Exploring the dynamics of mixed-species biofilms involving Candida spp. and bacteria in cystic fibrosis.

Cystic fibrosis (CF) is an inherited disease that results from mutations in the gene responsible for the cystic fibrosis transmembrane conductance regulator (CFTR). The airways become clogged with thick, viscous mucus that traps microbes in respiratory tracts, facilitating colonization, inflammation and infection. CF is recognized as a biofilm-associated disease, it is commonly polymicrobial and can develop in biofilms. This review discusses Candida spp. and both Gram-positive and Gram-negative bacterial biofilms that affect the airways and cause pulmonary infections in the CF context, with a particular focus on mixed-species biofilms. In addition, the review explores the intricate interactions between fungal and bacterial species within these biofilms and elucidates the underlying molecular mechanisms that govern their dynamics. Moreover, the review addresses the multifaceted issue of antimicrobial resistance in the context of CF-associated biofilms. By synthesizing current knowledge and research findings, this review aims to provide insights into the pathogenesis of CF-related infections and identify potential therapeutic approaches to manage and combat these complex biofilm-mediated infections.

Considerations for the use of inhaled antibiotics for Pseudomonas aeruginosa in people with cystic fibrosis receiving CFTR modulator therapy.

The major cause of mortality in people with cystic fibrosis (pwCF) is progressive lung disease characterised by acute and chronic infections, the accumulation of mucus, airway inflammation, structural damage and pulmonary exacerbations. The prevalence of Pseudomonas aeruginosa rises rapidly in the teenage years, and this organism is the most common cause of chronic lung infection in adults with cystic fibrosis (CF). It is associated with an accelerated decline in lung function and premature death. New P. aeruginosa infections are treated with antibiotics to eradicate the organism, while chronic infections require long-term inhaled antibiotic therapy. The prevalence of P. aeruginosa infections has decreased in CF registries since the introduction of CF transmembrane conductance regulator modulators (CFTRm), but clinical observations suggest that chronic P. aeruginosa infections usually persist in patients receiving CFTRm. This indicates that pwCF may still need inhaled antibiotics in the CFTRm era to maintain long-term control of P. aeruginosa infections. Here, we provide an overview of the changing perceptions of P. aeruginosa infection management, including considerations on detection and treatment, the therapy burden associated with inhaled antibiotics and the potential effects of CFTRm on the lung microbiome. We conclude that updated guidance is required on the diagnosis and management of P. aeruginosa infection. In particular, we highlight a need for prospective studies to evaluate the consequences of stopping inhaled antibiotic therapy in pwCF who have chronic P. aeruginosa infection and are receiving CFTRm. This will help inform new guidelines on the use of antibiotics alongside CFTRm.

Enzyme-Linked Lipid Nanocarriers for Coping Pseudomonal Pulmonary Infection. Would Nanocarriers Complement Biofilm Disruption or Pave Its Road?

Introduction: Cystic fibrosis (CF) is associated with pulmonary Pseudomonas aeruginosa infections persistent to antibiotics. Methods: To eradicate pseudomonal biofilms, solid lipid nanoparticles (SLNs) loaded with quorum-sensing-inhibitor (QSI, disrupting bacterial crosstalk), coated with chitosan (CS, improving internalization) and immobilized with alginate lyase (AL, destroying alginate biofilms) were developed. Results: SLNs (140-205 nm) showed prolonged release of QSI with no sign of acute toxicity to A549 and Calu-3 cells. The CS coating improved uptake, whereas immobilized-AL ensured >1.5-fold higher uptake and doubled SLN diffusion across the artificial biofilm sputum model. Respirable microparticles comprising SLNs in carbohydrate matrix elicited aerodynamic diameters MMAD (3.54, 2.48 µm) and fine-particle-fraction FPF (65, 48%) for anionic and cationic SLNs, respectively. The antimicrobial and/or antibiofilm activity of SLNs was explored in Pseudomonas aeruginosa reference mucoid/nonmucoid strains as well as clinical isolates. The full growth inhibition of planktonic bacteria was dependent on SLN type, concentration, growth medium, and strain. OD measurements and live/dead staining proved that anionic SLNs efficiently ceased biofilm formation and eradicated established biofilms, whereas cationic SLNs unexpectedly promoted biofilm progression. AL immobilization increased biofilm vulnerability; instead, CS coating increased biofilm formation confirmed by 3D-time lapse confocal imaging. Incubation of SLNs with mature biofilms of P. aeruginosa isolates increased biofilm density by an average of 1.5-fold. CLSM further confirmed the binding and uptake of the labeled SLNs in P. aeruginosa biofilms. Considerable uptake of CS-coated SLNs in non-mucoid strains could be observed presumably due to interaction of chitosan with LPS glycolipids in the outer cell membrane of P. aeruginosa. Conclusion: The biofilm-destructive potential of QSI/SLNs/AL inhalation is promising for site-specific biofilm-targeted interventional CF therapy. Nevertheless, the intrinsic/extrinsic fundamentals of nanocarrier-biofilm interactions require further investigation.

