Host Age Prediction from Fecal Microbiota Composition in Male C57BL/6J Mice.

The lifelong relationship between microorganisms and hosts has a profound impact on the overall health and physiology of the holobiont. Microbiome composition throughout the life span of a host remains largely understudied. Here, the fecal microbiota of conventionally raised C57BL/6J male mice was characterized throughout almost the entire adult life span, from "maturing" (9 weeks) until "very old" (112 weeks) age. Our results suggest that microbiota changes occur throughout life but are more pronounced in maturing to middle-age mice than in mice later in life. Phylum-level analysis indicates a shift of the Bacteroidota-to-Firmicutes ratio in favor of Firmicutes in old and very old mice. More Firmicutes amplicon sequence variants (ASVs) were transient with varying successional patterns than Bacteroidota ASVs, which varied primarily during maturation. Microbiota configurations from five defined life phases were used as training sets in a Bayesian model, which effectively enabled the prediction of host age. These results suggest that age-associated compositional differences may have considerable implications for the interpretation and comparability of animal model-based microbiome studies. The sensitivity of the age prediction to dietary perturbations was tested by applying this approach to two age-matched groups of C57BL/6J mice that were fed either a standard or western diet. The predicted age for the western diet-fed animals was on average 27 ± 11 (mean ± standard deviation) weeks older than that of standard diet-fed animals. This indicates that the fecal microbiota-based predicted age may be influenced not only by the host age and physiology but also potentially by other factors such as diet. IMPORTANCE The gut microbiome of a host changes with age. Cross-sectional studies demonstrate that microbiota of different age groups are distinct but do not demonstrate the temporal change that a longitudinal study is able to show. Here, we performed a longitudinal study of adult mice for over 2 years. We identified life stages where compositional changes were more dynamic and showed temporal changes for the more abundant species. Using a Bayesian model, we could reliably predict the life stages of the mice. Application of the same training set to mice fed different dietary regimens revealed that life-stage age predictions were possible for mice fed the same diet but less so for mice fed different diets. This study sheds light on the temporal changes that occur within the gut microbiota of laboratory mice over their life span and may inform researchers on the appropriate mouse age for their research.

Longitudinal Changes in Diet Cause Repeatable and Largely Reversible Shifts in Gut Microbial Communities of Laboratory Mice and Are Observed across Segments of the Entire Intestinal Tract.

Dietary changes are known to alter the composition of the gut microbiome. However, it is less understood how repeatable and reversible these changes are and how diet switches affect the microbiota in the various segments of the gastrointestinal tract. Here, a treatment group of conventionally raised laboratory mice is subjected to two periods of western diet (WD) interrupted by a period of standard diet (SD) of the same duration. Beta-diversity analyses show that diet-induced microbiota changes are largely reversible (q = 0.1501; PERMANOVA, weighted-UniFrac comparison of the treatment-SD group to the control-SD group) and repeatable (q = 0.032; PERMANOVA, weighted-UniFrac comparison of both WD treatments). Furthermore, we report that diet switches alter the gut microbiota composition along the length of the intestinal tract in a segment-specific manner, leading to gut segment-specific Firmicutes/Bacteroidota ratios. We identified prevalent and distinct Amplicon Sequencing Variants (ASVs), particularly in genera of the recently described Muribaculaceae, along the gut as well as ASVs that are differentially abundant between segments of treatment and control groups. Overall, this study provides insights into the reversibility of diet-induced microbiota changes and highlights the importance of expanding sampling efforts beyond the collections of fecal samples to characterize diet-dependent and segment-specific microbiome differences.

Muribaculum gordoncarteri sp. nov., an anaerobic bacterium from the faeces of C57BL/6J mice.

