Human Fecal Samples Research Articles

C-Glycoside-Metabolizing Human Gut Bacterium, Dorea sp. MRG-IFC3.

Biochemical gut metabolism of dietary bioactive compounds is of great significance in elucidating health-related issues at the molecular level. In this study, a human gut bacterium cleaving C-C glycosidic bond was screened from puerarin conversion to daidzein, and a new, gram-positive C-glycoside-deglycosylating strain, Dorea sp. MRG-IFC3, was isolated from human fecal sample under anaerobic conditions. Though MRG-IFC3 biotransformed isoflavone C-glycoside, it could not metabolize other C-glycosides, such as vitexin, bergenin, and aloin. As evident from the production of the corresponding aglycons from various 7-O-glucosides, MRG-IFC3 strain also showed 7-O-glycoside cleavage activity; however, flavone 3-O-glucoside icariside II was not metabolized. In addition, for mechanism study, C-glycosyl bond cleavage of puerarin by MRG-IFC3 strain was performed in D2O GAM medium. The complete deuterium enrichment on C-8 position of daidzein was confirmed by 1H NMR spectroscopy, and the result clearly proved for the first time that daidzein is produced from puerarin. Two possible reaction intermediates, the quinoids and 8-dehydrodaidzein anion, were proposed for the production of daidzein-8d. These results will provide the basis for the mechanism study of stable C-glycosidic bond cleavage at the molecular level.

Bacillus velezensis DSM 33864 reduces Clostridioides difficile colonization without disturbing commensal gut microbiota composition

Up to 25% of the US population harbor Clostridioides difficile in the gut. Following antibiotic disruption of the gut microbiota, C. difficile can act as an opportunistic pathogen and induce potentially lethal infections. Consequently, reducing the colonization of C. difficile in at-risk populations is warranted, prompting us to identify and characterize a probiotic candidate specifically targeting C. difficile colonization. We identified Bacillus velezensis DSM 33864 as a promising strain to reduce C. difficile levels in vitro. We further investigated the effects of B. velezensis DSM 33864 in an assay including human fecal medium and in healthy or clindamycin-treated mouse models of C. difficile colonization. The addition of B. velezensis DSM 33864 to human fecal samples was shown to reduce the colonization of C. difficile in vitro. This was supported in vivo where orally administered B. velezensis DSM 33864 spores reduced C. difficile levels in clindamycin-treated mice. The commensal microbiota composition or post-antibiotic reconstitution was not impacted by B. velezensis DSM 33864 in human fecal samples, short-, or long-term administration in mice. In conclusion, oral administration of B. velezensis DSM 33864 specifically reduced C. difficile colonization in vitro and in vivo without adversely impacting the commensal gut microbiota composition.

Identification of Intestinal Microbial Community in Gallstone Patients with Metagenomic Next-Generation Sequencing.

Gallstone disease (GD) is one of the most common gastrointestinal diseases worldwide. Nowadays, intestinal microbiota are thought to play important roles in the formation of gallstones. In our study, human fecal samples were extracted for metagenomic next-generation sequencing (mNGS) on the Illumina HiSeq platform, followed by bioinformatics analyses. Our results showed that there was a particular intestinal micro-ecosystem in GD patients. In contrast to healthy people, the sequences of Bacteroidetes, Bacteroides and Thetaiotaomicron were obviously more abundant in GD patients at phylum, genus and species levels, respectively. On the other hand, the glycan metabolism and drug resistance, especially for the β-lactams, were the most profound functions of gut microbes in GD patients compared to those in normal subjects. Furthermore, a correlation analysis drew out that there existed a significant relationship between the serum levels of biochemical indicators and abundances of intestinal microbes in GD patients. Our results illuminate both the composition and functions of intestinal microbiota in GD patients. All in all, our study can broaden the insight into the potential mechanism of how gut microbes affect the progression of gallstones to some extent, which may provide potential targets for the prevention, diagnosis or treatment of GD.

