Cystic Fibrosis Lung Infections Research Articles

Pseudomonas aeruginosa Lipid A Structural Variants Induce Altered Immune Responses.

Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection, not seen in any other disease state. Lipid A, the membrane anchor of lipopolysaccharide (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent toll-like receptor (TLR)4 agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4, and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human bronchoalveolar lavage fluid. This structure triggers increased pro-inflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CFTR function. Interestingly, there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lack PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobialrecalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

A role for the stringent response in ciprofloxacin resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major cause of nosocomial infections and the leading cause of chronic lung infections in cystic fibrosis and chronic obstructive pulmonary disease patients. Antibiotic treatment remains challenging because P. aeruginosa is resistant to high concentrations of antibiotics and has a remarkable ability to acquire mutations conferring resistance to multiple groups of antimicrobial agents. Here we report that when P. aeruginosa is plated on ciprofloxacin (cipro) plates, the majority of cipro-resistant (ciproR) colonies observed at and after 48 h of incubation carry mutations in genes related to the Stringent Response (SR). Mutations in one of the major SR components, spoT, were present in approximately 40% of the ciproR isolates. Compared to the wild-type strain, most of these isolates had decreased growth rate, longer lag phase and altered intracellular ppGpp content. Also, 75% of all sequenced mutations were insertions and deletions, with short deletions being the most frequently occurring mutation type. We present evidence that most of the observed mutations are induced on the selective plates in a subpopulation of cells that are not instantly killed by cipro. Our results suggests that the SR may be an important contributor to antibiotic resistance acquisition in P. aeruginosa.

Characterization of a new Pseudomonas aeruginosa Queuovirinae bacteriophage.

The ESKAPEE pathogen Pseudomonas aeruginosa is a common cause of chronic wound and cystic fibrosis lung infections, as well as acute burn and nosocomial infections. Many of these infections are recalcitrant to conventional antibiotic therapies due to both traditional antibiotic resistance mechanisms and antimicrobial tolerance. Recent successes with bacteriophage (phage) therapy to treat chronic human P. aeruginosa infections have led to a renewed interest in isolating and characterizing new P. aeruginosa phages. Here, we isolated and characterized a new lytic phage (termed PIP, pili-infecting phage) capable of infecting P. aeruginosa PA14. PIP is a tailed phage with an icosahedral head and flexible tail containing a genome that is 57,462 bp in length. Phylogenetic analysis reveals that PIP belongs to the subfamily Queuovirinae and genus Nipunavirus but is highly divergent in gene content from known Nipunaviruses. By isolating and characterizing a P. aeruginosa strain that spontaneously evolved resistance to PIP, we show that the receptor for PIP is Type IV pili. In summary, we isolated a new P. aeruginosa phage species with a unique genome, thus increasing the diversity of phages known to infect this important human pathogen.IMPORTANCEThe opportunistic pathogen Pseudomonas aeruginosa causes both acute and chronic human infections. These infections are notoriously difficult to treat due to both antibiotic resistance and antibiotic tolerance. The increasing frequency of antibiotic failure in P. aeruginosa infections has led scientists to explore other treatment options, including bacteriophage (phage) therapy. To this end, there has been a significant effort to identify new Pseudomonas phages. Here, we isolated and characterized a bacteriophage (termed PIP, pili-infecting phage) that infects P. aeruginosa PA14. Examination of the PIP genome revealed that this phage represents a new species in the subclass Queuovirinae. The isolation and characterization of spontaneous PA14 mutants that are resistant to PIP infection revealed Type IV pili as the PIP receptor. Ultimately, this study characterizes a new species of Pseudomonas phage, thus enhancing the known diversity of phages that infect this important pathogen.

Apical dehydration impairs the cystic fibrosis airway epithelium barrier via a β1-integrin/YAP1 pathway.

