Infection In Cystic Fibrosis Research Articles

Spray drying siRNA-lipid nanoparticles for dry powder pulmonary delivery.

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

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.

Amikacin Liposomal Inhalation Suspension in the Treatment of Mycobacterium abscessus Lung Infection: A French Observational Experience.

Background Mycobacterium abscessus infections remain difficult to manage in both cystic fibrosis (CF) and non-CF patients and reported clinical outcomes are largely unsatisfactory. Clinical trial data are limited and no approved therapies are currently available for the management of M abscessus lung diseases. As an alternative, cohort studies may provide insightful information into the management of M abscessus pulmonary disease.MethodsBased on a retrospective observational cohort study, we investigated the safety and efficacy of amikacin liposome inhaled suspension (ALIS) as an adjunct to a standard antibiotic regimen for M abscessus lung infection in both CF and non-CF patients. We also assessed the association of patient drug compliance with culture conversion and clinical outcomes.ResultsTwenty-six patients had long-term follow-up data available. Culture conversion was achieved in 54% (14/26) of the patients with no difference between CF and non-CF patients after an average treatment duration of 10 months. Patient treatment compliance was significantly better in the converter group compared to nonconverters with an odds ratio of 44.78 associated with good compared to poor patient compliance. Overall, 9 patients (35%) experienced an adverse event that led to treatment discontinuation.ConclusionsALIS appears beneficial in both CF and non-CF populations with M abscessus lung disease.

The Sense and Nonsense of Antimicrobial Susceptibility Testing in Cystic Fibrosis.

Antimicrobial susceptibility testing (AST) has been used to guide therapy of airway infection in persons with cystic fibrosis (CF) for decades. However, evidence that AST adds benefit to treatment outcomes in CF is lacking. In fact, the routine use of AST has potential to exacerbate inappropriate antibiotic use. Several features of airway infection in CF contribute to the limitations of AST in predicting treatment outcomes, providing rationale for abandoning this practice altogether. Other features of CF infection suggest, however, that select use of AST can provide worthwhile guidance to antibiotic selection.

Airway Infection in Cystic Fibrosis: Microbiology and Management.

Airway Infection in Cystic Fibrosis: Microbiology and Management.

Achromobacter spp. Adaptation in Cystic Fibrosis Infection and Candidate Biomarkers of Antimicrobial Resistance

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

Nitric oxide inhibits alginate biosynthesis in Pseudomonas aeruginosa and increases its sensitivity to tobramycin by downregulating algU gene expression

In the process of chronic cystic fibrosis (CF) infection, Pseudomonas aeruginosa (PA) is converted into a mucoid phenotype characterized by an overproduction of exopolysaccharide alginate. The alginate forms a thick mucus that causes difficulty in patient's breathing, drug resistance and contributes to both the morbidity and mortality of the patient. AlgU of PA, an extracytoplasmic function sigma factor, is responsible for the alginate overproduction and leads to mucoidy and chronic infection of CF patients. In this report, we found that endogenous and exogenous nitric oxide (NO) can significantly reduce algU expression, leading to down-regulation of a series of alginate synthesis-related genes (algD, alg8, algX, and algK), eventually down-regulated alginate synthesis. A fluorescent reporter strain was constructed to clarify the inhibitory effect of alginate synthesis through real-time monitoring in different conditions. The results showed that NO presented inhibitory effect on alginate synthesis in nine clinical PA isolates as in the PA reference strain, and the reduction of alginate was more significant in three mucoid strains (by about 51%, 70% and 61%, respectively, while 47% for the reference strain). In the co-culture system, effect of NO on PA fluorescence intensity is similar to that in monocultures, with the best effect at 10 μM NO donor sodium nitroprusside (SNP). Finally, we examined the changes in the antibiotic susceptibility of PA under NO-inhibited alginate conditions. In the presence of 10 μM SNP, the number of planktonic cells increased, and both adherent and planktonic PA cells showed increased susceptibility to tobramycin. We thus suggest that NO can potentially be employed as a therapeutic strategy to prevent cystic fibrosis lungs from PA infection.

Curvularin Isolated From Phoma macrostoma Is an Antagonist of RhlR Quorum Sensing in Pseudomonas aeruginosa.

