A GMR enzymatic assay for quantifying nuclease and peptidase activity.

Hydrolytic enzymes play crucial roles in cellular processes, and dysregulation of their activities is implicated in various physiological and pathological conditions. These enzymes cleave substrates such as peptide bonds, phosphodiester bonds, glycosidic bonds, and other esters. Detecting aberrant hydrolase activity is vital for understanding disease mechanisms and developing targeted therapeutic interventions. This study introduces a novel approach to measuring hydrolase activity using giant magnetoresistive (GMR) spin valve sensors. These sensors change resistance in response to magnetic fields, and here, they are functionalized with specific substrates for hydrolases conjugated to magnetic nanoparticles (MNPs). When a hydrolase cleaves its substrate, the tethered magnetic nanoparticle detaches, causing a measurable shift in the sensor's resistance. This design translates hydrolase activity into a real-time, activity-dependent signal. The assay is simple, rapid, and requires no washing steps, making it ideal for point-of-care settings. Unlike fluorescent methods, it avoids issues like autofluorescence and photobleaching, broadening its applicability to diverse biofluids. Furthermore, the sensor array contains 80 individually addressable sensors, allowing for the simultaneous measurement of multiple hydrolases in a single reaction. The versatility of this method is demonstrated with substrates for nucleases, Bcu I and DNase I, and the peptidase, human neutrophil elastase. To demonstrate a clinical application, we show that neutrophil elastase in sputum from cystic fibrosis patients hydrolyze the peptide-GMR substrate, and the cleavage rate strongly correlates with a traditional fluorogenic substrate. This innovative assay addresses challenges associated with traditional enzyme measurement techniques, providing a promising tool for real-time quantification of hydrolase activities in diverse biological contexts.

Early Evidence of Chronic Obstructive Pulmonary Disease Obscured by Race-Specific Prediction Equations.

Rationale: The identification of early chronic obstructive pulmonary disease (COPD) is essential to appropriately counsel patients regarding smoking cessation, provide symptomatic treatment, and eventually develop disease-modifying treatments. Disease severity in COPD is defined using race-specific spirometry equations. These may disadvantage non-White individuals in diagnosis and care. Objectives: Determine the impact of race-specific equations on African American (AA) versus non-Hispanic White individuals. Methods: Cross-sectional analyses of the COPDGene (Genetic Epidemiology of Chronic Obstructive Pulmonary Disease) cohort were conducted, comparing non-Hispanic White (n = 6,766) and AA (n = 3,366) participants for COPD manifestations. Measurements and Main Results: Spirometric classifications using race-specific, multiethnic, and "race-reversed" prediction equations (NHANES [National Health and Nutrition Examination Survey] and Global Lung Function Initiative "Other" and "Global") were compared, as were respiratory symptoms, 6-minute-walk distance, computed tomography imaging, respiratory exacerbations, and St. George's Respiratory Questionnaire. Application of different prediction equations to the cohort resulted in different classifications by stage, with NHANES and Global Lung Function Initiative race-specific equations being minimally different, but race-reversed equations moving AA participants to more severe stages and especially between the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage 0 and preserved ratio impaired spirometry groups. Classification using the established NHANES race-specific equations demonstrated that for each of GOLD stages 1-4, AA participants were younger, had fewer pack-years and more current smoking, but had more exacerbations, shorter 6-minute-walk distance, greater dyspnea, and worse BODE (body mass index, airway obstruction, dyspnea, and exercise capacity) scores and St. George's Respiratory Questionnaire scores. Differences were greatest in GOLD stages 1 and 2. Race-reversed equations reclassified 774 AA participants (43%) from GOLD stage 0 to preserved ratio impaired spirometry. Conclusions: Race-specific equations underestimated disease severity among AA participants. These effects were particularly evident in early disease and may result in late detection of COPD.

Returning incidentally discovered Hepatitis C RNA-seq results to COPDGene study participants.

