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The fate of inhaled antibiotics after deposition in cystic fibrosis: How to get drug to the bug?

Open ArchivePublished:October 26, 2016DOI:https://doi.org/10.1016/j.jcf.2016.10.001

      Abstract

      Background

      Chronic airway infections in patients with cystic fibrosis (CF) are most often treated with inhaled antibiotics of which deposition patterns have been extensively studied. However, the journey of aerosol particles does not end after deposition within the bronchial tree.

      Objectives

      To review how local conditions affect the clinical efficacy of antibiotic aerosol particles after deposition in the airways of patients with CF.

      Methods

      Electronic databases were searched from inception to September 2015. Original studies describing the effect of CF sputum or bacterial factors on antibiotic efficacy and formulations to increase efficacy were included.

      Results

      35 articles were included which mostly described in vitro studies and mainly investigated aminoglycosides. After deposition, diffusion through the mucus layer was reduced for aminoglycosides, β-lactam antibiotics and fluoroquinolones. Within CF mucus, low oxygen tension adversely affected aminoglycosides, β-lactam antibiotics, and chloramphenicol; and molecules inactivated aminoglycosides but not β-lactam antibiotics. Finally, the alginate layer surrounding Pseudomonas aeruginosa was an important factor in the resistance against all antibiotics.

      Conclusions

      After deposition in the airways, the local efficacy of inhaled antibiotics can be reduced by molecules within CF mucus and the alginate layer surrounding P. aeruginosa.

      Abbreviations:

      ABH glycoconjugates (ABO blood group antigens (A=type A, B=type B, H=type O)), AlgL (Alginate lyase), ALX-009&ALX-109 (Drug developed by Alaxia (Lyon, France) that is composed of lactoferrin and hypothiocyanite), CF (Cystic fibrosis), DNA (Deoxyribonucleic acid), ELF (Epithelial lining fluid), GRADE (Grading Recommendations Assessment Development and Evaluation), LPS (Lipopolysaccharides), LTA (Lipoteichoic acid), MBC (Minimal bactericidal concentration), MBEC (Minimum biofilm eradication concentration), MgCl2 (Magnesium chloride), MIC (Minimal inhibitory concentration), MIC50 (Minimum inhibitory concentration that inhibits 50% of the isolates), MIC90 (Minimum inhibitory concentration that inhibits 90% of the isolates), NAC (N-acetylcysteine), NaCl (Sodium chloride), Na2SO4 (Sodium sulfate), Pa (Pseudomonas aeruginosa), pH (Power of hydrogen)

      Keywords

      1. Introduction

      Patients with cystic fibrosis (CF) have difficulties clearing inhaled bacteria from the lungs due to the presence of thick, viscous mucus that obstructs the airways and impairs mucociliary clearance. This causes a relentless cycle of chronic infections and inflammation; leading to progressive lung disease, which is the primary cause of morbidity and mortality in patients with CF.
      Chronic lung infections in CF are mainly treated with inhaled antibiotics, most commonly tobramycin, aztreonam and colistin. Deposition patterns of inhaled antibiotics have been extensively studied. However, after deposition in the airways, the antibiotic must first overcome several challenges before it can perform its activity against the bacteria. Firstly, the aerosol particles need to dissolve in the epithelial lining fluid (ELF) and in mucus.
      Secondly, the antibiotic has to diffuse towards the bacteria of which the location of bacteria in the lungs of patients with CF varies. In case of chronic infections with Pseudomonas aeruginosa (Pa), the location is most likely intraluminal within the mucus and not directly at the airway epithelium or at the surface of the mucus [
      • Worlitzsch D.
      • Tarran R.
      • Ulrich M.
      • Schwab U.
      • Cekici A.
      • Meyer K.C.
      • et al.
      Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.
      ]. As antibiotics diffuse through the mucus layer, they may bind to mucus particles and this is thought to impair the antibiotic bioavailability at the site of infection.
      Thirdly, the antibiotic has to overcome barriers generated by the microorganisms. Multiple microorganisms play a role in CF-related pulmonary infections, although Pa has been studied most extensively. Pa produces a protective slimy layer called alginate [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ] and can grow in a biofilm within the mucus in CF airways. A biofilm is a microcolony of bacteria surrounded by a self-produced polymer matrix, which confers greater resistance against antibiotics [
      • Nichols W.W.
      • Dorrington S.M.
      • Slack M.P.
      • Walmsley H.L.
      Inhibition of tobramycin diffusion by binding to alginate.
      ].
      Hence, many factors influence the clinical efficacy of inhaled antibiotics after deposition and prior to reaching the bacteria. The aim of this systematic review is to provide a critical appraisal of the literature to answer the question, ‘what determines the effect of inhaled antibiotics after deposition into the lungs in patients with CF?’

