The MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to ...the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and ...characterise the genomic variation, pangenomic diversity, and key virulence traits of 56
strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that
has an "open" pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined
phylogeny (16S rRNA gene) in relation to some 25
species, with
and
determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised
-associated exotoxins genes including α-toxin (
), enterotoxin (
), and Perfringolysin O (
or
), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (
) and anti-defensins genes (
) were consistently detected
(
: 75%;
: 100%). However, pre-antibiotic era strain genomes did not encode for
, thus implying antimicrobial selective pressures in
evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen.
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.
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Bifidobacterium dominates the gut microbiota in supplemented preterm infantsSupplemented preterm infants have lower abundance of potential pathobiontsMetabolomic analysis show higher fecal acetate and lower pH in supplemented infantsIn 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Infants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic ...treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation.
Using ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a 'gold-standard'.
Results indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had altered diversity profiles. Both bioinformatics 16S rRNA pipelines used in this study (OTU and paired end) presented similar taxonomic profiles at genus level.
We determined that DNA extraction from ELBW faecal samples, particularly those infants receiving probiotic supplementation, should include a prolonged beat-beating step. Furthermore, use of the 16S rRNA (V1 + V2 + V3) and (V6 + V7 + V8) regions provides reliable representation of ELBW microbiota profiles, while inclusion of the (V4 + V5) region may not be appropriate for studies where Bifidobacterium constitutes a resident microbiota member.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Early detection of cancer will improve survival rates. The blood biomarker 5-hydroxymethylcytosine has been shown to discriminate cancer. In a large covariate-controlled study of over two ...thousand individual blood samples, we created, tested and explored the properties of a 5-hydroxymethylcytosine-based classifier to detect colorectal cancer (CRC). In an independent validation sample set, the classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (95% CI 87, 93). Sensitivity was 55% at 95% specificity. Performance was similar for early stage 1 (AUC 89%; 95% CI 83, 94) and late stage 4 CRC (AUC 94%; 95% CI 89, 98). The classifier could detect CRC even when the proportion of tumor DNA in blood was undetectable by other methods. Expanding the classifier to include information about cell-free DNA fragment size and abundance across the genome led to gains in sensitivity (63% at 95% specificity), with similar overall performance (AUC 91%; 95% CI 89, 94). We confirm that 5-hydroxymethylcytosine can be used to detect CRC, even in early-stage disease. Therefore, the inclusion of 5-hydroxymethylcytosine in multianalyte testing could improve sensitivity for the detection of early-stage cancer.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
represents an important early life microbiota member. Specific bifidobacterial components, exopolysaccharides (EPS), positively modulate host responses, with purified EPS also suggested to impact ...microbe-microbe interactions by acting as a nutrient substrate. Thus, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system.
Differential gene expression and growth characteristics were determined for each strain;
UCC2003 and corresponding isogenic EPS-deletion mutant (
UCC2003del). Model colon vessels were inoculated with
and microbiome dynamics monitored using 16S rRNA sequencing and metabolomics (NMR).
Transcriptomics of EPS mutant vs.
UCC2003 highlighted discrete differential gene expression (e.g.,
biosynthetic cluster), though overall growth dynamics between strains were unaffected. The EPS-positive vessel had significant shifts in microbiome and metabolite profiles until study end (405 h); with increases of
and
, and short-chain fatty acids, with further correlations between taxa and metabolites which were not observed within the EPS-negative vessel.
These data indicate that
UCC2003 EPS is potentially metabolized by infant microbiota members, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow development of EPS-specific strategies to promote infant health.
The gut microbiota’s function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). ...This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium, rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development.
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•Microbiota disturbances promote tumor growth in several breast cancer models•Increased tumor volume positively correlates with stromal mast cell density•Supplementation with a commensal restores tumor growth to normal
Pathophysiology; Microbiology; Cancer
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Background
Gut microbiomes, such as the rumen, greatly influence host nutrition due to their feed energy-harvesting capacity. We investigated temporal ecological interactions facilitating ...energy harvesting at the fresh perennial ryegrass (PRG)-biofilm interface in the rumen using an in sacco approach and prokaryotic metatranscriptomic profiling.