Defective hydration of airway surface mucosa is associated with lung infection in cystic fibrosis (CF), partly caused by disruption of the epithelial barrier integrity. Although rehydration of the CF airway surface liquid (ASL) alleviates epithelium vulnerability to infection by junctional protein expression, the mechanisms linking ASL to barrier integrity are unknown. We show here the strong degradation of YAP1 and TAZ proteins in well-polarized CF human airway epithelial cells (HAECs), a process that was prevented by ASL rehydration. Conditional silencing of YAP1 in rehydrated CF HAECs indicated that YAP1 expression was necessary for the maintenance of junctional complexes. A higher plasma membrane tension in CF HAECs reduced endocytosis, concurrent with the maintenance of active β1-integrin ectopically located at the apical membrane. Pharmacological inhibition of β1-integrin accumulation restored YAP1 expression in CF HAECs. These results indicate that dehydration of the CF ASL affects epithelial plasma membrane tension, resulting in ectopic activation of a β1-integrin/YAP1 signaling pathway associated with degradation of junctional proteins.

Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness.

Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR- genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR- strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR- strains. Citrate uptake by LasR- strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR- strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR- strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities.IMPORTANCECross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.

Co-occurring microflora and mucin drive Pseudomonas aeruginosa diversification and pathoadaptation.

While several environmental factors contribute to the evolutionary diversification of the pathogenic bacterium Pseudomonas aeruginosa during cystic fibrosis lung infections, relatively little is known about the impact of the surrounding microbiota. By using in vitro experimental evolution, we show that the presence of Stenotrophomonas maltophilia, Staphylococcus aureus, or them both, prevent the evolution of loss of virulence, which repeatedly occurs in the absence of these species due to mutations in regulators of the Pseudomonas Quinolone Signal quorum sensing system, vqsM and pqsR. Moreover, the strength of the effect of co-occurring species is attenuated through changes in the physical environment by the addition of mucin, resulting in selection for phenotypes resembling those evolved in the absence of the co-occurring species. Together, our findings show that variation in mucosal environment and the surrounding polymicrobial environment can determine the evolutionary trajectory of P. aeruginosa, partly explaining its diversification and pathoadaptation from acute to chronic phenotype during cystic fibrosis lung infections.

The role of hypermutation and collateral sensitivity in antimicrobial resistance diversity of Pseudomonas aeruginosa populations in cystic fibrosis lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient) and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity, (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure, (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection.IMPORTANCEUpon infection in the cystic fibrosis (CF) lung, Pseudomonas aeruginosa rapidly acquires genetic mutations, especially in genes involved in antimicrobial resistance (AMR), often resulting in diverse, treatment-resistant populations. However, the role of bacterial population diversity within the context of chronic infection is still poorly understood. In this study, we found that hypermutator strains of P. aeruginosa in the CF lung undergoing treatment with tobramycin evolved increased sensitivity to tobramycin relative to non-hypermutators within the same population. This finding suggests that antimicrobial treatment may only exert weak selection pressure on P. aeruginosa populations in the CF lung. We further found no evidence for collateral sensitivity in these clinical populations, suggesting that collateral sensitivity may not be a robust, naturally occurring phenomenon for this microbe.

Development and characterization of a spray-dried inhalable ternary combination for the treatment of Pseudomonas aeruginosa biofilm infection in cystic fibrosis

Cystic fibrosis (CF) is an inherited lung disease characterised by the accumulation of thick layers of dried mucus in the lungs which serve as a nidus for chronic infection. Pseudomonas aeruginosa is the predominant cause of chronic lung infection in cystic fibrosis. The dense mucus coupled with biofilm formation hinder antibiotic penetration and prevent them from reaching their target. Mucoactive agents are recommended in the treatment of CF in combination with antibiotics. In spite of the extensive research in developing novel drug combinations for the treatment of lung infection in CF, to our knowledge, there is no study that combines antibiotic, antibiofilm and mucoactive agent in a single inhaled dry powder formulation. In the present study, we investigate the possibility of adding a mucoactive agent to our previously developed ciprofloxacinquercetin (antibiotic-antibiofilm) dry powder for inhalation. Three mucoactive agents, namely mannitol (MAN), N-acetyl-L-cysteine (NAC) and ambroxol hydrochloride (AMB), were investigated for this purpose. The ternary combinations were prepared via spray drying without the addition of excipients. All ternary combinations conserved or improved the antibacterial and biofilm inhibition activities of ciprofloxacin against P. aeruginosa (ATCC 10145). The addition of AMB resulted in an amorphous ternary combination (SD-CQA) with superior physical stability as indicated by DSC and nonambient XRPD. Furthermore, SD-CQA displayed better in vitro aerosolization performance (ED ∼ 71 %; FPF ∼ 49 %) compared to formulations containing MAN and NAC (ED ∼ 64 % and 44 %; FPF ∼ 44 % and 29 %, respectively). In conclusion, a ternary drug combination powder with suitable aerosolization, physical stability and antibacterial/antibiofilm properties was prepared by a single spray drying step.

