Publications
30 results found
Shaw AG, Troman C, Akello JO, et al., 2023, Defining a research agenda for environmental wastewater surveillance of pathogens., Nat Med, Vol: 29, Pages: 2155-2157
Shaw AG, Mampuela TK, Lofiko EL, et al., 2023, Sensitive poliovirus detection using nested PCR and nanopore sequencing: a prospective validation study., Nat Microbiol, Vol: 8, Pages: 1634-1640
Timely detection of outbreaks is needed for poliovirus eradication, but gold standard detection in the Democratic Republic of the Congo takes 30 days (median). Direct molecular detection and nanopore sequencing (DDNS) of poliovirus in stool samples is a promising fast method. Here we report prospective testing of stool samples from suspected polio cases, and their contacts, in the Democratic Republic of the Congo between 10 August 2021 and 4 February 2022. DDNS detected polioviruses in 62/2,339 (2.7%) of samples, while gold standard combination of cell culture, quantitative PCR and Sanger sequencing detected polioviruses in 51/2,339 (2.2%) of the same samples. DDNS provided case confirmation in 7 days (median) in routine surveillance conditions. DDNS enabled confirmation of three serotype 2 circulating vaccine-derived poliovirus outbreaks 23 days (mean) earlier (range 6-30 days) than the gold standard method. The mean sequence similarity between sequences obtained by the two methods was 99.98%. Our data confirm the feasibility of implementing DDNS in a national poliovirus laboratory.
Shaw A, Troman C, Akello J, et al., 2023, The future of integrated environmental surveillance: defining a research agenda, Nature Medicine, ISSN: 1078-8956
Kiu R, Shaw AG, Sim K, et al., 2023, Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains, Nature Microbiology, Vol: 8, Pages: 1160-1175, ISSN: 2058-5276
Clostridium perfringens is an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link between C. perfringens and the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundant C. perfringens termed C. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272 C. perfringens isolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O, pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typical pfoA-encoding virulent lineages. We determined that infant-associated pfoA+ strains caused significantly more cellular damage than pfoA- strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance of pfoA+ C. perfringens as a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.
Akello J, Bujaki E, Shaw A, et al., 2023, Comparison of eleven RNA extraction methods for poliovirus direct molecular detection in stool samples, Microbiology Spectrum, Vol: 11, Pages: 1-13, ISSN: 2165-0497
Direct detection by PCR of poliovirus RNA in stool samples provides a rapid diagnostic and surveillance tool that can replace virus isolation by cell culture in global polio surveillance. The sensitivity of direct detection methods is likely to depend on the choice of RNA extraction method and sample volume. We report a comparative analysis of 11 nucleic acid extraction methods (7 manual and 4 semiautomated) for poliovirus molecular detection using stool samples (n = 59) that had been previously identified as poliovirus positive by cell culture. To assess the effect of RNA recovery methods, extracted RNA using each of the 11 methods was tested with a poliovirus-specific reverse transcription-quantitative PCR (RT-qPCR), a pan-poliovirus RT-PCR (near-whole-genome amplification), a pan-enterovirus RT-PCR (entire capsid region), and a nested VP1 PCR that is the basis of a direct detection method based on nanopore sequencing. We also assessed extracted RNA integrity and quantity. The overall effect of extraction method on poliovirus PCR amplification assays tested in this study was found to be statistically significant (P < 0.001), thus indicating that the choice of RNA extraction method is an important component that needs to be carefully considered for any diagnostic based on nucleic acid amplification. Performance of the methods was generally consistent across the different assays used. Of the 11 extraction methods tested, the MagMAX viral RNA isolation kit used manually or automatically was found to be the preferable method for poliovirus molecular direct detection considering performance, cost, and processing time.
