Genomic attributes of airway commensal bacteria and mucosa.

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

Delayed acquisition of airway commensals in antibiotic naïve children and its relationship with wheezing in rural Ecuador.

Introduction: The hygiene hypothesis identified a relationship between living in rural areas and acquiring protective environmental factors against the development of asthma and atopy. In our previous study, we found a correlation between particular bacterial species and early-onset wheezing in infants from the rural tropics of Ecuador who were corticosteroid-naïve and had limited antibiotic exposure. We now describe a longitudinal study of infants conducted to determine the age-related changes of the microbiome and its relationship with wheezing. Methods: We performed an amplicon sequencing of the 16S rRNA bacterial gene from the oropharyngeal samples obtained from 110 infants who had a history of recurrent episodic wheezing sampled at different ages (7, 12, and 24 months) and compared it to the sequencing of the oropharyngeal samples from 150 healthy infants sampled at the same time points. Bioinformatic analyses were conducted using QIIME and R. Results: As expected, the microbiota diversity consistently increased as the infants grew older. Considering age-based microbiota changes, we found that infants with wheeze had significantly lower species richness than the healthy infants at 7 months, but not at 12 or 24 months. Most of the core and accessory organisms increased in abundance and prevalence with age, except for a few which decreased. At 7 months of age, infants with wheeze had notably higher levels of a single Streptococcus operational taxonomic unit and core microbiota member than controls. Conclusions: In a cohort with limited antibiotic and corticosteroid use, a progressively more complex and diverse respiratory microbial community develops with age. The respiratory microbiota in early life is altered in infants with wheeze, but this does not hold true in older infants.

Investigating Metabolic and Molecular Ecological Evolution of Opportunistic Pulmonary Fungal Coinfections: Protocol for a Laboratory-Based Cross-Sectional Study.

BACKGROUND: Fungal-bacterial cocolonization and coinfections pose an emerging challenge among patients suspected of having pulmonary tuberculosis (PTB); however, the underlying pathogenic mechanisms and microbiome interactions are poorly understood. Understanding how environmental microbes, such as fungi and bacteria, coevolve and develop traits to evade host immune responses and resist treatment is critical to controlling opportunistic pulmonary fungal coinfections. In this project, we propose to study the coexistence of fungal and bacterial microbial communities during chronic pulmonary diseases, with a keen interest in underpinning fungal etiological evolution and the predominating interactions that may exist between fungi and bacteria. OBJECTIVE: This is a protocol for a study aimed at investigating the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections through determining and characterizing the burden, etiological profiles, microbial communities, and interactions established between fungi and bacteria as implicated among patients with presumptive PTB. METHODS: This will be a laboratory-based cross-sectional study, with a sample size of 406 participants. From each participant, 2 sputa samples (one on-spot and one early morning) will be collected. These samples will then be analyzed for both fungal and bacterial etiology using conventional metabolic and molecular (intergenic transcribed spacer and 16S ribosomal DNA-based polymerase chain reaction) approaches. We will also attempt to design a genome-scale metabolic model for pulmonary microbial communities to analyze the composition of the entire microbiome (ie, fungi and bacteria) and investigate host-microbial interactions under different patient conditions. This analysis will be based on the interplays of genes (identified by metagenomics) and inferred from amplicon data and metabolites (identified by metabolomics) by analyzing the full data set and using specific computational tools. We will also collect baseline data, including demographic and clinical history, using a patient-reported questionnaire. Altogether, this approach will contribute to a diagnostic-based observational study. The primary outcome will be the overall fungal and bacterial diagnostic profile of the study participants. Other diagnostic factors associated with the etiological profile, such as incidence and prevalence, will also be analyzed using univariate and multivariate schemes. Odds ratios with 95% CIs will be presented with a statistical significance set at P<.05. RESULTS: The study has been approved by the Mbarara University Research Ethic Committee (MUREC1/7-07/09/20) and the Uganda National Council of Science and Technology (HS1233ES). Following careful scrutiny, the protocol was designed to enable patient enrollment, which began in March 2022 at Mbarara University Teaching Hospital. Data collection is ongoing and is expected to be completed by August 2023, and manuscripts will be submitted for publication thereafter. CONCLUSIONS: Through this protocol, we will explore the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections among patients with presumptive PTB. Establishing key fungal-bacterial cross-kingdom synergistic relationships is crucial for instituting fungal bacterial coinfecting etiology. TRIAL REGISTRATION: ISRCTN Registry ISRCTN33572982; https://tinyurl.com/caa2nw69. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48014.

