Species-level Operational Taxonomic Units Research Articles

Enhancing metabarcoding efficiency and ecological insights through integrated taxonomy and DNA reference barcoding: Acase study on beach meiofauna.

Molecular techniques like metabarcoding, while promising for exploring diversity of communities, are often impeded by the lack of reference DNA sequences available for taxonomic annotation. Our study explores the benefits of combining targeted DNA barcoding and morphological taxonomy to improve metabarcoding efficiency, using beach meiofauna as a case study. Beaches are globally important ecosystems and are inhabited by meiofauna, microscopic animals living in the interstitial space between the sand grains, which play a key role in coastal biodiversity and ecosystem dynamics. However, research on meiofauna faces challenges due to limited taxonomic expertise and sparse sampling. We generated 775 new cytochrome c oxidase I DNA barcodes from meiofauna specimens collected along the Netherlands' west coast and combined them with the NCBI GenBank database. We analysed alpha and beta diversity in 561 metabarcoding samples from 24 North Sea beaches, a region extensively studied for meiofauna, using both the enriched reference database and the NCBI database without the additional reference barcodes. Our results show a 2.5-fold increase in sequence annotation and a doubling of species-level Operational Taxonomic Units (OTUs) identification when annotating the metabarcoding data with the enhanced database. Additionally, our analyses revealed a bell-shaped curve of OTU richness across the intertidal zone, aligning more closely with morphological analysis patterns, and more defined community dissimilarity patterns between supralittoral and intertidal sites. Our research highlights the importance of expanding molecular reference databases and combining morphological taxonomy with molecular techniques for biodiversity assessments, ultimately improving our understanding of coastal ecosystems.

Global Spore Sampling Project: A global, standardized dataset of airborne fungal DNA

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.

227 Comparative analysis of rumen bacterial communities from bison heifers fed two forage-based diets of different quality

Abstract Bison have a demonstrated ability to thrive on low-quality forage, an attribute that distinguishes them from their domestic ruminant counterparts. This could be attributed to the capability of their rumen symbiotic microorganisms in extracting more nutrients out of plant biomass. To gain further insight into the role of the bison microbiome in this beneficial adaptation, the primary objective of this study was to determine the diversity and composition of ruminal bacteria in bison fed poor versus good quality forage-based diets. Ruminal content was collected by stomach tubing from 25 bison heifers fed good quality forage (GQG) during the summer (last week of June 2023) at the McGinley Ranch (Gordon, Nebraska, USA). The same individuals were then transitioned to low quality hay (PQG) in pen confinement for approximately 4 to 5 wk before resampling. Data generated from Illumina MiSeq (2×300) sequencing of PCR amplicons targeting the V1-V3 region of the 16S rRNA gene were analyzed using a combination of custom Perl scripts, and publicly available software (Mothur v.1.40, RDP classifier and NCBI Blast). Principal Coordinate Analysis (PCoA) showed a clear distinction in bacterial composition between the two types of forages (Figure 1). Further analysis of species-level operational taxonomic units (OTUs) was performed, and a total of 78,808 OTUs were identified across all samples. From the 20 most highly represented OTUs, the respective abundance of twelve OTUs showed significant differences between the two forage types (Kruskal-Wallis’s sum-rank test; P < 0.05). For example, OTUs SD_Bb-00014 (µGQG = 1.06% vs µPQG = 0.19%), and SD_Bb-00049 (µGQG = 0.98% vs µPQG = 0.08%) showed significant decreases in abundance when animals switched to PQG. Conversely, OTUs SD_Bb-00071 (µGQG = 0.2% vs µPQG = 2.71%), SD_Bb-00049 (µGQG = 0.68% vs µPQG = 1.67%) and OTUs SD_Bb-00148 (µGQG = 0.19% vs µPQG = 1.40%) exhibited a significant increase in abundance in response to the forage transition. Notably, only one of the assessed OTUs, SD_Bb-00071, showed high sequence identity to its closest valid relative, Fibrobacter succinogenes (98.41%). In contrast, most of the other highly represented OTUs were found to be very dissimilar to known species, suggesting that they might represent novel or currently uncharacterized bacterial species (Table 1). Together, these results demonstrate the significant impact of variations in nutritional value and forage quality on the composition of rumen bacterial communities in the North American bison. Poor forage quality appeared to favor bacterial species such as Fibrobacter succinogenes, known for its cellulolytic capabilities, presumably because of its greater efficiency in breaking down more resistant plant cell walls. Its ability and that of other abundant OTUs to efficiently convert plant fibers into assimilable nutrients is likely critical in allowing bison to perform better on poor-quality forage compared with domesticated ruminants.

