Faculty Bibliography

Since alterations in the intestinal microbiota may induce systemic inflammation and polarization of macrophages to the M1 state, the microbiome role in atherosclerosis, an M1-driven disease, requires evaluation. We aimed to determine if antibiotic (Abx) induced alterations to the intestinal microbiota interferes with atherosclerotic plaque inflammation resolution after lipid-lowering in mice. Hyperlipidemic Apoe^-/- mice were fed a western diet to develop aortic atherosclerosis with aortas then transplanted into normolipidemic wild-type (WT) mice to model clinically aggressive lipid management and promote atherosclerosis inflammation resolution. Gut microbial composition pre and post-transplant was altered via an enteral antibiotic or not. Post aortic transplant, after Abx treatment, while plaque size did not differ, compared to Apoe^-/- mice, Abx^- WT recipient mice had a 32% reduction in CD68-expressing cells (p = 0.02) vs. a non-significant 12% reduction in Abx⁺ WT mice. A trend toward an M1 plaque CD68-expresing cell phenotype was noted in Abx⁺ mice. By 16S rRNA sequence analysis, the Abx⁺ mice had reduced alpha diversity and increased Firmicutes/Bacteroidetes relative abundance ratio with a correlation between gut Firmicutes abundance and plaque CD68-expressing cell content (p < 0.05). These results indicate that in a murine atherosclerotic plaque inflammation resolution model, antibiotic-induced microbiome perturbation may blunt the effectiveness of lipid-lowering to reduce the content of plaque inflammatory CD68-expressing cells.

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.

OBJECTIVES:Hypercoagulability may be a key mechanism for acute organ injury and death in patients with severe coronavirus disease 2019, but the relationship between elevated plasma levels of d-dimer, a biomarker of coagulation activation, and mortality has not been rigorously studied. We examined the independent association between d-dimer and death in critically ill patients with coronavirus disease 2019. DESIGN:Multicenter cohort study. SETTING:ICUs at 68 hospitals across the United States. PATIENTS:Critically ill adults with coronavirus disease 2019 admitted to ICUs between March 4, 2020, and May 25, 2020, with a measured d-dimer concentration on ICU day 1 or 2. INTERVENTIONS:None. MEASUREMENTS AND MAIN RESULTS:The primary exposure was the highest normalized d-dimer level (assessed in four categories: < 2×, 2-3.9×, 4-7.9×, and ≥ 8× the upper limit of normal) on ICU day 1 or 2. The primary endpoint was 28-day mortality. Multivariable logistic regression was used to adjust for confounders. Among 3,418 patients (63.1% male; median age 62 yr [interquartile range, 52-71 yr]), 3,352 (93.6%) had a d-dimer concentration above the upper limit of normal. A total of 1,180 patients (34.5%) died within 28 days. Patients in the highest compared with lowest d-dimer category had a 3.11-fold higher odds of death (95% CI, 2.56-3.77) in univariate analyses, decreasing to a 1.81-fold increased odds of death (95% CI, 1.43-2.28) after multivariable adjustment for demographics, comorbidities, and illness severity. Further adjustment for therapeutic anticoagulation did not meaningfully attenuate this relationship (odds ratio, 1.73; 95% CI, 1.36-2.19). CONCLUSIONS:In a large multicenter cohort study of critically ill patients with coronavirus disease 2019, higher d-dimer levels were independently associated with a greater risk of death.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

Mortality among patients with COVID-19 and respiratory failure is high and there are no known lower airway biomarkers that predict clinical outcome. We investigated whether bacterial respiratory infections and viral load were associated with poor clinical outcome and host immune tone. We obtained bacterial and fungal culture data from 589 critically ill subjects with COVID-19 requiring mechanical ventilation. On a subset of the subjects that underwent bronchoscopy, we also quantified SARS-CoV-2 viral load, analyzed the microbiome of the lower airways by metagenome and metatranscriptome analyses and profiled the host immune response. We found that isolation of a hospital-acquired respiratory pathogen was not associated with fatal outcome. However, poor clinical outcome was associated with enrichment of the lower airway microbiota with an oral commensal ( Mycoplasma salivarium ), while high SARS-CoV-2 viral burden, poor anti-SARS-CoV-2 antibody response, together with a unique host transcriptome profile of the lower airways were most predictive of mortality. Collectively, these data support the hypothesis that 1) the extent of viral infectivity drives mortality in severe COVID-19, and therefore 2) clinical management strategies targeting viral replication and host responses to SARS-CoV-2 should be prioritized.