Comparison of four DNA extraction methods for 16s rRNA microbiota profiling of human faecal samples

ObjectiveGrowth in large population-based studies assessing contributions of the gut microbiota to health and disease requires high-throughput sample processing and analysis methods. This study assessed the impact that modifications to a commercially available magnetic bead based, semi-automated DNA extraction kit had on determination of microbial composition, relative to an established in-house method involving a combination of mechanical and chemical lysis. DNA was extracted from faecal samples from healthy adults (n = 12; 34–69 years), microbial composition was determined by V3-V4 16s rRNA sequencing and compared between extraction methods.ResultsDiversity metrics did not differ between extraction methods. Differences in the relative abundance of key phyla, including a significantly lower abundance of the Firmicutes (p = 0.004) and higher relative abundance of the Bacteroidetes (p = 0.005) and Proteobacteria (p = 0.008) phyla were noted where the DNA extraction did not include additional chemical and mechanical lysis. Principal coordinate analysis of family and genera level data also suggested a potential for sample pre-processing to impact microbial composition. Observations of the potential for skewed microbial composition profiles from samples prepared using a semi-automated DNA extraction kit without additional sample pre-processing highlights a need for consideration of standardisation of methodological approaches to increase the comparability of microbial compositional data.

Diagnostic Performance of Toluidine Blue Stain for Direct Wet Mount Detection of Cryptosporidium Oocysts: Qualitative and Quantitative Comparison to the Modified Ziehl-Neelsen Stain.

(1) Background: The wet mount staining technique is a simple, economical, and rapid method for detecting parasitic stages. The objective of the current study was to evaluate wet mount diagnostic stains for Cryptosporidium infection in human faecal samples and to compare the sensitivity and qualitative performance of toluidine blue (TolB) and modified Ziehl-Neelsen (mZN) stain. (2) Methods: The collection, purification, and molecular amplification of Cryptosporidium oocysts were performed. TolB, malachite green, trypan blue, and crystal violet were evaluated qualitatively to diagnose Cryptosporidium oocysts. The outperforming stain was compared to mZN using a quantitative evaluation and qualitative scoring system. (3) Results: The oocysts of Cryptosporidium parvum were effectively purified and utilized for spiking. TolB was the most effective diagnostic stain among wet mount stains for detecting Cryptosporidium oocysts. TolB outperformed mZN in terms of sensitivity, with 100% versus 79% at a concentration of 104 and 49% versus 23% at a concentration of 102. TolB had the most favourable qualitative stain characteristics against mZN; however, sample freshness and being a temporary stain were crucial considerations. (4) Conclusions: This study emphasizes that TolB, as a routine wet mount technique for screening Cryptosporidium infection, will provide a more sensitive and faster method than mZN staining.

Carriage of linezolid-resistant enterococci (LRE) among humans and animals in Nigeria: coexistence of the cfr, optrA, and poxtA genes in Enterococcus faecium of animal origin

In contrast to increasing reports of the emergence of linezolid-resistant enterococci (LRE) emanating from many countries in Europe, Asia, and North America, data on its status and dissemination from the African continent remain scarce, with the information available limited to countries in North Africa. This study investigated the carriage of LRE and the genetic mechanism of resistance among Enterococcus faecium and Enterococcus faecalis strains recovered from humans and animals in Makurdi, Nigeria. We conducted a cross-sectional study between June 2020 and July 2021 during which 630 non-duplicate human and animal faecal samples were collected and processed for the recovery of LRE. The genetic mechanisms for resistance were investigated using polymerase chain reaction (PCR) and Sanger sequencing. Linezolid-resistant enterococci were recovered from 5.87% (37/630; 95% CI: 4.17-8.00) of the samples, with the prevalence in animals and humans being 6.22% [(28/450); 95% CI: 4.17-8.87] and 5.00% [(9/180); 95% CI: 2.31-9.28], respectively. All isolates remained susceptible to vancomycin. No known point mutation mediating linezolid resistance was detected in the 23S rRNA and ribosomal protein genes; however, acquisition of one or more potentially transferable genes (cfr, optrA, and poxtA) was observed in 26 of the 37 LRE isolates. Co-existence of all three transferable genes in a single isolate was found in four E. faecium strains of animal origin. This study provides baseline evidence for the emergence and active circulation of LRE driven majorly by the acquisition of the optrA gene in Nigeria. To the best of our knowledge, our study is the first to report a co-carriage of all three transferable linezolid resistance determinants in E. faecium. Active LRE surveillance is urgently required to understand the extent of LRE spread across sub-Saharan Africa and to develop tailored mitigation strategies.

