IntroductionMycobacterium abscessus (Mabsc), a nontuberculous mycobacterium (NTM), is readily cleared from healthy lungs but can cause infections in immunocompromised individuals and individuals with chronic airways diseases that disrupt mucociliary clearance, such as cystic fibrosis and bronchiectasis. In bronchiectatic airways, Mabsc can persist despite robust immune cell recruitment, raising the possibility that, in addition to impaired mucociliary clearance, local pulmonary immune defects contribute to NTM susceptibility. Since chronic infections result from failed eradication of acute infection, we sought to determine whether immune cell responses critical for control of acute Mabsc infection in healthy lungs remain relevant when Mabsc infection occurs in obstructed airways, rather than in the alveoli, as occurs in bronchiectasis.MethodsUsing an agar bead model of Mabsc infection that prolongs murine small airway infection, replicates factors associated with bronchiectasis, and mirrors pathology of human Mabsc lung disease, we tested the hypothesis that Mabsc infection in obstructed airways elicits a qualitatively different immune response that alveolar Mabsc infection. We compared myeloid cell responses in in the two infection models using flow cytometric analyses of lung cells, immunofluorescent imaging, and histopathologic evaluation of lung sections.ResultsDuring the first 2 weeks of the bead model of Mabsc infection, absolute abundance of neutrophils in the lungs was significantly higher and relative abundance of recruited macrophages was lower than in the alveolar Mabsc infection model. Additionally, most resident alveolar macrophages (CD11c+ RAMs) in the bead model upregulated CD11b, a marker of inflammation, by 1-week post-infection and maintained high levels of CD11b expression at 3 weeks post infection despite infection occurring in airways, not alveoli. To understand the role of macrophage subsets in controlling Mabsc infection, clodronate liposomes were administered oropharyngeally to deplete RAMs. RAMs were essential for early control of alveolar Mabsc infection, but depletion of RAMs had no effect on control of Mabsc burden during bead infection.ConclusionsThese studies demonstrate that murine Mabsc airway infection induced by obstructing small airways with Mabsc embedded in agar beads generates an immune milieu distinct from that induced by Mabsc infection in alveoli, altering the importance of different macrophage populations for control of infection.

RATIONALE:Elexacaftor-tezacaftor-ivacaftor (ETI) broadly improves outcomes in cystic fibrosis (CF). Cystic fibrosis transmembrane conductance regulator (CFTR) correction by ETI is incomplete, and patients with established disease continue to experience infection. We hypothesized that CFTR restoration by ETI would modify T cell memory, providing a basis for improved infectious outcomes after treatment. OBJECTIVES:To evaluate ETI modification of lymphocyte signatures using transcriptomics and metabolomics, in blood samples taken from adults with CF (awCF). METHODS AND MAIN RESULTS:Circulating CD3 + T cells from awCF (n = 18) before and after ETI initiation were evaluated by CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by sequencing). Single cell data profiled multiple T cell populations, including naive, memory, and effector states, with the most extensive changes occurring in CD4 + T populations. Simultaneous plasma metabolomic measurements were analyzed in association with CD4 + T cell metabolic gene signatures. The relationships between clinical outcomes, cell-specific gene expression and metabolomics were evaluated using generalized linear mixed models. The effect of reduced CFTR in lymphocytes was evaluated in vitro utilizing CRISPR/Cas9-mediated CFTR knockdown in primary human lymphocyte cultures, as well in splenocytes harvested from CFTR knockout mice. CONCLUSIONS:We reveal that altered metabolism occurs within the CD4 + T memory compartment following CFTR modulation, identifying multiple previously unrecognized changes in memory functions. Memory T cell metabolic switches are highly related to reduction in hospitalizations after treatment. Adaptive immune memory in awCF after modulator therapy is highly associated with improved outcomes from CF pulmonary infections, with variation seen in individuals experiencing lesser benefit after modulators.

