Longitudinal multicompartment characterization of host-microbiota interactions in patients with acute respiratory failure.

Critical illness can significantly alter the composition and function of the human microbiome, but few studies have examined these changes over time. Here, we conduct a comprehensive analysis of the oral, lung, and gut microbiota in 479 mechanically ventilated patients (223 females, 256 males) with acute respiratory failure. We use advanced DNA sequencing technologies, including Illumina amplicon sequencing (utilizing 16S and ITS rRNA genes for bacteria and fungi, respectively, in all sample types) and Nanopore metagenomics for lung microbiota. Our results reveal a progressive dysbiosis in all three body compartments, characterized by a reduction in microbial diversity, a decrease in beneficial anaerobes, and an increase in pathogens. We find that clinical factors, such as chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, are associated with specific patterns of dysbiosis. Interestingly, unsupervised clustering of lung microbiota diversity and composition by 16S independently predicted survival and performed better than traditional clinical and host-response predictors. These observations are validated in two separate cohorts of COVID-19 patients, highlighting the potential of lung microbiota as valuable prognostic biomarkers in critical care. Understanding these microbiome changes during critical illness points to new opportunities for microbiota-targeted precision medicine interventions.

Prognostic Insights from Longitudinal Multicompartment Study of Host-Microbiota Interactions in Critically Ill Patients.

Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.

Prognostic Insights from Longitudinal Multicompartment Study of Host-Microbiota Interactions in Critically Ill Patients.

Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.

Respiratory Fungal Communities are Associated with Systemic Inflammation and Predict Survival in Patients with Acute Respiratory Failure.

Rationale: Disruption of respiratory bacterial communities predicts poor clinical outcomes in critical illness; however, the role of respiratory fungal communities (mycobiome) is poorly understood. Objectives: We investigated whether mycobiota variation in the respiratory tract is associated with host-response and clinical outcomes in critically ill patients. Methods: To characterize the upper and lower respiratory tract mycobiota, we performed rRNA gene sequencing (internal transcribed spacer) of oral swabs and endotracheal aspirates (ETA) from 316 mechanically-ventilated patients. We examined associations of mycobiome profiles (diversity and composition) with clinical variables, host-response biomarkers, and outcomes. Measurements and Main Results: ETA samples with >50% relative abundance for C. albicans (51%) were associated with elevated plasma IL-8 and pentraxin-3 (p=0.05), longer time-to-liberation from mechanical ventilation (p=0.04) and worse 30-day survival (adjusted hazards ratio (adjHR): 1.96 [1.04-3.81], p=0.05). Using unsupervised clustering, we derived two clusters in ETA samples, with Cluster 2 (39%) showing lower alpha diversity (p<0.001) and higher abundance of C. albicans (p<0.001). Cluster 2 was significantly associated with the prognostically adverse hyperinflammatory subphenotype (odds ratio 2.07 [1.03-4.18], p=0.04) and predicted worse survival (adjHR: 1.81 [1.03-3.19], p=0.03). C. albicans abundance in oral swabs was also associated with the hyperinflammatory subphenotype and mortality. Conclusions: Variation in respiratory mycobiota was significantly associated with systemic inflammation and clinical outcomes. C. albicans abundance emerged as a negative predictor in both the upper and lower respiratory tract. The lung mycobiome may play an important role in the biological and clinical heterogeneity among critically ill patients and represent a potential therapeutic target for lung injury in critical illness.

Meta-omics profiling of the gut-lung axis illuminates metabolic networks and host-microbial interactions associated with elevated lung elastance in a murine model of obese allergic asthma.

Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.

Magnetic-activated cell sorting identifies a unique lung microbiome community.

