Respiratory Infections In Cystic Fibrosis Research Articles

Improvement of a mouse infection model to capture Pseudomonas aeruginosa chronic physiology in cystic fibrosis

Laboratory models are central to microbiology research, advancing the understanding of bacterial physiology by mimicking natural environments, from soil to the human microbiome. When studying host-bacteria interactions, animal models enable investigators to examine bacterial dynamics associated with a host, and in the case of human infections, animal models are necessary to translate basic research into clinical treatments. Efforts toward improving animal infection models are typically based on reproducing host genotypes/phenotypes and disease manifestations, leaving a gap in how well the physiology of microbes reflects their behavior in a human host. Understanding bacterial physiology is vital because it dictates host response and bacterial interactions with antimicrobials. Thus, our goal was to develop an animal model that accurately recapitulates bacterial physiology in human infection. The system we chose to model was a chronic Pseudomonas aeruginosa respiratory infection in cystic fibrosis (CF). To accomplish this goal, we leveraged a framework that we recently developed to evaluate model accuracy by calculating the percentage of bacterial genes that are expressed similarly in a model to how they are expressed in their infection environment. We combined two complementary models of P. aeruginosa infection-an in vitro synthetic CF sputum model (SCFM2) and a mouse acute pneumonia model. This combined model captured the chronic physiology of P. aeruginosa in CF better than the standard mouse infection model, showing the power of a data-driven approach to refining animal models. In addition, the results of this work challenge the assumption that a chronic infection model requires long-term colonization.

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 < 2years 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.

Complex ecological approach to cystic fibrosis respiratory infections

Complex ecological approach to cystic fibrosis respiratory infections

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.

19 DNA repair mutations aid in Mycobacterium avium adaptation to antibiotic stress during chronic cystic fibrosis respiratory infection

19 DNA repair mutations aid in Mycobacterium avium adaptation to antibiotic stress during chronic cystic fibrosis respiratory infection

Ceftolozane-Tazobactam against Pseudomonas aeruginosa Cystic Fibrosis Clinical Isolates in the Hollow-Fiber Infection Model: Challenges Imposed by Hypermutability and Heteroresistance.

Pseudomonas aeruginosa remains a challenge in chronic respiratory infections in cystic fibrosis (CF). Ceftolozane-tazobactam has not yet been evaluated against multidrug-resistant hypermutable P. aeruginosa isolates in the hollow-fiber infection model (HFIM). Isolates CW41, CW35, and CW44 (ceftolozane-tazobactam MICs of 4, 4, and 2 mg/L, respectively) from adults with CF were exposed to simulated representative epithelial lining fluid pharmacokinetics of ceftolozane-tazobactam in the HFIM. Regimens were continuous infusion (CI; 4.5 g/day to 9 g/day, all isolates) and 1-h infusions (1.5 g every 8 hours and 3 g every 8 hours, CW41). Whole-genome sequencing and mechanism-based modeling were performed for CW41. CW41 (in four of five biological replicates) and CW44 harbored preexisting resistant subpopulations; CW35 did not. For replicates 1 to 4 of CW41 and CW44, 9 g/day CI decreased bacterial counts to <3 log10 CFU/mL for 24 to 48 h, followed by regrowth and resistance amplification. Replicate 5 of CW41 had no preexisting subpopulations and was suppressed below ~3 log10 CFU/mL for 120 h by 9 g/day CI, followed by resistant regrowth. Both CI regimens reduced CW35 bacterial counts to <1 log10 CFU/mL by 120 h without regrowth. These results corresponded with the presence or absence of preexisting resistant subpopulations and resistance-associated mutations at baseline. Mutations in ampC, algO, and mexY were identified following CW41 exposure to ceftolozane-tazobactam at 167 to 215 h. Mechanism-based modeling well described total and resistant bacterial counts. The findings highlight the impact of heteroresistance and baseline mutations on the effect of ceftolozane-tazobactam and limitations of MIC to predict bacterial outcomes. The resistance amplification in two of three isolates supports current guidelines that ceftolozane-tazobactam should be utilized together with another antibiotic against P. aeruginosa in CF.