The consequences of returning infectious pathogen test results identified incidentally in research studies have not been well-studied. Concerns include identification of an important health issue for individuals, accuracy of research test results, public health impact, potential emotional distress for participants, and need for IRB permissions. Blood RNA-sequencing analysis for non-human RNA in 3984 participants from the COPDGene study identified 228 participants with evidence suggestive for hepatitis C virus (HCV) infection. We hypothesized that incidentally discovered HCV results could be effectively returned to COPDGene participants with attention to the identified concerns. In conjunction with a COPDGene Participant Advisory Panel, we developed and obtained IRB approval for a process of returning HCV research results and an HCV Follow-Up Study questionnaire to capture information about previous HCV diagnosis and treatment information and participant reactions to return of HCV results. During phone calls following the initial HCV notification letter, 84 of 124 participants who could be contacted (67.7%) volunteered that they had been previously diagnosed with HCV infection. Thirty-one of these 124 COPDGene participants were enrolled in the HCV Follow-Up Study. Five of the 31 HCV Follow-Up Study participants did not report a previous diagnosis of HCV. For four of these participants, subsequent clinical HCV testing confirmed HCV infection. Thus, 30/31 Follow-Up Study participants had confirmed HCV diagnoses, supporting the accuracy of the HCV research test results. However, the limited number of participants in the Follow-Up Study precludes an accurate assessment of the false-positive and false-negative rates of the research RNA sequencing evidence for HCV. Most HCV Follow-Up Study participants (29/31) were supportive of returning HCV research results, and most participants found the process for returning HCV results to be informative and not upsetting. Newly diagnosed participants were more likely to be pleased to learn about a potentially curable infection (p = 0.027) and showed a trend toward being more frightened by the potential health risks of HCV (p = 0.11). We conclude that HCV results identified incidentally during transcriptomic research studies can be successfully returned to research study participants with a carefully designed process.

Compounding Achromobacter Phages for Therapeutic Applications.

Achromobacter species colonization of Cystic Fibrosis respiratory airways is an increasing concern. Two adult patients with Cystic Fibrosis colonized by Achromobacter xylosoxidans CF418 or Achromobacter ruhlandii CF116 experienced fatal exacerbations. Achromobacter spp. are naturally resistant to several antibiotics. Therefore, phages could be valuable as therapeutics for the control of Achromobacter. In this study, thirteen lytic phages were isolated and characterized at the morphological and genomic levels for potential future use in phage therapy. They are presented here as the Achromobacter Kumeyaay phage collection. Six distinct Achromobacter phage genome clusters were identified based on a comprehensive phylogenetic analysis of the Kumeyaay collection as well as the publicly available Achromobacter phages. The infectivity of all phages in the Kumeyaay collection was tested in 23 Achromobacter clinical isolates; 78% of these isolates were lysed by at least one phage. A cryptic prophage was induced in Achromobacter xylosoxidans CF418 when infected with some of the lytic phages. This prophage genome was characterized and is presented as Achromobacter phage CF418-P1. Prophage induction during lytic phage preparation for therapy interventions require further exploration. Large-scale production of phages and removal of endotoxins using an octanol-based procedure resulted in a phage concentrate of 1 × 109 plaque-forming units per milliliter with an endotoxin concentration of 65 endotoxin units per milliliter, which is below the Food and Drugs Administration recommended maximum threshold for human administration. This study provides a comprehensive framework for the isolation, bioinformatic characterization, and safe production of phages to kill Achromobacter spp. in order to potentially manage Cystic Fibrosis (CF) pulmonary infections.

Race-Specific Spirometry Equations Do Not Improve Models of Dyspnea and Quantitative Chest CT Phenotypes.

