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We previously demonstrated that P. aeruginosa isolates that persisted in children with cystic fibrosis (CF) despite inhaled tobramycin treatment had increased anti-Psl antibody binding in vitro compared to those successfully eradicated. We aimed to validate these findings by directly visualizing P. aeruginosa in CF sputum. This was a prospective observational study of children with CF with new-onset P. aeruginosa infection who underwent inhaled tobramycin eradication treatment. Using microbial identification passive clarity technique (MiPACT), P. aeruginosa was visualized in sputum samples obtained before treatment and classified as persistent or eradicated based on outcomes. Pre-treatment isolates were also grown as biofilms in vitro. Of 11 patients enrolled, 4 developed persistent infection and 7 eradicated infection. P. aeruginosa biovolume and the number as well as size of P. aeruginosa aggregates were greater in the sputum of those with persistent compared with eradicated infections (p P. aeruginosa biovolume. When visualized in sputum, P. aeruginosa had a greater biovolume, with more expressed Psl, and formed more numerous, larger aggregates in CF children who failed eradication therapy compared to those who successfully cleared their infection.

Background Pulmonary exacerbations (PEx) in people with cystic fibrosis (PwCF) are associated with significant morbidity. While standard PEx treatment for PwCF with Pseudomonas aeruginosa infection includes two IV antipseudomonal antibiotics, little evidence exists to recommend this approach. This study aimed to compare clinical outcomes of single versus double antipseudomonal antibiotic use for PEx treatment. Methods Retrospective cohort study using the linked CF Foundation Patient Registry-Pediatric Health Information System dataset. PwCF were included if hospitalized between 2007 and 2018 and 6–21 years of age. Regression modeling accounting for repeated measures was used to compare lung function outcomes between single versus double IV antipseudomonal antibiotic regimens using propensity-score weighting to adjust for relevant confounding factors. Results Among 10,660 PwCF in the dataset, we analyzed 2,578 PEx from 1,080 PwCF, of which 455 and 2,123 PEx were treated with 1 versus 2 IV antipseudomonal antibiotics, respectively. We identified no significant differences between PEx treated with 1 versus 2 IV antipseudomonal antibiotics either in change between pre- and post-PEx percent predicted forced expiratory volume in one second (ppFEV1) (–0.84%, [95% CI –2.25, 0.56]; P = 0.24), odds of returning to ≥90% of baseline ppFEV1 within 3 months following PEx (Odds Ratio 0.83, [95% CI 0.61, 1.13]; P = 0.24) or time to next PEx requiring IV antibiotics (Hazard Ratio 1.04, [95% CI 0.87, 1.24]; P = 0.69). Conclusions Use of 2 IV antipseudomonal antibiotics for PEx treatment in young PwCF was not associated with greater improvements in measured respiratory and clinical outcomes compared to treatment with 1 IV antipseudomonal antibiotic.

The exopolysaccharide Psl contributes to biofilm structure and antibiotic tolerance and may play a role in the failure to eradicate Pseudomonas aeruginosa from cystic fibrosis (CF) airways. The study objective was to determine whether there were any differences in Psl in P. aeruginosa isolates that were successfully eradicated compared to those that persisted, despite inhaled tobramycin treatment, in children with CF. Initial P. aeruginosa isolates were collected from children with CF undergoing eradication treatment, grown as biofilms and labeled with 3 anti-Psl monoclonal antibodies (Cam003/Psl0096, WapR001, WapR016) before confocal microscopy visualization. When grown as biofilms, P. aeruginosa isolates from children who failed antibiotic eradication therapy, had significantly increased Psl0096 binding compared to isolates from those who cleared P. aeruginosa. This was confirmed in P. aeruginosa isolates from the SickKids Eradication Cohort as well as the Early Pseudomonas Infection Control (EPIC) trial. Increased anti-Psl antibody binding was associated with bacterial aggregation and tobramycin tolerance. The biofilm matrix represents a potential therapeutic target to improve P. aeruginosa eradication treatment.