Abstract 6204: Differential gut microbiome alpha diversity in endometrial and ovarian carcinoma patients

Abstract Background: Emerging research underscores the gut microbiome's critical role in cancer development, progression, and response to treatment. As a part of the “ASAP” study, which focuses on the implementation of comprehensive molecular profiling and deep clinical annotation of electronic health records in participants diagnosed with or at risk of developing cancer, we have established a microbiome-associated biobank and corresponding clinical dataset to systematically explore the dynamics of the gut microbiome as patients progress through cancer treatment. We describe a pilot project where we explore the baseline microbiome samples collected from ASAP participants, particularly from those with endometrial and ovarian carcinomas. Methods: Stool samples from patients were collected at the time of enrollment in the ASAP study and sampling is repeated every three months to align with the additional biosampling procedures. Microbial DNA was extracted from the baseline stool samples and subjected to sequencing of the V4 region of the 16S rRNA gene. After generating species level operational taxonomic units (OTUs), alpha diversity measures were generated in the form of the inverse Simpson index. In order to determine whether there was a difference in alpha diversity between individuals with ovarian (N = 37) and endometrial (N = 35) carcinomas, we employed a t-test with 100,000 permutations to derive an empirical p-value. Results: We present sequencing results from the initial n=297 samples. Our comparison of the alpha diversity between ASAP study participants with ovarian and endometrial carcinomas showed a significant difference between the two groups, with the ovarian cancer patients having a lower alpha diversity (empirical p-value = 0.03915). Due to the uniform sampling, biobanking, and analytical procedures, our study allows the ability to explore microbiome features amongst various cancer diagnoses while minimizing laboratory and analytical differences that plague microbiome reproducibility. Conclusions: We observed a significant difference in alpha diversity between patients with endometrial and ovarian carcinomas. Although the current results are preliminary, the wealth of information available in the ASAP protocol will allow us to continue exploring the association of the gut microbiome with cancer and the associated treatment. Citation Format: Casey T. Finnicum, Tobias Meissner, Christel Davis, Sarah Viet, Jason L. Petersen, McKenna Deaton, Benjamin Solomon, William Spanos, David Starks, Rachel Elsey, Erik A. Ehli, Casey Williams. Differential gut microbiome alpha diversity in endometrial and ovarian carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6204.

PSV-9 Characterization of the Rumen Bacterial Communities of Lambs That Transitioned from Forage to a Concentrate-Based Diet

Abstract Forage and concentrate-based diets are distinct strategies used in ruminant production. The transition from a forage-based diet to a concentrate-based diet has been shown to have a great impact on the rumen microbiome composition due to vast differences in substrates provided by each diet type. To gain further insight on the nature of the changes that occur in the rumen microbiome as a result of a switch in diet, ruminal bacterial composition was compared between lambs initially fed a forage-based diet and lambs that were switched to a concentrate-based diet for approximately 60 days. From rumen fluid that was collected after slaughter, data were generated by Illumina MiSeq 2X300 sequencing of PCR amplicons targeting the V1-V3 regions of the 16S rRNA gene. Next-Generation Sequence data were analyzed using a combination of custom Perl scripts, and publicly available software (Mothur (v.1.35.1), RDP classifier and NCBI Blast). A total of 10,693 species-level operational taxonomic units (OTUs) were identified across all samples, with 86 OTUs shared between diets. A comparative analysis using the non-parametric Kruskal-Wallis test identified 16 highly represented species-level OTUs that differed in abundance across dietary treatments (P < 0.05). Of these, three OTUs, RA-01223, RA1-35340, and RA1-55937, were found to be of particular interest. OTU RA-01223 was at its highest representation in samples from individuals given a concentrate-based diet, with an average abundance of 8.76% compared with 0.80% in samples from animals fed a forage-based diet. RA-01223 was predicted to be a novel species of the family Oscillospiraceae based on its low 16S rRNA gene sequence identity (94.8%) to its closest valid relative, Ruminococcoides bili. OTU RA1-35340, predicted to be a strain of Sharpea azabuensis, was also in much greater abundance in the concentrate-based diet as compared with the forage-based diet (2.83% and 0.05%, respectively). In contrast, RA1-55937, which was predicted to be a strain of Pectinatus haikarae, was at its greatest abundance in samples from individuals given a forage-based diet, with an average representation of 4.91% compared with 0.52% in samples from animals fed a concentrate-based diet. These and other specific ruminal OTUs whose abundance are affected by changes in diet can then be studied further to gain more insight on the effects of the transition from forage to concentrate on rumen physiology.