Efficacy of antimicrobial and anti-viral coated air filters to prevent the spread of airborne pathogens.

The COVID-19 pandemic has demonstrated the real need for mechanisms to control the spread of airborne respiratory pathogens. Thus, preventing the spread of disease from pathogens has come to the forefront of the public consciousness. This has brought an increasing demand for novel technologies to prioritise clean air. In this study we report on the efficacy of novel biocide treated filters and their antimicrobial activity against bacteria, fungi and viruses. The antimicrobial filters reported here are shown to kill pathogens, such as Candida albicans, Escherichia coli and MRSA in under 15 min and to destroy SARS-CoV-2 viral particles in under 30 s following contact with the filter. Through air flow rate testing, light microscopy and SEM, the filters are shown to maintain their structure and filtration function. Further to this, the filters are shown to be extremely durable and to maintain antimicrobial activity throughout the operational lifetime of the product. Lastly, the filters have been tested in field trials onboard the UK rail network, showing excellent efficacy in reducing the burden of microbial species colonising the air conditioning system.

Comparison of the airway microbiota in children with chronic suppurative lung disease.

RATIONALE: The airway microbiota is important in chronic suppurative lung diseases, such as primary ciliary dyskinesia (PCD) and cystic fibrosis (CF). This comparison has not previously been described but is important because difference between the two diseases may relate to the differing prognoses and lead to pathological insights and potentially, new treatments. OBJECTIVES: To compare the longitudinal development of the airway microbiota in children with PCD to that of CF and relate this to age and clinical status. METHODS: Sixty-two age-matched children (age range 0.5-17 years) with PCD or CF (n=31 in each group) were recruited prospectively and followed for 1.1 years. Throat swabs or sputum as well as clinical information were collected at routine clinical appointments. 16S rRNA gene sequencing was performed. MEASUREMENTS AND MAIN RESULTS: The microbiota was highly individual and more diverse in PCD and differed in community composition when compared with CF. While Streptococcus was the most abundant genus in both conditions, Pseudomonas was more abundant in CF with Haemophilus more abundant in PCD (Padj=0.0005). In PCD only, an inverse relationship was seen in the relative abundance of Streptococcus and Haemophilus with age. CONCLUSIONS: Bacterial community composition differs between children with PCD and those with CF. Pseudomonas is more prevalent in CF and Haemophilus in PCD, at least until infection with Pseudomonas supervenes. Interactions between organisms, particularly members of Haemophilus, Streptococcus and Pseudomonas genera appear important. Study of the interactions between these organisms may lead to new therapies or risk stratification.

Molecular advances in pancreatic cancer: A genomic, proteomic and metabolomic approach.

Pancreatic ductal adenocarcinoma (PDAC) represents a challenging pathology with very poor outcomes and is increasing in incidence within the general population. The majority of patients are diagnosed incidentally with insidious symptoms and hence present late in the disease process. This significantly affects patient outcomes: the only cure is surgical resection but only up to 20% of patients present with resectable disease at the time of clinical presentation. The use of "omic" technology is expanding rapidly in the field of personalised medicine - using genomic, proteomic and metabolomic approaches allows researchers and clinicians to delve deep into the core molecular processes of this difficult disease. This review gives an overview of the current findings in PDAC using these "omic" approaches and summarises useful markers in aiding clinicians treating PDAC. Future strategies incorporating these findings and potential application of these methods are presented in this review article.

Airway microbial communities, smoking and asthma in a general population sample.

