Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.

Two mycobacteriophages were administered intravenously to a male with treatment-refractory Mycobacterium abscessus pulmonary infection and severe cystic fibrosis lung disease. The phages were engineered to enhance their capacity to lyse M. abscessus and were selected specifically as the most effective against the subject's bacterial isolate. In the setting of compassionate use, the evidence of phage-induced lysis was observed using molecular and metabolic assays combined with clinical assessments. M. abscessus isolates pre and post-phage treatment demonstrated genetic stability, with a general decline in diversity and no increased resistance to phage or antibiotics. The anti-phage neutralizing antibody titers to one phage increased with time but did not prevent clinical improvement throughout the course of treatment. The subject received lung transplantation on day 379, and systematic culturing of the explanted lung did not detect M. abscessus. This study describes the course and associated markers of a successful phage treatment of M. abscessus in advanced lung disease.

Investigating Nontuberculous Mycobacteria Transmission at the Colorado Adult Cystic Fibrosis Program.

Rationale: Healthcare-associated transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been investigated at CF centers worldwide, with conflicting conclusions. We investigated transmission at the Colorado Adult CF Program. Objectives: To systematically investigate healthcare-associated transmission and/or acquisition of NTM to determine similarity among respiratory and environmental isolates, and to compare home residence watershed mapping among pwCF having genetically similar NTM isolates. Methods: Whole-genome sequencing of NTM isolates from 80 pwCF was conducted to identify genetically similar isolate clusters (⩽30 SNP differences). Epidemiology, comparison of respiratory and environmental isolates, and home residence watershed mapping were analyzed. Measurements and Main Results: Whole-genome sequencing analysis revealed 11 clusters of NTM [6 Mycobacterium abscessus subspecies (ssp.) abscessus, 1 M. abscessus ssp. massiliense, 2 Mycobacterium avium, and 2 Mycobacterium intracellulare] among pwCF. Epidemiologic investigation demonstrated opportunities for healthcare-associated transmission in two M. abscessus and two M. avium clusters. Respiratory and healthcare environmental isolate comparisons revealed no genetic similarity. Individuals comprising one M. abscessus cluster, with no plausible healthcare-associated transmission, resided in the same watershed. Conclusions: This study suggests healthcare-associated transmission of M. abscessus is rare and includes a report of potential healthcare-associated transmission of M. avium among pwCF. One M. abscessus cluster possibly had common acquisition arising from residing in the same watershed. The presence of genetically similar isolates is insufficient to demonstrate healthcare-associated NTM transmission. Standardizing epidemiologic investigation, combined with environmental sampling and watershed analysis, will improve understanding of the frequency and nature of healthcare-associated NTM transmission among pwCF.

Mycobacterium abscessus Clearance by Neutrophils Is Independent of Autophagy.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly prevalent in chronic lung disease, including cystic fibrosis, and infections are characterized by neutrophil-dominated environments. However, mechanisms of immune control are poorly understood. Azithromycin, a macrolide antibiotic with immunomodulatory effects, is used to treat M. abscessus infections. Recently, inhibition of macrophage bactericidal autophagy was described for azithromycin, which could be detrimental to the host. Therefore, we explored the role of autophagy in mycobactericidal neutrophils. Azithromycin did not affect M. abscessus-induced neutrophil reactive oxygen species formation, phagocytosis, or cytokine secretion, and neutrophils treated with azithromycin killed M. abscessus equally as well as untreated neutrophils from either healthy or cystic fibrosis subjects. One clinical isolate was killed more effectively in azithromycin-treated neutrophils, suggesting that pathogen-specific factors may interact with an azithromycin-sensitive pathway. Chloroquine and rapamycin, an inhibitor and an activator of autophagy, respectively, also failed to affect mycobactericidal activity, suggesting that autophagy was not involved. However, wortmannin, an inhibitor of intracellular trafficking, inhibited mycobactericidal activity, but as a result of inhibiting phagocytosis. The effects of these autophagy-modifying agents and azithromycin in neutrophils from healthy subjects were similar between the smooth and rough morphotypes of M. abscessus However, in cystic fibrosis neutrophils, wortmannin inhibited killing of a rough clinical isolate and not a smooth isolate, suggesting that unique host-pathogen interactions exist in cystic fibrosis. These studies increase our understanding of M. abscessus virulence and of neutrophil mycobactericidal mechanisms. Insight into the immune control of M. abscessus may provide novel targets of therapy.