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

BackgroundTaxonomic 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.ResultsWe 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.ConclusionsWe 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.EtaL3gdjB5XhyYuYaq3JvNVideo

Bacterial Communities in the Feces of Laboratory Reared Gampsocleis gratiosa (Orthoptera: Tettigoniidae) across Different Developmental Stages and Sexes.

Simple SummaryMany insects host a diverse gut microbial community, ranging from pathogenic to obligate mutualistic organisms. Little is known about the bacteria associated with katydids. Gampsocleis gratiosa (Orthoptera, Tettigoniidae) is an economically important singing pet in China. In the present study, the bacterial communities of the laboratory-reared G. gratiosa feces were characterized using Illumina sequencing of the 16S rDNA V3-V4 region.We used Illumina sequencing of the 16S rDNA V3-V4 region to identify the bacterial community in laboratory-reared G. gratiosa feces across different developmental stages (1st–7th instar nymph day 0, and 0-, 7-, 14-, and 21-day adult) and sexes. In total, 14,480,559 high-quality reads were clustered into 2982 species-level operational taxonomic units (OTUs), with an average of 481.197 (±137.366) OTUs per sample. These OTUs were assigned into 25 phyla, 42 classes, 60 orders, 116 families, 241 genera, and some unclassified groups. Only 21 core OTUs were shared by all samples. The most representative phylum was Proteobacteria, followed by Firmicutes, Bacteroidetes, and Acidobacteria. At the genus level, Kluyvera (387 OTUs), Obesumbacterium (339 OTUs), Buttiauxella (296 OTUs), Lactobacillus (286 OTUs), and Hafnia (152 OTUs) were dominant bacteria. The early-instar nymphs harbored a similar bacterial community with other developmental stages, which contain higher species diversity. Both principal coordinate analysis (PCoA) and non-metric multidimensional scaling analysis (NMDS) failed to provide a clear clustering based on the developmental stages and sexes. Overall, we assume that G. gratiosa transmits bacteria vertically by eating contaminated eggshells, and both developmental stages and sexes had no significant effect on the fecal bacterial community.

PSIV-5 Characterization of the Fecal Bacterial Communities of Grass-fed and Grain-fed Bison Heifers

Abstract In comparison to the rumen, the bacterial communities of the hindgut have remained largely unexplored in ruminants. They not only strongly influence host health, but also efficiency, notably by continuing fermentation of feed that escaped foregut digestion. In non-domesticated ruminants, such as the North American bison, they are thought to contribute to the higher efficiency of their host on feed of poor quality. To gain further insight on this gut microbial ecosystem, fecal bacterial community composition of bison heifers raised at two locations [Standing Butte (n=17), SD, and Blue Creek (n=17), NE] were investigated. Each animal was sampled once while on pasture, then after 100 days on a grain-based diet. Data generated from Illumina MiSeq (2×300) sequencing of PCR amplicons targeting the V1-V3 region of the 16S rRNA gene were analyzed using a combination of custom Perl scripts, and publicly available software (Mothur v.1.40, RDP classifier and NCBI Blast). A total of 26,379 and 13,294 species-level operational taxonomic units (OTUs) were identified from Standing Butte and Blue Creek samples, respectively, with 446 and 281 OTUs shared between diets at each respective location. Further analysis of the most highly represented OTUs from each ranch revealed that the abundance of six OTUs differed between diets in Standing Butte heifers compared to nine OTUs in Blue Creek heifers (Kruskal-Wallis sum-rank test; P < 0.05). These included OTUs SD_Bb-00727 (µgrass = < 0.01% vs µgrain = 13.13%) and SD_Bb-00728 (µgrass = 5.62% vs µgrain = < 0.01%) from the Standing Butte samples, as well as OTUs SD_Bb-00730 (µgrass = 0.01% vs µgrain = 10.21%) and SD_Bb-00745 (µgrass = < 0.01% vs µgrain = 3.5%) from the Blue Creek samples. Together, these results indicate that the composition of hindgut bacterial communities of the North American bison are greatly affected by changes in diet.

