Leah Cuthbertson - Academia.edu (original) (raw)

Papers by Leah Cuthbertson

Background: Asthma exacerbations are frequently associated with respiratory viruses. The role of ... more Background: Asthma exacerbations are frequently associated with respiratory viruses. The role of bacteria is unclear yet antibiotics are often prescribed. We examined the airway microbiota during a naturally-occurring cold (NC) and experimental rhinovirus (RV) infection. Methods: Two independent studies were performed. 46 asthmatic subjects experienced natural colds. Sputum samples were taken before and during cold. A further 11 asthmatic and 12 healthy subjects were experimentally infected with RV-16. Bronchoalveolar lavage (BAL) was obtained at baseline and post-infection. Sputum and BAL samples underwent 16S rRNA gene sequencing. Results: In both studies, increased Neisseria sp. relative abundance following cold/ RV-16 infection was significantly associated with greater peak flow (PEF) decline (NC: R 2 = 0.16, p 2 = 0.41, p Prevotella spp. relative abundance correlated with lower PEF decline (NC: R 2 = 0.14, p 2 = 0.56, p Conclusion: A microbiota dominated by Neisseria sp. was associated with greater PEF decline in both natural cold and experimental RV-16 infection, whilst ‘commensals’ dominated ( Prevotella spp.) community correlated with better outcomes. Further work is needed to evaluate if the microbiota actively contributes to increased airway inflammation.

EBioMedicine, May 1, 2022

The European respiratory journal, Oct 28, 2021

Respiratory syncytial virus (RSV) is the commonest cause of acute lower respiratory tract infecti... more Respiratory syncytial virus (RSV) is the commonest cause of acute lower respiratory tract infection (RTI) in infants, resulting in seasonal surges in hospital admissions [1]. In addition to its impact in childhood, RSV is increasingly recognised as a cause of morbidity and mortality in elderly persons [2]. The virus is highly contagious and regularly causes reinfections, despite limited genetic diversity [3]. Safe and effective vaccines have so far proven elusive [2]. Severe infantile bronchiolitis is associated with recurrent wheeze and asthma in later childhood [4]. One possible explanation is that RSV infection causes lasting changes in the respiratory microbial community leading to secondary effects on physiology and immunity. Alternatively, it has been proposed that disordered microbial communities predispose to severe RSV disease [5]. Longitudinal birth cohort studies have demonstrated that frequent RTIs in early life are associated with a perturbed respiratory microbiota, dominated by Moraxella, that may precede viral infections. These studies indicate associations between the respiratory microbiota and viral infection [6]. Nonetheless, the issues of cause and effect have been difficult to resolve in observational studies. To determine the effect of RSV infection on the respiratory microbiome, we inoculated 37 healthy non-smoking adults between 18 and 50 years of age with an established RSV challenge inoculum, Memphis 37 (RSV-A M37) [7]. We anticipated that infection would result in significant changes in the bacterial community within the upper respiratory tract. Baseline samples were collected prior to participant inoculation by intranasal drops of Memphis 37 [8]. Subjects were quarantined for 10 days post-infection and sampled daily [9]. Participants returned post-quarantine for further sampling at days 14 and 28. The study was approved by the UK National Research Ethics Services (study numbers 10/H0711/94 and 11/LO/1826) and written informed consent was provided by all subjects [10]. Endpoint titre of IgA against RSV was determined from nasal wash samples [7]. Cytokine and chemokine inflammatory mediators within nasosorption eluates were quantified by MSD multiplex immunoassay [10] (MesoScale Discovery, Rockville, MD, USA). DNA was extracted from oropharyngeal swabs; SYBR green qPCR and 16S rRNA gene sequencing was performed [11]. Sequences were submitted to the European Nucleotide Database, project number PRJEB28323. Sequence processing was carried out as previously described [11]. All further analysis was carried out in R, version 3.3.2. Enrolled volunteers showed one of three outcomes: clinical cold with RSV detection (determined by qPCR [10]) and/or virus-specific IgA production alongside a cumulative self-reported symptom score over a 14-day period (n=17) [7]; asymptomatic infection (n=6) or no infection (n=14) (figure 1). No significant differences in participant demographics (age, sex, ethnicity; p>0.05) were observed between outcome groups [10]. Between viral inoculation and day 3, only low levels of RSV were detected in the

Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal ... more Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA-and rRNA-based microbial community composition.. Applied and environmental microbiology.

Frontiers in Microbiology, Dec 23, 2021

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, ... more Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (<18 years) microbiota in acute and chronic respiratory conditions, with >10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha-and beta-diversity approaches, machine learning, and biomarker analyses. Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively. Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.