Prospective multicenter randomized patient recruitment and sample collection to enable future measurements of sputum biomarkers of inflammation in an observational study of cystic fibrosis.

BACKGROUND: Biomarkers of inflammation predictive of cystic fibrosis (CF) disease outcomes would increase the power of clinical trials and contribute to better personalization of clinical assessments. A representative patient cohort would improve searching for believable, generalizable, reproducible and accurate biomarkers. METHODS: We recruited patients from Mountain West CF Consortium (MWCFC) care centers for prospective observational study of sputum biomarkers of inflammation. After informed consent, centers enrolled randomly selected patients with CF who were clinically stable sputum producers, 12 years of age and older, without previous organ transplantation. RESULTS: From December 8, 2014 through January 16, 2016, we enrolled 114 patients (53 male) with CF with continuing data collection. Baseline characteristics included mean age 27 years (SD = 12), 80% predicted forced expiratory volume in 1 s (SD = 23%), 1.0 prior year pulmonary exacerbations (SD = 1.2), home elevation 328 m (SD = 112) above sea level. Compared with other patients in the US CF Foundation Patient Registry (CFFPR) in 2014, MWCFC patients had similar distribution of sex, age, lung function, weight and rates of exacerbations, diabetes, pancreatic insufficiency, CF-related arthropathy and airway infections including methicillin-sensitive or -resistant Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex, fungal and non-tuberculous Mycobacteria infections. They received CF-specific treatments at similar frequencies. CONCLUSIONS: Randomly-selected, sputum-producing patients within the MWCFC represent sputum-producing patients in the CFFPR. They have similar characteristics, lung function and frequencies of pulmonary exacerbations, microbial infections and use of CF-specific treatments. These findings will plausibly make future interpretations of quantitative measurements of inflammatory biomarkers generalizable to sputum-producing patients in the CFFPR.

The role of leptin in the development of pulmonary neutrophilia in infection and acute lung injury.

OBJECTIVES: One of the hallmarks of severe pneumonia and associated acute lung injury is neutrophil recruitment to the lung. Leptin is thought to be up-regulated in the lung following injury and to exert diverse effects on leukocytes, influencing both chemotaxis and survival. We hypothesized that pulmonary leptin contributes directly to the development of pulmonary neutrophilia during pneumonia and acute lung injury. DESIGN: Controlled human and murine in vivo and ex vivo experimental studies. SETTING: Research laboratory of a university hospital. SUBJECTS: Healthy human volunteers and subjects hospitalized with bacterial and H1N1 pneumonia. C57Bl/6 and db/db mice were also used. INTERVENTIONS: Lung samples from patients and mice with either bacterial or H1N1 pneumonia and associated acute lung injury were immunostained for leptin. Human bronchoalveolar lavage samples obtained after lipopolysaccharide-induced lung injury were assayed for leptin. C57Bl/6 mice were examined after oropharyngeal aspiration of recombinant leptin alone or in combination with Escherichia coli- or Klebsiella pneumoniae-induced pneumonia. Leptin-resistant (db/db) mice were also examined using the E. coli model. Bronchoalveolar lavage neutrophilia and cytokine levels were measured. Leptin-induced chemotaxis was examined in human blood- and murine marrow-derived neutrophils in vitro. MEASUREMENTS AND MAIN RESULTS: Injured human and murine lung tissue showed leptin induction compared to normal lung, as did human bronchoalveolar lavage following lipopolysaccharide instillation. Bronchoalveolar lavage neutrophilia in uninjured and infected mice was increased and lung bacterial load decreased by airway leptin administration, whereas bronchoalveolar lavage neutrophilia in infected leptin-resistant mice was decreased. In sterile lung injury by lipopolysaccharide, leptin also appeared to decrease airspace neutrophil apoptosis. Both human and murine neutrophils migrated toward leptin in vitro, and this required intact signaling through the Janus Kinase 2/phosphatidylinositol-4,5-bisphosphate 3-kinase pathway. CONCLUSIONS: We demonstrate that pulmonary leptin is induced in injured human and murine lungs and that this cytokine is effective in driving alveolar airspace neutrophilia. This action appears to be caused by direct effects of leptin on neutrophils.

