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Introduction\ud The COLO-COHORT study aims to produce a multi-factorial risk prediction model for colorectal neoplasia that will be able to be used to target colonoscopy to those at greatest risk of colorectal neoplasia, ensuring that people are not investigated unnecessarily and maximising the use of limited endoscopy resources. The study will also explore the link between neoplasia and the human gut microbiome. Additionally, the study aims to generate a cohort of colonoscopy patients who are ‘research ready’ through the development of a consent-for-contact platform (C4C), to facilitate a range of CRC prevention studies to be conducted at scale and speed.\ud \ud Methods and Analysis\ud This is a multi-centre observational study involving sites across the UK. Recruitment is over a 6-year period (2019 – 2025). Patients recruited to the study are those attending for colonoscopy. Patients are recruited into two groups, namely; observational Group A (10,000 patients) and C4C Group B (10,000 patients), known as COLO-SPEED (Colorectal Cancer Screening Prevention Endoscopy and Early Diagnosis; https://colospeed.uk).\ud \ud Patients complete a health questionnaire, provide anthropometric measurements and submit biosamples (blood and stool - depending on the part of the study they are recruited into). Patients’ colonoscopy and histology findings are also recorded. Models of factors associated with presence of neoplasia at colonoscopy will be developed using logistic or multinomial regression. For internal validation, model discrimination and calibration will be assessed and bootstrapping and cross-validation approaches used. To enable long-term follow up for outcomes related to CRC and polyps, patients are asked to consent to follow up through data linkage with national databases.\ud \ud Dissemination\ud In keeping with good research practice, following analysis by the study team the study investigators will make the anonymised dataset available to other researchers. The C4C platform will also be accessible to other researchers. The study findings will be submitted for publication in peer reviewed journals and lay summaries will be disseminated to participants and the wider public.

Background & Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologist grouped individuals by modified Bell’s staging (healthy, NEC1, NEC2/3) and faecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Bacillota (adj. p=0.0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of faecal cytokine levels revealed significantly higher concentrations of IL-1α (p=0.045), IL-5 (p=0.0074), and IL-10 (p=0.032) in Bell stage 1 NEC compared to healthy individuals. Conclusions: Differences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

ObjectivesNecrotizing enterocolitis (NEC) is a life-threatening disease, and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell’s staging is routinely used to guide diagnosis, but early diagnostic signs are non-specific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed in NEC infants. New techniques, using biomarkers as diagnostic tool to improve diagnosis of NEC, are emerging. Here we investigated faecal cytokine levels, coupled with gut microbiota profiles, as a non-invasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers.Study designPremature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary’s or Queen Charlotte’s & Chelsea) between January 2011 and December 2012. All but two babies received a first course of antibiotics from birth onwards. Faecal samples from diapers were collected consecutively during the NICU stay.ResultsEvaluation of microbiota profiles between the study groups revealed only minor differences. However, at later time points, significant changes in microbiota structure were observed for Firmicutes, withEnterococcusbeing the least abundant in Bell stage 2/3 NEC. Faecal cytokine levels were similar to those found in previous studies evaluating systemic cytokine concentrations in NEC settings, but measurement in faeces represents a non-invasive method to evaluate the early onset of the disease. For IL-1α, IL-5 and IL-10, a significantly rising gradient of levels were observed from healthy to NEC1 to NEC2/3.ConclusionsDifferences in certain faecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles, contribute to improving knowledge about NEC pathogenesis.

Background Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. Objectives We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. Methods Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC–tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. Results There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P 0.94, P

Within the human intestinal tract, dietary, microbial- and host-derived compounds are used as signals by many pathogenic organisms, including Clostridioides difficile. Trehalose has been reported to enhance virulence of certain C. difficile ribotypes; however, such variants are widespread and not correlated with clinical outcomes for patients suffering from C. difficile infection (CDI). Here, we make preliminary observations on how trehalose supplementation affects the microbiota in an in vitro model and show that trehalose-induced changes can reduce the outgrowth of C. difficile, preventing simulated CDI. Three clinically reflective human gut models simulated the effects of sugar (trehalose or glucose) or saline ingestion on the microbiota. Models were instilled with sugar or saline and further exposed to C. difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodelled to utilise the bioavailable trehalose. Clindamycin induction caused simulated CDI in models supplemented with either glucose or saline; however, trehalose supplementation did not result in CDI, although limited spore germination did occur. The absence of CDI in trehalose model was associated with enhanced abundances of Finegoldia, Faecalibacterium and Oscillospira, and reduced abundances of Klebsiella and Clostridium spp., compared with the other models. Functional analysis of the microbiota in the trehalose model revealed differences in the metabolic pathways, such as amino acid metabolism, which could be attributed to prevention of CDI. Our data show that trehalose supplementation remodelled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C. difficile.

