Extensive Unexplored Human Microbiome Diversity Revealed by Over 150,000 Genomes from Metagenomes Spanning Age, Geography, and Lifestyle
E Pasolli, F Asnicar, S Manara, M Zolfo, N Karcher, F Armanini, F Beghini, P Manghi, A Tett, P Ghensi, MC Collado, BL Rice, C DuLong, XC Morgan, CD Golden, C Quince, C Huttenhower, N Segata
Cell, In Press. DOI: 10.1016/j.cell.2019.01.001
The body-wide human microbiome plays a role in health, but its full diversity remains uncharacterized, particularly outside of the gut and in international populations. We leveraged 9,428 metagenomes to reconstruct 154,723 microbial genomes (45% of high quality) spanning body sites, ages, countries, and lifestyles. We recapitulated 4,930 species-level genome bins (SGBs), 77% without genomes in public repositories (unknown SGBs [uSGBs]). uSGBs are prevalent (in 93% of well-assembled samples), expand underrepresented phyla, and are enriched in non-Westernized populations (40% of the total SGBs). We annotated 2.85 M genes in SGBs, many associated with conditions including infant development (94,000) or Westernization (106,000). SGBs and uSGBs permit deeper microbiome analyses and increase the average mappability of metagenomic reads from 67.76% to 87.51% in the gut (median 94.26%) and 65.14% to 82.34% in the mouth. We thus identify thousands of microbial genomes from yet-to-be-named species, expand the pangenomes of human-associated microbes, and allow better exploitation of metagenomic technologies.
Impact of Fermentable Fibres on the Colonic Microbiota Metabolism of Dietary Polyphenols Rutin and Quercetin.
B Mansoorian, E Combet, A Alkhaldy, AL Garcia, CA Edwards
Int J Environ Res Public Health, 16(2), 2019. DOI: 10.3390/ijerph16020292
Dietary fibre and polyphenols are both metabolised to short-chain fatty acids (SCFAs) and phenolic acids (PA) by the colonic microbiota. These may alter microbiota growth/diversity, but their interaction is not understood. Interactions between rutin and raftiline, ispaghula or pectin were investigated in human faecal batch cultures (healthy participants; 19⁻33 years, 4 males, 6 females, BMI 18.4⁻27.4) after a low (poly)phenol diet three days prior to study. Phenolic acids were measured by gas chromatography-mass spectrometry and SCFAs by gas chromatography-flame ionisation after 2, 4, 6, and 24 h. Rutin fermentation produced Phenyl acetic acid (PAA), 4-Hydroxy benzoic acid (4-OHBA), 3-Hydroxy phenyl acetic acid (3-OHPAA), 4-Hydroxy phenyl acetic acid (4-OHPAA), 3,4-Dihydroxy phenyl acetic acid (3,4-diOHPAA), 3-Hydroxy phenyl propionic acid (3-OHPPA), and 4-Hydroxy phenyl propionic acid (4-OHPPA). 3,4-DiOHPAA and 3-OHPAA were predominant at 6 h (1.9 ± 1.8 µg/mL, 2.9 ± 2.5 µg/mL, and 0.05 ± 0.0 µg/mL, respectively) and 24 h (5.5 ± 3.3 µg/mL, 3.1 ± 4.2 µg/mL, and 1.2 ± 1.6 µg/mL). Production of all PA except 3-OHPPA and 4-OHPPA was reduced by at least one fibre. Inhibition of PA was highest for rutin (8-fold, p < 0.01), then pectin (5-fold, p < 0.01), and ispaghula (2-fold, p = 0.03). Neither rutin nor quercetin had a detectable impact on SCFA production. These interactions should be considered when assessing dietary polyphenols and potential health benefits.
Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples
F Cholet, UZ Ijaz, CJ Smith
Environmental Microbiology, In Press, 2019. DOI: 10.1111/1462-2920.14516
Reliability and reproducibility of transcriptomics‐based studies are dependent on RNA integrity. In microbial ecology, microfluidics‐based techniques, such as the Ribosomal Integrity Number (RIN), targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here we present an integrity index, the Ratio Amplicon, Ramp, adapted from human clinical studies, to directly monitor mRNA integrity from complex environmental samples. We show, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of 16S rRNA, amoA and glnA transcripts. We successfully sequenced transcripts for all three targets even from highly‐degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcript profiles. Since both RT‐Q‐PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.
Fecal Microbiota Transplantation for Recurrent Clostridium difficile Infection and Other Conditions in Children
ZH Davidovics, S Michail, MR Nicholson, LK Kociolek, N Pai, R Hansen, T Schwerd, A Maspons, R Shamir, H Szajewska, N Thapar, Tde Meij, A Mosca, Y Vandenplas, SA Kahn, R Kellermayer, and the FMT Special Interest Group of the NASPGHAN, ESPGHAN
Journal of Paediatric Gastroenterology and Nutrition, 68: 130–143, 2018. DOI: 10.1097/MPG.0000000000002205
ABSTRACT Fecal microbiota transplantation (FMT) is becoming part of the treatment algorithms against recurrent Clostridium difficile infection (rCDI) both in adult and pediatric gastroenterology practice. With our increasing recognition of the critical role the microbiome plays in human health and disease, FMT is also being considered as a potential therapy for other disorders, including inflammatory bowel disease (Crohn disease, ulcerative colitis), graft versus host disease, neuropsychiatric diseases, and metabolic syndrome. Controlled trials with FMT for rCDI have not been performed in children, and numerous clinical and regulatory considerations have to be considered when using this untraditional therapy. This report is intended to provide guidance for FMT in the treatment of rCDI in pediatric patients.
Salmonella enterica Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously
A Bravo-Blas, L Utriainen, SL Clay, V Kästele, V Cerovic, AF Cunningham, IR Henderson, DM Wall, SWF Milling
The Journal of Immunology, 202(1): 260–267, 2019. DOI: 10.4049/jimmunol.1701254
Salmonella infection is a globally important cause of gastroenteritis and systemic disease and is a useful tool to study immune responses in the intestine. Although mechanisms leading to immune responses against Salmonella have been extensively studied, questions remain about how bacteria travel from the intestinal mucosa to the mesenteric lymph nodes (MLN), a key site for Ag presentation.