Comparative analysis of full-length 16s ribosomal RNA genome sequencing in human fecal samples using primer sets with different degrees of degeneracy.

Next-generation sequencing has revolutionized the field of microbiology research and greatly expanded our knowledge of complex bacterial communities. Nanopore sequencing provides distinct advantages, combining cost-effectiveness, ease of use, high throughput, and high taxonomic resolution through its ability to process long amplicons, such as the entire 16s rRNA genome. We examine the performance of the conventional 27F primer (27F-I) included in the 16S Barcoding Kit distributed by Oxford Nanopore Technologies (ONT) and that of a more degenerate 27F primer (27F-II) in the context of highly complex bacterial communities in 73 human fecal samples. The results show striking differences in both taxonomic diversity and relative abundance of a substantial number of taxa between the two primer sets. Primer 27F-I reveals a significantly lower biodiversity and, for example, at the taxonomic level of the phyla, a dominance of Firmicutes and Proteobacteria as determined by relative abundances, as well as an unusually high ratio of Firmicutes/Bacteriodetes when compared to the more degenerate primer set (27F-II). Considering the findings in the context of the gut microbiomes common in Western industrial societies, as reported in the American Gut Project, the more degenerate primer set (27F-II) reflects the composition and diversity of the fecal microbiome significantly better than the 27F-I primer. This study provides a fundamentally relevant comparative analysis of the in situ performance of two primer sets designed for sequencing of the entire 16s rRNA genome and suggests that the more degenerate primer set (27F-II) should be preferred for nanopore sequencing-based analyses of the human fecal microbiome.

A preliminary study on the effects of human microRNAs on gut microbiota

MicroRNAs (miRNAs) play a key role in disease manifestation, metabolic processes and immune regulation and modulation of the host. MiRNAs can be found in abundance in fecal samples of humans and are present in intracellular, extracellular fluid and extracellular vesicles. Abundance of miRNAs is found to be in ileal lumen as compared to the colon, whereas gut microbiota is more in colon, not in ileum. Still the effect of miRNA is found where the gut microbiota is more, which is by co-localizing with the nucleic acid of the bacteria. Some human miRNAs can regulate the non-coding RNAs in bacterial species like Escherichia coli and Fusobacterium nucleatum by entering into the bacterial cell in the intestine. By repressing the activity of the noncoding RNAs of intestinal bacterial species, human miRNAs bring about the increase or decrease in population of gut microbiota. With the change in the population of the certain microbiota species in the intestine, the host becomes more susceptible to the dysbiosis and becomes vulnerable to intestinal localization of pathogenic bacteria causing diseases like inflammatory bowel disease. With the usage of bioinformatics tool, this study has deepened into interaction of human miRNA and E. coli non-coding RNA, thereby opening up a detailed analysis into interaction of host with its gut microbiome and how the interaction is responsible for diseases in host.

Fast profiling of primary, secondary, conjugated, and sulfated bile acids in human urine and murine feces samples.