BACKGROUND: Race-specific spirometry reference equations are used globally to interpret lung function for clinical, research, and occupational purposes, but inclusion of race is under scrutiny. RESEARCH QUESTION: Does including self-identified race in spirometry reference equation formation improve the ability of predicted FEV1 values to explain quantitative chest CT abnormalities, dyspnea, or Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification? STUDY DESIGN AND METHODS: Using data from healthy adults who have never smoked in both the National Health and Nutrition Survey (2007-2012) and COPDGene study cohorts, race-neutral, race-free, and race-specific prediction equations were generated for FEV1. Using sensitivity/specificity, multivariable logistic regression, and random forest models, these equations were applied in a cross-sectional analysis to populations of individuals who currently smoke and individuals who formerly smoked to determine how they affected GOLD classification and the fit of models predicting quantitative chest CT phenotypes or dyspnea. RESULTS: Race-specific equations showed no advantage relative to race-neutral or race-free equations in models of quantitative chest CT phenotypes or dyspnea. Race-neutral reference equations reclassified up to 19% of Black participants into more severe GOLD classes, while race-neutral/race-free equations may improve model fit for dyspnea symptoms relative to race-specific equations. INTERPRETATION: Race-specific equations offered no advantage over race-neutral/race-free equations in three distinct explanatory models of dyspnea and chest CT scan abnormalities. Race-neutral/race-free reference equations may improve pulmonary disease diagnoses and treatment in populations highly vulnerable to lung disease.

Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia.

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Bacteriophage Therapy of Multidrug-resistant Achromobacter in an 11-Year-old Boy With Cystic Fibrosis Assessed by Metagenome Analysis.

BACKGROUND: Cystic fibrosis (CF) is a genetic disease associated with lung disease characterized by chronic pulmonary infection, increasingly caused by multiple drug-resistant pathogens after repeated antibiotic exposure, limiting antibiotic treatment options. Bacteriophages can provide a pathogen-specific bactericidal treatment used with antibiotics to improve microbiologic and clinical outcomes in CF. METHODS: Achromobacter species isolates from sputum of a chronically infected person with CF, were assessed for susceptibility to bacteriophages: 2 highly active, purified bacteriophages were administered intravenously every 8 hours, in conjunction with a 14-day piperacillin/tazobactam course for CF exacerbation. Sputum and blood were collected for metagenome analysis during treatment, with sputum analysis at 1-month follow-up. Assessments of clinical status, pulmonary status and laboratory evaluation for safety were conducted. RESULTS: Bacteriophage administration was well-tolerated, with no associated clinical or laboratory adverse events. Metagenome analysis documented an 86% decrease in the relative proportion of Achromobacter DNA sequence reads in sputum and a 92% decrease in blood, compared with other bacterial DNA reads, comparing pretreatment and posttreatment samples. Bacteriophage DNA reads were detected in sputum after intravenous administration during treatment, and at 1-month follow-up. Reversal of antibiotic resistance to multiple antibiotics occurred in some isolates during treatment. Stabilization of lung function was documented at 1-month follow-up. CONCLUSIONS: Bacteriophage/antibiotic treatment decreased the host pulmonary bacterial burden for Achromobacter assessed by metagenome analysis of sputum and blood, with ongoing bacteriophage replication documented in sputum at 1-month follow-up. Prospective controlled studies are needed to define the dose, route of administration and duration of bacteriophage therapy for both acute and chronic infection in CF.

Is it time to end race and ethnicity adjustment for pediatric pulmonary function tests?

The continued inclusion of race in spirometry reference equations is a topic of intense debate for adult lung function, but less discussion has focused on implications for children. Obtaining accurate estimates of children's lung function is an important component of the diagnosis of childhood respiratory illnesses, including asthma, cystic fibrosis, and interstitial lung disease. Given the higher burden among racial/ethnic minorities for many respiratory illnesses, it is critical to avoid racial bias in interpreting lung function. We recommend against the continued use of race-specific reference equations for a number of reasons. The original reference populations used to develop the equations were comprised of children with restricted racial diversity, relatively small sample sizes, and likely included some unhealthy children. Moreover, there is no scientific justification for innate racial differences in lung function, as there is no clear physiological or genetic explanation for the disparities. Alternatively, many environmental factors harm lung development, including allergens from pests, asbestos, lead, prenatal smoking, and air pollution, as well as preterm birth and childhood respiratory illnesses, which are all more common among minority racial groups. Race-neutral equations may provide a temporary solution, but still rely on the racial diversity of the reference populations used to build them. Ultimately researchers must uncover the underlying factors truly driving racial differences in lung function.

Use of the Spirometric "Fixed-Ratio" Underdiagnoses COPD in African-Americans in a Longitudinal Cohort Study.