Within the human intestinal tract, dietary, microbial- and host-derived compounds are used as signals by many pathogenic organisms, including Clostridioides difficile. Trehalose has been reported to enhance virulence of certain C. difficile ribotypes; however, such variants are widespread and not correlated with clinical outcomes for patients suffering from C. difficile infection (CDI). Here, we make preliminary observations to how to trehalose supplementation affects the microbiota in an in vitro model and show that trehalose can reduce the outgrowth of C. difficile, preventing simulated CDI. Three clinically reflective human gut models simulated the effects of sugar (trehalose or glucose) or saline ingestion on the microbiota. Models were instilled with sugar or saline and further exposed to C. difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodeled to utilise the bioavailable trehalose. Clindamycin induction caused simulated CDI in models supplemented with either glucose or saline; however, trehalose supplementation did not result in CDI, although limited spore germination did occur. The absence of CDI in trehalose model was associated with enhanced abundances of Finegoldia, Faecalibacterium and Oscillospira, and reduced abundances of Klebsiella and Clostridium spp., compared with the other models. Functional analysis of the microbiota in the trehalose model revealed differences in the metabolic pathways, such as amino acid metabolism, which could be attributed to prevention of CDI. Our data show that trehalose supplementation remodelled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C. difficile.

Objective Individuals with newly diagnosed RA have a distinct microbiome when compared with healthy controls. However, little is known as to when these microbiome perturbations begin. Using a prospective at-risk cohort of individuals positive for anti-citrullinated protein (anti-CCP) antibody with new onset musculoskeletal symptoms, but without clinical arthritis, we investigated for the presence of a gut dysbiosis before the onset of RA. Methods The gut microbiota of 25 anti-CCP positive individuals without clinical synovitis were sequenced targeting the V4 region of the 16S rRNA gene. Using a publicly available database, a control population of 44 individuals, approximately matched in age, gender, diet and ethnicity was selected for comparison, using the same sequencing methodology. Median interval between sample collection and progression to RA was 188 days. Taxonomic analysis was performed using QIIME and MEGAN, and statistical analysis using R software. Results There were significant differences (P =0.01) at family level in gut microbiomes of anti-CCP positive individuals vs controls. The anti-CCP positive population had an overabundance of Lachnospiraceae, Helicobacteraceae, Ruminococcaceae, Erysipelotrichaceae and Bifidobacteriaceae, among others. Five individuals progressed to RA between sample collection and analysis. Clustering of the progressor population was observed on a phylogenetic network created using a probabilistic similarity index (Goodall’s index). Conclusions Anti-CCP positive at-risk individuals without clinical synovitis appear to have a distinct gut microbiome compared with healthy controls. Phylogenetic clustering was observed in individuals who progressed to RA, suggesting that distinct taxa are associated with the development of RA many months before its onset.

Summary: Supplementation with members of the early-life microbiota as “probiotics” is increasingly used in attempts to beneficially manipulate the preterm infant gut microbiota. We performed a large observational longitudinal study comprising two preterm groups: 101 infants orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 infants non-supplemented (control) matched by age, sex, and delivery method. 16S rRNA gene profiling on fecal samples (n = 592) showed a predominance of Bifidobacterium and a lower abundance of pathobionts in the Bif/Lacto group. Metabolomic analysis showed higher fecal acetate and lactate and a lower fecal pH in the Bif/Lacto group compared to the control group. Fecal acetate positively correlated with relative abundance of Bifidobacterium, consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides into acetate. This study demonstrates that microbiota supplementation is associated with a Bifidobacterium-dominated preterm microbiota and gastrointestinal environment more closely resembling that of full-term infants.