BACKGROUND: The advent of culture-independent, next-generation DNA sequencing has led to the discovery of distinct lung bacterial communities. Studies of lung microbiome taxonomy often reveal only subtle differences between health and disease, but host recognition and response may distinguish the members of similar bacterial communities in different populations. Magnetic-activated cell sorting has been applied to the gut microbiome to identify the numbers and types of bacteria eliciting a humoral response. We adapted this technique to examine the populations of immunoglobulin-bound bacteria in the lung. METHODS: Sixty-four individuals underwent bronchoalveolar lavage (BAL). We separated immunoglobulin G-bound bacteria using magnetic-activated cell sorting and sequenced the 16S rRNA gene on the Illumina MiSeq platform. We compared microbial sequencing data in IgG-bound bacterial communities compared to raw BAL then examined the differences in individuals with and without HIV as a representative disease state. RESULTS: Immunoglobulin G-bound bacteria were identified in all individuals. The community structure differed when compared to raw BAL, and there was a greater abundance of Pseudomonas and fewer oral bacteria in IgG-bound BAL. Examination of IgG-bound communities in individuals with HIV demonstrated the differences in Ig-bound bacteria by HIV status that were not seen in a comparison of raw BAL, and greater numbers of immunoglobulin-bound bacteria were associated with higher pulmonary cytokine levels. CONCLUSIONS: We report a novel application of magnetic-activated cell sorting to identify immunoglobulin G-bound bacteria in the lung. This technique identified distinct bacterial communities which differed in composition from raw bronchoalveolar lavage, revealing the differences not detected by traditional analyses. Cytokine response was also associated with differential immunoglobulin binding of lung bacteria, suggesting the functional importance of these communities. Video Abstract.

The upper and lower respiratory tract microbiome in severe aspiration pneumonia.

Uncertainty persists whether anaerobic bacteria represent important pathogens in aspiration pneumonia. In a nested case-control study of mechanically ventilated patients classified as macro-aspiration pneumonia (MAsP, n = 56), non-macro-aspiration pneumonia (NonMAsP, n = 91), and uninfected controls (n = 11), we profiled upper (URT) and lower respiratory tract (LRT) microbiota with bacterial 16S rRNA gene sequencing, measured plasma host-response biomarkers, analyzed bacterial communities by diversity and oxygen requirements, and performed unsupervised clustering with Dirichlet Multinomial Models (DMM). MAsP and NonMAsP patients had indistinguishable microbiota profiles by alpha diversity and oxygen requirements with similar host-response profiles and 60-day survival. Unsupervised DMM clusters revealed distinct bacterial clusters in the URT and LRT, with low-diversity clusters enriched for facultative anaerobes and typical pathogens, associated with higher plasma levels of SPD and sCD14 and worse 60-day survival. The predictive inter-patient variability in these bacterial profiles highlights the importance of microbiome study in patient sub-phenotyping and precision medicine approaches for severe pneumonia.

Low Diversity and Instability of the Sinus Microbiota over Time in Adults with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1ß [IL-1ß]). Increased IL-1ß was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1ß in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1ß. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Gut and oral microbiota associations with viral mitigation behaviors during the COVID-19 pandemic.

Imposition of social and health behavior mitigations are important control measures in response to the coronavirus disease 2019 (COVID-19) pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Although postulated that these measures may impact the human microbiota including losses in diversity from heightened hygiene and social distancing measures, this hypothesis remains to be tested. Other impacts on the microbiota and host mental and physical health status associations from these measures are also not well-studied. Here we examine changes in stool and oral microbiota by analyzing 16S rRNA gene sequence taxonomic profiles from the same individuals during pre-pandemic (before March 2020) and early pandemic (May-November 2020) phases. During the early pandemic phase, individuals were also surveyed using questionnaires to report health histories, anxiety, depression, sleep and other lifestyle behaviors in a cohort of predominantly Caucasian adults (mean age = 61.5 years) with the majority reporting at least one underlying co-morbidity. We identified changes in microbiota (stool n = 288; oral n = 89) between pre-pandemic and early pandemic time points from the same subject and associated these differences with questionnaire responses using linear statistical models and hierarchical clustering of microbiota composition coupled to logistic regression. While a trend in loss of diversity was identified between pre-pandemic and early pandemic time points it was not statistically significant. Paired difference analyses between individuals identified fewer significant changes between pre-pandemic and early pandemic microbiota in those who reported fewer comorbidities. Cluster transition analyses of stool and saliva microbiota determined most individuals remained in the same cluster assignments from the pre-pandemic to early pandemic period. Individuals with microbiota that shifted in composition, causing them to depart a pre-pandemic cluster, reported more health issues and pandemic-associated worries. Collectively, our study identified that stool and saliva microbiota from the pre-pandemic to early pandemic periods largely exhibited ecological stability (especially stool microbiota) with most associations in loss of diversity or changes in composition related to more reported health issues and pandemic-associated worries. Longitudinal observational cohorts are necessary to monitor the microbiome in response to pandemics and changes in public health measures.

