Whole genome sequencing (WGS) provides the highest resolution for genome-based species identification and can provide insight into the antimicrobial resistance and virulence potential of a single microbiological isolate during the diagnostic process. In contrast, metagenomic sequencing allows the analysis of DNA segments from multiple microorganisms within a community, either using an amplicon- or shotgun-based approach. However, WGS and shotgun metagenomic data are rarely combined, although such an approach may generate additive or synergistic information, critical for, e.g., patient management, infection control, and pathogen surveillance. To produce a combined workflow with actionable outputs, we need to understand the pre-to-post analytical process of both technologies. This will require specific databases storing interlinked sequencing and metadata, and also involves customized bioinformatic analytical pipelines. This review article will provide an overview of the critical steps and potential clinical application of combining WGS and metagenomics together for microbiological diagnosis.

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity.

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 - 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

High-throughput qPCR and 16S rRNA gene amplicon sequencing as complementary methods for the investigation of the cheese microbiota.

BACKGROUND: Next-generation sequencing (NGS) methods and especially 16S rRNA gene amplicon sequencing have become indispensable tools in microbial ecology. While they have opened up new possibilities for studying microbial communities, they also have one drawback, namely providing only relative abundances and thus compositional data. Quantitative PCR (qPCR) has been used for years for the quantification of bacteria. However, this method requires the development of specific primers and has a low throughput. The constraint of low throughput has recently been overcome by the development of high-throughput qPCR (HT-qPCR), which allows for the simultaneous detection of the most prevalent bacteria in moderately complex systems, such as cheese and other fermented dairy foods. In the present study, the performance of the two approaches, NGS and HT-qPCR, was compared by analyzing the same DNA samples from 21 Raclette du Valais protected designation of origin (PDO) cheeses. Based on the results obtained, the differences, accuracy, and usefulness of the two approaches were studied in detail. RESULTS: The results obtained using NGS (non-targeted) and HT-qPCR (targeted) show considerable agreement in determining the microbial composition of the cheese DNA samples studied, albeit the fundamentally different nature of these two approaches. A few inconsistencies in species detection were observed, particularly for less abundant ones. The detailed comparison of the results for 15 bacterial species/groups measured by both methods revealed a considerable bias for certain bacterial species in the measurements of the amplicon sequencing approach. We identified as probable origin to this PCR bias due to primer mismatches, variations in the number of copies for the 16S rRNA gene, and bias introduced in the bioinformatics analysis. CONCLUSION: As the normalized microbial composition results of NGS and HT-qPCR agreed for most of the 21 cheese samples analyzed, both methods can be considered as complementary and reliable for studying the microbial composition of cheese. Their combined application proved to be very helpful in identifying potential biases and overcoming methodological limitations in the quantitative analysis of the cheese microbiota.

Higher microbial diversity in raw than in pasteurized milk Raclette-type cheese enhances peptide and metabolite diversity after in vitro digestion.

Numerous bacteria are responsible for hydrolysis of proteins during cheese ripening. The raw milk flora is a major source of bacterial variety, starter cultures are needed for successful acidification of the cheese and proteolytic strains like Lactobacillus helveticus, are added for flavor improvement or acceleration of ripening processes. To study the impact of higher bacterial diversity in cheese on protein hydrolysis during simulated human digestion, Raclette-type cheeses were produced from raw or heat treated milk, with or without proteolytic L. helveticus and ripened for 120 days. Kinetic processes were studied with a dynamic (DIDGI®) in vitro protocol and endpoints with the static INFOGEST in vitro digestion protocol, allowing a comparison of the two in vitro protocols at the level of gastric and intestinal endpoints. Both digestion protocols resulted in comparable peptide patterns after intestinal digestion and higher microbial diversity in cheeses led to a more diverse peptidome after simulated digestion.

DAIRYdb: a manually curated reference database for improved taxonomy annotation of 16S rRNA gene sequences from dairy products.

BACKGROUND: Reads assignment to taxonomic units is a key step in microbiome analysis pipelines. To date, accurate taxonomy annotation of 16S reads, particularly at species rank, is still challenging due to the short size of read sequences and differently curated classification databases. The close phylogenetic relationship between species encountered in dairy products, however, makes it crucial to annotate species accurately to achieve sufficient phylogenetic resolution for further downstream ecological studies or for food diagnostics. Curated databases dedicated to the environment of interest are expected to improve the accuracy and resolution of taxonomy annotation. RESULTS: We provide a manually curated database composed of 10'290 full-length 16S rRNA gene sequences from prokaryotes tailored for dairy products analysis ( https://github.com/marcomeola/DAIRYdb ). The performance of the DAIRYdb was compared with the universal databases Silva, LTP, RDP and Greengenes. The DAIRYdb significantly outperformed all other databases independently of the classification algorithm by enabling higher accurate taxonomy annotation down to the species rank. The DAIRYdb accurately annotates over 90% of the sequences of either single or paired hypervariable regions automatically. The manually curated DAIRYdb strongly improves taxonomic annotation accuracy for microbiome studies in dairy environments. The DAIRYdb is a practical solution that enables automatization of this key step, thus facilitating the routine application of NGS microbiome analyses for microbial ecology studies and diagnostics in dairy products.

Metaxa2 Database Builder: enabling taxonomic identification from metagenomic or metabarcoding data using any genetic marker.

