Prevalence and Clinical Impact of Respiratory Viral Infections from the STOP2 Study of Cystic Fibrosis Pulmonary Exacerbations.

Rationale: Rates of viral respiratory infection (VRI) are similar in people with cystic fibrosis (CF) and the general population; however, the associations between VRI and CF pulmonary exacerbations (PEx) require further elucidation.Objectives: To determine VRI prevalence during CF PEx and evaluate associations between VRI, clinical presentation, and treatment response.Methods: The STOP2 (Standardized Treatment of Pulmonary Exacerbations II) study was a multicenter randomized trial to evaluate different durations of intravenous antibiotic therapy for PEx. In this ancillary study, participant sputum samples from up to three study visits were tested for respiratory viruses using multiplex polymerase chain reactions. Baselines and treatment-associated changes in mean lung function (percent predicted forced expiratory volume in 1 s), respiratory symptoms (Chronic Respiratory Infection Symptom Score), weight, and C-reactive protein were compared as a function of virus detection. Odds of PEx retreatment within 30 days and future PEx hazard were modeled by logistic and Cox proportional hazards regression, respectively.Results: A total of 1,254 sputum samples from 621 study participants were analyzed. One or more respiratory viruses were detected in sputum samples from 245 participants (39.5%). Virus-positive participants were more likely to be receiving CF transmembrane conductance regulator modulator therapy (45% vs. 34%) and/or chronic azithromycin therapy (54% vs. 44%) and more likely to have received treatment for nontuberculous Mycobacterium infection in the preceding 2 years (7% vs. 3%). At study visit 1, virus-positive participants were more symptomatic (mean Chronic Respiratory Infection Symptom Score, 53.8 vs. 51.1), had evidence of greater systemic inflammation (log10 C-reactive protein concentration, 1.32 log10 mg/L vs. 1.23 log10 mg/L), and had a greater drop in percent predicted forced expiratory volume in 1 second from the prior 6-month baseline (5.8 vs. 3.6). Virus positivity was associated with reduced risk of future PEx (hazard ratio, 0.82; 95% confidence interval, 0.69-0.99; P = 0.034) and longer median time to next PEx (255 d vs. 172 d; P = 0.021) compared with virus negativity.Conclusions: More than one-third of STOP2 participants treated for a PEx had a positive test result for a respiratory virus with more symptomatic initial presentation compared with virus-negative participants, but favorable long-term outcomes. More refined phenotyping of PEx, taking VRIs into account, may aid in optimizing personalized management of PEx.Clinical trial registered with www.clinicaltrials.gov (NCT02781610).

Microbial community organization designates distinct pulmonary exacerbation types and predicts treatment outcome in cystic fibrosis.

Polymicrobial infection of the airways is a hallmark of obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease (COPD). Intermittent pulmonary exacerbations (PEx) in these conditions are associated with lung function decline and higher mortality rates. An understanding of the microbial underpinnings of PEx is challenged by high inter-patient variability in airway microbial community profiles. We analyzed 880 near-daily CF sputum samples and developed non-standard microbiome descriptors to model community reorganization prior and during 18 PEx. We identified two communal microbial regimes with opposing ecology and dynamics. Whereas pathogen-governed dysbiosis showed hierarchical community organization and reduced diversity, anaerobic bloom dysbiosis displayed stochasticity and increased diversity. Microbiome organization modulated the relevance of pathogens and a simulation of antimicrobial treatment predicted better efficacy for hierarchically organized microbiota. This causal link between PEx, microbiome organization, and treatment success advances the development of personalized dysbiosis management in CF and, potentially, other obstructive lung diseases.

Quantifying variation in home spirometry in people with cystic fibrosis during baseline health, and associations with clinical outcomes.

