Global Growth Phase Response of the Gut Bacterium Phocaeicola vulgatus (Phylum Bacteroidota).

INTRODUCTION: Phocaeicola vulgatus (basonym Bacteroides vulgatus) belongs to the intestinal microbiome of healthy humans and animals, where it participates in the fermentative breakdown of biopolymers ingested with food. In doing so, P. vulgatus contributes to the shaping of the gut metabolome, which benefits the host health. Moreover, considering the fermentation product range (short chain fatty acids), P. vulgatus suggests itself as a potential nonstandard platform organism for a sustainable production of basic organic chemicals. Complementing a recent physiologic-proteomic report deciphering the strain's versatile fermentation network, the present study focusses on the global growth phase-dependent response of P. vulgatus. METHODS: P. vulgatus was anaerobically cultivated with glucose as sole source of carbon and energy in process-controlled bioreactors operated in parallel. Close sampling was conducted to measure growth parameters (OD, CDW, ATP-content, substrate/product profiles) as basis for determining growth stoichiometry in detail. A coarser sampling (½ODmax, ODmax, and ODstat) served the molecular analysis of the global growth phase-dependent response, studied by means of differential proteomics (soluble and membrane fractions, nanoLC-ESI-MS/MS) as well as targeted metabolite (GC-MS and LC-MS/MS) and untargeted exometabolome (FT-ICR-MS) analyses. RESULTS: The determined growth performance of P. vulgatus features 1.74 h doubling time, 0.06 gCDW/mmolGlc biomass yield, 0.36 (succinate) and 0.61 (acetate) mmolP/mmolGlc as predominant fermentation product yields, and 0.43 mmolATP/mmolC as theoretically calculated ATP yield. The fermentation pathway displayed distinct growth phase-dependent dynamics: the levels of proteins and their accompanying metabolites constituting the upper part of glycolysis peaked at ½ODmax, whereas those of the lower part of glycolysis and of the fermentation routes in particular toward the predominant products acetate and succinate were highest at ODmax and ODstat. While identified proteins of monomer biosynthesis displayed rather unspecific profiles, most of the intracellular amino acids peaked at ODmax. By contrast, proteins and metabolites related to stress response and quorum sensing showed increased abundances at ODmax and ODstat. Finally, the composition of the exometabolome expanded from 2,317 molecular formulas at ½ODmax via 4,258 at ODmax to 4,501 at ODstat, with growth phase-specific subsets increasing in parallel. CONCLUSIONS: The present study provides insights into the distinct growth phase-dependent behavior of P. vulgatus during cultivation in bioreactors on the physiological and molecular levels. This could serve as a valuable knowledge-base for further developing P. vulgatus as a nonconventional platform organism for biotechnological applications. In addition, the findings shed new light on the potential growth phase-dependent imprints of P. vulgatus on the gut microbiome environment, e.g., by indicating interactions via quorum sensing and by unraveling the complex exometabolic background against which fermentation products and secondary metabolites are formed.

Exploring the resilience and stability of a defined human gut microbiota consortium: An isothermal microcalorimetric study.

The gut microbiota significantly contributes to human health and well-being. The aim of this study was to evaluate the stability and resilience of a consortium composed of three next-generation probiotics (NGPs) candidates originally found in the human gut. The growth patterns of Akkermansia muciniphila, Bacteroides thetaiotaomicron, and Faecalibacterium prausnitzii were studied both individually and consortium. The growth kinetics of Akkermansia muciniphila (A. muciniphila), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Faecalibacterium prausnitzii (F. prausnitzii) were characterized both individually and in consortium using isothermal microcalorimetry and 16S ribosomal RNA next-generation sequencing. The consortium reached stability after three passages and demonstrated resilience to changes in its initial composition. The concentration of butyrate produced was nearly twice as high in the consortium compared to the monoculture of F. prausnitzii. The experimental conditions and methodologies used in this article are a solid foundation for developing further complex consortia.

Enrichment and characterization of human-associated mucin-degrading microbial consortia by sequential passage.

Mucin is a glycoprotein secreted throughout the mammalian gastrointestinal tract that can support endogenous microorganisms in the absence of complex polysaccharides. While several mucin-degrading bacteria have been identified, the interindividual differences in microbial communities capable of metabolizing this complex polymer are not well described. To determine whether community assembly on mucin is deterministic across individuals or whether taxonomically distinct but functionally similar mucin-degrading communities are selected across fecal inocula, we used a 10-day in vitro sequential batch culture fermentation from three human donors with mucin as the sole carbon source. For each donor, 16S rRNA gene amplicon sequencing was used to characterize microbial community succession, and the short-chain fatty acid profile was determined from the final community. All three communities reached a steady-state by day 7 in which the community composition stabilized. Taxonomic comparisons amongst communities revealed that one of the final communities had Desulfovibrio, another had Akkermansia, and all three shared other members, such as Bacteroides. Metabolic output differences were most notable for one of the donor's communities, with significantly less production of acetate and propionate than the other two communities. These findings demonstrate the feasibility of developing stable mucin-degrading communities with shared and unique taxa. Furthermore, the mechanisms and efficiencies of mucin degradation across individuals are important for understanding how this community-level process impacts human health.

A cohort study in family triads: impact of gut microbiota composition and early life exposures on intestinal resistome during the first two years of life.

Antibiotic resistance genes (ARGs) are prevalent in the infant gut microbiota and make up the intestinal resistome, representing a community ARG reservoir. This study focuses on the dynamics and persistence of ARGs in the early gut microbiota, and the effect of early exposures therein. We leveraged 2,328 stool metagenomes from 475 children in the HELMi cohort and the available parental samples to study the diversity, dynamics, and intra-familial sharing of the resistome during the first two years of life. We found higher within-family similarity of the gut resistome composition and ARG load in infant-mother pairs, and between spouses, but not in father-infant pairs. Early gut microbiota composition and development correlated with the ARG load; Bacteroides correlated positively and Bifidobacterium negatively with the load, reflecting the typical resistance levels in these taxa. Caesarean delivered infants harbored lower ARG loads, partly reflecting the scarcity of Bacteroides compared to vaginally delivered. Exposure to intrapartum or post-natal antibiotics showed only modest associations with the ARG load and composition, mainly before 12 months. Our results indicate that the resistome is strongly driven by the normal development of the microbiota in early life, and suggest importance of longer evolution of ARGs over effects of recent antibiotic exposure.