Background: Rheumatoid arthritis (RA) is now recognised as the end point of a disease continuum. Anti-citrullinated peptide antibodies (ACPA) mark the presence of RA-related systemic autoimmunity, but the production of auto-antibodies alone is not sufficient to develop disease. Data on progression between the stages of RA disease are limited, but accumulating evidence suggests a microbial dysbiosis in the gut may trigger RA development. Using a prospective ‘at-risk’ cohort of ACPA positive individuals without arthritis, we investigated for the presence of a gut dysbiosis before the onset of RA. Materials/methods: Faecal samples from 25 ACPA-positive individuals, with non-specific musculoskeletal pain, were sequenced using16S rRNA gene. A control population was selected from publicly available data on the NCBI database, matched for rRNA V4 amplification region, sequencing technique, and approximately for age, gender, diet and ethnicity. Taxonomic analysis was performed using QIIME and MEGAN, and statistical analysis using R software. Results: Comparison of the ACPA-positive and control populations shows large clustering at bacterial family level. The ACPA population has an abundance of Lachnospiraceae, Helicobacteraceae, Ruminococcaceae, Erysipelotrichaceae and Bifidobacteriaceae, and a lower abundance of Bacteroidaceae, Barnesiellaceae, Methanobacteriaceae amongst others. The relative abundance of bacterial taxa between the ACPA positive and control population was significantly different (P value 0.01, permutation MANOVA). Conclusions: The gut microbiome of individuals ‘at-risk’ of developing RA is distinct from heathy controls. These pilot data can inform further studies, and are an important step in the attribution of causality to the gut microbiome in the development of RA.

Summary Supplementation with members of the early-life microbiota as “probiotics” is increasingly used in attempts to beneficially manipulate the preterm infant gut microbiota. We performed a large observational longitudinal study comprising two preterm groups: 101 infants orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 infants non-supplemented (control) matched by age, sex, and delivery method. 16S rRNA gene profiling on fecal samples (n = 592) showed a predominance of Bifidobacterium and a lower abundance of pathobionts in the Bif/Lacto group. Metabolomic analysis showed higher fecal acetate and lactate and a lower fecal pH in the Bif/Lacto group compared to the control group. Fecal acetate positively correlated with relative abundance of Bifidobacterium, consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides into acetate. This study demonstrates that microbiota supplementation is associated with a Bifidobacterium-dominated preterm microbiota and gastrointestinal environment more closely resembling that of full-term infants., Graphical Abstract, Highlights Bifidobacterium dominates the gut microbiota in supplemented preterm infants Supplemented preterm infants have lower abundance of potential pathobionts Metabolomic analysis show higher fecal acetate and lower pH in supplemented infants In vitro and genomic analysis confirm HMO metabolism in Bifidobacterium supplement, Alcon-Giner et al. show that probiotic supplementation in preterm infants leads to a Bifidobacterium-dominated gut microbiota, elevated acetate and lactate levels, and lower fecal pH, with concurrent reduction in pathobionts. Bifidobacterium isolation confirms strain presence; genomic and in vitro studies indicate strain encoded enzymatic clusters allowing growth on HMOs.

Rifaximin is a low solubility antibiotic with activity against a wide range of bacterial pathogens. It accumulates in the intestine and is suitable for prolonged use. Three chemostat models (A, B and C) were used to investigate the effects of three rifaximin formulations (α, β and κ, respectively) on the gut microbiome. Bacterial populations were monitored by bacterial culture and 16S rRNA gene amplicon (16S) sequencing. Limited disruption of bacterial populations was observed for rifaximin α, β and κ. All formulations caused declines in total spores (∼2 log10 cfu ml-1), Enterococcus spp. (∼2 log10 cfu ml-1 in models A and C, and ∼1 log10 cfu ml-1 in model B), and Bacteroides spp. populations (∼3 log10 cfu ml-1 in models A and C, and ∼4 log10 cfu ml-1 in model B). Bacterial populations fully recovered during antibiotic dosing in model C, and before the end of the experiment in models A and B. According to the taxonomic analysis, prior to rifaximin exposure, Bifidobacteriaceae, Ruminococcaceae, Acidaminococcaceae, Lachnospiraceae and Rikenellaceae families represented >92% of the total relative abundance, in all models. Within these families, 15 bacterial genera represented >99% of the overall relative abundance. Overall, the 16S sequencing and culture data showed similar variations in the bacterial populations studied. Among the three formulations, rifaximin κ appeared to have the least disruptive effect on the colonic microbiota, with culture populations showing recovery in a shorter period and the taxonomic analysis revealing the least global variation in relative abundance of prevalent groups.

