Michael J Cox

Imperial College London, Londinium, England, United Kingdom

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Publications (37)166.52 Total impact


  • No preview · Conference Paper · Dec 2015

  • No preview · Article · Aug 2015 · American Journal of Respiratory and Critical Care Medicine
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    ABSTRACT: Background: Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants. Method: Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity. Results: From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis. Conclusions: The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes.
    Preview · Article · Jul 2015 · PLoS ONE
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    ABSTRACT: C21 CONFLICT OR PEACEFUL CO-EXISTENCE? THE BACTERIAL LUNG MICROBIOME AND HOST IMMUNITY / Poster Discussion Session / Tuesday, May 19/9:30 AM-11:30 AM / Mile High Ballroom 1 A-B (Lower Level) Colorado Convention Center The Genus Haemophilus Was Dominant In A Cohort With Community Acquired Pneumonia And Low Species Diversity Was Related To Age And Prior Pulmonary Disease D. G. Wootton1, M. J. Cox2, G. L. Hickey3, O. Eneje4, J. Court4, 5, L. Macfarlane4, S. Wilks4, M. Woodhead6, W. O. Cookson2, M. F. Moffatt2, P. J. Diggle3, S. B. Gordon4 University of Liverpool, Liverpool, United Kingdom, 2Imperial College London, 6LY, United Kingdom, University of Liverpool, 7BE, United Kingdom, 4Liverpool School of Tropical Medicine, 5QA, United Kingdom, 5, 6Central Manchester University Hospitals NHS Foundation Trust, 9WM, United Kingdom Corresponding author's email: dwootton@liverpool.ac.uk Rationale The causative organism(s) responsible for the syndrome of community acquired pneumonia (CAP) are frequently not defined. Culture-independent techniques offer the possibility of new insights into the range and interactions of pathogens in CAP. Here we explored the bacteria present in sputum from patients with CAP by 16SrRNA gene sequencing. Methods Between February 2011 and March 2013, and as part of a prospective observational cohort study, CAP admissions were recruited within 24 hours of admission at two hospitals in Liverpool. Eligibility criteria were:- Aged ≥ 16yrs; Treatment for CAP (British Thoracic Society in-hospital definition) Exclusion Criteria were:- Admission within the last 14 days; Bronchiectasis or cystic fibrosis; Immunocompromised Self-expectorated sputum was frozen at -80oC in sealed, gamma irradiated pots with no prior processing. DNA was extracted using FastDNATM extraction kits and a bead-beater. Pooled, barcoded products from quadruplicate PCRs of the V3-5 region of the 16SrRNA gene were sequenced using the Roche 454FLX platform. Compositional analysis of relationships between individual taxa and clinical variables was performed using the R package ALDeX2. Bacterial species diversity was explored with the R package Phyloseq. Results 169 subjects provided 86 acute sputum samples of which 76 were analysed (44.7% women, median age 68.5yrs (IQR 49 – 76), median CURB65 score 2 (IQR 0-2.25), median pro-calcitonin 0.73 ng/ml (IQR 0.18-4.30), median length of stay 5 days (IQR 2.75 – 7.25)). Although several individual bacterial operational taxonomic units (OTUs) appeared to be associated with clinical parameters none were significant after correction for multiple testing (Benjamini-Hochberg method). Following rarefaction to 549 reads and removal of singletons, 774 OTUs were identified across all sputum samples; median per-sample was 43 (IQR 23-62). Multiple-regression revealed age (P=0.014) and underlying pulmonary disease (P=0.006) were independently associated with the diversity (Shannon) of bacterial species in a subject’s sputum. Haemophilus_617 was the most abundant OTU in these samples (Figure). Image.png Conclusions In this cohort of patients with CAP, increasing age and underlying pulmonary disease were associated with lower bacterial species diversity. These are well documented risk factors for CAP and are associated with worse outcomes. In culture-based studies pneumococcus is the most commonly implicated bacterial pathogen in CAP. In this study streptococcal OTUs were amongst the most abundant but Haemophilus_617 was twice as abundant. This raises the possibility that bacteria of the genus Haemophilus may be more important than has previously been thought in the context of CAP. This abstract is funded by: This work was supported by a Doctoral Research Fellowship awarded to D.Wootton by the UK National Institute of Health Research (NIHR) and by the Wellcome Trust. Am J Respir Crit Care Med 191;2015:A3954 Internet address: www.atsjournals.org Online Abstracts Issue
    Full-text · Conference Paper · May 2015
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    ABSTRACT: Introduction The prevalence and diversity of fungal airway isolates is increasing in cystic fibrosis (CF). Amidst an extending spectrum of fungal complications, lack of standardised mycology methods and poor sensitivity of culture-dependent techniques renders interpretation of isolates challenging. Aims To evaluate the diagnostic utility of fungal cytology and microbiology stains in addition to prolonged sputum culture from adult CF patients in comparison to standard mycology techniques. Secondly, to develop a novel, next-generation sequencing assay targeting the ITS2 region of the fungal ribosomal-RNA gene to comprehensively profile the sputum fungal microbiota. Methods Sputum samples were investigated by a panel of three mycology techniques: prolonged fungal culture (each examined at: Day7, D14, D21, D28); Calcofluor White (CFW) stain; Grocott’s Methenamine Silver (GMS) stain. A cohort of samples was also subject to broad-spectrum fungal next-generation sequencing. Results 25 adult patients provided 45 sputum samples. Four fungal species were cultivatable: Candida species (26.6%); Aspergillus fumigatus (4.4%); Scedosporium apiospermum (15.5%) and Exophiala dermatitidis (11.1%). Prolonged culture significantly increased overall fungal prevalence by 22% compared to standard duration (D7) p = 0.008). A significant increase of 11.1% in S. apiopspermum prevalence was observed p = 0.02), whilst all E. dermatitidis isolates required prolonged culture. The sensitivity of GMS and CFW stains (85% and 93%) compared favourably to standard duration culture (29%). DNA extracted from a pilot group of these sputum samples (n = 14/45) was subject to PCR using barcode-indexed ITS2 primers designed for Illumina-MiSeq amplicon sequencing. Fungal taxa were detected in all samples, of which seven samples (50%) were negative after prolonged culture. Preliminary sequencing analysis of an extended sample cohort (n = 30) has detected 89 fungal taxa, from which only four species were cultured. Conclusions Prolonged fungal culture is associated with a significant increase in fungal prevalence. The increased sensitivity is restricted to less common filamentous fungi associated with increasing pathogenicity: S. apiospermum and E. dermatitidis. The predictive value of stains in identifying samples positive at prolonged culture, but negative at standard duration illustrates their clinical utility. Illumina-MiSeq ITS2-amplicon sequencing directly from sputum has identified a more diverse CF airways fungal microbiota. Preliminary analysis suggests that this is a highly sensitive tool for detecting fungi from sputum, including species which are refractory to standard and enhanced culture.