Bronchoscopy-guided antimicrobial therapy for cystic fibrosis.

BACKGROUND: Early diagnosis and treatment of lower respiratory tract infections is the mainstay of management of lung disease in cystic fibrosis (CF). When sputum samples are unavailable, diagnosis relies mainly on cultures from oropharyngeal specimens; however, there are concerns about whether this approach is sensitive enough to identify lower respiratory organisms. Bronchoscopy and related procedures such as bronchoalveolar lavage (BAL) are invasive but allow the collection of lower respiratory specimens from non-sputum producers. Cultures of bronchoscopic specimens provide a higher yield of organisms compared to those from oropharyngeal specimens. Regular use of bronchoscopy and related procedures may increase the accuracy of diagnosis of lower respiratory tract infections and improve the selection of antimicrobials, which may lead to clinical benefits. This is an update of a previous review that was first published in 2013 and was updated in 2016 and in 2018. OBJECTIVES: To evaluate the use of bronchoscopy-guided (also known as bronchoscopy-directed) antimicrobial therapy in the management of lung infection in adults and children with cystic fibrosis. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis Trials Register, compiled from electronic database searches and handsearching of journals and conference abstract books. We also searched three registries of ongoing studies and the reference lists of relevant articles and reviews. The date of the most recent searches was 1 November 2023. SELECTION CRITERIA: We included randomised controlled studies involving people of any age with CF that compared the outcomes of antimicrobial therapies guided by the results of bronchoscopy (and related procedures) versus those guided by any other type of sampling (e.g. cultures from sputum, throat swab and cough swab). DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies, assessed their risk of bias and extracted data. We contacted study investigators for further information when required. We assessed the certainty of the evidence using the GRADE criteria. MAIN RESULTS: We included two studies in this updated review. One study enrolled 170 infants under six months of age who had been diagnosed with CF through newborn screening. Participants were followed until they were five years old, and data were available for 157 children. The study compared outcomes for pulmonary exacerbations following treatment directed by BAL versus standard treatment based on clinical features and oropharyngeal cultures. The second study enrolled 30 children with CF aged between five and 18 years and randomised participants to receive treatment based on microbiological results of BAL triggered by an increase in lung clearance index (LCI) of at least one unit above baseline or to receive standard treatment based on microbiological results of oropharyngeal samples collected when participants were symptomatic. We judged both studies to have a low risk of bias across most domains, although the risk of bias for allocation concealment and selective reporting was unclear in the smaller study. In the larger study, the statistical power to detect a significant difference in the prevalence of Pseudomonas aeruginosa was low because Pseudomonas aeruginosa isolation in BAL samples at five years of age in both groups were much lower than the expected rate that was used for the power calculation. We graded the certainty of evidence for the key outcomes as low, other than for high-resolution computed tomography scoring and cost-of-care analysis, which we graded as moderate certainty. Both studies reported similar outcomes, but meta-analysis was not possible due to different ways of measuring the outcomes and different indications for the use of BAL. Whether antimicrobial therapy is directed by the use of BAL or standard care may make little or no difference in lung function z scores after two years (n = 29) as measured by the change from baseline in LCI and forced expiratory volume in one second (FEV1) (low-certainty evidence). At five years, the larger study found little or no difference between groups in absolute FEV1 z score or forced vital capacity (FVC) (low-certainty evidence). BAL-directed therapy probably makes little or no difference to any measure of chest scores assessed by computed tomography (CT) scan at either two or five years (different measures used in the two studies; moderate-certainty evidence). BAL-directed therapy may make little or no difference in nutritional parameters or in the number of positive isolates of P aeruginosa per participant per year, but may lead to more hospitalisations per year (1 study, 157 participants; low-certainty evidence). There is probably no difference in average cost of care per participant (either for hospitalisations or total costs) at five years between BAL-directed therapy and standard care (1 study, 157 participants; moderate-certainty evidence). We found no difference in health-related quality of life between BAL-directed therapy and standard care at either two or five years, and the larger study found no difference in the number of isolates of Pseudomonas aeruginosa per child per year. The eradication rate following one or two courses of eradication treatment and the number of pulmonary exacerbations were comparable in the two groups. Mild adverse events, when reported, were generally well tolerated. The most common adverse event reported was transient worsening of cough after 29% of procedures. Significant clinical deterioration was documented during or within 24 hours of BAL in 4.8% of procedures. AUTHORS' CONCLUSIONS: This review, limited to two well-designed randomised controlled studies, shows no evidence to support the routine use of BAL for the diagnosis and management of pulmonary infection in preschool children with CF compared to the standard practice of providing treatment based on results of oropharyngeal culture and clinical symptoms. No evidence is available for adults.