151: PLANT PATHOGENS CAUSING PERICARDITIS

Introduction: Typical organisms associated with purulent pericarditis include numerous bacteria, such as Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Salmonella sp., and anaerobes, as well as various fungal organisms. We present an unusual case of pericarditis resulting from Burkholderia spp. Description: 79-year-old male with history of esophageal cancer (8 years ago, status post chemotherapy and radiation, currently in remission), esophageal stricture (status post stent placement), atrial fibrillation, arthritis, hypothyroidism, and anemia presented with fatigue, poor oral intake, lethargy, shortness of breath and productive cough. On arrival, he was hypotensive with focused cardiac ultrasound demonstrating a large circumferential pericardial effusion with right ventricular diastolic collapse and arterial line showing pulsus paradoxus on waveform analysis. White cell count was 11.62 k/mm3, hemoglobin 12.7 g/dl, potassium 7.1 mEq/L, bicarbonate 15.8 mEq/L, blood urea nitrogen 158 mg/dl, creatinine 7.79 mg/dl, brain natriuretic peptide 23,141 pg/ml. Computed tomography scan showed large pericardial effusion, bilateral pleural effusions and ascites which was confirmed on transthoracic echocardiogram (along with tamponade physiology). Empiric antibiotics, intravenous fluids and renal replacement therapy were started. Pericardiocentesis was performed (with removal of 200 ml of purulent fluid) and bilateral chest tubes were inserted. Pericardial fluid analysis showed 497,520 white cells/mm3; culture grew pansensitive Burkholderia gladioli/glumae/plantarii; fungal/tubercular cultures were negative, and cytology did not show any malignant cells. Course complicated by shock needing vasopressors and atrial fibrillation with rapid ventricular rate. Given worsening clinical condition, the patient opted for comfort care measures and succumbed to his illness shortly thereafter. Discussion: Burkholderia plantarii/glumae/gladioli are gram negative bacilli which are mainly pathogenic for plants. They have been shown to cause respiratory infections in cystic fibrosis and lung transplant patients. This patient with complex esophageal issues demonstrates an exceedingly rare case of Burkholderia-associated purulent pericarditis resulting from common rice pathogens.

S10.5a Fungal respiratory infections in cystic fibrosis patients in the Middle East

S10.5 Fungal respiratory infections in Cystic Fibrosis, September 24, 2022, 10:30 AM - 12:00 PMCystic Fibrosis (CF) is among the most common genetic disorders, which involves multiple organs including the respiratory tract. CF is thought to be an uncommon disease in the Middle East (ME). However, the prevalence is estimated at 1 in 30 000-50 000, while the incidence is estimated at 1 in 2000-5800 live births. Several studies from ME revealed that many children with CF in these populations probably remain undiagnosed due to lack of clinical suspicion and proper diagnostic facilities. According to the experts’ idea, CF may be more common in Iran than expected before. Chronic colonization of the airways of CF patients and infections due to a wide variety of opportunistic fungal pathogens including Aspergillus, Candida, Scedosporium species, Exophiala dermatitidis, Rasamsonia argillacea complex, and Lomentospora prolificans are currently increasing. On the other hand, the resistance of these opportunistic pathogens to commonly available antifungals challenges therapeutic options and consequently endanger the CF patients’ life. Dissimilar to bacterial colonization or infections, the epidemiology and pathogenicity of colonization and fungal respiratory infections in CF are less known. According to our recent study, the prevalence rate of respiratory colonization was reported as 73.3%. Among mold isolates, Aspergillus was also the most common genus followed by Scedosporium species. In contrast to the reports from western countries, A. flavus was also identified as the most prevalent species of Aspergillus from ME countries including Iran and India. In some studies, allergic bronchopulmonary aspergillosis (ABPA) in CF patients from ME was evaluated. In our recent experience from Iran, of 86 patients with CF, 9 (10.5%) cases were met ABPA diagnosis. A. flavus was the most common agent followed by A. fumigatus, A. terreus and A. tubingensis. A significant resistance of Scedosporium and Aspergillus isolates from CF patients against the main antifungal agents in invasive fungal infections therapy was reported in different studies. According to these realities, there are a few reports on the Research Topic ‘Fungal Respiratory Infections and Colonization in CF’ from Iran and some other ME countries. Therefore, in this presentation, we are going to highlight our experiences and other published data from Iran and ME in this field including clinical presentations, fungal species involved, diagnosis strategies, and in vitro antifungal susceptibility patterns of fungal isolates from CF patients, and common treatments and prophylactic strategies.

