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.

Molecular Epidemiologic Investigation of Mycobacterium intracellulare Subspecies chimaera Lung Infections at an Adult Cystic Fibrosis Program.

Rationale: Outbreaks of nontuberculous mycobacteria (NTM) among people with cystic fibrosis (pwCF) have been reported at CF centers with conflicting conclusions. The occurrence of NTM at the UVMC (University of Vermont Medical Center) adult CF program was investigated. Objectives: Use the HALT NTM (Healthcare-associated Links in Transmission of NTM) toolkit to investigate the healthcare-associated transmission and/or acquisition of NTM among pwCF having genetically similar NTM isolates. Methods: Whole genome sequencing of NTM isolates from 23 pwCF was conducted to identify genetically similar NTM isolate clusters (30 or fewer single-nucleotide polymorphism differences). The epidemiological investigation, comparison of respiratory and healthcare environmental isolates, and home residence watershed mapping were analyzed. Results: Whole genome sequencing analysis revealed two clusters of NTM isolates (Mycobacterium avium and M. intracellulare ssp. chimaera) among pwCF. The epidemiologic investigation demonstrated opportunities for healthcare-associated transmission within both clusters. Healthcare environmental M. avium isolates revealed no genetic similarity to respiratory isolates. However, M. intracellulare ssp. chimaera respiratory isolates revealed greater genetic similarity to a hospital water biofilm isolate than to each other. Neither cluster had all subjects residing in the same watershed. Conclusions: This study suggests the healthcare-associated transmission of M. avium among pwCF is unlikely at UVMC but supports the healthcare-associated environmental acquisition of M. intracellulare ssp. chimaera. The presence of genetically similar isolates alone is insufficient to confirm healthcare-associated transmission and/or acquisition. The HALT NTM toolkit standardizes outbreak investigation with genetic analysis, epidemiologic investigation, healthcare environmental sampling, and home of residence watershed identification to test the frequency and nature of healthcare-associated NTM transmission among pwCF.

Specificity of Immunoglobulin Response to Nontuberculous Mycobacteria Infection in People with Cystic Fibrosis.

Nontuberculous mycobacteria (NTM) infections are increasingly prevalent in chronic lung diseases, including cystic fibrosis (CF). Mycobacterium abscessus is of particular concern due to relatively greater virulence and intrinsic antimicrobial resistance. Airway culture identification, the standard method for detecting pulmonary infection, is hindered by low sensitivity, long culture times, and reliance on sputum production or lavage. A culture-independent test for detecting NTM infection could complement, or replace, sputum culture, which is becoming more difficult to obtain with reduced sputum production by people with CF (pwCF) on highly effective modulator therapy. We describe an assay for the detection of plasma anti-M. abscessus antibodies of pwCF to antigens from M. abscessus lysates. Anti-M. abscessus IgG and IgA, but not IgM, discriminated with high specificity subjects infected with M. abscessus from those infected by M. avium complex, and from those with distant or no NTM infections. The IgG3 subclass predominated with minor contributions by other subclasses. Both aqueous and organic soluble antigens were recognized by plasma IgG. A validation cohort measuring IgG and IgG3 identified M. abscessus positive subjects, and elevated IgG was sustained over several years. These studies show the benefit of M. abscessus cell lysates to detect plasma IgG of subjects with CF and M. abscessus infections. Subclass analysis suggests that IgG3 is the predominant subtype in these subjects with chronic bacterial infections suggesting a defect in class maturation. Serodiagnosis could be useful to monitor M. abscessus group infections in chronic lung disease as an adjunct or alternative to culture. IMPORTANCE Lung infections with nontuberculous mycobacteria (NTM), and particularly Mycobacterium abscessus, a pathogen with high antibiotic resistance, are of great concern due to poor clinical outcomes and challenging detection in people with cystic fibrosis and other diseases. Standard detection methods are insensitive and increasingly difficult. We describe the measurement of NTM-specific antibodies from plasma to identify subjects infected with M. abscessus. The assay is sensitive and provides information on the immune response to NTM infections. This assay could be used to help identify subjects with NTM pulmonary infections and track disease progression, either alone or in conjunction with other tests.