In this study, we used a mouse model of infection with Salmonella enterica serovar Typhimurium (STM) to identify changes in intestinal immune cells induced during early infection. We then used fluorescently labeled STM to identify interactions with immune cells from the site of infection through migration in lymph to the MLN. We show that viable STM can be carried in the lymph by any subset of migrating dendritic cells but not by macrophages. Moreover, approximately half of the STM in lymph are not associated with cells at all and travel autonomously. Within the MLN, STM associates with dendritic cells and B cells but predominantly with MLN-resident macrophages.
In conclusion, we describe the routes used by STM to spread systemically in the period immediately postinfection. This deeper understanding of the infection process could open new avenues for controlling it.
Treatment of Active Crohn’s Disease With an Ordinary Food-based Diet That Replicates Exclusive Enteral Nutrition
V Svolos, R Hansen, B Nichols, C Quince, UZ Ijaz, RT Papadopoulou, CA Edwards, D Watson, A Alghamdi, A Brejnrod, C Ansalone, H Duncan, L Gervais, R Tayler, J Salmond, D Bolognini, R Klopfleisch, DR Gaya, S Milling, RK Russell, K Gerasimidis
Gastroenterology, In Press, DOI: 10.1053/j.gastro.2018.12.002
Background & Aims:
Exclusive enteral nutrition (EEN) is the only established dietary treatment for Crohn’s disease (CD), but its acceptability is limited. There is a need for novel dietary treatments for CD.
Methods:
We evaluated the effects of an individualized, food-based diet (CD-TREAT), with similar composition to EEN, on the gut microbiome, inflammation and clinical response in a rat model,healthy adults, and children with relapsing CD. Twenty-five healthy adults randomly received EEN or CD-TREAT for 7 days, followed by a 14-day washout period, followed by the alternate diet. Fecal microbiome and metabolome were assessed before and after each diet. HLA-B7 and HLA-B27 transgenic rats with gut inflammation received EEN, CD-TREAT, or standard chow for 4 weeks. Fecal, luminal and tissue microbiome, fecal metabolites and gut inflammation were assessed. Five children received CD-TREAT with clinical activity and fecal calprotectin evaluated after 8-week treatment.
Results:
Among healthy adults, CD-TREAT was easier to comply with and more acceptable than EEN. CD-TREAT induced similar effects to EEN (EEN vs CD-TREAT) on fecal microbiome composition, metabolome, mean total sulfide (increase 133.0±80.5 vs 54.3±47.0 nmol/g), pH (increase 1.3±0.5 vs 0.9±0.6), the short-chain fatty acids (µmol/g) acetate (decrease 27.4±22.6 vs 21.6±20.4), propionate (decrease 5.7±7.8 vs 5.2±7.9), and butyrate (decrease 7.0±7.4 vs 10.2±8.5). In the rat model, CD-TREAT and EEN produced similar changes in bacterial load (decrease 0.3±0.3 log10 16S rRNA gene copies/g), short-chain fatty acids, microbiome, and ileitis severity (meanhistopathology score reductions 1.25 for EEN (P=.015) and 1.0 for CD-TREAT (P=.044) vs chow).Among the children receiving CD-TREAT, 4 (80%) had a clinical response and 3 (60%) enteredremission, with significant concurrent reductions in fecal calprotectin (mean decrease 918±555 mg/kg, (P=.002)).
Conclusion:
CD-TREAT replicates EEN changes in the microbiome, reduces gut inflammation, is well-tolerated and is potentially effective in patients with active CD.
Eating disorder or disordered eating: undiagnosed inflammatory bowel disease mimicking eating disorder
RE Harris, R Tayler, RK Russell
Archives of Disease in Childhood, 316324, 2018. DOI: 10.1136/archdischild-2018-316324
Impact of frequency of denture cleaning on microbial and clinical parameters – a bench to chairside approach
G Ramage, L O’Donnell, L Sherry, S Culshaw, J Bagg, M Czesnikiewicz-Guzik, C Brown, D McKenzie, L Cross, A MacInnes, D Bradshaw, R Varghese, PG Pereira, A Jose, S Sanyal, D Robertsona
Journal of Oral Microbiology, 11(1): 1538437, 2019. DOI: 10.1080/20002297.2018.1538437
Objective:
Robust scientific and clinical evidence of how to appropriately manage denture plaque is lacking. This two-part study (i) developed an in vitro model of denture plaque removal, and (ii) assessed effectiveness of these approaches in a randomised clinical trial.
Method:
(i) a complex denture plaque model was developed using the dominant microbial genera from a recent microbiome analyses. Biofilms formed on polymethylmethacrylate were brushed daily with a wet toothbrush, then either treated daily for 5 days or only on Days 1 and 5 with Polident® denture cleanser tablets (3 min soaking). Quantitative and qualitative microbiological assessments were performed. (ii), an examiner-blind, randomised, crossover study of complete maxillary denture wearers was performed (n = 19). Either once-daily for 7 days or on Day 7 only, participants soaked dentures for 15 min using Corega® denture cleansing tables, then brushed. Denture plaque microbiological assessment used sterilized filter paper discs.
Results:
The in vitro model showed daily cleaning with denture cleanser plus brushing significantly reduced microbial numbers compared to intermittent denture cleaning with daily brushing (p < 0.001). The clinical component of the study showed a statistically significant reduction in denture plaque microbial numbers in favour of daily versus weekly treatment (aerobic bacteria p = 0.0144). Both in vitro and in vivo studies showed that denture plaque biofilm composition were affected by different treatment arms.
Conclusions:
This study demonstrated that daily denture cleansing regimens are superior to intermittent denture cleansing, and that cleansing regimens can induce denture plaque compositional changes. Clinicaltrials.gov registration: NCT02780661.