Background Cystic Fibrosis (CF) Centers worldwide have reported healthcare-associated outbreaks of nontuberculous mycobacteria (NTM). We report a retrospective investigation of shared Mycobacterium abscessus strains among people with cystic fibrosis (pwCF) receiving care at Dell Children’s/Ascension combined Pediatric and Adult CF Program (DCMC). Methods Whole genome sequencing (WGS) was used to identify genetically similar isolates among 167 NTM isolates from 57 pwCF. Epidemiological investigation, respiratory and environmental isolate comparisons, and watershed mapping were performed. Results WGS analysis revealed four M. abscessus clusters, two ssp. abscessus and two ssp. massiliense. One subject was infected with two distinct clustered M. abscessus (ssp. abscessus and ssp. massiliense). Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission within all clusters. Two ssp. massiliense subject pairs had healthcare overlaps and high genomic relatedness, including one cohabitating sibling pair. M. abscessus recovered from DCMC revealed genetic similarity to a respiratory isolate from one patient who was never exposed to the hospital environment. Conclusions We identified shared M. abscessus strains via genomic analysis among pwCF at DCMC. None of the clustered patient isolates matched hospital environmental isolates at the genomic level. One hospital environmental isolate had genomic similarity to a respiratory isolate of M. abscessus, but the epidemiologic investigation revealed no evidence of subject exposure to the hospital setting. One ssp. massiliense subject pair had the same level of pangenome relatedness as the sibling pair and epidemiological investigation revealed overlap in the clinic, supporting healthcare-associated person-to-person transmission among the pair within a cluster. One pwCF had polyclonal clustered infections, suggesting multiple environmental sources of acquisition outside the healthcare environment.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that cause pulmonary disease (PD) in people with bronchiectasis and other chronic airways diseases. Difficulty treating and eradicating NTM-PD highlights the need for improved understanding of bacterial mechanisms to establish chronic infections. People with the genetic disorder cystic fibrosis (CF) develop bronchiectasis and are the population at highest risk of NTM-PD, caused mainly by Mycobacterium avium or Mycobacterium abscessus (Mabsc). The majority of people with CF (pwCF) and bronchiectasis develop chronic Pseudomonas aeruginosa airway infections. We hypothesized that antibiotics used to treat P. aeruginosa infections could enhance Mabsc persistence in the CF airway. Here we demonstrate that clinically relevant concentrations of tobramycin, which does not kill Mabsc but is frequently administered to pwCF with chronic P. aeruginosa infections, induced Mabsc expression of whiB7, a transcription factor that activates genes associated with resistance to host defenses. Tobramycin promoted Mabsc resistance to killing by hydrogen peroxide (H2O2) and survival in both human macrophages and mice. Deletion of whiB7 increased Mabsc susceptibility to killing by H2O2, decreased ability of Mabsc to persist in macrophages, and disrupted ability of tobramycin to enhance Mabsc survival. Transcriptomic data defining the tobramycin associated WhiB7 regulon revealed differential gene expression of factors that could enhance Mabsc resistance to stress conditions such as those in found in the CF lung. Overall, our data indicate that administration of tobramycin to pwCF may have unexpected off-target effects, enhancing Mabsc whiB7 expression and promoting Mabsc persistent infection.

Nontuberculous mycobacteria (NTM) cause pulmonary disease in people with cystic fibrosis (pwCF). Recommended prolonged antibiotic therapy has significant toxicity. Non-antibiotic therapies for NTM pulmonary disease and supporting evidence were identified. Online databases and trials registries were searched for randomised or non-randomised studies (NRSIs). Primary outcomes were microbiology, quality of life (QoL), adverse events, lung function and pulmonary exacerbations. Bias was assessed using the relevant Joanna Briggs Institute tool. Seven NRSIs assessed: phage therapy, CF transmembrane conductance regulator (CFTR) modulator therapy (ETI: elexacaftor/tezacaftor/ivacaftor), inhaled nitric oxide (INO) and surgical lung resection. All had varying response definitions and very low-certainty evidence. The phage therapy study reported NTM culture conversion (n=4), partial conversion (n=4), inconclusive response (n=4) or no response (n=4). In one study of ETI with antibiotics (n=7) or without (n=8), nine participants eradicated NTM after a year, with positive cultures decreasing during treatment compared to pre-treatment. In another study (n=91) NTM infection fell by approximately 75% after at least two months of ETI (no information on concomitant treatment). In three INO studies (n=16), NTM was eradicated (n=2), showed transient negative cultures (n=6) and reductions in bacterial burden (n=11). Comparing lung resection (n=3) to antibiotic therapy (n=6), all surgical patients cleared M abscessus remaining negative at 24 months; 4 patients taking antibiotics cleared NTM. All M avium complex (MAC)-positive patients (surgery, n=1; antibiotic, n=2) cleared infection. Currently, non-antibiotic therapies are considered case by case. Randomised studies are needed to increase evidence quality and inform clinical practice for interventions appearing safe and feasible in pilot studies.