BACKGROUND: Normal airway microbial communities play a central role in respiratory health but are poorly characterized. Cigarette smoking is the dominant global environmental influence on lung function, and asthma has become the most prevalent chronic respiratory disease worldwide. Both conditions have major microbial components that are incompletely defined. METHODS: We investigated airway bacterial communities in a general population sample of 529 Australian adults. Posterior oropharyngeal swabs were analyzed by sequencing of the 16S rRNA gene. The microbiota were characterized according to their prevalence, abundance and network memberships. FINDINGS: The microbiota were similar across the general population, and were strongly organized into co-abundance networks. Smoking was associated with diversity loss, negative effects on abundant taxa, profound alterations to network structure and expansion of Streptococcus spp. By contrast, the asthmatic microbiota were selectively affected by an increase in Neisseria spp. and by reduced numbers of low abundance but prevalent organisms. INTERPRETATION: Our study shows that the healthy airway microbiota in this population were contained within a highly structured ecosystem, suggesting balanced relationships between the microbiome and human host factors. The marked abnormalities in smokers may contribute to chronic obstructive pulmonary disease (COPD) and lung cancer. The narrow spectrum of abnormalities in asthmatics encourages investigation of damaging and protective effects of specific bacteria. FUNDING: The study was funded by the Asmarley Trust and a Wellcome Joint Senior Investigator Award to WOCC and MFM (WT096964MA and WT097117MA). The Busselton Healthy Ageing Study is supported by the Government of Western Australia (Office of Science, Department of Health) the City of Busselton, and private donations.

The fungal airway microbiome in cystic fibrosis and non-cystic fibrosis bronchiectasis.

BACKGROUND: The prevalence of fungal disease in cystic fibrosis (CF) and non-CF bronchiectasis is increasing and the clinical spectrum is widening. Poor sensitivity and a lack of standard diagnostic criteria renders interpretation of culture results challenging. In order to develop effective management strategies, a more accurate and comprehensive understanding of the airways fungal microbiome is required. The study aimed to use DNA sequences from sputum to assess the load and diversity of fungi in adults with CF and non-CF bronchiectasis. METHODS: Next generation sequencing of the ITS2 region was used to examine fungal community composition (n = 176) by disease and underlying clinical subgroups including allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, non-tuberculous mycobacteria, and fungal bronchitis. Patients with no known active fungal disease were included as disease controls. RESULTS: ITS2 sequencing greatly increased the detection of fungi from sputum. In patients with CF fungal diversity was lower, while burden was higher than those with non-CF bronchiectasis. The most common operational taxonomic unit (OTU) in patients with CF was Candida parapsilosis (20.4%), whereas in non-CF bronchiectasis sputum Candida albicans (21.8%) was most common. CF patients with overt fungal bronchitis were dominated by Aspergillus spp., Exophiala spp., Candida parapsilosis or Scedosporium spp. CONCLUSION: This study provides a framework to more accurately characterize the extended spectrum of fungal airways diseases in adult suppurative lung diseases.

Post-acute COVID-19 associated with evidence of bystander T-cell activation and a recurring antibiotic-resistant bacterial pneumonia.

Here, we describe the case of a COVID-19 patient who developed recurring ventilator-associated pneumonia caused by Pseudomonas aeruginosa that acquired increasing levels of antimicrobial resistance (AMR) in response to treatment. Metagenomic analysis revealed the AMR genotype, while immunological analysis revealed massive and escalating levels of T-cell activation. These were both SARS-CoV-2 and P. aeruginosa specific, and bystander activated, which may have contributed to this patient's persistent symptoms and radiological changes.

Tales of 1,008 small molecules: phenomic profiling through live-cell imaging in a panel of reporter cell lines.

Phenomic profiles are high-dimensional sets of readouts that can comprehensively capture the biological impact of chemical and genetic perturbations in cellular assay systems. Phenomic profiling of compound libraries can be used for compound target identification or mechanism of action (MoA) prediction and other applications in drug discovery. To devise an economical set of phenomic profiling assays, we assembled a library of 1,008 approved drugs and well-characterized tool compounds manually annotated to 218 unique MoAs, and we profiled each compound at four concentrations in live-cell, high-content imaging screens against a panel of 15 reporter cell lines, which expressed a diverse set of fluorescent organelle and pathway markers in three distinct cell lineages. For 41 of 83 testable MoAs, phenomic profiles accurately ranked the reference compounds (AUC-ROC ≥ 0.9). MoAs could be better resolved by screening compounds at multiple concentrations than by including replicates at a single concentration. Screening additional cell lineages and fluorescent markers increased the number of distinguishable MoAs but this effect quickly plateaued. There remains a substantial number of MoAs that were hard to distinguish from others under the current study's conditions. We discuss ways to close this gap, which will inform the design of future phenomic profiling efforts.

