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Background High-throughput sequencing of the full-length 16S rRNA gene has improved the taxonomic classification of prokaryotes found in natural environments. However, sequencing of shorter regions from the same gene, like the V4-V5 region, can provide more cost-effective high throughput. It is unclear which approach best describes prokaryotic communities from underexplored environments. In this study, we hypothesize that high-throughput full-length 16S rRNA gene sequencing combined with adequate taxonomic databases improves the taxonomic description of prokaryotic communities from underexplored environments in comparison with high-throughput sequencing of a short region of the 16S rRNA gene. Results To test our hypothesis, we compared taxonomic profiles of seawater samples from the Arctic Ocean using: full-length and V4-V5 16S rRNA gene sequencing in combination with either the Genome Taxonomy Database (GTDB) or the Silva taxonomy database. Our results show that all combinations of sequencing strategies and taxonomic databases present similar results at higher taxonomic levels. However, at lower taxonomic levels, namely family, genus, and most notably species level, the full-length approach led to higher proportions of Amplicon Sequence Variants (ASVs) assigned to formally valid taxa. Hence, the best taxonomic description was obtained by the full-length and GTDB combination, which in some cases allowed for the identification of intraspecific diversity of ASVs. Conclusions We conclude that coupling high-throughput full-length 16S rRNA gene sequencing with GTDB improves the description of microbiome profiling at lower taxonomic ranks. The improvements reported here provide more context for scientists to discuss microbial community dynamics within a solid taxonomic framework in environments like the Arctic Ocean with still underrepresented microbiome sequences in public databases.

Background Childhood malnutrition is a global health challenge associated with multiple adverse consequences, including delayed maturation of the gut microbiota (GM) which might induce long-term immune dysfunction and stunting. To understand GM dynamics during malnutrition and subsequent re-feeding, we used a piglet model with a malnutrition-induced phenotype similar to humans. Piglets were weaned at the age of 4 weeks, fed a nutritionally optimal diet for 1 week post-weaning before being fed a pure maize diet for 7 weeks to induce symptoms of malnutrition. After malnourishment, the piglets were re-fed using different regimes all based on general food aid products, namely Corn-Soy blend (CSB) fortified with phosphorus (CSB+), CSB fortified with phosphorus and skim milk powder (CSB++) and CSB fortified with phosphorus and added whey permeate (CSB + P). Results Malnourishment had profound impact on the GM of the piglets leading to a less diverse GM dominated especially by Akkermansia spp. as determined by 16S rRNA gene amplicon sequencing. All three re-feeding regimes partly restored GM, leading to a more diverse GM compositionally closer to that of well-nourished piglets. This effect was even more pronounced for CSB++ compared to CSB+ and CSB + P. Conclusion The GM of piglets were profoundly disturbed by malnourishment resulting in significantly increased abundance of Akkermansia spp. CSB++ may have superior effect on recovering GM diversity compared to the two other food aid products used in this study.

Saline-alkalisation of the soil environment and microorganism is a global challenge. However, relevant studies on the effects of saline-alkali stress on soil bacterial communities are limited. In this study, we investigated the effects of saline-alkali stress on the carbon source metabolic utilisation of the microbial community, bacterial diversity, and composition in soil using Biolog Ecoplate and 16S rRNA gene amplicon sequencing. Biolog Ecoplate results showed that saline-alkali stress decreased the metabolic activity and functional diversity, and changed the utilisation characteristics of carbon sources in soil microorganisms. Particularly, high level of saline-alkali stress significantly decreased the utilisation of carbohydrates and amino acids carbon sources. The results of 16S rRNA gene amplicon sequencing showed that high level of saline-alkali stress significantly reduced the diversity of soil bacterial communities. In addition, high level of saline-alkali stress significantly decreased the relative abundances of some key bacterial taxa, such as Gemmatimonas, Sphingomonas, and Bradyrhizobium. Furthermore, as saline-alkali content increased, the soil catalase, protease, urease, and sucrase activities also significantly decreased. Collectively, these results provide new insight for studies on the changes in the soil bacterial community and soil enzyme activity under saline-alkali stress.