      2. Methods

      An extensive electronic literature search was conducted to identify as many relevant articles as possible. These articles were published from inception of the databases to September 25 2015, as indexed by Embase, Medline, Web-of-Science, Scopus, Cochrane, PubMed publisher and Google Scholar (Table 1).
      Table 1Search terms.
      DatabaseSearches
      Embase(‘cystic fibrosis’/de OR ‘lung fibrosis’/exp OR (((cyst* OR lung OR pulmonar*) NEAR/3 fibro*) OR fibrocyst* OR mucoviscid* OR cf))

      AND ((((‘antibiotic agent’/exp OR ‘antibiotic therapy’/de OR ‘antiinfective agent’/de OR ‘antibiotic sensitivity’/exp OR ‘antimicrobial activity’/exp OR levofloxacin/de OR (antibiotic* OR antimicrob* OR antibact* OR (anti NEXT/1 (biotic* OR microb* OR bact*)) OR tobramycin* OR colisti* OR colisiti* OR colomycin* OR colymycin* OR (coly NEXT/1 mycin*) OR tadim OR aztreonam OR aminoglycoside* OR amikacin* OR levofloxacin* OR ‘mp 376’ OR azithromycin* OR vancomycin* OR gentamicin OR bactericid*):ab,ti)

      AND (inhalation/de OR ‘oral spray’/de OR ‘inhalational drug administration’/de OR ‘nebulization’/de OR inhaler/exp OR nebulizer/exp OR aerosol/de OR powder/exp OR (inhal* OR vapor* OR vapour* OR aerosol* OR spray* OR mist OR atomi* OR nebuli* OR compressor* OR powder* OR dry OR dried OR jet OR ultraso*):ab,ti)) OR (gernebcin OR tobi OR tsi OR bramitob OR cayston OR azli OR (liposom* NEAR/3 amikacin*)):ab,ti)