ObjectiveOmega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22).DesignA randomised, open-label, cross-over trial of 8 weeks’ treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week ‘washout’ period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry.ResultsBoth omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or β diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects.ConclusionOmega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure.Trial registration numberISRCTN18662143.

Ultrasound-guided Pecs block can provide perioperative analgesia for breast surgery. A single-injection technique (COMBIPECS) combines both Pecs 1 and Pecs 2 blocks in a single needle pass. This technique saves time and is equally effective as the modified Pecs block which uses 2 needle passes. We present a case series of 21 patients who received the COMBIPECS block as a part of multimodal analgesia for breast cancer surgery. The block was administered before the surgery after induction of general anesthesia. The patients needed minimal intraoperative analgesics and remained pain free in the postoperative period up to 24 hours after surgery.

Supplementation with members of the early-life microbiota or ‘probiotics’ is becoming increasingly popular to attempt to beneficially manipulate the preterm gut microbiota. We performed a large longitudinal study comprising two preterm groups; 101 orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 non-supplemented (Control) matched by age, sex, birth-mode, and diet. 16S rRNA metataxonomic profiling on stool samples (n = 592) indicated a predominance of Bifidobacterium, and a reduction of pathobionts in the Bif/Lacto group. Metabolic phenotyping found a parallel increase in fecal acetate and lactate in the Bif/Lacto group compared to the Control group, which positively correlated with Bifidobacterium abundance consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides and reduced gut pH. This study demonstrates that microbiota supplementation can modify the preterm microbiome and the gastrointestinal environment to more closely resemble that of a full-term infant.

Metagenomics, also known as environmental genomics, is the study of the genomic content of a sample of organisms (microbes) obtained from a common habitat. Metagenomics and other "omics" disciplines have captured the attention of researchers for several decades. The effect of microbes in our body is a relevant concern for health studies. There are plenty of studies using metagenomics which examine microorganisms that inhabit niches in the human body, sometimes causing disease, and are often correlated with multiple treatment conditions. No matter from which environment it comes, the analyses are often aimed at determining either the presence or absence of specific species of interest in a given metagenome or comparing the biological diversity and the functional activity of a wider range of microorganisms within their communities. The importance increases for comparison within different environments such as multiple patients with different conditions, multiple drugs, and multiple time points of same treatment or same patient. Thus, no matter how many hypotheses we have, we need a good understanding of genomics, bioinformatics, and statistics to work together to analyze and interpret these datasets in a meaningful way. This chapter provides an overview of different data analyses and statistical approaches (with example scenarios) to analyze metagenomics samples from different medical projects or clinical trials.

Metagenomics, also known as environmental genomics, is the study of the genomic content of a sample of organisms (microbes) obtained from a common habitat. Metagenomics and other "omics" disciplines have captured the attention of researchers for several decades. The effect of microbes in our body is a relevant concern for health studies. There are plenty of studies using metagenomics which examine microorganisms that inhabit niches in the human body, sometimes causing disease, and are often correlated with multiple treatment conditions. No matter from which environment it comes, the analyses are often aimed at determining either the presence or absence of specific species of interest in a given metagenome or comparing the biological diversity and the functional activity of a wider range of microorganisms within their communities. The importance increases for comparison within different environments such as multiple patients with different conditions, multiple drugs, and multiple time points of same treatment or same patient. Thus, no matter how many hypotheses we have, we need a good understanding of genomics, bioinformatics, and statistics to work together to analyze and interpret these datasets in a meaningful way. This chapter provides an overview of different data analyses and statistical approaches (with example scenarios) to analyze metagenomics samples from different medical projects or clinical trials.

Archaea remain important players in global biogeochemical cycles worldwide, including in the highly productive mangrove estuarine ecosystems. In the present study, we have explored the diversity, distribution, and function of the metabolically active fraction of the resident archaeal community of the Sundarban mangrove ecosystem, using both culture-independent and culture-dependent approaches. To evaluate the diversity and distribution pattern of the active archaeal communities, RNA based analysis of the 16S rRNA gene was performed on an Illumina platform. The active Crenarchaeal community was observed to remain constant while active Euryarchaeal community underwent considerable change across the sampling sites depending on varying anthropogenic factors. Haloarchaea were the predominant group in hydrocarbon polluted sediments, leading us to successfully isolate eleven p-hydroxybenzoic acid degrading haloarchaeal species. The isolates could also survive in benzoic acid, naphthalene, and o-phthalate. Quantitative estimation of p-hydroxybenzoic acid degradation was studied on select isolates, and their ability to reduce COD of polluted saline waters of Sundarban was also evaluated. To our knowledge, this is the first ever study combining culture-independent (Next Generation sequencing and metatranscriptome) and culture-dependent analyses for an assessment of archaeal function in the sediment of Sundarban.