Quorum sensing (QS) is an attractive target for the treatment of multidrug-resistant Pseudomonas aeruginosa, against which new antibiotics are urgently needed. Because LasR is at the top of the QS hierarchy controlling Rhl and PQS systems, most QS inhibitors have been targeted to LasR. However, it has recently been reported that in clinical isolates of P. aeruginosa, LasR is frequently mutated and nonfunctional, and RhlR independently acts to produce virulent factors that maintain toxicity. Thus, for effective treatment of chronic cystic fibrosis infections, RhlR antagonists is needed to prevent the LasR-independent Rhl system, but RhlR antagonists have rarely been reported. In this study, we found that curvularin, an aromatic compound with a cyclized alkyl side chain isolated from Phoma macrostoma, at a low micromolar concentration of 1–30 μM potently and selectively inhibited pyocyanin and rhamnolipid production without affecting the cell viability of P. aeruginosa. Only high concentration (more over 100 μM) curvularin negligibly inhibited biofilm formation and elastase production, suggesting that curvularin at low concentrations selectively inhibits RhlR. The QS antagonism by curvularin was investigated in experiments using QS competition and signaling molecules assays with QS gene expression analysis, and the results showed that, indeed, at low concentrations, curvularin selectively antagonized RhlR; in contrast, it negligibly antagonized LasR only when applied at a high concentration. The exclusive RhlR antagonizing activity of curvularin at low concentrations was confirmed using QS mutants; specifically, curvularin at low concentrations inhibited pyocyanin and rhamnolipid production by selectively antagonizing N-butanoyl homoserine lactone (BHL)-activated RhlR. Moreover, by targeting RhlR, curvularin reduced the in vivo virulence of wild-type P. aeruginosa as well as lasR mutants in Caenorhabditis elegans. Overall, low-concentration curvularin is a pure RhlR antagonist in P. aeruginosa, and to the best of our knowledge, this is the first report describing an RhlR antagonist from natural resources. Hence, curvularin has great potential for the development of chronic P. aeruginosa infection therapeutics and for the study of RhlR function in the complex QS system.

Persistent Aspergillus fumigatus infection in cystic fibrosis: impact on lung function and role of treatment of asymptomatic colonization—a registry-based case–control study

BackgroundAspergillus fumigatus is the most common filamentous fungus isolated from the airways of people with cystic fibrosis (CF). The aim of this study was to investigate how chronic A. fumigatus colonization affects lung function in people with CF, to identify risk factors for colonization, and to evaluate antifungal treatment of asymptomatic Aspergillus colonization.MethodsData from 2014–2018 was collected from the Swedish CF registry and medical records. Baseline data before the start of A. fumigatus colonization was compared with the two succeeding years to evaluate how colonization and treatment affected lung function and other clinical aspects.ResultsA total of 437 patients were included, of which 64 (14.6%) became colonized with A. fumigatus during the study period. Inhaled antibiotics was associated with A. fumigatus colonization (adjusted OR 3.1, 95% CI 1.6–5.9, p < 0.05). Fungal colonization was not associated with a more rapid lung function decline or increased use of IV-antibiotics compared to the non-colonized group, but patients with A. fumigatus had more hospital days, a higher increase of total IgE, and higher eosinophil counts. In the Aspergillus group, 42 patients were considered to be asymptomatic. Of these, 19 patients received antifungal treatment. Over the follow up period, the treated group had a more pronounced decrease in percent predicted Forced Expiratory Volume in one second (ppFEV1) compared to untreated patients (− 8.7 vs − 1.4 percentage points, p < 0.05).ConclusionInhaled antibiotics was associated with A. fumigatus colonization, but no association was found between persistent A. fumigatus and subsequent lung function decline. No obvious benefits of treating asymptomatic A. fumigatus colonization were demonstrated.

Tissue-localized immune responses in people with cystic fibrosis and respiratory nontuberculous mycobacteria infection.

Nontuberculous mycobacteria (NTM) are an increasingly common cause of respiratory infection in people with cystic fibrosis (PwCF). Relative to those with no history of NTM infection (CF-NTMNEG), PwCF and a history of NTM infection (CF-NTMPOS) are more likely to develop severe lung disease and experience complications over the course of treatment. In other mycobacterial infections (e.g., tuberculosis), an overexuberant immune response causes pathology and compromises organ function; however, since the immune profiles of CF-NTMPOS and CF-NTMNEG airways are largely unexplored, it is unknown which, if any, immune responses distinguish these cohorts or concentrate in damaged tissues. Here, we evaluated lung lobe-specific immune profiles of 3 cohorts (CF-NTMPOS, CF-NTMNEG, and non-CF adults) and found that CF-NTMPOS airways are distinguished by a hyperinflammatory cytokine profile. Importantly, the CF-NTMPOS airway immune profile was dominated by B cells, classical macrophages, and the cytokines that support their accumulation. These and other immunological differences between cohorts, including the near absence of NK cells and complement pathway members, were enriched in the most damaged lung lobes. The implications of these findings for our understanding of lung disease in PwCF are discussed, as are how they may inform the development of host-directed therapies to improve NTM disease treatment.

