Phillip James

Host–Microbial Interactions in Idiopathic Pulmonary Fibrosis

Rationale: Changes in the respiratory microbiome are associated with disease progression in idiopathic pulmonary fibrosis (IPF). The role of the host response to the respiratory microbiome remains unknown. Objectives: To explore the host‐microbial interactions in IPF. Methods: Sixty patients diagnosed with IPF were prospectively enrolled together with 20 matched control subjects. Subjects underwent bronchoalveolar lavage (BAL), and peripheral whole blood was collected into PAXgene tubes for all subjects at baseline. For subjects with IPF, additional samples were taken at 1, 3, and 6 months and (if alive) 1 year. Gene expression profiles were generated using Affymetrix Human Gene 1.1 ST arrays. Measurements and Main Results: By network analysis of gene expression data, we identified two gene modules that strongly associated with a diagnosis of IPF, BAL bacterial burden (determined by 16S quantitative polymerase chain reaction), and specific microbial operational taxonomic units, as well as with lavage and peripheral blood neutrophilia. Genes within these modules that are involved in the host defense response include NLRC4, PGLYRP1, MMP9, and DEFA4. The modules also contain two genes encoding specific antimicrobial peptides (SLPI and CAMP). Many of these particular transcripts were associated with survival and showed longitudinal overexpression in subjects experiencing disease progression, further strengthening the relationship of the transcripts with disease. Conclusions: Integrated analysis of the host transcriptome and microbial signatures demonstrated an apparent host response to the presence of an altered or more abundant microbiome. These responses remained elevated in longitudinal follow‐up, suggesting that the bacterial communities of the lower airways may act as persistent stimuli for repetitive alveolar injury in IPF.

Longitudinal assessment of sputum microbiome by sequencing of the 16S rRNA gene in non-cystic fibrosis bronchiectasis patients.

Background Bronchiectasis is accompanied by chronic bronchial infection that may drive disease progression. However, the evidence base for antibiotic therapy is limited. DNA based methods offer better identification and quantification of microbial constituents of sputum than standard clinical culture and may help inform patient management strategies. Our study objective was to determine the longitudinal variability of the non-cystic fibrosis (CF) bronchiectasis microbiome in sputum with respect to clinical variables. Eighty-five patients with non-CF bronchiectasis and daily sputum production were recruited from outpatient clinics and followed for six months. Monthly sputum samples and clinical measurements were taken, together with additional samples during exacerbations. 16S rRNA gene sequencing of the sputum microbiota was successful for 381 samples from 76 patients and analysed in conjunction with clinical data. Results Microbial communities were highly individual in composition and stability, usually with limited diversity and often containing multiple pathogens. When compared to DNA sequencing, microbial culture had restricted sensitivity in identifying common pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae, Moraxella catarrhalis. With some exceptions, community characteristics showed poor correlations with clinical features including underlying disease, antibiotic use and exacerbations, with the subject showing the strongest association with community structure. When present, the pathogens mucoid Pseudomonas aeruginosa and Haemophilus influenzae may also shape the structure of the rest of the microbial community. Conclusions The use of microbial community analysis of sputum added to information from microbial culture. A simple model of exacerbations driven by bacterial overgrowth was not supported, suggesting a need for revision of principles for antibiotic therapy. In individual patients, the management of chronic bronchial infection may be improved by therapy specific to their microbiome, taking into account pathogen load, community stability, and acute and chronic community responses to antibiotics.

Effects of different antibiotic classes on airway bacteria in stable COPD using culture and molecular techniques: a randomised controlled trial

Background Long-term antibiotic therapy is used to prevent exacerbations of COPD but there is uncertainty over whether this reduces airway bacteria. The optimum antibiotic choice remains unknown. We conducted an exploratory trial in stable patients with COPD comparing three antibiotic regimens against placebo. Methods This was a single-centre, single-blind, randomised placebo-controlled trial. Patients aged ≥45 years with COPD, FEV1<80% predicted and chronic productive cough were randomised to receive either moxifloxacin 400 mg daily for 5 days every 4 weeks, doxycycline 100 mg/day, azithromycin 250 mg 3 times a week or one placebo tablet daily for 13 weeks. The primary outcome was the change in total cultured bacterial load in sputum from baseline; secondary outcomes included bacterial load by 16S quantitative PCR (qPCR), sputum inflammation and antibiotic resistance. Results 99 patients were randomised; 86 completed follow-up, were able to expectorate sputum and were analysed. After adjustment, there was a non-significant reduction in bacterial load of 0.42 log10 cfu/mL (95% CI −0.08 to 0.91, p=0.10) with moxifloxacin, 0.11 (−0.33 to 0.55, p=0.62) with doxycycline and 0.08 (−0.38 to 0.54, p=0.73) with azithromycin from placebo, respectively. There were also no significant changes in bacterial load measured by 16S qPCR or in airway inflammation. More treatment-related adverse events occurred with moxifloxacin. Of note, mean inhibitory concentrations of cultured isolates increased by at least three times over placebo in all treatment arms. Conclusions Total airway bacterial load did not decrease significantly after 3 months of antibiotic therapy. Large increases in antibiotic resistance were seen in all treatment groups and this has important implications for future studies. Trial registration number clinicaltrials.gov (NCT01398072).