Journal of Cystic Fibrosis, Mar 1, 2023

Scientific Reports, Mar 26, 2019

The pathogenesis of airway infection in cystic fibrosis (CF) is poorly understood. We performed a... more The pathogenesis of airway infection in cystic fibrosis (CF) is poorly understood. We performed a longitudinal study coupling clinical information with frequent sampling of the microbiota to identify changes in the airway microbiota in infancy that could underpin deterioration and potentially be targeted therapeutically. Thirty infants with CF diagnosed on newborn screening (NBS) were followed for up to two years. Two hundred and forty one throat swabs were collected as a surrogate for lower airway microbiota (median 35 days between study visits) in the largest longitudinal study of the CF oropharyngeal microbiota. Quantitative PCR and Illumina sequencing of the 16S rRNA bacterial gene were performed. Data analyses were conducted in QIIME and Phyloseq in R. Streptococcus spp. and Haemophilus spp. were the most common genera (55% and 12.5% of reads respectively) and were inversely related. Only beta (between sample) diversity changed with age (Bray Curtis r 2 = 0.15, P = 0.03). Staphylococcus and Pseudomonas were rarely detected. These results suggest that Streptococcus spp. and Haemophilus spp., may play an important role in early CF. Whether they are protective against infection with more typical CF microorganisms , or pathogenic and thus meriting treatment needs to be determined.

Science, Oct 9, 2020

Mucosal neutrophil activation predisposes to respiratory viral infection Running title: Roles of ... more Mucosal neutrophil activation predisposes to respiratory viral infection Running title: Roles of neutrophils & IL-17 in RSV infection One sentence summary: Mucosal neutrophil activation at the time of respiratory virus exposure enhances infection and opposes early prodromal mucosal inflammatory responses that prevent disease.

Frontiers in Immunology, Feb 12, 2018

Alterations in the composition of the gut microbiota have profound effects on human health. Conse... more Alterations in the composition of the gut microbiota have profound effects on human health. Consequently, there is great interest in identifying, characterizing, and understanding factors that initiate these changes. Despite their high prevalence, studies have only recently begun to investigate how viral lung infections have an impact on the gut microbiota. There is also considerable interest in whether the gut microbiota could be manipulated during vaccination to improve efficacy. In this highly controlled study, we aimed to establish the effect of viral lung infection on gut microbiota composition and the gut environment using mouse models of common respiratory pathogens respiratory syncytial virus (RSV) and influenza virus. This was then compared to the effect of live attenuated influenza virus (LAIV) vaccination. Both RSV and influenza virus infection resulted in significantly altered gut microbiota diversity, with an increase in Bacteroidetes and a concomitant decrease in Firmicutes phyla abundance. Although the increase in the Bacteroidetes phylum was consistent across several experiments, differences were observed at the family and operational taxonomic unit level. This suggests a change in gut conditions after viral lung infection that favors Bacteroidetes outgrowth but not individual families. No change in gut microbiota composition was observed after LAIV vaccination, suggesting that the driver of gut microbiota change is specific to live viral infection. Viral lung infections also resulted in an increase in fecal lipocalin-2, suggesting low-grade gut inflammation, and colonic Muc5ac levels. Owing to the important role that mucus plays in the gut environment, this may explain the changes in microbiota composition observed. This study demonstrates that the gut microbiota and the gut environment are altered following viral lung infections and that these changes are not observed during vaccination. Whether increased mucin levels and gut inflammation drive, or are a result of, these changes is still to be determined.

Gut, Apr 8, 2015

of patients with Crohn's disease exhibit a biologically relevant dysbiosis in mucosal microbial m... more of patients with Crohn's disease exhibit a biologically relevant dysbiosis in mucosal microbial metacommunities.

The ISME Journal, Nov 29, 2012

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accur... more High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing 'core' and 'satellite' taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.

, periostin (POSTN: r=0.79) and nitric oxide synthase 2 (NOS2: r=0.72). Correlations in controls ... more , periostin (POSTN: r=0.79) and nitric oxide synthase 2 (NOS2: r=0.72). Correlations in controls were not significant (not shown). Conclusion Eotaxin-3 gene expression is upregulated in the airway epithelium in eosinophilic asthma and highly correlates with IL-13 signature genesincluding biomarker genes POSTN and NOS2.

Journal of Cystic Fibrosis, Jun 1, 2013

Objectives: P. aeruginosa constitutes a particular problem in CF patients due to its ability to p... more Objectives: P. aeruginosa constitutes a particular problem in CF patients due to its ability to persist in the airways. Two dominating clones with different genotypes, DK1 and DK2, have been found to infect many CF patients in the Copenhagen CF clinic. We investigated the adaptation and evolution in patient of these transmissible P. aeruginosa clones. Methods: Since 1973 P. aeruginosa isolates from sputum of CF patients have been stored. These isolates represent bacterial evolution and adaptation covering up to 200,000 bacterial generations of growth in CF airways. We have combined fullgenome sequencing of longitudinal isolates of P. aeruginosa from a number of chronically infected patients with global gene expression analysis and other types of phenotypic characterization. Results: Genome sequencing showed that mutations in regulatory genes were frequent. Generation of AlgT regulated population diversity and colonization of different niches in the CF airways was found to constitute a specific route of adaptation for DK1. In contrast, generation of AlgT variants with conditional phenotypes and colonization of different niches in the CF airways was found to be the dominant platform for DK2 adaptation. Conclusion: The two transmissible clones have followed different evolutionary trajectories, suggesting that there could be several routes towards persistence of colonization of the CF airways. In the DK1 clone several genetic alterations in regulatory genes have resulted in diverse populations within the same patient. In the DK2 clone a small number of early mutations in global regulatory genes fixed in the population seem to be important for the colonization success. WS1.2 Three clinically distinct chronic pediatric airway infections share a common core microbiota