Networks of engineered waterways are critical in meeting the growing water demands in megacities. To capture and treat rainwater in an energy-efficient manner, approaches can be developed for such networks that use ecological services from microbial communities. Traditionally, engineered waterways were regarded as homogeneous systems with little responsiveness of ecological communities and ensuing processes. This study provides ecogenomics-derived key information to explain the complexity of urban aquatic ecosystems in well-managed watersheds with densely interspersed land-use patterns. Overall, sedimentary microbial communities had higher richness and evenness compared to the suspended communities in water phase. On the basis of PERMANOVA analysis, variation in structure and functions of microbial communities over space within same land-use type was not significant. In contrast, this difference was significant between different land-use types, which had similar chemical profiles. Of the 36 environmental parameters from spatial analysis, only three metals, namely potassium, copper and aluminum significantly explained between 7% and 11% of the variation in taxa and functions, based on distance-based linear models (DistLM). The ecogenomics approach adopted here allows the identification of key drivers of microbial communities and their functions at watershed-scale. These findings can be used to enhance microbial services, which are critical to develop ecologically friendly waterways in rapidly urbanizing environments.

Evidence suggests a correlation between the gut microbiota composition and weight loss caused by caloric restriction. Laparoscopic sleeve gastrectomy (LSG), a surgical intervention for obesity, is classified as predominantly restrictive procedure. In this study we investigated functional weight loss mechanisms with regard to gut microbial changes and energy harvest induced by LSG and a very low calorie diet in ten obese subjects (n=5per group) demonstrating identical weight loss during a follow-up period of six months. For gut microbiome analysis next generation sequencing was performed and faeces were analyzed for targeted metabolomics. The energy-reabsorbing potential of the gut microbiota decreased following LSG, indicated by the Bacteroidetes/Firmicutes ratio, but increased during diet. Changes in butyrate-producing bacterial species were responsible for the Firmicutes changes in both groups. No alteration of faecal butyrate was observed, but the microbial capacity for butyrate fermentation decreased following LSG and increased following dietetic intervention. LSG resulted in enhanced faecal excretion of nonesterified fatty acids and bile acids. LSG, but not dietetic restriction, improved the obesity-associated gut microbiota composition towards a lean microbiome phenotype. Moreover, LSG increased malabsorption due to loss in energy-rich faecal substrates and impairment of bile acid circulation. This trial is registered with ClinicalTrials.govNCT01344525.

One important question in microbiome analysis is how to assess the homogeneity of the microbial composition in a given environment, with respect to a given analysis method. Do different microbial samples taken from the same environment follow the same taxonomic distribution of organisms, or the same distribution of functions? Here we provide a non-parametric statistical “triangulation test” to address this type of question. The test requires that multiple replicates are available for each of the biological samples, and it is based on three-way computational comparisons of samples. To illustrate the application of the test, we collected three biological samples taken from different locations in one piece of human stool, each represented by three replicates, and analyzed them using MEGAN. (Despite its name, the triangulation test does not require that the number of biological samples or replicates be three.) The triangulation test rejects the null hypothesis that the three biological samples exhibit the same distribution of taxa or function (error probability ≤0.05), indicating that the microbial composition of the investigated human stool is not homogenous on a macroscopic scale, suggesting that pooling material from multiple locations is a reasonable practice. We provide an implementation of the test in our open source program MEGAN Community Edition.