183 Effects of Protein Supplementation on the Gut Bacterial Community Composition of Hybrid Striped Bass, Morone Chrysops X M. Saxatilis

Abstract Plant-based protein ingredients have become an attractive substitute for traditional animal sources in the aquaculture feed industry. However, inclusion in carnivorous fish diets is limited due to reduced digestibility, presence of anti-nutritional factors, and increased risk of digestive tract inflammation. To gain further insight on the adaptation of the fish digestive tract environment to plant-based protein sources, intestinal bacterial communities from Hybrid Striped Bass, Morone chrysops x M. saxatilis, fed diets supplemented with different protein sources were compared. Data were generated by Illumina MiSeq 2X300 sequencing of PCR generated amplicons targeting the V1–V3 regions of the 16S rRNA gene. A comparative analysis using the non-parametric Kruskal-Wallis test identified 17 highly represented species-level Operational Taxonomic Units (OTUs) that differed in abundance across dietary treatments (P < 0.05). Notably, OTU SD_McMs-0001 was at its highest abundance in samples from individuals fed poultry-fishmeal (PFM; 47.61% ± 0.92%) and plant protein-based (PP; 43.13% ± 1.76%) diets, while it was found in much lower abundance in the non-supplemented control diet samples (B; 4.29% ± 0.92%). It was predicted to be a novel species of the family Peptostreptococcaceae based on its limited 16S rRNA gene sequence identity to its closest valid relative (Peptostreptococcus russellii, 91%). In contrast, three Proteobacteria-affiliated OTUs (SD_McMs-0002, SD_McMs-0003, and SD_McMs-0004) were most highly represented in B diet samples, with averages of 30.28%, 27.22%, and 13.54%, respectively. They were in much lower abundance in the PFM and PP samples, with averages ranging between 0.18% and 4.82%, respectively. Based on 16S rRNA sequence comparisons, they were predicted to be strains of Plesiomonas shigelloides (99%), Ralstonia pickettii (99%) and Sphingomonas elodea (99%), respectively. These results indicate that protein supplementation affects gut bacterial community composition of Hybrid Striped Bass, but that the type of protein used has minimal or no detectable impact.

Diversification of the mygalomorph spider genus Aname (Araneae: Anamidae) across the Australian arid zone: Tracing the evolution and biogeography of a continent-wide radiation

The assembly of the Australian arid zone biota has long fascinated biogeographers. Covering over two-thirds of the continent, Australia’s vast arid zone biome is home to a distinctive fauna and flora, including numerous lineages which have diversified since the Eocene. Tracing the origins and speciation history of these arid zone taxa has been an ongoing endeavour since the advent of molecular phylogenetics, and an increasing number of studies on invertebrate animals are beginning to complement a rich history of research on vertebrate and plant taxa. In this study, we apply continent-wide genetic sampling and one of the largest phylogenetic data matrices yet assembled for a genus of Australian spiders, to reconstruct the phylogeny and biogeographic history of the open-holed trapdoor spider genus Aname L. Koch, 1873. This highly diverse lineage of Australian mygalomorph spiders has a distribution covering the majority of Australia west of the Great Dividing Range, but apparently excluding the high rainfall zones of eastern Australia and Tasmania. Original and legacy sequences were obtained for three mtDNA and four nuDNA markers from 174 taxa in seven genera, including 150 Aname specimen terminals belonging to 102 species-level operational taxonomic units, sampled from 32 bioregions across Australia. Reconstruction of the phylogeny and biogeographic history of Aname revealed three radiations (Tropical, Temperate-Eastern and Continental), which could be further broken into eight major inclusive clades. Ancestral area reconstruction revealed the Pilbara, Monsoon Tropics and Mid-West to be important ancestral areas for the genus Aname and its closest relatives, with the origin of Aname itself inferred in the Pilbara bioregion. From these origins in the arid north-west of Australia, our study found evidence for a series of subsequent biome transitions in separate lineages, with at least eight tertiary incursions back into the arid zone from more mesic tropical, temperate or eastern biomes, and only two major clades which experienced widespread (primary) in situ diversification within the arid zone. Based on our phylogenetic results, and results from independent legacy divergence dating studies, we further reveal the importance of climate-driven biotic change in the Miocene and Pliocene in shaping the distribution and composition of the Australian arid zone biota, and the value of continent-wide studies in revealing potentially complex patterns of arid zone diversification in dispersal-limited invertebrate taxa.