      AND (pharmacodynamics/exp OR pharmacokinetics/exp OR ‘drug efficacy’/de OR ‘concentration (parameters)’/exp OR ‘concentration response’/de OR ‘drug sputum level’/de OR ‘sputum analysis’/de OR clearance/exp OR (pharmacodynam* OR pharmacokinet* OR effectiv* OR efficien* OR efficac* OR concentrat* OR sputum* OR mucocilliar* OR mucus OR ((lining OR surface) NEAR/3 (fluid* OR liquid*)) OR clearance* OR ‘half life’):ab,ti))
      MedlineModelled search strategy designed for Embase
      Web-of-ScienceModelled search strategy designed for Embase
      ScopusModelled search strategy designed for Embase
      CochraneModelled search strategy designed for Embase
      PubMed publisherModelled search strategy designed for Embase
      Google ScholarModelled search strategy designed for Embase
      Two independent reviewers (ACB and KP) screened the titles and abstracts for initial eligibility. Every article, considered useful by at least one of the authors was included. Articles were selected based on the following inclusion criteria: (i) effect of CF sputum or (ii) bacterial factors on antibiotic efficacy; (iii) local efficacy determined by antibiotic concentration or number of molecules; (iv) liposomal formulations or co-medication to increase efficacy; and (v) original research. The exclusion criteria are as follows: (i) article not in English or Dutch; (ii) data solely on clinical efficacy of inhaled antibiotics; (iii) no abstract or full text available; (iv) inhaled non-antibiotic drugs; (v) antibiotic combinations; (vi) nanoparticles to increase efficacy and (vii) review/overview.
      Both reviewers assessed the full text of each selected article to ensure that they met the eligibility criteria, developed to critically appraise the selected publications. These criteria were based on the Grading Recommendations Assessment Development and Evaluation (GRADE) criteria by the GRADE working group and the scoring methods of descriptive studies by Slim et al. [
      • Atkins D.
      • Best D.
      • Briss P.A.
      • Eccles M.
      • Falck-Ytter Y.
      • Flottorp S.
      • et al.
      Grading quality of evidence and strength of recommendations.
      ,
      • Slim K.
      • Nini E.
      • Forestier D.
      • Kwiatkowski F.
      • Panis Y.
      • Chipponi J.
      Methodological index for non-randomized studies (minors): development and validation of a new instrument.
      ]. The set of eligibility criteria was more extensive for comparative studies than for non-comparative studies. Using these criteria the relevance and validity of the selected articles were scored independently by the two reviewers. Each criterion received 0, 1 or 2 points (online supplementary Table S1) and the total score was made up by the sum of all criteria. This score was used as a measure of data quality and a total score of 20 and 38 could be obtained for non-comparative and comparative studies, respectively. For both non-comparative and comparative studies, studies were considered relevant for this review and included if more than half of the points for the non-comparative criteria were obtained; thus a total score on critical appraisal of ≥11. For the completed critical appraisal table, see Table S2., Table S3.. Discrepancies between reviewers during the review process were resolved by discussion until a consensus was obtained. Reference lists of the included articles were searched for additional potentially relevant articles, assessed using the same set of criteria.