Recent studies of human respiratory secretions using culture-independent techniques have found a surprisingly diverse array of microbes. Interactions among these community members can profoundly impact microbial survival, persistence and antibiotic susceptibility and, consequently, disease progression. Studies of polymicrobial interactions in the human microbiota have shown that the taxonomic and structural compositions, and resulting behaviours, of microbial communities differ substantially from those of the individual constituent species and in ways of clinical importance. These studies primarily involved oral and gastrointestinal microbiomes. While the field of polymicrobial respiratory disease is relatively young, early findings suggest that respiratory tract microbiota members also compete and cooperate in ways that may influence disease outcomes. Ongoing efforts therefore focus on how these findings can inform more ‘enlightened’, rational approaches to combat respiratory infections. Among the most common respiratory diseases involving polymicrobial infections are cystic fibrosis (CF), non-CF bronchiectasis, COPD and ventilator-associated pneumonia. While respiratory microbiota can be diverse, two of the most common and best-studied members are Staphylococcus aureus and Pseudomonas aeruginosa, which exhibit a range of competitive and cooperative interactions. Here, we review the state of research on pulmonary coinfection with these pathogens, including their prevalence, combined and independent associations with patient outcomes, and mechanisms of those interactions that could influence lung health. Because P. aeruginosa–S. aureus coinfection is common and well studied in CF, this disease serves as the paradigm for our discussions on these two organisms and inform our recommendations for future studies of polymicrobial interactions in pulmonary disease.

Rationale: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. Objectives: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. Methods: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. Measurements and Main Results: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. Conclusions: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments.

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations., Author summary Microbiologists have known for decades that Pseudomonas aeruginosa mutates during chronic respiratory infection of cystic fibrosis (CF) patients. One of the most reported functions lost during these infections is flagellar motility. A long-standing interpretation of this observation is that the flagellum is disadvantageous for the bacterium in the CF environment. We report the surprising finding that mutation of a wide range of flagellar genes results in the overproduction of the biofilm matrix polysaccharides Psl and Pel. We propose, therefore, that flagellar mutations represent a gain-of-function that would help the bacterium to form biofilms and persist in the CF airways.

Azithromycin has been shown to improve lung function and reduce the number of pulmonary exacerbations in patients with cystic fibrosis. Concerns remain, however, regarding the potential emergence of treatment-related respiratory pathogens.To determine whether chronic azithromycin use (defined as three-times weekly administration) is associated with increased rates of detection of eight specific respiratory pathogens.We performed a new-user, propensity score-matched retrospective cohort study utilizing data from the Cystic Fibrosis Foundation Patient Registry. Incident azithromycin users were propensity score matched 1:1 with contemporaneous nonusers. Kaplan-Meier curves and Cox proportional hazards regression were used to evaluate the association between chronic azithromycin use and incident respiratory pathogen detection. Analyses were performed separately for each pathogen, limited to patients among whom that pathogen had not been isolated in the 2 years before cohort entry.After propensity score matching, the mean age of the cohorts was approximately 12 years. Chronic azithromycin users had a significantly lower risk of detection of new methicillin-resistant Staphylococcus aureus, nontuberculous mycobacteria, and Burkholderia cepacia complex compared with nonusers. The risk of acquiring the remaining five pathogens was not significantly different between users and nonusers.Using an innovative new-user, propensity score-matched study design to minimize indication and selection biases, we found in a predominantly pediatric cohort that chronic azithromycin users had a lower risk of acquiring several cystic fibrosis-related respiratory pathogens. These results may ease concerns that chronic azithromycin exposure increases the risk of acquiring new respiratory pathogens among pediatric patients with cystic fibrosis.

Pseudomonas aeruginosa undergoes phenotypic changes during cystic fibrosis (CF) lung infection. Although mucoidy is traditionally associated with transition to chronic infection, we hypothesized that additional in vitro phenotypes correlate with this transition and contribute to disease.To characterize the relationships between in vitro P. aeruginosa phenotypes, infection stage, and clinical outcomes.A total of 649 children with CF and newly identified P. aeruginosa were followed for a median 5.4 years during which a total of 2,594 P. aeruginosa isolates were collected. Twenty-six in vitro bacterial phenotypes were assessed among the isolates, including measures of motility, exoproduct production, colony morphology, growth, and metabolism.P. aeruginosa phenotypes present at the time of culture were associated with both stage of infection (new onset, intermittent, or chronic) and the primary clinical outcome, occurrence of a pulmonary exacerbation (PE) in the subsequent 2 years. Two in vitro P. aeruginosa phenotypes best distinguished infection stages: pyoverdine production (31% of new-onset cultures, 48% of intermittent, 69% of chronic) and reduced protease production (31%, 39%, and 65%, respectively). The best P. aeruginosa phenotypic predictors of subsequent occurrence of a PE were mucoidy (odds ratio, 1.75; 95% confidence interval, 1.19-2.57) and reduced twitching motility (odds ratio, 1.43; 95% confidence interval, 1.11-1.84).In this large epidemiologic study of CF P. aeruginosa adaptation, P. aeruginosa isolates exhibited two in vitro phenotypes that best distinguished early and later infection stages. Among the many phenotypes tested, mucoidy and reduced twitching best predicted subsequent PE. These phenotypes indicate potentially useful prognostic markers of transition to chronic infection and advancing lung disease.