Ceragenins exhibit bactericidal properties that are independent of the ionic strength in the environment mimicking cystic fibrosis sputum.

The purpose of the work was to investigate the impact of sodium chloride (NaCl) on the antimicrobial efficacy of ceragenins (CSAs) and antimicrobial peptides (AMPs) against bacterial and fungal pathogens associated with cystic fibrosis (CF) lung infections. CF-associated bacterial (Pseudomonas aeruginosa, Ochrobactrum spp., and Staphylococcus aureus), and fungal pathogens (Candida albicans, and Candida tropicalis) were used as target organisms for ceragenins (CSA-13 and CSA-131) and AMPs (LL-37 and omiganan). Susceptibility to the tested compounds was assessed using minimal inhibitory concentrations (MICs) and bactericidal concentrations (MBCs), as well as by colony counting assays in CF sputum samples supplemented with various concentrations of NaCl. Our results demonstrated that ceragenins exhibit potent antimicrobial activity in CF sputum regardless of the NaCl concentration when compared to LL-37 and omiganan. Given the broad-spectrum antimicrobial activity of ceragenins in the microenvironments mimicking the airways of CF patients, ceragenins might be promising agents in managing CF disease.

Improving health literacy of antibiotic use in people with cystic fibrosis (CF)-comparison of the readability of patient information leaflets (PILs) from the EU, USA and UK of 23 CF-related antibiotics used in the treatment of CF respiratory infections.

Antibiotic adherence is poor amongst people with cystic fibrosis (CF). Low-quality patient information leaflets (PILs), which accompany prescription antibiotics, with poor readability may contribute to poor antibiotic adherence, with the potential for antimicrobial resistance (AMR) development. The aim of this study was to examine the readability of antibiotic PILs used to treat CF lung infections. CF-related antibiotics (n = 23; seven classes: aminoglycosides, β-lactams, fluoroquinolones, macrolides/lincosamides, oxazolidinones, tetracyclines, trimethoprim/sulfamethoxazole) were investigated. Readability of PILs (n = 141; 23 antibiotics) from the EU (n = 40), USA (n = 42) and UK (n = 59) was calculated. Mean [± standard error of mean (SEM)] values for the Flesch Reading Ease (FRE) for EU, USA and UK were 50.0 ± 1.1, 56.2 ± 1.3 and 51.7 ± 1.1, respectively (FRE target ≥60). Mean (± SEM) values for the Flesch Kinkaid Grade Level (FKGL) for the EU, USA and UK were 9.0 ± 0.2, 7.5 ± 0.2 and 9.6 ± 0.2, respectively (FKGL target ≤8). US PILs were significantly shorter (P < 0.0001) in words (mean ± SEM = 1365 ± 52), than either UK or EU PILs, with fewer sentences (P < 0.0001), fewer words per sentence (P < 0.0001) and fewer syllables per word. The mean ( ± SEM) reading time of UK PILs (n = 59) was 12.7 ± 0.55 mins . Readability of antibiotic PILs is poor. Improving PIL readability may lead to improved health literacy, which may translate to increased antibiotic adherence and AMR avoidance. Authors preparing written materials for the lay/patient CF community are encouraged to employ readability calculators, so that final materials are within recommended readability reference parameters, to support the health (antibiotic) literacy of their readers.

High prevalence of lipopolysaccharide mutants and R2-pyocin susceptible variants in Pseudomonas aeruginosa populations sourced from cystic fibrosis lung infections.