Skin staining due to intravenous iron extravasation in a teenager with Crohn’s disease
RE Harris, V Garrick, L Curtis, RK Russell
Archives of Disease in Childhood, 316405, 2018. DOI: 10.1136/archdischild-2018-316405
Metabolomic profiling using high resolution mass spectrometry with hydrophilic interaction chromatography was applied to 11 faecal extracts from eleven healthy children and to 43 faecal extracts from eleven children undergoing exclusive enteral nutrition for the treatment of active Crohn’s disease (CD) at timepoints before, during (15, 30, and 60 days), and after treatment. Differences between the control and CD samples were identified at each timepoint. An orthogonal partial least square-discriminant analysis (OPLS-DA) model identified eight metabolites that were normally distributed according to Q-Q plots. The OPLS-DA model was able to discriminate the CD samples from the controls at every timepoint, but the model was not able to differentiate the CD samples from one another at the different timepoints during treatment with exclusive enteral nutrition. The differentiated metabolites identified in the CD samples included tyrosine, an ornithine isomer, arachidonic acid, eicosatrienoic acid, docosatetraenoic acid, a sphingomyelin, a ceramide, and dimethylsphinganine. Despite successful treatment, underlying differences remained in the metabolome of the CD patients. These differences dominated the separation of the samples when multivariate methods were applied.
Response to treatment is more important than disease severity at diagnosis for prediction of early relapse in new‐onset paediatric Crohn’s disease
T Ziv‐Baran, S Hussey, M Sladek, JA Dias, JM Carpi-Erasmo-Miele, G Veres, P Lionetti, S Koletzko, F Nuti, A Paerregaard, KL Kolho, RK Russell, R Shaoul, D Weiner, RS Boneh, J Escher, L, Finnby, D Turner, A Levine
Alimentary Pharmacology and Therapeutics, 48:1242–1250, 2018. DOI: 10.1111/apt.15016
Background
Paediatric Crohn's disease is characteried by frequently relapsing disease which may lead to hospitalisations and complications.
Aim
To develop predictive models for early relapse following first remission.
Methods
The GROWTH CD prospective inception cohort was designed to predict risk for early disease relapse and poor outcomes. Newly diagnosed children underwent endoscopies and imaging. They were phenotyped and followed at scheduled visits through 78 weeks for relapses. Twenty‐eight dichotomous and continuous variables were assessed at baseline and week 12, including phenotype, inflammatory markers, disease activity (PCDAI) and other markers. Clinical relapses defined as PCDAI >10 after remission were recorded using a relapse form. Logistic regression & risk modelling was performed.
Results
We enrolled 282 eligible patients of whom 178 (63.6%) patients achieved steroid free remission by week 12. Disease complications developed in 22/76(29%) of patients with relapse compared to 20/206 (9.7%) without relapse (P = 0.01). Multivariable analysis demonstrated that while variables from age/gender at diagnosis were not predictive, week 12 variables including PCDAI >5 (P = 0.02), CRP >20 mg/L (P = 0.02), and faecal calprotectin >400 µg/g (P = 0.03) as optimal cut‐offs were associated with increased risk of relapse. A prediction model for patients in remission including gender, age, week 12 PCDAI, calprotectin and CRP had sensitivity 43%, specificity 92%, PPV 78%, NPV 71% for relapse.
Conclusions
Early relapses were associated with a higher risk for disease complications at followup. Relapse prediction based on week 12 disease activity or inflammation is superior to prediction using data from diagnosis.
Comprehensive Longitudinal Microbiome Analysis of the Chicken Cecum Reveals a Shift From Competitive to Environmental Drivers and a Window of Opportunity for Campylobacter
UZ Ijaz, L Sivaloganathan, A McKenna, A Richmond, C Kelly, M Linton, AC Stratakos, U Lavery, A Elmi, BW Wren, N Dorrell, N Corcionivoschi, O Gundogdu2
Frontiers in Biology, 9: 2452, 2018. DOI: 10.3389/fmicb.2018.02452
Chickens are a key food source for humans yet their microbiome contains bacteria that can be pathogenic to humans, and indeed potentially to chickens themselves. Campylobacter is present within the chicken gut and is the leading cause of bacterial foodborne gastroenteritis within humans worldwide. Infection can lead to secondary sequelae such as Guillain-Barré syndrome and stunted growth in children from low-resource areas.
Despite the global health impact and economic burden of Campylobacter, how and when Campylobacter appears within chickens remains unclear. The lack of day to day microbiome data with replicates, relevant metadata, and a lack of natural infection studies have delayed our understanding of the chicken gut microbiome and Campylobacter.
Here, we performed a comprehensive day to day microbiome analysis of the chicken cecum from day 3 to 35 (12 replicates each day; final n = 379). We combined metadata such as chicken weight and feed conversion rates to investigate what the driving forces are for the microbial changes within the chicken gut over time, and how this relates to Campylobacter appearance within a natural habitat setting. We found a rapidly increasing microbial diversity up to day 12 with variation observed both in terms of genera and abundance, before a stabilization of the microbial diversity after day 20. In particular, we identified a shift from competitive to environmental drivers of microbial community from days 12 to 20 creating a window of opportunity whereby Campylobacter can appear. Campylobacter was identified at day 16 which was 1 day after the most substantial changes in metabolic profiles observed. In addition, microbial variation over time is most likely influenced by the diet of the chickens whereby significant shifts in OTU abundances and beta dispersion of samples often corresponded with changes in feed.
This study is unique in comparison to the most recent studies as neither sampling was sporadic nor Campylobacter was artificially introduced, thus the experiments were performed in a natural setting. We believe that our findings can be useful for future intervention strategies and help reduce the burden of Campylobacter within the food chain.
Patients with inflammatory bowel disease have higher abdominal adiposity and less skeletal mass than healthy controls
T Barroso, F Conway, S Emel, D McMillan, D Young, H Karteszi, DR Gaya, K Gerasimidis
Annals of gastroenterology,31(5): 566–571, 2018. DOI: 10.20524/aog.2018.0280
Background
Abdominal fat type and distribution have been associated with complicated Crohn’s disease and adverse postoperative outcomes. Few studies have assessed the abdominal distribution of fat and lean stores in patients with inflammatory bowel disease (IBD) and compared this with healthy controls. This retrospective study aimed to compare the abdominal body composition in IBD patients who failed medical treatment and who underwent computed tomography (CT) imaging prior to gastrointestinal surgery with healthy controls. Associations between preoperative abdominal body composition and postoperative outcomes within a year of surgery were explored.
