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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., (© 2021. The Author(s).)

Pseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1β by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1β expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration., Competing Interests: CA, CL, JP, Jd, AX, KZ, BT, LH, FJ, CH, GW, MP No competing interests declared, (© 2019, Armbruster et al.)

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., (Copyright © 2016 Feltner et al.)

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility. Despite an impaired fitness in rich media, melanogenic mutants can inhibit their wild-type parents and compete with them in coculture. Surprisingly, melanogenic mutants became highly resistant to two intraspecific toxins, the S-pyocins AP41 and S1. Our results suggest that pyocins produced within a population of infecting P. aeruginosa may have selected for bacterial mutants that underwent massive gene losses and that were adapted to the life in diverse bacterial communities in the human host. Intraspecific interactions may therefore be an important factor driving the continuing evolution of pathogens during host infections., Competing Interests: Conflict of interests The authors have declared that no competing interests exist., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Unlabelled: 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., (Copyright © 2016 Armbruster et al.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Rationale: 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., Objectives: To characterize the relationships between in vitro P. aeruginosa phenotypes, infection stage, and clinical outcomes., Methods: 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., Measurements and Main Results: 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)., Conclusions: 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.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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 0 days 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., (Copyright © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

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.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) regulates cellular motility and the synthesis of organelles and molecules that promote adhesion to a variety of biological and nonbiological surfaces. These properties likely require tight spatial and temporal regulation of c-di-GMP concentration. We have developed genetically encoded fluorescence resonance energy transfer (FRET)-based biosensors to monitor c-di-GMP concentrations within single bacterial cells by microscopy. Fluctuations of c-di-GMP were visualized in diverse Gram-negative bacterial species and observed to be cell cycle dependent. Asymmetrical distribution of c-di-GMP in the progeny correlated with the time of cell division and polarization for Caulobacter crescentus and Pseudomonas aeruginosa. Thus, asymmetrical distribution of c-di-GMP was observed as part of cell division, which may indicate an important regulatory step in extracellular organelle biosynthesis or function.

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.

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.

Opportunistic infections are often polymicrobial. Two of the most important bacterial opportunistic pathogens of humans, Pseudomonas aeruginosa and Staphylococcus aureus, frequently are coisolated from infections of catheters, endotracheal tubes, skin, eyes, and the respiratory tract, including the airways of people with cystic fibrosis (CF). Here, we show that suppression of S. aureus respiration by a P. aeruginosa exoproduct, 4-hydroxy-2-heptylquinoline-N-oxide (HQNO), protects S. aureus during coculture from killing by commonly used aminoglycoside antibiotics such as tobramycin. Furthermore, prolonged growth of S. aureus with either P. aeruginosa or with physiological concentrations of pure HQNO selects for typical S. aureus small-colony variants (SCVs), well known for stable aminoglycoside resistance and persistence in chronic infections, including those found in CF. We detected HQNO in the sputum of CF patients infected with P. aeruginosa, but not in uninfected patients, suggesting that this HQNO-mediated interspecies interaction occurs in CF airways. Thus, in all coinfections with P. aeruginosa, S. aureus may be underappreciated as a pathogen because of the formation of antibiotic-resistant and difficult to detect small-colony variants. Interspecies microbial interactions, analogous to those mediated by HQNO, commonly may alter not only the course of disease and the response to therapy, but also the population structure of bacterial communities that promote the health of host animals, plants, and ecosystems.

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.

ABSTRACTPseudomonas aeruginosa, a bacterium causing infections in immunocompromised individuals, regulates several of its virulence functions using three interlinked quorum sensing (QS) systems (las, rhl, and pqs). Despite its presumed importance in regulating virulence, dysfunction of the las system regulator LasR occurs frequently in strains isolated from various environments, including clinical infections. This newfound abundance of LasR-defective strains calls into question existing hypotheses regarding their selection. Indeed, current assumptions concerning factors driving the emergence of LasR-deficient isolates and the role of LasR in the QS hierarchy must be reconsidered. Here, we propose that LasR is not the primary master regulator of QS in all P. aeruginosa genetic backgrounds, even though it remains ecologically significant. We also revisit and complement current knowledge on the ecology of LasR-dependent QS in P. aeruginosa, discuss the hypotheses explaining the putative adaptive benefits of selecting against LasR function, and consider the implications of this renewed understanding.

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition between planktonic and biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. Current thinking in the field is that once cells attach to a surface, they uniformly respond by producing c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.

