144 results found
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.
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
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
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.
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
Faraday C, Hamad S, Jones KD, et al., 2018, Characteristics and incidence of transfusion-associated necrotizing enterocolitis in the UK, Journal of Maternal-Fetal and Neonatal Medicine, Vol: 33, Pages: 398-403, ISSN: 1476-4954
BACKGROUND AND AIMS: The etiology of necrotizing enterocolitis (NEC) is unclear and postulated as being multifactorial. It has been suggested that one causative factor is the transfusion of packed red blood cells (PRBCs) leading to the disease entity commonly referred to as transfusion-associated NEC (TANEC). TANEC has been reported in North America but its incidence has not been formally investigated in the UK. Our aims were to identify the incidence of NEC and TANEC in tertiary-level UK neonatal units and to describe characteristics of TANEC cases. MATERIALS AND METHODS: Using strict case definitions for NEC and TANEC, we undertook a retrospective review to estimate the incidence of TANEC cases occurring in four UK tertiary-level centers during a 38-month period. RESULTS: Of 8007 consecutive neonatal admissions of all gestations to the four centers, 68 babies went on to develop NEC and all affected infants were of very low birth weight (VLBW); 34 of these had previously received a transfusion of PRBCs but did not fit the diagnostic criteria for TANEC, whereas 15 (22%) of the 68 babies with NEC qualified as TANEC cases. UK cases occurred at an earlier postnatal age than cases reported in multiple large North American series and were of a lower birth weight. CONCLUSIONS: We have confirmed the presence of TANEC in the UK VLBW neonatal population. Its incidence lies within the wide range described in previous reports of this phenomenon globally, though with some local variation in characteristics. Further work is needed to clarify causation, pathophysiology, and possible mechanisms of prevention of TANEC.
Bidmos FA, Nadel S, Screaton GR, et al., 2018, Cross-reactive bactericidal antimeningococcal antibodies can be isolated from convalescing invasive Meningococcal disease patients using reverse vaccinology 2.0, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224
The threat from invasive meningococcal disease remains a serious source of concern despite the licensure and availability of vaccines. A limitation of currently-available serogroup B vaccines is the breadth of coverage afforded, resulting from the capacity for extensive variation of the meningococcus and its huge potential for the generation of further diversity. Thus, the continuous search for candidate antigens that will compose supplementary or replacement vaccines is mandated. Here, we describe successful efforts to utilize the reverse vaccinology 2.0 approach to identify novel functionally-immunogenic meningococcal antigens. In this study, eight broadly cross-reactive sequence-specific anti-meningococcal human monoclonal antibodies (hmAbs) were cloned from 4 ml of blood taken from a 7-month old sufferer of invasive meningococcal disease (IMD). Three of these hmAbs possessed human complement-dependent bactericidal activity against meningococcal serogroup B strains of disparate PorA and 4CMenB antigen sequence types, strongly suggesting that the target(s) of these bactericidal hmAbs are not PorA (the immunodominant meningococcal antigen), factor-H binding protein (fHbp) or other components of currently-available meningococcal vaccines. Reactivity of the bactericidal hmAbs was confirmed to a single ca. 35 kDa protein in western blots. Unequivocal identification of this antigen is currently ongoing. Collectively, our results provide proof-of-principle for the use of reverse vaccinology 2.0 as a powerful tool in the search for alternative meningococcal vaccine candidate antigens.
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.
Kroll J, Månsson V, Gilsdorf J, et al., 2018, Capsule Typing of Haemophilus influenzae by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry, Emerging Infectious Diseases, Vol: 24, Pages: 443-452, ISSN: 1080-6040
Pammi M, Cope J, Tarr P, et al., 2017, Intestinal dysbiosis in preterm infants preceding necrotizing enterocolitis: a systematic review and meta-analysis, Microbiome, Vol: 5, ISSN: 2049-2618
BackgroundNecrotizing enterocolitis (NEC) is a catastrophic disease of preterm infants, and microbial dysbiosis has been implicated in its pathogenesis. Studies evaluating the microbiome in NEC and preterm infants lack power and have reported inconsistent results.Methods and resultsOur objectives were to perform a systematic review and meta-analyses of stool microbiome profiles in preterm infants to discern and describe microbial dysbiosis prior to the onset of NEC and to explore heterogeneity among studies. We searched MEDLINE, PubMed, CINAHL, and conference abstracts from the proceedings of Pediatric Academic Societies and reference lists of relevant identified articles in April 2016. Studies comparing the intestinal microbiome in preterm infants who developed NEC to those of controls, using culture-independent molecular techniques and reported α and β-diversity metrics, and microbial profiles were included. In addition, 16S ribosomal ribonucleic acid (rRNA) sequence data with clinical meta-data were requested from the authors of included studies or searched in public data repositories. We reprocessed the 16S rRNA sequence data through a uniform analysis pipeline, which were then synthesized by meta-analysis.We included 14 studies in this review, and data from eight studies were available for quantitative synthesis (106 NEC cases, 278 controls, 2944 samples). The age of NEC onset was at a mean ± SD of 30.1 ± 2.4 weeks post-conception (n = 61). Fecal microbiome from preterm infants with NEC had increased relative abundances of Proteobacteria and decreased relative abundances of Firmicutes and Bacteroidetes prior to NEC onset. Alpha- or beta-diversity indices in preterm infants with NEC were not consistently different from controls, but we found differences in taxonomic profiles related to antibiotic exposure, formula feeding, and mode of delivery. Exploring heterogeneity revealed differences in micr