Bacteriophages (phages) are viruses that selectively infect bacteria and have been utilized to treat Mycobacterium abscessus (Mab) with varying success. The POSTSTAMP study is an ongoing, multi-site phage therapy protocol for treatment-refractory pulmonary Mab disease in people with cystic fibrosis (pwCF). Participants (n = 10) are prospectively assessed while utilizing FDA investigational new drug (IND) approval for compassionate use. Participants are >6 years old, able to produce sputum, have been treated with guideline-based antibiotic therapy (GBT) for >12 months without culture conversion, and are currently receiving GBT with at least 3 and ≥ 80 % positive Mab cultures in the prior year. At enrollment, an isolate is assessed for the availability of lytic phage(s). Open-label phage therapy consists of 1 or 2 phages administered intravenously twice daily for 52 weeks. Participants without a phage match will be followed on GBT as a comparison group. Follow-up visits will occur monthly, with one follow-up visit at completion and intermittent visits for a year after phage therapy. Efficacy will be assessed by culture, standard clinical measures and a patient-reported quality-of-life instrument. Frequency of Mab detection 12 months prior to treatment will be compared with the 12-month period beginning 6 months after treatment initiation. Individual-level tests of difference in percent positive cultures within subjects will be used to identify "responders". Collectively and including all persons, a mixed-effect model will be used to test for a difference in frequency of Mab detection following treatment or without treatment. The trial will also test for markers of treatment failure and pathogen adaptation in participants who did not achieve microbiological response, and will monitor for safety and tolerance.

Mycobacterium abscessus is a nontuberculous mycobacterium emerging as a significant pathogen in individuals with chronic lung diseases, including cystic fibrosis and chronic obstructive pulmonary disease. Current therapeutics have poor efficacy. Strategies of bacterial control based on host defenses are appealing; however, antimycobacterial immunity remains poorly understood and is further complicated by the appearance of smooth and rough morphotypes, which elicit distinct host responses. We investigated the role of serum components in neutrophil-mediated clearance of M. abscessus morphotypes. M. abscessus opsonization with complement enhanced bacterial killing compared to complement-deficient opsonization. Killing of rough isolates was less reliant on complement. Complement C3 and mannose-binding lectin 2 (MBL2) were deposited on M. abscessus morphotypes in distinct patterns, with a greater association of MBL2 on rough M. abscessus. Killing was dependent on C3; however, depletion and competition experiments indicate that canonical complement activation pathways are not involved. Complement-mediated killing relied on natural IgG and IgM for smooth morphotypes and on IgG for rough morphotypes. Both morphotypes were recognized by complement receptor 3 in a carbohydrate- and calcium-dependent manner. These findings indicate a role for noncanonical C3 activation pathways for M. abscessus clearance by neutrophils and link smooth-to-rough adaptation to complement activation.

Background. The duration of time a person with cystic fibrosis (pwCF) spends on the lung transplant waitlist is dependent on waitlist and post-transplant survival probabilities and can extend up to 2 years. Understanding the characteristics involved with lung transplant and survival prognoses may help guide decision making by the patient, the referring CF Center and the transplant team. Methods. This study seeks to identify clinical predictors of lung transplant and survival of individuals with CF using 29,847 subjects from 2003-2014 entered in the Cystic Fibrosis Foundation Patient Registry (CFFPR). Results. Predictors significant (p <= 0.05) in the final logistic regression model predicting probability of lung transplant/death were: FEV1 (% predicted), BMI, age of diagnosis, age, number of pulmonary exacerbations, race, sex, CF-related diabetes (CFRD), corticosteroid use, infections with B. cepacia, P. aeruginosa, S. aureus, MRSA, pancreatic enzyme use, insurance status, and consecutive ibuprofen use for at least 4 years. The final Cox regression model predicting time to lung transplant identified these predictors as significant FEV1 (% predicted), BMI, age of diagnosis, age, number of pulmonary exacerbations, race, sex, CF-related diabetes (CFRD), corticosteroid use, infections with B. cepacia, P. aeruginosa, S. aureus, MRSA, pancreatic enzyme use, and consecutive ibuprofen use for at least 4 years. The concordance indices were 0.89 and 0.92, respectively. Conclusions. The models are translated into nomograms to simplify investigation of how various characteristics relate to lung transplant and survival prognosis individuals with CF not receiving highly effective CFTR modulator therapy.