Background and aims Plant breeding activities shape the rhizosphere microbiome but less is known about the relationship of both with the seed microbiome. We analyzed the composition of bacterial communities of seeds and rhizospheres of Styrian oil pumpkin genotypes in comparison to bulk soil to elucidate specific microbial signatures to support a concept involving plant-microbe interactions in breeding strategies. Methods The seed and rhizosphere microbiomes of 14 genotypes of oilseed pumpkin and relatives were analyzed using a 16S rRNA gene amplicon sequencing approach, which was assessed by bioinformatics and statistical methods. Results All analyzed microhabitats were characterized by diverse bacterial communities, but the relative proportions of phyla and the overall diversity was different. Seed microbiomes were characterized by the lowest diversity and dominant members of Enterobacteriaceae including potential pathogens (Erwinia, Pectobacterium). Potential plant-beneficial bacteria like Lysobacter, Paenibacillus and Lactococcus contributed to the microbial communities in significant abundances. Interestingly, strong genotype-specific microbiomes were detected for seeds but not for the rhizospheres. Conclusions Our study indicates a strong impact of the Cucurbita pepo genotype on the composition of the seed microbiome. This should be considered in breeding of new cultivars that are more capable of exploiting beneficial indigenous microbial communities.

Background Immune-mediated inflammatory disease (IMID) represents a substantial health concern. It is widely recognized that IMID patients are at a higher risk for developing secondary inflammation-related conditions. While an ambiguous etiology is common to all IMIDs, in recent years, considerable knowledge has emerged regarding the plausible role of the gut microbiome in IMIDs. This study used 16S rRNA gene amplicon sequencing to compare the gut microbiota of patients with Crohn’s disease (CD; N  = 20), ulcerative colitis (UC; N  = 19), multiple sclerosis (MS; N = 19), and rheumatoid arthritis (RA; N  = 21) versus healthy controls (HC; N  = 23). Biological replicates were collected from participants within a 2-month interval. This study aimed to identify common (or unique) taxonomic biomarkers of IMIDs using both differential abundance testing and a machine learning approach. Results Significant microbial community differences between cohorts were observed (pseudo F  = 4.56; p  = 0.01). Richness and diversity were significantly different between cohorts (pFDR < 0.001) and were lowest in CD while highest in HC. Abundances of Actinomyces , Eggerthella, Clostridium III , Faecalicoccus , and Streptococcus (pFDR < 0.001) were significantly higher in all disease cohorts relative to HC, whereas significantly lower abundances were observed for Gemmiger , Lachnospira , and Sporobacter (pFDR < 0.001). Several taxa were found to be differentially abundant in IMIDs versus HC including significantly higher abundances of Intestinibacter in CD, Bifidobacterium in UC, and unclassified Erysipelotrichaceae in MS and significantly lower abundances of Coprococcus in CD, Dialister in MS, and Roseburia in RA. A machine learning approach to classify disease versus HC was highest for CD (AUC = 0.93 and AUC = 0.95 for OTU and genus features, respectively) followed by MS, RA, and UC. Gemmiger and Faecalicoccus were identified as important features for classification of subjects to CD and HC. In general, features identified by differential abundance testing were consistent with machine learning feature importance. Conclusions This study identified several gut microbial taxa with differential abundance patterns common to IMIDs. We also found differentially abundant taxa between IMIDs. These taxa may serve as biomarkers for the detection and diagnosis of IMIDs and suggest there may be a common component to IMID etiology.

Herbivorous fishes play important ecological roles in coral reefs by consuming algae that can otherwise outcompete corals, but we know little about the gut microbiota that facilitates this process. This study focussed on the gut microbiota of an ecologically important coral reef fish, the convict surgeonfish Acanthurus triostegus. We sought to understand how the microbiome of this species varies along its gastrointestinal tract and how it varies between juvenile and adult fish. Further, we examined if the bacteria associated with the diet consumed by juveniles contribute to the gut microbiota. 16S rRNA gene amplicon sequencing showed that bacterial communities associated with the midgut and hindgut regions were distinct between adults and juveniles; however, no significant differences were seen for gut wall samples. The microbiota associated with the epilithic algal food source was similar to that of the juvenile midgut and gut wall but differed from the microbiome of the hindgut. A core bacterial community including members of taxa Epulopiscium and Brevinemataceae was observed across all gastrointestinal and diet samples, suggesting that these bacterial symbionts can be acquired by juvenile convict surgeonfish horizontally via their diet and then are retained into adulthood.