The variability and seasonality of the environmental reservoir of Mycobacterium bovis shed by wild European badgers

The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, has been increasing in UK cattle herds resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir of infection. One likely route of transmission to cattle is through exposure to infected badger urine and faeces. The relative importance of the environment in transmission remains unknown, in part due to the lack of information on the distribution and magnitude of environmental reservoirs. Here we identify potential infection hotspots in the badger population and quantify the heterogeneity in bacterial load; with infected badgers shedding between 1 × 103 − 4 × 105 M. bovis cells g−1 of faeces, creating a substantial and seasonally variable environmental reservoir. Our findings highlight the potential importance of monitoring environmental reservoirs of M. bovis which may constitute a component of disease spread that is currently overlooked and yet may be responsible for a proportion of transmission amongst badgers and onwards to cattle.

Performance of a Noninvasive Test for Detecting Mycobacterium bovis Shedding in European Badger (Meles meles) Populations

ABSTRACT The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, in cattle herds in the United Kingdom is increasing, resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir and is the subject of control measures aimed at reducing the incidence of infection in cattle populations. Understanding the epidemiology of M. bovis in badger populations is essential for directing control interventions and understanding disease spread; however, accurate diagnosis in live animals is challenging and currently uses invasive methods. Here we present a noninvasive diagnostic procedure and sampling regimen using field sampling of latrines and detection of M. bovis with quantitative PCR tests, the results of which strongly correlate with the results of immunoassays in the field at the social group level. This method allows M. bovis infections in badger populations to be monitored without trapping and provides additional information on the quantities of bacterial DNA shed. Therefore, our approach may provide valuable insights into the epidemiology of bovine tuberculosis in badger populations and inform disease control interventions.

T1 Fluticasone propionate alters the resident airway microbiota and impairs anti-viral and anti-bacterial immune responses in the airways

Background Inhaled corticosteroids are the cornerstone of therapy in asthma and COPD but cause only modest reduction in exacerbations and are associated with increased pneumonia frequency. This has raised concern about potential detrimental effects on host-defence against respiratory pathogens. The aim of this study was to evaluate the effects of fluticasone propionate on airway anti-viral and anti-bacterial host-defence. Methods C57BL/6 mice were intranasally treated with fluticasone propionate (1 mg/kg) or vehicle control. 16S Quantitative PCR was used to evaluate total bacterial loads and pyrosequencing was used to evaluate microbiota community composition in lung tissue. Using mouse models of infection with rhinovirus 1B and S. pneumoniae D39, effects of fluticasone administration on anti-viral and anti-bacterial immune responses, airway inflammation and pathogen control were evaluated. Results Mice treated with fluticasone had increased lung bacterial loads compared to vehicle-treated controls at 8 h post administration (p < 0.05). Evaluation of community composition revealed that fluticasone treatment was associated with significant increases in Stenotrophomonas genera (p < 0.05). In a mouse model of S. pneumoniae infection, fluticasone administration suppressed anti-bacterial responses including expression of cytokines IL-6 and TNF-α (4 h post-infection; p < 0.001) and airway neutrophil recruitment (8 h post-infection; p < 0.001) and was also associated with increased lung bacterial loads measured by quantitative culture (8 h post-infection; p < 0.001). In a mouse model of rhinovirus infection, fluticasone suppressed innate anti-viral responses including BAL protein levels of interferon-β and -λ2/3 (day 1 post-infection; p < 0.001). Virus clearance was impaired by fluticasone with increased viral RNA copies observed in lung tissue (day 1&2 post-infection; p < 0.001). The late expression of rhinovirus-induced airway mucins MUC5AC and MUC5B BAL proteins was increased by fluticasone (p < 0.01 and p < 0.05 respectively at day 7). Administration of recombinant interferon-β in combination with fluticasone and rhinovirus led to upregulation of interferon-stimulated genes and improved virus clearance, thereby demonstrating that adverse effects of fluticasone on RV clearance are causally related to interferon suppression. Recombinant IFN-β did not alter the increased mucins observed with fluticasone treatment. Conclusion Fluticasone alters the airway microbiota and impairs airway anti-viral and anti-bacterial host-defence in mice. Human studies are required to confirm the relevance of these effects in the context of inflammatory airway diseases.