Methane-Oxidizing Communities in Lichen-Dominated Forested Tundra Are Composed Exclusively of High-Affinity USCα Methanotrophs.

Upland soils of tundra function as a constant sink for atmospheric CH4 but the identity of methane oxidizers in these soils remains poorly understood. Methane uptake rates of −0.4 to −0.6 mg CH4-C m−2 day−1 were determined by the static chamber method in a mildly acidic upland soil of the lichen-dominated forested tundra, North Siberia, Russia. The maximal CH4 oxidation activity was localized in an organic surface soil layer underlying the lichen cover. Molecular identification of methanotrophic bacteria based on retrieval of the pmoA gene revealed Upland Soil Cluster Alpha (USCα) as the only detectable methanotroph group. Quantification of these pmoA gene fragments by means of specific qPCR assay detected ~107 pmoA gene copies g−1 dry soil. The pmoA diversity was represented by seven closely related phylotypes; the most abundant phylotype displayed 97.5% identity to pmoA of Candidatus Methyloaffinis lahnbergensis. Further analysis of prokaryote diversity in this soil did not reveal 16S rRNA gene fragments from well-studied methanotrophs of the order Methylococcales and the family Methylocystaceae. The largest group of reads (~4% of all bacterial 16S rRNA gene fragments) that could potentially belong to methanotrophs was classified as uncultivated Beijerinckiaceae bacteria. These reads displayed 96–100 and 95–98% sequence similarity to 16S rRNA gene of Candidatus Methyloaffinis lahnbergensis and “Methylocapsa gorgona” MG08, respectively, and were represented by eight species-level operational taxonomic units (OTUs), two of which were highly abundant. These identification results characterize subarctic upland soils, which are exposed to atmospheric methane concentrations only, as a unique habitat colonized mostly by USCα methanotrophs.

206 Characterization of the Rumen Bacterial Communities of Grass-fed and Grain-fed Bison Heifers

Abstract The ruminal bacterial environment of the North American bison has remained largely unexplored. The current study aimed to compare the diversity and composition of ruminal bacteria between bison heifers on two different diets. Stomach tubing was used to collect rumen fluid from lifetime grass-fed heifers between 27 and 30 months of age distributed between 2 ranches located in Standing-Butte (n = 17), SD, and Blue-Creek (n = 17), NE, respectively. A second set of samples was collected after the same individuals had been transitioned to a grain-based diet for 100 days. Bacterial composition was determined by Illumina MiSeq (2×300) sequencing of PCR amplicons generated from the V1-V3 region of the 16S rRNA gene. Next-Generation Sequence data was analyzed using a combination of custom Perl scripts, and publicly available software (Mothur v.1.40, RDP classifier and NCBI Blast). Taxonomic analysis identified Bacteroidetes and Firmicutes as the dominant phyla across all samples analyzed. A total of 61,746 and 68,437 species-level Operational Taxonomic Units (OTUs) were identified in grass-fed heifers, in contrast to 15,003 and 23,458 OTUs that were found in the same animals on a grain-based diet. A comparative analysis using the most abundant OTUs from each group was conducted using the Kruskal-Wallis sum-rank test. In the Standing Butte heifers, 24 abundant OTUs were found to be different between diets (P < 0.05), including BP_31-10446 (µ grass = 0.3% vs µ grain = 3.2%) and BP_25-00404 (µ grass = 0.4% vs µ grain = 4.1%). In the Blue Creek heifers, 20 of the most abundant OTUs were found to be different between diets (P < 0.05), including BP_18-00869 (µ grass = 1.4% vs µ grain = 0.4%) and BCRC_1-00422 (µ grass = 0.07% vs µ grain = 4.4%). Together, these results indicate that the rumen of the North American bison harbors highly diverse bacterial communities that undergo dramatic changes in response to changes in diet.