Sim K, Powell E, Emma C, et al., 2023, Development of the gut microbiota during early life in premature and term infants, Gut Pathogens, Vol: 15, ISSN: 1757-4749
Background:The gastrointestinal (GI) microbiota has been linked to health consequences throughout life, from early life illnesses (e.g. sepsis and necrotising enterocolitis) to lifelong chronic conditions such as obesity and inflammatory bowel disease. It has also been observed that events in early life can lead to shifts in the microbiota, with some of these changes having been documented to persist into adulthood. A particularly extreme example of a divergent early GI microbiota occurs in premature neonates, who display a very different GI community to term infants. Certain characteristic patterns have been associated with negative health outcomes during the neonatal period, and these patterns may prove to have continual damaging effects if not resolved.Results:In this study we compared a set of premature infants with a paired set of term infants (n = 37 pairs) at 6 weeks of age and at 2 years of age. In the samples taken at 6 weeks of age we found microbial communities differing in both diversity and specific bacterial groups between the two infant cohorts. We identified clinical factors associated with over-abundance of potentially pathogenic organisms (e.g. Enterobacteriaceae) and reduced abundances of some beneficial organisms (e.g. Bifidobacterium). We contrasted these findings with samples taken at 2 years of age, which indicated that despite a very different initial gut microbiota, the two infant groups converged to a similar, more adult-like state. We identified clinical factors, including both prematurity and delivery method, which remain associated with components of the gut microbiota. Both clinical factors and microbial characteristics are compared to the occurrence of childhood wheeze and eczema, revealing associations between components of the GI microbiota and the development of these allergic conditions.Conclusions:The faecal microbiota differs greatly between infants born at term and those born prematurely during early life, yet it c
Zenner C, Chalklen L, Adjei H, et al., 2023, Noninvasive Fecal Cytokine and Microbiota Profiles Predict Commencement of Necrotizing Enterocolitis in a Proof-of-Concept Study., Gastro Hep Adv, Vol: 2, Pages: 666-675
BACKGROUND AND 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 nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive 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 neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal 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 composition were observed for Bacillota (adj. P = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α (P = .045), IL-5 (P = .0074), and IL-10 (P = .032) in Bell stage 1 NEC compared to healthy individuals. CONCLUSION: Differences in certain fecal 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.
Karampatsas K, Faal A, Jaiteh M, et al., 2022, Gastrointestinal, vaginal, nasopharyngeal, and breast milk microbiota profiles and breast milk metabolomic changes in Gambian infants over the first two months of lactation: a prospective cohort study, Medicine, Vol: 101, Pages: 1-10, ISSN: 0025-7974
Background: Microbiota composition in breast milk affects intestinal and respiratory microbiota colonization and the mucosal immune system's development in infants. The metabolomic content of breast milk is thought to interact with the microbiota and may influence developing infant immunity.Methods: 107 Gambian mothers and their healthy, vaginally delivered, exclusively breastfed infants were included in our study. We analyzed 32 breast milk samples, 51 maternal rectovaginal swabs and 30 infants' rectal swabs at birth. We also analyzed 9 breast milk samples and 18 infants' nasopharyngeal swabs 60 days post-delivery. We used 16S rRNA genesequencing to determine the microbiota composition. Metabolomic profiling analysis was performed on colostrum and mature breast milk samples using a multiplatform approach combining 1-H Nuclear Magnetic Resonance Spectroscopy and Gas Chromatography-Mass Spectrometry.Results: Bacterial communities were distinct in composition and diversity across different sample types. Breast milk composition changed over the first 60 days of lactation. α-1,4- and α-1,3-fucosylated human milk oligosaccharides, and other 33 key metabolites in breast milk(monosaccharides, sugar alcohols and fatty acids) increased between birth and day of 60 life.Conclusions: This study's results indicate that infant gut and respiratory microbiota are unique bacterial communities, distinct from maternal gut and breast milk, respectively. Breast milk microbiota composition and metabolomic profile change throughout lactation. These changesmay contribute to the infant's immunological, metabolic, and neurological development and could consist the basis for future interventions to correct disrupted early life microbial colonization.
