Faculty Bibliography

The respiratory microbiome and its impact in health and disease is now well characterised. With the development of next-generation sequencing and the use of other techniques such as metabolomics, the functional impact of microorganisms in different host environments can be elucidated. It is now clear that the respiratory microbiome plays an important role in respiratory disease. In some diseases, such as bronchiectasis, examination of the microbiome can even be used to identify patients at higher risk of poor outcomes. Furthermore, the microbiome can aid in phenotyping. Finally, development of multi-omic analysis has revealed interactions between the host and microbiome in some conditions. This review, although not exhaustive, aims to outline how the microbiome is investigated, the healthy respiratory microbiome and its role in respiratory disease.

INTRODUCTION/BACKGROUND:There are few existing severity scoring systems in the literature, and no formally widely accepted chest X-ray template for reporting COVID-19 infection. We aimed to modify the chest X-ray COVID-19 severity scoring system from the Brixia scoring system with placement of more emphasis on consolidation and to assess if the scoring tool could help predict intubation. METHODS:A severity chest X-ray scoring system was modified from the Brixia scoring system. PCR positive COVID-19 positive patient's chest X-rays admitted to our hospital over 3 months were reviewed and correlated with; non-invasive ventilation, intubation and death. An analysis was performed using a receiver operating curve to predict intubation from all admission chest X-rays. RESULTS:The median score of all 325 admission chest X-rays was 3 (Interquartile range (IQR) 0-6.5). The median score of admission chest X-rays of those who did not require ICU admission and survived was 1.5 (IQR 0-5); and 9 (IQR 4.75-12) was median admission score of those requiring intubation. The median scores of the pre-intubation ICU chest X-rays was 11.5 (IQR 9-14.125), this increased from a median admission chest X-ray score for this group of 9 (P-value < 0.01). A cut-off score of 6 had a sensitivity of 77% and specificity of 73% in predicting the need for intubation. CONCLUSION/CONCLUSIONS:Higher chest X-ray severity scores are associated with intubation, need for non-invasive ventilation and death. This tool may also be helpful in predicting intubation.

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.

Rational Recent investigations support that the gut microbiota influences anti-PD-1 cancer immunotherapy. Lower airway dysbiosis with enrichment with oral commensals are associated with lung cancer. Recently we had shown, in both a prospective human cohort and preclinical mice model, that lung dysbiotic signatures were associated with clinical lung cancer prognosis and progression. To further understand the role of lung dysbiosis in lung cancer, we examined the role of PD-1 expression and anti- PD treatment in a lung cancer and lung dysbiotic model. Method KrasLSL-G12D/+;p53fl/fl Non-small cell Lung Cancer mice (KP) were challenged with an oral commensal, Veillonella parvula, through intra-tracheal inoculation and exposed to immune inhibition (anti- PD-1). Measurements included tumor burden and lower airway inflammatory markers (PD-1 expression and neutrophils) by FACS. Results In a preclinical lung cancer model, inoculation with Veillonella parvula, a marker taxon for the dysbiotic signature found in humans, led to: 1) decrease survival with increase tumor burden; 2) dysbiosis with oral commensal is associated with elevated level of PD-1 expression and neutrophils level compared to control. With exposure to PD-1 inhibition we observe a reverse of tumor growth (at day 7); there was significant decrease in tumor growth compared with Isotype-control (p=0.030, day7-14) and observed that PD-1+ level (p=0.0007) and Neutrophil level (p=0.0027) were lower as well. Discussion Our study suggests that lower airway dysbiosis induced by microaspiration of oral commensals may affect lung carcinogenesis due to increase in inflammatory markers and increase in the checkpoint inhibitor tone in the lower airways that may lead to suboptimal immune surveillance. These effects of lower airway dysbiosis can be partially blunted by PD-1 blockade. These data supports that treatment in lung cancer may be influenced by lower airway dysbiosis and dynamic changes in the microbial-host interaction in the lower airways

Rationale: Chronic airway colonization and recurrent infections are common in advanced stage chronic obstructive pulmonary disease (COPD). However, changes in the lung microbiota in early stages of this disease remain unclear. Here, we characterized the upper and lower airway microbiota of patients with early stage COPD and smoker controls.
Method(s): Upper and lower airway samples (plus appropriate environmental and technical controls) were obtained from patients with GOLD 1-2 COPD (n = 26) and smoker controls (n = 31). Bacterial load was measured with droplet digital PCR while microbiota profiling was performed using 16S rRNA gene sequencing. Data was analyzed using QIIME, Phyloseq, Vegan and DESeq. Parallel RNA metatranscriptome sequencing and host Transcriptome approach were just completed and data is becoming available.
Result(s): Characterization of the lower airway microbial communities with 16S rRNA gene sequencing showed that compared to smoker controls, COPD patients exhibited lower alpha Shannon diversity (Fig.1a, p = 0.0037). Beta diversity analysis based on Bray Curtis Dissimilarity index showed that the composition of the microbial communities in the lower airway samples were clearly distinct from background and upper airway as a whole. Some samples overlapped with both of those areas suggesting that for some subjects their lower airway microbiota was enriched with taxa commonly found in the oral cavity. We then evaluated for differentially enriched taxa in BAL samples using DESeq. The lower airway microbiota of subjects with COPD was enriched with oral commensals such as Veillonella, Prevotella (Fig 1c). Comparison of bacterial load based on bacterial composition was performed based on cluster determination of lower airway samples enriched with oral commensals (SPT for supraglottic predominant taxa) or enriched with background taxa (BPT for background predominant taxa). The bacterial load of lower airway samples categorized as SPT was one log higher than those categorized as BPT among the COPD group but not among the smoker controls (Fig.1d, p < 0.001).
Conclusion(s): Our results suggest that lower airway exposure to oral commensals occurs more frequently among subjects with COPD. Further investigation with functional microbiome approaches such as metatranscriptomics are warranted. This may be of importance given significant data showing that these taxa may contribute to an increase in lower airway inflammatory tone (especially in the Th17 pathway) that may lead to airway/parenchymal inflammatory damage and/or affect treatment response and clinical outcome in this disease

BACKGROUND:The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood. METHODS:To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB. FINDINGS/RESULTS:Cases and SCs each had similar α- and β-diversities in oral washes and sputum, however, β-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways. INTERPRETATION/CONCLUSIONS:TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB. FUNDING/BACKGROUND:European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.