Prospective evaluation of nontuberculous mycobacteria disease in cystic fibrosis: The design of the PREDICT study.

BACKGROUND: Nontuberculous mycobacteria (NTM) are an important cause of airway infections in people with cystic fibrosis (pwCF). Isolation of NTM from respiratory specimens of pwCF do not mandate treatment in the absence of clinical and radiologic features of NTM pulmonary disease (NTM-PD), as some pwCF clear the infection without treatment and others do not appear to progress to NTM-PD despite persistent infection. An evidence-based protocol to standardize diagnosis of NTM-PD is needed to systematically identify pwCF who may benefit from treatment. METHODS: In this multicenter observational study, eligible pwCF who are 6 years of age and older and who have had a recent positive NTM culture are systematically evaluated for NTM-PD. Participants are identified based on positive NTM culture results obtained during routine clinical care and following enrollment are evaluated for NTM-PD and CF-related comorbidities. Participants are followed in PREDICT until they meet NTM-PD diagnostic criteria and are ready to initiate NTM treatment, or until study termination. Active participants who have not met these criteria are re-consented every 5 years to enable long-term participation. RESULTS: The primary endpoint will summarize the proportion of participants who meet the NTM-PD diagnosis definition. The time from enrollment to NTM-PD diagnosis will be derived from Kaplan-Meier estimates. CONCLUSION: A prospective protocol to identify NTM-PD in pwCF will test if this standardized approach defines a cohort with signs and symptoms associated with NTM-PD, to assist with clinical decision making and to build a framework for future therapeutic trials. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02073409.

Genomic epidemiology of Mycobacterium abscessus at an adult cystic fibrosis programme reveals low potential for healthcare-associated transmission.

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.

Remote endpoints for clinical trials in cystic fibrosis: Report from the U.S. CF foundation remote endpoints task force.

The COVID-19 pandemic necessitated a rapid shift in clinical research to perform virtual visits and remote endpoint assessments, providing a key opportunity to optimize the use of remote endpoints for clinical trials in cystic fibrosis. The use of remote endpoints could allow more diverse participation in clinical trials while minimizing participant burden but must be robustly evaluated to ensure adequate performance and feasibility. In response, the Cystic Fibrosis Foundation convened the Remote Endpoint Task Force (Supplemental Table 1), a multidisciplinary group of CF researchers with remote endpoint expertise and community members tasked to better understand the current and future use of remote endpoints for clinical research. Here, we describe the current use of remote endpoints in CF clinical research, address key unanswered questions regarding their use and feasibility, and discuss the next steps to determine clinical trial readiness.

Factors that predict pursuing sinus surgery in the era of highly effective modulator therapy.

BACKGROUND: Comorbid chronic rhinosinusitis (CRS) remains unresolved for many people with cystic fibrosis (PwCF). While highly effective modulator therapy improves quality-of-life and symptom severity, the impact of this intervention and other factors associated with pursuing endoscopic sinus surgery (ESS) remains understudied. METHODS: Adult PwCF + CRS were enrolled into a prospective, observational, multi-institutional study. Participants completed validated outcome measures to evaluate respiratory symptom severity, depression, headache, and sleep quality, as well as nasal endoscopy, sinus computed tomography (CT), and olfactory testing. Bivariate comparisons and regression modeling evaluated treatment cofactors, disease characteristics, and outcome measures associated with pursuing ESS. RESULTS: Sixty PwCF were analyzed, including 24 (40%) who elected ESS. Pursuing ESS was associated with worse SinoNasal Outcome Test (SNOT-22) total, rhinologic, psychological, and sleep dysfunction domain scores; worse Patient Health Questionnaire-9-Revised depression scores; worse Pittsburgh Sleep Quality Index total scores; worse weight, role, emotion, and eating domain scores on the Cystic Fibrosis Questionnaire-Revised; more severe disease on nasal endoscopy; and lack of modulator therapy (all p < 0.050). Multivariable regression identified that worse SNOT-22 total score was associated with electing ESS (odds ratio [OR] 1.09, 95% confidence interval [CI] 1.02-1.16, p = 0.015) and elexacaftor/tezacaftor/ivacaftor (ETI) treatment (OR 0.04, 95% CI 0.004-0.34, p = 0.004) was associated with pursing medical therapy. CONCLUSIONS: Worse sinonasal symptom burden, lack of ETI treatment, sleep quality, depression, and nasal endoscopy scores were associated with electing ESS, while lung disease severity and sinus CT scores were not. ETI use was associated with lower odds of pursuing ESS independent of sinonasal symptom burden.