A genomic catalog of Earth\u2019s microbiomes

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.

Taxonomic and functional profiling of nitrifying biofilms in freshwater, brackish and marine RAS biofilters

In recirculating aquaculture systems (RAS), the crucial step of eliminating toxic N compounds like ammonia and nitrite is mediated via nitrifying microorganisms and takes place in biofilters. In this study, analyses of microorganisms colonizing biocarriers of nine moving-bed biofilters of three different RAS operated with freshwater, brackish or marine process water uncovered site specific communities. Illumina-based amplicon sequencing of the V4-region of the 16S rRNA gene revealed a high microbial diversity with 1000–2500 species-level operational taxonomic units (OTUs) in all biofilters with the highest diversity in the brackish RAS. Proteobacteria, Bacteriodetes, Plantomycetes, Chloroflexi and Nitrospirae represented the most abundant phyla. 76 out of 674 known genera occurred in all nine biofilters and were defined as core-taxa, including nitrifying bacteria (Nitrosomonas and Nitrospira) as well as members of the (heterotrophic) genera Planctomyces, Blastopirellula, Nannocystis and Lewinella. Nitrifying communities composed of different, closely related and so far uncultured members of Nitrosomonas and Nitrospira were identified, strongly indicating that several potentially novel ammonia and nitrite oxidizing species are present in RAS biofilters. Relatives of known comammox Nitrospira were detected in the brackish biofilters, revealing 94–99 % identity of the 16S rRNA gene sequence to Ns. inopinata. Salinity tolerance tests with biocarriers derived from biofilters of the three distinct RAS showed an unexpected broad physiological flexibility with regard to salinity. Nitrification performance of freshwater nitrifiers was drastically reduced with increasing salinity and nearly completely inhibited at 15 PSU, while the brackish and marine nitrifiers showed a high resistance and maintained nitrification activity in a broad range of salt concentrations. This data can help to improve the nitrification process in RAS with changing salinity of the process water.

Gestational diabetes and the human salivary microbiota: a longitudinal study during pregnancy and postpartum

BackgroundAn aberrant composition of the salivary microbiota has been found in individuals with type 2 diabetes, and in pregnant women salivary microbiota composition has been associated with preeclampsia and pre-term birth. Pregnant women, who develop gestational diabetes (GDM), have a high risk of developing type 2 diabetes after pregnancy. In the present study we assessed whether GDM is linked to variation in the oral microbial community by examining the diversity and composition of the salivary microbiota.MethodIn this observational study the salivary microbiota of pregnant women with GDM (n = 50) and normal glucose regulation (n = 160) in third trimester and 9 months postpartum was assessed by 16S rRNA gene amplicon sequencing of the V1-V3 region. GDM was diagnosed in accordance with the International Association of the Diabetes and Pregnancy Study Groups (IADPSG) criteria. Cross-sectional difference in alpha diversity was assessed using Student’s t-test and longitudinal changes were assessed by mixed linear regression. Cross-sectional and longitudinal difference in beta diversity was assessed by permutational multivariate analyses of variance. Differentially abundant genera and OTUs were identified by negative binomial regression.ResultsIn the third trimester, two species-level operational taxonomic units (OTUs), while eight OTUs postpartum were differentially abundant in women with GDM compared with normoglycaemic women. OTU richness, Shannon diversity and Pielou evenness decreased from late pregnancy to 9 months after delivery regardless of glycaemic status.ConclusionGDM is associated with a minor aberration of the salivary microbiota during late pregnancy and postpartum. For unknown reasons richness of the salivary microbiota decreased from late pregnancy to postpartum, which might be explained by the physiological changes of the immune system during human pregnancy.

