Alexander G. Shaw

Dysbiosis Anticipating Necrotizing Enterocolitis in Very Premature Infants

Using 16S rRNA gene sequencing and targeted culture, we compared microbiota in fecal samples from infants with necrotizing enterocolitis (NEC) and controls. Two significant signatures were associated with NEC: 1 with dominant Clostridium perfringens and 1 with dominant Enterobacteriaceae.

Changed Genome Heterochromatinization Upon Prolonged Activation of the Raf/ERK Signaling Pathway

The Raf/ERK (Extracellular Signal Regulated Kinase) signal transduction pathway controls numerous cellular processes, including growth, differentiation, cellular transformation and senescence. ERK activation is thought to involve complex spatial and temporal regulation, to achieve a high degree of specificity, though precisely how this is achieved remains to be confirmed. We report here that prolonged activation of a conditional form of c-Raf-1 (BXB-ER) leads to profound changes in the level and distribution of a heterochromatic histone mark. In mouse fibroblasts, the heterochromatic trimethylation of lysine 9 in histone H3 (H3K9Me3) is normally confined to pericentromeric regions. However, following ERK activation a genome-wide redistribution of H3K9Me3 correlates with loss of the histone modification from chromocentres and the appearance of numerous punctuate sites throughout the interphase nucleus. These epigenetic changes during interphase correlate with altered chromosome structure during mitosis, where robust H3K9Me3 signals appear within telomeric heterochromatin. This pattern of heterochromatinization is distinct from previously described oncogene induced senescence associated heterochromatin foci (SAHF), which are excluded from telomeres. The H3K9Me3 histone mark is known to bind the major heterochromatin protein HP1 and we show that the alterations in the distribution of this histone epistate correlate with redistribution of HP1β throughout the nucleus. Interestingly while ERK activation is fully reversible, the observed chromatin changes induced by epigenetic modifications are not reversible once established. We describe for the first time a link from prolonged ERK activation to stable changes in genome organization through redistribution of heterochromatic domains involving the telomeres. These epigenetic changes provide a possible mechanism through which prolonged activation of Raf/ERK can lead to growth arrest or the induction of differentiation, senescence and cancer.

S Phase Progression in Human Cells Is Dictated by the Genetic Continuity of DNA Foci

DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.

Late-Onset Bloodstream Infection and Perturbed Maturation of the Gastrointestinal Microbiota in Premature Infants

Background Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants. Method Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity. Results From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis. Conclusions The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes.

S-phase progression in mammalian cells: modelling the influence of nuclear organization

The control of DNA replication is of fundamental importance as cell proliferation demands that identical copies of the genetic material are passed to the two daughter cells that form during mitosis. These genetic copies are generated in the preceding S phase, where the entire DNA complement of the mother cell must be copied exactly once. As part of this process, it is known that different regions of mammalian genomes are replicated at specific times of a temporally defined replication programme. The key feature of this programme is that active genes in euchromatin are replicated before inactive ones in heterochromatin. This separation of S phase into periods where different classes of chromatin are duplicated is important in maintaining changes in gene expression that define individual cell types. Recent attempts to understand the structure of the S-phase timing programme have focused on the use of genome-wide strategies that inevitably use DNA isolated from large cell populations for analysis. However, this approach provides a composite view of events that occur within a population without knowledge of the cell-to-cell variability across the population. In this review, we attempt to combine information generated using genome-wide and single cell strategies in order to develop a coherent molecular understanding of S-phase progression. During this integration, we have explored how available information can be introduced into a modelling environment that best describes S-phase progression in mammalian cells.

The neonatal gastrointestinal microbiota: the foundation of future health?

There is a developing appreciation of the abundance and diversity of the trillions of micro-organisms that live on and within us,1 ,2 and how they influence human health and disease.3 Outnumbering human cells in our bodies by 10:1, and their genes outnumbering ours by 100:1,4 bacteria in the gastrointestinal (GI) tract have the potential to significantly modulate human metabolism.5 Previous studies of the microbiota (all the microbes in a given environment) have been hampered by the difficulties of culturing complex samples containing fastidious or unculturable organisms, resulting in inaccurate depictions of microbial communities. Microbiota studies have now progressed to identifying organisms by their DNA sequence. A particular focus has been on the bacterial component, exploiting sequence variation in the ubiquitous 16S rRNA gene. By careful choice of primers hybridising to highly conserved domains within 16S rRNA ,6 a sufficiently large and variable region of this gene can be amplified and sequenced for organisms to be identified at least at genus level, without the bias arising from the need for culture. Utilising next-generation sequencing, which allows millions of sequencing reactions to be performed in parallel, we can determine in quantitative fashion, as never before, the composition of complex bacterial communities,7 such as exist in faecal samples, a pragmatic surrogate for the microbiota of the GI tract mucosal surface.8 Two large international initiatives, The Human Microbiome Project (HMP)9 and Metagenomics of the Human Intestinal Tract (MetaHIT),10 have used this approach to characterise the adult human microbiota at different body sites in health and disease. There is a burgeoning interest in the neonatal GI microbiota, and its association with diseases of prematurity,11 normal child development4 and future health.12 Our group is conducting a longitudinal prospective study assessing the importance of …