Respiratory Viral Infection Alters the Gut Microbiota by Inducing Inappetence.

Respiratory viral infections are extremely common, but their impacts on the composition and function of the gut microbiota are poorly understood. We previously observed a significant change in the gut microbiota after viral lung infection. Here, we show that weight loss during respiratory syncytial virus (RSV) or influenza virus infection was due to decreased food consumption, and that the fasting of mice altered gut microbiota composition independently of infection. While the acute phase tumor necrosis factor alpha (TNF-α) response drove early weight loss and inappetence during RSV infection, this was not sufficient to induce changes in the gut microbiota. However, the depletion of CD8+ cells increased food intake and prevented weight loss, resulting in a reversal of the gut microbiota changes normally observed during RSV infection. Viral infection also led to changes in the fecal gut metabolome, with a significant shift in lipid metabolism. Sphingolipids, polyunsaturated fatty acids (PUFAs), and the short-chain fatty acid (SCFA) valerate were all increased in abundance in the fecal metabolome following RSV infection. Whether this and the impact of infection-induced anorexia on the gut microbiota are part of a protective anti-inflammatory response during respiratory viral infections remains to be determined.IMPORTANCE The gut microbiota has an important role in health and disease: gut bacteria can generate metabolites that alter the function of immune cells systemically. Understanding the factors that can lead to changes in the gut microbiome may help to inform therapeutic interventions. This is the first study to systematically dissect the pathway of events from viral lung infection to changes in gut microbiota. We show that the cellular immune response to viral lung infection induces inappetence, which in turn alters the gut microbiome and metabolome. Strikingly, there was an increase in lipids that have been associated with the resolution of disease. This opens up new paths of investigation: first, what is the (presumably secreted) factor made by the T cells that can induce inappetence? Second, is inappetence an adaptation that accelerates recovery from infection, and if so, does the microbiome play a role in this?

Methods in Lung Microbiome Research.

The lung microbiome is associated with host immune response and health outcomes in experimental models and patient cohorts. Lung microbiome research is increasing in volume and scope; however, there are no established guidelines for study design, conduct, and reporting of lung microbiome studies. Standardized approaches to yield reliable and reproducible data that can be synthesized across studies will ultimately improve the scientific rigor and impact of published work and greatly benefit microbiome research. In this review, we identify and address several key elements of microbiome research: conceptual modeling and hypothesis framing; study design; experimental methodology and pitfalls; data analysis; and reporting considerations. Finally, we explore possible future directions and research opportunities. Our goal is to aid investigators who are interested in this burgeoning research area and hopefully provide the foundation for formulating consensus approaches in lung microbiome research.

Viral respiratory infections and the oropharyngeal bacterial microbiota in acutely wheezing children.

Acute viral wheeze in children is a major cause of hospitalisation and a major risk factor for the development of asthma. However, the role of the respiratory tract microbiome in the development of acute wheeze is unclear. To investigate whether severe wheezing episodes in children are associated with bacterial dysbiosis in the respiratory tract, oropharyngeal swabs were collected from 109 children with acute wheezing attending the only tertiary paediatric hospital in Perth, Australia. The bacterial community from these samples was explored using next generation sequencing and compared to samples from 75 non-wheezing controls. No significant difference in bacterial diversity was observed between samples from those with wheeze and healthy controls. Within the wheezing group, attendance at kindergarten or preschool was however, associated with increased bacterial diversity. Rhinovirus (RV) infection did not have a significant effect on bacterial community composition. A significant difference in bacterial richness was observed between children with RV-A and RV-C infection, however this is likely due to the differences in age group between the patient cohorts. The bacterial community within the oropharynx was found to be diverse and heterogeneous. Age and attendance at day care or kindergarten were important factors in driving bacterial diversity. However, wheeze and viral infection were not found to significantly relate to the bacterial community. Bacterial airway microbiome is highly variable in early life and its role in wheeze remains less clear than viral influences.

Longitudinal development of the airway microbiota in infants with cystic fibrosis.

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 r2 = 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 micro-organisms, or pathogenic and thus meriting treatment needs to be determined.

A Haemophilus sp. dominates the microbiota of sputum from UK adults with non-severe community acquired pneumonia and chronic lung disease.