The Use of Artificial Sputum Media to Enhance Investigation and Subsequent Treatment of Cystic Fibrosis Bacterial Infections.

In cystic fibrosis (CF), mutations in the CF transmembrane conductance regulator protein reduce ionic exchange in the lung, resulting in thicker mucus, which impairs mucociliary function, airway inflammation and infection. The mucosal and nutritional environment of the CF lung is inadequately mimicked by commercially available growth media, as it lacks key components involved in microbial pathogenesis. Defining the nutritional composition of CF sputum has been a long-term goal of in vitro research into CF infections to better elucidate bacterial growth and infection pathways. This narrative review highlights the development of artificial sputum medium, from a viable in vitro method for understanding bacterial mechanisms utilised in CF lung, to uses in the development of antimicrobial treatment regimens and examination of interactions at the epithelial cell surface and interior by the addition of host cell layers. The authors collated publications based on a PubMed search using the key words: “artificial sputum media” and “cystic fibrosis”. The earliest iteration of artificial sputum media were developed in 1997. Formulations since then have been based either on published data or chemically derived from extracted sputum. Formulations contain combinations of mucin, extracellular DNA, iron, amino acids, and lipids. A valuable advantage of artificial sputum media is the ability to standardise media composition according to experimental requirements.

Synergistic Activity of Repurposed Peptide Drug Glatiramer Acetate with Tobramycin against Cystic Fibrosis Pseudomonas aeruginosa.

ABSTRACTPseudomonas aeruginosa is the most common pathogen infecting the lungs of people with cystic fibrosis (CF), causing both acute and chronic infections. Intrinsic and acquired antibiotic resistance, coupled with the physical barriers resulting from desiccated CF sputum, allow P. aeruginosa to colonize and persist in spite of antibiotic treatment. As well as the specific difficulties in eradicating P. aeruginosa from CF lungs, P. aeruginosa is also subject to the wider, global issue of antimicrobial resistance. Glatiramer acetate (GA) is a peptide drug, used in the treatment of multiple sclerosis (MS), which has been shown to have moderate antipseudomonal activity. Other antimicrobial peptides (AMPs) have been shown to be antibiotic resistance breakers, potentiating the activities of antibiotics when given in combination, restoring and/or enhancing antibiotic efficacy. Growth, viability, MIC determinations, and synergy analysis showed that GA improved the efficacy of tobramycin (TOB) against reference strains of P. aeruginosa, reducing TOB MICs and synergizing with the aminoglycoside. This was also the case for clinical strains from people with CF. GA significantly reduced the MIC50 of TOB for viable cells from 1.69 mg/L (95% confidence interval [CI], 0.26 to 8.97) to 0.62 mg/L (95% CI, 0.15 to 3.94; P = 0.002) and the MIC90 for viable cells from 7.00 mg/L (95% CI, 1.18 to 26.50) to 2.20 mg/L (95% CI, 0.99 to 15.03; P = 0.001), compared to results with TOB only. Investigation of mechanisms of GA activity showed that GA resulted in significant disruption of outer membranes, depolarization of cytoplasmic membranes, and permeabilization of P. aeruginosa and was the only agent tested (including cationic AMPs) to significantly affect all three mechanisms.IMPORTANCE The antimicrobial resistance crisis urgently requires solutions to the lost efficacy of antibiotics. The repurposing of drugs already in clinical use, with strong safety profiles, as antibiotic adjuvants to restore the efficacy of antibiotics is an important avenue to alleviating the resistance crisis. This research shows that a clinically used drug from outside infection treatment, glatiramer acetate, reduces the concentration of tobramycin required to be effective in treating Pseudomonas aeruginosa, based on analyses of both reference and clinical respiratory isolates from people with cystic fibrosis. The two agents acted synergistically against P. aeruginosa, being more effective combined in vitro than predicted for their combination. As a peptide drug, glatiramer acetate functions similarly to many antimicrobial peptides, interacting with and disrupting the P. aeruginosa cell wall and permeabilizing bacterial cells, thereby allowing tobramycin to work. Our findings demonstrate that glatiramer acetate is a strong candidate for repurposing as an antibiotic resistance breaker of pathogenic P. aeruginosa.

