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.

Bacteriophage infection and killing of intracellular Mycobacterium abscessus.

IMPORTANCE: As we rapidly approach a post-antibiotic era, bacteriophage (phage) therapy may offer a solution for treating drug-resistant bacteria. Mycobacterium abscessus is an emerging, multidrug-resistant pathogen that causes disease in people with cystic fibrosis, chronic obstructive pulmonary disease, and other underlying lung diseases. M. abscessus can survive inside host cells, a niche that can limit access to antibiotics. As current treatment options for M. abscessus infections often fail, there is an urgent need for alternative therapies. Phage therapy is being used to treat M. abscessus infections as an option of last resort. However, little is known about the ability of phages to kill bacteria in the host environment and specifically in an intracellular environment. Here, we demonstrate the ability of phages to enter mammalian cells and to infect and kill intracellular M. abscessus. These findings support the use of phages to treat intracellular bacterial pathogens.

Determining effects of nitrate, arginine, and ferrous on antibiotic recalcitrance of clinical strains of Pseudomonas aeruginosa in biofilm-inspired alginate encapsulates.

BACKGROUND: Biofilms play a role in recalcitrance and treatability of bacterial infections, but majority of known antibiotic resistance mechanisms are biofilm-independent. Biofilms of Pseudomonas aeruginosa, especially in cystic fibrosis patients infected with the alginate producing strains in their lungs, are hard to treat. Changes in growth-related bacterial metabolism in biofilm affect their antibiotic recalcitrance which could be considered for new therapies designed based on these changes. In this study, effects of nitrate, arginine, and ferrous were investigated on antibiotic recalcitrance in alginate-encapsulated P. aeruginosa strains isolated from cystic fibrosis patients in the presence of amikacin, tobramycin, and ciprofloxacin. Also, expression of an efflux pump gene, mexY, was analyzed in selected strains in the presence of amikacin and ferrous. METHODS: Clinical P. aeruginosa strains were isolated from cystic fibrosis patients and minimum inhibitory concentration of amikacin, tobramycin, and ciprofloxacin was determined against all the strains. For each antibiotic, a susceptible and a resistant or an intermediate-resistant strain were selected, encapsulated into alginate beads, and subjected to minimal biofilm eradication concentration (MBEC) test. After determining MBECs, sub-MBEC concentrations (antibiotics at concentrations one level below the determined MBEC) for each antibiotic were selected and used to study the effects of nitrate, arginine, and ferrous on antibiotic recalcitrance of encapsulated strains. Effects of ferrous and amikacin on expression of the efflux pump gene, mexY, was studied on amikacin sensitive and intermediate-resistant strains. One-way ANOVA and t test were used as the statistical tests. RESULTS: According to the results, the supplements had a dose-related effect on decreasing the number of viable cells; maximal effect was noted with ferrous, as ferrous supplementation significantly increased biofilm susceptibility to both ciprofloxacin and amikacin in all strains, and to tobramycin in a resistant strain. Also, treating an amikacin-intermediate strain with amikacin increased the expression of mexY gene, which has a role in P. aeruginosa antibiotic recalcitrance, while treating the same strain with ferrous and amikacin significantly decreased the expression of mexY gene, which was a promising result. CONCLUSIONS: Our results support the possibility of using ferrous and arginine as an adjuvant to enhance the efficacy of conventional antimicrobial therapy of P. aeruginosa infections.

Repurposing High-Throughput Screening Identifies Unconventional Drugs with Antibacterial and Antibiofilm Activities against Pseudomonas aeruginosa under Experimental Conditions Relevant to Cystic Fibrosis.