Methods
Abdominal body composition was evaluated in 22 presurgical patients with medically refractory IBD (18 with Crohn’s disease) and 22 healthy controls, using routinely acquired CT. Total fat, subcutaneous fat, visceral fat, and skeletal muscle cross-sectional area were measured.
Results
An independent disease effect was observed, explaining a fat deposition excess of 38 cm2 and a skeletal muscle deficit of 15 cm2 in IBD. Abdominal skeletal muscle correlated with visceral fat for the control (rho=0.51, P=0.015), but not for the IBD group (rho=-0.13, P=0.553). A positive correlation observed between subcutaneous fat with skeletal muscle in the controls (rho=0.47, P=0.026) was inverted in the IBD group (rho=-0.43, P=0.045). Preoperative abdominal body composition was not predictive of postoperative outcomes.
Conclusions
A higher degree of abdominal adiposity, a lower skeletal mass and a larger body size for the same anthropometry can be expected in IBD patients. Preoperative abdominal body composition is not associated with surgical outcomes.
A multicentre development and evaluation of a dietetic referral score for nutritional risk in sick infants
K Gerasimidis, S Milani, A Tester, O Purcell, C Woodley, M Tsiountsioura, A Koulieri, O Zerva, K Loizou, M Rafeey, M Kontogianni, C Wright
Clinical Nutrition, In Press, 2018. DOI: 10.1016/j.clnu.2018.08.017
Background & aims
Unrecognized nutritional issues may delay recovery in hospitalized infants. It has been proposed that nutritional risk screening should be performed at hospital admission, but few tools include infants. The aim of this study was to develop and test a tool to identify sick infants in need of dietetic input.
Methods
Hospitalised infants were recruited from hospitals in the United Kingdom (UK), Greece and Iran. Weight, skinfold thickness and mid upper arm circumference (MUAC) were measured, with detailed dietetic assessment in the UK and Greece. Simple screening questions were used in the UK cohort to formulate a score (infant early nutrition warning score-iNEWS) which was then validated in the Greek and Iranian groups.
Results
After dietetic assessment, 20 (9.6%) UK and 22 (22%) Greek infants were rated as needing dietetic input. Underweight, poor weight gain/loss and reduced intake were all independent predictors of perceived need for dietetic input in stepwise multivariate regression analysis. The score based on these items (iNEWS), had 84% sensitivity, 91% specificity and 49% positive predictive value to predict need for dietetic input in the UK cohort. In the Greek cohort this was 86%, 78% and 53% respectively. In all three countries, infants with high iNEWS had significantly lower average skinfold thickness (between −1 and −1.8 SD, p < 0.0001) and MUAC (between −1.8 and −2 SD, p < 0.0001) than those at low risk.
Conclusions
iNEWS, a simple nutritional risk tool, identifies most hospitalised infants who need dietetic input.
Use of Biosimilars in Paediatric Inflammatory Bowel Disease: An Updated Position Statement of the Paediatric IBD Porto Group of ESPGHAN
L De Ridder, A Assa, J Bronsky, C Romano, RK Russell, NA Afzal, C Hauer, D Knafelz, P Lionetti, C Strisciuglio, G Veres, H Winter, VM Wolters, M Sladek, AG Vulto, JA Dias
Journal of Pediatric Gastroenterology and Nutrition, In Press, 2018. DOI: 10.1097/MPG.0000000000002141
Biologic therapies have changed the outcome of both adult and pediatric patients with Inflammatory Bowel Disease (IBD). In September 2013, the first biosimilar of infliximab was introduced into the pharmaceutical market. In 2015, a first position paper on the use of biosimilars in pediatric IBD was published by the ESPGHAN IBD Porto group. Since then, more data have accumulated for both adults and children demonstrating biosimilars are an effective and safe alternative to the originator. In this updated position statement, we summarize current evidence and provide joint consensus statements regarding the recommended practice of biosimilar use in children with IBD.
Effects of Recombinant Human Growth Hormone in Children with Crohn’s Disease on the Muscle-Bone Unit: A Preliminary Study
MA Altowati, S Shepherd, P McGrogan, RK Russell, SF Ahmed, SC Wong
Hormone Research in Paediatrics, 90 (2) pp: 1-4 2018. DOI: 10.1159/000492398
Background/Aims:
There is limited information on the impact of recombinant human growth hormone (rhGH) on the muscle-bone unit in children with Crohn’s disease (CD). In this pilot study, we report on the effects of rhGH on bone formation, dual-energy X-ray absorptiometry (DXA) total body (TB) bone mineral density adjusted for height and lumbar spine (LS) bone mineral apparent density (BMAD), and body composition.
Methods:
Prospective study of 8 children with CD (6 male), aged 14.8 years (9.0–16.4), who received rhGH for 24 months. Serum procollagen type 1 N-terminal propeptide (P1NP) was measured at baseline and at 6 months. DXA was performed every 6 months.
Results:
Six months of rhGH led to improvement in P1NP SDS adjusted for bone age from –3.6 (–7.9 to –0.9) to –2.4 (–3.7 to 0.4) (p = 0.01). At baseline, reduction in LS-BMAD and TB lean mass SDS was observed being –1.2 (–3.6 to 0.8) (p = 0.01 vs. zero) and –0.8 (–2.4 to 3.0) (p = 0.11 vs. zero), respectively. No significant changes were seen in DXA bone and muscle parameters over the 24 months.
