Search

Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus . Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile., Importance: Next-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe-host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields., Competing Interests: E.M. is currently working as Medical Advisor for BioCodex Nordics. A.J.H. reports receiving personal fees for lectures from BioCodex, Merck, and Pfizer. All other authors declare no competing interests.

The lack of standardization in the methods of DNA extraction from fecal samples represents the major source of experimental variation in the microbiome research field. In this study, we aimed to compare the metagenomic profiles and microbiome-phenotype associations obtained by applying two commercially available DNA extraction kits: the AllPrep DNA/RNA Mini Kit (APK) and the QIAamp Fast DNA Stool Mini Kit (FSK). Using metagenomic sequencing data available from 745 paired fecal samples from two independent population cohorts, Lifelines-DEEP (LLD, n = 292) and the 500 Functional Genomics project (500FG, n = 453), we confirmed significant differences in DNA yield and the recovered microbial communities between protocols, with the APK method resulting in a higher DNA concentration and microbial diversity. Further, we observed a massive difference in bacterial relative abundances at species-level between the APK and the FSK protocols, with > 75% of species differentially abundant between protocols in both cohorts. Specifically, comparison with a standard mock community revealed that the APK method provided higher accuracy in the recovery of microbial relative abundances, with the absence of a bead-beating step in the FSK protocol causing an underrepresentation of gram-positive bacteria. This heterogeneity in the recovered microbial composition led to remarkable differences in the association with anthropometric and lifestyle phenotypes. The results of this study further reinforce that the choice of DNA extraction method impacts the metagenomic profile of human gut microbiota and highlight the importance of harmonizing protocols in microbiome studies., (© 2024. The Author(s).)

The impact of therapeutic interventions on the human gut microbiota (GM) is a clinical issue of paramount interest given the strong interconnection between microbial dynamics and human health. Orally administered antibiotics are known to reduce GM biomass and modify GM taxonomic profile. However, the impact of antimicrobial therapies on GM functions and biochemical pathways has scarcely been studied. Here, we characterized the fecal metaproteome of 10 Helicobacter pylori -infected patients before (T0) and after 10 days (T1) of a successful quadruple therapy (bismuth, tetracycline, metronidazole, and rabeprazole) and 30 days after therapy cessation (T2), to investigate how GM and host functions change during the eradication and healing processes. At T1, the abundance ratio between microbial and host proteins was reversed compared with that at T0 and T2. Several pathobionts (including Klebsiella , Proteus , Enterococcus , Muribaculum , and Enterocloster ) were increased at T1. Therapy reshaped the relative contributions of the functions required to produce acetate, propionate, and butyrate. Proteins related to the uptake and processing of complex glycans were increased. Microbial cross-feeding with sialic acid, fucose, and rhamnose was enhanced, whereas hydrogen sulfide production was reduced. Finally, microbial proteins involved in antibiotic resistance and inflammation were more abundant after therapy. Moreover, a reduction in host proteins with known roles in inflammation and H. pylori -mediated carcinogenesis was observed. In conclusion, our results support the use of metaproteomics to monitor drug-induced remodeling of GM and host functions, opening the way for investigating new antimicrobial therapies aimed at preserving gut environmental homeostasis.

The Djungarian hamster ( Phodopus sungorus ) is a prominent model organism for seasonal acclimatization, showing drastic whole-body physiological adjustments to an energetically challenging environment, which are considered to also involve the gut microbiome. Fecal samples of hamsters in long photoperiod and again after twelve weeks in short photoperiod were analyzed by 16S-rRNA sequencing to evaluate seasonal changes in the respective gut microbiomes. In both photoperiods, the overall composition was stable in the major superordinate phyla of the microbiota, with distinct and delicate changes of abundance in phyla representing each <1% of all. Elusimicrobia, Tenericutes, and Verrucomicrobia were exclusively present in short photoperiod hamsters. In contrast to Elusimicrobium and Aneroplasma as representatives of Elusimicrobia and Tenericutes, Akkermansia muciniphila is a prominent gut microbiome inhabitant well described as important in the health context of animals and humans, including neurodegenerative diseases and obesity. Since diet was not changed, Akkermansia enrichment appears to be a direct consequence of short photoperiod acclimation. Future research will investigate whether the Djungarian hamster intestinal microbiome is responsible for or responsive to seasonal acclimation, focusing on probiotic supplementation.