Pseudomonas aeruginosa is the most common pathogen infecting cystic fibrosis (CF) lungs, causing acute and chronic infections. Intrinsic and acquired antibiotic resistance allow P. aeruginosa to colonize and persist despite antibiotic treatment, making new therapeutic approaches necessary. Combining high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses for drugs. This study screened a drug library of 3,386 drugs, mostly FDA approved, to identify antimicrobials against P. aeruginosa under physicochemical conditions relevant to CF-infected lungs. Based on the antibacterial activity, assessed spectrophotometrically against the prototype RP73 strain and 10 other CF virulent strains, and the toxic potential evaluated toward CF IB3-1 bronchial epithelial cells, five potential hits were selected for further analysis: the anti-inflammatory and antioxidant ebselen, the anticancer drugs tirapazamine, carmofur, and 5-fluorouracil, and the antifungal tavaborole. A time-kill assay showed that ebselen has the potential to cause rapid and dose-dependent bactericidal activity. The antibiofilm activity was evaluated by viable cell count and crystal violet assays, revealing carmofur and 5-fluorouracil as the most active drugs in preventing biofilm formation regardless of the concentration. In contrast, tirapazamine and tavaborole were the only drugs actively dispersing preformed biofilms. Tavaborole was the most active drug against CF pathogens other than P. aeruginosa, especially against Burkholderia cepacia and Acinetobacter baumannii, while carmofur, ebselen, and tirapazamine were particularly active against Staphylococcus aureus and B. cepacia. Electron microscopy and propidium iodide uptake assay revealed that ebselen, carmofur, and tirapazamine significantly damage cell membranes, with leakage and cytoplasm loss, by increasing membrane permeability. IMPORTANCE Antibiotic resistance makes it urgent to design new strategies for treating pulmonary infections in CF patients. The repurposing approach accelerates drug discovery and development, as the drugs' general pharmacological, pharmacokinetic, and toxicological properties are already well known. In the present study, for the first time, a high-throughput compound library screening was performed under experimental conditions relevant to CF-infected lungs. Among 3,386 drugs screened, the clinically used drugs from outside infection treatment ebselen, tirapazamine, carmofur, 5-fluorouracil, and tavaborole showed, although to different extents, anti-P. aeruginosa activity against planktonic and biofilm cells and broad-spectrum activity against other CF pathogens at concentrations not toxic to bronchial epithelial cells. The mode-of-action studies revealed ebselen, carmofur, and tirapazamine targeted the cell membrane, increasing its permeability with subsequent cell lysis. These drugs are strong candidates for repurposing for treating CF lung P. aeruginosa infections.

Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression.

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift.

AtbFinder Diagnostic Test System Improves Optimal Selection of Antibiotic Therapy in Persons with Cystic Fibrosis.

Cystic fibrosis (CF) is characterized by mutations of CFTR that lead to increased viscous secretions, bacterial colonization, and recurrent infections. Chronic Pseudomonas aeruginosa infection in persons with CF is associated with progressive and accelerated lung function decline despite aggressive antibiotic treatment. We report the management of respiratory infections in persons with CF with antibiotic therapy that was based on the recommendations of AtbFinder, a novel, rapid, culture-based diagnostic test system that employs a novel paradigm of antibiotic selection. AtbFinder mimics bacterial interactions with antibiotics at concentrations that can be achieved in affected tissues or organs and models conditions of interbacterial interactions within polymicrobial biofilms. This open-label, single-arm, investigator-initiated clinical study was designed to identify the efficacy of antibiotics selected using AtbFinder in persons with CF. Microbiological and clinical parameters were assessed following the change of antibiotic therapy to antibiotics selected with AtbFinder between January 2016 and December 2018 and retrospectively compared with clinical data collected between January 2013 and December 2015. We enrolled 35 persons with CF (33 with chronic P. aeruginosa colonization). Antibiotics selected using AtbFinder resulted in clearance of P. aeruginosa in 81.8% of subsequent cultures, decreased pulmonary exacerbations from 1.21 per patient per annum to 0, and an increase in predicted percent predicted forced expiratory volume in 1 s up to 28.4% from baseline. The number of systemic antibiotic courses used in patients after switching to the AtbFinder-selected therapy was reduced from 355 to 178. These findings describe the superiority of antibiotic regimens selected with AtbFinder compared with routine antimicrobial susceptibility testing.

