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Journal articleRose G, Shaw AG, Sim K, et al., 2017,
Antibiotic resistance potential of the healthy preterm infant gut microbiome
, PeerJ, Vol: 5, ISSN: 2167-8359Background: 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.
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Journal articleMcArdle 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: 1932-6203Carboxyhemoglobin 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.
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Conference paperWopereis 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 -
Journal articleShaw 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: 2049-2618BackgroundIn 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.
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Journal articleShaw 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: 1536-4801ObjectivesInflammatory 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.
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Journal articleCunnington 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 -
Journal articlePratt 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: 1098-5530Bacterial 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
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Journal articleKroll JS, Pratt AJ, DiDonato M, et al.,
Structural, Functional, and Immunogenic Insights on Cu,ZnSuperoxide Dismutase Pathogenic Virulence Factors from Neisseriameningitidis and Brucella abortus.
, Journal of Bacteriology, ISSN: 1098-5530 -
Journal articleShaw 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 -
Journal articleSim 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: 1537-6591Background. 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.
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