P130 Achromobacter spp. phenotypic differences between chronic and occasional lung infection in cystic fibrosis

P130 Achromobacter spp. phenotypic differences between chronic and occasional lung infection in cystic fibrosis

EPS3.06 Isolation and characterisation of nebulised phage for treatment of chronic Pseudomonas aeruginosa (Pa) pulmonary infections in cystic fibrosis (CF) patients

ePS3.06 Isolation and characterisation of nebulised phage for treatment of chronic Pseudomonas aeruginosa (Pa) pulmonary infections in cystic fibrosis (CF) patients

DNA Analysis of qacE Gene in Pseudomonas Aeruginosa Isolated from Iraqi Patient

Pseudomonas aeruginosa is an opportunistic pathogen that can cause different infections such as nosocomial bacterial infection, Urinary tract infection, Meningitis, Eye infection, Otitis Media, Respiratory infections, and Pneumonia, especially in people with Cystic fibrosis, Bone and joints infection, gastrointestinal infection, Skin infection and soft tissue inflammation. Resistance of P. aeruginosa to antibiotics and disinfectants is a major problem all over the world due to the random and unrestricted use of antibiotics. This resistance is also likely to occur due to several reasons, including the production of beta-lactamase enzymes and the lack of permeability of their cell wall, possibility of it containing resistance genes and the occurrence of genetic mutations leading to the development of resistance to antiseptics and causing failure in treating infections that caused by P. aeruginosa. This study aims to detect and sequence analysis of qacE gene in P. aeruginosa that responds to the resistance of Quaternary ammonium compounds and detect the mutations in this sequence and detection protein synthesis in this gene. A hundred swabs were collected from patients with burns and wounds infection from many Hospital in Baghdad including Al- Kindi Teaching Hospital, Al-Yarmouk Teaching Hospital, Baghdad Teaching Hospital, National Centre for Educational Laboratories, Al-Shahid Ghazi Hariri Specialist Specialized Burns Centre (Medical City), and Al-Zaafarania General Hospital during the period October 2017 for the end of December 2017.The current study detected 69 isolates of P. aeruginosa from wound 68.6% (24/35) and burn samples 69.2% (45/65). The study was investigated QacE gene in 97.1% that responses of P. aeruginosa resistance to Quaternary ammonium compounds. The sequencing analysis for QacE genes were determined and the results appeared multiple mutations including Missense mutations, insertion mutations and several silent mutations, some mutations effected in the translation of protein while others not affected. This study also included the analysis of phylogenetic tree of QacE gene and the results showed that the tree consisted of only one species which is P. aeruginosa, and the isolates in this study did not deviate of this bacteria even if their mutations were more than 22.

Polymicrobial infections can select against Pseudomonas aeruginosa mutators because of quorum-sensing trade-offs.

Bacteria with increased mutation rates (mutators) are common in chronic infections and are associated with poorer clinical outcomes, especially in the case of Pseudomonas aeruginosa infecting cystic fibrosis (CF) patients. There is, however, considerable between-patient variation in both P. aeruginosa mutator frequency and the composition of co-infecting pathogen communities. We investigated whether community context might affect selection of mutators. Using an in vitro CF model community, we show that P. aeruginosa mutators were favoured in the absence of other species but not in their presence. This was because there were trade-offs between adaptation to the biotic and abiotic environments (for example, loss of quorum sensing and associated toxin production was beneficial in the latter but not the former in our in vitro model community) limiting the evolvability advantage of an elevated mutation rate. Consistent with a role of co-infecting pathogens selecting against P. aeruginosa mutators in vivo, we show that the mutation frequency of P. aeruginosa population was negatively correlated with the frequency and diversity of co-infecting bacteria in CF infections. Our results suggest that co-infecting taxa can select against P. aeruginosa mutators, which may have potentially beneficial clinical consequences.

Genomics of Diversification of Pseudomonas aeruginosa in Cystic Fibrosis Lung-like Conditions.

Pseudomonas aeruginosa is among the most problematic opportunistic pathogens for adults with cystic fibrosis (CF), causing repeated and resilient infections in the lung and surrounding airways. Evidence suggests that long-term infections are associated with diversification into specialized types but the underlying cause of that diversification and the effect it has on the persistence of infections remains poorly understood. Here, we use evolve-and-resequence experiments to investigate the genetic changes accompanying rapid, de novo phenotypic diversification in lab environments designed to mimic two aspects of human lung ecology: spatial structure and complex nutritional content. After ∼220 generations of evolution, we find extensive genetic variation present in all environments, including those that most closely resemble the CF lung. We use the abundance and frequency of nonsynonymous and synonymous mutations to estimate the ratio of mutations that are selectively neutral (hitchhikers) to those that are under positive selection (drivers). A significantly lower proportion of driver mutations in spatially structured populations suggests that reduced dispersal generates subpopulations with reduced effective population size, decreasing the supply of beneficial mutations and causing more divergent evolutionary trajectories. In addition, we find mutations in a handful of genes typically associated with chronic infection in the CF lung, including one gene associated with antibiotic resistance. This demonstrates that many of the genetic changes considered to be hallmarks of CF lung adaptation can arise as a result of adaptation to a novel environment and do not necessarily require antimicrobial treatment, immune system suppression, or competition from other microbial species to occur.