BACKGROUND: Home spirometry is increasingly used to monitor lung function in people with cystic fibrosis (pwCF). Although decreases in lung function in the setting of increased respiratory symptoms are consistent with a pulmonary exacerbation (PEx), the interpretation of home spirometry during asymptomatic periods of baseline health is unclear. The aims of this study were to determine the variation in home spirometry in pwCF during asymptomatic periods of baseline health and to identify associations between this variation and PEx. METHODS: Near-daily home spirometry measurements were obtained from a cohort of pwCF enrolled in a long-term study of the airway microbiome. Associations between the degree of variation in home spirometry and the time to next PEx were evaluated. RESULTS: Thirteen subjects (mean age of 29 years and mean percent predicted forced expiratory volume in one second [ppFEV1] of 60) provided a median of 204 spirometry readings taken during 40 periods of baseline health. The mean week-to-week within-subject level of variation in ppFEV1 was 15.2 ± 6.2%. The degree of variation in ppFEV1 during baseline health was not associated with time to PEx. CONCLUSIONS: Variation in ppFEV1 measured with near-daily home spirometry in pwCF during periods of baseline health exceeded the variation in ppFEV1 expected in clinic spirometry (based on ATS guidelines). The degree of variation in ppFEV1 during baseline health was not associated with time to PEx. These data are relevant for guiding interpretation of home spirometry.

Airway bacterial community composition in persons with advanced cystic fibrosis lung disease.

BACKGROUND: The progression of lung disease in people with cystic fibrosis (pwCF) has been associated with a decrease in the diversity of airway bacterial communities. How often low diversity communities occur in advanced CF lung disease and how they may be associated with clinical outcomes is not clear, however. METHODS: We sequenced a region of the bacterial 16S ribosomal RNA gene to characterize bacterial communities in sputum from 190 pwCF with advanced lung disease (FEV1≤40% predicted), with particular attention to the prevalence and relative abundance of dominant genera. We evaluated relationships between community diversity and clinical outcomes. RESULTS: Although most of the 190 pwCF with advanced lung disease had airway bacterial communities characterized by low diversity with a dominant genus, a considerable minority (40%) did not. The absence of a dominant genus, presence of methicillin-susceptible Staphylococcus aureus, and greater bacterial richness positively correlated with lung function. Higher relative abundance of the dominant genus and greater antimicrobial use negatively correlated with lung function. PwCF with a low diversity community and dominant genus had reduced lung transplant-free survival compared to those without (median survival of 1.6 vs 2.9 years). CONCLUSIONS: A considerable proportion of pwCF with advanced lung disease do not have airway bacterial communities characterized by low diversity and a dominant genus and these individuals had better survival. An understanding of the antecedents of low diversity airway communities- and the impact these may have on lung disease trajectory - may provide avenues for improved management strategies.

Changes in airway bacterial communities occur soon after initiation of antibiotic treatment of pulmonary exacerbations in cystic fibrosis.

Chronic polymicrobial airway infections are a hallmark of cystic fibrosis (CF) lung disease. Antibiotic therapy is a primary treatment of CF pulmonary exacerbations (PEx); however, the impact of episodic antibiotic treatment on airway bacterial communities has not been well described. We analyzed sputum samples from adults with CF obtained immediately before and during antibiotic treatment of PEx. Sequencing of the V4 region of the bacterial 16S ribosomal RNA gene was used to assess changes in bacterial community structure during antibiotic treatment. The peak impact of antibiotic treatment was observed by day four or five of treatment. These findings advance our understanding of bacterial community dynamics during antibiotic treatment of PEx and complement recent and ongoing studies evaluating the optimal duration of antibiotic therapy for PEx.

Association of bacterial community types, functional microbial processes and lung disease in cystic fibrosis airways.

Bacterial infection and inflammation of the airways are the leading causes of morbidity and mortality in persons with cystic fibrosis (CF). The ecology of the bacterial communities inhabiting CF airways is poorly understood, especially with respect to how community structure, dynamics, and microbial metabolic activity relate to clinical outcomes. In this study, the bacterial communities in 818 sputum samples from 109 persons with CF were analyzed by sequencing bacterial 16S rRNA gene amplicons. We identified eight alternative community types (pulmotypes) by using a Dirichlet multinomial mixture model and studied their temporal dynamics in the cohort. Across patients, the pulmotypes displayed chronological patterns in the transition among each other. Furthermore, significant correlations between pulmotypes and patient clinical status were detected by using multinomial mixed effects models, principal components regression, and statistical testing. Constructing pulmotype-specific metabolic activity profiles, we found that pulmotype microbiota drive distinct community functions including mucus degradation or increased acid production. These results indicate that pulmotypes are the result of ordered, underlying drivers such as predominant metabolism, ecological competition, and niche construction and can form the basis for quantitative, predictive models supporting clinical treatment decisions.