    No preview · Article · Dec 2014 · Thorax
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    ABSTRACT: Background The study of microbial communities has been revolutionised in recent years by the widespread adoption of culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder of these sequence-based approaches is the presence of contamination in DNA extraction kits and other laboratory reagents. Results In this study we demonstrate that contaminating DNA is ubiquitous in commonly used DNA extraction kits and other laboratory reagents, varies greatly in composition between different kits and kit batches, and that this contamination critically impacts results obtained from samples containing a low microbial biomass. Contamination impacts both PCR-based 16S rRNA gene surveys and shotgun metagenomics. We provide an extensive list of potential contaminating genera, and guidelines on how to mitigate the effects of contamination. Conclusions These results suggest that caution should be advised when applying sequence-based techniques to the study of microbiota present in low biomass environments. Concurrent sequencing of negative control samples is strongly advised. Electronic supplementary material The online version of this article (doi:10.1186/s12915-014-0087-z) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2014 · BMC Biology
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    ABSTRACT: Introduction The lower airway microbiota is important in chronic lung disease but young children do not expectorate, making longitudinal studies difficult. Upper airway samples, of uncertain reliability, are used as a surrogate. Whilst throat swabs (TS) have shown differences in the microbiota between healthy and wheezing children [PLoS One 2012;7(10):e46803], the role of cough swabs (CS) is at present unclear. This study assessed the correlation between upper and lower airway samples as a prelude to a longitudinal study. We hypothesised that upper airway samples reflect the lower airway microbiota. Methods TS and lower airway samples (bronchoalveolar lavage fluid, bronchial brushings or both) were collected from 55 children undergoing a clinically indicated fibre-optic bronchoscopy (FOB), including CS from 35 children. Bacterial DNA was extracted for quantitative PCR (qPCR) and 454 FLX pyrosequencing of the V3-V5 region of the 16S rRNA bacterial gene. Data analysis was performed with Quantitative Insights Into Microbial Ecology (QIIME) and Phyloseq in R. Results FOB indications included: recurrent lower respiratory tract infections (47%); cystic fibrosis (CF) (35%), and Primary Ciliary Dyskinesia (PCD) (11%). Only seven CS amplified successfully. 375,268 high quality 16S rRNA sequences were obtained from 132 upper and lower airway samples. No significant difference was seen in richness (H = 1.352, 2 d.f., p = 0.509), evenness (H = 3.942, 2 d.f, p = 0.139), Shannon’s diversity index (H = 1.38, 2 d.f., p = 0.501), and Inverse Simpson’s diversity index (F (2,91) = 0.547, p = 0.581) between TS and lower airway samples. Beta-diversity (diversity between samples) was significantly different; ≤7.2% of variation in diversity attributed to the sampling method (p = 0.002). Greater variation was observed between underlying pathologies and between patients (≤41.2% and 68.3% respectively, p = 0.001). Conclusions CS are not useful for pyrosequencing. TS are predictive of the lower airway microbiota and can differentiate diseases. TS are therefore potentially useful in studying longitudinal changes in the microbiota in children with chronic lung diseases.
    No preview · Article · Dec 2014 · Thorax
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    ABSTRACT: 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.
    Preview · Article · Oct 2014 · Clinical Infectious Diseases
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    ABSTRACT: Rationale: IPF is a progressive lung disease of unknown cause that leads to respiratory failure and death within 5 years of diagnosis. Overt respiratory infection and immunosuppression carry a high morbidity and mortality, while polymorphisms in genes related to epithelial integrity and host defence predispose to IPF. Objectives: To investigate the role of bacteria in the pathogenesis and progression of IPF. Methods: We prospectively enrolled patients diagnosed with IPF according to international criteria together with healthy smokers, non-smokers and subjects with moderate Chronic Obstructive Pulmonary Disease (COPD) as controls. Subjects underwent bronchoalveolar lavage (BAL) from which genomic DNA was isolated. The V3-V5 region of the bacterial 16S rRNA gene was amplified, allowing quantification of bacterial load and identification of communities by 16S rRNA qPCR and pyrosequencing. Measurements and Main Results: Sixty five IPF patients had double the burden of bacteria in Bronchoalveolar lavage (BAL) fluid compared to 44 controls. Baseline bacterial burden predicted the rate of decline in lung volume, risk of death and associated independently with the rs35705950 polymorphism of the MUC5B mucin gene, a proven host susceptibility factor for IPF. Sequencing yielded 912,883 high quality reads from all subjects. We identified Haemophilus, Streptococcus, Neisseria and Veillonella spp. to be more abundant in cases than controls. Regression analyses indicated that these specific OTUs as well as bacterial burden associated independently with IPF. Conclusions: IPF is characterised by an increased bacterial burden in BAL that predicts decline in lung function and death. Trials of antimicrobial therapy are needed to determine if microbial burden is pathogenic in the disease.
    Full-text · Article · Sep 2014 · American Journal of Respiratory and Critical Care Medicine