Antibiotic Tolerance and Treatment Outcomes in Cystic Fibrosis Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent in U.S. cystic fibrosis (CF) patients and is associated with worse clinical outcomes in CF. These infections often become chronic despite repeated antibiotic therapy. Here, we assessed whether bacterial phenotypes, including antibiotic tolerance, can predict the clinical outcomes of MRSA infections. MRSA isolates (n = 90) collected at the incident (i.e., acute) and early infection states from 57 patients were characterized for growth rates, biofilm formation, hemolysis, pigmentation, and vancomycin tolerance. The resistance profiles were consistent with those in prior studies. Isolates from the early stage of infection were found to produce biofilms, and 70% of the isolates exhibited delta-hemolysis, an indicator of agr activity. Strong vancomycin tolerance was present in 24% of the isolates but was not associated with intermediate vancomycin susceptibility. There were no associations between these phenotypic measures, antibiotic tolerance, and MRSA clearance. Our research suggests that additional factors may be relevant for predicting the clearance of MRSA. IMPORTANCE Chronic MRSA infections remain challenging to treat in patients with cystic fibrosis (CF). The ability of the bacterial population to survive high concentrations of bactericidal antibiotics, including vancomycin, despite lacking resistance is considered one of the main reasons for treatment failures. The connection between antibiotic tolerance and treatment outcomes remains unexplored and can be crucial for prognosis and regimen design toward eradication. In this study, we measured the capacity of 90 MRSA isolates from CF patients to form vancomycin-tolerant persister cells and evaluated their correlation with the clinical outcomes. Additionally, various traits that could reflect the metabolism and/or virulence of those MRSA isolates were systematically phenotyped and included for their predictive power. Our research highlights that despite the importance of antibiotic tolerance, additional factors need to be considered for predicting the clearance of MRSA.

Effectiveness of different eradication treatment protocols for new-onset Pseudomonas aeruginosa in children with cystic fibrosis.

OBJECTIVES: While eradicating new-onset Pseudomonas aeruginosa in children with cystic fibrosis is an important issue, there is no clear evidence about the best treatment approach. This retrospective observational cohort study aims to compare the effectiveness of intravenous therapy versus inhalation with/without oral therapy in the eradication of new-onset P. aeruginosa, determine the factors affecting the treatment success and assess lung function at baseline and posttreatment. METHODS: Of 399 children, 110 (140 episodes) with either the first P. aeruginosa isolation or a new isolation after at least 1 year free of infection were included. Different eradication regimens (intravenous therapy or inhaled tobramycin or inhaled tobramycin plus oral ciprofloxacin) were compared. Eradication success was accepted as remaining free of infection with a negative culture for 12 months. Demographic, clinical, and microbiological characteristics of children, effectiveness of different eradication strategies, time to a new P. aeruginosa isolation, and the relationship between lung function and the type of eradication regimen were determined. RESULTS: Of 140 episodes, intravenous therapy was administered in 53 and inhalation therapy (in combination with or without oral ciprofloxacin) in 87. Total success rate of eradication was 60.7%. Eradication was achieved in 56.6% of children with intravenous therapy, 59.7% with inhaled tobramycin therapy, and 72% with inhaled tobramycin plus oral ciprofloxacin therapy. Success rates of different eradication regimens did not differ significantly (p = 0.419). Lung function by the end of the first year was worse in the intravenous group compared to the inhalation group (p = 0.017 for forced expiratory volume in 1 s, p = 0.015 for forced vital capacity). CONCLUSION: No advantage of intravenous therapy was demonstrated compared to inhalation therapy in terms of eradication success.

Does Cefiderocol Have a Potential Role in Cystic Fibrosis Pulmonary Exacerbation Management?

Cystic fibrosis (CF) is associated with frequent pulmonary exacerbations and the need for novel antibiotics against antimicrobial resistance. Cefiderocol is a newly approved therapeutic option active against a variety of multidrug resistant (MDR) bacteria such as gram-negative species commonly encountered by CF patients. This review describes the potential role of cefiderocol against Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and Burkholderia cepacia complex. Cefiderocol is a potential therapeutic option for MDR pathogens with minimum inhibitory concentrations (MICs) of ≤4 mg/L. Due to the lack of in vivo evidence in the CF population, cefiderocol may be utilized in patients in which alternative options are lacking due to MDR organisms or rapid pulmonary decline.

In-Host Emergence of Linezolid Resistance in a Complex Pattern of Toxic Shock Syndrome Toxin-1-Positive Methicillin-Resistant Staphylococcus aureus Colonization in Siblings with Cystic Fibrosis.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause chronic lung infections in patients with Cystic Fibrosis (CF). One option for managing them is the use of linezolid. We hereby report the in-host emergence of linezolid resistance (LR) in MRSA in CF siblings via a population analysis. A collection of 171 MRSA strains from 68 samples were characterized by determining their linezolid Minimal Inhibitory Concentrations (MICs), analyzing the locus of staphylococcal protein A (spa) and whole genome sequencing. Courses of linezolid were retraced. Strains belonged to three spa types (t002, t045, t127) and two sequence types (ST1, ST5). Emergence of LR occurred under treatment, one year apart in both siblings, in the CC5-MRSA-I Geraldine clone harboring the toxic shock syndrome toxin-1-encoding gene. Resistance was related to a G2576T substitution present in a variable number of 23S rRNA gene copies. Susceptible and resistant strains were co-isolated within samples. Single Nucleotide Polymorphism-based analysis revealed complex colonizations by highly diversified, clonally related populations. LR remains rare in MRSA and there are very few longitudinal analyses documenting its emergence. Analyzing a large MRSA collection revealed new aspects of LR emergence: it emerges in specific subclonal lineages resulting from adaptive diversification of MRSA in the CF lung and this heterogeneity of intra-sample resistance may contribute to compromising antibiotic management.