Conclusion:
Twenty-four months of therapy with rhGH in CD did not lead to an improvement in DXA BMD and lean mass, despite improvement in P1NP and linear growth
Diversity converges during community assembly in methanogenic granules, suggesting a biofilm life-cycle
A. C. Trego, C. Morabito, S. Mills, S. Connelly, I. Bourven, G. Guibaud, C. Quince, U. Z. Ijaz, G. Collins
bioRxiv, preprint. DOI: 10.1101/484642
Anaerobic biological decomposition of organic matter is ubiquitous in nature wherever anaerobic environments prevail, and is catalysed by hydrolytic, fermentative, acetogenic, methanogenic, and various other groups, including syntrophic bacteria. It is also harnessed in innovative ways in engineered systems that may rely on small (0.1-4.0 mm), spherical, anaerobic granules, which we have found to be highly-replicated, whole-ecosystems harbouring the entire community necessary to mineralise complex organics.
We hypothesised distinct granule sizes correspond to stages in a biofilm life-cycle, in which small granules are young and larger ones are old. Here, granules were separated into 10 size fractions used for physico-chemical and ecological characterisation. Gradients of volatile solids, density, settleability, biofilm morphology, methanogenic activity, and EPS profiles were observed across size fractions.
Sequencing of 16S rRNA genes indicated linear convergence of diversity during community assembly as granules increased in size. A total of 155 discriminant OTUs were identified, and correlated strongly with physico-chemical parameters. Community assembly across sizes was influenced by a niche effect, whereby Euryarchaeota dominated a core microbiome presumably as granules became more anaerobic. The findings indicate opportunities for precision management of environmental biotechnologies, and the potential of aggregates as playgrounds to study assembly and succession in whole microbiomes.
NanoAmpli-Seq: a workflow for amplicon sequencing for mixed microbial communities on the nanopore sequencing platform
ST Calus, UZ Ijaz, AJ Pinto
bioRxiv, Volume 7, Issue 12, December 2018. DOI: 10.1093/gigascience/giy140
Background
Amplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences sequencing platforms overcome this limitation, their application has been limited due to higher error rates or lower data output.
Results
In this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on the intramolecular-ligated nanopore consensus sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the INC-Seq protocol that reduces sample processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain accurate full-length 16S rRNA gene sequences.
Conclusions
NanoAmpli-Seq accurately estimates the diversity of tested mock communities with average consensus sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware base calling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.
Binning metagenomic contigs by coverage and composition
J Alneberg, B Smári Bjarnason, I de Bruijn, M Schirmer, J Quick, UZ Ijaz, L Lahti, NJ Loman, AF Andersson, C Quince
Nature Methods, 11, pages 1144–1146; 2014. DOI: 10.1038/nmeth.3103
Shotgun sequencing enables the reconstruction of genomes from complex microbial communities, but because assembly does not reconstruct entire genomes, it is necessary to bin genome fragments. Here we present CONCOCT, a new algorithm that combines sequence composition and coverage across multiple samples, to automatically cluster contigs into genomes. We demonstrate high recall and precision on artificial as well as real human gut metagenome data sets.
The distinct features of microbial ‘dysbiosis’ of Crohn’s disease do not occur to the same extent in their unaffected, genetically-linked kindred
UZ Ijaz, C Quince, L Hanske, N Loman, ST Calus, M Bertz, CA Edwards, DR Gaya, R Hansen, P McGrogan, RK Russell, K Gerasimidis
PLoS ONE, 12(2); 2017. DOI: 10.1371/journal.pone.0172605
Background/Aims
Studying the gut microbiota in unaffected relatives of people with Crohn’s disease (CD) may advance our understanding of the role of bacteria in disease aetiology.
Methods
Faecal microbiota composition (16S rRNA gene sequencing), genetic functional capacity (shotgun metagenomics) and faecal short chain fatty acids (SCFA) were compared in unaffected adult relatives of CD children (CDR, n = 17) and adult healthy controls, unrelated to CD patients (HUC, n = 14). The microbiota characteristics of 19 CD children were used as a benchmark of CD ‘dysbiosis’.
Results
The CDR microbiota was less diverse (p = 0.044) than that of the HUC group. Local contribution of β-diversity analysis showed no difference in community structure between the CDR and HUC groups. Twenty one of 1,243 (1.8%) operational taxonomic units discriminated CDR from HUC. The metagenomic functional capacity (p = 0.207) and SCFA concentration or pattern were similar between CDR and HUC (p>0.05 for all SCFA). None of the KEGG metabolic pathways were different between these two groups. Both of these groups (HUC and CDR) had a higher microbiota α-diversity (CDR, p = 0.026 and HUC, p<0.001) with a community structure (β-diversity) distinct from that of children with CD.
Conclusions
While some alterations were observed, a distinct microbial ‘dysbiosis’, characteristic of CD patients, was not observed in their unaffected, genetically linked kindred.
Comparison of the human gastric microbiota in hypochlorhydric states arising as a result of Helicobacter pylori-induced atrophic gastritis, autoimmune atrophic gastritis and proton pump inhibitor use
BN Parsons, UZ Ijaz, R D’Amore, MD Burkitt, R Eccles, L Lenzi, CA Duckworth, AR Moore, L Tiszlavicz, A Varro, N Hall, D. Mark Pritchard
PLoS Pathogens, 13(11): e1006653; 2017. DOI: 10.1371/journal.ppat.1006653
Several conditions associated with reduced gastric acid secretion confer an altered risk of developing a gastric malignancy. Helicobacter pylori-induced atrophic gastritis predisposes to gastric adenocarcinoma, autoimmune atrophic gastritis is a precursor of type I gastric neuroendocrine tumours, whereas proton pump inhibitor (PPI) use does not affect stomach cancer risk.
We hypothesised that each of these conditions was associated with specific alterations in the gastric microbiota and that this influenced subsequent tumour risk. 95 patients (in groups representing normal stomach, PPI treated, H. pylori gastritis, H. pylori-induced atrophic gastritis and autoimmune atrophic gastritis) were selected from a cohort of 1400. RNA extracted from gastric corpus biopsies was analysed using 16S rRNA sequencing (MiSeq). Samples from normal stomachs and patients treated with PPIs demonstrated similarly high microbial diversity. Patients with autoimmune atrophic gastritis also exhibited relatively high microbial diversity, but with samples dominated by Streptococcus.