Blood mRNA biomarkers distinguish variable systemic and sputum inflammation at treatment initiation of inhaled antibiotics in cystic fibrosis: A prospective non-randomized trial.

Inhaled antibiotics control chronic airway infection and maintain respiratory health in cystic fibrosis (CF). Given variation in patient responses to inhaled antibiotics, the ability to identify distinct responder phenotypes would facilitate the delivery of personalized care. Previously, a 10-gene panel was identified, measured directly from blood leukocytes, which predicted host response to intravenous antibiotic treatment during pulmonary exacerbations. In the current study, we tested whether the same panel predicted clinical response in subjects receiving a month of inhaled antibiotic therapy with aztreonam lysine (AZLI; Cayston®). A small cohort of CF subjects infected with Pseudomonas aeruginosa were enrolled at baseline health, prior to initiating one month's treatment with AZLI using the Altera® nebulizer system. Eighteen CF subjects underwent blood leukocyte gene panel measurements, sputum quantitative microbiology, spirometry, and C-reactive protein (CRP) measurement prior to onset and at completion of 4 weeks of AZLI therapy. Mean absolute improvement in percent predicted Forced Expiratory Volume in one second (ppFEV1) was 3%. Significant reductions in sputum bacterial colony counts were detected with treatment. CRP increased following treatment. While single genes within the panel did not change significantly following treatment, the analysis of multigene panel data demonstrated that HCA112 gene predicted ppFEV1 improvement. Hierarchical clustering based on gene expression yielded two distinctive molecular clusters before and after AZLI therapy. In conclusion, peripheral blood leukocyte genes quantifying inflammation are associated with responses to inhaled antibiotic therapy. Molecular quantification of systemic inflammation may indicate subgroups of CF subjects with variations in underlying inflammation and with variable clinical responses to inhaled antibiotics. Given the size limitation of the study, larger studies are needed in order to evaluate whether molecular measures may add precision to the determination of infectious and inflammatory outcomes following courses of inhaled antimicrobial therapies. Clinical Trials.gov Identifier: NCT01736839.

Breath biomarkers associated withnontuberculosis mycobacteriadisease status in persons with cystic fibrosis: a pilot study.

Pulmonary infections caused by mycobacteria cause significant mortality and morbidity in the human population. Diagnosing mycobacterial infections is challenging. An infection can lead to active disease or remain indolent with little clinical consequence. In patients with pulmonarynontuberculosis mycobacteria(PNTM) identification of infection and diagnosis of disease can take months to years. Our previous studies showed the potential diagnostic power of volatile molecules in the exhaled breath samples to detect active pulmonaryM. tuberculosisinfection. Herein, we demonstrate the ability to detect the disease status of PNTM in the breath of persons with cystic fibrosis (PwCF). We putatively identified 17 volatile molecules that could discriminate between active-NTM disease (n= 6), indolent patients (n= 3), and those patients who have never cultured an NTM (n= 2). The results suggest that further confirmation of the breath biomarkers as a non-invasive and culture-independent tool for diagnosis of NTM disease in a larger cohort of PwCF is warranted.

Urine lipoarabinomannan as a marker for low-risk of NTM infection in the CF airway.