Journal of Cystic Fibrosis, Jul 1, 2015

Background: Best practice when performing culture-independent microbiological analysis of sputum ... more Background: Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at − 80°C. However, accessing biobanked collections can mean that material has been passed through repeated freeze-thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles. Methods: Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze-thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing. Results: The impact of freeze-thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles. Conclusion: Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.

Microbiome, Apr 2, 2020

Background: Chronic infection and concomitant airway inflammation is the leading cause of morbidi... more Background: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. Results: We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa.

bioRxiv (Cold Spring Harbor Laboratory), May 23, 2023

Antimicrobial peptides (AMPs) are key components of innate immunity across all kingdoms of life. ... more Antimicrobial peptides (AMPs) are key components of innate immunity across all kingdoms of life. Both natural and synthetic AMPs are receiving renewed attention in the efforts to combat the antimicrobial resistance (AMR) crisis and the loss of antibiotic efficacy. The gramnegative pathogen Pseudomonas aeruginosa is one of the most concerning infectious bacteria in AMR, particularly in people with cystic fibrosis (CF) where respiratory infections are difficult to eradicate and are associated with increased morbidity and mortality. Cationic AMPs exploit the negative charge of lipopolysaccharides (LPS) on P. aeruginosa to bind to and disrupt the bacterial membrane(s) and cause lethal damage. P. aeruginosa modifies its LPS, via environmental or genetic factors, to neutralise the charge of the cell and evade AMP killing. Free-LPS is also a component of CF sputum, as is anionic extracellular DNA (eDNA), each of which can bind AMPs by electrostatic interaction. Both free LPS and eDNA also feed into pro-inflammatory cycles. Glatiramer acetate (GA) is a random peptide co-polymer of glycine, lysine, alanine, and tyrosine and used as drug in the treatment of multiple sclerosis (MS); we have previously shown GA to be an AMP which synergises with tobramycin against P. aeruginosa from CF, functioning via bacterial membrane disruption. Here, we demonstrate direct binding and sequestration/neutralisation of P. aeruginosa LPS in keeping with GA's ability to disrupt the outer membrane. Binding and neutralisation of eDNA was also seen. At CF-relevant concentrations, however, neither strongly inhibited membrane disruption by GA. Furthermore, in both type strains and clinical CF isolates of P. aeruginosa, exposure to GA did not result in increased modification of the Lipid A portion of LPS or in increased expression of genetically encoded systems involved in AMP sensing and LPS modification. With this low selective pressure on P. aeruginosa for known AMP resistance mechanisms, the potential to neutralise pro-inflammatory CF sputum components, as well as the previously described enhancement of antibiotic function, GA is a promising candidate for drug repurposing. .

bioRxiv (Cold Spring Harbor Laboratory), Aug 29, 2017

Introduction Persistent bacterial bronchitis (PBB) is a leading cause of chronic wet cough in you... more Introduction Persistent bacterial bronchitis (PBB) is a leading cause of chronic wet cough in young children. This study aimed to characterise the respiratory bacterial microbiota of healthy children and to assess the impact of the changes associated with the development of PBB. Blind, protected brushings were obtained from 20 healthy controls and 24 children with PBB, with an additional directed sample obtained from PBB patients. DNA was extracted, quantified using a 16S rRNA gene quantitative PCR assay prior to microbial community analysis by 16S rRNA gene sequencing. Results No significant difference in bacterial diversity or community composition (R 2 = 0.01, P = 0.36) was observed between paired blind and non-blind brushes, showing that blind brushings are a valid means of accessing the airway microbiota. This has important implications for collecting lower respiratory samples from healthy children. A significant decrease in bacterial diversity (P < 0.001) and change in community composition (R 2 = 0.08, P = 0.004) was observed among controls, in comparison with patients. Bacterial communities within patients with PBB were dominated by Proteobacteria, and indicator species analysis showed that Haemophilus and Neisseria were significantly associated with the patient group. In 15 (52.9%) cases the dominant organism by sequencing was not identified by standard routine clinical culture. Conclusion The bacteria present in the lungs of patients with PBB were less diverse in terms of richness and evenness. The results validate the clinical diagnosis, and suggest that more attention to bacterial communities in children with chronic cough may lead to more rapid recognition of this condition with earlier treatment and reduction in disease burden.