BACKGROUND: COPD diagnosis is tightly linked to the fixed-ratio spirometry criteria of FEV1/FVC < 0.7. African-Americans are less often diagnosed with COPD. OBJECTIVE: Compare COPD diagnosis by fixed-ratio with findings and outcomes by race. DESIGN: Genetic Epidemiology of COPD (COPDGene) (2007-present), cross-sectional comparing non-Hispanic white (NHW) and African-American (AA) participants for COPD diagnosis, manifestations, and outcomes. SETTING: Multicenter, longitudinal US cohort study. PARTICIPANTS: Current or former smokers with ≥ 10-pack-year smoking history enrolled at 21 clinical centers including over-sampling of participants with known COPD and AA. Exclusions were pre-existing non-COPD lung disease, except for a history of asthma. MEASUREMENTS: Subject diagnosis by conventional criteria. Mortality, imaging, respiratory symptoms, function, and socioeconomic characteristics, including area deprivation index (ADI). Matched analysis (age, sex, and smoking status) of AA vs. NHW within participants without diagnosed COPD (GOLD 0; FEV1 ≥ 80% predicted and FEV1/FVC ≥ 0.7). RESULTS: Using the fixed ratio, 70% of AA (n = 3366) were classified as non-COPD, versus 49% of NHW (n = 6766). AA smokers were younger (55 vs. 62 years), more often current smoking (80% vs. 39%), with fewer pack-years but similar 12-year mortality. Density distribution plots for FEV1 and FVC raw spirometry values showed disproportionate reductions in FVC relative to FEV1 in AA that systematically led to higher ratios. The matched analysis demonstrated GOLD 0 AA had greater symptoms, worse DLCO, spirometry, BODE scores (1.03 vs 0.54, p < 0.0001), and greater deprivation than NHW. LIMITATIONS: Lack of an alternative diagnostic metric for comparison. CONCLUSIONS: The fixed-ratio spirometric criteria for COPD underdiagnosed potential COPD in AA participants when compared to broader diagnostic criteria. Disproportionate reductions in FVC relative to FEV1 leading to higher FEV1/FVC were identified in these participants and associated with deprivation. Broader diagnostic criteria for COPD are needed to identify the disease across all populations.

Medicaid Integrated Purchasing for Physical and Behavioral Health: Early Adopters' Perceptions of Payment Reform Implementation in Washington State.

The Centers for Medicare and Medicaid Innovation (CMMI) gave rise to the State Innovation Models (SIMs). Medicaid Integrated Purchasing for Physical and Behavioral Health, referred to as Payment Model 1 (PM1), was a core payment redesign area of the Washington State SIM project under which our research team was contracted to provide an evaluation. In doing so, we leveraged an open systems conceptual model to assess qualitatively Early Adopter stakeholders' perceived effects of implementation. Between 2017 and 2019, we conducted three rounds of interviews, examining themes of care coordination, common facilitators and barriers to integration, and potential concerns for sustaining the initiative into the future. Further, we noted the initiative's complexity may require the establishment of enduring partnerships, secure funding sources, and committed regional leadership to ensure longer-term success.

Cystic Fibrosis Screen Positive, Inconclusive Diagnosis Genotypes in People with Cystic Fibrosis from the U.S. Patient Registry.