Reintroduction is an effective strategy in the conservation of endangered species under scientific monitoring. Intestinal flora plays an important role in the envir onmental adaptation of endangered Père David's deer ( Elaphurus davidianus ). In this study, 34 fecal samples from E. davidianus were collected from different habitats in Tianjin city of China to investigate differences in the intestinal flora under captive and semi-free-ranging conditions. Based on 16S rRNA high-throughput sequencing technology, a total of 23 phyla and 518 genera were obtained. Firmicutes was dominant in all individuals. At the genus level, UCG-005 (13.05%) and Rikenellaceae&#95;RC9&#95;gut&#95;group (8.94%) were dominant in captive individuals, while Psychrobacillus (26.53%) and Pseudomonas (11.33%) were dominant in semi-free-ranging individuals. Alpha diversity results showed that the intestinal flora richness and diversity were significantly ( P < 0.001) higher in captive individuals than in semi-free-ranging individuals. Beta diversity analysis also showed a significant difference ( P = 0.001) between the two groups. In addition, some age- and sex-related genera such as Monoglobus were identified. In summary, the structure and diversity of intestinal flora showed significant habitat variation. This is the first time an analysis has been undertaken of the structural differences of the intestinal flora in Père David's deer, under different habitats in the warm temperate zone, providing a reference basis for the conservation of endangered species., Competing Interests: The authors declare that they have no competing interests., (© 2023 Yao et al.)

The dietary patterns are closely associated with gut microbiota, which has been proved associated with kidney stones. To assess the association among the dietary patterns, gut microbiota, and kidney stones, patients with calcium oxalate stones and participants without kidney stones were recruited in West China Hospital and were divided into the low nephrolithiasis risk (LNR) and high nephrolithiasis risk (HNR) dietary pattern group based on the results of food frequency questionnaires. The genomic DNA of the fecal samples were extracted for 16S ribosomal RNA gene sequencing. The non-kidney stone (NS) group comprised 39 LNR and 45 HNR individuals, while the kidney stone (KS) group consisted of 19 LNR and 50 HNR individuals. The distribution of oxalate in urine (p < 0.01) but not calcium (p = 0.741) was significantly varied among the four groups. Significant difference was found in the dietary patterns of people with KS and NS controls (X 2  = 5.744, p = 0.017). Forty-six discriminative bacteria were found among different dietary patterns groups in KS patients and NS controls. Not only gut bacteria such as Pseudomonas, Sphingomonas, Hydrogenoanaerobacterium, Faecalitalea, etc., but also metabolic pathways associated with inflammation, lipid, and mineral metabolism were found more abundant in KS patients with HNR dietary pattern. It is noteworthy that g&#95;&#95;Prevotellaceae&#95;UCG&#95;001, g&#95;&#95;hgcI&#95;clade, and g&#95;&#95;Bradyrhizobium were negatively related to water intake but instead had a positive correlation with salt and meat intake. Our study revealed that gut microbiota with significantly different abundance existed in the HNR dietary patterns compared to the LNR counterparts in both calcium oxalate KS and NS individuals. The dietary patterns may affect the prevention and management of calcium oxalate stones by regulating the homeostasis of gut microbiota., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bottom-up proteomics is a powerful method for the functional characterization of mouse gut microbiota. To date, most of the bottom-up proteomics studies of the mouse gut rely on limited amounts of fecal samples. With mass-limited samples, the performance of such analyses is highly dependent on the protein extraction protocols and contaminant removal strategies. Here, protein extraction protocols (using different lysis buffers) and contaminant removal strategies (using different types of filters and beads) were systematically evaluated to maximize quantitative reproducibility and the number of identified proteins. Overall, our results recommend a protein extraction method using a combination of sodium dodecyl sulfate (SDS) and urea in Tris-HCl to yield the greatest number of protein identifications. These conditions led to an increase in the number of proteins identified from gram-positive bacteria, such as Firmicutes and Actinobacteria, which is a challenging task. Our analysis further confirmed these conditions led to the extraction of non-abundant bacterial phyla such as Proteobacteria. In addition, we found that, when coupled to our optimized extraction method, suspension trap (S-Trap) outperforms other contaminant removal methods by providing the most reproducible method while producing the greatest number of protein identifications. Overall, our optimized sample preparation workflow is straightforward and fast, and requires minimal sample handling. Furthermore, our approach does not require high amounts of fecal samples, a vital consideration in proteomics studies where mice produce smaller amounts of feces due to a particular physiological condition. Our final method provides efficient digestion of mouse fecal material, is reproducible, and leads to high proteomic coverage for both host and microbiome proteins., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

Species of fungi belonging to the order Mucorales can be found everywhere in the environment. Gilbertella persicaria, which belongs to this order, have often been isolated from fruits and in water systems. However, there has been no report of isolation of this fungus from human samples. During a gut mycobiome study, from the Segamat community, Gilbertella persicaria was isolated from a human fecal sample and was characterized through a series of morphological assessment, biochemical tests, and molecular techniques. The isolate produced a white velvety surface that turned grayish after 24 h. Although no biofilm production was observed, the results indicated that the isolate could form calcium oxalate crystals, produced urease, and was resistant to low pH. The isolate was sensitive to amphotericin but resistant to voriconazole and itraconazole. The features of this fungus that could help in its survival in the human gut are also discussed.