New insights from uncultivated genomes of the global human gut microbiome

The genome sequences of many species of the human gut microbiome remain unknown, largely owing to challenges in cultivating microorganisms under laboratory conditions. Here we address this problem by reconstructing 60,664 draft prokaryotic genomes from 3,810 faecal metagenomes, from geographically and phenotypically diverse humans. These genomes provide reference points for 2,058 newly identified species-level operational taxonomic units (OTUs), which represents a 50% increase over the previously known phylogenetic diversity of sequenced gut bacteria. On average, the newly identified OTUs comprise 33% of richness and 28% of species abundance per individual, and are enriched in humans from rural populations. A meta-analysis of clinical gut-microbiome studies pinpointed numerous disease associations for the newly identified OTUs, which have the potential to improve predictive models. Finally, our analysis revealed that uncultured gut species have undergone genome reduction that has resulted in the loss of certain biosynthetic pathways, which may offer clues for improving cultivation strategies in the future.

Assessing the Microbiota of Black Soldier Fly Larvae (Hermetia illucens) Reared on Organic Waste Streams on Four Different Locations at Laboratory and Large Scale.

This study aimed to gain insight into the microbial quality, safety and bacterial community composition of black soldier fly larvae (Hermetia illucens) reared at different facilities on a variety of organic waste streams. For seven rearing cycles, both on laboratory-scale and in large-scale facilities at several locations, the microbiota of the larvae was studied. Also samples of the substrate used and the residue (= leftover substrate after rearing, existing of non-consumed substrate, exuviae and faeces) were investigated. Depending on the sample, it was subjected to plate counting, Illumina Miseq sequencing and/or detection of specific food pathogens. The results revealed that the substrates applied at the various locations differed substantially in microbial numbers as well as in the bacterial community composition. Furthermore, little similarity was observed between the microbiota of the substrate and that of the larvae reared on that substrate. Despite substantial differences between the microbiota of larvae reared at several locations, 48 species-level operational taxonomic units (OTUs) were shared by all larvae, among which most belonged to the phyla Firmicutes and Proteobacteria. Although the substrate is assumed to be an important source of bacteria, our results suggest that a variety of supposedly interacting factors-both abiotic and biotic-are likely to affect the microbiota in the larvae. In some larvae and/or residue samples, potential foodborne pathogens such as Salmonella and Bacillus cereus were detected, emphasising that decontamination technologies are required when the larvae are used in feed, just as for other feed ingredients, or eventually in food.

Microbial Dynamics during Industrial Rearing, Processing, and Storage of Tropical House Crickets (Gryllodes sigillatus) for Human Consumption.

In this study, the microbiota during industrial rearing, processing, and storage of the edible tropical house cricket, Gryllodessigillatus, was investigated. To this end, we analyzed samples from the cricket feed, obtained before feeding as well as from the cages, and from the crickets during rearing, after harvest, and after processing into frozen, oven-dried, and smoked and oven-dried (smoked/dried) end products. Although the feed contained lower microbial numbers than the crickets, both were dominated by the same species-level operational taxonomic units, as determined by Illumina MiSeq sequencing. They corresponded, among others, to members of Porphyromonadaceae, Fusobacterium, Parabacteroides, and Erwinia The harvested crickets contained high microbial numbers, but none of the investigated food pathogens Salmonella spp., Listeria monocytogenes, Bacillus cereus, or coagulase-positive staphylococci. However, some possible mycotoxin-producing fungi were isolated from the crickets. A postharvest heat treatment, shortly boiling the crickets, reduced microbial numbers, but an endospore load of 2.4 log CFU/g remained. After processing, an increase in microbial counts was observed for the dried and smoked/dried crickets. Additionally, in the smoked/dried crickets, a high abundance of a Bacillus sp. was observed. Considering the possible occurrence of food-pathogenic species from this genus, it is advised to apply a heat treatment which is sufficient to eliminate spores. Nevertheless, the microbial numbers remained constant over a 6-month storage period, whether frozen (frozen end product) or at ambient temperature (oven-dried and smoked/dried end products).IMPORTANCE The need for sustainable protein sources has led to the emergence of a new food sector, producing and processing edible insects into foods. However, insight into the microbial quality of this new food and into the microbial dynamics during rearing, processing, and storage of edible insects is still limited. Samples monitored for their microbiota were obtained in this study from an industrial rearing and processing cycle. The results lead first to the identification of process steps which are critical for microbial food safety. Second, they can be used in the construction of a Hazard Analysis and Critical Control Points (HACCP) plan and of a Novel Food dossier, which is required in Europe for edible insects. Finally, they confirm the shelf-life period which was determined by the rearer.

