Optimization of microbial DNA extraction in 96-format from fecal samples for Next Generation Sequencing

Gut microbiota composition is often determined by using Next Generation Sequencing methods. The demand for rapid, efficient, and reliable microbial profiling is continuously increasing, thus making the optimization of high-throughput 96-format DNA extraction integral for downstream applications. In this study, we analyzed the impact of four pre-treatment methods to the DNA extraction and 16S metabarcoding results. More specifically, pre-treatments with and without bead-beating were compared. Fecal DNA was extracted from infant, adult and senior fecal samples. The extraction also included negative controls and ZymoBIOMICS Gut Microbiome Standards to test the resolution of the sequencing target and assess DNA yield. The optimized kit was DNA Stool 200 Kit special H96 with Chemagic MSM I extraction robot. Four pre-treatments, including bead-beating and chemical lysis, were applied to the samples to assess the impact of the sample lysis and homogenization. The microbial composition was determined using 16S sequencing targeting V3V4 and V4 regions with Illumina Miseq platform. The observed compositions of the Gut Microbiome Standards differed considerably from the expected theoretical compositions with all methods and sequencing targets. The fecal samples showed different degrees of microbial diversity across different pre-treatment groups; however, the inclusion of bead-beating generally lead to higher degrees of microbial diversity. The extraction in 96-format proved to be? feasible. As the DNA extraction methods are advancing, it is important to validate new procedures with the existing NGS-methods. The application of 96-format could reduce the hands-on time as well as human error in clinical microbiology. The 96-format extraction systems proved to be suitable for fecal DNA extraction. However, the results indicate the need of a standardized methods for microbial profiling.Suoliston mikrobikoostumuksen selvittämiseksi käytetään usein uuden sukupolven sekvensointimenetelmiä (engl. NGS). Korkean kapasiteetin 96-formaatissa olevan DNA-eristyksen optimoiminen alavirran sovelluksille on tärkeää, sillä kysyntä nopealle, tehokkaalle ja luotettavalle mikrobiologiselle profiloinnille on alati kasvamassa. Tässä tutkimuksessa analysoimme neljän esikäsittelytavan vaikutusta DNA-eristys -ja 16S viivakoodaustuloksiin. Tarkemmin sanottuna esikäsittelyjä ilman -ja helmiravistelun kanssa vertailtiin. Uloste-DNA:ta eristettiin aikuisen, vauvan ja seniorin ulostenäytteistä. Eristykseen kuului myös negatiivisia kontrolleja sekä ZymoBIOMICS Gut Standard -standardi sekvensointimenetelmän tarkkuuden testaamiseksi ja DNA:n saannon arvioimiseksi. Optimoitu kitti oli DNA Stool 200 Kit special H96 ja eristysrobotti Chemagic MSM I. Lyysiksen ja homogenisoinnin vaikutuksen tutkimiseksi näytteille tehtiin neljä eri esikäsittelyä, jotka sisälsivät kemiallisen -ja helmiravistelulyysiksen (engl. bead-beating). Mikrobikoostumus selvitettiin sekvensoimalla 16S-ribosomin V3V4 -ja V4-alueet käyttämällä Illumina Miseq-alustaa. Havaitut Gut Standard -standardien mikrobikoostumukset erosivat huomattavasti odotetuista teoreettisista koostumuksista kaikkien esikäsittelyjen ja sekvensointikohteiden suhteen. Eri esikäsittelymenetelmät tuottivat ulostenäytteille vaihtelevia diversiteettejä. Kuitenkin helmikäsittelyn sisällyttäminen johti yleensä korkeimpiin diversiteettilukuihin. DNA:n eristys 96-formaatissa todettiin mahdolliseksi On tärkeää validoida uusia menetelmiä vanhojen toimintatapojen valossa, koska DNA eristysmenetelmät kehittyvät. 96-formaatin käyttöönottaminen voisi vähentää käytännön työaikaa sekä ihmisestä johtuvia virhelähteitä kliinisen mikrobiologian alalla. Tämä 96- formaatti todettiin toteutettavissa olevaksi ulostenäytteiden DNA-eristyksessä. Kuitenkin tulokset viittaavat siihen, että standardisoituja menetelmiä tarvitaan mikrobikoostumuksen selvittämisessä