BACKGROUND: Individuals with Cystic fibrosis (CF) are the most vulnerable population for pulmonary infection with nontuberculous mycobacteria (NTM). Screening, diagnosis, and assessment of treatment response currently depend on traditional culture techniques, but sputum analysis for NTM in CF is challenging, and associated with a low sensitivity. The cell wall lipoarabinomannan (LAM), a lipoglycan found in all mycobacterial species, and has been validated as a biomarker in urine for active Mycobacterium tuberculosis infection. METHODS: Urine from a CF cohort (n = 44) well-characterized for NTM infection status by airway cultures was analyzed for LAM by gas chromatography/mass spectrometry. All subjects with positive sputum cultures for NTM had varying amounts of LAM in their urine. No LAM was detected in subjects who never had a positive culture (14/45). One individual initially classified as NTM sputum negative subsequently developed NTM disease 657 days after the initial urine LAM testing. Repeat urine LAM testing turned positive, correlating to her positive NTM status. Subjects infected with subspecies of M. abscessus had greater LAM quantities than those infected with M. avium complex (MAC). There was no correlation with disease activity or treatment status and LAM quantity. A TB Capture ELISA using anti-LAM antibodies demonstrated very poor sensitivity in identifying individuals with positive NTM sputum cultures. CONCLUSION: These findings support the conclusion that urine LAM related to NTM infection may be a useful screening test to determine patients at low risk for having a positive NTM sputum culture, as part of a lifetime screening strategy in the CF population.

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.

Neutrophil killing of Mycobacterium abscessus by intra- and extracellular mechanisms.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, are increasingly present in soft tissue infections and chronic lung diseases, including cystic fibrosis, and infections are characterized by growth in neutrophil-rich environments. M. abscessus is observed as two distinct smooth and rough morphotypes. The environmental smooth morphotype initiates infection and has a relatively limited ability to activate neutrophils. The rough morphotype has increased virulence and immunogenicity. However, the neutrophil response to the rough morphotype has not been explored. Killing of the smooth and rough strains, including cystic fibrosis clinical isolates, was equivalent. Neutrophil uptake of M. abscessus was similar between morphotypes. Mechanistically, both rough and smooth morphotypes enhanced neutrophil reactive oxygen species generation but inhibition of NADPH oxidase activity did not affect M. abscessus viability. However, inhibition of phagocytosis and extracellular traps reduced killing of the smooth morphotype with lesser effects against the rough morphotype. Neutrophils treated with M. abscessus released a heat-labile mycobactericidal activity against the rough morphotype, but the activity was heat-tolerant against the smooth morphotype. Overall, M. abscessus stimulates ineffective neutrophil reactive oxygen species generation, and key mechanisms differ in killing of the smooth (phagocytosis-dependent, extracellular traps, and heat-tolerant secreted factor) and rough (extracellular traps and a heat-labile secreted factor) morphotypes. These studies represent an essential advancement in understanding the host response to M. abscessus, and help explain the recalcitrance of infection.

Whole Blood Gene Expression Profiling Predicts Severe Morbidity and Mortality in Cystic Fibrosis: A 5-Year Follow-Up Study.

RATIONALE: Cystic fibrosis pulmonary exacerbations accelerate pulmonary decline and increase mortality. Previously, we identified a 10-gene leukocyte panel measured directly from whole blood, which indicates response to exacerbation treatment. We hypothesized that molecular characteristics of exacerbations could also predict future disease severity. OBJECTIVES: We tested whether a 10-gene panel measured from whole blood could identify patient cohorts at increased risk for severe morbidity and mortality, beyond standard clinical measures. METHODS: Transcript abundance for the 10-gene panel was measured from whole blood at the beginning of exacerbation treatment (n = 57). A hierarchical cluster analysis of subjects based on their gene expression was performed, yielding four molecular clusters. An analysis of cluster membership and outcomes incorporating an independent cohort (n = 21) was completed to evaluate robustness of cluster partitioning of genes to predict severe morbidity and mortality. RESULTS: The four molecular clusters were analyzed for differences in forced expiratory volume in 1 second, C-reactive protein, return to baseline forced expiratory volume in 1 second after treatment, time to next exacerbation, and time to morbidity or mortality events (defined as lung transplant referral, lung transplant, intensive care unit admission for respiratory insufficiency, or death). Clustering based on gene expression discriminated between patient groups with significant differences in forced expiratory volume in 1 second, admission frequency, and overall morbidity and mortality. At 5 years, all subjects in cluster 1 (very low risk) were alive and well, whereas 90% of subjects in cluster 4 (high risk) had suffered a major event (P = 0.0001). In multivariable analysis, the ability of gene expression to predict clinical outcomes remained significant, despite adjustment for forced expiratory volume in 1 second, sex, and admission frequency. The robustness of gene clustering to categorize patients appropriately in terms of clinical characteristics, and short- and long-term clinical outcomes, remained consistent, even when adding in a secondary population with significantly different clinical outcomes. CONCLUSIONS: Whole blood gene expression profiling allows molecular classification of acute pulmonary exacerbations, beyond standard clinical measures, providing a predictive tool for identifying subjects at increased risk for mortality and disease progression.