Signature changes in gut microbiome are associated with increased susceptibility to HIV-1 infection in MSM.

BACKGROUND: Men who have sex with men (MSM) have been disproportionately affected by HIV-1 since the beginning of the AIDS pandemic, particularly in the USA and Europe. Compared to men who have sex with women (MSW), MSM have a distinct fecal microbiome regardless of HIV-1 infection. However, it is unclear whether the MSM-associated gut microbiome affects the susceptibility and progression of HIV-1 infection. We studied fecal microbiome profiles, short-chain fatty acids, and blood plasma inflammatory cytokines of 109 HIV-1 seroconverters (SC) from the early, 1984-1985 phase of the HIV-1 pandemic in the Multicenter AIDS Cohort Study (MACS) before and after HIV-1 infection compared to 156 HIV-1-negative MACS MSM (negative controls [NC]). RESULTS: We found that family Succinivibrionaceae, S24-7, Mogibacteriaceae, Coriobacteriaceae, and Erysipelotrichaceae were significantly higher (p<0.05), whereas Odoribacteraceae, Verucomicrobiaceae, Bacteroidaceae, Barnesiellaceae, and Rikenellaceae were significantly lower (p<0.05), in SC before HIV-1 infection compared to NC. At the species level, Prevotella stercorea, Eubacterium biforme, and Collinsella aerofaciens were significantly higher (p<0.05), and Eubacterium dolichum, Desulfovibrio D168, Alistipes onderdonkii, Ruminococcus torques, Bacteroides fragilis, Bacteroides caccae, Alistipes putredinis, Akkermansia muciniphila, Bacteroides uniformis, and Bacteroides ovatus were significantly lower (p<0.05) in SC before HIV-1 infection compared to NC. After HIV-1 infection, family Prevotellaceae and Victivallaceae and species Bacteroides fragilis and Eubacterium cylindroides were significantly higher (p<0.05) in SC who developed AIDS within 5 years compared to the SC who were AIDS free for more than 10 years without antiretroviral therapy (ART). In addition, family Victivallaceae and species Prevotella stercorea, Coprococcus eutactus, and Butyrivibrio crossotus were significantly higher (p<0.05) and Gemmiger formicilis and Blautia obeum were significantly lower (p<0.05) after HIV-1 infection in SC who developed AIDS within 5-10 years compared to the SC who were AIDS-free for more than 10 years without ART. Furthermore, plasma inflammatory cytokine levels of sCD14, sCD163, interleukin 6, and lipopolysaccharide binding protein were significantly higher in SC with p<0.05 before HIV-1 infection compared to NC. CONCLUSIONS: Our results suggest that pathogenic changes in the gut microbiome were present in MSM several months prior to infection with HIV-1 in the early phase of the AIDS pandemic in the USA. This was associated with increased inflammatory biomarkers in the blood and risk for development of AIDS. Video abstract.

The microbiome of pediatric patients with chronic rhinosinusitis.

BACKGROUND: This study aimed to compare the microbiota of pediatric patients with chronic rhinosinusitis (CRS) who are undergoing adenoidectomy to treat their disease with that of healthy control patients. METHODS: Patients undergoing adenoidectomy-only for obstructive sleep apnea (n = 50) and CRS (n = 37) were recruited. Preoperative 22-item Sino-Nasal Outcome Test (SNOT-22) or Sinus and Nasal Quality of Life Survey (SN-5) were collected. Each patient had samples collected from their nasopharynx (adenoid bed) and nasal cavity (sinus) at the onset of surgery. 16S ribosomal ribonucleic acid (rRNA) gene sequencing was subsequently performed to obtain per sample taxonomic abundances. Statistical analyses included permutational multivariate analysis of variance (PERMANOVA), alpha (within sample) diversity measures, and changes in taxonomic abundance. RESULTS: Moraxella was the most abundant organism. Nasopharyngeal swabs demonstrated higher alpha diversity compared to the nasal cavity. The diversity was not different based on CRS vs obstructive history. There was an increase in diversity with increasing age, and eczema contributed to a greater difference in diversity between the nasopharynx and nasal cavity. Diversity was not affected by adenoid size; however, use of nasal steroids, inhaled steroids, and antihistamines influenced diversity in both the nasopharynx and nasal cavity. Nasopharyngeal samples were higher in relative abundance for Fusobacterium, Prevotella, Porphyromonas, and Campylobacter compared to the nasal cavity. CONCLUSION: The nasopharynx and nasal cavity differed in both microbiota composition and diversity. In contrast, no significant difference in composition or diversity were found in CRS vs control patients. Ecological changes in the nasopharyngeal and sinus site may contribute to the etiology for adenoid hypertrophy in both healthy controls and CRS patients.