Parallel Analysis of Cystic Fibrosis Sputum and Saliva Reveals Overlapping Communities and an Opportunity for Sample Decontamination.

Culture-independent studies of the cystic fibrosis (CF) airway microbiome typically rely on expectorated sputum to assess the microbial makeup of lower airways. These studies have revealed rich bacterial communities. There is often considerable overlap between taxa observed in sputum and those observed in saliva, raising questions about the reliability of expectorated sputum as a sample representing lower airway microbiota. These concerns prompted us to compare pairs of sputum and saliva samples from 10 persons with CF. Using 16S rRNA gene sequencing and droplet digital PCR (ddPCR), we analyzed 37 pairs of sputum and saliva samples, each collected from the same person on the same day. We developed an in silico postsequencing decontamination procedure to remove from sputum the fraction of DNA reads estimated to have been contributed by saliva during expectoration. We demonstrate that while there was often sizeable overlap in community membership between sample types, expectorated sputum typically contains a higher bacterial load and a less diverse community compared to saliva. The differences in diversity between sputum and saliva were more pronounced in advanced disease stage, owing to increased relative abundance of the dominant taxa in sputum. Our effort to model saliva contamination of sputum in silico revealed generally minor effects on community structure after removal of contaminating reads. Despite considerable overlap in taxa observed between expectorated sputum and saliva samples, the impact of saliva contamination on measures of lower airway bacterial community composition in CF using expectorated sputum appears to be minimal.IMPORTANCE Cystic fibrosis is an inherited disease characterized by chronic respiratory tract infection and progressive lung disease. Studies of cystic fibrosis lung microbiology often rely on expectorated sputum to reflect the microbiota present in the lower airways. Passage of sputum through the oropharynx during collection, however, contributes microbes present in saliva to the sample, which could confound interpretation of results. Using culture-independent DNA sequencing-based analyses, we characterized the bacterial communities in pairs of expectorated sputum and saliva samples to generate a model for "decontaminating" sputum in silico Our results demonstrate that salivary contamination of expectorated sputum does not have a large effect on most sputum samples and that observations of high bacterial diversity likely accurately reflect taxa present in cystic fibrosis lower airways.

Measures of Cystic Fibrosis Airway Microbiota during Periods of Clinical Stability.

Rationale: Differences in cystic fibrosis (CF) airway microbiota between periods of clinical stability and exacerbation of respiratory symptoms have been investigated in efforts to better understand microbial triggers of CF exacerbations. Prior studies have often relied on a single sample or a limited number of samples to represent airway microbiota. However, the variability in airway microbiota during periods of clinical stability is not well known.Objectives: To determine the temporal variability of measures of airway microbiota during periods of clinical stability, and to identify factors associated with this variability.Methods: Sputum samples (N = 527), obtained daily from six adults with CF during 10 periods of clinical stability, underwent sequencing of the V4 region of the bacterial 16S ribosomal RNA gene. The variability in airway microbiota among samples within each period of clinical stability was calculated as the average of the Bray-Curtis similarity measures of each sample to every other sample within the same period. Outlier samples were defined as samples outside 1.5 times the interquartile range within a baseline period with respect to the average Bray-Curtis similarity. Total bacterial load was measured with droplet digital polymerase chain reaction.Results: The variation in Bray-Curtis similarity and total bacterial load among samples within the same baseline period was greater than the variation observed in technical replicate control samples. Overall, 6% of samples were identified as outliers. Within baseline periods, changes in bacterial community structure occurred coincident with changes in maintenance antibiotics (P < 0.05, analysis of molecular variance). Within subjects, bacterial community structure changed between baseline periods (P < 0.01, analysis of molecular variance). Sample-to-sample similarity within baseline periods was greater with fewer interval days between sampling.Conclusions: During periods of clinical stability, airway bacterial community structure and bacterial load vary among daily sputum samples from adults with CF. This day-to-day variation has bearing on study design and interpretation of results, particularly in analyses that rely on single samples to represent periods of interest (e.g., clinical stability vs. pulmonary exacerbation). These data also emphasize the importance of accounting for maintenance antibiotic use and granularity of sample collection in studies designed to assess the dynamics of CF airway microbiota relative to changes in clinical state.

