Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status.

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

Application of a quantitative framework to improve the accuracy of a bacterial infection model.

Laboratory models are critical to basic and translational microbiology research. Models serve multiple purposes, from providing tractable systems to study cell biology to allowing the investigation of inaccessible clinical and environmental ecosystems. Although there is a recognized need for improved model systems, there is a gap in rational approaches to accomplish this goal. We recently developed a framework for assessing the accuracy of microbial models by quantifying how closely each gene is expressed in the natural environment and in various models. The accuracy of the model is defined as the percentage of genes that are similarly expressed in the natural environment and the model. Here, we leverage this framework to develop and validate two generalizable approaches for improving model accuracy, and as proof of concept, we apply these approaches to improve models of Pseudomonas aeruginosa infecting the cystic fibrosis (CF) lung. First, we identify two models, an in vitro synthetic CF sputum medium model (SCFM2) and an epithelial cell model, that accurately recapitulate different gene sets. By combining these models, we developed the epithelial cell-SCFM2 model which improves the accuracy of over 500 genes. Second, to improve the accuracy of specific genes, we mined publicly available transcriptome data, which identified zinc limitation as a cue present in the CF lung and absent in SCFM2. Induction of zinc limitation in SCFM2 resulted in accurate expression of 90% of P. aeruginosa genes. These approaches provide generalizable, quantitative frameworks for microbiological model improvement that can be applied to any system of interest.

Endotypes of Exacerbation in Bronchiectasis: An Observational Cohort Study.

Rationale: Bronchiectasis is characterized by acute exacerbations, but the biological mechanisms underlying these events are poorly characterized. Objectives: To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. Methods: A total of 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation before receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral, or both. Sputum inflammatory assessments included label-free liquid chromatography-tandem mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16 s rRNA sequencing was used to characterize the microbiome. Measurements and Main Results: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacterium was identified in 103 samples (86%), and a high bacterial load (total bacterial load > 107 copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients, with rhinovirus being the most common virus (31%). PCR testing was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, IL-1ß, and CXCL8. These markers were particularly associated with bacterial and bacterial plus viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral, and eosinophilic events in both hypothesis-led and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating four subtypes of exacerbation. Conclusions: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses, and inflammatory dysregulation.

Airway "Resistotypes" and Clinical Outcomes in Bronchiectasis.

Rationale: Chronic infection and inflammation shapes the airway microbiome in bronchiectasis. Utilizing whole-genome shotgun metagenomics to analyze the airway resistome provides insight into interplay between microbes, resistance genes, and clinical outcomes. Objectives: To apply whole-genome shotgun metagenomics to the airway microbiome in bronchiectasis to highlight a diverse pool of antimicrobial resistance genes: the "resistome," the clinical significance of which remains unclear. Methods: Individuals with bronchiectasis were prospectively recruited into cross-sectional and longitudinal cohorts (n = 280), including the international multicenter cross-sectional Cohort of Asian and Matched European Bronchiectasis 2 (CAMEB 2) study (n = 251) and two independent cohorts, one describing patients experiencing acute exacerbation and a further cohort of patients undergoing Pseudomonas aeruginosa eradication treatment. Sputum was subjected to metagenomic sequencing, and the bronchiectasis resistome was evaluated in association with clinical outcomes and underlying host microbiomes. Measurements and Main Results: The bronchiectasis resistome features a unique resistance gene profile and increased counts of aminoglycoside, bicyclomycin, phenicol, triclosan, and multidrug resistance genes. Longitudinally, it exhibits within-patient stability over time and during exacerbations despite between-patient heterogeneity. Proportional differences in baseline resistome profiles, including increased macrolide and multidrug resistance genes, associate with shorter intervals to the next exacerbation, whereas distinct resistome archetypes associate with frequent exacerbations, poorer lung function, geographic origin, and the host microbiome. Unsupervised analysis of resistome profiles identified two clinically relevant "resistotypes," RT1 and RT2, the latter characterized by poor clinical outcomes, increased multidrug resistance, and P. aeruginosa. Successful targeted eradication in P. aeruginosa-colonized individuals mediated reversion from RT2 to RT1, a more clinically favorable resistome profile demonstrating reduced resistance gene diversity. Conclusions: The bronchiectasis resistome associates with clinical outcomes, geographic origin, and the underlying host microbiome. Bronchiectasis resistotypes link to clinical disease and are modifiable through targeted antimicrobial therapy.