Mycobacterium abscessus Displays Fitness for Fomite Transmission.

Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterium (NTM) increasingly reported in soft tissue infections and chronic lung diseases, including cystic fibrosis. The environmental source of M. abscessus has not been definitively identified, but NTM have been detected in soil and water. To determine the potential of soil-derived M. abscessus as an infectious source, we explored the association, growth, and survival of M. abscessus with defined mineral particulates, including kaolin, halloysite, and silicone dioxide, and house dust as possible M. abscessus fomites. M. abscessus physically associated with particulates, and the growth of M. abscessus was enhanced in the presence of both kaolin and house dust. M. abscessus survived desiccation for 2 weeks but was not viable after 3 weeks. The rate of decline of M. abscessus viability during desiccation was reduced in the presence of house dust. The evidence for enhanced growth and survival of M. abscessus during alternating growth and drying periods suggests that dissemination could occur when in wet or dry environments. These studies are important to understand environmental survival and acquisition of NTM.IMPORTANCE The environmental source of pulmonary Mycobacterium abscessus infections is not known. Fomites are nonliving carriers of infectious agents and may contribute to acquisition of M. abscessus This study provides evidence that M. abscessus growth is enhanced in the presence of particulates, using kaolin, an abundant natural clay mineral, and house dust as experimental fomites. Moreover, M. abscessus survived desiccation for up to 2 weeks in the presence of house dust, kaolin, and several chemically defined mineral particulates; mycobacterial viability during extended periods of dessication was enhanced by the presence of house dust. The growth characteristics of M. abscessus with particulates suggest that a fomite mechanism of transmission may contribute to M. abscessus acquisition, which may lead to strategies to better control infections by M. abscessus and related organisms.

Impact of azithromycin on the clinical and antimicrobial effectiveness of tobramycin in the treatment of cystic fibrosis.

BACKGROUND: Concomitant use of oral azithromycin and inhaled tobramycin occurs in approximately half of US cystic fibrosis (CF) patients. Recent data suggest that this combination may be antagonistic. METHODS: Test the hypothesis that azithromycin reduces the clinical benefits of tobramycin by analyses of clinical trial data, in vitro modeling of P. aeruginosa antibiotic killing, and regulation of the MexXY efflux pump. RESULTS: Ongoing administration of azithromycin associates with reduced ability of inhaled tobramycin, as compared with aztreonam, to improve lung function and quality of life in a completed clinical trial. In users of azithromycin FEV1 (L) increased 0.8% during a 4-week period of inhaled tobramycin and an additional 6.4% during a subsequent 4-week period of inhaled aztreonam (P<0.005). CFQ-R respiratory symptom score decreased 1.8 points during inhaled tobramycin and increased 8.3 points during subsequent inhaled aztreonam (P<0.001). A smaller number of trial participants not using azithromycin had similar improvement in lung function and quality of life scores during inhaled tobramycin and inhaled aztreonam. In vitro, azithromycin selectively reduced the bactericidal effects tobramycin in cultures of clinical strains of P. aeruginosa, while up regulating antibiotic resistance through MexXY efflux. CONCLUSIONS: Azithromycin appears capable of reducing the antimicrobial benefits of tobramycin by inducing adaptive bacterial stress responses in P. aeruginosa, suggesting that these medications together may not be optimal chronic therapy for at least some patients.

Extremes of Interferon-Stimulated Gene Expression Associate with Worse Outcomes in the Acute Respiratory Distress Syndrome.