This is the third paper in the series providing updated information and recommendations for people with cystic fibrosis transmembrane conductance regulator (CFTR)-related disorder (CFTR-RD). This paper covers the individual disorders, including the established conditions - congenital absence of the vas deferens (CAVD), diffuse bronchiectasis and chronic or acute recurrent pancreatitis - and also other conditions which might be considered a CFTR-RD, including allergic bronchopulmonary aspergillosis, chronic rhinosinusitis, primary sclerosing cholangitis and aquagenic wrinkling. The CFTR functional and genetic evidence in support of the condition being a CFTR-RD are discussed and guidance for reaching the diagnosis, including alternative conditions to consider and management recommendations, is provided. Gaps in our knowledge, particularly of the emerging conditions, and future areas of research, including the role of CFTR modulators, are highlighted.

Rationale Nontuberculous mycobacteria (NTM) has been reported to be transmitted between people with cystic fibrosis (CF) attending CF centres. A suspected Mycobacterium abscessus outbreak was investigated at the University of Texas Southwestern (UTSW) Adult CF Program using a combination of pathogen genomic sequencing and epidemiologic methods. The objectives of the present study were to apply the Healthcare-Associated Links in Transmission of NTM (HALT NTM) study to investigate the occurrence of potential healthcare-associated transmission and/or acquisition of NTM among people with CF infected with genetically similar NTM isolates. Methods Whole-genome sequencing of respiratory M. abscessus isolates from 50 people with CF receiving care at UTSW was performed to identify genetically similar isolates. Epidemiologic investigation, comparison of respiratory and environmental isolates, and home residence watershed mapping were studied. Measurements and main results Whole-genome sequencing analysis demonstrated seven clusters of genetically similar M. abscessus (four ssp . abscessus and three ssp. massiliense ). Epidemiologic investigation revealed potential opportunities for healthcare-associated transmission within three of these clusters. Healthcare environmental sampling did not recover M. abscessus , but did recover four human disease-causing species of NTM. No subjects having clustered infections lived in the same home residence watershed. Some subjects were infected with more than one M. abscessus genotype, both within and outside of the dominant circulating clones. Conclusions Healthcare-associated person-to-person transmission of M. abscessus appears to be rare at this centre. However, polyclonal infections of M. abscessus species and subspecies, not originating from the endemic hospital environment, suggest multiple shared modes of acquisition outside the healthcare setting.

Headspace solid-phase micro-extraction (HS-SPME) is a prevalent technique in metabolomics and volatolomics research. However, the performance of HS-SPME can vary considerably depending on the sample matrix. As a result, fine-tuning the parameters for each specific sample matrix is crucial to maximize extraction efficacy. In this context, we conducted comprehensive HS-SPME optimization for bronchoalveolar lavage fluid (BALF) samples using two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-ToFMS). Our exploration spanned several HS-SPME parameters, including vial size, dilution factor, extraction time, extraction temperature, and ionic strength. The 10 mL vial size, no sample dilution, extraction time of 50 min, extraction temperature of 45 °C, and 40% salt were identified as the optimized parameters. The optimized method was then evaluated by a pair-wise comparison of ten sets of samples. The results revealed that the optimized method yielded an increase of 340% in total peak area and an increase of 80% in total peak number. Moreover, enhancements were observed across nine major chemical classes in both peak area and number. Notably, the optimized method also doubled the number of volatile compounds consistently detected across BALF samples, from 52 to 108.