Rationale: Variants within the cystic fibrosis (CF) transmembrane conductance regulator gene, CFTR, that are of unknown significance or are categorized as non-CF causing may be observed in persons with CF. These variants are frequently detected in children with inconclusive newborn screen results and, in some cases, may be associated with a benign presentation in early childhood that progresses to a CF phenotype later in life. Objectives: To analyze data from individuals enrolled in the U.S. Cystic Fibrosis Foundation Patient Registry who have received a diagnosis of CF and who have variants found in a population of children with a CF screen positive, inconclusive diagnosis (CFSPID). Methods: This retrospective review analyzed registry data from individuals with a diagnosis of CF who also harbor one or more variants of interest because of their frequency within a CFSPID population and/or their interpretation as non-CF causing. Three groups were defined by the number of CF-causing variants identified (CF-Cx2, CF-Cx1, and CF-Cx0), which were reported in addition to the variant(s) of interest. Multivariate quantile regression modeling of the outcome for forced expiratory volume in 1 second (FEV1) generated a disease severity score for each person determined by six selected variables. Median scores were calculated for the three groups. Results: Patients carrying one CF-causing variant and at least one variant of interest (CF-Cx1) had higher median disease severity scores compared with those carrying CF-Cx2, suggesting a milder phenotype (P < 0.05). However, there was no statistically significant difference in scores between CF-Cx2 and the two other groups combined (CF-Cx1 and CF-Cx0; P = 0.33). Analysis revealed that the CF-Cx1 and CF-Cx0 groups, when compared with the CF-Cx2 group, had later median diagnoses (8 years vs. newborn; P < 0.0001), lower median sweat chloride (48 mmol/L vs. 94.5 mmol/L; P < 0.0001), lower prevalence of pancreatic insufficiency (29% vs. 78%; P < 0.0001), and higher median FEV1% predicted (95% vs. 87%; P = 0.0002). Conclusions: Individuals with CF who have specific variants frequently identified in children with CFSPID have a similar range of disease severity scores compared with those who have two CF-causing variants, but a milder phenotype overall. Variants that should be given careful scrutiny because of their high prevalence are G576A+R668C, T854T, R75Q, F1052V, R1070W, R31C, and L967S.

Safety and microbiological activity of phage therapy in persons with cystic fibrosis colonized with Pseudomonas aeruginosa: study protocol for a phase 1b/2, multicenter, randomized, double-blind, placebo-controlled trial.

BACKGROUND: Bacteriophages (phages) are a promising anti-infective option for human disease. Major gaps remain in understanding their potential utility. METHODS: This is a randomized, placebo-controlled, double-blind study of a single dose of intravenous phage in approximately 72 clinically stable adult cystic fibrosis volunteers recruited from up to 20 US sites with Pseudomonas aeruginosa airway colonization. The single dose of phage consists of a mixture of four anti-pseudomonal phages. Six sentinel participants will be sequentially enrolled with dose escalation of the phage mixture by one log10 beginning with 4 × 107 plaque-forming units in an unblinded stage 1. If no serious adverse events related to the study product are identified, the trial will proceed to a double-blinded stage 2. In stage 2a, 32 participants will be randomly assigned to one of three phage dosages or placebo in a 1:1:1:1 allocation. An interim analysis will be performed to determine the phage dosage with the most favorable safety and microbiological activity profile to inform phage dosing in stage 2b. During stage 2b, up to 32 additional volunteers will be randomized 1:1 to the phage or placebo arm. Primary outcomes include (1) the number of grade 2 or higher treatment-emergent adverse events, (2) change in log10 P. aeruginosa total colony counts in sputum, and (3) the probability of a randomly selected subject having a more favorable outcome ranking if assigned to receive phage therapy versus placebo. Exploratory outcomes include (1) sputum and serum phage pharmacokinetics, (2) the impact of phage on lung function, (3) the proportion of P. aeruginosa isolates susceptible to the phage mixture before and after study product administration, and (4) changes in quality of life. DISCUSSION: This trial will investigate the activity of phages in reducing P. aeruginosa colony counts and provide insights into the safety profile of phage therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT05453578. Registered on 12 July 2022.

Integrating specialist palliative care to improve care and reduce suffering: cystic fibrosis (InSPIRe:CF) - study protocol for a multicentre randomised clinical trial.

INTRODUCTION: Cystic fibrosis (CF) is a life-limiting genetic disorder estimated to affect more than 160 000 individuals and their families worldwide. People living with CF commonly experience significant physical and emotional symptom burdens, disruptions to social roles and complex treatment decision making. While palliative care (PC) interventions have been shown to relieve many such burdens in other serious illnesses, no rigorous evidence exists for palliative care in CF. Thus, this study aims to compare the effect of specialist palliative care plus usual CF care vs usual CF care alone on patient quality of life. METHODS AND ANALYSIS: This is a five-site, two-arm, partially masked, randomised superiority clinical trial. 264 adults with CF will be randomly assigned to usual CF care or usual CF care plus a longitudinal palliative care intervention delivered by a palliative care specialist. The trial's primary outcome is patient quality of life (measured with the Functional Assessment of Chronic Illness Therapy-Palliative care instrument). Secondary outcomes include symptom burden, satisfaction with care and healthcare utilisation. Outcomes will be measured at 12 months (primary endpoint) and 15 months (secondary endpoint). In addition, we will conduct qualitative interviews with patient participants, caregivers, and palliative care and CF care team members to explore perceptions of the intervention's impact and barriers and facilitators to dissemination. ETHICS AND DISSEMINATION: Human subjects research ethics approval was obtained from all participating sites, and all study participants gave informed consent. We will publish the results of this trial in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: ISRCTN53323164.