Topographic heterogeneity of lung microbiota in end-stage idiopathic pulmonary fibrosis: the Microbiome in Lung Explants-2 (MiLEs-2) study.

BACKGROUND: Lung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown. METHODS: We performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene. FINDINGS: Explants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death. INTERPRETATION: IPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions.

Respiratory Tract Dysbiosis Is Associated with Worse Outcomes in Mechanically Ventilated Patients.

Rationale: Host inflammatory responses have been strongly associated with adverse outcomes in critically ill patients, but the biologic underpinnings of such heterogeneous responses have not been defined.Objectives: We examined whether respiratory tract microbiome profiles are associated with host inflammation and clinical outcomes of acute respiratory failure.Methods: We collected oral swabs, endotracheal aspirates (ETAs), and plasma samples from mechanically ventilated patients. We performed 16S ribosomal RNA gene sequencing to characterize upper and lower respiratory tract microbiota and classified patients into host-response subphenotypes on the basis of clinical variables and plasma biomarkers of innate immunity and inflammation. We derived diversity metrics and composition clusters with Dirichlet multinomial models and examined our data for associations with subphenotypes and clinical outcomes.Measurements and Main Results: Oral and ETA microbial communities from 301 mechanically ventilated subjects had substantial heterogeneity in α and ß diversity. Dirichlet multinomial models revealed a cluster with low α diversity and enrichment for pathogens (e.g., high Staphylococcus or Pseudomonadaceae relative abundance) in 35% of ETA samples, associated with a hyperinflammatory subphenotype, worse 30-day survival, and longer time to liberation from mechanical ventilation (adjusted P < 0.05), compared with patients with higher α diversity and relative abundance of typical oral microbiota. Patients with evidence of dysbiosis (low α diversity and low relative abundance of "protective" oral-origin commensal bacteria) in both oral and ETA samples (17%, combined dysbiosis) had significantly worse 30-day survival and longer time to liberation from mechanical ventilation than patients without dysbiosis (55%; adjusted P < 0.05).Conclusions: Respiratory tract dysbiosis may represent an important, modifiable contributor to patient-level heterogeneity in systemic inflammatory responses and clinical outcomes.

A prospective cohort analysis of gut microbial co-metabolism in Alaska Native and rural African people at high and low risk of colorectal cancer.

BACKGROUND: Alaska Native (AN) people have the world's highest recorded incidence of sporadic colorectal cancer (CRC) (∼91:100,000), whereas rural African (RA) people have the lowest risk (<5:100,000). Previous data supported the hypothesis that diet affected CRC risk through its effects on the colonic microbiota that produce tumor-suppressive or -promoting metabolites. OBJECTIVES: We investigated whether differences in these metabolites may contribute to the high risk of CRC in AN people. METHODS: A cross-sectional observational study assessed dietary intake from 32 AN and 21 RA healthy middle-aged volunteers before screening colonoscopy. Analysis of fecal microbiota composition by 16S ribosomal RNA gene sequencing and fecal/urinary metabolites by 1H-NMR spectroscopy was complemented with targeted quantification of fecal SCFAs, bile acids, and functional microbial genes. RESULTS: Adenomatous polyps were detected in 16 of 32 AN participants, but not found in RA participants. The AN diet contained higher proportions of fat and animal protein and less fiber. AN fecal microbiota showed a compositional predominance of Blautia and Lachnoclostridium, higher microbial capacity for bile acid conversion, and low abundance of some species involved in saccharolytic fermentation (e.g., Prevotellaceae, Ruminococcaceae), but no significant lack of butyrogenic bacteria. Significantly lower concentrations of tumor-suppressive butyrate (22.5 ± 3.1 compared with 47.2 ± 7.3 SEM µmol/g) coincided with significantly higher concentrations of tumor-promoting deoxycholic acid (26.7 ± 4.2 compared with 11 ± 1.9 µmol/g) in AN fecal samples. AN participants had lower quantities of fecal/urinary metabolites than RA participants and metabolite profiles correlated with the abundance of distinct microbial genera in feces. The main microbial and metabolic CRC-associated markers were not significantly altered in AN participants with adenomatous polyps. CONCLUSIONS: The low-fiber, high-fat diet of AN people and exposure to carcinogens derived from diet or environment are associated with a tumor-promoting colonic milieu as reflected by the high rates of adenomatous polyps in AN participants.