Klapsa D, Wilton T, Zealand A, et al., 2022, Sustained detection of type 2 poliovirus in London sewage between February and July, 2022, by enhanced environmental surveillance, The Lancet, Vol: 400, Pages: 1531-1538, ISSN: 0140-6736
BACKGROUND: The international spread of poliovirus exposes all countries to the risk of outbreaks and is designated a Public Health Emergency of International Concern by WHO. This risk can be exacerbated in countries using inactivated polio vaccine, which offers excellent protection against paralysis but is less effective than oral vaccine against poliovirus shedding, potentially allowing circulation without detection of paralytic cases for long periods of time. Our study investigated the molecular properties of type 2 poliovirus isolates found in sewage with an aim to detect virus transmission in the community. METHODS: We performed environmental surveillance in London, UK, testing sewage samples using WHO recommended methods that include concentration, virus isolation in cell culture, and molecular characterisation. We additionally implemented direct molecular detection and determined whole-genome sequences of every isolate using novel nanopore protocols. FINDINGS: 118 genetically linked poliovirus isolates related to the serotype 2 Sabin vaccine strain were detected in 21 of 52 sequential sewage samples collected in London between Feb 8 and July 4, 2022. Expansion of environmental surveillance sites in London helped localise transmission to several boroughs in north and east London. All isolates have lost two key attenuating mutations, are recombinants with a species C enterovirus, and an increasing proportion (20 of 118) meet the criterion for a vaccine-derived poliovirus, having six to ten nucleotide changes in the gene coding for VP1 capsid protein. INTERPRETATION: Environmental surveillance allowed early detection of poliovirus importation and circulation in London, permitting a rapid public health response, including enhanced surveillance and an inactivated polio vaccine campaign among children aged 1-9 years. Whole-genome sequences generated through nanopore sequencing established linkage of isolates and confirmed transmission of a unique recombinant poliov
Shaw A, Cooper L, Gumede N, et al., 2022, Time taken to detect and respond to polio outbreaks in Africa and the potential impact of direct molecular detection and nanopore sequencing, Journal of Infectious Diseases, Vol: 226, Pages: 453-462, ISSN: 0022-1899
BackgroundDetection of poliovirus outbreaks relies on a complex laboratory algorithm of cell-culture, PCR and sequencing to distinguish wild-type and vaccine-derived polioviruses (VDPV) from Sabin-like strains. We investigated the potential for direct molecular detection and nanopore sequencing (DDNS) to accelerate poliovirus detection.MethodsWe analysed laboratory data for time required to analyse and sequence serotype-2 VDPV (VDPV2) in stool collected from children with acute flaccid paralysis in Africa (May 2016-February 2020). Impact of delayed detection on VDPV2 outbreak size was assessed through negative binomial regression.ResultsVDPV2 confirmation in 525 stools required a median of 49 days from paralysis onset (10th-90th percentile: 29-74), comprising collection and transport (median: 16 days), cell-culture (7 days), intratypic differentiation RT-qPCR (3 days) and sequencing (including shipping if required) (15 days). New VDPV2 outbreaks were confirmed a median of 35 days (27-60) after paralysis onset, which we estimate could be reduced to 16 days by DDNS (9-37). Because longer delays in confirmation and response were positively associated with more cases (p<0.001), we estimate that DDNS could reduce the number of VDPV2 cases before a response by 28% (95% CrI 12-42%).ConclusionsDDNS could accelerate poliovirus outbreak response, reducing their size and the cost of eradication.