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 antimicrobial recalcitrance 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.

Persistence and evolution of Pseudomonas aeruginosa following initiation of highly effective modulator therapy in cystic fibrosis.

Today, more than 90% of people with cystic fibrosis (pwCF) are eligible for the highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy called elexacaftor/tezacaftor/ivacaftor (ETI) and its use is widespread. Given the drastic respiratory symptom improvement experienced by many post-ETI, clinical studies are already underway to reduce the number of respiratory therapies, including antibiotic regimens, that pwCF historically relied on to combat lung disease progression. Early studies suggest that bacterial burden in the lungs is reduced post-ETI, yet it is unknown how chronic Pseudomonas aeruginosa populations are impacted by ETI. We found that pwCF remain infected throughout their upper and lower respiratory tract with their same strain of P. aeruginosa post-ETI, and these strains continue to evolve in response to the newly CFTR-corrected airway. Our work underscores the continued importance of CF airway microbiology in the new era of highly effective CFTR modulator therapy. IMPORTANCE: The highly effective cystic fibrosis transmembrane conductance regulator modulator therapy Elexakaftor/Tezacaftor/Ivacaftor (ETI) has changed cystic fibrosis (CF) disease for many people with cystic fibrosis. While respiratory symptoms are improved by ETI, we found that people with CF remain infected with Pseudomonas aeruginosa. How these persistent and evolving bacterial populations will impact the clinical manifestations of CF in the coming years remains to be seen, but the role and potentially changing face of infection in CF should not be discounted in the era of highly effective modulator therapy.

The changing epidemiology of pulmonary infection in children and adolescents with cystic fibrosis: an 18-year experience.