S10.5d Disseminated pulmonary infection due to Mortierella wolfii in a 6-year-old patient with X-linked CGD receiving MUD-HSCT

S10.5 Fungal respiratory infections in Cystic Fibrosis, September 24, 2022, 10:30 AM - 12:00 PM ObjectivesInvasive fungal infections represent one of the major limiting factors for the successful outcome of patients receiving hematopoietic stem cell transplantation (HSCT). The identification and successful treatment of pretransplant fungal colonization/infections may allow for risk modifications before or at the time of HSCT. Here, we report a case of disseminated pulmonary infection due to a hyaline non-septate mold, Mortierella wolfii in an X-linked chronic granulomatous disease (X-linked CGD) patient that was successfully treated with a combination of terbinafine and posaconazole antifungal therapy.MethodsA 6-year-old male with X-linked CGD from Sri Lanka was admitted to NIH Clinical Center, Bethesda, MD, USA to receive a matched unrelated donor (MUD) hematopoietic stem cell transplantation (HSCT). During pre-transplant immunosuppressive conditioning, the patient developed complicated pulmonary signs resulting in diffuse lymphadenitis and meningitis. Upon further radiologic evaluation, a lung biopsy was performed. The lung biopsy sample was submitted to Microbiology Service of Department of Microbiology at NIH Clinical Center for Fungal culture. Antifungal susceptibility testing was conducted in accordance with the Clinical and Laboratory Standards Institute CLSI M38-A3 guidelines.ResultsA pure heavy growth of white mold grew within 2 days on Sabouraud's Dextrose Agar. Microscopic examination showed hyaline (non-pigmented), non-septate branched hyphae. Sporangiophore-like structures were also present. The species-level identification of the isolate was confirmed as M. wolfii by PCR-sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA.Minimum inhibitory/effective concentrations (μg/ml) were as follows in increasing order: terbinafine = 0.25, amphotericin B = 1, isavuconazole = 4, micafungin >8, itraconazole >16, voriconazole >16, and posaconazole >16. To evaluate the interactions between antifungal drugs, the activity of the posaconazole in combination with terbinafine were also evaluated M. wolfii using agar diffusion test. A combination of posaconazole and terbinafine, significantly inhibited the mycelial growth, which indicates synergism. The patient’s treatment was started on terbinafine in combination with posaconazole. On several follow-up examinations following treatment on day 30, 90 and 120, the infection had not recurred.ConclusionThe species of M. wolfii a is an environmental mold belongs to the order Mortierellales within the subphylum Mortierellomycotina of Kingdom Fungi. This fungus has been mostly associated with fungal infections leading to abortion in dairy cows feeding moldy hays and ensilage.Although posaconazole exhibited high MICs against M. wolfii, our in vitro combination study demonstrated that posaconazole and terbinafine combined are significantly more potent than either drug alone. As a suggestion, combination therapy could provide an option for the treatment of severe cases of M. wolfii in patients with underlying primary immunodeficiencies.As molecular identification and sequencing techniques continue to develop and become more available, we will likely see more diverse pathogens emerge in patients with underlying primary immunodeficiencies. In this current case. Additional study is warranted to explore insight into human immunity and the efficacy of combination therapy against rare fungal species in CGD patients.

