The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray-Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.
To highlight the contribution of the gut microbiota to the modulation of host metabolism by dietary inulin-type fructans (ITF prebiotics) in obese women.
Methods
A double blind, placebo controlled, intervention study was performed with 30 obese women treated with ITF prebiotics (inulin/oligofructose 50/50 mix; n=15) or placebo (maltodextrin; n=15) for 3 months (16 g/day). Blood, faeces and urine sampling, oral glucose tolerance test, homeostasis model assessment and impedancemetry were performed before and after treatment. The gut microbial composition in faeces was analysed by phylogenetic microarray and qPCR analysis of 16S rDNA. Plasma and urine metabolic profiles were analysed by 1H-NMR spectroscopy.
Results
Treatment with ITF prebiotics, but not the placebo, led to an increase in Bifidobacterium and Faecalibacterium prausnitzii; both bacteria negatively correlated with serum lipopolysaccharide levels. ITF prebiotics also decreased Bacteroides intestinalis, Bacteroides vulgatus and Propionibacterium, an effect associated with a slight decrease in fat mass and with plasma lactate and phosphatidylcholine levels. No clear treatment clustering could be detected for gut microbial analysis or plasma and urine metabolomic profile analyses. However, ITF prebiotics led to subtle changes in the gut microbiota that may importantly impact on several key metabolites implicated in obesity and/or diabetes.
Conclusions
ITF prebiotics selectively changed the gut microbiota composition in obese women, leading to modest changes in host metabolism, as suggested by the correlation between some bacterial species and metabolic endotoxaemia or metabolomic signatures.
People aged 60 years or older are at high risk for respiratory infections, one of the leading causes of mortality worldwide. Vaccination is the main way to protect against these infections; however, vaccination is less effective in older adults than in younger adults due to ageing of the immune system, so innovative strategies that improve vaccine responses could provide a major public health benefit. The gut microbiota regulates host immune homoeostasis and response against pathogens, but human studies showing the effects of the gut microbiota on respiratory infections in older adults are sparse. We aimed to investigate the composition of the microbiota in relation to respiratory infections and local and systemic immune markers in older adults during an influenza season.In this observational study, participants were selected from an influenza-like illness (ILI) prospective surveillance cohort in which community-dwelling adults aged 60 years and older in the Netherlands were recruited through their general practitioner or the Civil Registry. Inclusion criteria have been described elsewhere. Participants completed questionnaires and self-reported symptoms. To measure microbiota composition, faecal samples were collected from participants registering an ILI event, with a follow-up (recovery) sample collected 7-9 weeks after the ILI event, and from asymptomatic participants not reporting any event throughout the season. We tested associations between microbiota profiles and a set of health-related variables, patient characteristics, and local and systemic immune markers. We cultured identified bacterial biomarkers for ILI with CaCo-2 cells in an in vitro intestinal epithelial model and measured the induced immune response. This study is registered with http://www.trialregister.nl, NL4666.Between Oct 1, 2014, and April 30, 2015, 2425 older adults were recruited into the ILI surveillance cohort. From Oct 1, 2014, to June 15, 2015, faecal samples were collected from 397 participants, of whom 213 (54%) reported an ILI event once throughout the season and 184 (46%) did not. 192 ILI participants recovered and provided follow-up samples. Microbiota composition was altered during an ILI event. The Bacteroidetes (mean relative abundance 17·51% [SD 11·41] in the ILI group and 14·19% [10·02] in the control group; adjusted p=0·014) and the Proteobacteria (3·40% [8·10] in the ILI group and 1·57% [3·69] in the control group; adjusted p=0·015) were more abundant in the ILI group than in the control group. The abundance of Ruminococcus torques was positively associated with ILI and the abundance of Escherichia/Shigella, negatively correlated with alpha diversity, and negatively co-occurred with beneficial taxa, including butyrate producers. R torques was associated with pro-inflammatory profiles, both locally in faeces and systemically in blood. ILI-associated taxa (R torques and Escherichia coli) had symbiotic effects on the cellular immune response when cultured together in an in vitro model.The abundances of specific bacteria could be used as potential biomarkers for susceptibility to respiratory infections and as targets for intervention in the ageing population.The Dutch Ministry of Health, Welfare and Sport, and the Strategic Program of the National Institute for Public Health and the Environment.