An anaerobic bacterial strain, named TLL-A4T, was isolated from fecal pellets of conventionally raised C57BL/6J mice. Analysis of the 16S rRNA gene indicated that the strain belongs to the phylum Bacteroidetes and, more specifically, to the recently proposed Muribaculaceae (also known as S24-7 clade or Candidatus Homeothermaceae). Strain TLL-A4T's 16S rRNA gene shared 92.8 % sequence identity with the type strain of the only published species of the genus Muribaculum, Muribaculum intestinale DSM 28989T. Genome-sequencing of TLL-A4T was performed to compare average amino acid identity (AAI) value and percentage of conserved proteins (POCP) between both strains. The AAI analysis revealed that strain TLL-A4T had high identity (69.8 %) with M. intestinale DSM 28989T, while the POCP was 56 %. These values indicate that strain TLL-A4T could be considered a member of the genus Muribaculum but not belonging to the species M. intestinale. Quinone analysis indicated MK10 (63 %) and MK11 (32 %) as major quinones in the membrane, while MK9 was only present as a minor component (5 %). The main cellular fatty acid was anteiso-C15 : 0 (42.8 %); summed feature 11 (17.5 %), C15 : 0 iso (13.4 %), C18 : 1 ω9c (5.6 %), C16.0 3-OH (4.5 %) and C15 : 0 (4.2 %) were detected in minor amounts. Analysis of enzyme activities using the API 32A and API 20A kits indicated major differences between strain TLL-A4T and Muribaculum intestinale DSM 28989T. Based on genotypic, phylogenetic and phenotypic differences, strain TLL-A4T is considered to represent a novel species of the genus Muribaculum, for which the name Muribaculum gordoncarteri sp. nov. is proposed. The type strain is TLL-A4T (=DSM 108194T=KCTC 15770T).

Cultivation and description of Duncaniella dubosii sp. nov., Duncaniella freteri sp. nov. and emended description of the species Duncaniella muris.

Three bacterial strains, C9, H5 and TLL-A3, were isolated from fecal pellets of conventionally raised C57BL/6J mice. Analysis of 16S rRNA genes indicated that the strains belonged to the Muribaculaceae, and shared 91.6-99.9 % sequence identity with the recently described Duncaniella muris DSM 103720T. Genome-sequencing of the isolates was performed to compare average nucleotide identities (ANI) between strains. The ANI analysis revealed that all isolates shared highest ANI with D. muris DSM 103720T, with strain C9 being most similar (ANI: 98.0 %) followed by strains H5 (ANI: 76.4 %) and TLL-A3 (ANI: 74.4 %). Likewise, digital DNA-DNA hybridization (dDDH) indicated high similarity of strain C9 (dDDH: 86.6 %) to D. muris DSM 103720T, but strains H5 and TLL-A3 showed lower similarity (dDDH <35 %) to either of the three type species of the Muribaculaceae (Muribaculum intestinale DSM 28989T , Paramuribaculum intestinale DSM 100749T, D. muris DSM 103720T). MK-10 and MK-11 were abundant in all three isolates, but concentrations varied between species. Based on genotypic, phylogenetic and phenotypic differences, the strains TLL-A3 and H5 are considered to represent novel species of the genus Duncaniella, for which the names Duncaniella freteri sp. nov., and Duncaniella dubosii sp. nov., are proposed. The respective type strains are TLL-A3T (=DSM 108168T=KCTC 15769T), and H5T (=DSM 107170T=KCTC 15734T). Strain C9 reveals limited sequence dissimilarity and minor differences in morphological properties with Duncaniella muris DSM 103720T and is therefore proposed to belong to the same species. The respective strain is C9 (=DSM 107165=KCTC 15733).

Insights into the microbiome of farmed Asian sea bass (Lates calcarifer) with symptoms of tenacibaculosis and description of Tenacibaculum singaporense sp. nov.