Antimicrobial activity of dalbavancin and comparators against Staphylococcus aureus causing pneumonia in patients with and without cystic fibrosis.

The activities of dalbavancin and comparator agents were evaluated against Staphylococcus aureus isolated from the lower respiratory tract of cystic fibrosis (CF) and non-CF patients with pneumonia. Bacterial isolates (n = 357) were collected from CF patients in 36 medical centers worldwide (2018-2019) and susceptibility tested using reference broth microdilution. Susceptibility results from these isolates were compared with those for 725 S. aureus isolates consecutively collected from non-CF patients with pneumonia from the same medical centers over the same period. Only isolates determined to be the probable cause of pneumonia were included in the study. Susceptibility profiles were very similar among isolates from CF and non-CF patients. Dalbavancin exhibited potent activity (MIC50/90, 0.03/0.03 mg/L) and complete coverage (100.0% susceptibility) against isolates from CF and non-CF patients. Ceftaroline (MIC50/90, 0.25/1 mg/L) was active against 97.8% and 98.1% of isolates from CF and non-CF patients, respectively. Oxacillin resistance (MRSA) rates were 27.7% among CF and 28.7% among non-CF patients. Among MRSA isolates from CF/non-CF patients (n = 99/208), susceptibility to ceftaroline, clindamycin, levofloxacin, and tetracycline were 91.9%/93.3%, 58.6%/64.4%, 40.4%/29.3%, and 83.8%/89.4%, respectively. Dalbavancin demonstrated high potency against S. aureus from CF and non-CF patients and may represent a valuable treatment option for CF patients with MRSA pulmonary infection.

Ceftolozane/tazobactam for Pseudomonas aeruginosa pulmonary exacerbations in cystic fibrosis adult patients: a case series.

Management of cystic fibrosis (CF) patients colonized with Pseudomonas aeruginosa is challenging due to its virulence and multi-drug resistance. Ceftolozane/tazobactam (C/T) is a promising new antipseudomonal agent, and clinical data on CF are limited. We describe our experience in the use of C/T for P. aeruginosa-related pulmonary exacerbations (PE) in CF adults admitted within 2016 and 2019 at Careggi Hospital, Florence, Italy. PE was diagnosed as deterioration of respiratory function, worsening cough, and increasing of sputum. C/T was given at the dose of 3 g every 8 h. C/T was used in ten patients. Mean length of C/T treatment was 16.3 days, and tobramycin was the most frequently combined antipseudomonal agent. All patients were successfully treated although susceptibility testing on sputum sample showed C/T resistance in two cases. No adverse effects related to C/T were reported. To our knowledge this is the largest case series on CF patients treated with C/T. Clinical responses were encouraging even where C/T resistant P. aeruginosa was isolated, probably due to multiple phenotypes colonizing CF lungs. C/T could play a promising role in combination therapy against P. aeruginosa as a part of a colistin-sparing regime.

Pulmonary infection secondary to Blastobotrys raffinosifermentans in a cystic fibrosis patient: Review of the literature.