Background. Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized. Methods. We measured 22 easily assayed in vitro phenotypes among the baseline P. aeruginosa isolates collected from 194 participants in the 18-month EPIC clinical trial, which assessed outcomes after antibiotic eradication therapy for newly identified P. aeruginosa. We then evaluated the associations between these baseline isolate phenotypes and subsequent outcomes during the trial, including failure to eradicate after antipseudomonal therapy, emergence of mucoidy, and occurrence of an exacerbation. Results. Baseline P. aeruginosa isolates frequently exhibited phenotypes thought to represent chronic adaptation, including mucoidy. Wrinkly colony surface and irregular colony edges were both associated with increased risk of eradication failure (hazard ratios [95% confidence intervals], 1.99 [1.03–3.83] and 2.14 [1.32–3.47], respectively). Phenotypes reflecting defective quorum sensing were significantly associated with subsequent mucoidy, but no phenotype was significantly associated with subsequent exacerbations during the trial. Conclusions. Pseudomonas aeruginosa phenotypes commonly considered to reflect chronic adaptation were observed frequently among isolates at early detection. We found that 2 easily assayed colony phenotypes were associated with failure to eradicate after antipseudomonal therapy, both of which have been previously associated with altered biofilm formation and defective quorum sensing.

Chronic Pseudomonas aeruginosa infections cause significant morbidity in patients with cystic fibrosis (CF). Over years to decades, P. aeruginosa adapts genetically as it establishes chronic lung infections. Nonsynonymous mutations in lasR, the quorum-sensing (QS) master regulator, are common in CF. In laboratory strains of P. aeruginosa, LasR activates transcription of dozens of genes, including that for another QS regulator, RhlR. Despite the frequency with which lasR coding variants have been reported to occur in P. aeruginosa CF isolates, little is known about their consequences for QS. We sequenced lasR from 2,583 P. aeruginosa CF isolates. The lasR sequences of 580 isolates (22%) coded for polypeptides that differed from the conserved LasR polypeptides of well-studied laboratory strains. This collection included 173 unique lasR coding variants, 116 of which were either missense or nonsense mutations. We studied 31 of these variants. About one-sixth of the variant LasR proteins were functional, including 3 with nonsense mutations, and in some LasR-null isolates, genes that are LasR dependent in laboratory strains were nonetheless expressed. Furthermore, about half of the LasR-null isolates retained RhlR activity. Therefore, in some CF isolates the QS hierarchy is altered such that RhlR quorum sensing is independent of LasR regulation. Our analysis challenges the view that QS-silent P. aeruginosa is selected during the course of a chronic CF lung infection. Rather, some lasR sequence variants retain functionality, and many employ an alternate QS strategy involving RhlR., IMPORTANCE Chronic Pseudomonas aeruginosa infections, such as those in patients with the genetic disease cystic fibrosis, are notable in that mutants with defects in the quorum-sensing transcription factor LasR frequently arise. In laboratory strains of P. aeruginosa, quorum sensing activates transcription of dozens of genes, many of which encode virulence factors, such as secreted proteases and hydrogen cyanide synthases. In well-studied laboratory strains, LasR-null mutants have a quorum-sensing-deficient phenotype. Therefore, the presence of LasR variants in chronic infections has been interpreted to indicate that quorum-sensing-regulated products are not important for those infections. We report that some P. aeruginosa LasR variant clinical isolates are not LasR-null mutants, and others have uncoupled a second quorum-sensing system, the RhlR system, from LasR regulation. In these uncoupled isolates, RhlR independently activates at least some quorum-sensing-dependent genes. Our findings suggest that quorum sensing plays a role in chronic P. aeruginosa infections, despite the emergence of LasR coding variants.