Antibiotic resistance potential of the healthy preterm infant gut microbiome

Background Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. Results Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. Conclusions We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

Intestinal microbiota in infants at high risk for allergy: Effects of prebiotics and role in eczema development

Background: Development of the gut microbiota in infancy is important in maturation of the immune system. Deviations in colonization patterns have been associated with allergic manifestations such as eczema, but exact microbiome dysfunctions underlying allergies remain unclear. We studied the gut microbiota of 138 infants at increased risk of allergy, participating in a clinical trial investigating the effectiveness of a partially hydrolyzed protein formula supplemented with nondigestible oligosaccharides on the prevention of eczema. Objective: The effects of interventions and breast‐feeding on fecal microbiota were investigated. Additionally, we aimed to identify microbial patterns associated with the onset of eczema. Methods: Bacterial taxonomic compositions in the first 26 weeks of life were analyzed by using 16S rRNA gene sequencing. Additionally, fecal pH and microbial metabolite levels were measured. Results: Fecal microbial composition, metabolites, and pH of infants receiving partially hydrolyzed protein formula supplemented with nondigestible oligosaccharides was closer to that of breast‐fed infants than that of infants receiving standard cows milk formula. Infants with eczema by 18 months showed discordant development of bacterial genera of Enterobacteriaceae and Parabacteroides species in the first 26 weeks, as well as decreased acquisition of lactate‐utilizing bacteria producing butyrate, namely Eubacterium and Anaerostipes species, supported by increased lactate and decreased butyrate levels. Conclusions: We showed that a partially hydrolyzed protein infant formula with specific prebiotics modulated the gut microbiota closer to that of breast‐fed infants. Additionally, we identified a potential link between microbial activity and onset of eczema, which might reflect a suboptimal implementation of gut microbiota at specific developmental stages in infants at high risk for allergy. GRAPHICAL ABSTRACT Figure. No caption available.

Assessing the Colonic Microbiota in Children: Effects of Sample Site and Bowel Preparation

Objectives: Inflammatory bowel disease states are associated with gastrointestinal dysbiosis. Mucosal biopsy sampling, retrieving the bacterial community that most directly interacts with the host, is an invasive procedure that, we hypothesis, may be sufficiently approximated by other sampling methods. We investigate the relatedness of samples obtained by different methods and the effects of bowel preparation on the gastrointestinal community in a paediatric population. Methods: We recruited a cohort of patients undergoing colonoscopy, collecting serial samples via differing methods (rectal swabs, biopsies, and faecal matter/luminal contents) prebowel preparation, during colonoscopy and after colonoscopy. Next-generation sequencing was used to determine the structure of the microbial community. Results: The microbial community in luminal contents collected during colonoscopy was found to be more similar to that of mucosal biopsies than rectal swabs. Community traits of the mucosal biopsies could be used to segregate patients with inflammatory bowel disease from other patients, and the similarity of the communities in the luminal contents was sufficient for the segregation to be reproduced. Microbial communities sampled by rectal swabs and prebowel preparation faeces were less similar to mucosal biopsies. Bowel preparation was found to have no significant long-term effects on the microbial community, despite the transient effects evident during colonoscopy. Conclusions: A clinically relevant description of the mucosal microbial community can be obtained via the noninvasive collection of luminal contents after bowel cleansing. Bowel preparation in a paediatric population results in no consistent sustained alterations to the gastrointestinal microbiota.

Streaming histogram sketching for rapid microbiome analytics

Motivation The growth in publically available microbiome data in recent years has yielded an invaluable resource for genomic research; allowing for the design of new studies, augmentation of novel datasets and reanalysis of published works. This vast amount of microbiome data, as well as the widespread proliferation of microbiome research and the looming era of clinical metagenomics, means there is an urgent need to develop analytics that can process huge amounts of data in a short amount of time. To address this need, we propose a new method for the compact representation of microbiome sequencing data using similarity-preserving sketches of streaming k-mer spectra. These sketches allow for dissimilarity estimation, rapid microbiome catalogue searching, and classification of microbiome samples in near real-time. Results We apply streaming histogram sketching to microbiome samples as a form of dimensionality reduction, creating a compressed ‘histosketch’ that can be used to efficiently represent microbiome k-mer spectra. Using public microbiome datasets, we show that histosketches can be clustered by sample type using pairwise Jaccard similarity estimation, consequently allowing for rapid microbiome similarity searches via a locality sensitive hashing indexing scheme. Furthermore, we show that histosketches can be used to train machine learning classifiers to accurately label microbiome samples. Specifically, using a collection of 108 novel microbiome samples from a cohort of premature neonates, we trained and tested a Random Forest Classifier that could accurately predict whether the neonate had received antibiotic treatment (95% accuracy, precision 97%) and could subsequently be used to classify microbiome data streams in less than 12 seconds. We provide our implementation, Histosketching Using Little K-mers (HULK), which can histosketch a typical 2GB microbiome in 50 seconds on a standard laptop using 4 cores, with the sketch occupying 3000 bytes of disk space. Availability Our implementation (HULK) is written in Go and is available at: https://github.com/will-rowe/hulk (MIT License)