Fluctuations in airway bacterial communities associated with clinical states and disease stages in cystic fibrosis.

Bacteria that infect the airways of persons with cystic fibrosis (CF) include a group of well-described opportunistic pathogens as well as numerous, mainly obligate or facultative anaerobic species typically not reported by standard sputum culture. We sequenced the V3-V5 hypervariable region of the bacterial 16S rRNA gene in DNA derived from 631 sputum specimens collected from 111 CF patients over 10 years. We describe fluctuations in the relative abundances of typical CF pathogens, as well as anaerobic species, in relation to changes in patients' clinical state and lung disease stage. Both bacterial community diversity and the relative abundance of anaerobes increased during exacerbation of symptoms (prior to antibiotic treatment), although this trend was not observed uniformly across disease stages. Community diversity and the relative abundance of anaerobic species decreased during antibiotic treatment. These results support current hypotheses regarding the role of anaerobes in CF pulmonary exacerbations and lung disease progression.

Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples.

Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log10 copies/mL for M. abscessus complex and by a mean of 0.43 log10 copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.

Culture-Based and Culture-Independent Bacteriologic Analysis of Cystic Fibrosis Respiratory Specimens.

Cystic fibrosis (CF) is characterized by chronic infection and inflammation of the airways. In vitro culture of select bacterial species from respiratory specimens has been used to guide antimicrobial therapy in CF for the past few decades. More recently, DNA sequence-based, culture-independent approaches have been used to assess CF airway microbiology, although the role that these methods will (or should) have in routine microbiologic analysis of CF respiratory specimens is unclear. We performed DNA sequence analyses to detect bacterial species in 945 CF sputum samples that had been previously analyzed by selective CF culture. We determined the concordance of results based on culture and sequence analysis, highlighting the comparison of the results for the most prevalent genera. Although overall prevalence rates were comparable between the two methods, results varied by genus. While sequence analysis was more likely to detect Achromobacter, Stenotrophomonas, and Burkholderia, it was less likely to detect Staphylococcus. Streptococcus spp. were rarely reported in culture results but were the most frequently detected species by sequence analysis. A variety of obligate and facultative anaerobic species, not reported by culture, was also detected with high prevalence by sequence analysis. Sequence analysis indicated that in a considerable proportion of samples, taxa not reported by selective culture constituted a relatively high proportion of the total bacterial load, suggesting that routine CF culture may underrepresent significant segments of the bacterial communities inhabiting CF airways.

The daily dynamics of cystic fibrosis airway microbiota during clinical stability and at exacerbation.

BACKGROUND: Recent work indicates that the airways of persons with cystic fibrosis (CF) typically harbor complex bacterial communities. However, the day-to-day stability of these communities is unknown. Further, airway community dynamics during the days corresponding to the onset of symptoms of respiratory exacerbation have not been studied. RESULTS: Using 16S rRNA amplicon sequencing of 95 daily sputum specimens collected from four adults with CF, we observed varying degrees of day-to-day stability in airway bacterial community structures during periods of clinical stability. Differences were observed between study subjects with respect to the degree of community changes at the onset of exacerbation. Decreases in the relative abundance of dominant taxa were observed in three subjects at exacerbation. We observed no relationship between total bacterial load and clinical status and detected no viruses by multiplex PCR. CONCLUSION: CF airway microbial communities are relatively stable during periods of clinical stability. Changes in microbial community structure are associated with some, but not all, pulmonary exacerbations, supporting previous observations suggesting that distinct types of exacerbations occur in CF. Decreased abundance of species that are dominant at baseline suggests a role for less abundant taxa in some exacerbations. Daily sampling revealed patterns of change in microbial community structures that may prove useful in the prediction and management of CF pulmonary exacerbations.