Comparison of taxonomic assignments among the 16S rRNA gene hypervariable regions tested using QIIME approach. Heat map displaying number of reads assigned to the most common bacterial taxa found in the study samples using QIIME bioinformatics pipeline. Top panel row divides the figure in the different regions of the 16 s rRNA gene analysed, namely: V1 + V2 + V3 (primers 27F-519R), V4 + V5 (primers 530F-926R) and V6 + V7 + V8 (primers 926F-1394R). The vertical axis of the panel indicates a selection of the 13 most common bacterial taxa found. The horizontal axis labels the different samples used in the study: preterms without supplementation (AP1E, AP5D, AP8C, AP25C), preterms with supplementation (P29F, P30N, P31B, P35C), and term baby samples (V2A, V3 J). The intensity of the green colour highlights the abundance of the number of reads found. Probiotic supplementation has been abbreviated to supplem. in the figure. Further information on the number of reads obtained for this study can be found in Additional file 19. (PDF 262 kb)

Number of reads detected by PE and QIIME pipelines for the different hypervariable regions. (PDF 181Â kb)

DNA yield from different DNA extraction methods. (PDF 306Â kb)

Shannon diversity index on 16S rRNA gene sequencing data analysed using OTU and PE protocol. Shannon diversity index was calculated using 16S rRNA bacterial community profiles for sample AP1E (ELBW infant without probiotic supplementation) and sample V3 J (term infant). a Shannon diversity indexes comparison of three different 16S rRNA libraries (27F-519R (region V1 + V2 + V3), 530F-926R (region V4 + V5) and 926F-1394R (region V6 + V7 + V8)) using OTU and PE protocol pipelines for sample AP1E (preterm no supplementation). b Shannon diversity indexes comparison of three different 16S rRNA libraries (27F-519R (region V1 + V2 + V3), 530F-926R (region V4 + V5) and 926F-1394R (region V6 + V7 + V8)) using OTU and PE protocol pipelines for sample V3 J (term infant). (PDF 74 kb)

Subject details and metadata. (PDF 202Â kb)

Bifidobacterium strains used for validating primers 530F-926R using PCR (PDF 198Â kb)

Objective. To compare the safety, efficacy, and direct cost during the introduction of laparoscopic radical hysterectomy within an enhanced recovery pathway. Methods. A 1 : 1 single centre retrospective case control study of 36 propensity matched pairs of patients receiving open or laparoscopic surgery for early cervical cancer. Results. There were no significant differences in the baseline characteristics of the two cohorts. Open surgery cohort had significantly higher intraoperative blood loss (189 versus 934 mL) and longer postoperative hospital stay (2.3 versus 4.1 days). Although no significant difference in the intraoperative or postoperative complications was found more urinary tract injuries were recorded in the laparoscopic cohort. Laparoscopic surgery had significantly longer duration (206 versus 159 minutes), lower lymph node harvest (12.6 versus 16.9), and slower bladder function recovery. The median direct hospital cost was £4850 for laparoscopic radical hysterectomy and £4400 for open surgery. Conclusions. Laparoscopic radical hysterectomy can be safely introduced in an enhanced recovery environment without significant increase in perioperative morbidity. The 10% higher direct hospital cost is not statistically significant and is expected to even out when indirect costs are included.

Primers used in 16S rRNA sequencing library. (PDF 137Â kb)

Number of reads shotgun and 16S rRNA data (PE versus QIIME). (PDF 286Â kb)

Rarefaction curves 16S rRNA gene sequencing data. Rarefaction curves representing number of species (leaves) detected at genus level versus number of reads sampled. Three different 16S rRNA gene sequencing data were used for this study: i) green curves represent sequencing data from 16S rRNA library prepared using primers 27F-519R, ii) red curves represent sequencing data from 16S rRNA library prepared using primers 530F-926R and iii) blue curves represent sequencing data from 16S rRNA library prepared using primers 926F-1394R. Rarefaction curves are labelled with numbers to differentiate among the samples used in the study: 1 (V2A.530F), 2 (V2A.926F), 3 (V3 J.926F), 4 (AP8C.530F), 5 (V2AJ.27F), 6 (AP5D.530F), 7 (P35C.530F), 8 (V3 J.530F), 9 (P29F.530F), 10 (P31B.530F), 11 (P30N.530F), 12 (AP25E.530F), 13 (AP25E.926F), 14 (AP25E. 27F), 15 (AP8C.926F), 16 (P35C.926F), 17 (V3 J.27F), 18 (P31B.926F), 19 (AP1E.530F), 20 (P31B.27F), 21(P29F.27F), 22 (P30N.27F), 23 (AP5D.926F), 24 (AP5D.27F), 25 (P29F.926F), 26 (AP1E.926F),27 (AP1E.27F), 28 (AP8C.27F), 29 (P30N.926F), 30 (P35C.27F). Numbers 12, 13 and 14 correspond to sample AP25E where majority of sequenced reads assigned at family level (i.e. Enterobacteriaceae). (PDF 62 kb)