The activity of aminoglycosides, which are used to treat Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF) patients, is reduced under the anaerobic conditions that reflect the CF lung in vivo. In contrast, a 4:1 (wt/wt) combination of fosfomycin and tobramycin (F:T), which is under investigation for use in the treatment of CF lung infection, has increased activity against P. aeruginosa under anaerobic conditions. The aim of this study was to elucidate the mechanisms underlying the increased activity of F:T under anaerobic conditions. Microarray analysis was used to identify the transcriptional basis of increased F:T activity under anaerobic conditions, and key findings were confirmed by microbiological tests, including nitrate utilization assays, growth curves, and susceptibility testing. Notably, growth in subinhibitory concentrations of F:T, but not tobramycin or fosfomycin alone, significantly downregulated (P < 0.05) nitrate reductase genes narG and narH, which are essential for normal anaerobic growth of P. aeruginosa. Under anaerobic conditions, F:T significantly decreased (P < 0.001) nitrate utilization in P. aeruginosa strains PAO1, PA14, and PA14 lasR::Gm, a mutant known to exhibit increased nitrate utilization. A similar effect was observed with two clinical P. aeruginosa isolates. Growth curves indicate that nitrate reductase transposon mutants had reduced growth under anaerobic conditions, with these mutants also having increased susceptibility to F:T compared to the wild type under similar conditions. The results of this study suggest that downregulation of nitrate reductase genes resulting in reduced nitrate utilization is the mechanism underlying the increased activity of F:T under anaerobic conditions.

Lung disease associated with chronic airway infection is the main determinant of longevity and morbidity in people with cystic fibrosis (CF) [1]. Staphylococcus aureus is the bacterium cultured most commonly from the respiratory tracts of children with CF [2], and the earliest descriptions of CF lung infections focused on this species [3]. Subsequently, Pseudomonas aeruginosa was increasingly isolated from children with CF, and studies established an association between P. aeruginosa and CF lung disease [4, 5], largely shifting the focus of CF microbiological research and therapy. Recently, increases in S. aureus prevalence, both methicillin susceptible and methicillin resistant (MRSA), have been described in CF [6]. Recent studies of children with CF noted similar inflammation and lung function decline during infection with S. aureus compared with P. aeruginosa [7–9]. These observations have led to a reexamination of S. aureus and its role in CF lung disease [10]. Phenotypic variants of S. aureus called small-colony variants (SCVs) emerge during many chronic infections [11], including in CF [12–17]. Staphylococcus aureus SCVs grow slowly on most laboratory media due to metabolic defects, which also confer resistance to many antibiotics [11]. Staphylococcus aureus SCVs can be selected in vitro either by long-term exposure to specific antibiotics [11, 18] or growth with P. aeruginosa [19, 20], both of which occur commonly in CF airways. However, these various conditions select for phenotypically different SCVs, with distinct metabolic defects and antibiotic resistance profiles. As a result of their slow growth, S. aureus SCVs are difficult to detect unless specifically looked for. Currently, most clinical laboratories do not use culture methods required to detect SCVs or to estimate their prevalence and clinical impact. Recent studies of adults and children with CF in Europe [12–16] reported S. aureus SCV prevalences of between 8% [16] and 33% [13] and unadjusted associations with lower lung function [14, 15], but for several reasons those results cannot necessarily be extrapolated to other CF populations. For example, US and European CF centers differ substantially both in antibiotic treatment and prophylaxis practices [1, 2, 21, 22] and in rates of P. aeruginosa culture positivity [2, 6, 14, 22], 2 factors predicted to influence S. aureus SCV prevalence and antibiotic resistances. Children with CF also differ from adults in those 2 factors. We hypothesized that S. aureus SCVs would be common in US pediatric CF patients, but with different predicted antibiotic resistances from those found in previous (European) studies, warranting routine surveillance. To test these hypotheses, we performed a 2-year study of our hospital's CF population using specialized culture methods and detailed logs of antibiotic use. We focused on children due to their relatively mild disease, high rates of S. aureus, relatively little antibiotic exposure, and less frequent P. aeruginosa infection compared with adults, strengthening our ability to determine associations with specific selection pressures and disease severity.