Evidence for Tuberculosis in Individuals With Xpert Ultra "Trace" Sputum During Screening of High-Burden Communities.

BACKGROUND: "Trace" results on Xpert MTB/RIF Ultra ("Ultra"; Cepheid) -a molecular diagnostic test for tuberculosis (TB)-are often interpreted as an indication for TB treatment, but may also represent detection of nonviable bacilli or analytical error. In community-screening settings where individual TB risk is low, there is limited guidance on how to interpret Ultra-trace results. METHODS: We conducted systematic Ultra TB screening of adults and adolescents (≥15 years) in Kampala, Uganda, through door-to-door and event-based sputum collection. We enrolled individuals with trace-positive sputum for detailed clinical, radiographic, and microbiological (including 2 sputum cultures, repeat Ultra, and for people with HIV, urine lipoarabinomannan) evaluation, and compared those findings with similar evaluations in controls with Ultra-negative and Ultra-positive (non-trace) sputum. RESULTS: Of 21 957 people screened with Ultra, 211 (1.0%) tested positive, including 96 (46% of positives) with trace results. Of 92 people enrolled with trace-positive sputum; 12% (11/92) were HIV-positive and 14% (13/92) had prior TB. The prevalence of TB among participants with trace-positive sputum results was 14% (13/92) by culture, 24% (22/92) using broader microbiological criteria, and 26% (24/92) after accounting for clinical diagnosis. The prevalence of cough and of abnormal chest computed tomography (CT) findings were 32% and 26%, respectively, if Ultra-negative; 34% and 54% if trace-positive/non-microbiologically confirmed; 72% and 95% if trace-positive/microbiologically confirmed; and 71% and 93% if Ultra-positive (more than trace). CONCLUSIONS: Most individuals with trace-positive sputum in Ugandan communities did not have microbiologically confirmed TB but had more symptoms and chest CT abnormalities than people with Ultra-negative sputum.

New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

BACKGROUND: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain. METHODS: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester). RESULTS: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently. CONCLUSIONS: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

From Bench to Clinic: A Nitroreductase Rv3368c-Responsive Cyanine-Based Probe for the Specific Detection of Live Mycobacterium tuberculosis.

Tuberculosis (TB), characterized by high mortality and low diagnosis, is caused by a single pathogen, Mycobacterium tuberculosis (Mtb). Imaging tools that can be used to track Mtb without pre-labeling and to diagnose live Mtb in clinical samples can shorten the gap between bench and clinic, fuel the development of novel anti-TB drugs, strengthen TB prevention, and improve patient treatment. In this study, we report an unprecedented novel nitroreductase-responsive cyanine-based fluorescent probe (Cy3-NO2-tre) that rapidly and specifically labels Mtb and detects it in clinical samples. Cy3-NO2-tre generated fluorescence after activation by a specific nitroreductase, Rv3368c, which is conserved in the Mycobacteriaceae. Cy3-NO2-tre effectively imaged mycobacteria within infected host cells, tracked the infection process, and visualized Mycobacterium smegmatis being endocytosed by macrophages. Cy3-NO2-tre also detected Mtb in the sputum of patients with TB and exhibited excellent photostability. Furthermore, the Cy3-NO2-tre/auramine O percentage change within 7 ± 2 days post drug treatment in the sputum of inpatients was closely correlated with the reexamination results of the chest computed tomography, strongly demonstrating the clinical application of Cy3-NO2-tre as a prognostic indicator in monitoring the therapeutic efficacy of anti-TB drugs in the early patient care stage.

A Portable Nucleic Acid Testing Platform with Photosensitization, a Three-Dimensionally Printed Multipiece Chip, and Digital Color Sensing.

Portable nucleic acid testing (NAT) holds great promise for point-of-care disease diagnosis and field-based applications but remains difficult to achieve. Herein, we describe a portable NAT that streamlines loop-mediated isothermal amplification with photosensitization-based color development in a fully sealed 3D-printed multipiece chip. Using a smartphone accessory and an APP, we also introduce a calibration-free quantification approach via digital color sensing and library matching. With these innovative approaches, our detection platform is highly accessible, allowing for rapid and sensitive NAT without requiring sophisticated instruments and well-trained personnel. The field applicability of our NAT platform was demonstrated by detecting tuberculosis infections in clinical sputum samples and food adulteration in commercial salmon meat products.