Results
Network analysis identified two distinct sub-microbiomes primarily representing primary (≤ 4 h) and secondary (≥ 4 h) colonisation phases and the most transcriptionally active bacterial families (i.e
Fibrobacteriaceae
,
Selemondaceae
and
Methanobacteriaceae
) did not interact with either sub-microbiome, indicating non-cooperative behaviour. Conversely,
Prevotellaceae
had most transcriptional activity within the primary sub-microbiome (focussed on protein metabolism) and
Lachnospiraceae
within the secondary sub-microbiome (focussed on carbohydrate degradation). Putative keystone taxa, with low transcriptional activity, were identified within both sub-microbiomes, highlighting the important synergistic role of minor bacterial families; however, we hypothesise that they may be ‘cheating’ in order to capitalise on the energy-harvesting capacity of other microbes. In terms of chemical cues underlying transition from primary to secondary colonisation phases, we suggest that AI-2-based quorum sensing plays a role, based on LuxS gene expression data, coupled with changes in PRG chemistry.
Conclusions
In summary, we show that fresh PRG-attached prokaryotes are resilient and adapt quickly to changing niches. This study provides the first major insight into the complex temporal ecological interactions occurring at the plant-biofilm interface within the rumen. The study also provides valuable insights into potential plant breeding strategies for development of the utopian plant, allowing optimal sustainable production of ruminants.
The underlying health-driving mechanisms of Bifidobacterium during early life are not well understood, particularly how this microbiota member may modulate the intestinal barrier via programming of ...intestinal epithelial cells (IECs). We investigated the impact of Bifidobacterium breve UCC2003 on the transcriptome of neonatal murine IECs. Small IECs from two-week-old neonatal mice administered B. breve UCC2003 or PBS (control) were subjected to global RNA sequencing, and differentially expressed genes, pathways, and affected cell types were determined. We observed extensive regulation of the IEC transcriptome with ∼4,000 genes significantly up-regulated, including key genes linked with epithelial barrier function. Enrichment of cell differentiation pathways was observed, along with an overrepresentation of stem cell marker genes, indicating an increase in the regenerative potential of the epithelial layer. In conclusion, B. breve UCC2003 plays a central role in driving intestinal epithelium homeostatic development during early life and suggests future avenues for next-stage clinical studies.
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•B. breve administration significantly alters the murine neonatal IEC transcriptome•Genes/pathways involved in epithelial barrier function are particularly impacted•Bifidobacterium may target the IEC stem cell compartment to induce regeneration
Microbiology; Microbiome; Transcriptomics
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
is a key pathogen in poultry-associated necrotic enteritis (NE). To date there are limited Whole Genome Sequencing based studies describing broiler-associated
in healthy and diseased birds. Moreover, ...changes in the caecal microbiome during NE is currently not well characterised. Thus, the aim of this present study was to investigate
virulence factors linked to health and diseased chickens, including identifying putative caecal microbiota signatures associated with NE.
We analysed 88 broiler chicken
genomes (representing 66 publicly available genomes and 22 newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and globally-distributed clone spanning 20-year time-frame (1993-2013). These isolates harbored a greater number of virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including
and
in NE-associated broiler isolates. Secondary virulence genes including
,
, and collagen adhesin genes
,
and
were also enriched in the NE-linked
genomes. Moreover, an environmental isolate obtained from farm animal feeds was found to encode
, suggesting potential reservoirs of NetB-positive
strains (toxinotype G). We also analysed caecal samples from a small sub-set of 11 diseased and healthy broilers for exploratory microbiome investigation using 16S rRNA amplicon sequencing, which indicated a significant and positive correlation in genus
within the wider microbiota of those broilers diagnosed with NE, alongside reductions in beneficial microbiota members.
These data indicate a positive association of virulence genes including
,
and
variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide sample collection from healthy and NE-associated birds.
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