Chronic, highly antibiotic-resistant infections in cystic fibrosis (CF) lungs contribute to increasing morbidity and mortality. Pseudomonas aeruginosa, a common CF pathogen, exhibits resistance to multiple antibiotics, contributing to antimicrobial resistance (AMR). These bacterial populations display genetic and phenotypic diversity, but it is unclear how this diversity affects susceptibility to bacteriocins. R-pyocins, i.e., bacteriocins produced by P. aeruginosa, are phage-tail-like antimicrobials. R-pyocins have potential as antimicrobials, however, recent research suggests the diversity of P. aeruginosa variants within CF lung infections leads to varying susceptibility to R-pyocins. This variation may be linked to changes in lipopolysaccharide (LPS), acting as the R-pyocin receptor. Currently, it is unknown how frequently R-pyocin-susceptible strains are in chronic CF lung infection, particularly when considering the heterogeneity within these strains. In this study, we tested the R2-pyocin susceptibility of 139 P. aeruginosa variants from 17 sputum samples of 7 CF patients and analyzed LPS phenotypes. We found that 83% of sputum samples did not have R2-pyocin-resistant variants, while nearly all samples contained susceptible variants. There was no correlation between LPS phenotype and R2-pyocin susceptibility, though we estimate that about 76% of sputum-derived variants lack an O-specific antigen, 40% lack a common antigen, and 24% have altered LPS cores. The absence of a correlation between LPS phenotype and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Our research supports the potential of R-pyocins as therapeutic agents, as many infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations. IMPORTANCE Cystic fibrosis (CF) patients often experience chronic, debilitating lung infections caused by antibiotic-resistant Pseudomonas aeruginosa, contributing to antimicrobial resistance (AMR). The genetic and phenotypic diversity of P. aeruginosa populations in CF lungs raises questions about their susceptibility to non-traditional antimicrobials, like bacteriocins. In this study, we focused on R-pyocins, a type of bacteriocin with high potency and a narrow killing spectrum. Our findings indicate that a large number of infectious CF variants are susceptible to R2-pyocins, even within diverse bacterial populations, supporting their potential use as therapeutic agents. The absence of a clear correlation between lipopolysaccharide (LPS) phenotypes and R-pyocin susceptibility suggests that LPS packing density may play a significant role in R-pyocin susceptibility among CF variants. Understanding the relationship between LPS phenotypes and R-pyocin susceptibility is crucial for developing effective treatments for these chronic infections.

Pseudomonas aeruginosa Soluble Pyocins as Antibacterial Weapons.

Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections and associated with lung infections in cystic fibrosis (CF) patients (Lyczak et al., Microbes Infect 2:1051-1060, 2000). Multiple drug-resistant P. aeruginosa strains pose a serious problem because of antibiotic treatment failure. There is therefore a need for alternative anti-Pseudomonas molecules. Soluble pyocins (S-pyocins) are bacteriocins produced by P. aeruginosa strains that kill sensitive strains of the same species. These bacteriocins and their immunity gene are easily cloned and expressed in E. coli and their activity spectrum against different P. aeruginosa strains can be tested. In this chapter, we describe the procedures for cloning, expression, and sensitivity testing of two different S-pyocins. We also describe how to identify their receptor binding domain in sensitive strains, how to construct chimeric pyocins with extended activity spectra, and how to identify new pyocins in genomes by multiplex PCR.

223 Single cell–level pro-resolving actions of Resolvin D1 and D2 on Staphylococcus aureus lung infection and inflammation in cystic fibrosis

223 Single cell–level pro-resolving actions of Resolvin D1 and D2 on Staphylococcus aureus lung infection and inflammation in cystic fibrosis

26 A birth cohort approach to understanding cystic fibrosis lung infections

26 A birth cohort approach to understanding cystic fibrosis lung infections

Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.

Involvement of Acquired Tobramycin Resistance in the Shift to the Viable but Non-Culturable State in Pseudomonas aeruginosa.