Bile acids (BAs) are a complex class of metabolites that have been described as specific biomarkers of gut microbiota activity. The development of analytical methods allowing the quantification of an ample spectrum of BAs in different biological matrices is needed to enable a wider implementation of BAs as complementary measures in studies investigating the functional role of the gut microbiota. This work presents results from the validation of a targeted ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 28 BAs and six sulfated BAs, covering primary, secondary, and conjugated BAs. The analysis of 73 urine and 20 feces samples was used to test the applicability of the method. Concentrations of BAs in human urine and murine feces were reported, ranging from 0.5 to 50nmol/g creatinine and from 0.012 to 332nmol/g, respectively. Seventy-nine percent of BAs present in human urine samples corresponded to secondary conjugated BAs, while 69% of BAs present in murine feces corresponded to primary conjugated BAs. Glycocholic acid sulfate (GCA-S) was the most abundant BA in human urine samples, while taurolithocholic acid was the lowest concentrated compound detected. In murine feces, the most abundant BAs were α-murocholic, deoxycholic, dehydrocholic, and β-murocholic acids, while GCA-S was the lowest concentrated BA. The presented method is a non-invasive approach for the simultaneous assessment of BAs and sulfated BAs in urine and feces samples, and the results will serve as a knowledge base for future translational studies focusing on the role of the microbiota in health.

Epidemiology of extended-spectrum beta-lactamase-producingEscherichia coli at the human-animal-environment interface in a farming community of central Uganda.

Extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) represents a significant global public health concern. The epidemiology of ESBL-Ec in Uganda is not well understood although it is harbored by humans, animals, and the environment. This study explains the epidemiology of ESBL-Ec using a one health approach in selected farming households in Wakiso district, Uganda. Environmental, human, and animal samples were collected from 104 households. Additional data were obtained using observation checklists and through interviews with household members using a semi-structured questionnaire. Surface swabs, soil, water, human and animal fecal samples were introduced onto ESBL chromogenic agar. The isolates were identified using biochemical tests and double-disk synergy tests. To assess associations, prevalence ratios (PRs) were computed using a generalized linear model (GLM) analysis with modified Poisson and a log link with robust standard errors in R software. Approximately 83% (86/104) households had at least one positive ESBL-Ec isolate. The overall prevalence of ESBL-Ec at the human-animal-environment interface was approximately 25.0% (95% CI: 22.7-28.3). Specifically, humans, animals and the environment had an ESBL-Ec prevalence of 35.4%, 55.4%, and 9.2% respectively. Having visitors (adj PR = 1.19, 95% CI: 1.04-1.36), utilizing veterinary services (adj PR = 1.39, 95% CI: 1.20-1.61) and using animal waste for gardening (adj PR = 1.29, 95% CI: 1.05-1.60) were positively associated with household ESBL-Ec contamination. Covering the drinking water container with a lid (adj PR = 0.84 95% CI: 0.73-0.96) was associated with absence of ESBL-Ec in a household. There is wider dissemination of ESBL-Ec in the environment, humans, and animals, indicating poor infection prevention and control (IPC) measures in the area. Improved collaborative one health mitigation strategies such as safe water chain, farm biosecurity, household and facility-based IPC measures are recommended to reduce the burden of antimicrobial resistance at community level.

Are Microplastics Toxic? A Review from Eco-Toxicity to Effects on the Gut Microbiota.

Emerging studies have presented an initial picture of the toxic effects of exposure to environmental micro- and nanoplastics. They have indicated that micro- and nanoplastics may induce toxicity by leading to oxidative stress, energy metabolism disorders, gene damage, and so forth in environmental organisms, marine invertebrates and vertebrates, and laboratory mouse models. In recent years, micro- and nanoplastics have been discovered in human fecal samples, placentas, lung tissue, and even blood; thus, micro- and nanoplastics pose an alarming and ever-increasing threat to global public health. However, current research on the health effects of micro- and nanoplastics and the possible adverse outcomes in humans has only presented the tip of the iceberg. More robust clinical data and basic experiments are still warranted to elucidate the specific relationships and mechanisms. In this paper, we review studies on micro- and nanoplastic toxicity from the perspectives of eco-toxicity, the adverse effects on invertebrates and vertebrates, and the impact of micro- and nanoplastics on the gut microbiota and its metabolites. In addition, we evaluate the toxicological role of micro- and nanoplastic exposure and its potential implications in respect to human health. We also summarize studies regarding preventive strategies. Overall, this review provides insights on micro- and nanoplastic toxicity and its underlying mechanisms, opening up scientific avenues for future in-depth studies.