Shaw A, Sim K, Rose G, et al., 2021, Premature neonatal gut microbial community patterns supporting an epithelial TLR-mediated pathway for necrotizing enterocolitis, BMC Microbiology, Vol: 21, Pages: 1-11, ISSN: 1471-2180
Background: Necrotising enterocolitis (NEC) is a devastating bowel disease, primarilyaffecting premature infants, with a poorly understood aetiology. Prior studies havefound associations in different cases with an overabundance of particular elements ofthe faecal microbiota (in particular Enterobacteriaceae or Clostridium perfringens ),but there has been no explanation for the different results found in different cohorts.Immunological studies have indicated that stimulation of the TLR4 receptor is involvedin development of NEC, with TLR4 signalling being antagonised by the activated TLR9receptor. We speculated that differential stimulation of these two components of thesignalling pathway by different microbiota might explain the dichotomous findings ofmicrobiota-centered NEC studies. Here we used shotgun metagenomic sequencingand qPCR to characterise the faecal microbiota community of infants prior to NEConset and in a set of matched controls. Bayesian regression was used to segregatecases from control samples using both microbial and clinical data.Results: We found that the infants suffering from NEC fell into two groups based ontheir microbiota; one with low levels of CpG DNA in bacterial genomes and the otherwith high abundances of organisms expressing LPS. The identification of thesecharacteristic communities was reproduced using an external metagenomic validationdataset. We propose that these two patterns represent the stimulation of a commonpathway at extremes; the LPS-enriched microbiome suggesting overstimulation ofTLR4, whilst a microbial community with low levels of CpG DNA suggests reduction ofthe counterbalance to TLR4 overstimulation.Conclusions: The identified microbial community patterns support the concept of NECresulting from TLR-mediated pathways. Identification of these signals suggestscharacteristics of the gastrointestinal microbial community to be avoided to preventNEC. Potential pre- or pro-biotic treatments may be designed to optimise TLRsig
Shaw AG, Troman C, Grassly N, 2020, Rapid and sensitive direct detection and identification of poliovirus from stool and environmental surveillance samples using nanopore sequencing, Journal of Clinical Microbiology, Vol: 58, Pages: 1-13, ISSN: 0095-1137
Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.
Kroll JS, Hall L, Kiu R, et al., 2020, Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome: an observational study, Cell Reports Medicine, Vol: 1, ISSN: 2666-3791
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.
Jorgensen D, Pons Salort M, Shaw A, et al., 2020, The role of genetic sequencing and analysis in the polio eradication program, Virus Evolution, Vol: 6, ISSN: 2057-1577
Genetic sequencing of polioviruses detected through clinical and environmental surveillance is used to confirm detection, identify their likely origin, track geographic patterns of spread and determine the appropriate vaccination response. The critical importance of genetic sequencing and analysis to the Global Polio Eradication Initiative has grown with the increasing incidence of vaccine-derived poliovirus infections in Africa specifically (470 reported cases in 2019), and globally, alongside persistent transmission of serotype 1 wild-type poliovirus in Pakistan and Afghanistan (197 reported cases in 2019). Adapting what has been learned about the virus genetics and evolution to address these threats has been a major focus of recent work. Here we review how phylogenetic and phylogeographic methods have been used to trace the spread of wild-type polioviruses and identify the likely origins of vaccine-derived polioviruses. We highlight the analysis methods and sequencing technology currently used and the potential for new technologies to speed up poliovirus detection and the interpretation of genetic data. At a pivotal point in the eradication campaign with the threat of anti-vaccine sentiment and donor and public fatigue, innovation is critical to maintain drive and overcome the last remaining circulating virus.
Shaw A, 2020, Rapid and sensitive direct detection and identification of poliovirus using nanopore sequencing
Shaw A, Cornwell E, Sim K, et al., 2020, Dynamics of toxigenic Clostridium perfringens colonisation in a cohort of prematurely born neonatal infants, BMC Pediatrics, Vol: 20, ISSN: 1471-2431
BackgroundClostridium perfringens forms part of the human gut microbiota and has been associated with life-threatening necrotising enterocolitis (NEC) in premature infants. Whether specific toxigenic strains are responsible is unknown, as is the extent of diversity of strains in healthy premature babies. We investigated the C. perfringens carrier status of premature infants in the neonatal intensive care unit, factors influence this status, and the toxic potential of the strains.MethodsC. perfringens was isolated by culture from faecal samples from 333 infants and their toxin gene profiles analysed by PCR. A survival analysis was used to identify factors affecting probability of carriage. Competitive growth experiments were used to explore the results of the survival analysis.Results29.4% of infants were colonized with C. perfringens before they left hospital. Three factors were inversely associated with probability of carriage: increased duration of maternal milk feeds, CPAP oxygen treatment and antibiotic treatment. C. perfringens grew poorly in breast milk and was significantly outperformed by Bifidobacterium infantis, whether grown together or separately. Toxin gene screening revealed that infants carried isolates positive for collagenase, perfringolysin O, beta 2, beta, becA/B, netB and enterotoxin toxin genes, yet none were observed to be associated with the development of NEC.ConclusionsApproximately a third of preterm infants are colonised 3 weeks after birth with toxin gene-carrying C. perfringens. We speculate that increased maternal breast milk, oxygen and antibiotic treatment creates an environment in the gut hostile to growth of C. perfringens. Whilst potentially toxigenic C. perfringens isolates were frequent, no toxin type was associated with NEC.