Persistent and viable but non-culturable (VBNC) Pseudomonas aeruginosa cells are mainly responsible for the recurrence and non-responsiveness to antibiotics of cystic fibrosis (CF) lung infections. The sub-inhibitory antibiotic concentrations found in the CF lung in between successive therapeutic cycles can trigger the entry into the VBNC state, albeit with a strain-specific pattern. Here, we analyzed the VBNC cell induction in the biofilms of two CF P. aeruginosa isolates, exposed to starvation with/without antibiotics, and investigated the putative genetic determinants involved. Total viable bacterial cells were quantified by the validated ecfX-targeting qPCR protocol and the VBNC cells were estimated as the difference between qPCR and cultural counts. The isolates were both subjected to whole genome sequencing, with attention focused on their carriage of aminoglycoside resistance genes and on identifying mutated toxin-antitoxin and quorum sensing systems. The obtained results suggest the variable contribution of different antibiotic resistance mechanisms to VBNC cell abundance, identifying a major contribution from tobramycin efflux, mediated by MexXY efflux pump overexpression. The genome analysis evidenced putative mutation hotspots, which deserve further investigation. Therefore, drug efflux could represent a crucial mechanism through which the VBNC state is entered and a potential target for anti-persistence strategies.

Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung.

The genetic disease cystic fibrosis (CF) frequently leads to chronic lung infections by bacteria and fungi. We identified three individuals with CF with persistent lung infections dominated by Clavispora (Candida) lusitaniae. Whole-genome sequencing analysis of multiple isolates from each infection found evidence for selection for mutants in the gene MRS4 in all three distinct lung-associated populations. In each population, we found one or two unfixed, non-synonymous mutations in MRS4 relative to the reference allele found in multiple environmental and clinical isolates including the type strain. Genetic and phenotypic analyses found that all evolved alleles led to loss of function (LOF) of Mrs4, a mitochondrial iron transporter. RNA-seq analyses found that Mrs4 variants with decreased activity led to increased expression of genes involved in iron acquisition mechanisms in both low iron and replete iron conditions. Furthermore, surface iron reductase activity and intracellular iron were much higher in strains with Mrs4 LOF variants. Parallel studies found that a subpopulation of a CF-associated Exophiala dermatitidis infection also had a non-synonymous LOF mutation in MRS4. Together, these data suggest that MRS4 mutations may be beneficial during chronic CF lung infections in diverse fungi, perhaps, for the purposes of adaptation to an iron-restricted environment with chronic infections. IMPORTANCE The identification of MRS4 mutations in Clavispora (Candida) lusitaniae and Exophiala dermatitidis in individuals with cystic fibrosis (CF) highlights a possible adaptive mechanism for fungi during chronic CF lung infections. The findings of this study suggest that loss of function of the mitochondrial iron transporter Mrs4 can lead to increased activity of iron acquisition mechanisms, which may be advantageous for fungi in iron-restricted environments during chronic infections. This study provides valuable information for researchers working toward a better understanding of the pathogenesis of chronic lung infections and more effective therapies to treat them.

Preclinical testing of antimicrobials for cystic fibrosis lung infections: current needs and future priorities.

A workshop was held by the PIPE-CF strategic research centre to consider preclinical testing of antimicrobials for cystic fibrosis (CF). The workshop brought together groups of people from the CF community to discuss current challenges and identify priorities when developing CF therapeutics. This paper summarizes the key points from the workshop from the different sessions, including talks given by presenters on the day and round table discussions. Currently, it is felt that there is a large disconnect throughout the community, with communication between patients, clinicians and researchers being the main issue. This leads to little consideration being given to factors such as treatment regimes, routes of administration and side effects when developing new therapies, that could alter the day-to-day lifestyles of people living with CF. Translation of numerical data that are obtained in the laboratory to successful outcomes of clinical trials is also a key challenge facing researchers today. Laboratory assays in preclinical testing involve basing results on bacterial clearance and decrease in viable cells, when these are not factors that are considered when determining the success of a treatment in the clinic. However, there are several models currently in development that seek to tackle some of these issues, such as the organ-on-a-chip technology and adaptation of a hollow-fibre model, as well as the development of media that aim to mimic the niche environments of a CF respiratory tract. It is hoped that by summarizing these opinions and discussing current research, the communication gap between groups can begin to close.

Genomic and Functional Characterization of Longitudinal Pseudomonas aeruginosa Isolates from Young Patients with Cystic Fibrosis.

Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient's lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. IMPORTANCE Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.