Impact of sociodemographic status and sex on chronic rhinosinusitis and olfaction in people with cystic fibrosis.

BACKGROUND: Sociodemographic status (SDS) including race/ethnicity and socioeconomic status as approximated by education, income, and insurance status impact pulmonary disease in people with cystic fibrosis (PwCF). The relationship between SDS and chronic rhinosinusitis (CRS) remains understudied. METHODS: In a prospective, multi-institutional study, adult PwCF completed the 22-Question SinoNasal Outcome Test (SNOT-22), Smell Identification Test (SIT), Questionnaire of Olfactory Disorder Negative Statements (QOD-NS), and Cystic Fibrosis Questionnaire-Revised (CFQ-R). Lund-Kennedy scores, sinus computed tomography, and clinical data were collected. Data were analyzed across race/ethnicity, sex, and socioeconomic factors using multivariate regression. RESULTS: Seventy-three PwCF participated with a mean age of 34.7 ± 10.9 years and 49 (67.1%) were female. Linear regression identified that elexacaftor/tezacaftor/ivacaftor (ETI) use (ß = â€’4.09, 95% confidence interval [CI] [‒6.08, ‒2.11], p < 0.001), female sex (ß = â€’2.14, 95% CI [‒4.11, ‒0.17], p = 0.034), and increasing age (ß = â€’0.14, 95% CI [‒0.22, ‒0.05], p = 0.003) were associated with lower/better endoscopy scores. Private health insurance (ß = 17.76, 95% CI [5.20, 30.32], p = 0.006) and >16 educational years (ß = 13.50, 95% CI [2.21, 24.80], p = 0.020) were associated with higher baseline percent predicted forced expiratory volume in one second (ppFEV1). Medicaid/Medicare insurance was associated with worse endoscopy scores, CFQ-R respiratory scores, and ppFEV1 (all p < 0.017), and Hispanic/Latino ethnicity was associated with worse SNOT-22 scores (p = 0.047), prior to adjustment for other cofactors. No other SDS factors were associated with SNOT-22, QOD-NS, or SIT scores. CONCLUSIONS: Differences in objective measures of CRS severity exist among PwCF related to sex, age, and ETI use. Variant status and race did not influence patient-reported CRS severity measures or olfaction in this study. Understanding how these factors impact response to treatment may improve care disparities among PwCF. CLINICAL TRIALS: NCT04469439.

Determining the minimal clinically important difference for the questionnaire of olfactory disorders in people with cystic fibrosis and factors associated with improvement after highly effective modulator therapy.