Gestational diabetes is associated with change in the gut microbiota composition in third trimester of pregnancy and postpartum

BackgroundImbalances of gut microbiota composition are linked to a range of metabolic perturbations. In the present study, we examined the gut microbiota of women with gestational diabetes mellitus (GDM) and normoglycaemic pregnant women in late pregnancy and about 8 months postpartum.MethodsGut microbiota profiles of women with GDM (n = 50) and healthy (n = 157) pregnant women in the third trimester and 8 months postpartum were assessed by 16S rRNA gene amplicon sequencing of the V1-V2 region. Insulin and glucose homeostasis were evaluated by a 75 g 2-h oral glucose tolerance test during and after pregnancy.ResultsGut microbiota of women with GDM was aberrant at multiple levels, including phylum and genus levels, compared with normoglycaemic pregnant women. Actinobacteria at phylum level and Collinsella, Rothia and Desulfovibrio at genus level had a higher abundance in the GDM cohort. Difference in abundance of 17 species-level operational taxonomic units (OTUs) during pregnancy was associated with GDM. After adjustment for pre-pregnancy body mass index (BMI), 5 of the 17 OTUs showed differential abundance in the GDM cohort compared with the normoglycaemic pregnant women with enrichment of species annotated to Faecalibacterium and Anaerotruncus and depletion of species annotated to Clostridium (sensu stricto) and to Veillonella. OTUs assigned to Akkermansia were associated with lower insulin sensitivity while Christensenella OTUs were associated with higher fasting plasma glucose concentration. OTU richness and Shannon index decreased from late pregnancy to postpartum regardless of metabolic status. About 8 months after delivery, the microbiota of women with previous GDM was still characterised by an aberrant composition. Thirteen OTUs were differentially abundant in women with previous GDM compared with women with previous normoglycaemic pregnancy.ConclusionGDM diagnosed in the third trimester of pregnancy is associated with a disrupted gut microbiota composition compared with normoglycaemic pregnant women, and 8 months after pregnancy, differences in the gut microbiota signatures are still detectable. The gut microbiota composition of women with GDM, both during and after pregnancy, resembles the aberrant microbiota composition reported in non-pregnant individuals with type 2 diabetes and associated intermediary metabolic traits.

Impact of a vegan diet on the human salivary microbiota

Little is known about the effect of long-term diet patterns on the composition and functional potential of the human salivary microbiota. In the present study, we sought to contribute to the ongoing elucidation of dietary effects on the oral microbial community by examining the diversity, composition and functional potential of the salivary microbiota in 160 healthy vegans and omnivores using 16S rRNA gene amplicon sequencing. We further sought to identify bacterial taxa in saliva associated with host inflammatory markers. We show that compositional differences in the salivary microbiota of vegans and omnivores is present at all taxonomic levels below phylum level and includes upper respiratory tract commensals (e.g. Neisseria subflava, Haemophilus parainfluenzae, and Rothia mucilaginosa) and species associated with periodontal disease (e.g. Campylobacter rectus and Porphyromonas endodontalis). Dietary intake of medium chain fatty acids, piscine mono- and polyunsaturated fatty acids, and dietary fibre was associated with bacterial diversity, community structure, as well as relative abundance of several species-level operational taxonomic units. Analysis of imputed genomic potential revealed several metabolic pathways differentially abundant in vegans and omnivores indicating possible effects of macro- and micro-nutrient intake. We also show that certain oral bacteria are associated with the systemic inflammatory state of the host.