Alterations in Oral Microbiota in HIV Are Related to Decreased Pulmonary Function.

Rationale: Mechanisms of HIV-associated chronic obstructive pulmonary disease (COPD) are poorly understood. The oral microbiome shapes the lung microbiome, and gut dysbiosis can affect lung diseases; however, relationships of the oral and gut microbiome to COPD in HIV have not been explored.Objectives: To examine alterations in the oral and gut microbiome associated with pulmonary disease in persons with HIV (PWH).Methods: Seventy-five PWH and 93 HIV-uninfected men from the MACS (Multicenter AIDS Cohort Study) performed pulmonary function testing. Sequencing of bacterial 16S ribosomal RNA in saliva and stool was performed. We used nonmetric multidimensional scaling, permutational multivariate ANOVA, and linear discriminant analysis to analyze communities by HIV and lung function.Measurements and Main Results: Oral microbiome composition differed by HIV and smoking status. Alterations of oral microbial communities were observed in PWH with abnormal lung function with increases in relative abundance of Veillonella, Streptococcus, and Lactobacillus. There were no significant associations between the oral microbiome and lung function in HIV-uninfected individuals. No associations with HIV status or lung function were seen with the gut microbiome.Conclusions: Alterations of oral microbiota in PWH were related to impaired pulmonary function and to systemic inflammation. These results suggest that the oral microbiome may serve as a biomarker of lung function in HIV and that its disruption may contribute to COPD pathogenesis.

Intestinal IL-17R Signaling Constrains IL-18-Driven Liver Inflammation by the Regulation of Microbiome-Derived Products.

Interleukin (IL)-17 signaling to the intestinal epithelium regulates the intestinal microbiome. Given the reported links between intestinal dysbiosis, bacterial translocation, and liver disease, we hypothesize that intestinal IL-17R signaling plays a critical role in mitigating hepatic inflammation. To test this, we study intestinal epithelium-specific IL-17RA-deficient mice in an immune-driven hepatitis model. At the naive state, these mice exhibit microbiome dysbiosis and increased translocation of bacterial products (CpG DNA), which drives liver IL-18 production. Upon disease induction, absence of enteric IL-17RA signaling exacerbates hepatitis and hepatocyte cell death. IL-18 is necessary for disease exacerbation and is associated with increased activated hepatic lymphocytes based on Ifng and Fasl expression. Thus, intestinal IL-17R regulates translocation of TLR9 ligands and constrains susceptibility to hepatitis. These data connect enteric Th17 signaling and the microbiome in hepatitis, with broader implications on the effects of impaired intestinal immunity and subsequent release of microbial products observed in other extra-intestinal pathologies.

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study.

BACKGROUND: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. METHODS: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. RESULTS: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. CONCLUSIONS: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

Rectal Swabs from Critically Ill Patients Provide Discordant Representations of the Gut Microbiome Compared to Stool Samples.

The role of the gut microbiome in critical illness is being actively investigated, but the optimal sampling methods for sequencing studies of gut microbiota remain unknown. Stool samples are generally considered the reference standard but are not practical to obtain in the intensive care unit (ICU), and thus, rectal swabs are often used. However, the reliability of rectal swabs for gut microbiome profiling has not been established in the ICU setting. In this study, we compared 16S rRNA gene sequencing results between rectal swab and stool samples collected at three time points from mechanically ventilated critically ill adults. Rectal swabs comprised 89% of the samples collected at the baseline time point, but stool samples became more extensively available at later time points. Significant differences in alpha-diversity and beta-diversity between rectal swabs and stool samples were observed, but these differences were primarily due to baseline samples. Higher relative abundances of members of the Actinobacteria phylum (typically skin microbes) were present in rectal swabs than in stool samples (P = 0.05), a difference that was attenuated over time. The progressively increasing similarity of rectal swabs and stool samples likely resulted from increasing levels of stool coating of the rectal vault and direct soiling of the rectal swabs taken at later time points. Therefore, inferences about the role of the gut microbiome in critical illness should be drawn cautiously and should take into account the type and timing of samples analyzed.IMPORTANCE Rectal swabs have been proposed as potential alternatives to stool samples for gut microbiome profiling in outpatients or healthy adults, but their reliability in assessment of critically ill patients has not been defined. Because stool sampling is not practical and often not feasible in the intensive care unit, we performed a detailed comparison of gut microbial sequencing profiles between rectal swabs and stool samples in a longitudinal cohort of critically ill patients. We identified systematic differences in gut microbial profiles between rectal swabs and stool samples and demonstrated that the timing of the rectal swab sampling had a significant impact on sequencing results. Our methodological findings should provide valuable information for the design and interpretation of future investigations of the role of the gut microbiome in critical illness.