Detection of Antimicrobial Resistance Genes from Shiga Toxin Producing Escherichia coli by Multiplex PCR

Background: Shiga toxin-producing Escherichia coli (STEC) strains are considered to be most common food-borne enteric zoonotic pathogen, causing various disease conditions in both animals and humans and are highly pathogenic to human in low infectious doses. Resistance against antibiotics by STEC is also a big concern now a days. Hence in view of the public health significance of STEC, present work was planned to know the combination of phenotypic and genotypic resistance patterns against certain most commonly used antibiotics by using disk diffusion method and multiplex PCR respectively. Methods: A total of 426 PCR confirmed STEC isolates isolated from pooled samples (animal faecal(179), farm water(122) and human faecal samples (125) of different livestock farms in and around Proddatur andhra Pradesh, were subjected to antibiotic sensitivity test by using disc diffusion method. The isolates that showed resistance for tetracycline, streptomycin, sulphonamides and ampicillin were selected and subjected to multiplex PCR for the detection of resistance genes. Result: Disk diffusion assay revealed highest phenotypic resistance for STEC isolates against Cepahlothin (100%), followed by Ampicillin (99.06%), Tetracycline (97.2%), Streptomycin (94.3%), Sulphonamides (90.8%) and Trimethoprim (84.5%). Pooled samples also revealed the presence of antimicrobial resistance genes like tetA(59.2%), tetB(43.5%), tetC (9.2%), strA (39.3%), strB(54.1%), sul1 (40.8%), sul2 (58.7%), sul3 (3.8%) and blaTEM(83.4%). These findings indicate the highest prevalence of antimicrobial resistance among the STEC isolates, which alarms indiscriminate use of antibiotics both for therapeutic purpose and as growth promoters. Strict hygienic, sanitation and HACCP programmes should be applied to counter STEC prevalence.

Improvement of crAssphage detection/quantification method and its extensive application for food safety.

Water-borne diseases are usually caused by the fecal-oral transmission of human fecal pathogens. Traditionally, coliforms and enterococci are widely used as indicator bacteria, but they do not allow to differentiate between human and animal fecal contamination. Owing to its presence only in the human gut environment, crAssphage has been suggested as an alternative indicator of human fecal contamination to overcome the above challenges. In this study, 139 human and 89 animal fecal samples (e.g., chicken, cow, dog, pig, pigeon, and mouse) were collected. For the rapid detection of human crAssphage in fecal samples, quantitative real-time PCR (qPCR) was performed using five different oligonucleotide primer/probe combinations. These included three previously reported oligonucleotide primer/probe combinations (RQ, CPQ056, and CrAssBP) and two newly developed combinations (ORF00018-targeting CrAssPFL1 and ORF00044-targeting CrAssPFL2). The detection rate (crAssphage-positive rate) in human fecal samples were 23.0, 30.2, 28.8, 20.1, and 30.9%, respectively, suggesting CrAssPFL2 showed the highest detection rate. Furthermore, the lowest copy numbers (436.16 copy numbers) could be detected using the CrAssPFL2 combination. Interestingly, no difference in crAssphage detection rates was found between healthy people and intestinal inflammatory patients. As expected, no crAssphage was detected in any animal fecal samples, indicating its human specificity. Furthermore, qPCR analysis of sewage samples collected from five different sewage treatment plants revealed that they were all contaminated with 105.71 copy numbers/mL of crAssphage on average. The simulation test of crAssphage-contaminated food samples also confirmed that the detection limit was from 107.55 copy numbers of crAssphage in foods. Therefore, the newly developed and optimized qPCR would be useful for the sensitive detection of crAssphage while identifying the source of human fecal contamination.