      3. Results

      3.1 Overview of the included studies

      Out of 2669 articles, 35 articles were included in this review (Fig. 1), 31 full text articles and 4 abstracts. None of the articles described studies in humans. Therefore, all results were drawn from in vitro experiments, except for the efficacy of liposomal formulations of antibiotics, which were investigated both in vitro as well as in vivo in animals. The main factors having an impact on each antibiotic class are described in Table 2. An overview of the main results per step in the pathway of the inhaled antibiotic is described in Table S4 and details of the 35 studies are shown in the online supplementary Table S5.
      Table 2Impact per antibiotic class.
      FactorAntibiotic classImpact
      CF mucus in generalAminoglycosides
      • Reduces diffusion
        • Russo P.
        • Stigliani M.
        • Prota L.
        • Auriemma G.
        • Crescenzi C.
        • Porta A.
        • et al.
        Gentamicin and leucine inhalable powder: what about antipseudomonal activity and permeation through cystic fibrosis mucus?.
      • Strong binding
        • Mendelman P.M.
        • Smith A.L.
        • Levy J.
        • Weber A.
        • Ramsey B.
        • Davis R.L.
        Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum.
        ,
        • Levy J.
        • Smith A.L.
        • Kenny M.A.
        • Ramsey B.
        • Schoenknecht F.D.
        Bioactivity of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis: comparison with activity in serum.
        ,
        • Davis S.D.
        • Bruns W.T.
        Effects of sputum from patients with cystic fibrosis on the activity in vitro of 5 antimicrobial drugs on Pseudomonas aeruginosa.
      β-Lactam AB
      • Reduces diffusion
        • Bolister N.
        • Basker M.
        • Hodges N.A.
        • Marriott C.
        The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      • Negligible binding
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
        ,
        • Davis S.D.
        • Bruns W.T.
        Effects of sputum from patients with cystic fibrosis on the activity in vitro of 5 antimicrobial drugs on Pseudomonas aeruginosa.
      Fluoroquinolones
      • Reduces diffusion
        • Yang Y.
        • Tsifansky M.D.
        • Shin S.
        • Lin Q.
        • Yeo Y.
        Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
        ,
        • Yang Y.
        • Tsifansky M.D.
        • Wu C.J.
        • Yang H.I.
        • Schmidt G.
        • Yeo Y.
        Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis.
      Other (Polymyxin B)
      • Strong binding
        • Davis S.D.
        • Bruns W.T.
        Effects of sputum from patients with cystic fibrosis on the activity in vitro of 5 antimicrobial drugs on Pseudomonas aeruginosa.
      MucinAminoglycosides
      • Strong binding
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
        ,
        • Bataillon V.
        • Lhermitte M.
        • Lafitte J.J.
        • Pommery J.
        • Roussel P.
        The binding of amikacin to macromolecules from the sputum of patients suffering from respiratory diseases.
      • Reduces efficacy of liposomal forms more strongly than free forms
        • Alipour M.
        • Suntres Z.E.
        • Omri A.
        Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      β-Lactam AB
      • Reduces diffusion
        • Bolister N.
        • Basker M.
        • Hodges N.A.
        • Marriott C.
        The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      • No binding
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      DNAAminoglycosides
      • Strong binding
        • Purdy Drew K.R.
        • Sanders L.K.
        • Culumber Z.W.
        • Zribi O.
        • Wong G.C.
        Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
        ,
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
        ,
        • Bataillon V.
        • Lhermitte M.
        • Lafitte J.J.
        • Pommery J.
        • Roussel P.
        The binding of amikacin to macromolecules from the sputum of patients suffering from respiratory diseases.
        ,
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      β-Lactam AB
      • Reduces diffusion
        • Bolister N.
        • Basker M.
        • Hodges N.A.
        • Marriott C.
        The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      • No binding
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      Other (Polymyxin B)
      • No binding
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      Nucleic acidsAminoglycosides
      • Strong binding
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
        ,
        • Bataillon V.
        • Lhermitte M.
        • Lafitte J.J.
        • Pommery J.
        • Roussel P.
        The binding of amikacin to macromolecules from the sputum of patients suffering from respiratory diseases.
      Bacterial endotoxins (LPS, LTA)Aminoglycosides
      • Strong binding
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      Other (Polymyxin B)
      • Binding
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      Anaerobic conditions in mucusAminoglycosides
      • Reduces efficacy
        • Hill D.
        • Rose B.
        • Pajkos A.
        • Robinson M.
        • Bye P.
        • Bell S.
        • et al.
        Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
        ,
        • King P.
        • Citron D.M.
        • Griffith D.C.
        • Lomovskaya O.
        • Dudley M.N.
        Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
        ,
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      β-Lactam AB
      • Reduces efficacy
        • Hill D.
        • Rose B.
        • Pajkos A.
        • Robinson M.
        • Bye P.
        • Bell S.
        • et al.
        Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
        ,
        • King P.
        • Citron D.M.
        • Griffith D.C.
        • Lomovskaya O.
        • Dudley M.N.
        Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
        ,