BACKGROUND: The genus Blastobotrys consists of at least 20 species. Disease in humans has been reported with B adeninivorans, B raffinosifermentans, B proliferans and B serpentis, mostly in immunocompromised patients and those with cystic fibrosis. OBJECTIVE: We report a lung infection secondary to B raffinosifermentans in a cystic fibrosis patient successfully treated with isavuconazole and review the literature of invasive infections caused this genus. We also evaluated clinical isolates in our laboratory for species identification and antifungal susceptibility. METHODS: Phylogenetic analysis was performed on a collection of 22 Blastobotrys isolates in our reference laboratory, and antifungal susceptibility patterns were determined for nine clinically available antifungals against 19 of these isolates. RESULTS: By phylogenetic analysis, 21 of the 22 isolates in our collection were identified as B raffinosifermentans and only 1 as B adeninivorans. Most were cultured from the respiratory tract, although others were recovered from other sources, including CSF and blood. Isavuconazole, caspofungin and micafungin demonstrated the most potent in vitro activity, followed by amphotericin B. In contrast, fluconazole demonstrated poor activity. The patient in this case responded to isavuconazole treatment for breakthrough infection due to B raffinosifermentans that was cultured from pleural fluid while on posaconazole prophylaxis post-bilateral lung transplantation for cystic fibrosis. CONCLUSIONS: Blastobotrys species are rare causes of infections in humans and primarily occur in immunocompromised hosts. In our collection, the majority of isolates were identified as B raffinosifermentans. To our knowledge, this is the first report of successful treatment of such an infection with isavuconazole.

Understanding Pseudomonas aeruginosa-Host Interactions: The Ongoing Quest for an Efficacious Vaccine.

Pseudomonas aeruginosa is a leading cause of chronic respiratory infections in people with cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD), and acute infections in immunocompromised individuals. The adaptability of this opportunistic pathogen has hampered the development of antimicrobial therapies, and consequently, it remains a major threat to public health. Due to its antimicrobial resistance, vaccines represent an alternative strategy to tackle the pathogen, yet despite over 50 years of research on anti-Pseudomonas vaccines, no vaccine has been licensed. Nevertheless, there have been many advances in this field, including a better understanding of the host immune response and the biology of P. aeruginosa. Multiple antigens and adjuvants have been investigated with varying results. Although the most effective protective response remains to be established, it is clear that a polarised Th2 response is sub-optimal, and a mixed Th1/Th2 or Th1/Th17 response appears beneficial. This comprehensive review collates the current understanding of the complexities of P. aeruginosa-host interactions and its implication in vaccine design, with a view to understanding the current state of Pseudomonal vaccine development and the direction of future efforts. It highlights the importance of the incorporation of appropriate adjuvants to the protective antigen to yield optimal protection.

Pseudo-Bartter syndrome in Chinese children with cystic fibrosis: Clinical features and genotypic findings.

OBJECTIVES: To characterize the clinical and genotypic features of cystic fibrosis-associated pseudo-Bartter syndrome (CF-PBS) in Chinese children. METHODS: We recruited and characterized the clinical manifestations of 12 Chinese children with CF-PBS. Sweat test, blood and urinary analysis, sputum culture, chest and sinus computed tomography, and abdominal ultrasonography were obtained. Whole-exome sequencing, bioinformatics analysis, and Sanger sequencing validation were performed to define the genotypes. RESULTS: CF-PBS was accompanied by recurrent and/or persistent pneumonia (91.7%), pancreatitis (83.3%), vomiting and/or diarrhea (66.7%), failure to thrive and liver disease (58.3% respectively), among our patients. The predominant organisms found in the airways were Pseudomonas aeruginosa (83.3%) and Staphylococcus aureus (75.0%). The mean concentrations of blood gas and electrolytes were pH 7.58, bicarbonate 40.8 mmol/L, sodium 125.9 mmol/L, chloride 77.5 mmol/L, and potassium 2.6 mmol/L. A high recurrence rate (50.0%) of CF-PBS was observed despite continued electrolyte supplementation during follow-up. In all, 19 different variants of CFTR gene were identified, and 10 of these were found to be novel observations (c.262_266delTTATA[p.L88FfsX21], c.579+2insACAT, c.1210-3C>G, c.1733T>C[p.L578P], c.2236_2246delGAGGCGATACTinsAAAAATC[p.E746KfsX8], c.3068T>G [p.I1023R], c.3635delT[p.V1212AfsX16], c.3859delG[p.G1287EfsX2], c.3964-7A>G and ΔE23 [c.3718-?_3873+?del]). The c.2909G>A[p.G970D] was the most common variant, with an allele frequency of 16.6%. A homozygous genotype of c.1521_1523delCTT[p.F508del] was discovered for the first time in patients of Chinese origin. CONCLUSIONS: In China, CF-PBS usually presents early and recurs frequently in infancy, accompanied by multiple comorbidities. Recurrence of CF-PBS in school-going patients does occur but is rare. The p.G970D is the most frequent variant, with a significant ethnic tendency of Chinese origin.