Colonization with Escherichia coli ST131-H30R (H30R) Corresponds with Increased Serum Anti-O25 IgG Levels and Decreased TNFα and IL-10 Responsiveness to H30R.

An exceptional gut-colonizing ability may underlie the dramatic epidemiological success of the multidrug-resistant H30R subclone of Escherichia coli sequence type 131 (O25b:K+:H4). In order to inform the development of colonization-preventing measures, we studied systemic immune correlates of H30R intestinal colonization. Human volunteers' fecal samples were screened for H30R by selective culture and PCR. Subjects were assessed by enzyme immunoassay for serum levels of anti-O25 IgG (representing H30R) and anti-O6 IgG (representing non-H30 E. coli generally), initially and for up to 14 months. Whole blood was tested for the antigen-stimulated release of IFNγ, TNFα, IL-4, IL-10, and IL-17 after incubation with E. coli strains JJ1886 (H30R; O25b:K+:H4) or CFT073 (non-H30; O6:K2:H1). Three main findings were obtained. First, H30R-colonized subjects had significantly higher anti-O25 IgG levels than controls, but similar anti-O6 IgG levels, suggesting an IgG response to H30R colonization. Second, anti-O25 and anti-O6 IgG levels were stable over time. Third, H30R-colonized subjects exhibited a lower TNFα and IL-10 release than controls in response to strain JJ1886 (H30R) relative to strain CFT073 (non-H30R), consistent with TNFα hypo-responsiveness to H30R possibly predisposing to H30R colonization. Thus, H30R-colonized hosts exhibit a sustained serum anti-O25 IgG response and an underlying deficit in TNFα responsiveness to H30R that could potentially be addressed for colonization prevention.

Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Molecular characterization and comparative genomic analysis of Acinetobacter baumannii isolated from the community and the hospital: an epidemiological study in Segamat, Malaysia.

Acinetobacter baumannii is a common cause of multidrug-resistant (MDR) nosocomial infections around the world. However, little is known about the persistence and dynamics of A. baumannii in a healthy community. This study investigated the role of the community as a prospective reservoir for A. baumannii and explored possible links between hospital and community isolates. A total of 12 independent A. baumannii strains were isolated from human faecal samples from the community in Segamat, Malaysia, in 2018 and 2019. Another 15 were obtained in 2020 from patients at the co-located tertiary public hospital. The antimicrobial resistance profile and biofilm formation ability were analysed, and the relatedness of community and hospital isolates was determined using whole-genome sequencing (WGS). Antibiotic profile analysis revealed that 12 out of 15 hospital isolates were MDR, but none of the community isolates were MDR. However, phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) and a pangenome analysis of core genes showed clustering between four community and two hospital strains. Such clustering of strains from two different settings based on their genomes suggests that these strains could persist in both. WGS revealed 41 potential resistance genes on average in the hospital strains, but fewer (n=32) were detected in the community strains. In contrast, 68 virulence genes were commonly seen in strains from both sources. This study highlights the possible transmission threat to public health posed by virulent A. baumannii present in the gut of asymptomatic individuals in the community.