  • No preview · Article · Nov 2013 · Thorax
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    ABSTRACT: Rationale: Rhinovirus (RV) infection is followed by significantly increased frequencies of positive, potentially pathogenic, sputum cultures in chronic obstructive pulmonary disease (COPD). It however remains unclear whether these represent de novo infections or an increased load of organisms from the complex microbial communities (microbiome) in the lower airways. Objectives: To investigate the effect of RV infection on the airway bacterial microbiome. Methods: Subjects with COPD (n=14) and healthy controls with normal lung function (n=17) were infected with rhinovirus. Induced sputum was collected at baseline prior to rhinovirus inoculation and again at days 5, 15 and 42 post rhinovirus infection and DNA extracted. The V3-V5 region of the bacterial 16S rRNA gene was amplified and pyrosequenced resulting in 370,849 high quality reads from 112 of the possible 124 time points. Measurements and Main Results: Fifteen days post rhinovirus infection there was a six-fold increase in 16S copy number (P=0.007) and a 16% rise in numbers of Proteobacterial sequences, in particular potentially pathogenic Haemophilus influenzae (P=2.7e-20), from a pre-existing community. These changes only occurred in the sputum microbiome of COPD subjects and were still evident 42 days post infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and non-smokers. Conclusions: Following rhinovirus infection there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of COPD subjects. This is not observed in healthy individuals. Our findings suggest that RV infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.
    No preview · Article · Aug 2013 · American Journal of Respiratory and Critical Care Medicine
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    ABSTRACT: Molecular techniques have revolutionised the practise of standard microbiology. In particular, 16S rRNA sequencing, whole microbial genome sequencing and metagenomics are revealing the extraordinary diversity of microorganisms on Earth and their vast genetic and metabolic repertoire.The increase in length, accuracy and number of reads generated by high-throughput sequencing has coincided with a surge in interest in the human microbiota, the totality of bacteria associated with the human body, in both health and disease. Traditional views of host/pathogen interactions are being challenged as the human microbiota are being revealed to be important in normal immune system function, to diseases not previously thought to have a microbial component and to infectious diseases with unknown aetiology.In this review, we introduce the nature of the human microbiota and application of these three key sequencing techniques for its study, highlighting both advances and challenges in the field. We go on to discuss how further adoption of additional techniques, also originally developed in environmental microbiology, will allow the establishment of disease causality against a background of numerous, complex and interacting microorganisms within the human host.
    No preview · Article · Aug 2013 · Human Molecular Genetics