Dispersal of Epithelium-Associated Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa grows in highly antibiotic-tolerant biofilms during chronic airway infections. Dispersal of bacteria from biofilms may restore antibiotic susceptibility or improve host clearance. We describe models to study biofilm dispersal in the nutritionally complex environment of the human airway. P. aeruginosa was cocultured in the apical surface of airway epithelial cells (AECs) in a perfusion chamber. Dispersal, triggered by sodium nitrite, a nitric oxide (NO) donor, was tracked by live cell microscopy. Next, a static model was developed in which biofilms were grown on polarized AECs without flow. We observed that NO-triggered biofilm dispersal was an energy-dependent process. From the existing literature, NO-mediated biofilm dispersal is regulated by DipA, NbdA, RbdA, and MucR. Interestingly, altered signaling pathways appear to be used in this model, as deletion of these genes failed to block NO-induced biofilm dispersal. Similar results were observed using biofilms grown in an abiotic model on glass with iron-supplemented cell culture medium. In cystic fibrosis, airway mucus contributes to the growth environment, and a wide range of bacterial phenotypes are observed; therefore, we tested biofilm dispersal in a panel of late cystic fibrosis clinical isolates cocultured in the mucus overlying primary human AECs. Finally, we examined dispersal in combination with the clinically used antibiotics ciprofloxacin, aztreonam and tobramycin. In summary, we have validated models to study biofilm dispersal in environments that recapitulate key features of the airway and identified combinations of currently used antibiotics that may enhance the therapeutic effect of biofilm dispersal.IMPORTANCE During chronic lung infections, Pseudomonas aeruginosa grows in highly antibiotic-tolerant communities called biofilms that are difficult for the host to clear. We have developed models for studying P. aeruginosa biofilm dispersal in environments that replicate key features of the airway. We found that mechanisms of biofilm dispersal in these models may employ alternative or additional signaling mechanisms, highlighting the importance of the growth environment in dispersal events. We have adapted the models to accommodate apical fluid flow, bacterial clinical isolates, antibiotics, and primary human airway epithelial cells, all of which are relevant to understanding bacterial behaviors in the context of human disease. We also examined dispersal agents in combination with commonly used antipseudomonal antibiotics and saw improved clearance when nitrite was combined with the antibiotic aztreonam.

Outcomes of pulmonary Mycobacterium abscessus infection.

Background: Treatment of Mycobacterium abscessus pulmonary disease (PD) is challenging with frequent side effects and uncertain rates of success. Methods: We performed a retrospective review of all patients at our center with at least one respiratory sample positive for M. abscessus between 2014 and 2019. Electronic health records were reviewed to determine factors associated with M. abscessus infection and clinical outcomes. Results: Thirty-seven patients were identified including 24 with cystic fibrosis (CF), 10 with bronchiectasis, two with chronic obstructive PD (COPD), and one with asthma. American Thoracic Society/Infectious Diseases Society of America criteria for nontuberculous mycobacteria PD were met in 21/37 (56.8%) of cases. Evidence of Aspergillus lung disease was noted in 18 (75.0%) CF patients compared with 3 (23.1%) non-CF patients (P = 0.005). Induction therapy for M. abscessus was given to 22/37 (59.5%) patients (18/24 [75%] with CF and 4/13 [30.8%] without CF). Median duration of induction therapy was 6 weeks (range 3-12). Maintenance antibiotic therapy was prescribed to 17/22 (77.3%) of treated patients. Culture conversion was seen in 15/24 (62.5%) of CF patients compared with 3/13 (23.1%) in the non-CF group (P = 0.034). Culture conversion occurred in 10/22 (45.5%) of treated patients compared with 8/15 (53.3%) untreated patients. Three patients (8.1%) died during follow-up: one with CF and two with COPD. Conclusions: Culture conversion following isolation of M. abscessus from respiratory samples not only is more common in CF than in patients without CF but also frequently occurs spontaneously in both groups. Targeted treatment for M. abscessus did not clearly impact rates of culture conversion.

Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in Mycobacterium abscessus.