S10.5b Bacterial and fungal coinfections in cystic fibrosis

S10.5 Fungal respiratory infections in Cystic Fibrosis, September 24, 2022, 10:30 AM - 12:00 PMLung infections due to Aspergillus fumigatus are in constant augmentation in cystic fibrosis (CF) patients. Up to 55% of CF patients show respiratory complications and exacerbated inflammation due to A. fumigatus. Allergic bronchopulmonary aspergillosis is the major complication found in up to 15% of these CF patients. Moreover, up to 60% of CF patients who are already infected chronically by Pseudomonas aeruginosa are also colonized by A. fumigatus. The interactions between A. fumigatus and P. aeruginosa showed a balance between inhibitory and stimulatory effects on fungal growth in mixed A. fumigatus-P. aeruginosa cultures. Moreover, the interaction between the bacteria and A. fumigatus modulates exposure to fungal patterns and the innate immune response to the pathogens. Indeed, superinfection by A. fumigatus of patients already colonized by P. aeruginosa leads to hyperinflammation. Therefore, studying the interaction between bacteria and fungus in cystic fibrosis is crucial.

S10.5c Insight into the role of secondary metabolism in the pathogenesis of Scedosporium apiospermum

S10.5 Fungal respiratory infections in Cystic Fibrosis, September 24, 2022, 10:30 AM - 12:00 PMSecondary metabolism is a general term defining biosynthetic pathways that occur in plants, bacteria, and fungi and lead to the production of highly diversified molecular structures. Among the diverse functions that were attributed to these molecules, it is now obvious that they are predominantly involved in chemical warfare with competitors in their environments. In fungi, the two main classes of fungal secondary metabolites are polyketides (PKs) and nonribosomal peptides (NRPs). The biosynthesis of such bioactive molecules is performed by large multifunctional enzymes (NRP synthases, NRPS, and polyketide synthases, PKS) encoded by genes usually located within clusters. Conversely to Aspergillus fumigatus, only a few data are currently available for other pathogenic fungi. In this context, our research group is interested in delineating the role of secondary metabolism in Scedosporium apiospermum, a multi-resistant mold known to colonize chronically the airways of patients with cystic fibrosis (CF). Taking advantage of the availability of the S. apiospermum genome sequence, we first conducted an in silico analysis aiming at exploring the PKs and NRPs battery of the fungus. A total of 9 genes encoding PKs, 9 encoding NRPs, and 5 encoding hybrid NRPs/PKs enzymes were identified. All 3 of the PKs gene clusters presented homologies with those involved in the biosynthesis of pseurotin A. transbergamotene, and ovalicin, or the tremorgenic toxin b-aflatrem while a fourth one is involved in the biosynthesis of melanin. Among the NRPs encoding genes, 6 exhibited sufficient similarity scores with other fungal NRPs to predict the class of the generated peptide: siderophores (2), epidithiodioxopiperazines (2), and cyclopeptides (2). Nevertheless, substrate prediction methods for NRPs domains failed, thus questioning about the nature of the produced peptides. We thus focused our attention on the characterization of some NRP and PK biosynthetic pathways. Since iron acquisition is known to be crucial for the survival of microorganisms as for the virulence of numerous pathogens, we first investigated clusters predicted to be responsible for the biosynthesis of siderophores in S. apiospermum. For instance, we disrupted the SAPIO_CDS2806 gene, an ortholog of sidD which drives the production of the extracellular hydroxamate-type siderophore fusarinin C in Aspergillus fumigatus. A comparison of culture supernatants from sidD mutants and their parent strain revealed that S. apiospermum secretes a unique extracellular siderophore, namely Nα‐methylcoprogen B and that sidD gene was essential for the biosynthesis of this siderophore. sidD mutation resulted in the lack of growth under iron limiting conditions. Interestingly, pyoverdine supported the growth of the parent strain only, suggesting that Nα‐methylcoprogen B is required for iron acquisition from this Pseudomonas aeruginosa siderophore. Finally, the deletion of sidD resulted in the loss of virulence in a murine model of scedosporiosis. Altogether, our results demonstrate that S. apiospermum sidD gene drives the synthesis of a unique extracellular siderophore, namely Nα‐methylcoprogen B, which is essential for fungal growth and virulence. Above all, we also provide unprecedented data suggesting that this fungal siderophore scavenges iron from pyoverdine, which might explain the antagonism between S. apiospermum and P. aeruginosa in CF.