Kiu R, Sim K, Shaw A, et al., 2019, Genomic analysis of clostridium perfringens BEC/CPILE-positive, toxinotype D and E strains isolated from healthy children, Toxins, Vol: 11, Pages: 1-14, ISSN: 2072-6651
Clostridium perfringens toxinotype D, toxinotype E, and gastroenteritis-linked BEC/CPILE-positive strains have never been reported in healthy children. We isolated, whole-genome sequenced and bioinformatically characterised three C. perfringens isolates—type D (IQ1), type E (IQ2) and BEC/CPILE-positive (IQ3), recovered from the stools of three healthy two-year-olds, which were further compared to 128 C. perfringens genomes available from NCBI. The analysis uncovered a previously under-described putative toxin gene alv (alveolysin) encoded by isolates IQ2 and IQ3, which appeared to be a clade-specific trait associated with strains from domestic animals. A plasmid analysis indicated that the iota-toxin was encoded on a near-intact previously described plasmid pCPPB-1 in type E strain IQ2. The BEC genes becA and becB were carried on a near-identical pCPOS-1 plasmid previously associated with Japanese gastroenteritis outbreaks. Furthermore, a close phylogenetic relatedness was inferred between the French C. perfringens type E isolates cp515.17 and newly sequenced IQ2, suggesting geographical links. This study describes novel C. perfringens isolates from healthy individuals which encode important toxin genes, indicating the potential spread of these veterinary and clinically important strains and mobile genetic elements, and highlights areas for future research.
Powell E, Fontanella S, Boakes E, et al., 2019, Temporal association of the development of oropharyngeal microbiota with early life wheeze in a population-based birth cohort, EBioMedicine, Vol: 46, Pages: 486-498, ISSN: 2352-3964
Background A critical window in infancy has been proposed, during which the microbiota may affect subsequent health. The longitudinal development of the oropharyngeal microbiota is under-studied and may be associated with early-life wheeze. We aimed to investigate the temporal association of the development of the oropharyngeal microbiota with early-life wheeze.Methods A population-based birth cohort based in London, UK was followed for 24 months. We collected oropharyngeal swabs at six time-points. Microbiota was determined using sequencing of the V3-V5 region of the 16S rRNA-encoding gene. Medical records were reviewed for the outcome of doctor diagnosed wheeze. We used a time-varying model to investigate the temporal association between the development of microbiota and doctor-diagnosed wheeze. Findings 159 participants completed the study to 24 months and for 98 there was complete sequencing data at all timepoints and outcome data. Of these, 26 had doctor-diagnosed wheeze. We observed significant increase in the abundance of Neisseria between 9 and 24 months in children who developed wheeze (p=0∙003), while in those without wheezing there was a significant increment in the abundance of Granulicatella (p=0 ∙012) between 9 and 12 months, and of Prevotella (p=0 ∙018) after 18 months. Interpretation A temporal association between the respiratory commensal Granulicatella and also Prevotella with wheeze (negative), and between Neisseria and wheeze (positive) was identified in infants prior to one year of age. This adds to evidence for the proposed role of the microbiota in the development of wheeze.