Acute Respiratory Distress Syndrome (ARDS) severity may be influenced by heterogeneity of neutrophil activation. Interferon-stimulated genes (ISG) are a broad gene family induced by Type I interferons, often as a response to viral infections, which evokes extensive immunomodulation. We tested the hypothesis that over- or under-expression of immunomodulatory ISG by neutrophils is associated with worse clinical outcomes in patients with ARDS. Genome-wide transcriptional profiles of circulating neutrophils isolated from patients with sepsis-induced ARDS (n = 31) and healthy controls (n = 19) were used to characterize ISG expression. Hierarchical clustering of expression identified 3 distinct subject groups with Low, Mid and High ISG expression. ISG accounting for the greatest variability in expression were identified (MX1, IFIT1, and ISG15) and used to analyze a prospective cohort at the Colorado ARDS Network site. One hundred twenty ARDS patients from four urban hospitals were enrolled within 72 hours of initiation of mechanical ventilation. Circulating neutrophils were isolated from patients and expression of ISG determined by PCR. Samples were stratified by standard deviation from the mean into High (n = 21), Mid, (n = 82) or Low (n = 17) ISG expression. Clinical outcomes were compared between patients with High or Low ISG expression to those with Mid-range expression. At enrollment, there were no differences in age, gender, co-existing medical conditions, or type of physiologic injury between cohorts. After adjusting for age, race, gender and BMI, patients with either High or Low ISG expression had significantly worse clinical outcomes than those in the Mid for number of 28-day ventilator- and ICU-free days (P = 0.0006 and 0.0004), as well as 90-day mortality and 90-day home with unassisted breathing (P = 0.02 and 0.004). These findings suggest extremes of ISG expression by circulating neutrophils from ARDS patients recovered early in the syndrome are associated with poorer clinical outcomes.

Mycobacterium abscessus morphotype comparison in a murine model.

Pulmonary infections with Mycobacterium abscessus (M. abscessus) are increasingly prevalent in patients with lung diseases such as cystic fibrosis. M. abscessus exists in two morphotypes, smooth and rough, but the impact of morphotype on virulence is unclear. We developed an immune competent mouse model of pulmonary M. abscessus infection and tested the differences in host inflammatory response between the morphotypes of M. abscessus. Smooth and rough morphotypes of M. abscessus were isolated from the same American Type Culture Collection strain. Wild type and cystic fibrosis mice were intratracheally inoculated with known quantities of M. abscessus suspended in fibrin plugs. At the time of sacrifice lung and splenic tissues and bronchoalveolar lavage fluid were collected and cultured. Bronchoalveolar lavage fluid was analyzed for leukocyte count, differential and cytokine expression. Pulmonary infection with M. abscessus was present at both 3 days and 14 days post-inoculation in all groups at greater levels than systemic infection. Inoculation with M. abscessus rough morphotype resulted in more bronchoalveolar lavage fluid neutrophils compared to smooth morphotype at 14 days post-inoculation in both wild type (p = 0.01) and cystic fibrosis (p<0.01) mice. Spontaneous in vivo conversion from smooth to rough morphotype occurred in 12/57 (21%) of mice. These mice trended towards greater weight loss than mice in which morphotype conversion did not occur. In the described fibrin plug model of M. abscessus infection, pulmonary infection with minimal systemic dissemination is achieved with both smooth and rough morphotypes. In this model M. abscessus rough morphotype causes a greater host inflammatory response than the smooth based on bronchoalveolar lavage fluid neutrophil levels.

Enhanced in vitro formation and antibiotic resistance of nonattached Pseudomonas aeruginosa aggregates through incorporation of neutrophil products.