In vitro gut models, such as the mucosal artificial colon (M-ARCOL), provide timely and cost-efficient alternatives to in vivo assays allowing mechanistic studies to better understand the role of human microbiome in health and disease. Using such models inoculated with human fecal samples may require a critical step of stool storage. The effects of preservation methods on microbial structure and function in in vitro gut models have been poorly investigated. This study aimed to assess the impact of three commonly used preserving methods, compared with fresh fecal samples used as a control, on the kinetics of lumen and mucus-associated microbiota colonization in the M-ARCOL model. Feces from two healthy donors were frozen 48 h at - 80 °C with or without cryoprotectant (10% glycerol) or lyophilized with maltodextrin and trehalose prior to inoculation of four parallel bioreactors (e.g., fresh stool, raw stool stored at - 80 °C, stool stored at - 80 °C with glycerol and lyophilized stool). Microbiota composition and diversity (qPCR and 16S metabarcoding) as well as metabolic activity (gases and short chain fatty acids) were monitored throughout the fermentation process (9 days). All the preservative treatments allowed the maintaining inside the M-ARCOL of a complex and functional microbiota, but considering stabilization time of microbial profiles and activities (and not technical constraints associated with the supply of frozen material), our results highlighted 48 h freezing at - 80 °C without cryoprotectant as the most efficient method. These results will help scientists to determine the most accurate method for fecal storage prior to inoculation of in vitro gut microbiome models. KEY POINTS: • In vitro ARCOL model reproduces luminal and mucosal human microbiome. • Short-term storage of fecal sample influences microbial stabilization and activity. • 48 h freezing at - 80°C: most efficient method to preserve microbial ecosystem. • Scientific and technical requirements: influencers of preservation method.

Objectives : Emerging evidence suggests that gut microbiota dysbiosis plays a critical role in chronic kidney disease (CKD). However, the relationship between altered gut microbiome profiles and disease severity remains unclear. In this study, we sought to characterize the gut microbiota in CKD patients compared to healthy controls, and to explore potential relationships between gut microbiota composition and disease severity. Methods : Fecal samples were collected from 95 patients at different stages of CKD (non-dialysis patients from stage 1 to 5) and 20 healthy controls. Bacterial DNA was extracted for 16S ribosomal DNA sequencing targeting the V3-V4 region. The diversity and relative abundance of gut microbiota were analyzed as outcome indicators. Results : Differences were observed in the microbial composition and diversity of fecal samples from CKD patients and healthy controls. Specifically, disease severity was found to alter gut microbiota composition. Compared to that in healthy controls, CKD patients showed an increased abundance of Proteobacteria and decreased Synergistetes, most notably in disease stage 5. Lower levels of butyrate-producing bacteria and higher levels of potential pathogens were also detected in CKD patients. Further, Pyramidobacter and Prevotellaceae&#95;UCG-001 were significantly decreased in the CKD1 group compared with healthy controls. Notably, nine microbial genera, including Escherichia-Shigella, Parabacteroides, Roseburia, rectale&#95;group, Ruminococcaceae&#95;NK4A214&#95;group, Prevotellaceae&#95;UCG.001, Hungatella, Intestinimonas , and Pyramidobacter , identified using a random forest model, distinguished between patients with CKD and healthy controls with high accuracy. Functional analysis also revealed that fatty acid and inositol phosphate metabolism were enriched in the CKD group, while aminoacyl-tRNA biosynthesis, oxidative phosphorylation, phenylalanine, tyrosine, and tryptophan biosynthesis, thiamine metabolism, pantothenate, and CoA biosynthesis, as well as valine, leucine, and isoleucine biosynthesis were enriched in healthy controls. Conclusion : Gut microbiota composition and function are associated with CKD severity. And, specific gut microbes are potentially helpful for CKD early diagnosis and prognosis monitoring.

Background: The number of large-scale studies on the gut microbiota in human cohorts is rapidly increasing. However, the few and expensive options for storage of fecal samples at room temperature have been an obstacle for large-scale metagenomic studies and the development of clinical/commercial personal metagenomic sequencing., Results: In this study, we systematically tested a novel N-octylpyridinium bromide-based fecal sample preservation method and compared it with other currently used storage methods. We found that the N-octylpyridinium bromide-based method enabled preservation of the bacterial composition in fecal samples transported and stored at room temperature for up to at least 14 days., Conclusions: We describe a novel chemical stabilizer that allows cost-effective transportation and storage at room temperature for several days with preservation of bacterial composition. This method will facilitate sample collection even in remote area and also enable transport via normal commercial transportation routes.