Rowe WPM, Carrieri AP, Alcon-Giner C, et al., 2019, Streaming histogram sketching for rapid microbiome analytics, Microbiome, Vol: 7, ISSN: 2049-2618
BackgroundThe 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.ResultsWe apply streaming histogram sketching to microbiome samples as a form of dimensionality reduction, creating a compressed ‘histosketch’ that can efficiently represent microbiome k-mer spectra. Using public microbiome datasets, we show that histosketches can be clustered by sample type using the pairwise Jaccard similarity estimation, consequently allowing for rapid microbiome similarity searches via a locality sensitive hashing indexing scheme.Furthermore, we use a ‘real life’ example to show that histosketches can 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 (97% accuracy, 96% precision) and could subsequently be used to classify microbiome data streams in less than 3 s.ConclusionsOur method offers a new approach to rapidly process microbiome data streams, allowing samples to be rapidly clustered, indexed and classified. We also provide our implementation, Histosk
Wopereis H, Sim K, Shaw A, et al., 2018, Intestinal microbiota in infants at high risk for allergy: effects of prebiotics and role in eczema development, Journal of Allergy and Clinical Immunology, Vol: 141, Pages: 1334-1342.e5, ISSN: 1097-6825
Background: The development of gut microbiota in infancy is important in the maturation of the immune system. Deviations in colonization patterns have been associated with allergic manifestations (e.g. eczema), but exact microbiome dysfunctions underlying allergies remain unclear. We studied the gut microbiota of 138 infants at increased risk of developing allergy, participating in a clinical trial investigating the effectiveness of a partially hydrolyzed protein formula supplemented with non-digestible oligosaccharides (pHF-OS) on the prevention of eczema. Objective: The effects of the interventions and breastfeeding 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 using 16S rRNA-gene sequencing. Additionally, fecal pH and microbial metabolites were measured. Results: Fecal microbial composition, metabolites and pH of infants receiving pHF-OS was closer to breastfed infants than to infants receiving standard cow’s milk formula. Infants developing eczema by 18 months showed temporal differences that were marked by decreased relative abundances of Parabacteroides and Enterobacteriaceae at 4 weeks, and decreased relative abundances of lactate-utilizing bacteria producing butyrate at 26 weeks, namely Eubacterium and Anaerostipes spp., supported by increased lact ate and decreased butyrate levels. Conclusions: We showed that a pHF with specific prebiotics modulated the gut microbiota closer to that of breastfed infants. Additionally, we identified a potential link between the microbial activity and onset of eczema, which may reflect a suboptimal implementation of gut microbiota at specific developmental stages in infants at high-risk for allergy.
Rose G, Shaw AG, Sim K, et al., 2017, Antibiotic resistance potential of the healthy preterm infant gut microbiome, PeerJ, Vol: 5, ISSN: 2167-8359
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 13 genes per preterm infant, ranging across 8 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.
Shaw AG, Sim K, Powell E, et al., 2016, Latitude in Sample Handling and Storage for Infant Faecal Microbiota Studies: The Elephant in the Room?, Microbiome, Vol: 4, ISSN: 2049-2618
BackgroundIn this manuscript we investigate the “stones best left unturned” of sample storage and preparation and their implications for the next-generation sequencing of infant faecal microbial communities by the 16S rRNA gene.We present a number of experiments that investigate the potential effects of often overlooked methodology factors, establishing a “normal” degree of variation expected between replica sequenced samples. Sources of excess variation are then identified, as measured by observation of alpha diversity, taxonomic group counts and beta diversity magnitudes between microbial communities. ResultsExtraction of DNA from samples on different dates, by different people and even using varied sample weights results in little significant difference in downstream sequencing data. A key assumption in many studies is the stability of samples stored long term at -80°C prior to extraction. After two years, we see relatively few changes; increased abundances of lactobacilli and bacilli and a reduction in the overall OTU count. Where samples cannot be frozen, we find that storing samples at room temperature does lead to significant changes in the microbial community after two days. Mailing of samples during this time period (a common form of sample collection from out-patients for example) does not lead to any additional variation.ConclusionsImportant methodological standards can be drawn from these results; painstakingly created archives of infant faecal samples stored at -80 °C are still largely representative of the original community and varying factors in DNA extraction methodology have comparatively little effect on overall results. Samples taken should ideally be either frozen at -80 °C or extracted within two days if stored at room temperature, with mail samples being mailed on the day of collection.