Pseudomonas aeruginosa is a major pathogen in cystic fibrosis (CF) lung disease. Children with CF are routinely exposed to P. aeruginosa from the natural environment, and by adulthood, 80% of patients are chronically infected. P. aeruginosa in the CF airway exhibits a unique biofilm-like structure, where it grows in small clusters or aggregates of bacteria in association with abundant polymers of neutrophil-derived components F-actin and DNA, among other components. These aggregates differ substantially in size and appearance compared to surface-attached in vitro biofilm models classically utilized for studies but are believed to share properties of surface-attached biofilms, including antibiotic resistance. However, little is known about the formation and function of surface-independent modes of biofilm growth, how they might be eradicated, and quorum sensing communication. To address these issues, we developed a novel in vitro model of P. aeruginosa aggregates incorporating human neutrophil-derived products. Aggregates grown in vitro and those found in CF patients' sputum samples were morphologically similar; viable bacteria were distributed in small pockets throughout the aggregate. The lasA quorum sensing gene was differentially expressed in the presence of neutrophil products. Importantly, aggregates formed in the presence of neutrophils acquired resistance to tobramycin, which was lost when the aggregates were dispersed with DNase, and antagonism of tobramycin and azithromycin was observed. This novel yet simple in vitro system advances our ability to model infection of the CF airway and will be an important tool to study virulence and test alternative eradication strategies against P. aeruginosa.

Expression of antimicrobial drug tolerance by attached communities of Mycobacterium tuberculosis.

There is an urgent need to improve methods used to screen antituberculosis drugs. An in vitro assay was developed to test drug treatment strategies that specifically target drug-tolerant Mycobacterium tuberculosis. The H37Rv strain of M. tuberculosis survived antimicrobial treatment as attached microbial communities when maintained in tissue culture media (RPMI-1640) with or without lysed human peripheral blood leukocytes. When cultured planktonically in the presence of Tween-80, bacilli failed to form microbial communities or reach logarithmic phase growth yet remained highly susceptible to antimicrobial drugs. In the absence of Tween, bacilli tolerated drug therapy by forming complex microbial communities attached to untreated well surfaces or to the extracellular matrix derived from lysed human leukocytes. Treatment of microbial communities with DNase I or Tween effectively dispersed bacilli and restored drug susceptibility. These data demonstrate that in vitro expression of drug tolerance by M. tuberculosis is linked to the establishment of attached microbial communities and that dispersion of bacilli targeting the extracellular matrix including DNA restores drug susceptibility. Modifications of this in vitro assay may prove beneficial in a high-throughput platform to screen new antituberculosis drugs especially those that target drug-tolerant bacilli.

Mycobacterium abscessus induces a limited pattern of neutrophil activation that promotes pathogen survival.

Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy.

Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR.

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.

Bacteria-specific neutrophil dysfunction associated with interferon-stimulated gene expression in the acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a poorly understood condition with greater than 30% mortality. Massive recruitment of neutrophils to the lung occurs in the initial stages of the ARDS. Significant variability in the severity and duration of ARDS-associated pulmonary inflammation could be linked to heterogeneity in the inflammatory capacity of neutrophils. Interferon-stimulated genes (ISGs) are a broad gene family induced by Type I interferons. While ISGs are central to anti-viral immunity, the potential exists for these genes to evoke extensive modification in cellular response in other clinical settings. In this prospective study, we sought to determine if ISG expression in circulating neutrophils from ARDS patients is associated with changes in neutrophil function. Circulating neutrophil RNA was isolated, and hierarchical clustering ranked patients' expression of three ISGs. Neutrophil response to pathogenic bacteria was compared between normal and high ISG-expressing neutrophils. High neutrophil ISG expression was found in 25 of 95 (26%) of ARDS patients and was associated with reduced migration toward interleukin-8, and altered responses to Staphylococcus aureus, but not Pseudomonas aeruginosa, which included decreased p38 MAP kinase phosphorylation, superoxide anion release, interleukin-8 release, and a shift from necrotic to apoptotic cell death. These alterations in response were reflected in a decreased capacity to kill S. aureus, but not P. aeruginosa. Therefore, the ISG expression signature is associated with an altered circulating neutrophil response phenotype in ARDS that may predispose a large subgroup of patients to increased risk of specific bacterial infections.