Background Microorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning. Results Here, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria . We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria , we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter. Conclusions Our study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs. 9DpTXjuT--dysnV-FwKdBe Video Abstract

Ruminants are dependent on their gut microbiomes for nutrient extraction from plant diets. However, knowledge about the composition, diversity, function, and spatial structure of gut microbiomes, especially in wild ruminants, is limited, largely because analysis has been restricted to faeces or the rumen. In two geographically separated reindeer subspecies, 16S rRNA gene amplicon sequencing revealed strong spatial structuring, and pronounced differences in microbial diversity of at least 33 phyla across the stomach, small intestine, and large intestine (including faeces). The main structural feature was the Bacteroidota to Firmicutes ratio, which declined from the stomach to the large intestine, likely reflecting functional adaptation. Metagenome shotgun sequencing also revealed highly significant structuring in the relative occurrence of carbohydrate-active enzymes (CAZymes). CAZymes were enriched in the rumen relative to the small and large intestines. Interestingly, taxonomic diversity was highest in the large intestine, suggesting an important and understudied role for this organ. Despite the two study populations being separated by an ocean and six millennia of evolutionary history, gut microbiome structuring was remarkably consistent. Our study suggests a strong selection for gut microbiome biogeography along the gastrointestinal tract in reindeer subspecies.

Expanding our knowledge of microbial communities across diverse environments includes collecting samples in places far from the laboratory. Identifying cost-effective preservatives that will enable room temperature storage of microbial communities for sequencing analysis is crucial to enabling microbiome analyses across diverse populations. As the number of human microbiome studies expand, it is increasingly important to identify cost-effective, practical preservatives that allow for room temperature sample storage. Here, we reanalyzed 16S rRNA gene amplicon sequencing data from a large sample storage study published in 2016 and performed shotgun metagenomic sequencing on remnant DNA from this experiment. Both results support the initial findings that 95% ethanol, a nontoxic, cost-effective preservative, is effective at preserving samples at room temperature for weeks. We expanded on this analysis by collecting a new set of fecal, saliva, and skin samples to determine the optimal ratio of 95% ethanol to sample. We identified optimal collection protocols for fecal samples (storing a fecal swab in 95% ethanol) and saliva samples (storing unstimulated saliva in 95% ethanol at a ratio of 1:2). Storing skin swabs in 95% ethanol reduced microbial biomass and disrupted community composition, highlighting the difficulties of low biomass sample preservation. The results from this study identify practical solutions for large-scale analyses of fecal and oral microbial communities. IMPORTANCE Expanding our knowledge of microbial communities across diverse environments includes collecting samples in places far from the laboratory. Identifying cost-effective preservatives that will enable room temperature storage of microbial communities for sequencing analysis is crucial to enabling microbiome analyses across diverse populations. Here, we validate findings that 95% ethanol efficiently preserves microbial composition at room temperature for weeks. We also identified the optimal ratio of 95% ethanol to sample for stool and saliva to preserve both microbial load and composition. These results provide rationale for an accessible, nontoxic, cost-effective solution that will enable crowdsourcing microbiome studies, such as The Microsetta Initiative, and lower the barrier for collecting diverse samples.

Background Gut microbiota has been increasingly acknowledged to shape significantly human health, contributing to various autoimmune diseases, both intestinal and non-intestinal, including multiple sclerosis (MS). Gut microbiota studies in patients with relapsing remitting MS strongly suggested its possible role in immunoregulation; however, the profile and potential of gut microbiota involvement in patients with primary progressive MS (PPMS) patients has received much less attention due to the rarity of this disease form. We compared the composition and structure of faecal bacterial assemblage using Illumina MiSeq sequencing of V3-V4 hypervariable region of 16S rRNA genes amplicons in patients with primary progressive MS and in the healthy controls. Results Over all samples 12 bacterial phyla were identified, containing 21 classes, 25 orders, 54 families, 174 genera and 1256 operational taxonomic units (OTUs). The Firmicutes phylum was found to be ultimately dominating both in OTUs richness (68% of the total bacterial OTU number) and in abundance (71% of the total number of sequence reads), followed by Bacteroidetes (12 and 16%, resp.) and Actinobacteria (7 and 6%, resp.). Summarily in all samples the number of dominant OTUs, i.e. OTUs with ≥1% relative abundance, was 13, representing much less taxonomic richness (three phyla, three classes, four orders, six families and twelve genera) as compared to the total list of identified OTUs and accounting for 30% of the sequence reads number in the healthy cohort and for 23% in the PPMS cohort. Human faecal bacterial diversity profiles were found to differ between PPMS and healthy cohorts at different taxonomic levels in minor or rare taxa. Marked PPMS-associated increase was found in the relative abundance of two dominant OTUs ( Gemmiger sp. and an unclassified Ruminococcaceae ) . The MS-related differences were also found at the level of minor and rare OTUs (101 OTUs). These changes in OTUs’ abundance translated into increased bacterial assemblage diversity in patients. Conclusion The findings are important for constructing a more detailed global picture of the primary progressive MS-associated gut microbiota, contributing to better understanding of the disease pathogenesis.