INTRODUCTION: Olfactory dysfunction (OD) is common among people with cystic fibrosis (PwCF). The Questionnaire of Olfactory Disorders (QOD) is a validated instrument that evaluates olfactory-specific quality-of-life. The QOD minimal clinically important difference (MCID) and factors associated with olfactory improvement after elexacaftor/tezacaftor/ivacaftor have not been determined for PwCF. METHODS: Prospective observational data were pooled from three studies that enrolled adult PwCF with chronic rhinosinusitis (CRS). QOD scores and disease characteristics were assessed. To evaluate internal consistency and calculate the QOD MCID, Cronbach's alpha and four distribution-based methods were employed. For participants who enrolled prior to elexacaftor/tezacaftor/ivacaftor, QOD scores were obtained at baseline and after elexacaftor/tezacaftor/ivacaftor initiation. Multivariable regression was used to identify factors associated with QOD improvement. RESULTS: Of 129 PwCF included, 65 had QOD scores before and 3-6 months after starting elexacaftor/tezacaftor/ivacaftor. Mean baseline QOD score was 6.5 ± 7.9. Mean Cronbach's alpha was ≥0.85. The MCID estimates were as follows: Cohen's effect size = 1.6, standard error of measurement = 2.5, ½ baseline standard deviation = 4.0, and minimal detectable change = 6.9. Mean MCID was 3.7. Of those with pre/post elexacaftor/tezacaftor/ivacaftor QOD scores, the mean change in QOD was -1.3 ± 5.4. After elexacaftor/tezacaftor/ivacaftor, QOD improvement surpassed the MCID in 22% of participants (14/65). Worse baseline QOD scores and nasal polyps were associated with improved QOD scores after elexacaftor/tezacaftor/ivacaftor (both p < 0.04). CONCLUSION: The QOD MCID in PwCF was estimated to be 3.7. Elexacaftor/tezacaftor/ivacaftor led to qualitative but not clinically meaningful improvements in QOD score for most PwCF; PwCF with worse baseline QOD scores and nasal polyps improved in a clinically significant manner.

Patient perspectives on chronic rhinosinusitis in cystic fibrosis: Symptom prioritization in the era of highly effective modulator therapy.

BACKGROUND: Chronic rhinosinusitis (CRS) is common in people with cystic fibrosis (PwCF). Rhinologic symptom prioritization and areas that influence CRS treatment choices, including pursuing endoscopic sinus surgery (ESS), remain understudied. METHODS: Adult PwCF + CRS were enrolled at eight centers into a prospective, observational study (2019-2023). Participants were administered the 22-SinoNasal Outcome Test (SNOT-22) survey and a modified SNOT-22 instrument examining symptom importance. We determined importance rankings for individual symptoms and SNOT-22 symptom importance subdomains in two sets of subgroups-those pursuing ESS versus continuing medical management (CMT), and those on elexacaftor/tezacaftor/ivacaftor (ETI) versus not on ETI. RESULTS: Among 69 participants, the highest priorities were nasal congestion (n = 48, 69.6% important), post-nasal discharge (32, 46.4%), facial pain (29, 43.3%), waking up tired (27, 39.1%), and fatigue (26, 37.7%). Those electing surgery (n = 23) prioritized sleep and psychological dysfunction symptoms compared to those pursuing CMT (n = 49) (sleep median score = 19.0 [interquartile range: 12.0, 25.0] vs. 4.5 [0.0, 12.8]; p < 0.0001; psychological = 17.0 [7.0, 26.0] vs. 7.0 [0.0, 15.8]; p = 0.002). ETI users had comparable SNOT-22 total symptom importance scores to non-ETI users (p = 0.14). Non-ETI users (n = 34) showed a trend toward prioritizing sleep symptoms compared to ETI users (n = 35) (13.0 [2.8, 22.3] vs. 6.0 [2.0, 17.0]; p = 0.055). CONCLUSIONS: Nasal congestion and post-nasal discharge were top priorities reported by PwCF + CRS. Those electing surgery prioritized sleep and psychological symptoms, highlighting their importance in pre-operative discussions. Non-ETI users' prioritization of sleep improvement may highlight their unique disease impact and therapeutic needs; however, additional investigation is required.

Airway inflammation accelerates pulmonary exacerbations in cystic fibrosis.