Kroll J, Li M-S, langford P, 2017, Inactivation of NMB0419 encoding a Sel1-like repeat (SLR) protein in Neisseria meningitidis is associated with differential expression of genes belonging to the Fur regulon and reduced intra-epithelial replication, Infection and Immunity, Vol: 85, ISSN: 1098-5522
Neisseria meningitidis is a commensal microbe that colonizes the human nasopharynx but occasionally invades the bloodstream to cause life-threatening infection. N. meningitidis MC58 NMB0419 encodes a Sel1-like repeat (SLR)-containing protein, previously implicated in invasion of epithelial cells. A gene-regulatory function was revealed in Escherichia coli expressing plasmid-borne NMB0419 showing significantly increased epithelial adherence compared to wild type, due to increased expression of mannose-sensitive type 1 pili. While a meningococcal NMB0419 mutant did not have altered epithelial adherence, in a transcriptome-wide comparison of wild type and an NMB0419 mutant, a large proportion of genes differentially regulated in the mutant were involved in iron acquisition and metabolism. Fifty one and 38% of genes respectively up- and down-regulated in the NMB0419 mutant had previously been identified as being induced and repressed by meningococcal Fur. An in vitro growth defect of the NMB0419 mutant under iron-restriction was consistent with the down-regulation of tbpAB and hmbR, while an intra-epithelial replication defect was consistent with the down-regulation of tonB, exbB and exbD, based on a known phenotype of a meningococcal tonB mutant. Disruption of the N-terminal NMB0419 signal peptide, predicted to export the protein beyond the cytoplasmic membrane, resulted in loss of functional traits in N. meningitidis and E. coli. Our study indicates that the expression of NMB0419 is associated with transcriptional changes counterbalancing the regulatory function of Fur, offering a new perspective on regulatory mechanisms involved in meningococcal interaction with epithelial cells, and suggests new insights into the roles of SLR-containing genes in other bacteria.
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.
McArdle AJ, Webbe J, Sim K, et al., 2016, Determinants of Carboxyhemoglobin Levels and Relationship with Sepsis in a Retrospective Cohort of Preterm Neonates, PLOS One, Vol: 11, ISSN: 1932-6203
Carboxyhemoglobin levels in blood reflect endogenous carbon monoxide production and are often measured during routine blood gas analysis. Endogenous carbon monoxide production has been reported to be increased during sepsis, but carboxyhemoglobin levels have not been thoroughly evaluated as a biomarker of sepsis. We sought to determine whether carboxyhemoglobin levels were elevated during sepsis in a high risk population of premature neonates. We conducted a retrospective cohort study of 30 infants in two neonatal intensive care units using electronic medical and laboratory records. The majority of infants were extremely premature and extremely low birth weight, and 25 had at least one episode of sepsis. We collected all carboxyhemoglobin measurements during their in-patient stay and examined the relationship between carboxyhemoglobin and a variety of clinical and laboratory parameters, in addition to the presence or absence of sepsis, using linear mixed-effect models. We found that postnatal age had the most significant effect on carboxyhemoglobin levels, and other significant associations were identified with gestational age, hemoglobin concentration, oxyhemoglobin saturation, and blood pH. Accounting for these covariates, there was no significant relationship between the onset of sepsis and carboxyhemoglobin levels. Our results show that carboxyhemoglobin is unlikely to be a clinically useful biomarker of sepsis in premature infants, and raise a note of caution about factors which may confound the use of carbon monoxide as a clinical biomarker for other disease processes such as hemolysis.
Wopereis H, Sim K, Shaw A, et al., 2016, Decreased microbial conversion of lactic acid into butyrate in infants developing eczema, Meeting of the European-Academy-of-Allergy-and-Clinical-Immunology, Publisher: Wiley, Pages: 168-169, ISSN: 0105-4538
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.
Cunnington A, Sim K, Deierl A, et al., 2016, “Vaginal seeding” of infants born by Caesarean section.How should health professionals engage with this increasingly popular but unproven practice?, BMJ, Vol: 352, ISSN: 0959-8138
Pratt AJ, DiDonato M, Shin DS, et al., 2015, Structural, functional and immunogenic insights on Cu,Zn Superoxide Dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus, Journal of Bacteriology, Vol: 197, Pages: 3834-3847, ISSN: 1098-5530
Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens. IMPORTANCE: By protecting microbes against reactive oxygen insults
Kroll JS, Pratt AJ, DiDonato M, et al., 2015, Structural, Functional, and Immunogenic Insights on Cu,ZnSuperoxide Dismutase Pathogenic Virulence Factors from Neisseriameningitidis and Brucella abortus., Journal of Bacteriology, ISSN: 1098-5530
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
Laydon DJ, Melamed A, Sim A, et al., 2014, Quantification of HTLV-1 Clonality and TCR Diversity, PLOS COMPUTATIONAL BIOLOGY, Vol: 10
Hey A, Li M-S, Hudson MJ, et al., 2013, Transcriptional Profiling of <i>Neisseria meningitidis</i> Interacting with Human Epithelial Cells in a Long-Term <i>In Vitro</i> Colonization Model, INFECTION AND IMMUNITY, Vol: 81, Pages: 4149-4159, ISSN: 0019-9567
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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