Outbreaks of diseases in farmed fish remain a recurring problem despite the development of vaccines and improved hygiene standards on aquaculture farms. One commonly observed bacterial disease in tropical aquaculture of the South-East Asian region is tenacibaculosis, which is attributed to members of the genus Tenacibaculum (family Flavobacteriaceae, phylum Bacteroidetes), most notably Tenacibaculum maritimum. The impact of tenacibaculosis on the fish microbiota remains poorly understood. In this study, we analysed the microbiota of different tissues of commercially reared Asian seabass (Lates calcarifer) that showed symptoms of tenacibaculosis and compared the microbial communities to those of healthy and experimentally infected fish that were exposed to diseased farmed fish. The relative abundance of Tenacibaculum species in experimentally infected fish was significantly lower than in commercially reared diseased fish and revealed a higher prevalence of different Tenacibaculum species. One isolated strain, TLL-A2T, shares 98.7% 16S rRNA gene identity with Tenacibaculum mesophilum DSM 13764T. The genome of strain TLL-A2T was sequenced and compared to that of T. mesophilum DSM 13764T. Analysis of average nucleotide identity and comparative genome analysis revealed only 92% identity between T. mesophilum DSM 13764T and strain TLL-A2T and differences between the two strains in predicted carbohydrate activating enzymes respectively. Phenotypic comparison between strain TLL-A2T and T. mesophilum DSM 13764T indicated additional differences, such as growth response at different salt concentrations. Based on molecular and phenotypic differences, strain TLL-A2T (=DSM 106434T, KCTC 62393T) is proposed as the type strain of Tenacibaculum singaporense sp. nov.

Complete Genome Sequence of Sponge-Associated Tenacibaculum mesophilum DSM 13764T.

Here, the complete genome sequence of sponge-associated Tenacibaculum mesophilum DSM 13764T is presented. T. mesophilum is a close relative of the fish pathogen T. maritimum, which causes significant fish disease outbreaks in aquaculture facilities. The T. mesophilum genome sequence will serve as an important resource for comparative genomics approaches.

Schaedlerella arabinosiphila gen. nov., sp. nov., a D-arabinose-utilizing bacterium isolated from faeces of C57BL/6J mice that is a close relative of Clostridium species ASF 502.

The use of gnotobiotics has attracted wide interest in recent years due to technological advances that have revealed the importance of host-associated microbiomes for host physiology and health. One of the oldest and most important gnotobiotic mouse model, the altered Schaedler flora (ASF) has been used for several decades. ASF comprises eight different bacterial strains, which have been characterized to different extent, but only a few are available through public strain collections. Here, the isolation of a close relative of one of the less-studied ASF strains, Clostridium species ASF 502, from faeces of C57BL/6J mice is reported. Isolate TLL-A1T shares 99.5 % 16S rRNA gene sequence identity with Clostridium species ASF 502 and phylogenetic analyses indicate that both strains belong to the uncultured so-called 'Lachnospiraceae UCG 006' clade. The rare sugar d-arabinose was used as a sole carbon source in the anaerobic isolation medium. Results of growth experiments with TLL-A1T on different carbon sources and analysis of its ~6.5 Gb indicate that TLL-A1T harbours a large gene repertoire that enables it to utilize a variety of carbohydrates for growth. Comparative genome analyses of TLL-A1T and Clostridium species ASF 502 reveal differences in genome content between the two strains, in particular with regards to carbohydrate-activating enzymes. Based on genomic, molecular and phenotypic differences, we propose to classify strain TLL-A1T (DSM 106076T=KCTC 15657T) as a representative of a new genus and a new species, for which we propose the name Schaedlerella arabinosiphila gen. nov., sp. nov.

Complete Genome Sequence of Duncaniella muris Strain B8, Isolated from the Feces of C57/BL6 Mice.

Here, the complete genome sequence of Duncaniella muris strain B8 is presented. The anaerobic strain was isolated from the feces of C57/BL6 mice and is closely related to D. muris strain DSM 103720, which is the type strain of the recently proposed genus Duncaniella of the Muribaculaceae.

Genomic diversification of giant enteric symbionts reflects host dietary lifestyles.

Herbivorous surgeonfishes are an ecologically successful group of reef fish that rely on marine algae as their principal food source. Here, we elucidated the significance of giant enteric symbionts colonizing these fishes regarding their roles in the digestive processes of hosts feeding predominantly on polysiphonous red algae and brown Turbinaria algae, which contain different polysaccharide constituents. Using metagenomics, single-cell genomics, and metatranscriptomic analyses, we provide evidence of metabolic diversification of enteric microbiota involved in the degradation of algal biomass in these fishes. The enteric microbiota is also phylogenetically and functionally simple relative to the complex lignocellulose-degrading microbiota of terrestrial herbivores. Over 90% of the enzymes for deconstructing algal polysaccharides emanate from members of a single bacterial lineage, "Candidatus Epulopiscium" and related giant bacteria. These symbionts lack cellulases but encode a distinctive and lineage-specific array of mostly intracellular carbohydrases concurrent with the unique and tractable dietary resources of their hosts. Importantly, enzymes initiating the breakdown of the abundant and complex algal polysaccharides also originate from these symbionts. These are also highly transcribed and peak according to the diel lifestyle of their host, further supporting their importance and host-symbiont cospeciation. Because of their distinctive genomic blueprint, we propose the classification of these giant bacteria into three candidate genera. Collectively, our findings show that the acquisition of metabolically distinct "Epulopiscium" symbionts in hosts feeding on compositionally varied algal diets is a key niche-partitioning driver in the nutritional ecology of herbivorous surgeonfishes.