Impact of storage conditions on metabolite profiles of sputum samples from persons with cystic fibrosis.

BACKGROUND: Although recent studies have begun to elucidate how airway microbial community structure relates to lung disease in cystic fibrosis (CF), microbial community activity and the host's response to changes in this activity are poorly understood. Metabolomic profiling provides a means to investigate microbial activity and human cell activity within diseased airways. However, variables in sample storage and shipping likely affect downstream analyses and standards for sample handling are lacking. METHODS: We assessed the impact of sample storage conditions on liquid chromatography mass spectrometry analysis of CF sputum samples. RESULTS: Significant changes in global metabolomic profiles occurred in samples stored at room temperature or at 4°C for longer than one day. Untargeted metabolomic profiles were stable in sputum samples stored at -20°C or -80°C for at least 28 days. Quorum sensing molecules and phenazines, both considered important to the in vivo activity of Pseudomonas during airway infection, were detected after sample storage at room temperature for five days. CONCLUSIONS: Sputum samples can be stored at -20°C or -80°C for weeks with minimal effect on global metabolomic profiles. This observation provides guidance in designing metabolomic studies that have the potential to deepen our understanding of how airway microbial communities impact lung disease progression in CF.

Changes in cystic fibrosis airway microbiota at pulmonary exacerbation.

RATIONALE: In persons with cystic fibrosis (CF), repeated exacerbations of pulmonary symptoms are associated with a progressive decline in lung function. Changes in the airway microbiota around the time of exacerbations are not well understood. OBJECTIVES: To characterize changes in airway bacterial communities around the time of exacerbations and to identify predictors for these changes. METHODS: DNA prepared from 68 paired baseline and exacerbation sputum samples collected from 28 patients with CF were subjected to barcoded 16S rRNA gene pyrosequencing. Bacterial density was calculated by quantitative PCR. MEASUREMENTS AND MAIN RESULTS: Overall, significant differences in bacterial community diversity and bacterial density between baseline and exacerbation samples were not observed. However, considerable changes in community structures were observed in a subset of patients. In these patients, the dominant taxa and initial level of community diversity were significant predictors of the magnitude of community structure changes at exacerbation. Pseudomonas-dominant communities became more diverse at exacerbation compared with communities with other or no dominant species. The relative abundance of Gemella increased in 24 (83%) of 29 samples at exacerbation and was found to be the most discriminative genus between baseline and exacerbation samples. CONCLUSIONS: The magnitude of changes in the CF lung microbiota around the time of exacerbation was found to be largely dependent on community diversity and composition at baseline. Certain genera appear to play important roles in driving change in airway bacterial community composition at exacerbation. Gemella might play a direct role in and/or be a biomarker for pulmonary exacerbation.

Decade-long bacterial community dynamics in cystic fibrosis airways.

The structure and dynamics of bacterial communities in the airways of persons with cystic fibrosis (CF) remain largely unknown. We characterized the bacterial communities in 126 sputum samples representing serial collections spanning 8-9 y from six age-matched male CF patients. Sputum DNA was analyzed by bar-coded pyrosequencing of the V3-V5 hypervariable region of the 16S rRNA gene, defining 662 operational taxonomic units (OTUs) from >633,000 sequences. Bacterial community diversity decreased significantly over time in patients with typically progressive lung disease but remained relatively stable in patients with a mild lung disease phenotype. Antibiotic use, rather than patient age or lung function, was the primary driver of decreasing diversity. Interpatient variability in community structure exceeded intrapatient variability in serial samples. Antibiotic treatment was associated with pronounced shifts in community structure, but communities showed both short- and long-term resilience after antibiotic perturbation. There was a positive correlation between OTU occurrence and relative abundance, with a small number of persistent OTUs accounting for the greatest abundance. Significant changes in community structure, diversity, or total bacterial density at the time of pulmonary exacerbation were not observed. Despite decreasing community diversity in patients with progressive disease, total bacterial density remained relatively stable over time. These findings show the critical relationship between airway bacterial community structure, disease stage, and clinical state at the time of sample collection. These features are the key parameters with which to assess the complex ecology of the CF airway.