Multilocus Sequence Typing Reveals Extensive Genetic Diversity of the Emerging Fungal Pathogen Scedosporium aurantiacum

Scedosporium spp. are the second most prevalent filamentous fungi after Aspergillus spp. recovered from cystic fibrosis (CF) patients in various regions of the world. Although invasive infection is uncommon prior to lung transplantation, fungal colonization may be a risk factor for invasive disease with attendant high mortality post-transplantation. Abundant in the environment, Scedosporium aurantiacum has emerged as an important fungal pathogen in a range of clinical settings. To investigate the population genetic structure of S. aurantiacum, a MultiLocus Sequence Typing (MLST) scheme was developed, screening 24 genetic loci for polymorphisms on a tester strain set. The six most polymorphic loci were selected to form the S. aurantiacum MLST scheme: actin (ACT), calmodulin (CAL), elongation factor-1α (EF1α), RNA polymerase subunit II (RPB2), manganese superoxide dismutase (SOD2), and β-tubulin (TUB). Among 188 global clinical, veterinary, and environmental strains, 5 to 18 variable sites per locus were revealed, resulting in 8 to 23 alleles per locus. MLST analysis observed a markedly high genetic diversity, reflected by 159 unique sequence types. Network analysis revealed a separation between Australian and non-Australian strains. Phylogenetic analysis showed two major clusters, indicating correlation with geographic origin. Linkage disequilibrium analysis revealed evidence of recombination. There was no clustering according to the source of the strains: clinical, veterinary, or environmental. The high diversity, especially amongst the Australian strains, suggests that S. aurantiacum may have originated within the Australian continent and was subsequently dispersed to other regions, as shown by the close phylogenetic relationships between some of the Australian sequence types and those found in other parts of the world. The MLST data are accessible at http://mlst.mycologylab.org. This is a joined publication of the ISHAM/ECMM working groups on “Scedosporium/Pseudallescheria Infections” and “Fungal Respiratory Infections in Cystic Fibrosis”.

Pseudomonas aeruginosa in Bronchiectasis.

Pseudomonas aeruginosa (PA) in patients with bronchiectasis (BE) is associated with a poor outcome and quality of life, and its presence is considered a marker of disease severity. This opportunistic pathogen is known for its ability to produce biofilms on biotic or abiotic surfaces and to survive environmental stress exerted by antimicrobials, inflammation, and nutrient or oxygen depletion. The presence of PA biofilms has been linked to chronic respiratory infection in cystic fibrosis but not in BE. There is considerable inconsistency in the reported infection/eradication rates of PA and chronic PA. In addition, inadequate antimicrobial treatment may potentiate the progression from intermittent to chronic infection and also the emergence of antibiotic resistance. A better comprehension of the pathophysiology of PA infections and its implications for BE is urgently needed. This can drive improvements in diagnostic accuracy, can move us toward a new consensus definition of chronic infection, can better define the follow-up of patients at risk of PA, and can achieve more successful eradication rates. In addition, the new technological advances regarding molecular diagnostics, -omics, and biomarkers require us to reconsider our traditional concepts.