RATIONALE/BACKGROUND:Microbiome studies of the lower airway based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. METHODS:Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. MEASUREMENTS/METHODS:We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole genome (WGS) and RNA metatranscriptome sequencing. Short chain fatty acids (SCFA) were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. MAIN RESULTS/RESULTS:Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFAs levels were compared with WGS and metatranscriptome. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. CONCLUSIONS:Functional characterisation of the lower airway microbiota through metatranscriptome identify metabolically active organisms capable of producing metabolites with immunomodulatory capacity such as SCFAs.

Rationale Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with increased Th17 inflammatory phenotype. In this study we evaluated the microbial and host immune response dynamics after aspiration with a oral commensals using a preclinical mouse model. Methods Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of sacrifice. Genetic background of mice included WT, MyD88 knock out and STAT3C. Measurements 16S rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host transcriptome sequencing was used to characterize host immune phenotype. Main Results While MOC aspiration correlated with lower airway dysbiosis that resolved within five days, it induced an extended inflammatory response associated with IL17-producing T-cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration prior to a respiratory challenge with S. pneumoniae led to a decreased in host's susceptibility to this pathogen. Conclusions Thus, in otherwise healthy mice, a single aspiration event with oral commensals are rapidly cleared from the lower airways, but induce a prolonged Th17 response that secondarily decreased susceptibility to respiratory pathogens. Translationally, these data implicate an immuno-protective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower airway pathogens.

In lung cancer, enrichment of the lower airway microbiota with oral commensals commonly occurs and ex vivo models support that some of these bacteria can trigger host transcriptomic signatures associated with carcinogenesis. Here, we show that this lower airway dysbiotic signature was more prevalent in group IIIB-IV TNM stage lung cancer and is associated with poor prognosis, as shown by decreased survival among subjects with early stage disease (I-IIIA) and worse tumor progression as measured by RECIST scores among subjects with IIIB-IV stage disease. In addition, this lower airway microbiota signature was associated with upregulation of IL-17, PI3K, MAPK and ERK pathways in airway transcriptome, and we identified Veillonella parvula as the most abundant taxon driving this association. In a KP lung cancer model, lower airway dysbiosis with V. parvula led to decreased survival, increased tumor burden, IL-17 inflammatory phenotype and activation of checkpoint inhibitor markers.

INTRODUCTION/BACKGROUND:Workers' exposure to metalworking fluid (MWF) has been associated with respiratory disease. As part of a public health investigation of a manufacturing facility, we performed paired environmental and human sampling to evaluate cross-pollination of microbes between environment and host and possible effects on lung pathology present among workers. METHODS:Workplace environmental microbiota was evaluated in air and MWF samples. Human microbiota was evaluated in lung tissue samples from workers with respiratory symptoms found to have lymphocytic bronchiolitis and alveolar ductitis with B-cell follicles and emphysema, lung tissue controls, and in skin, nasal and oral samples from 302 workers from different areas of the facility. In vitro effects of MWF exposure on murine B-cells were assessed. RESULTS:Increased similarity of microbial composition was found between MWF samples and lung tissue samples of case workers compared to controls. Among workers in different locations within the facility, those that worked in machine shop area had skin, nasal and oral microbiota more closely related to the microbiota present in MWF samples. Lung samples from four index cases, and skin and nasal samples from workers in machine shop area were enriched with Pseudomonas, the dominant taxa in MWF. Exposure to used MWF stimulated murine B-cell proliferation in vitro, a hallmark cell subtype found in pathology of index cases. CONCLUSIONS:Evaluation of a manufacturing facility with a cluster of workers with respiratory disease supports cross-pollination of microbes from MWF to humans and suggests the potential for exposure to these microbes to be a health hazard.