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      Macrolides
      • Reduces efficacy
        • Hill D.
        • Rose B.
        • Pajkos A.
        • Robinson M.
        • Bye P.
        • Bell S.
        • et al.
        Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      Fluoroquinolones
      • Reduces efficacy
        • Hill D.
        • Rose B.
        • Pajkos A.
        • Robinson M.
        • Bye P.
        • Bell S.
        • et al.
        Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
        ,
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
        , remain bactericidal
        • King P.
        • Citron D.M.
        • Griffith D.C.
        • Lomovskaya O.
        • Dudley M.N.
        Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      Tetracyclines
      • Reduces efficacy
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      Other
      • Cotrimoxazol: Reduces efficacy
        • Hill D.
        • Rose B.
        • Pajkos A.
        • Robinson M.
        • Bye P.
        • Bell S.
        • et al.
        Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      • Chloramphenicol: Reduces efficacy
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      • Colistin: Reduces efficacy
        • Hill D.
        • Rose B.
        • Pajkos A.
        • Robinson M.
        • Bye P.
        • Bell S.
        • et al.
        Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
        , increases efficacy
        • Pompilio A.
        • Crocetta V.
        • Pomponio S.
        • Fiscarelli E.
        • Di Bonaventura G.
        In vitro activity of colistin against biofilm by Pseudomonas aeruginosa is significantly improved under “cystic fibrosis-like” physicochemical conditions.
      Salt content in mucusAminoglycosides
      • Reduces efficacy (magnesium, calcium, nitrate)
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
        ,
        • Beggs W.H.
        • Andrews F.A.
        Role of ionic strength in salt antagonism of aminoglycoside action on Escherichia coli and Pseudomonas aeruginosa.
      • Decreases binding to alginate (sodium chloride, potassium, calcium, phosphate)
        • Gordon C.A.
        • Hodges N.A.
        • Marriott C.
        Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
        ,
        • Tannenbaum C.S.
        • Hastie A.T.
        • Higgins M.L.
        • Kueppers F.
        • Weinbaum G.
        Inability of purified Pseudomonas aeruginosa exopolysaccharide to bind selected antibiotics.
      β-Lactam AB
      • Carbenicillin: nitrate increases efficacy if aerobic, but effect abolished if anaerobic
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      • Ceftazidime: nitrate no effect
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      Fluoroquinolones
      • Nitrate decreases susceptibility of organisms to AB
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      Tetracyclines
      • Nitrate no effect
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      Other (Chloramphenicol)
      • Nitrate no effect
        • Borriello G.
        • Werner E.
        • Roe F.
        • Kim A.M.
        • Ehrlich G.D.
        • Stewart P.S.
        Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      AlginateAminoglycosides
      • Reduces diffusion
        • Nichols W.W.
        • Dorrington S.M.
        • Slack M.P.
        • Walmsley H.L.
        Inhibition of tobramycin diffusion by binding to alginate.
        ,
        • Slack M.P.
        • Nichols W.W.
        The penetration of antibiotics through sodium alginate and through the exopolysaccharide of a mucoid strain of Pseudomonas aeruginosa.
        ,
        • Gordon C.A.
        • Hodges N.A.
        • Marriott C.
        Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
        ,
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      • Binding
        • Nichols W.W.
        • Dorrington S.M.
        • Slack M.P.
        • Walmsley H.L.
        Inhibition of tobramycin diffusion by binding to alginate.
        ,
        • Gordon C.A.
        • Hodges N.A.
        • Marriott C.
        Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
        ,
        • Tannenbaum C.S.
        • Hastie A.T.
        • Higgins M.L.
        • Kueppers F.
        • Weinbaum G.
        Inability of purified Pseudomonas aeruginosa exopolysaccharide to bind selected antibiotics.
        ,
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      β-Lactam AB
      • Reduces diffusion
        • Bolister N.
        • Basker M.
        • Hodges N.A.
        • Marriott C.
        The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      Tetracyclines
      • Reduces diffusion
        • Bolister N.
        • Basker M.
        • Hodges N.A.
        • Marriott C.
        The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      Other (Polymyxin B)
      • Reduces diffusion
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      • Binding
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      Liposomal formulationAminoglycosides
      • Increases efficacy
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
        ,
        • Meers P.
        • Neville M.
        • Malinin V.
        • Scotto A.W.
        • Sardaryan G.
        • Kurumunda R.
        • et al.
        Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections.
        ,
        • Halwani M.
        • Mugabe C.
        • Azghani A.O.
        • Lafrenie R.M.
        • Kumar A.
        • Omri A.
        Bactericidal efficacy of liposomal aminoglycosides against Burkholderia cenocepacia.
        ,
        • Mugabe C.
        • Azghani A.O.
        • Omri A.
        Liposome-mediated gentamicin delivery: development and activity against resistant strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients.
        , decreases efficacy
        • Alipour M.
        • Suntres Z.E.
        • Omri A.
        Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      • Reduces binding
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      Other (Polymyxin B)