Airway inflammation underlies cystic fibrosis (CF) pulmonary exacerbations. In a prospective multicenter study of randomly selected, clinically stable adolescents and adults, we assessed relationships between 24 inflammation-associated molecules and the future occurrence of CF pulmonary exacerbation using proportional hazards models. We explored relationships for potential confounding or mediation by clinical factors and assessed sensitivities to treatments including CF transmembrane regulator (CFTR) protein synthesis modulators. Results from 114 participants, including seven on ivacaftor or lumacaftor-ivacaftor, representative of the US CF population during the study period, identified 10 biomarkers associated with future exacerbations mediated by percent predicted forced expiratory volume in 1 s. The findings were not sensitive to anti-inflammatory, antibiotic, and CFTR modulator treatments. The analyses suggest that combination treatments addressing RAGE-axis inflammation, protease-mediated injury, and oxidative stress might prevent pulmonary exacerbations. Our work may apply to other airway inflammatory diseases such as bronchiectasis and the acute respiratory distress syndrome.

Predictors of Sinonasal Improvement After Highly Effective Modulator Therapy in Adults with Cystic Fibrosis.

OBJECTIVES: The 22-question SinoNasal Outcome Test (SNOT-22) assesses chronic rhinosinusitis (CRS) severity. We aimed to identify predictors of SNOT-22 score improvement following highly effective modulator therapy (HEMT) initiation and to corroborate the SNOT-22 minimal clinically important difference (MCID) in adults with cystic fibrosis (CF). METHODS: Prospective observational data was pooled from four studies across 10 US centers investigating people with CF (PwCF) and CRS. Three studies evaluated HEMT's impact on CRS. For participants enrolled prior to HEMT initiation, SNOT-22 scores were obtained at baseline and after 3-6 months of HEMT. Multivariate regression identified predictors of improvement. Cronbach's alpha and four distribution-based methods were used to assess internal consistency and calculate the MCID of the SNOT-22. RESULTS: A total of 184 PwCF participated with mean baseline SNOT-22 scores ranging from 18.1 to 56.7. Cronbach's alpha was ≥0.90 across sites. Participants at sites with pre- and post-HEMT data reported improvement in SNOT-22 scores after initiating HEMT (all p < 0.05). Worse baseline SNOT-22 score (odds ratio (OR): 1.05, p < 0.001, 95% CI: 1.02-1.08), F508del homozygosity (OR: 4.30, p = 0.040, 95% CI: 1.14-18.99), and absence of prior modulator therapy (OR: 4.99, p = 0.017, 95% CI: 1.39-20.11) were associated with greater SNOT-22 improvement. The mean MCID calculated via distribution-based methods was 8.5. CONCLUSION: Worse baseline sinonasal symptoms, F508del homozygosity, and absence of prior modulator therapy predicted greater improvement after HEMT initiation. The mean MCID for SNOT-22 in PwCF is 8.5 points, similar to non-CF individuals with CRS, and provides a threshold specifically for PwCF. The SNOT-22 has strong internal consistency in PwCF. LEVEL OF EVIDENCE: 3 Laryngoscope, 2024.

Effects of azithromycin in Pseudomonas aeruginosa burn wound infection.

BACKGROUND: Cutaneous thermal injuries (i.e., burns) remain a common form of debilitating trauma, and outcomes are often worsened by wound infection with environmental bacteria, chiefly Pseudomonas aeruginosa. MATERIALS AND METHODS: We tested the effects of early administration of a single dose of azithromycin, with or without subsequent antipseudomonal antibiotics, in a mouse model of standardized thermal injury infected with P aeruginosa via both wound site and systemic infection. We also tested the antimicrobial effects of these antibiotics alone or combined in comparative biofilm and planktonic cultures in vitro. RESULTS: In our model, early azithromycin administration significantly reduced wound and systemic infection without altering wound site or circulating neutrophil activity. The antimicrobial effect of azithromycin was additive with ciprofloxacin but significantly reduced the antimicrobial effect of tobramycin. This pattern was reproduced in biofilm cultures and not observed in planktonic cultures of P aeruginosa. CONCLUSION: These data suggest that early administration of azithromycin following burn-related trauma and infection may reduce P aeruginosa infection and potential interactions with other antibiotics should be considered when designing future studies.

Deficient Complement Opsonization Impairs Mycobacterium avium Killing by Neutrophils in Cystic Fibrosis.