Objective sputum colour assessment and clinical outcomes in bronchiectasis: data from the European Bronchiectasis Registry (EMBARC).

BACKGROUND: A validated 4-point sputum colour chart can be used to objectively evaluate the levels of airway inflammation in bronchiectasis patients. In the European Bronchiectasis Registry (EMBARC), we tested whether sputum colour would be associated with disease severity and clinical outcomes. METHODS: We used a prospective, observational registry of adults with bronchiectasis conducted in 31 countries. Patients who did not produce spontaneous sputum were excluded from the analysis. The Murray sputum colour chart was used at baseline and at follow-up visits. Key outcomes were frequency of exacerbations, hospitalisations for severe exacerbations and mortality during up to 5-year follow-up. RESULTS: 13 484 patients were included in the analysis. More purulent sputum was associated with lower forced expiratory volume in 1 s (FEV1), worse quality of life, greater bacterial infection and a higher bronchiectasis severity index. Sputum colour was strongly associated with the risk of future exacerbations during follow-up. Compared to patients with mucoid sputum (reference group), patients with mucopurulent sputum experienced significantly more exacerbations (incident rate ratio (IRR) 1.29, 95% CI 1.22-1.38; p<0.0001), while the rates were even higher for patients with purulent (IRR 1.55, 95% CI 1.44-1.67; p<0.0001) and severely purulent sputum (IRR 1.91, 95% CI 1.52-2.39; p<0.0001). Hospitalisations for severe exacerbations were also associated with increasing sputum colour with rate ratios, compared to patients with mucoid sputum, of 1.41 (95% CI 1.29-1.56; p<0.0001), 1.98 (95% CI 1.77-2.21; p<0.0001) and 3.05 (95% CI 2.25-4.14; p<0.0001) for mucopurulent, purulent and severely purulent sputum, respectively. Mortality was significantly increased with increasing sputum purulence, hazard ratio 1.12 (95% CI 1.01-1.24; p=0.027), for each increment in sputum purulence. CONCLUSION: Sputum colour is a simple marker of disease severity and future risk of exacerbations, severe exacerbations and mortality in patients with bronchiectasis.

Retrospective validation of MetaSystems' deep-learning-based digital microscopy platform with assistance compared to manual fluorescence microscopy for detection of mycobacteria.

This study aimed to validate Metasystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module (Neon Metafer) with assistance on respiratory and pleural samples, compared to conventional manual fluorescence microscopy (MM). Analytical parameters were assessed first, followed by a retrospective validation study. In all, 320 archived auramine-O-stained slides selected non-consecutively [85 originally reported as AFB-smear-positive, 235 AFB-smear-negative slides; with an overall mycobacterial culture positivity rate of 24.1% (77/320)] underwent whole-slide imaging and were analyzed by the Metafer Neon AFB Module (version 4.3.130) using a predetermined probability threshold (PT) for AFB detection of 96%. Digital slides were then examined by a trained reviewer blinded to previous AFB smear and culture results, for the final interpretation of assisted digital microscopy (a-DM). Paired results from both microscopic methods were compared to mycobacterial culture. A scanning failure rate of 10.6% (34/320) was observed, leaving 286 slides for analysis. After discrepant analysis, concordance, positive and negative agreements were 95.5% (95%CI, 92.4%-97.6%), 96.2% (95%CI, 89.2%-99.2%), and 95.2% (95%CI, 91.3%-97.7%), respectively. Using mycobacterial culture as reference standard, a-DM and MM had comparable sensitivities: 90.7% (95%CI, 81.7%-96.2%) versus 92.0% (95%CI, 83.4%-97.0%) (P-value = 1.00); while their specificities differed 91.9% (95%CI, 87.4%-95.2%) versus 95.7% (95%CI, 92.1%-98.0%), respectively (P-value = 0.03). Using a PT of 96%, MetaSystems' platform shows acceptable performance. With a national laboratory staff shortage and a local low mycobacterial infection rate, this instrument when combined with culture, can reliably triage-negative AFB-smear respiratory slides and identify positive slides requiring manual confirmation and semi-quantification. IMPORTANCE: This manuscript presents a full validation of MetaSystems' automated acid-fast bacilli (AFB) smear microscopy scanning and deep-learning-based image analysis module using a probability threshold of 96% including accuracy, precision studies, and evaluation of limit of AFB detection on respiratory samples when the technology is used with assistance. This study is complementary to the conversation started by Tomasello et al. on the use of image analysis artificial intelligence software in routine mycobacterial diagnostic activities within the context of high-throughput laboratories with low incidence of tuberculosis.

Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction.

IMPORTANCE: In this study, we successfully established a new One-Pot method, named TB One-Pot, for detecting Mtb in sputum by combining CRISPR-cas12b-mediated trans-cleavage with cross-priming amplification (CPA). Our study evaluated the diagnostic performance of TB One-Pot in clinical sputum samples for tuberculosis. The findings provide evidence for the potential of TB One-Pot as a diagnostic tool for tuberculosis.

Diagnostic accuracy of tongue swab testing on two automated tuberculosis diagnostic platforms, Cepheid Xpert MTB/RIF Ultra and Molbio Truenat MTB Ultima.

Tongue dorsum swabbing is a potential alternative to sputum collection for tuberculosis (TB) testing. Previous studies showed that Cepheid Xpert MTB/RIF Ultra (Xpert Ultra) can detect Mycobacterium tuberculosis DNA on tongue swabs stored in buffer, with 72% sensitivity and 100% specificity relative to a sputum microbiological reference standard (sputum MRS). The present study evaluated a more convenient sample collection protocol (dry swab storage), combined with streamlined sample processing protocols, for evaluating two commercial TB diagnostic tests: Xpert Ultra and Molbio Truenat MTB Ultima (MTB Ultima). Copan FLOQSwabs were self-collected or collected by study workers from 321 participants in Western Cape, South Africa. All participants had symptoms suggestive of TB, and 245 of them had sputum MRS-confirmed TB (by sputum MGIT culture and/or Xpert Ultra). One tongue swab per participant was tested on Xpert Ultra, and another tongue swab was tested with MTB Ultima. Xpert Ultra was 75.5% sensitive and 100% specific relative to sputum MRS, similar to previous methods that used swabs stored in buffer. MTB Ultima was 71.6% sensitive and 96.9% specific relative to sputum MRS. When sample lysates that were false-negative or invalid by MTB Ultima were frozen, thawed, and re-tested, MTB Ultima sensitivity rose to 79.1%. Both tests were more sensitive with swabs from participants with higher sputum Xpert Ultra semi-quantitative results. Although additional development could improve diagnostic accuracy, these results further support tongue swabs as easy-to-collect samples for TB testing. IMPORTANCE: Tongue dorsum swabbing is a promising alternative to sputum collection for tuberculosis (TB) testing. Our results lend further support for tongue swabs as exceptionally easy-to-collect samples for high-throughput TB testing.

The characteristics of microbiome in the upper respiratory tract of COVID-19 patients.

BACKGROUND: Co-infection with other pathogens in coronavirus disease 2019 (COVID-19) patients exacerbates disease severity and impacts patient prognosis. Clarifying the exact pathogens co-infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is premise of the precise treatment for COVID-19 patients. METHODS: Sputum samples were collected from 17 patients in the COVID-19 positive group and 18 patients in the COVID-19 negative group. DNA extraction was performed to obtain the total DNA. Sequencing analysis using 16S and ITS rRNA gene was carried out to analyze the composition of bacterial and fungal communities. Meanwhile, all the samples were inoculated for culture. RESULTS: We did not observe significant differences in bacterial composition between the COVID-19 positive and negative groups. However, a significantly higher abundance of Candida albicans was observed in the upper respiratory tract samples from the COVID-19 positive group compared to the COVID-19 negative group. Moreover, the Candida albicans strains isolated from COVID-19 positive group exhibited impaired secretion of aspartyl proteinases. CONCLUSION: COVID-19 positive patients demonstrate a notable increase in the abundance of Candida albicans, along with a decrease in the levels of aspartyl proteinases, indicating the alteration of microbiota composition of upper respiratory tract.

Comparison of the sputum microbiome between patients with stable nontuberculous mycobacterial pulmonary disease and patients requiring treatment.