Impact of enhanced Staphylococcus DNA extraction on microbial community measures in cystic fibrosis sputum.

Staphylococcus aureus is a common constituent of the bacterial community inhabiting the airways of persons with cystic fibrosis (CF). Culture-independent studies have shown that this species is often present in relatively high abundance and would therefore be expected to exert a pronounced effect on measures of CF airway bacterial community structure. We investigated the impact of DNA extraction method on pyrosequencing-based measures of Staphylococcus abundance and bacterial community structure in 17 sputum samples from five CF patients. Staphylococcus was detected in fewer samples when DNA was extracted using a standard bacterial lysis method compared to when DNA was extracted using a lysis buffer amended with lysostaphin and lysozyme. The standard lysis method resulted in significantly lower measures of Staphylococcus relative abundance and higher levels of community diversity, richness, and evenness compared to the lysostaphin-lysozyme modified method. Measures of community dynamics in serial sputum samples from the same individual were nevertheless highly concordant between the two DNA extraction methods. These results illustrate the impact of DNA preparation method on measures of Staphylococcus abundance and bacterial community structures in studies of the airways microbiota in CF.

Genomes and characterization of phages Bcep22 and BcepIL02, founders of a novel phage type in Burkholderia cenocepacia.

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.

Reassessment of Stenotrophomonas maltophilia phenotype.

Standard microbiology references describe Stenotrophomonas maltophilia as oxidase negative and variable with respect to utilization of lactose and sucrose. Analysis of a collection of 766 S. maltophilia isolates indicated that approximately 20% are oxidase positive and that this species should be reevaluated for other phenotypes, including oxidative fermentation of lactose and sucrose.

Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection.

The therapeutic potential of bacteriophages (phages) in a mouse model of acute Burkholderia cenocepacia pulmonary infection was assessed. Phage treatment was administered by either intranasal inhalation or intraperitoneal injection. Bacterial density, macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor alpha (TNF-alpha) levels were significantly reduced in lungs of mice treated with intraperitoneal phages (P < .05). No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with intranasal phages, intraperitoneal ultraviolet-inactivated phages, or intraperitoneal lambda phage control mice. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNF-alpha levels compared with mock-infected/mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic phage administration was more effective than inhalational administration, suggesting that circulating phages have better access to bacteria in lungs than do topical phages.

A type IV secretion system contributes to intracellular survival and replication of Burkholderia cenocepacia.

Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis (CF). Recent studies indicate that B. cenocepacia survives within macrophages and airway epithelial cells in vitro by evading endosome-lysosome fusion. We investigated the role of a plasmid-encoded type IV secretion system in the intracellular survival, replication, and processing of B. cenocepacia. Both a wild-type strain (K56-2) and its type IV secretion system mutant (designated LC101) entered and replicated in CF airway epithelial cells and monocyte-derived macrophages. However, significantly more intracellular K56-2 than LC101 bacteria were found in both cell types at 24 h postinfection. Colocalization of bacteria with markers of the classical endocytic pathway indicated that although both K56-2 and LC101 reside transiently in early endosomes, a greater proportion of the mutant bacteria are targeted to lysosomal degradation. In contrast, wild-type bacteria escape from the classical endocytic pathway and traffic to the endoplasmic reticulum, where they replicate. Our results show that the intracellular processing of B. cenocepacia is similar in both professional and nonprofessional phagocytes and that a functional plasmid-encoded type IV secretion system contributes to the survival and replication of B. cenocepacia in eukaryotic cells.