The impact of evolving treatment regimens, airway clearance strategies, and antibiotic combinations on the incidence and prevalence of respiratory infection in cystic fibrosis (CF) in children and adolescents remains unclear. The incidence, prevalence, and prescription trends from 2002 to 2019 with 18,339 airway samples were analysed. Staphylococcus aureus [- 3.86% (95% CI - 5.28-2.43)] showed the largest annual decline in incidence, followed by Haemophilus influenzae [- 3.46% (95% CI - 4.95-1.96)] and Pseudomonas aeruginosa [- 2.80%95% CI (- 4.26-1.34)]. Non-tuberculous mycobacteria and Burkholderia cepacia showed a non-significant increase in incidence. A similar pattern of change in prevalence was observed. No change in trend was observed in infants < 2 years of age. The mean age of the first isolation of S. aureus (p < 0.001), P. aeruginosa (p < 0.001), H. influenza (p < 0.001), Serratia marcescens (p = 0.006) and Aspergillus fumigatus (p = 0.02) have increased. Nebulised amikacin (+ 3.09 ± 2.24 prescription/year, p = 0.003) and colistin (+ 1.95 ± 0.3 prescriptions/year, p = 0.032) were increasingly prescribed, while tobramycin (- 8.46 ± 4.7 prescriptions/year, p < 0.001) showed a decrease in prescription. Dornase alfa and hypertonic saline nebulisation prescription increased by 16.74 ± 4.1 prescriptions/year and 24 ± 4.6 prescriptions/year (p < 0.001). There is a shift in CF among respiratory pathogens and prescriptions which reflects the evolution of cystic fibrosis treatment strategies over time.

A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

Ecological patterns and processes of temporal turnover within lung infection microbiota.

BACKGROUND: Chronic infection and consequent airway inflammation are the leading causes of morbidity and early mortality for people living with cystic fibrosis (CF). However, lower airway infections across a range of chronic respiratory diseases, including in CF, do not follow classical 'one microbe, one disease' concepts of infection pathogenesis. Instead, they are comprised of diverse and temporally dynamic lung infection microbiota. Consequently, temporal dynamics need to be considered when attempting to associate lung microbiota with changes in disease status. Set within an island biogeography framework, we aimed to determine the ecological patterns and processes of temporal turnover within the lung microbiota of 30 paediatric and adult CF patients prospectively sampled over a 3-year period. Moreover, we aimed to ascertain the contributions of constituent chronic and intermittent colonizers on turnover within the wider microbiota. RESULTS: The lung microbiota within individual patients was partitioned into constituent chronic and intermittent colonizing groups using the Leeds criteria and visualised with persistence-abundance relationships. This revealed bacteria chronically infecting a patient were both persistent and common through time, whereas intermittently infecting taxa were infrequent and rare; respectively representing the resident and transient portions of the wider microbiota. It also indicated that the extent of chronic colonization was far greater than could be appreciated with microbiological culture alone. Using species-time relationships to measure temporal turnover and Vellend's rationalized ecological processes demonstrated turnover in the resident chronic infecting groups was conserved and underpinned principally by the deterministic process of homogenizing dispersal. Conversely, intermittent colonizing groups, representing newly arrived immigrants and transient species, drove turnover in the wider microbiota and were predominately underpinned by the stochastic process of drift. For adult patients, homogenizing dispersal and drift were found to be significantly associated with lung function. Where a greater frequency of homogenizing dispersal was observed with worsening lung function and conversely drift increased with better lung function. CONCLUSIONS: Our work provides a novel ecological framework for understanding the temporal dynamics of polymicrobial infection in CF that has translational potential to guide and improve therapeutic targeting of lung microbiota in CF and across a range of chronic airway diseases. Video Abstract.

Airway commensal bacteria in cystic fibrosis inhibit the growth of P. aeruginosa via a released metabolite.

In cystic fibrosis (CF), Pseudomonas aeruginosa infection plays a critical role in disease progression. Although multiple studies suggest that airway commensals might be able to interfere with pathogenic bacteria, the role of the distinct commensals in the polymicrobial lung infections is largely unknown. In this study, we aimed to identify airway commensal bacteria that may inhibit the growth of P. aeruginosa. Through a screening study with more than 80 CF commensal strains across 21 species, more than 30 commensal strains from various species have been identified to be able to inhibit the growth of P. aeruginosa. The underlying mechanisms were investigated via genomic, metabolic and functional analysis, revealing that the inhibitory commensals may affect the growth of P. aeruginosa by releasing a large amount of acetic acid. The data provide information about the distinct roles of airway commensals and provide insights into novel strategies for controlling airway infections.

Challenges and mitigation strategies associated with Burkholderia cepacia complex contamination in pharmaceutical manufacturing.