Current Trends and Challenges of Microbiome Research in Prostate Cancer

Purpose of review: The role of the gut microbiome in prostate cancer is an emerging area of research interest. However, no single causative organism has yet been identified. The goal of this paper is to examine the role of the microbiome in prostate cancer and summarize the challenges relating to methodology in specimen collection, sequencing technology, and interpretation of results. Recent findings: Significant heterogeneity still exists in methodology for stool sampling/storage, preservative options, DNA extraction, and sequencing database selection/in silico processing. Debate persists over primer choice in amplicon sequencing as well as optimal methods for data normalization. Statistical methods for longitudinal microbiome analysis continue to undergo refinement. While standardization of methodology may help yield more consistent results for organism identification in prostate cancer, this is a difficult task due to considerable procedural variation at each step in the process. Further reproducibility and methodology research is required

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

Anorexia nervosa and microbiota: systematic review and critical appraisal

Purpose: Recent studies have reported a gut microbiota imbalance or dysbiosis associated with anorexia nervosa (AN), which has prompted an appraisal of its aetiological role, and the reformulation of AN as a metabo-psychiatric disorder. Thus, the aim of this paper was to critically review the current scientific findings regarding the role of microbiota in anorexia nervosa. Methods: A systematic study of peer-reviewed literature published in four databases between 2009 and 2022 was conducted according to PRISMA guidelines. Both human and animal studies were included.Results: A total of 18 studies were included. In animal models, both the preclinical and clinical findings were inconsistent regarding microbiota composition, faecal metabolite concentrations, and the effects of human faecal microbiota transplants. Conclusion: The methodological limitations, lack of standardisation, and conceptual ambiguity hinder the analysis of microbiota as a key explanatory factor for ANOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer NatureS

An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF

What Pediatricians Should Know Before Studying Gut Microbiota

Billions of microorganisms, or "microbiota", inhabit the gut and affect its homeostasis, influencing, and sometimes causing if altered, a multitude of diseases. The genomes of the microbes that form the gut ecosystem should be summed to the human genome to form the hologenome due to their influence on human physiology; hence the term "microbiome" is commonly used to refer to the genetic make-up and gene-gene interactions of microbes. This review attempts to provide insight into this recently discovered vital organ of the human body, which has yet to be fully explored. We herein discuss the rhythm and shaping of the microbiome at birth and during the first years leading up to adolescence. Furthermore, important issues to consider for conducting a reliable microbiome study including study design, inclusion/exclusion criteria, sample collection, storage, and variability of different sampling methods as well as the basic terminology of molecular approaches, data analysis, and clinical interpretation of results are addressed. This basic knowledge aims to provide the pediatricians with a key tool to avoid data dispersion and pitfalls during child microbiota study

Non-Invasive Mapping of the Gastrointestinal Microbiota Identifies Children with Inflammatory Bowel Disease

Background: Pediatric inflammatory bowel disease (IBD) is challenging to diagnose because of the non-specificity of symptoms; an unequivocal diagnosis can only be made using colonoscopy, which clinicians are reluctant to recommend for children. Diagnosis of pediatric IBD is therefore frequently delayed, leading to inappropriate treatment plans and poor outcomes. We investigated the use of 16S rRNA sequencing of fecal samples and new analytical methods to assess differences in the microbiota of children with IBD and other gastrointestinal disorders. Methodology/Principal Findings: We applied synthetic learning in microbial ecology (SLiME) analysis to 16S sequencing data obtained from i) published surveys of microbiota diversity in IBD and ii) fecal samples from 91 children and young adults who were treated in the gastroenterology program of Children’s Hospital (Boston, USA). The developed method accurately distinguished control samples from those of patients with IBD; the area under the receiver-operating-characteristic curve (AUC) value was 0.83 (corresponding to 80.3% sensitivity and 69.7% specificity at a set threshold). The accuracy was maintained among data sets collected by different sampling and sequencing methods. The method identified taxa associated with disease states and distinguished patients with Crohn’s disease from those with ulcerative colitis with reasonable accuracy. The findings were validated using samples from an additional group of 68 patients; the validation test identified patients with IBD with an AUC value of 0.84 (e.g. 92% sensitivity, 58.5% specificity). Conclusions/Significance: Microbiome-based diagnostics can distinguish pediatric patients with IBD from patients with similar symptoms. Although this test can not replace endoscopy and histological examination as diagnostic tools, classification based on microbial diversity is an effective complementary technique for IBD detection in pediatric patients.Natural Sciences and Engineering Research Council of Canada (Award NSERC PGS D)National Institutes of Health (U.S.) (1-R21-A1084032-01A1