New carbohydrate binding domains identified by phage display based functional metagenomic screens of human gut microbiota

Uncultured microbes represent a huge untapped biological resource of novel genes and gene products. Although recent genomic and metagenomic sequencing efforts have led to the identification of numerous genes that are homologous to existing annotated genes, there remains, yet, an enormous pool of unannotated genes that do not find significant sequence homology to existing annotated genes. Functional metagenomics offers a way to identify and annotate novel gene products. Here, we use functional metagenomics to mine novel carbohydrate binding domains that might aid human gut commensals in adherence, gut colonization, and metabolism of complex carbohydrates. We report the construction and functional screening of a metagenomic phage display library from healthy human fecal samples against dietary, microbial and host polysaccharides/glycoconjugates. We identify several protein sequences that do not find a hit to any known protein domain but are predicted to contain carbohydrate binding module-like folds. We heterologously express, purify and biochemically characterize some of these protein domains and demonstrate their carbohydrate-binding function. Our study reveals several previously unannotated carbohydrate-binding domains, including a levan binding domain and four complex N-glycan binding domains that might be useful for the labeling, visualization, and isolation of these glycans.

An in vitro platform for study of the human gut microbiome under an oxygen gradient

The complex, dynamic environment of the human lower gastrointestinal tract is colonized by hundreds of bacterial species that impact health and performance. Ex vivo study of the functional interactions between microbial community members in conditions representative of those in the gut is an ongoing challenge. We have developed an in vitro 40-plex platform that provides an oxygen gradient to support simultaneous maintenance of microaerobic and anaerobic microbes from the gut microbiome that can aid in rapid characterization of microbial interactions and direct comparison of individual microbiome samples. In this report, we demonstrate that the platform more closely maintained the microbial diversity and composition of human donor fecal microbiome samples than strict anaerobic conditions. The oxygen gradient established in the platform allowed the stratification and subsequent sampling of diverse microbial subpopulations that colonize microaerobic and anaerobic micro-environments. With the ability to run forty samples in parallel, the platform has the potential to be used as a rapid screening tool to understand how the gut microbiome responds to environmental perturbations such as toxic compound exposure, dietary changes, or pharmaceutical treatments.

Temperate bacteriophages infecting the mucin-degrading bacterium Ruminococcus gnavus from the human gut

ABSTRACT Ruminococcus gnavus is a prevalent gut microbe reported to occur in higher abundance among individuals with inflammatory bowel disease (IBD). This study reports the isolation and characterization of six bacteriophages (phages) isolated from human fecal material and environmental samples that infect this species. Isolated phages have a siphovirus morphology, with genomes ranging between 36.5 and 37.8 kbp. Genome analysis indicates that the phages have a temperate lifestyle, which was confirmed by their ability to form lysogens on their host bacterial species. In contrast to the finding that phages lyse their host in liquid medium, results from a mouse trial indicate these phages can co-exist with the host bacterium in the gut without causing a significant reduction of R. gnavus. The bacterial counts in the feces of phage-treated mice did not significantly differ in the presence of phage. Furthermore, analysis of publicly available gut virome sequence data indicates a high abundance of these phages among individuals suffering from IBD. This work provides the first insight into how phages interact with R. gnavus in the human gut microbiome.

Reducing bias in microbiome research: Comparing methods from sample collection to sequencing.

Microbiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. To investigate and identify potential biases introduced by technical variations, we compared several approaches throughout the entire workflow of a microbiome study, from sample collection to sequencing, using commercially available mock communities (from bacterial strains as well as from DNA) and multiple human fecal samples, including a large set of positive controls created as a random mix of several participant samples. Human fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene·GUT and Zymo Research) in comparison to samples frozen immediately upon collection. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls. Samples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). These stabilized samples stored at RT were different from immediately frozen samples, where the relative abundance of Bacteroidota was higher and Actinobacteriota and Firmicutes were lower. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. The DNA extraction had a significant impact on the microbial composition, also observed with the use of different Illumina barcodes during library preparation and sequencing, while no batch effect was observed in replicate runs. Our study reaffirms the importance of the mechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison of storage conditions revealed that the bias was limited in RT samples preserved in stabilization systems, and these may be a suitable compromise when logistics are challenging due to the size or location of a study. Moreover, to reduce the effect of contaminants in fecal microbiota profiling studies, we suggest the use of ~125 pg input DNA and 25 PCR cycles as optimal parameters during library preparation.