Respiratory Disease following Viral Lung Infection Alters the Murine Gut Microbiota

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.

Metal worker’s lung: spatial association with Mycobacterium avium

Background Outbreaks of hypersensitivity pneumonitis (HP) are not uncommon in workplaces where metal working fluid (MWF) is used to facilitate metal turning. Inhalation of microbe-contaminated MWF has been assumed to be the cause, but previous investigations have failed to establish a spatial relationship between a contaminated source and an outbreak. Objectives After an outbreak of five cases of HP in a UK factory, we carried out blinded, molecular-based microbiological investigation of MWF samples in order to identify potential links between specific microbial taxa and machines in the outbreak zone. Methods Custom-quantitative PCR assays, microscopy and phylogenetic analyses were performed on blinded MWF samples to quantify microbial burden and identify potential aetiological agents of HP in metal workers. Measurements and main results MWF from machines fed by a central sump, but not those with an isolated supply, was contaminated by mycobacteria. The factory sump and a single linked machine at the centre of the outbreak zone, known to be the workstation of the index cases, had very high levels of detectable organisms. Phylogenetic placement of mycobacterial taxonomic marker genes generated from these samples indicated that the contaminating organisms were closely related to Mycobacterium avium. Conclusions We describe, for the first time, a close spatial relationship between the abundance of a mycobacterium-like organism, most probably M. avium, and a localised outbreak of MWF-associated HP. The further development of sequence-based analytic techniques should assist in the prevention of this important occupational disease.

Environmental reservoirs of pathogenic mycobacteria across the Ethiopian biogeographical landscape

The Mycobacterium genus comprises over one-hundred-and-fifty recognised species, the majority of which reside in the environment and many of which can be pathogenic to mammals. Some species of environmental mycobacteria may interfere with BCG vaccination efficacy and in tuberculin test interpretation. Examining biogeographic trends in the distribution of members of the mycobacteria across a number of physicochemical and spatial gradients in soil and water environments across Ethiopia using oligotyping identified differential distributions of pathogenic and significant species. The tuberculosis complex was identified in more than 90% of water samples and taxonomic groups implicated in lower BCG vaccine efficiency were core in both soil and water Mycobacterium communities. A reservoir of Mycobacterium bovis was identified in water, with up to 7.3×102 genome equivalents per ml. Elevation, temperature, habitat and vegetation type were important predictors of both soil and water Mycobacterium communities. These results represent the first step in understanding the potential risk of exposure to environmental mycobacteria that may undermine efforts to reduce disease incidence.

Longitudinal assessment of sputum microbiome by sequencing of the 16S rRNA gene in non-CF bronchiectasis patients

Background Bronchiectasis is accompanied by chronic bronchial infection that may drive disease progression. However, the evidence base for antibiotic therapy is limited. DNA based methods offer better identification and quantification of microbial constituents of sputum than standard clinical culture and may help inform patient management strategies. Our study objective was to determine the longitudinal variability of the non-CF bronchiectasis microbiome in sputum with respect to clinical variables. Eighty-five patients with non-cystic fibrosis (CF) bronchiectasis and daily sputum production were recruited from outpatient clinics and followed for six months. Monthly sputum samples and clinical measurements were taken, together with additional samples during exacerbations. 16S rRNA gene sequencing of the sputum microbiota was successful for 381 samples from 76 patients and analysed in conjunction with clinical data. Results Microbial communities were highly individual in composition and stability, usually with limited diversity and often containing multiple pathogens. When compared to DNA sequencing, microbial culture had restricted sensitivity in identifying common pathogens. With some exceptions, community characteristics showed poor correlations with clinical features including underlying disease, antibiotic use and exacerbations, with the subject showing the strongest association with community structure. When present, certain pathogens may also shape the structure of the rest of the microbial community. Conclusions The use of microbial community analysis of sputum added to information from microbial culture. A simple model of exacerbations driven by bacterial overgrowth was not supported, suggesting a need for revision of principles for antibiotic therapy. In individual patients, the management of chronic bronchial infection may be improved by therapy specific to their microbiome, taking into account pathogen load, community stability, and acute and chronic community responses to antibiotics.