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing ‘core’ and ‘satellite’ taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.

While considerable research has focused on the properties of individual bacteria, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. Staphylococcus aureus frequently coinfects with other pathogens in a range of different infectious diseases. For example, coinfection by S. aureus with Pseudomonas aeruginosa occurs commonly in people with cystic fibrosis and is associated with higher lung disease morbidity and mortality. S. aureus secretes numerous exoproducts that are known to interact with host tissues, influencing inflammatory responses. The abundantly secreted S. aureus staphylococcal protein A (SpA) binds a range of human glycoproteins, immunoglobulins, and other molecules, with diverse effects on the host, including inhibition of phagocytosis of S. aureus cells. However, the potential effects of SpA and other S. aureus exoproducts on coinfecting bacteria have not been explored. Here, we show that S. aureus-secreted products, including SpA, significantly alter two behaviors associated with persistent infection. We found that SpA inhibited biofilm formation by specific P. aeruginosa clinical isolates, and it also inhibited phagocytosis by neutrophils of all isolates tested. Our results indicate that these effects were mediated by binding to at least two P. aeruginosa cell surface structures—type IV pili and the exopolysaccharide Psl—that confer attachment to surfaces and to other bacterial cells. Thus, we found that the role of a well-studied S. aureus exoproduct, SpA, extends well beyond interactions with the host immune system. Secreted SpA alters multiple persistence-associated behaviors of another common microbial community member, likely influencing cocolonization and coinfection with other microbes., IMPORTANCE Bacteria rarely exist in isolation, whether on human tissues or in the environment, and they frequently coinfect with other microbes. However, relatively little is known about how microbial interspecies interactions alter bacterial behaviors and pathogenesis. We identified a novel interaction between two bacterial species that frequently infect together—Staphylococcus aureus and Pseudomonas aeruginosa. We show that the S. aureus-secreted protein staphylococcal protein A (SpA), which is well-known for interacting with host targets, also binds to specific P. aeruginosa cell surface molecules and alters two persistence-associated P. aeruginosa behaviors: biofilm formation and uptake by host immune cells. Because S. aureus frequently precedes P. aeruginosa in chronic infections, these findings reveal how microbial community interactions can impact persistence and host interactions during coinfections.

Background Culture-independent analysis of the respiratory secretions of people with cystic fibrosis (CF) has identified many bacterial species not previously detected using culture in this context. However, little is known about their clinical significance or persistence in CF airways. Methods The authors characterised the viable bacterial communities in the sputum collected from 14 patients at monthly intervals over 1 year using a molecular community profiling technique—terminal restriction fragment length polymorphism. Clinical characteristics were also collected, including lung function and medications. Ecological community measures were determined for each sample. Microbial community change over time within subjects was defined using ecological analytical tools, and these measures were compared between subjects and to clinical features. Results Bacterial communities were stable within subjects over time but varied between subjects, despite similarities in clinical course. Antibiotic therapy temporarily perturbed these communities which generally returned to pretreatment configurations within 1 month. Species usually considered CF pathogens and those not previously regarded as such exhibited similar patterns of persistence. Less diverse sputum bacterial communities were correlated to lung disease severity and relative abundance of Pseudomonas aeruginosa . Conclusion Whilst not true in all cases, the microbial communities that chronically infect the airways of patients with CF can vary little over a year despite antibiotic perturbation. The species present tended to vary more between than within subjects, suggesting that each CF airway infection is unique, with relatively stable and resilient bacterial communities. The inverse relationship between community richness and disease severity is similar to findings reported in other mucosal infections.

Cystic fibrosis–adapted Pseudomonas aeruginosa lasR quorum sensing mutants cause hyperinflammation contributing to chronic lung disease., Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease–dependent cytokine degradation. In subacute pulmonary infections, lasR mutant–infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

The opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4-hydroxy-2-heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss-of-function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased beta-lactamase activity that increased tolerance to ceftazidime, a widely used beta-lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.