Motivation: Correct taxonomic identification of DNA sequences is central to studies of biodiversity using both shotgun metagenomic and metabarcoding approaches. However, no genetic marker gives sufficient performance across all the biological kingdoms, hampering studies of taxonomic diversity in many groups of organisms. This has led to the adoption of a range of genetic markers for DNA metabarcoding. While many taxonomic classification software tools can be re-trained on these genetic markers, they are often designed with assumptions that impair their utility on genes other than the SSU and LSU rRNA. Here, we present an update to Metaxa2 that enables the use of any genetic marker for taxonomic classification of metagenome and amplicon sequence data. Results: We evaluated the Metaxa2 Database Builder on 11 commonly used barcoding regions and found that while there are wide differences in performance between different genetic markers, our software performs satisfactorily provided that the input taxonomy and sequence data are of high quality. Availability and implementation: Freely available on the web as part of the Metaxa2 package at http://microbiology.se/software/metaxa2/. Supplementary information: Supplementary data are available at Bioinformatics online.

Bacterial Composition and Survival on Sahara Dust Particles Transported to the European Alps.

Deposition of Sahara dust (SD) particles is a frequent phenomenon in Europe, but little is known about the viability and composition of the bacterial community transported with SD. The goal of this study was to characterize SD-associated bacteria transported to the European Alps, deposited and entrapped in snow. During two distinct events in February and May 2014, SD particles were deposited and promptly covered by falling snow, thus preserving them in distinct ochre layers within the snowpack. In June 2014, we collected samples at different depths from a snow profile at the Jungfraujoch (Swiss Alps; 3621 m a.s.l.). After filtration, we performed various microbiological and physicochemical analyses of the snow and dust particles therein that originated in Algeria. Our results show that bacteria survive and are metabolically active after the transport to the European Alps. Using high throughput sequencing, we observed distinct differences in bacterial community composition and structure in SD-layers as compared to clean snow layers. Sporulating bacteria were not enriched in the SD-layers; however, phyla with low abundance such as Gemmatimonadetes and Deinococcus-Thermus appeared to be specific bio-indicators for SD. Since many members of these phyla are known to be adapted to arid oligotrophic environments and UV radiation, they are well suited to survive the harsh conditions of long-range airborne transport.

Diversity, resistance and resilience of the bacterial communities at two alpine glacier forefields after a reciprocal soil transplantation.

In this study, we determined the driving key factor determining variability in bacterial community structures in soils at two unvegetated alpine glacier forefields with different bedrock geology (calcareous and siliceous). We further assessed the resistance and resilience capacities of the bacterial communities through reciprocal soil transplantations. Sterilized and unsterilized soils were incubated locally ('home') or transplanted ('away') for 15 months (July 2011-October 2012) and sampled regularly during the snow-free seasons. Changes in bacterial community structures were determined through fingerprinting of the 16S rRNA gene and correlated with several environmental factors. This study demonstrates that bacterial community structures at our field sites were shaped by distinct mineralogical soil properties. Soil moisture and pH appeared to not be the major driving key factors. Calcareous soil was more selective to bacteria, thus diversity was higher in siliceous soils as a positive effect of its more diverse mineralogical composition. Bacterial community in the calcareous soil exhibited stronger resistance to transplantation than the community in the siliceous soil. In fact, siliceous soil was more easily invaded by extrinsic taxa. Bacterial communities of both soil types were equally resilient at home, although different resilience patterns were observed between calcareous and siliceous soils incubated away.

The sperm-oocyte switch in the C. elegans hermaphrodite is controlled through steady-state levels of the fem-3 mRNA.

Post-transcriptional control regulates many aspects of germline development in the Caenorhabditis elegans hermaphrodite. This nematode switches from spermatogenesis to oogenesis and is, therefore, capable of self-fertilization. This sperm-oocyte switch requires 3' UTR-mediated repression of the fem-3 mRNA. Loss of fem-3 repression results in continuous spermatogenesis in hermaphrodites. Although several factors regulating fem-3 have been identified, little is known about the mechanisms that control fem-3. Here, we investigate the steady-state levels of the fem-3 transcript and the expression pattern of its protein product. We show that FEM-3 is exclusively present in germ cells that are committed to spermatogenesis. We found that in fem-3(gf)/+ heterozygotes, mutant fem-3 gain-of-function transcripts are more abundant than their wild-type counterpart. Furthermore, we show that the penetrance of the fem-3(gf) allele correlates with inefficient FBF binding and extended poly(A) tail size of fem-3 mRNAs. Finally, we show that wild-type and gain-of-function mutated fem-3 mRNAs associate equally well with polyribosomes. We propose that the fem-3 mRNA is regulated through stabilization rather than through translatability.

Role of the C. elegans U2 snRNP protein MOG-2 in sex determination, meiosis, and splice site selection.

In Caenorhabditis elegans, germ cells develop as spermatids in the larva and as oocytes in the adult. Such fundamentally different gametes are produced through a fine-tuned balance between feminizing and masculinizing genes. For example, the switch to oogenesis requires repression of the fem-3 mRNA through the mog genes. Here we report on the cloning and characterization of the sex determination gene mog-2. MOG-2 is the worm homolog of spliceosomal protein U2A'. We found that MOG-2 is expressed in most nuclei of somatic and germ cells. In addition to its role in sex determination, mog-2 is required for meiosis. Moreover, MOG-2 binds to U2B″/RNP-3 in the absence of RNA. We also show that MOG-2 associates with the U2 snRNA in the absence of RNP-3. Therefore, we propose that MOG-2 is a bona fide component of the U2 snRNP. Albeit not being required for general pre-mRNA splicing, MOG-2 increases the splicing efficiency to a cryptic splice site that is located at the 5' end of the exon.