Burkholderia cepacia complex (BCC) is a Gram-negative, non-spore-forming bacterium with more than 20 opportunistic pathogenic species, most commonly found in soil and water. Due to their rapid mutation rates, these organisms are adaptable and possess high genomic plasticity. BCC can cause life-threatening infections in immunocompromised individuals, such as those with cystic fibrosis, chronic granulomatous disease, and neonates. BCC contamination is a significant concern in pharmaceutical manufacturing, frequently causing non-sterile product recalls. BCC has been found in purified water, cosmetics, household items, and even ultrasound gel used in veterinary practices. Pharmaceuticals, personal care products, and cleaning solutions have been implicated in numerous outbreaks worldwide, highlighting the risks associated with intrinsic manufacturing site contamination. Regulatory compliance, product safety, and human health protection depend on testing for BCC in pharmaceutical manufacturing. Identification challenges exist, with BCC often misidentified as other bacteria like non-lactose fermenting Escherichia coli or Pseudomonas spp., particularly in developing countries where reporting BCC in pharmaceuticals remains limited. This review comprehensively aims to address the organisms causing BCC contamination, genetic diversity, identification challenges, regulatory requirements, and mitigation strategies. Recommendations are proposed to aid pharmaceutical chemists in managing BCC-associated risks and implementing prevention strategies within manufacturing processes.

Antibacterial peptide Reg4 ameliorates Pseudomonas aeruginosa-induced pulmonary inflammation and fibrosis.

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative facultative anaerobe that has become an important cause of severe infections in humans, particularly in patients with cystic fibrosis. The development of efficacious methods or mendicants against P. aeruginosa is still needed. We previously reported that regenerating islet-derived family member 4 (Reg4) has bactericidal activity against Salmonella Typhimurium, a Gram-negative flagellated bacterium. We herein explore whether Reg4 has bactericidal activity against P. aeruginosa. In the P. aeruginosa PAO1-chronic infection model, Reg4 significantly inhibits the colonization of PAO1 in the lung and subsequently ameliorates pulmonary inflammation and fibrosis. Reg4 recombinant protein suppresses the growth motility and biofilm formation capability of PAO1 in vitro. Mechanistically, Reg4 not only exerts bactericidal action via direct binding to the P. aeruginosa cell wall but also enhances the phagocytosis of alveolar macrophages in the host. Taken together, our study demonstrates that Reg4 may provide protection against P. aeruginosa-induced pulmonary inflammation and fibrosis via its antibacterial activity.IMPORTANCEChronic lung infection with Pseudomonas aeruginosa is a leading cause of morbidity and mortality in patients with cystic fibrosis. Due to the antibiotic resistance of Pseudomonas aeruginosa, antimicrobial peptides appear to be a potential alternative to combat its infection. In this study, we report an antimicrobial peptide, regenerating islet-derived 4 (Reg4), that showed killing activity against clinical strains of Pseudomonas aeruginosa PAO1 and ameliorated PAO1-induced pulmonary inflammation and fibrosis. Experimental data also showed Reg4 directly bound to the bacterial cell membrane and enhanced the phagocytosis of host alveolar macrophages. Our presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

The longitudinal microbial and metabolic landscape of infant cystic fibrosis: the gut-lung axis.

BACKGROUND AND AIM: In cystic fibrosis, gastrointestinal dysfunction and lower airway infection occur early and are independently associated with poorer outcomes in childhood. This study aimed to define the relationship between the microbiota at each niche during the first 2 years of life, its association with growth and airway inflammation, and explanatory features in the metabolome. MATERIALS AND METHODS: 67 bronchoalveolar lavage fluid (BALF), 62 plasma and 105 stool samples were collected from 39 infants with cystic fibrosis between 0 and 24 months who were treated with prophylactic antibiotics. 16S rRNA amplicon and shotgun metagenomic sequencing were performed on BALF and stool samples, respectively; metabolomic analyses were performed on all sample types. Sequencing data from healthy age-matched infants were used as controls. RESULTS: Bacterial diversity increased over the first 2 years in both BALF and stool, and microbial maturation was delayed in comparison to healthy controls from the RESONANCE cohort. Correlations between their respective abundance in both sites suggest stool may serve as a noninvasive alternative for detecting BALF Pseudomonas and Veillonella. Multisite metabolomic analyses revealed age- and growth-related changes, associations with neutrophilic airway inflammation, and a set of core systemic metabolites. BALF Pseudomonas abundance was correlated with altered stool microbiome composition and systemic metabolite alterations, highlighting a complex gut-plasma-lung interplay and new targets with therapeutic potential. CONCLUSION: Exploration of the gut-lung microbiome and metabolome reveals diverse multisite interactions in cystic fibrosis that emerge in early life. Gut-lung metabolomic links with airway inflammation and Pseudomonas abundance warrant further investigation for clinical utility, particularly in non-expectorating patients.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