      • Increases efficacy
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      • Reduces binding
        • Alipour M.
        • Suntres Z.E.
        • Halwani M.
        • Azghani A.O.
        • Omri A.
        Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      Dornase alfaAminoglycosides
      • Increases binding
        • Hunt B.E.
        • Weber A.
        • Berger A.
        • Ramsey B.
        • Smith A.L.
        Macromolecular mechanisms of sputum inhibition of tobramycin activity.
        , decreases binding
        • Ramphal R.
        • Lhermitte M.
        • Filliat M.
        • Roussel P.
        The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      • Enhances bactericidal efficacy
        • Alipour M.
        • Suntres Z.E.
        • Omri A.
        Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
        , reduces bactericidal efficacy
        • Hunt B.E.
        • Weber A.
        • Berger A.
        • Ramsey B.
        • Smith A.L.
        Macromolecular mechanisms of sputum inhibition of tobramycin activity.
      Fluoroquinolones
      • Enhances bactericidal efficacy
        • Yang Y.
        • Tsifansky M.D.
        • Wu C.J.
        • Yang H.I.
        • Schmidt G.
        • Yeo Y.
        Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis.
      • Enhances diffusion
        • Yang Y.
        • Tsifansky M.D.
        • Wu C.J.
        • Yang H.I.
        • Schmidt G.
        • Yeo Y.
        Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis.
      MannitolAminoglycosides
      • Enhances bactericidal efficacy
        • Barraud N.
        • Buson A.
        • Jarolimek W.
        • Rice S.A.
        Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      Fluoroquinolones
      • Enhances bactericidal efficacy
        • Ong H.X.
        • Loo C.Y.
        • Lee W.H.
        • Traini D.
        • Whitchurch C.
        • Young P.
        Late-breaking abstract: sweetening antibiotic treatment for eradication of bacteria biofilm.
      • Enhances diffusion
        • Yang Y.
        • Tsifansky M.D.
        • Shin S.
        • Lin Q.
        • Yeo Y.
        Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
      Alginate lyaseAminoglycosides
      • Improves diffusion
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      • Tobramycin and amikacin: Enhances bactericidal efficacy
        • Alipour M.
        • Suntres Z.E.
        • Omri A.
        Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
        ,
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      • Gentamicin: Enhances bactericidal efficacy
        • Hatch R.A.
        • Schiller N.L.
        Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
        , no effect on bactericidal efficacy
        • Alipour M.
        • Suntres Z.E.
        • Omri A.
        Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      NaClAminoglycosides
      • Enhances bactericidal efficacy of tobramycin
        • Barraud N.
        • Buson A.
        • Jarolimek W.
        • Rice S.A.
        Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
        , antagonistic effect on tobramycin
        • Potter R.
        • Hatley R.H.M.
        Effect of saline concentration on the minimum inhibitory concentration of colistimethate sodium and tobramycin.
      Fluoroquinolones
      • Enhances bactericidal efficacy
        • Yang Y.
        • Tsifansky M.D.
        • Shin S.
        • Lin Q.
        • Yeo Y.
        Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
      • No effect on diffusion
        • Yang Y.
        • Tsifansky M.D.
        • Shin S.
        • Lin Q.
        • Yeo Y.
        Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
      Other (Colistin)
      • Synergistic effect
        • Potter R.
        • Hatley R.H.M.
        Effect of saline concentration on the minimum inhibitory concentration of colistimethate sodium and tobramycin.
      GlucoseAminoglycosides
      • Enhances bactericidal efficacy
        • Barraud N.
        • Buson A.
        • Jarolimek W.
        • Rice S.A.
        Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      ALX-109&ALX-009Aminoglycosides
      • Enhances bactericidal efficacy
        • Moreau-Marquis S.
        • Coutermarsh B.
        • Stanton B.A.
        Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.
        ,
        • Moreau-Marquis S.
        • Perrotto S.
        • Stanton B.A.
        ALX-009 potentiates the effect of tobramycin at killing Pseudomonas aeruginosa biofilms on human airway cells.
        ,
        • Moreau-Marquis S.
        • Drexinger J.K.
        • Perrotto S.
        • Stanton B.A.
        Treatment with ALX-009 in combination with aztreonam potentiates the killing of Pseudomonas aeruginosa biofilms on human CF airway cells.
      β-Lactam AB
      • Enhances bactericidal efficacy
        • Moreau-Marquis S.
        • Coutermarsh B.
        • Stanton B.A.
        Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.
        ,
        • Moreau-Marquis S.
        • Perrotto S.
        • Stanton B.A.
        ALX-009 potentiates the effect of tobramycin at killing Pseudomonas aeruginosa biofilms on human airway cells.
        ,
        • Moreau-Marquis S.
        • Drexinger J.K.
        • Perrotto S.
        • Stanton B.A.
        Treatment with ALX-009 in combination with aztreonam potentiates the killing of Pseudomonas aeruginosa biofilms on human CF airway cells.
      Cationic amphiphilesAminoglycosides
      • Reduces binding with DNA
        • Purdy Drew K.R.
        • Sanders L.K.
        • Culumber Z.W.
        • Zribi O.
        • Wong G.C.
        Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
      CitrateAminoglycosides
      Macrolides
      Other
      Succinic acidAminoglycosides
      Other
      AB = antibiotic; CF = cystic fibrosis; LPS = lipopolysaccharides; LTA = lipoteichoic acid.
      The following antibiotic classes were studied: aminoglycosides (n = 28 studies), β-lactam antibiotics (n = 12), fluoroquinolones (n = 2) and other antibiotics (n = 9).