Improved Detection of Culprit Pathogens by Bacterial DNA Sequencing Affects Antibiotic Management Decisions in Severe Pneumonia.

BACKGROUND Severe pneumonia requiring admission to an intensive care unit carries high morbidity and mortality. Evidence-based management includes early administration of empiric antibiotics against plausible bacterial pathogens while awaiting results of microbiologic cultures. However, in over 60% of pneumonia cases, no causative pathogen is identified with conventional diagnostic techniques. In this case report, we demonstrate how direct-from-sample sequencing of bacterial DNA could have identified the multiple culprit pathogens early in the disease course to guide appropriate antibiotic management. CASE REPORT A previously healthy, 21-year-old man presented with neck pain and fever and rapidly developed acute respiratory distress syndrome (ARDS) requiring mechanical ventilation. He was started on broad-spectrum antibiotics and was found to have septic thrombophlebitis of the left internal jugular vein (Lemierre syndrome), with blood cultures growing Fusobacterium necrophorum. While his antibiotics were narrowed to piperacillin-tazobactam monotherapy, his clinical condition worsened, but repeated efforts to define an additional/alternative respiratory pathogen resulted in negative cultures. He eventually developed bilateral empyemas growing Mycoplasma hominis. Once azithromycin was added to the patient's regimen, he improved dramatically. Retrospective sequencing of consecutive endotracheal aspirates showed Fusobacterium as the dominant pathogen early in the course, but with significant and increasing Mycoplasma abundance several days prior to clinical detection. CONCLUSIONS Had sequencing information been available to the treating clinicians, the causative pathogens could have been detected earlier, guiding appropriate antibiotic therapy and perhaps preventing his clinical complications. Real-time bacterial DNA sequencing has the potential to shift the diagnostic paradigm in severe pneumonia.

Respiratory Microbiome Profiling for Etiologic Diagnosis of Pneumonia in Mechanically Ventilated Patients.

Etiologic diagnosis of bacterial pneumonia relies on identification of causative pathogens by cultures, which require extended incubation periods and have limited sensitivity. Next-generation sequencing of microbial DNA directly from patient samples may improve diagnostic accuracy for guiding antibiotic prescriptions. In this study, we hypothesized that enhanced pathogen detection using sequencing can improve upon culture-based diagnosis and that certain sequencing profiles correlate with host response. We prospectively collected endotracheal aspirates and plasma within 72 h of intubation from patients with acute respiratory failure. We performed 16S rRNA gene sequencing to determine pathogen abundance in lung samples and measured plasma biomarkers to assess host responses to detected pathogens. Among 56 patients, 12 patients (21%) had positive respiratory cultures. Sequencing revealed lung communities with low diversity (p < 0.02) dominated by taxa (>50% relative abundance) corresponding to clinically isolated pathogens (concordance p = 0.009). Importantly, sequencing detected dominant pathogens in 20% of the culture-negative patients exposed to broad-spectrum empiric antibiotics. Regardless of culture results, pathogen dominance correlated with increased plasma markers of host injury (receptor of advanced glycation end-products-RAGE) and inflammation (interleukin-6, tumor necrosis factor receptor 1-TNFR1) (p < 0.05), compared to subjects without dominant pathogens in their lung communities. Machine-learning algorithms identified pathogen abundance by sequencing as the most informative predictor of culture positivity. Thus, enhanced detection of pathogenic bacteria by sequencing improves etiologic diagnosis of pneumonia, correlates with host responses, and offers substantial opportunity for individualized therapeutic targeting and antimicrobial stewardship. Clinical translation will require validation with rapid whole meta-genome sequencing approaches to guide real-time antibiotic prescriptions.