H. pylori colonisation was associated with decreased microbial diversity and reduced complexity of co-occurrence networks. H. pylori-induced atrophic gastritis resulted in lower bacterial abundances and diversity, whereas autoimmune atrophic gastritis resulted in greater bacterial abundance and equally high diversity compared to normal stomachs. Pathway analysis suggested that glucose-6-phospahte1-dehydrogenase and D-lactate dehydrogenase were over represented in H. pylori-induced atrophic gastritis versus autoimmune atrophic gastritis, and that both these groups showed increases in fumarate reductase. Autoimmune and H. pylori-induced atrophic gastritis were associated with different gastric microbial profiles. PPI treated patients showed relatively few alterations in the gastric microbiota compared to healthy subjects.
Host–microbe interactions have shaped the genetic architecture of inflammatory bowel disease
L Jostins, S Ripke, RK Weersma, et al
Nature, 491, 119–124; 2012. DOI: 10.1038/nature11582
Crohn’s disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn’s disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.
Current clinical trials in paediatrics: Report of the ESPEN special interest group in paediatrics
RD Eveleens, K Gerasimidis, M Chourdakis, K Huysentruyt, B Koletzko, JM Hulst, KFM Joosten
Clinical Nutrition ESPEN, 27 pp: 75-78, 2018. DOI: 10.1016/j.clnesp.2018.06.006
Background & aims
At the 38th annual ESPEN congress in The Hague, the Netherlands, the Special Interest Group (SIG) in Paediatrics presented data about current research activities in the field of paediatric nutrition which are performed worldwide and translated this to future research perspectives.
Methods
Extensive search of all registered observational and interventional clinical trials in the database ClinicalTrials.gov using the search terms: children nutrition, paediatrics nutrition and children feeding.
Results
A total of 717 studies were found; 173 were duplicates and 114 included adult participants and were therefore excluded. Hence, 430 remained for analysis, of which 69% were randomized controlled trials. The most investigated research topic was nutrition in specific diseases (n = 98), followed by obesity (n = 92), and studies including premature infants (n = 48). The overall median estimated enrolment of children in the trials was 150 children [IQR 50–365]. There were 44 studies in which >1000 participants will be enrolled and six studies with >10,000 participants. Studies including >1000 participants were primarily performed in North America (39%), Africa (27%), and Europe (16%).
Conclusions
This SIG report showed that 430 clinical nutrition trials in paediatrics are registered and current research focusses primarily on specific diseases and obesity. The SIG paediatrics encourages future research to invest in well-controlled interventional trials.
Evaluating Streptococcus mutans Strain Dependent Characteristics in a Polymicrobial Biofilm Community
Y Zhou, E Millhouse, T Shaw, DF Lappin, R Rajendran, J Bagg, H Lin, G Ramage
Frontiers in Microbiology, 9 pp: 1498, 2018. DOI: 10.3389/fmicb.2018.01498
Aim:
The purpose of this study was to investigate strain dependent differences of the cariogenic biofilm forming Streptococcus mutans within both simple and complex communities.
Methods:
A mono-species containing representative S. mutans clinical isolates (caries and non-caries), and a multispecies in vitro caries biofilm model containing Lactobacillus casei, Veillonella dispar, Fusobacterium nucleatum and Actinomyces naeslundii, and either of two representative S. mutans clinical isolates (caries and non-caries), was developed as a comparison model. Compositional analysis of total and live bacteria within biofilms, and transcriptional analysis of biofilm associated virulence factors were evaluated by live/dead PCR and quantitative PCR, respectively. Scanning electron microscopy (SEM) was used to analyze the architecture of biofilm. One-way analysis of variance and t-tests were used to investigate significant differences between independent groups of data.
Results:
Within a mono-species biofilm, different S. mutans strains responded similarly to one another during biofilm formation in different carbohydrate sources, with sucrose showing the highest levels of biofilm biomass and galactose showing the lowest. Within the polymicrobial biofilm system, compositional analysis of the bacteria within the biofilm showed that S. mutans derived from a caries-free patient was preferentially composed of both total and viable L. casei, whereas S. mutans derived from a caries patient was dominated by both total and viable S. mutans (p < 0.001). Normalized gene expression analysis of srtA, gtfB, ftf, spaP, gbpB, and luxS, showed a general upregulation within the S. mutans dominant biofilm.
Conclusion:
We were able to demonstrate that individual strains derived from different patients exhibited altered biofilm characteristics, which were not obvious within a simple mono-species biofilm model. Influencing the environmental conditions changed the composition and functionality S. mutans within the polymicrobial biofilm. The biofilm model described herein provides a novel and reproducible method of assessing the impact on the biofilm microbiome upon different environmental influences.
The Gut Microbiome in Patients with Intestinal Failure: Current Evidence and Implications for Clinical Practice
E Neelis, B de Koning, E Rings, R Wijnen, B Nichols, J Hulst, K Gerasimidis
Journal of Parenteral and Enteral Nutrition, In Press, 2018. DOI: 10.1002/jpen.1423
Intestinal failure (IF) is the reduction of gut function or mass below a minimum needed to absorb nutrients and fluids, such that patients are dependent on parenteral nutrition (PN). Patients with IF have an altered gut microbiome. Our aim was to review and evaluate the current evidence on gut microbiome and its metabolic activity, as well as its association with disease characteristics in adults and children with IF. We performed a PubMed literature search for articles published after 2000 using the following terms: intestinal, microbiome, microbiota, short‐chain fatty acids, short bowel syndrome, and PN. Literature search was restricted to human studies only. The gut microbiome diversity is remarkably reduced, and community structure is altered with a noticeable overabundance of Proteobacteria, especially the Enterobacteriaceae family. A substantial increase in Lactobacillus level is often reported in patients with IF. Gut microbiome characteristics have been associated with poor growth, liver disease, D‐lactic acidosis, and duration of intestinal adaptation. Differences in microbiome characteristics have been found between patients receiving PN and those whose guts have adapted and have been weaned off PN. Future research with prospective sample collection should explore the value of the gut microbiome as a biomarker to guide clinical practice and as a modifiable therapeutic target to optimize outcomes of patients with IF.