MEK1/2 inhibition decreases pro-inflammatory responses in macrophages from people with cystic fibrosis and mitigates severity of illness in experimental murine methicillin-resistant Staphylococcus aureus infection.

Chronic pulmonary bacterial infections and associated inflammation remain a cause of morbidity and mortality in people with cystic fibrosis (PwCF) despite new modulator therapies. Therapies targeting host factors that dampen detrimental inflammation without suppressing immune responses critical for controlling infections remain limited, while the development of lung infections caused by antimicrobial resistant bacteria is an increasing global problem, and a significant challenge in CF. Pharmacological compounds targeting the mammalian MAPK proteins MEK1 and MEK2, referred to as MEK1/2 inhibitor compounds, have potential combined anti-microbial and anti-inflammatory effects. Here we examined the immunomodulatory properties of MEK1/2 inhibitor compounds PD0325901, trametinib, and CI-1040 on CF innate immune cells. Human CF macrophage and neutrophil phagocytic functions were assessed by quantifying phagocytosis of serum opsonized pHrodo red E. coli, Staphylococcus aureus, and zymosan bioparticles. MEK1/2 inhibitor compounds reduced CF macrophage pro-inflammatory cytokine production without impairing CF macrophage or neutrophil phagocytic abilities. Wild-type C57BL6/J and Cftr tm1kth (F508del homozygous) mice were used to evaluate the in vivo therapeutic potential of PD0325901 compared to vehicle treatment in an intranasal methicillin-resistant Staphylococcus aureus (MRSA) infection with the community-acquired MRSA strain USA300. In both wild-type and CF mice, PD0325901 reduced inflammation associated body mass loss. Wild-type mice treated with PD0325901 had significant reduction in neutrophil-mediated inflammation compared to vehicle treatment groups, with preserved clearance of bacteria in lung, liver, or spleen 1 day after infection in either wild-type or CF mouse models. In summary, this study provides the first data evaluating the therapeutic potential of MEK1/2 inhibitor to modulate CF immune cells and demonstrates that MEK1/2 inhibitors diminish pro-inflammatory responses without impairing host defense mechanisms required for acute pathogen clearance.

Genomic editing in Burkholderia multivorans by CRISPR/Cas9.

Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.

CFTR is required for zinc-mediated antibacterial defense in human macrophages.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.

Cystic Fibrosis Mice Are Highly Susceptible to Repeated Acute Pseudomonas aeruginosa Pneumonia after Intranasal Inoculation.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that controls chloride current. A number of different CFTR transgenic mouse lines have been developed and subjected to both acute and chronic infection models. However, prior studies showed no substantial differences in bacterial clearance between CF and non-CF mice after single inoculations. Here, using F508del transgenic CF mice, we examined the role of repeated acute Pseudomonas aeruginosa (PA) infection, with the second inoculation 7 days after the first. We found that CF mice were more susceptible to PA infection than non-CF mice following the second inoculation, with non-CF mice showing better neutrophil recruitment and effector functions. We further investigated the characteristics of lung immune cells using single-cell RNA sequencing, finding that non-CF lung neutrophils had more prominent upregulation of adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) compared to CF lung neutrophils. Although people with CF are often colonized with bacteria and have high numbers of neutrophils in the airways during chronic infection, these data suggest that CF neutrophils have deficient effector functions in the setting of repeated acute infection.