The effect of particle size of inhaled tobramycin dry powder on the eradication of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa is the predominant opportunistic bacterium that causes chronic respiratory infections in cystic fibrosis (CF) patients. This bacterium can form biofilms, which are structured communities of cells encased within a self-produced matrix. Such biofilms have a high level of resistance to multiple classes of antibiotics. A widely used treatment of P. aeruginosa lung infections in CF patients is tobramycin dry powder inhalation. The behaviour of particles in the lung has been well studied, and dry powder inhalers are optimised for optimal dispersion of the drug into different zones of the lung. However, one question that has not been addressed is whether the size of an antibiotic particle influences the antibiofilm activity against P. aeruginosa. We investigated this by fractionating tobramycin particles using a Next Generation Impactor (NGI). The fractions obtained were then tested in an in vitro model on P. aeruginosa biofilms. The results indicate that the antibiofilm activity of tobramycin dry powder inhaler can indeed be influenced by the particle size. Against P. aeruginosa biofilms of two clinical isolates, smaller tobramycin particles (aerodynamic diameter <2.82 µm) showed better efficacy by approximately 20% as compared to larger tobramycin particles (aerodynamic diameter <11.7 µm) However, this effect was only observed when biofilms were treated for 3 hours, whereas there was no difference after treatment for 24 hours. This suggests that in our model the rate of dissolution of larger particles limits the effectiveness of tobramycin over a 3-hour time period, which is relevant as this is equivalent to the time in which most tobramycin is cleared from the lung.

Cystic fibrosis microbiome: analysis of nasal middle meatus and sputum in different lung disease stages

BACKGROUND: Culture independent methods of molecular detection of microbiome have shown the polymicrobial nature of respiratory infections in cystic fibrosis, with pathogenic agents undetectable in conventional culture methods. Composition and diversity of the airway microbiome are still poorly understood. METHODOLOGY: This study evaluated the airway microbiome in 31 adult cystic fibrosis patients via the analysis of 16S rRNA se- quences by next generation sequencing. RESULTS: Staphylococcus, Streptococcus and Corynebacterium were the most abundant genera in the middle meatus, and Pseudo- monas, Haemophilus and Prevotella were the most abundant in sputum. In patients with advanced disease (FEV1&lt; 50%), there was an increase in the prevalence of Pseudomonas in both sample types when studied separately. In each patient, in a paired analysis, the sputum and middle meatus showed similar microbiome composition in mild or moderate disease (FEV1≥ 50%). In patients with severe lung disease, the relative abundance of Pseudomonas had a positive correlation in both collection sites. CONCLUSIONS: This is the first Brazilian study to evaluate the airway microbiome in cystic fibrosis patients. Our findings agree with those in the international literature and indicate the role of Pseudomonas in the sputum and middle meatus in patients with advanced disease.

The implementation of an aminoglycoside induced ototoxicity algorithm for people with cystic fibrosis

Aminoglycoside antibiotics treat respiratory infections in cystic fibrosis (CF). An aminoglycoside induced ototoxicity algorithm (AIOA) was implemented to assess ototoxicity among people with CF treated with intravenous and/or inhaled aminoglycosides. Prior to AIOA implementation, 14 of 52 patients (27%) treated with intravenous aminoglycosides had an audiogram. In the 24 months post AIOA implementation, 43 of 44 patients (98%) treated with intravenous aminoglycosides had an audiogram, with 27 (63%) demonstrating abnormalities. Prior to AIOA development, 18 of 70 patients (26%) who received at least two courses of inhaled aminoglycosides had an audiogram. Post AIOA implementation, 33 patients qualified for an audiogram after receiving inhaled aminoglycosides. Of these, 19 (58%) had an audiogram and 10 (53%) had abnormalities. Overall, we identified 46 (61%) people with CF with hearing abnormalities compared to 2.4% in the CF Foundation Patient Registry. This suggests an urgent need for CF programs to implement AIOAs.

Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms

BackgroundNovel methods are necessary to reduce morbidity and mortality of patients suffering from infections with Pseudomonas aeruginosa. Being the most common infectious species of the Pseudomonas genus, P. aeruginosa is the primary Gram-negative etiology responsible for nosocomial infections. Due to the ubiquity and high adaptability of this species, an effective universal treatment method for P. aeruginosa infection still eludes investigators, despite the extensive research in this area.ResultsWe report bacterial inhibition by iron-oxide (nominally magnetite) nanoparticles (NPs) alone, having a mean hydrodynamic diameter of ~ 16 nm, as well as alginate-capped iron-oxide NPs. Alginate capping increased the average hydrodynamic diameter to ~ 230 nm. We also investigated alginate-capped iron-oxide NP-drug conjugates, with a practically unchanged hydrodynamic diameter of ~ 232 nm. Susceptibility and minimum inhibitory concentration (MIC) of the NPs, NP-tobramycin conjugates, and tobramycin alone were determined in the PAO1 bacterial colonies. Investigations into susceptibility using the disk diffusion method were done after 3 days of biofilm growth and after 60 days of growth. MIC of all compounds of interest was determined after 60-days of growth, to ensure thorough establishment of biofilm colonies.ConclusionsPositive inhibition is reported for uncapped and alginate-capped iron-oxide NPs, and the corresponding MICs are presented. We report zero susceptibility to iron-oxide NPs capped with polyethylene glycol, suggesting that the capping agent plays a major role in enabling bactericidal ability in of the nanocomposite. Our findings suggest that the alginate-coated nanocomposites investigated in this study have the potential to overcome the bacterial biofilm barrier. Magnetic field application increases the action, likely via enhanced diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate barriers, which are characteristic of cystic-fibrosis respiratory infections. We demonstrate that iron-oxide NPs coated with alginate, as well as alginate-coated magnetite–tobramycin conjugates inhibit P. aeruginosa growth and biofilm formation in established colonies. We have also determined that susceptibility to tobramycin decreases for longer culture times. However, susceptibility to the iron-oxide NP compounds did not demonstrate any comparable decrease with increasing culture time. These findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in antibacterial coatings, solutions, and drugs, as well as other applications in which microbial abolition or infestation prevention is sought.

The OmpR Regulator of Burkholderia multivorans Controls Mucoid-to-Nonmucoid Transition and Other Cell Envelope Properties Associated with Persistence in the Cystic Fibrosis Lung.

Bacteria from the Burkholderia cepacia complex grow in different natural and man-made environments and are feared opportunistic pathogens that cause chronic respiratory infections in cystic fibrosis patients. Previous studies showed that Burkholderia mucoid clinical isolates grown under stress conditions give rise to nonmucoid variants devoid of the exopolysaccharide cepacian. Here, we determined that a major cause of the nonmucoid morphotype involves nonsynonymous mutations and small indels in the ompR gene encoding a response regulator of a two-component regulatory system. In trans complementation of nonmucoid variants (NMVs) with the native gene restored exopolysaccharide production. The loss of functional Burkholderia multivorans OmpR had positive effects on growth, adhesion to lung epithelial cells, and biofilm formation in high-osmolarity medium, as well as an increase in swimming and swarming motilities. In contrast, phenotypes such as antibiotic resistance, biofilm formation at low osmolarity, and virulence in Galleria mellonella were compromised by the absence of functional OmpR. Transcriptomic studies indicated that loss of the ompR gene affects the expression of 701 genes, many associated with outer membrane composition, motility, stress response, iron acquisition, and the uptake of nutrients, consistent with starvation tolerance. Since the stresses here imposed on B. multivorans may strongly resemble the ones found in the cystic fibrosis (CF) airways and mutations in the ompR gene from longitudinally collected CF isolates have been found, this regulator might be important for the production of NMVs in the CF environment.IMPORTANCE Within the cystic fibrosis (CF) lung, bacteria experience high-osmolarity conditions due to an ion unbalance resulting from defects in CF transmembrane conductance regulator (CFTR) protein activity in epithelial cells. Understanding how bacterial CF pathogens thrive in this environment might help the development of new therapeutic interventions to prevent chronic respiratory infections. Here, we show that the OmpR response regulator of one of the species found in CF respiratory infections, Burkholderia multivorans, is involved in the emergence of nonmucoid colony variants and is important for osmoadaptation by regulating several cell envelope components. Specifically, genetic, phenotypic, genomic, and transcriptomic approaches uncover OmpR as a regulator of cell wall remodeling under stress conditions, with implications in several phenotypes such as exopolysaccharide production, motility, antibiotic resistance, adhesion, and virulence.

Evolution of the Pseudomonas aeruginosa Aminoglycoside Mutational Resistome In Vitro and in the Cystic Fibrosis Setting.

Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.