Erratum: Automated CT Staging of Chronic Obstructive Pulmonary Disease Severity for Predicting Disease Progression and Mortality with a Deep Learning Convolutional Neural Network.

[This corrects the article DOI: 10.1148/ryct.2021200477.].

Unsupervised Learning Identifies Computed Tomographic Measurements as Primary Drivers of Progression, Exacerbation, and Mortality in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome with phenotypic manifestations that tend to be distributed along a continuum. Unsupervised machine learning based on broad selection of imaging and clinical phenotypes may be used to identify primary variables that define disease axes and stratify patients with COPD. Objectives: To identify primary variables driving COPD heterogeneity using principal component analysis and to define disease axes and assess the prognostic value of these axes across three outcomes: progression, exacerbation, and mortality. Methods: We included 7,331 patients between 39 and 85 years old, of whom 40.3% were Black and 45.8% were female smokers with a mean of 44.6 pack-years, from the COPDGene (Genetic Epidemiology of COPD) phase I cohort (2008-2011) in our analysis. Out of a total of 916 phenotypes, 147 continuous clinical, spirometric, and computed tomography (CT) features were selected. For each principal component (PC), we computed a PC score based on feature weights. We used PC score distributions to define disease axes along which we divided the patients into quartiles. To assess the prognostic value of these axes, we applied logistic regression analyses to estimate 5-year (n = 4,159) and 10-year (n = 1,487) odds of progression. Cox regression and Kaplan-Meier analyses were performed to estimate 5-year and 10-year risk of exacerbation (n = 6,532) and all-cause mortality (n = 7,331). Results: The first PC, accounting for 43.7% of variance, was defined by CT measures of air trapping and emphysema. The second PC, accounting for 13.7% of variance, was defined by spirometric and CT measures of vital capacity and lung volume. The third PC, accounting for 7.9% of the variance, was defined by CT measures of lung mass, airway thickening, and body habitus. Stratification of patients across each disease axis revealed up to 3.2-fold (95% confidence interval [CI] 2.4, 4.3) greater odds of 5-year progression, 5.4-fold (95% CI 4.6, 6.3) greater risk of 5-year exacerbation, and 5.0-fold (95% CI 4.2, 6.0) greater risk of 10-year mortality between the highest and lowest quartiles. Conclusions: Unsupervised learning analysis of the COPDGene cohort reveals that CT measurements may bolster patient stratification along the continuum of COPD phenotypes. Each of the disease axes also individually demonstrate prognostic potential, predictive of future forced expiratory volume in 1 second decline, exacerbation, and mortality.

Complex and unexpected outcomes of antibiotic therapy against a polymicrobial infection.

Antibiotics are our primary approach to treating complex infections, yet we have a poor understanding of how these drugs affect microbial communities. To better understand antimicrobial effects on host-associated microbial communities we treated cultured sputum microbiomes from people with cystic fibrosis (pwCF, n = 24) with 11 different antibiotics, supported by theoretical and mathematical modeling-based predictions in a mucus-plugged bronchiole microcosm. Treatment outcomes we identified in vitro that were predicted in silico were: 1) community death, 2) community resistance, 3) pathogen killing, and 4) fermenter killing. However, two outcomes that were not predicted when antibiotics were applied were 5) community profile shifts with little change in total bacterial load (TBL), and 6) increases in TBL. The latter outcome was observed in 17.8% of samples with a TBL increase of greater than 20% and 6.8% of samples with an increase greater than 40%, demonstrating significant increases in community carrying capacity in the presence of an antibiotic. An iteration of the mathematical model showed that TBL increase was due to antibiotic-mediated release of pH-dependent inhibition of pathogens by anaerobe fermentation. These dynamics were verified in vitro when killing of fermenters resulted in a higher community carrying capacity compared to a no antibiotic control. Metagenomic sequencing of sputum samples during antibiotic therapy revealed similar dynamics in clinical samples. This study shows that the complex microbial ecology dictates the outcomes of antibiotic therapy against a polymicrobial infection.