The demographics and comorbidities of patients with community acquired pneumonia (CAP) vary enormously but stratified treatment is difficult because aetiological studies have failed to comprehensively identify the pathogens. Our aim was to describe the bacterial microbiota of CAP and relate these to clinical characteristics in order to inform future trials of treatment stratified by co-morbidity. CAP patients were prospectively recruited at two UK hospitals. We used 16S rRNA gene sequencing to identify the dominant bacteria in sputum and compositional data analysis to determine associations with patient characteristics. We analysed sputum samples from 77 patients and found a Streptococcus sp. and a Haemophilus sp. were the most relatively abundant pathogens. The Haemophilus sp. was more likely to be dominant in patients with pre-existing lung disease, and its relative abundance was associated with qPCR levels of Haemophilus influenzae. The most abundant Streptococcus sp. was associated with qPCR levels of Streptococcus pneumoniae but dominance could not be predicted from clinical characteristics. These data suggest chronic lung disease influences the microbiota of sputum in patients with CAP. This finding could inform a trial of stratifying empirical CAP antibiotics to target Haemophilus spp. in addition to Streptococcus spp. in those with chronic lung disease.

The effect of astigmatic axis on visual acuity measured with different alphabets in Roman alphabet readers.

OBJECTIVE: Astigmatism produces meridional variations in the retinal blur pattern, thus interacting with object spatial detail and altering visual performance as the axis changes. This study investigates the influence of astigmatic axis orientation on visual acuity (VA) for four alphabets used worldwide. METHODS: Visual acuity was measured monocularly in 25 Roman alphabet users (mean age: 25.6±7.5 years) using computer-presented logarithm of the minimum angle of resolution (log-MAR) charts with letters from four different alphabets (Arabic, Chinese, Roman, and Tamil). VA was assessed under the effect of four optical conditions: best distance correction and three astigmatic conditions (using a +2.00 cylindrical diopter trial case lens with its axis oriented at 180, 45, or 90 degrees). For each alphabet, single optotypes were presented on a monitor viewed from a distance of 4.0 m, and a matching technique was used to identify the letters. RESULTS: The degradation in VA with astigmatic defocus was influenced by the alphabet used (p<0.001) and by the astigmatic axis (p<0.001). Interactions in VA degradation between astigmatic axes and alphabet (p<0.001) showed differences within 0.10 logMAR. These interactions were more pronounced in alphabets with higher dominance of curves and vertical (Tamil) and horizontal (Arabic) detail. CONCLUSION: Interactions between alphabet and type of astigmatism indicate that the effects of meridional blur on letter discrimination differ between alphabets. These findings have relevance in the way VA is assessed in populations using different typographies, and ultimately in the impact of astigmatic axis on their visual performance.

Comparison of the upper and lower airway microbiota in children with chronic lung diseases.

RATIONALE: The lower airway microbiota is important in normal immunological development and chronic lung diseases (CLDs). Young children cannot expectorate and because of the uncertainty whether upper airway samples reflect the lower airway microbiota, there have been few longitudinal paediatric studies to date. OBJECTIVES: To assess whether throat swabs (TS) and cough swabs (CS) are representative of the lower airway microbiota. METHODS: TS, CS, bronchoalveolar lavage and bronchial brushings were prospectively collected from 49 children undergoing fibreoptic bronchoscopy for CLDs. Bacterial DNA was extracted and the 16S rRNA gene V4 region sequenced using the Illumina MiSeq. RESULTS: 5.97 million high quality reads were obtained from 168 samples (47 TS, 37 CS, 42 BALF and 42 bronchial brushings). CS sequenced poorly. At a community level, no difference in alpha diversity (richness, evenness or Shannon Diversity Index) was seen between lower airway samples and TS (P > 0.05). Less than 6.31% of beta diversity variation related to sampling method for TS (P = 0.001). Variation between pathologies and individual patients was greater (20%, 54% respectively P ≤ 0.001) than between TS and lower airway samples. There was strong correlation in the relative abundance of genera between samples (r = 0.78, P < 0.001). Similarity between upper and lower airway samples was observed to be less for individuals where one sample type was dominated by a single organism. CONCLUSIONS: At the community structure level, TS correlate with lower airway samples and distinguish between different CLDs. TS may be a useful sample for the study of the differences in longitudinal changes in the respiratory microbiota between different CLDs. Differences are too great however for TS to be used for clinical decision making.