Background: Mycobacterium abscessus is notorious for being intrinsically resistant to most antibiotics. Antibiotic efflux is one of the mechanisms used by M. abscessus to pump out antibiotics from their cells. Inhibiting efflux pumps (EPs) can be an attractive strategy to enhance the activity of drugs. The objective of this study is to determine the activity of EP inhibitors (EPIs) to enhance the efficacy of the new drug bedaquiline against M. abscessus clinical isolates. Methods: A total of 31 phenotypically and genotypically identified M. abscessus subsp. abscessus, M. abscesss subsp. massiliense, and M. abscessus subsp. bolletii clinical isolates were studied. The contribution of EPs was determined by investigating the minimum inhibitory concentration (MIC) levels of bedaquiline reduction in the absence and presence of EPIs verapamil and reserpine using the resazurin microtiter assay. Results: The observed bedaquiline MIC reduction by verapamil was observed in 100% isolates and by reserpine in 54.8% isolates. Bedaquiline MIC was 4-32-fold using verapamil with M. abscessus subsp. bolletii showing the highest fold change and between 2- and 4-fold using reserpine. Conclusions: The results obtained in this study confirm that bedaquiline MIC decreased in the presence of EPIs verapamil and reserpine in clinical isolates of M. abscessus. Verapamil was the most effective EPI. As shown in previous studies, verapamil may have clinical potential as adjunctive therapy to enhance the effect of bedaquiline.

Combination antimicrobial susceptibility testing for acute exacerbations in chronic infection of Pseudomonas aeruginosa in cystic fibrosis.

BACKGROUND: Antibiotic therapy for acute pulmonary exacerbations in people with cystic fibrosis is usually chosen based on the results of antimicrobial susceptibility testing of individual drugs. Combination antimicrobial susceptibility testing assesses the efficacy of drug combinations including two or three antibiotics in vitro and can often demonstrate antimicrobial efficacy against bacterial isolates even when individual antibiotics have little or no effect. Therefore, choosing antibiotics based on combination antimicrobial susceptibility testing could potentially improve response to treatment in people with cystic fibrosis with acute exacerbations. This is an updated version of a previously published review. OBJECTIVES: To compare antibiotic therapy based on conventional antimicrobial susceptibility testing to antibiotic therapy based on combination antimicrobial susceptibility testing in the treatment of acute pulmonary exacerbations in people with cystic fibrosis and chronic infection with Pseudomonas aeruginosa. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Cystic Fibrosis Trials Register which comprises of references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Date of latest search: 19 March 2020. We also searched ongoing trials registries. Date of latest search: 07 April 2020. SELECTION CRITERIA: Randomised and quasi-randomised controlled studies of antibiotic therapy based on conventional antimicrobial susceptibility testing compared to antibiotic therapy based on combination antimicrobial susceptibility testing in the treatment of acute pulmonary exacerbations in cystic fibrosis due to chronic infection with Pseudomonas aeruginosa. DATA COLLECTION AND ANALYSIS: Both authors independently selected studies, assessed their quality and extracted data from eligible studies. Additionally, the authors contacted the study investigators to obtain further information. MAIN RESULTS: The search identified one multicentre study eligible for inclusion in the review. This study prospectively assessed whether the use of multiple combination bactericidal antibiotic testing improved clinical outcomes in participants with acute pulmonary exacerbations of cystic fibrosis who were infected with multiresistant bacteria. A total of 132 participants were randomised in the study. The study investigators provided data specific to the 82 participants who were only infected with Pseudomonas aeruginosa for their primary outcome of time until next pulmonary exacerbation. For participants specifically infected with only Pseudomonas aeruginosa, the hazard ratio of a subsequent exacerbation was 0.82, favouring the control group (95% confidence interval 0.44 to 1.51) (P = 0.52). No further data for any of this review's outcomes specific to participants infected with Pseudomonas aeruginosa were available. The risk of bias for the included study was deemed to be low. The quality of the evidence was moderate for the only outcome providing data solely for individuals with infection due to Pseudomonas aeruginosa. For other outcomes, we were unable to judge the quality of the evidence as no data were available for the relevant subset of participants. AUTHORS' CONCLUSIONS: The current evidence, limited to one study, shows that there is insufficient evidence to determine effect of choosing antibiotics based on combination antimicrobial susceptibility testing compared to choosing antibiotics based on conventional antimicrobial susceptibility testing in the treatment of acute pulmonary exacerbations in people with cystic fibrosis with chronic Pseudomonas aeruginosa infection. A large international and multicentre study is needed to further investigate this issue. The only study included in the review was published in 2005, and we have not identified any further relevant studies up to March 2017. We therefore do not plan to update this review until new studies are published.