Stenotrophomonas maltophilia in patients with bronchiectasis: An analysis of the US bronchiectasis and NTM Research Registry.

INTRODUCTION: Little information is available about Stenotrophomonas maltophilia in patients with bronchiectasis. We analyzed data from the US Bronchiectasis and NTM Research Registry to determine its prevalence and association with patient characteristics and severity of disease. METHODS: Baseline and follow-up data were entered into a central web-based database. Patients were grouped into four cohorts based on their baseline cultures: 1) S. maltophilia, no Pseudomonas aeruginsosa, 2) P. aeruginosa, no S. maltophilia, 3) No pathogens, 4) Pathogens other than P. aeruginosa and S. maltophilia. The association between S. maltophilia, demographic characteristics, pulmonary function, exacerbations and hospitalizations was assessed at baseline and one year follow-up. RESULTS: Among 2659 patients, 134 (5.0%) had grown S. maltophilia at baseline. The prior exacerbation rate at baseline was similar in patients with S. maltophilia and P. aeruginosa, but significantly higher than the other two groups. Hospitalizations were more frequent in patients with S. maltophilia or P. aeruginosa. Pre-bronchodilator FEV1 among S. maltophilia patients was between that of Pseudomonas patients and patients without either organism, but was not significantly different from any of the other groups. For all risk-adjusted one-year outcomes, patients with S. maltophilia had a non-significant trend towards worse outcomes compared to patients without P. aeruginosa, but were more similar to patients with P aeruginosa. DISCUSSION: Bronchiectasis patients with S. maltophilia may have worse outcomes than patients without this organism or without P. aeruginosa; further study is needed to determine if the non-significant trends we note are clinically significant.

Bone Marrow Transplantation Rescues Monocyte Recruitment Defect and Improves Cystic Fibrosis in Mice.

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.

Multi-Omics Study of Keystone Species in a Cystic Fibrosis Microbiome.

Ecological networking and in vitro studies predict that anaerobic, mucus-degrading bacteria are keystone species in cystic fibrosis (CF) microbiomes. The metabolic byproducts from these bacteria facilitate the colonization and growth of CF pathogens like Pseudomonas aeruginosa. Here, a multi-omics study informed the control of putative anaerobic keystone species during a transition in antibiotic therapy of a CF patient. A quantitative metagenomics approach combining sequence data with epifluorescence microscopy showed that during periods of rapid lung function loss, the patient's lung microbiome was dominated by the anaerobic, mucus-degrading bacteria belonging to Streptococcus, Veillonella, and Prevotella genera. Untargeted metabolomics and community cultures identified high rates of fermentation in these sputa, with the accumulation of lactic acid, citric acid, and acetic acid. P. aeruginosa utilized these fermentation products for growth, as indicated by quantitative transcriptomics data. Transcription levels of P. aeruginosa genes for the utilization of fermentation products were proportional to the abundance of anaerobic bacteria. Clindamycin therapy targeting Gram-positive anaerobes rapidly suppressed anaerobic bacteria and the accumulation of fermentation products. Clindamycin also lowered the abundance and transcription of P. aeruginosa, even though this patient's strain was resistant to this antibiotic. The treatment stabilized the patient's lung function and improved respiratory health for two months, lengthening by a factor of four the between-hospitalization time for this patient. Killing anaerobes indirectly limited the growth of P. aeruginosa by disrupting the cross-feeding of fermentation products. This case study supports the hypothesis that facultative anaerobes operated as keystone species in this CF microbiome. Personalized multi-omics may become a viable approach for routine clinical diagnostics in the future, providing critical information to inform treatment decisions.