Cystic fibrosis lung disease is characterized by chronic airway infections with the opportunistic pathogen Pseudomonas aeruginosa and severe neutrophilic pulmonary inflammation. P. aeruginosa undergoes extensive genetic adaptation to the cystic fibrosis (CF) lung environment, and adaptive mutations in the quorum sensing regulator gene lasR commonly arise. We sought to define how mutations in lasR alter host-pathogen relationships. We demonstrate that lasR mutants induce exaggerated host inflammatory responses in respiratory epithelial cells, with increased accumulation of proinflammatory cytokines and neutrophil recruitment due to the loss of bacterial protease–dependent cytokine degradation. In subacute pulmonary infections, lasR mutant–infected mice show greater neutrophilic inflammation and immunopathology compared with wild-type infections. Finally, we observed that CF patients infected with lasR mutants have increased plasma interleukin-8 (IL-8), a marker of inflammation. These findings suggest that bacterial adaptive changes may worsen pulmonary inflammation and directly contribute to the pathogenesis and progression of chronic lung disease in CF patients.

Rationale: The airways of people with cystic fibrosis (CF) are chronically infected with a variety of bacterial species. Although routine culture methods are usually used to diagnose these infections, culture-independent, DNA-based methods have identified many bacterial species in CF respiratory secretions that are not routinely cultured. Many prior culture-independent studies focused either on microbiota in explanted CF lungs, reflecting end-stage disease, or those in oropharyngeal swabs, which likely sample areas in addition to the lower airways. Therefore, it was unknown whether the lower airways of children with CF, well before end-stage but with symptomatic lung disease, truly contained diverse microbiota. Objectives: To define the microbiota in the diseased lung tissue of a child who underwent lobectomy for severe, localized CF lung disease. Methods: After pathologic examination verified that this child’s lung tissue reflected CF lung disease, we used bacterial ribosomal RNA gene pyrosequencing and computational phylogenetic analysis to identify the microbiota in serial sections of the tissue. Measurements and Main Results: This analysis identified diverse, and anatomically heterogeneous, bacterial populations in the lung tissue that contained both culturable and nonculturable species, including abundant Haemophilus, Ralstonia, and Propionibacterium species. Routine clinical cultures identified only Staphylococcus aureus, which represented only a small fraction of the microbiota found by sequencing. Microbiota analysis of an intraoperative oropharyngeal swab identified predominantly Streptococcus species. The oropharyngeal findings therefore represented the lung tissue microbiota poorly, in agreement with findings from earlier studies of oropharyngeal swabs in end-stage disease. Conclusions: These results support the concept that diverse and spatially heterogeneous microbiota, not necessarily dominated by “traditional CF pathogens,” are present in the airways of young, symptomatic children with early CF lung disease.

Exposure to aminoglycoside antibiotics induces biofilm formation by a laboratory strain of the cystic fibrosis (CF) pathogen Pseudomonas aeruginosa . Here, we detected this effect among about half of the clinical isolates from CF patients in a cross-sectional collection, suggesting that biofilm induction may represent a common mechanism of inducible aminoglycoside resistance in CF infections. This induction always occurred at the same tobramycin concentration regardless of MIC, suggesting that the mechanisms of killing and induction may be separable.

Complex microbiota are being reported increasingly across a range of chronic infections, including those of the cystic fibrosis airways. Such diversity fits poorly into classical models of sterile tissue infections, which generally involve one species, and where microbe–outcome associations usually imply causality. It has been suggested that microbiota at sites of infection could represent pathogenic entities, analogous to individual species. We argue that our ability to identify causality in microbiota–disease associations is, however, inherently confounded. Although particular microbiota may be associated with clinical outcomes, niche characteristics at sites of infection will shape microbiota composition through exerting selective pressures. Here, we suggest that ecological theory can inform clinical understanding.

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues., Author Summary Chronic infections are distinguished from many other infections in that they are difficult to eradicate with antibiotics. Thus, the microbes that cause chronic infections persist within host tissues for long periods despite our best treatment efforts. During the course of these chronic infections, the causative microbes often change genetically. For example, a bacterium that commonly infects the lungs of people with the genetic disease cystic fibrosis (CF) undergoes several known changes that affect the growth of this pathogen. However, the causes and clinical impact of the changes undergone by this and other chronically infecting microbes are unclear. We show that a common, early mutation found in bacteria isolated from chronically infected CF airways renders these bacteria better able to grow in the nutrients found in CF lung secretions. Interestingly, these same changes also confer resistance to several antibiotics used commonly to treat CF patients. Many of the characteristics conferred by this mutation are exhibited by other microbes found in chronic infections, suggesting that adaptation of these microbes to host tissue nutrient environments may be a common mechanism of antibiotic resistance in chronic infections.