  • No preview · Article · Apr 2013 · European Respiratory Journal
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    Full-text · Dataset · Dec 2012
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    ABSTRACT: Correspondence noise is a major factor limiting direction discrimination performance in random-dot kinematograms [1]. In the current study we investigated the influence of correspondence noise on Dmax, which is the upper limit for the spatial displacement of the dots for which coherent motion is still perceived. Human direction discrimination performance was measured, using 2-frame kinematograms having leftward/rightward motion, over a 200-fold range of dot-densities and a four-fold range of dot displacements. From this data Dmax was estimated for the different dot densities tested. A model was proposed to evaluate the correspondence noise in the stimulus. This model summed the outputs of a set of elementary Reichardt-type local detectors that had receptive fields tiling the stimulus and were tuned to the two directions of motion in the stimulus. A key assumption of the model was that the local detectors would have the radius of their catchment areas scaled with the displacement that they were tuned to detect; the scaling factor k linking the radius to the displacement was the only free parameter in the model and a single value of k was used to fit all of the psychophysical data collected. This minimal, correspondence-noise based model was able to account for 91% of the variability in the human performance across all of the conditions tested. The results highlight the importance of correspondence noise in constraining the largest displacement that can be detected.
    Preview · Article · Oct 2012 · PLoS ONE
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    ABSTRACT: Observations that the airway microbiome is disturbed in asthma may be confounded by the widespread use of antibiotics and inhaled steroids. We have therefore examined the oropharyngeal microbiome in early onset wheezing infants from a rural area of tropical Ecuador where antibiotic usage is minimal and glucocorticoid usage is absent. We performed pyrosequencing of amplicons of the polymorphic bacterial 16S rRNA gene from oropharyngeal samples from 24 infants with non-infectious early onset wheezing and 24 healthy controls (average age 10.2 months). We analyzed microbial community structure and differences between cases and controls by QIIME software. We obtained 76,627 high quality sequences classified into 182 operational taxonomic units (OTUs). Firmicutes was the most common and diverse phylum (71.22% of sequences) with Streptococcus being the most common genus (49.72%). Known pathogens were found significantly more often in cases of infantile wheeze compared to controls, exemplified by Haemophilus spp. (OR = 2.12, 95% Confidence Interval (CI) 1.82-2.47; P = 5.46×10(-23)) and Staphylococcus spp. (OR = 124.1, 95%CI 59.0-261.2; P = 1.87×10(-241)). Other OTUs were less common in cases than controls, notably Veillonella spp. (OR = 0.59, 95%CI = 0.56-0.62; P = 8.06×10(-86)). The airway microbiota appeared to contain many more Streptococci than found in Western Europe and the USA. Comparisons between healthy and wheezing infants revealed a significant difference in several bacterial phylotypes that were not confounded by antibiotics or use of inhaled steroids. The increased prevalence of pathogens such as Haemophilus and Staphylococcus spp. in cases may contribute to wheezing illnesses in this age group.
    Full-text · Article · Oct 2012 · PLoS ONE
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    ABSTRACT: Methyl halides have a significant impact on atmospheric chemistry, particularly in the degradation of stratospheric ozone. Bacteria are known to contribute to the degradation of methyl halides in the oceans and marine bacteria capable of using methyl bromide and methyl chloride as sole carbon and energy source have been isolated. A genetic marker for microbial degradation of methyl bromide ( cmuA ) was used to examine the distribution and diversity of these organisms in the marine environment. Three novel marine clades of cmuA were identified in unamended seawater and in marine enrichment cultures degrading methyl halides. Two of these cmuA clades are not represented in extant bacteria, demonstrating the utility of this molecular marker in identifying uncultivated marine methyl halide-degrading bacteria. The detection of populations of marine bacteria containing cmuA genes suggests that marine bacteria employing the CmuA enzyme contribute to methyl halide cycling in the ocean.
    Full-text · Article · Jun 2012 · FEMS Microbiology Letters

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Publication Stats

812 Citations
166.52 Total Impact Points

Institutions

  • 2011-2015
    • Imperial College London
      Londinium, England, United Kingdom
  • 2012
    • University of Bradford
      • Department of Optometry and Vision Science
      Bradford, England, United Kingdom
  • 2005-2012
    • The University of Warwick
      • • School of Life Sciences
      • • Biological Sciences
      Coventry, ENG, United Kingdom
  • 2010
    • University of California, San Francisco
      • Division of Gastroenterology
      San Francisco, CA, United States
  • 2007
    • University of Waterloo
      • Department of Biology
      Ватерлоо, Ontario, Canada