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 < 0.01). The amount of Psl antibody binding in sputum was also greater overall (p < 0.05) in samples with increased 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. [ABSTRACT FROM AUTHOR]

Rationale: Antibiotic selection for pulmonary exacerbation (PEx) management in children with cystic fibrosis is typically guided by prior respiratory culture results. Although antipseudomonal antibiotics are often used in children with chronic Pseudomonas aeruginosa (Pa) airway infection, no data exist to guide antibiotic selection in children who are culture negative for Pa for ≥1 year. Objectives: To determine among children classified as 1, 2, or 3 years' Pa negative if PEx treatment with at least one oral and/or intravenous antipseudomonal antibiotic is associated with improved clinical outcomes compared with treatment with antibiotics not effective against Pa. Methods: A retrospective cohort study was conducted using the linked Cystic Fibrosis Foundation Patient Registry-Pediatric Health Information System database. We included children 6-21 years old hospitalized between 2008 and 2018 consistently culture negative for Pa 1 year before a study PEx. Children were classified as 1 or 2 years' Pa negative if their last Pa-positive culture occurred in the 13-24 months or 25-36 months before a study PEx, respectively, with all subsequent cultures negative for Pa. Children classified as 3 years' Pa negative had no Pa-positive cultures in the 36 months before a study PEx. Inverse probability of treatment weighted linear or logistic regression models were used to compare clinical outcomes (pre- to post-PEx forced expiratory volume in 1 s, odds of returning to ≥90% of baseline lung function, and odds of having a future PEx) between antipseudomonal and non-antipseudomonal antibiotic strategies. Results: Among all children included in the linked data set, 1,290 children with 2,347 PExs were eligible for analysis. Among all study PExs, 530, 326, and 1,491 were classified as 1, 2, and 3 years' Pa negative, respectively, and antipseudomonal antibiotics were administered in 79%, 67%, and 66% of all PExs classified as 1, 2, and 3 years' Pa negative, respectively. For all Pa-negative groups, when compared with non-antipseudomonal antibiotic regimens, antipseudomonal antibiotic treatment was not associated with greater improvement in any studied clinical outcome. Conclusions: Despite their common use, including antibiotics effective against Pa may provide no additional benefit for PEx treatment among children who are Pa negative for at least 1 year prior. Prospective trials are warranted to directly test this hypothesis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We sought to investigate the prevalence of potentially pathogenic bacteria in secretions and tonsillar tissues of children with chronic adenotonsillitis hypertrophy compared to controls. Prospective case-control study comparing patients between 2 and 12 years old who underwent adenotonsillectomy due to chronic adenotonsillar hypertrophy to children without disease. We compared detection of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Moraxella catarrhalis by real-time PCR in palatine tonsils, adenoids, and nasopharyngeal washes obtained from 37 children with and 14 without adenotonsillar hypertrophy. We found high frequency (>50%) of Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa in both groups of patients. Although different sampling sites can be infected with more than one bacterium and some bacteria can be detected in different tissues in the same patient, adenoids, palatine tonsils, and nasopharyngeal washes were not uniformly infected by the same bacteria. Adenoids and palatine tonsils of patients with severe adenotonsillar hypertrophy had higher rates of bacterial coinfection. There was good correlation of detection of Moraxella catarrhalis in different sampling sites in patients with more severe tonsillar hypertrophy, suggesting that Moraxella catarrhalis may be associated with the development of more severe hypertrophy, that inflammatory conditions favor colonization by this agent. Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis are frequently detected in palatine tonsils, adenoids, and nasopharyngeal washes in children. Simultaneous detection of Moraxella catarrhalis in adenoids, palatine tonsils, and nasopharyngeal washes was correlated with more severe tonsillar hypertrophy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Background: Pseudomonas 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. Methods: In 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. Results: lasR 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. Conclusions: Culture 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. [Copyright &y& Elsevier]

Biofilms are adherent aggregates of bacterial cells that form on biotic and abiotic surfaces, including human tissues. Biofilms resist antibiotic treatment and contribute to bacterial persistence in chronic infections. Hence, the elucidation of the mechanisms by which biofilms are formed may assist in the treatment of chronic infections, such as Pseudomonas aeruginosa in the airways of patients with cystic fibrosis. Here we show that subinhibitory concentrations of aminoglycoside antibiotics induce biofilm formation in P. aeruginosa and Escherichia coli. In P. aeruginosa, a gene, which we designated aminoglycoside response regulator (arr), was essential for this induction and contributed to biofilm-specific aminoglycoside resistance. The arr gene is predicted to encode an inner-membrane phosphodiesterase whose substrate is cyclic di-guanosine monophosphate (c-di-GMP)—a bacterial second messenger that regulates cell surface adhesiveness. We found that membranes from arr mutants had diminished c-di-GMP phosphodiesterase activity, and P. aeruginosa cells with a mutation changing a predicted catalytic residue of Arr were defective in their biofilm response to tobramycin. Furthermore, tobramycin-inducible biofilm formation was inhibited by exogenous GTP, which is known to inhibit c-di-GMP phosphodiesterase activity. Our results demonstrate that biofilm formation can be a specific, defensive reaction to the presence of antibiotics, and indicate that the molecular basis of this response includes alterations in the level of c-di-GMP. [ABSTRACT FROM AUTHOR]