ABSTRACT Background Critically ill patients hospitalized in an Intensive Care Unit (ICU) are at increased risk of acquiring potentially life-threatening infections with opportunistic pathogens. The gut microbiota of ICU patients forms an important reservoir for these infectious agents. To suppress gut colonization with opportunistic pathogens, a prophylactic antibiotic regimen, termed ‘Selective decontamination of the digestive tract’ (SDD), may be used. SDD has previously been shown to improve clinical outcome in ICU patients, but the impact of ICU hospitalization and SDD on the gut microbiota remains largely unknown. Here, we characterize the composition of the gut microbiota and its antimicrobial resistance genes (‘the resistome’) of ICU patients during SDD. Results During ICU-stay, 30 fecal samples of ten patients were collected. Additionally, feces were collected from five of these patients after transfer to a medium-care ward and cessation of SDD. As a control group, feces from ten healthy subjects were collected twice, with a one-year interval. Gut microbiota and resistome composition were determined using 16S rRNA phylogenetic profiling and nanolitre-scale quantitative PCRs. The microbiota of the ICU patients differed from the microbiota of healthy subjects and was characterized by low microbial diversity, decreased levels of E. coli and of anaerobic Gram-positive, butyrate-producing bacteria of the Clostridium clusters IV and XIVa, and an increased abundance of Bacteroidetes and enterococci. Four resistance genes ( aac(6′)-Ii , ermC , qacA , tetQ ), providing resistance to aminoglycosides, macrolides, disinfectants and tetracyclines respectively, were significantly more abundant among ICU patients than in healthy subjects, while a chloramphenicol resistance gene ( catA ) and a tetracycline resistance gene ( tetW ) were more abundant in healthy subjects. Conclusions The microbiota and resistome of ICU patients and healthy subjects were noticeably different, but importantly, levels of E. coli remained low during ICU hospitalization, presumably due to SDD therapy. Selection for four antibiotic resistance genes was observed, but none of these are of particular concern as they do not contribute to clinically relevant resistance. Our data support the ecological safety of SDD, at least in settings with low levels of circulating antibiotic resistance.
Abstract The early-life microbiome appears to be affected by mode of delivery, but this effect may depend on intrapartum antibiotic exposure. Here, we assess the effect of delivery mode on gut microbiota, independent of intrapartum antibiotics, by postponing routine antibiotic administration to mothers until after cord clamping in 74 vaginally delivered and 46 caesarean section born infants. The microbiota differs between caesarean section born and vaginally delivered infants over the first year of life, showing enrichment of Bifidobacterium spp., and reduction of Enterococcus and Klebsiella spp. in vaginally delivered infants. The microbiota composition at one week of life is associated with the number of respiratory infections over the first year. The taxa driving this association are more abundant in caesarean section born children, providing a possible link between mode of delivery and susceptibility to infectious outcomes.
The colonization and development of gut microbiota immediately after birth is highly variable and depends on several factors, such as delivery mode and modality of feeding during the first months of life. A cohort of 31 mother and neonate pairs, including 25 at-term caesarean (CS) and 6 vaginally (V) delivered neonates (DNs), were included in this study and 121 meconium/faecal samples were collected at days 1 through 30 following birth. Operational taxonomic units (OTUs) were assessed in 69 stool samples by phylogenetic microarray HITChip and inter- and intra-individual distributions were established by inter-OTUs correlation matrices and OTUs co-occurrence or co-exclusion networks. 1H-NMR metabolites were determined in 70 stool samples, PCA analysis was performed on 55 CS DNs samples, and metabolome/OTUs co-correlations were assessed in 45 CS samples, providing an integrated map of the early microbiota OTUs-metabolome. A microbiota "core" of OTUs was identified that was independent of delivery mode and lactation stage, suggesting highly specialized communities that act as seminal colonizers of microbial networks. Correlations among OTUs, metabolites, and OTUs-metabolites revealed metabolic profiles associated with early microbial ecological dynamics, maturation of milk components, and host physiology.
Poster: SERAM 2014 / S-0013 / Implantacion antebraquial de cateteres venosos centrales tunelizados con reservorio para administracion de quimioterapia. by: A. M. Fernandez Martinez , I. Alvarez Silva, S. Molnar Fuentes, J. Gomez Morinigo, U. Sobrino Castro, �. Balboa Arregui; Leon/ES
Abstract The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilu s when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature.
OBJECTIVES: To provide a comprehensive analysis of the fecal microbiota in infants with colic, as compared with control infants, during their first 100 days of life. METHODS: Microbial DNA of >200 samples from 12 infants with colic and 12 age-matched control infants was extracted and hybridized to a phylogenetic microarray. RESULTS: Microbiota diversity gradually increased after birth only in the control group; moreover, in the first weeks, the diversity of the colic group was significantly lower than that of the control group. The stability of the successive samples also appeared to be significantly lower in the infants with colic for the first weeks. Further analyses revealed which bacterial groups were responsible for colic-related differences in microbiota at age 1 or 2 weeks, the earliest ages with significant differences. Proteobacteria were significantly increased in infants with colic compared with control infants, with a relative abundance that was more than twofold. In contrast, bifidobacteria and lactobacilli were significantly reduced in infants with colic. Moreover, the colic phenotype correlated positively with specific groups of proteobacteria, including bacteria related to Escherichia, Klebsiella, Serratia, Vibrio, Yersinia, and Pseudomonas, but negatively with bacteria belonging to the Bacteroidetes and Firmicutes phyla, the latter of which includes some lactobacilli and canonical groups known to produce butyrate and lactate. CONCLUSIONS: The results indicate the presence of microbial signatures in the first weeks of life in infants who later develop colic. These microbial signatures may be used to understand the excessive crying. The results offer opportunities for early diagnostics as well as for developing specific therapies.