Shaw AG, Black N, Rushd A, et al., 2016, Assessing the colonic microbiota in children: effects of sample site and bowel preparation, Journal of Pediatric Gastroenterology and Nutrition, Vol: 64, Pages: 230-237, ISSN: 1536-4801
ObjectivesInflammatory bowel disease (IBD) states are associated with gastrointestinal dysbiosis. Mucosal biopsy sampling, retrieving the bacterial community that most directly interacts with the host, is an invasive procedure, and we hypothesize 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. MethodsWe recruited a cohort of patients undergoing colonoscopy, collecting serial samples via differing methods (rectal swabs, biopsies and faecal matter/luminal contents) pre-bowel preparation, during colonoscopy and post-colonoscopy. Next generation sequencing was used to determine the structure of the microbial community. ResultsThe 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 IBD patients 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 pre-bowel 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. ConclusionsA clinically relevant description of the mucosal microbial community can be obtained via the non-invasive collection of luminal contents after bowel cleansing. Bowel preparation in a paediatric population results in no consistent sustained alterations to the gastrointestinal microbiota.
Shaw AG, Sim K, Randell P, et al., 2015, Late-onset bloodstream infection and perturbed maturation of the gastrointestinal microbiota in premature infants, PLOS One, Vol: 10, ISSN: 1932-6203
Sim K, Shaw AG, Randell P, et al., 2015, Dysbiosis anticipating necrotizing enterocolitis in very premature infants, Clinical Infectious Diseases, Vol: 60, Pages: 389-397, ISSN: 1537-6591
Background. Necrotizing enterocolitis (NEC) is a devastating inflammatory bowel disease of premature infants speculatively associated with infection. Suspected NEC can be indistinguishable from sepsis, and in established cases an infant may die within hours of diagnosis. Present treatment is supportive. A means of presymptomatic diagnosis is urgently needed. We aimed to identify microbial signatures in the gastrointestinal microbiota preceding NEC diagnosis in premature infants.Methods. Fecal samples and clinical data were collected from a 2-year cohort of 369 premature neonates. Next-generation sequencing of 16S ribosomal RNA gene regions was used to characterize the microbiota of prediagnosis fecal samples from 12 neonates with NEC, 8 with suspected NEC, and 44 controls. Logistic regression was used to determine clinical characteristics and operational taxonomic units (OTUs) discriminating cases from controls. Samples were cultured and isolates identified using matrix-assisted laser desorption/ionization–time of flight. Clostridial isolates were typed and toxin genes detected.Results. A clostridial OTU was overabundant in prediagnosis samples from infants with established NEC (P = .006). Culture confirmed the presence of Clostridium perfringens type A. Fluorescent amplified fragment-length polymorphism typing established that no isolates were identical. Prediagnosis samples from NEC infants not carrying profuse C. perfringens revealed an overabundance of a Klebsiella OTU (P = .049). Prolonged continuous positive airway pressure (CPAP) therapy with supplemental oxygen was also associated with increased NEC risk.Conclusions. Two fecal microbiota signatures (Clostridium and Klebsiella OTUs) and need for prolonged CPAP oxygen signal increased risk of NEC in presymptomatic infants. These biomarkers will assist development of a screening tool to allow very early diagnosis of NEC.Clinical Trials Registration. NCT01102738.
Wopereis H, Sim K, Shaw A, et al., 2014, The developmental gut microbiota is modulated towards a pattern closer to breastfed infants by a partially hydrolyzed cow's milk formula supplemented with specific prebiotic oligosaccharides, European-Academy-of-Allergy-and-Clinical-Immunology Congress, Publisher: WILEY-BLACKWELL, Pages: 315-315, ISSN: 0105-4538
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Sim K, Powell E, Shaw AG, et al., 2013, The neonatal gastrointestinal microbiota: the foundation of future health?, ARCHIVES OF DISEASE IN CHILDHOOD-FETAL AND NEONATAL EDITION, Vol: 98, Pages: F362-F364, ISSN: 1359-2998
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- Citations: 9
Martin C, Chen S, Heilos D, et al., 2010, Changed genome heterochromatinization upon prolonged activation of the Raf/ERK signaling pathway., PLoS One, Vol: 5
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
Maya-Mendoza A, Olivares-Chauvet P, Shaw A, et al., 2010, S phase progression in human cells is dictated by the genetic continuity of DNA foci., PLoS Genet, Vol: 6
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.
Shaw A, Olivares-Chauvet P, Maya-Mendoza A, et al., 2010, S-phase progression in mammalian cells: modelling the influence of nuclear organization., Chromosome Res, Vol: 18, Pages: 163-178
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.
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