BACKGROUND: We evaluated whether the sputum bacterial microbiome differs between nontuberculous mycobacteria pulmonary disease (NTM-PD) patients with stable disease not requiring antibiotic treatment and those requiring antibiotics. METHODS: We collected sputum samples from 21 clinically stable NTM-PD patients (stable group) and 14 NTM-PD patients needing antibiotic treatment (treatment group). We also obtained 13 follow-up samples from the stable group. We analyzed the 48 samples using 16S rRNA gene sequencing (V3-V4 region) and compared the groups. RESULTS: In the linear discriminant analysis effect size (LEfSe) analysis, the species Porphyromonas pasteri, Haemophilus parahaemolyticus, Prevotella nanceiensis, and Gemella haemolysans were significantly more prevalent in the sputum of the stable group compared to the treatment group. No taxa showed significant differences in alpha-/beta-diversity or LEfSe between the 21 baseline and 13 follow-up sputum samples in the stable group. In the stable group, the genus Bergeyella and species Prevotella oris were less common in patients who achieved spontaneous culture conversion (n = 9) compared to those with persistent NTM positivity (n = 12) (effect size 3.04, p = 0.039 for Bergeyella; effect size 3.64, p = 0.033 for P. oris). In the treatment group, H. parainfluenzae was more common in patients with treatment success (n = 7) than in treatment-refractory patients (n = 7) (effect size 4.74, p = 0.013). CONCLUSIONS: Our study identified distinct bacterial taxa in the sputum of NTM-PD patients based on disease status. These results suggest the presence of a microbial environment that helps maintain disease stability.

Bacterial interactome disturbance in chronic obstructive pulmonary disease clinical stability and exacerbations.

RATIONALE: Our understanding of airway dysbiosis in chronic obstructive pulmonary disease (COPD) remains incomplete, which may be improved by unraveling the complexity in microbial interactome. OBJECTIVES: To characterize reproducible features of airway bacterial interactome in COPD at clinical stability and during exacerbation, and evaluate their associations with disease phenotypes. METHODS: We performed weighted ensemble-based co-occurrence network analysis of 1742 sputum microbiomes from published and new microbiome datasets, comprising two case-control studies of stable COPD versus healthy control, two studies of COPD stability versus exacerbation, and one study with exacerbation-recovery time series data. RESULTS: Patients with COPD had reproducibly lower degree of negative bacterial interactions, i.e. total number of negative interactions as a proportion of total interactions, in their airway microbiome compared with healthy controls. Evaluation of the Haemophilus interactome showed that the antagonistic interaction networks of this established pathogen rather than its abundance consistently changed in COPD. Interactome dynamic analysis revealed reproducibly reduced antagonistic interactions but not diversity loss during COPD exacerbation, which recovered after treatment. In phenotypic analysis, unsupervised network clustering showed that loss of antagonistic interactions was associated with worse clinical symptoms (dyspnea), poorer lung function, exaggerated neutrophilic inflammation, and higher exacerbation risk. Furthermore, the frequent exacerbators (≥ 2 exacerbations per year) had significantly reduced antagonistic bacterial interactions while exhibiting subtle compositional changes in their airway microbiota. CONCLUSIONS: Bacterial interactome disturbance characterized by reduced antagonistic interactions, rather than change in pathogen abundance or diversity, is a reproducible feature of airway dysbiosis in COPD clinical stability and exacerbations, which suggests that we may target interactome rather than pathogen alone for disease treatment.

Comparative microbiome analysis in cystic fibrosis and non-cystic fibrosis bronchiectasis.