Phylogenetic Diversity, Distribution, and Cophylogeny of Giant Bacteria (Epulopiscium) with their Surgeonfish Hosts in the Red Sea.

Epulopiscium is a group of giant bacteria found in high abundance in intestinal tracts of herbivorous surgeonfish. Despite their peculiarly large cell size (can be up to 600 µm), extreme polyploidy (some with over 100,000 genome copies per cell) and viviparity (whereby mother cells produce live offspring), details about their diversity, distribution or their role in the host gut are lacking. Previous studies have highlighted the existence of morphologically distinct Epulopiscium cell types (defined as morphotypes A to J) in some surgeonfish genera, but the corresponding genetic diversity and distribution among other surgeonfishes remain mostly unknown. Therefore, we investigated the phylogenetic diversity of Epulopiscium, distribution and co-occurrence in multiple hosts. Here, we identified eleven new phylogenetic clades, six of which were also morphologically characterized. Three of these novel clades were phylogenetically and morphologically similar to cigar-shaped type A1 cells, found in a wide range of surgeonfishes including Acanthurus nigrofuscus, while three were similar to smaller, rod-shaped type E that has not been phylogenetically classified thus far. Our results also confirmed that biogeography appears to have relatively little influence on Epulopiscium diversity, as clades found in the Great Barrier Reef and Hawaii were also recovered from the Red Sea. Although multiple symbiont clades inhabited a given species of host surgeonfish and multiple host species possessed a given symbiont clade, statistical analysis of host and symbiont phylogenies indicated significant cophylogeny, which in turn suggests co-evolutionary relationships. A cluster analysis of Epulopiscium sequences from previously published amplicon sequencing dataset revealed a similar pattern, where specific clades were consistently found in high abundance amongst closely related surgeonfishes. Differences in abundance may indicate specialization of clades to certain gut environments reflected by inferred differences in the host diets. Overall, our analysis identified a large phylogenetic diversity of Epulopiscium (up to 10% sequence divergence of 16S rRNA genes), which lets us hypothesize that there are multiple species that are spread across guts of different host species.

Diet strongly influences the gut microbiota of surgeonfishes.

Intestinal tracts are among the most densely populated microbial ecosystems. Gut microbiota and their influence on the host have been well characterized in terrestrial vertebrates but much less so in fish. This is especially true for coral reef fishes, which are among the most abundant groups of vertebrates on earth. Surgeonfishes (family: Acanthuridae) are part of a large and diverse family of reef fish that display a wide range of feeding behaviours, which in turn has a strong impact on the reef ecology. Here, we studied the composition of the gut microbiota of nine surgeonfish and three nonsurgeonfish species from the Red Sea. High-throughput pyrosequencing results showed that members of the phylum Firmicutes, especially of the genus Epulopiscium, were dominant in the gut microbiota of seven surgeonfishes. Even so, there were large inter- and intraspecies differences in the diversity of surgeonfish microbiota. Replicates of the same host species shared only a small number of operational taxonomic units (OTUs), although these accounted for most of the sequences. There was a statistically significant correlation between the phylogeny of the host and their gut microbiota, but the two were not completely congruent. Notably, the gut microbiota of three nonsurgeonfish species clustered with some surgeonfish species. The microbiota of the macro- and microalgavores was distinct, while the microbiota of the others (carnivores, omnivores and detritivores) seemed to be transient and dynamic. Despite some anomalies, both host phylogeny and diet were important drivers for the intestinal microbial community structure of surgeonfishes from the Red Sea.