The opportunistic pathogen Pseudomonas aeruginosa undergoes genetic change during chronic airway infection of cystic fibrosis (CF) patients. One common change is a mutation inactivating lasR, which encodes a transcriptional regulator that responds to a homoserine lactone signal to activate expression of acute virulence factors. Colonies of lasR mutants visibly accumulated the iridescent intercellular signal 4-hydroxy-2-heptylquinoline. Using this colony phenotype, we identified P. aeruginosa lasR mutants that emerged in the airway of a CF patient early during chronic infection, and during growth in the laboratory on a rich medium. The lasR loss-of-function mutations in these strains conferred a growth advantage with particular carbon and nitrogen sources, including amino acids, in part due to increased expression of the catabolic pathway regulator CbrB. This growth phenotype could contribute to selection of lasR mutants both on rich medium and within the CF airway, supporting a key role for bacterial metabolic adaptation during chronic infection. Inactivation of lasR also resulted in increased beta-lactamase activity that increased tolerance to ceftazidime, a widely used beta-lactam antibiotic. Loss of LasR function may represent a marker of an early stage in chronic infection of the CF airway with clinical implications for antibiotic resistance and disease progression.

In many human infections, hosts and pathogens coexist for years or decades. Important examples include HIV, herpes viruses, tuberculosis, leprosy, and malaria. With the exception of intensively studied viral infections such as HIV/AIDs, little is known about the extent to which the clonal expansion that occurs during long-term infection by pathogens involves important genetic adaptations. We report here a detailed, whole-genome analysis of one such infection, that of a cystic fibrosis (CF) patient by the opportunistic bacterial pathogen Pseudomonas aeruginosa . The bacteria underwent numerous genetic adaptations during 8 years of infection, as evidenced by a positive-selection signal across the genome and an overwhelming signal in specific genes, several of which are mutated during the course of most CF infections. Of particular interest is our finding that virulence factors that are required for the initiation of acute infections are often selected against during chronic infections. It is apparent that the genotypes of the P. aeruginosa strains present in advanced CF infections differ systematically from those of “wild-type” P. aeruginosa and that these differences may offer new opportunities for treatment of this chronic disease.

BackgroundPseudomonas aeruginosa with mutations in the transcriptional regulator LasR chronically infect the airways of people with cystic fibrosis (CF), yet the prevalence and clinical implications of lasR mutant infection are unknown.MethodsIn an exploratory study, we screened 166 P. aeruginosa isolates from 58 CF patients for LasR inactivation and mucoidy, and compared clinical characteristics among source patients.ResultslasR mutation prevalence was comparable to that of mucoidy, the best-described CF-adapted phenotype, but affected patients were on average approximately 2 years younger. In a regression analysis, lung function decline with age was worse among patients with lasR mutant infection than in those without, similar to the effect of mucoidy.ConclusionsCulture positivity for lasR mutant P. aeruginosa may serve as a marker of early CF adaptive change of prognostic significance. Furthermore, as LasR inactivation alters susceptibility to antibiotics, infection with lasR mutant P. aeruginosa may impact response to therapy.

Background Cystic Fibrosis (CF) lung disease is characterised by an inexorable decline in lung function, punctuated by periods of symptomatic worsening known as pulmonary exacerbations (referred to here as CFPE). Despite their clinical significance, the cause of CFPE remains undetermined. It has been suggested that an increase in bacterial density may be a trigger, although this has not been shown empirically. Methods Here, a previously validated quantitative PCR-based approach was used to assess numbers of Pseudomonas aeruginosa and of total bacteria in respiratory secretions from patients during the period leading up to CFPE. Sputum samples collected from 12 adult CF patients were selected retrospectively to fall approximately 21, 14, 7 and 0days prior to CFPE diagnosis. In addition, the relationships between clinical parameters (FEV 1 , temperature and patient reported outcome measures) and microbiological data were investigated. Results No significant changes either in total bacterial or P . aeruginosa numbers were identified prior to CFPE. Of all the correlations tested, only temperature showed a significant correlation with total bacterial numbers in the period leading to CFPE. Conclusions These findings strongly suggest that CFPE do not generally result from increased bacterial density within the airways. Instead, data presented here are consistent with alternative models of pulmonary exacerbation.