BACKGROUND: Bronchiectasis is a condition characterized by abnormal and irreversible bronchial dilation resulting from lung tissue damage and can be categorized into two main groups: cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). Both diseases are marked by recurrent infections, inflammatory exacerbations, and lung damage. Given that infections are the primary drivers of disease progression, characterization of the respiratory microbiome can shed light on compositional alterations and susceptibility to antimicrobial drugs in these cases compared to healthy individuals. METHODS: To assess the microbiota in the two studied diseases, 35 subjects were recruited, comprising 10 NCFB and 13 CF patients and 12 healthy individuals. Nasopharyngeal swabs and induced sputum were collected, and total DNA was extracted. The DNA was then sequenced by the shotgun method and evaluated using the SqueezeMeta pipeline and R. RESULTS: We observed reduced species diversity in both disease cohorts, along with distinct microbial compositions and profiles of antimicrobial resistance genes, compared to healthy individuals. The nasopharynx exhibited a consistent microbiota composition across all cohorts. Enrichment of members of the Burkholderiaceae family and an increased Firmicutes/Bacteroidetes ratio in the CF cohort emerged as key distinguishing factors compared to NCFB group. Staphylococcus aureus and Prevotella shahii also presented differential abundance in the CF and NCFB cohorts, respectively, in the lower respiratory tract. Considering antimicrobial resistance, a high number of genes related to antibiotic efflux were detected in both disease groups, which correlated with the patient's clinical data. CONCLUSIONS: Bronchiectasis is associated with reduced microbial diversity and a shift in microbial and resistome composition compared to healthy subjects. Despite some similarities, CF and NCFB present significant differences in microbiome composition and antimicrobial resistance profiles, suggesting the need for customized management strategies for each disease.

Highly diverse sputum microbiota correlates with the disease severity in patients with community-acquired pneumonia: a longitudinal cohort study.

BACKGROUND: Community-acquired pneumonia (CAP) is a common and serious condition that can be caused by a variety of pathogens. However, much remains unknown about how these pathogens interact with the lower respiratory commensals, and whether any correlation exists between the dysbiosis of the lower respiratory microbiota and disease severity and prognosis. METHODS: We conducted a retrospective cohort study to investigate the composition and dynamics of sputum microbiota in patients diagnosed with CAP. In total, 917 sputum specimens were collected consecutively from 350 CAP inpatients enrolled in six hospitals following admission. The V3-V4 region of the 16 S rRNA gene was then sequenced. RESULTS: The sputum microbiota in 71% of the samples were predominately composed of respiratory commensals. Conversely, 15% of the samples demonstrated dominance by five opportunistic pathogens. Additionally, 5% of the samples exhibited sterility, resembling the composition of negative controls. Compared to non-severe CAP patients, severe cases exhibited a more disrupted sputum microbiota, characterized by the highly dominant presence of potential pathogens, greater deviation from a healthy state, more significant alterations during hospitalization, and sparser bacterial interactions. The sputum microbiota on admission demonstrated a moderate prediction of disease severity (AUC = 0.74). Furthermore, different pathogenic infections were associated with specific microbiota alterations. Acinetobacter and Pseudomonas were more abundant in influenza A infections, with Acinetobacter was also enriched in Klebsiella pneumoniae infections. CONCLUSION: Collectively, our study demonstrated that pneumonia may not consistently correlate with severe dysbiosis of the respiratory microbiota. Instead, the degree of microbiota dysbiosis was correlated with disease severity in CAP patients.

Inhaler Steroid Use Changes Oral and Airway Bacterial and Fungal Microbiome Profile in Asthma Patients.

INTRODUCTION: The full spectrum of bacterial and fungal species in adult asthma and the effect of inhaled corticosteroid use is not well described. The aim was to collect mouthwash and induced sputum samples from newly diagnosed asthma patients in the pretreatment period and in chronic asthma patients while undergoing regular maintenance inhaled corticosteroid therapy, in order to demonstrate the bacterial and fungal microbiome profile. METHODS: The study included 28 asthmatic patients on inhaler steroid therapy, 25 steroid-naive asthmatics, and 24 healthy controls. Genomic DNA was isolated from induced sputum and mouthwash samples. Analyses were performed using bacterial primers selected from the 16S rRNA region for the bacterial genome and "panfungal" primers selected from the 5.8S rRNA region for the fungal genome. RESULTS: Dominant genera in mouthwash samples of steroid-naive asthmatics were Neisseria, Haemophilus, and Rothia. The oral microbiota of asthmatic patients on inhaler steroid treatment included Neisseria, Rothia, and Veillonella species. Abundant genera in induced sputum samples of steroid-naive asthma patients were Actinomyces, Granulicatella, Fusobacterium, Peptostreptococcus, and Atopobium. Sputum microbiota of asthma patients taking inhaler steroids were dominated by Prevotella and Porphyromonas. Mucor plumbeus and Malassezia restricta species were abundant in the airways of steroid-naive asthma patients. Choanephora infundibulifera and Malassezia restricta became dominant in asthma patients taking inhaled steroids. CONCLUSION: The oral and airway microbiota consist of different bacterial and fungal communities in healthy and asthmatic patients. Inhaler steroid use may influence the composition of the oral and airway microbiota.