An automated identification and analysis of ontological terms in gastrointestinal diseases and nutrition-related literature provides useful insights
O Koci, M Logan, V Svolos, RK Russell, K Gerasimidis, UZ Ijaz
PeerJ, 6 pp: e5047, 2018 . DOI: 10.7717/peerj.5047
With an unprecedented growth in the biomedical literature, keeping up to date with the new developments presents an immense challenge. Publications are often studied in isolation of the established literature, with interpretation being subjective and often introducing human bias. With ontology-driven annotation of biomedical data gaining popularity in recent years and online databases offering metatags with rich textual information, it is now possible to automatically text-mine ontological terms and complement the laborious task of manual management, interpretation, and analysis of the accumulated literature with downstream statistical analysis. In this paper, we have formulated an automated workflow through which we have identified ontological information, including nutrition-related terms in PubMed abstracts (from 1991 to 2016) for two main types of Inflammatory Bowel Diseases: Crohn’s Disease and Ulcerative Colitis; and two other gastrointestinal (GI) diseases, namely, Coeliac Disease and Irritable Bowel Syndrome. Our analysis reveals unique clustering patterns as well as spatial and temporal trends inherent to the considered GI diseases in terms of literature that has been accumulated so far. Although automated interpretation cannot replace human judgement, the developed workflow shows promising results and can be a useful tool in systematic literature reviews. The workflow is available at https://github.com/KociOrges/pytag.
Management of Paediatric Ulcerative Colitis, Part 2: Acute Severe Colitis—An Evidence-based Consensus Guideline From the European Crohn's and Colitis Organization and the European Society of Paediatric Gastroenterology, Hepatology and Nutrition
D Turner, FM Ruemmele, E Orlanski-Meyer, AM Griffiths, JM Carpi, J Bronsky, G Veres, M Aloi, C Strisciuglio, CP Braegger, A Assa, C Romano, S Hussey, M Stanton, M Pakarinen, L de Ridder, Lissy, KH Katsanos, NM Croft, VM Navas-Lopez, Victor Manuel, DC Wilson, S Lawrence, RK Russell
Journal of Pediatric Gastroenterology and Nutrition, 67(2):292–310, 2018. DOI: 10.1097/MPG.0000000000002036
Background and aim:
Acute severe colitis (ASC) is one of the few emergencies in pediatric gastroenterology. Tight monitoring and timely medical and surgical interventions may improve outcomes and minimize morbidity and mortality. We aimed to standardize daily treatment of ASC in children through detailed recommendations and practice points which are based on a systematic review of the literature and consensus of experts.
Methods:
These guidelines are a joint effort of the European Crohn's and Colitis Organization (ECCO) and the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN). Fifteen predefined questions were addressed by working subgroups. An iterative consensus process, including 2 face-to-face meetings, was followed by voting of the national representatives of ECCO and all members of the Paediatric Inflammatory Bowel Disease (IBD) Porto group of ESPGHAN (43 voting experts).
Results:
A total of 24 recommendations and 43 practice points were endorsed with a consensus rate of at least 91% regarding diagnosis, monitoring, and management of ASC in children. A summary flowchart is presented based on daily scoring of the Paediatric Ulcerative Colitis Activity Index. Several topics have been altered since the previous 2011 guidelines and from those published in adults.
Discussion:
These guidelines standardize the management of ASC in children in an attempt to optimize outcomes of this intensive clinical scenario.
Management of Paediatric Ulcerative Colitis, Part 1: Ambulatory Care-An Evidence-based Guideline From European Crohn's and Colitis Organization and European Society of Paediatric Gastroenterology, Hepatology and Nutrition
D Turner, FM Ruemmele, E Orlanski-Meyer, AM Griffiths, JM Carpi, J Bronsky, G Veres, M Aloi, C Strisciuglio, CP Braegger, A Assa, C Romano, S Hussey, M Stanton, M Pakarinen, L de Ridder, Lissy, KH Katsanos, NM Croft, VM Navas-Lopez, Victor Manuel, DC Wilson, S Lawrence, RK Russell
Journal of Pediatric Gastroenterology and Nutrition, 67(2):257–291, 2018. DOI: 10.1097/MPG.0000000000002035
BACKGROUND:
The contemporary management of ambulatory ulcerative colitis (UC) continues to be challenging with ∼20% of children needing a colectomy within childhood years. We thus aimed to standardize daily treatment of paediatric UC and inflammatory bowel diseases (IBD)-unclassified through detailed recommendations and practice points.
METHODS:
These guidelines are a joint effort of the European Crohn's and Colitis Organization (ECCO) and the Paediatric IBD Porto group of European Society of Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN). An extensive literature search with subsequent evidence appraisal using robust methodology was performed before two face-to-face meetings. All 40 included recommendations and 86 practice points, were endorsed by 43 experts in Paediatric IBD with at least an 88% consensus rate.
RESULTS:
These guidelines centre on initial use of mesalamine (including topical), before using steroids, thiopurines and, for more severe disease, anti-TNF. The use of other emerging therapies and the role of surgery are also covered. Algorithms are provided to aid therapeutic decision making based on clinical assessment and the paediatric UC activity index (PUCAI). Advice on contemporary therapeutic targets incorporating the use of calprotectin and the role of therapeutic drug monitoring are presented, as well as other management considerations around pouchitis, extraintestinal manifestations, nutrition, growth, psychology and transition. A brief section on disease classification using the PIBD-classes criteria and IBDU is also part of these guidelines.
CONCLUSION:
These guidelines provide a guide to clinicians managing children with UC and IBDU to provide modern management strategies while maintaining vigilance around appropriate outcomes and safety issues.