Nontuberculous mycobacteria (NTM), including Mycobacterium avium, are clinically important pathogens in cystic fibrosis (CF). The innate immune response to M. avium remains incompletely understood. We evaluated the role of complement opsonization in neutrophil-mediated killing of M. avium. Killing assays were performed using neutrophils from healthy donors (HDs) and persons with CF (pwCF). Clinical isolates of M. avium were opsonized with plasma from HDs or pwCF, which was intact or heat-treated to inactivate complement. HD neutrophils had killing activity against M. avium opsonized with intact HD plasma and killing was significantly reduced when M. avium was opsonized with heat-inactivated HD plasma. When opsonized with HD plasma, CF neutrophils had killing activity against M. avium that was not different than HD neutrophils. When opsonized with intact plasma from pwCF, HD neutrophil killing of M. avium was significantly reduced. Opsonization of M. avium with C3-depleted serum or IgM-depleted plasma resulted in significantly reduced killing. Plasma C3 levels were elevated in pwCF with NTM infection compared to pwCF without NTM infection. These studies demonstrate that human neutrophils efficiently kill M. avium when opsonized in the presence of plasma factors from HD that include C3 and IgM. Killing efficiency is significantly lower when the bacteria are opsonized with plasma from pwCF. This indicates a novel role for opsonization in neutrophil killing of M. avium and a deficiency in complement opsonization as a mechanism of impaired M. avium killing in CF. IMPORTANCE Mycobacterium avium is a member of a group of bacterial species termed nontuberculous mycobacteria (NTM) that cause lung disease in certain populations, including persons with cystic fibrosis (CF). NTM infections are challenging to diagnose and can be even more difficult to treat. This study investigated how the immune system responds to M. avium infection in CF. We found that neutrophils, the most abundant immune cell in the lungs in CF, can effectively kill M. avium in individuals both with and without CF. Another component of the immune response called the complement system is also required for this process. Levels of complement proteins are altered in persons with CF who have a history of NTM compared to those without a history of NTM infection. These results add to our understanding of how the immune system responds to M. avium, which can help pave the way toward better diagnostic and treatment strategies.

Divergent host innate immune response to the smooth-to-rough M. abscessus adaptation to chronic infection.

Mycobacterium abscessus is a nontuberculous mycobacterium emerging as a significant pathogen for individuals with chronic lung disease, including cystic fibrosis and chronic obstructive pulmonary disease. Current therapeutics have poor efficacy. New strategies of bacterial control based on host defenses are appealing, but anti-mycobacterial immune mechanisms are poorly understood and are complicated by the appearance of smooth and rough morphotypes with distinct host responses. We explored the role of the complement system in the clearance of M. abscessus morphotypes by neutrophils, an abundant cell in these infections. M. abscessus opsonized with plasma from healthy individuals promoted greater killing by neutrophils compared to opsonization in heat-inactivated plasma. Rough clinical isolates were more resistant to complement but were still efficiently killed. Complement C3 associated strongly with the smooth morphotype while mannose-binding lectin 2 was associated with the rough morphotype. M. abscessus killing was dependent on C3, but not on C1q or Factor B; furthermore, competition of mannose-binding lectin 2 binding with mannan or N-acetyl-glucosamine during opsonization did not inhibit killing. These data suggest that M. abscessus does not canonically activate complement through the classical, alternative, or lectin pathways. Complement-mediated killing was dependent on IgG and IgM for smooth and on IgG for rough M. abscessus. Both morphotypes were recognized by Complement Receptor 3 (CD11b), but not CR1 (CD35), and in a carbohydrate- and calcium-dependent manner. These data suggest the smooth-to-rough adaptation changes complement recognition of M. abscessus and that complement is an important factor for M. abscessus infection.

Systems serology in cystic fibrosis: Anti-Pseudomonas IgG1 responses and reduced lung function.