Nocardia implantans sp. nov. isolated from a patient with pulmonary infection.

TwoGram-stain-positive and rod-shaped actinomycetes (strains CDC186T and CDC192) were isolated from sputum samples of a patient in Chongqing, PR China, and were investigated to determine their taxonomic status. The results of phylogenetic analysis based on the 16S rRNA gene indicated that CDC186T and CDC192 represented members of the genus Nocardia, and the sequence similarity with Nocardia beijingensis DSM 44636T was the highest, at 99.71 and 99.78 %, respectively. The DNA G+C content of both CDC186T and CDC192 was 69.1 %. Genomic diversity analysis revealed that the average nucleotide identity and in silico DNA‒DNA hybridisation values between the two novel strains and closely related species were significantly below the thresholds of 95-96 and 70 %, respectively, but these values between the two novel strains were 99.96 and 99.90 %, respectively. The phylogenetic relationship based on the dapb1 gene and the single-copy core genes further indicated that the two novel strains were clustered in separate branch adjacent to N. beijingensis DSM 44636T. Growth occurred within the ranges of 20-42 °C, pH 6.0-9.0 and NaCl concentrations of 0.5-4.5 % (w/v). The major fatty acids of CDC186T and CDC192 were C16 : 0 and C18 : 0 10-methyl [tuberculostearic acid (TBSA)]. The predominant respiratory menaquinone was MK-9. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannoside, one unidentified glycolipid, one unidentified phospholipid and one unidentified phosphoglycolipid. All the genomes of the studied strains were annotated with virulence factor (VF)-associated genes homologous to those of Mycobacterium tuberculosis, and the results of susceptibility testing indicated that CDC186T and CDC192 were resistant to amoxicillin-clavulanic acid and tigecycline. On the basis of chemotaxonomic characteristics and the results of phylogenetic analyses, strains CDC186T and CDC192 represent a novel species within the genus Nocardia, for which the name Nocardia implantans sp. nov. is proposed. The type strain is CDC186T (=GDMCC 4.206T= JCM 34959T).

Effect of Bedaquiline and Delamanid Pharmacokinetics on Sputum Culture Conversion and Adverse Events in Drug-Resistant Tuberculosis.

BACKGROUND: Pharmacokinetic studies of bedaquiline and delamanid in patients with pre-extensively drug-resistant tuberculosis (pre-XDR TB) will help in the optimization of these drugs for both culture conversion and adverse events. METHODS: A prospective cohort of 165 adult patients (56% male with mean [SD] age 29 [9.7] years) with pre-XDR TB was treated with bedaquiline, delamanid, clofazimine, and linezolid for 24 weeks at 5 sites in India. Bedaquiline was administered at 400 mg daily for 2 weeks followed by 200 mg thrice weekly for 22 weeks, whereas delamanid was administered at 100 mg twice daily. In 23 consenting participants at 8 and 16 weeks of treatment, blood was collected at 0, 2, 4, 5, 6, 8, 12, and 24 hours postdosing for an intense pharmacokinetic study. Pharmacokinetic parameters were correlated with sputum culture conversion and adverse events. RESULTS: The mean (SD) age and weight of patients were 30 (10) years and 54 kg, respectively. The median minimum concentration (C min ) and time-concentration curve (AUC) for bedaquiline, respectively, were 0.6 mcg/mL and 27 mcg/mL·h at week 8 and 0.8 mcg/mL and 36 mcg/mL·h at week 16, suggesting drug accumulation over time. The median C min and AUC of delamanid, respectively, were 0.17 mcg/mL and 5.1 mcg/mL·h at week 8 and 0.20 mcg/mL and 7.5 mcg/mL·h at week 16. Delay in sputum conversion was observed in patients with drug concentrations lower than the targeted concentration. At weeks 8 and 16, 13 adverse events were observed. Adverse events were resolved through symptomatic treatment. Body mass index was found to be significantly associated with drug-exposure parameters. CONCLUSIONS: Bedaquiline and delamanid when co-administered exhibit plasma drug levels within the targeted concentrations, showing an exposure-response relationship.