Transcriptome Assembly and Profiling of Candida auris Reveals Novel Insights into Biofilm-Mediated Resistance
R Kean, C Delaney, L Sherry, A Borman, EM Johnson, MD Richardson, R Rautemaa-Richardson, C Williams, G Ramage
mSphere, 3(4): e00334-18, 2018. DOI: 10.1128/mSphere.00334-18
Candida auris has emerged as a significant global nosocomial pathogen. This is primarily due to its antifungal resistance profile but also its capacity to form adherent biofilm communities on a range of clinically important substrates. While we have a comprehensive understanding of how other Candida species resist and respond to antifungal challenge within the sessile phenotype, our current understanding of C. auris biofilm-mediated resistance is lacking. In this study, we are the first to perform transcriptomic analysis of temporally developing C. auris biofilms, which were shown to exhibit phase- and antifungal class-dependent resistance profiles. A de nov transcriptome assembly was performed, where sequenced sample reads were assembled into an ~11.5-Mb transcriptome consisting of 5,848 genes. Differential expression (DE) analysis demonstrated that 791 and 464 genes were upregulated in biofilm formation and planktonic cells, respectively, with a minimum 2-fold change. Adhesin-related glycosylphosphatidylinositol (GPI)-anchored cell wall genes were upregulated at all time points of biofilm formation. As the biofilm developed into intermediate and mature stages, a number of genes encoding efflux pumps were upregulated, including ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. When we assessed efflux pump activity biochemically, biofilm efflux was greater than that of planktonic cells at 12 and 24 h. When these were inhibited, fluconazole sensitivity was enhanced 4- to 16-fold. This study demonstrates the importance of efflux-mediated resistance within complex C. auris communities and may explain the resistance of C. auris to a range of antimicrobial agents within the hospital environment.
Optimized R functions for analysis of ecological community data using the R virtual laboratory (RvLab)
C Varsos, T Patkos, A Oulas, C Pavloudi, A Gougousis, UZ Ijaz, I Filiopoulou, N Pattakos, EV Berghe, A Fernandez-Guerra, S Faulwetter, E Chatzinikolaou, E Pafilis, C Beikiari, M Doerr, and C Arvantidis
Biodiversity Data Journal, 4:e8357, 2016. DOI:10.3897/BDJ.4.e8357
Accompanying Software: RvLab
Background
Parallel data manipulation using R has previously been addressed by members of the R community, however most of these studies produce ad hoc solutions that are not readily available to the average R user. Our targeted users, ranging from the expert ecologist/microbiologists to computational biologists, often experience difficulties in finding optimal ways to exploit the full capacity of their computational resources. In addition, improving performance of commonly used R scripts becomes increasingly difficult especially with large datasets. Furthermore, the implementations described here can be of significant interest to expert bioinformaticians or R developers. Therefore, our goals can be summarized as: (i) description of a complete methodology for the analysis of large datasets by combining capabilities of diverse R packages, (ii) presentation of their application through a virtual R laboratory (RvLab) that makes execution of complex functions and visualization of results easy and readily available to the end-user.
New information
In this paper, the novelty stems from implementations of parallel methodologies which rely on the processing of data on different levels of abstraction and the availability of these processes through an integrated portal. Parallel implementation R packages, such as the pbdMPI (Programming with Big Data – Interface to MPI) package, are used to implement Single Program Multiple Data (SPMD) parallelization on primitive mathematical operations, allowing for interplay with functions of the vegan package. The dplyr and RPostgreSQL R packages are further integrated offering connections to dataframe like objects (databases) as secondary storage solutions whenever memory demands exceed available RAM resources.
Host-microbiome interactions in human type 2 diabetes following prebiotic dietary fibre (galacto-oligosaccharide) intake
C Pedersen, E Gallagher, F Horton, RJ Ellis, UZ Ijaz, H Wu, E Jaiyeola, O Diribe, T Duparc, PD Cani, GR Gibson, P Hinton, J Wright, D Russell-Jones, R La Ragione, and MD Robertson
British Journal of Nutrition, 116(11):1869-1877, 2016. DOI: 10.1017/S0007114516004086
Aberrant microbiota composition and function have been linked to several pathologies, including type 2 diabetes. In animal models, prebiotics induce favourable changes in the intestinal microbiota, intestinal permeability (IP) and endotoxaemia, which are linked to concurrent improvement in glucose tolerance.
This is the first study to investigate the link between IP, glucose tolerance and intestinal bacteria in human type 2 diabetes. In all, twenty-nine men with well-controlled type 2 diabetes were randomised to a prebiotic (galacto-oligosaccharide mixture) or placebo (maltodextrin) supplement (5·5 g/d for 12 weeks). Intestinal microbial community structure, IP, endotoxaemia, inflammatory markers and glucose tolerance were assessed at baseline and post intervention. IP was estimated by the urinary recovery of oral 51Cr-EDTA and glucose tolerance by insulin-modified intravenous glucose tolerance test.
Intestinal microbial community analysis was performed by high-throughput next-generation sequencing of 16S rRNA amplicons and quantitative PCR. Prebiotic fibre supplementation had no significant effects on clinical outcomes or bacterial abundances compared with placebo; however, changes in the bacterial family Veillonellaceae correlated inversely with changes in glucose response and IL-6 levels (r −0·90, P=0·042 for both) following prebiotic intake. The absence of significant changes to the microbial community structure at a prebiotic dosage/length of supplementation shown to be effective in healthy individuals is an important finding.
We propose that concurrent metformin treatment and the high heterogeneity of human type 2 diabetes may have played a significant role. The current study does not provide evidence for the role of prebiotics in the treatment of type 2 diabetes.
The effect of DNA extraction methodology on gut microbiota research applications
K Gerasimidis, M Bertz, C Quince, K Brunner, A Bruce, E Combet, ST Calus, N Loman, and UZ Ijaz
BMC Research Notes, 9:365, 2016. DOI:10.1186/s13104-016-2171-7
Background
The effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated.
Results
The CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used.
Conclusions
We have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods.
Extensive modulation of the fecal metagenome in children with Crohn's disease during exclusive enteral nutrition
C Quince, UZ Ijaz, N Loman, AM Eren, D Saulnier, J Russell, SJ Haig, ST Calus, J Quick, A Barclay, M Bertz, M Blaut, R Hansen, P McGrogan, RK Russell, C Edwards, and K Gerasimidis
American Journal of Gastroenterology, 110:1718-1729, 2015. DOI:10.1038/ajg.2015.357
OBJECTIVES:
Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn’s disease (CD).
METHODS:
Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.
RESULTS:
Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.
CONCLUSIONS:
Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.