Nearly one-half of patients with cystic fibrosis (CF) carry the homozygous F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene but exhibit variable lung function phenotypes. How adaptive immunity influences their lung function remains unclear, particularly the serological antibody responses to antigens from mucoid Pseudomonas in sera from patients with CF with varying lung function. Sera from patients with CF with reduced lung function show higher anti-outer membrane protein I (OprI) immunoglobulin G1 (IgG1) titers and greater antibody-mediated complement deposition. Induction of anti-OprI antibody isotypes with complement activity enhances lung inflammation in preclinical mouse models. This enhanced inflammation is absent in immunized Rag2-/- mice and is transferrable to unimmunized mice through sera. In a CF cohort undergoing treatment with elexacaftor-tezacaftor-ivacaftor, the declination in anti-OprI IgG1 titers is associated with lung function improvement and reduced hospitalizations. These findings suggest that antibody responses to specific Pseudomonas aeruginosa (PA) antigens worsen lung function in patients with CF.

Culture independent markers of nontuberculous mycobacterial (NTM) lung infection and disease in the cystic fibrosis airway.

Nontuberculous mycobacteria (NTM) are opportunistic pathogens that affect a relatively small but significant portion of the people with cystic fibrosis (CF), and may cause increased morbidity and mortality in this population. Cultures from the airway are the only test currently in clinical use for detecting NTM. Culture techniques used in clinical laboratories are insensitive and poorly suited for population screening or to follow progression of disease or treatment response. The lack of sensitive and quantitative markers of NTM in the airway impedes patient care and clinical trial design, and has limited our understanding of patterns of acquisition, latency and pathogenesis of disease. Culture-independent markers of NTM infection have the potential to overcome many of the limitations of standard NTM cultures, especially the very slow growth, inability to quantitate bacterial burden, and low sensitivity due to required decontamination procedures. A range of markers have been identified in sputum, saliva, breath, blood, urine, as well as radiographic studies. Proposed markers to detect presence of NTM or transition to NTM disease include bacterial cell wall products and DNA, as well as markers of host immune response such as immunoglobulins and the gene expression of circulating leukocytes. In all cases the sensitivity of culture-independent markers is greater than standard cultures; however, most do not discriminate between various NTM species. Thus, each marker may be best suited for a specific clinical application, or combined with other markers and traditional cultures to improve diagnosis and monitoring of treatment response.

Prospective healthcare-associated links in transmission of nontuberculous mycobacteria among people with cystic fibrosis (pHALT NTM) study: Rationale and study design.

BACKGROUND: Healthcare-associated acquisition and transmission of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) has been described, and remains a concern for both patients and providers. This report describes the design of a prospective observational study utilizing the standardized epidemiologic investigation toolkit for healthcare-associated links in transmission of NTM among pwCF. METHODS: This is a parallel multi-site study of pwCF who have infections with respiratory NTM isolates and receive healthcare within a common CF Care Center. Participants have a history of one or more NTM positive airway cultures and have been identified as having NTM infections suggestive of a possible outbreak within a single Center, based on NTM isolate genomic analysis. Participants are enrolled in the study over a 3-year period. Primary endpoints are identification of shared healthcare-associated source(s) among pwCF in a Center, identification of healthcare environmental dust and water biofilm NTM isolates that are genetically highly-related to respiratory isolates, and identification of common home of residence watersheds among pwCF infected with clustered isolates. Secondary endpoints include characterization of healthcare-associated transmission and/or acquisition modes and settings as well as description of incidence and prevalence of healthcare-associated environmental NTM species/subspecies by geographical region. DISCUSSION: We hypothesize that genetically highly-related isolates of NTM among pwCF cared for at the same Center may arise from healthcare sources including patient-to-patient transmission and/or acquisition from health-care environmental dust and/or water biofilms. This study design utilizes a published, standardized, evidence-based epidemiologic toolkit to facilitate confidential, independent healthcare-associated NTM outbreak investigations within CF Care Centers. This study will facilitate real-time, rapid detection and mitigation of healthcare-associated NTM outbreaks to reduce NTM risk, inform infection prevention and control guidelines, and characterize the prevalence and origin of NTM outbreaks from healthcare-associated patient-to-patient transmission and/or environmental acquisition. This study will systematically characterize human disease causing NTM isolates from serial collection of healthcare environmental dust and water biofilms and define the most common healthcare environmental sources harboring NTM biofilms. TRIAL REGISTRATION: ClinicalTrials.gov NCT05686837.