      3.2 Physiology of antibiotic availability in airway secretions

      3.2.1 Solubilization of antibiotic in mucus layer (Table S5, part 1)

      After deposition on the mucus layer, local bioavailability of the inhaled antibiotic first depends on the solubility of the drug in the mucus. With solubilization we refer to the absorption of antibiotic aerosols by CF mucus. Within CF patients the secretor and non-secretor phenotypes are described. This refers to a genetic trait defining whether there is or there is no expression of ABH glycoconjugates in exocrine secretions. These are glycoproteins and glycolipids structurally related to antigens which define ABO blood groups. The presence or absence of these molecules alters the biochemical properties of exocrine secretions [
      • Barboza M.A.
      • Brandao de Mattos C.C.
      • Barja P.R.
      • Franco de Oliveira L.V.
      • de Mattos L.C.
      Influence of secretor and non-secretor phenotypes on the solubilization of pulmonary mucus by three common medicines in cystic fibrosis patients assessed using photoacoustic analysis.
      ]. Tobramycin was shown to dissolve more effective in mucus of secretors relative to non-secretors. Carriers of this secretor phenotype reached the typical interaction time of solubilization quicker than non-secretors. However, the question is if this is clinically relevant as the difference was 5 min and the total time it took to dissolve in mucus was similar [
      • Barboza M.A.
      • Brandao de Mattos C.C.
      • Barja P.R.
      • Franco de Oliveira L.V.
      • de Mattos L.C.
      Influence of secretor and non-secretor phenotypes on the solubilization of pulmonary mucus by three common medicines in cystic fibrosis patients assessed using photoacoustic analysis.
      ,
      • Barja P.R.
      • Coelho C.C.
      • Paiva R.F.
      • Barboza M.A.
      • Matos L.C.
      • Matos C.C.B.
      • et al.
      Photoacoustic analysis of the solubilization kinetics of pulmonary secretions from cystic fibrosis patients – secretor and non-secretor phenotypes.
      ].

      3.2.2 Diffusion through mucus layer (Table S5, part 2)

      Mucus primarily consists of mucin (glycoprotein) but also contains proteins, DNA, lipids and cellular debris. After solubilization, the drug needs to diffuse through the mucus layer to reach the bacteria, whereby the viscosity and elasticity of CF mucus are increased due to various factors. With diffusion we refer to the process in which there is movement of aerosols from an area of high concentration of aerosol to an area of lower concentration. Mucin molecules in CF mucus are very long, extensively branched and have been shown to interact with other macromolecules in mucus secretions, including: albumin which increased its viscosity, and DNA which increased its elasticity [
      • Bhat P.G.
      • Flanagan D.R.
      • Donovan M.D.
      Drug diffusion through cystic fibrotic mucus: steady-state permeation, rheologic properties, and glycoprotein morphology.
      ]. Additionally, elevated concentrations of actin and alginate contributed to the increased viscoelasticity of CF mucus [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ]. Due to the length of the mucin glycoprotein backbone and the presence of branching, CF mucins had a higher tendency to interact and obstruct drug transport in vitro [
      • Bhat P.G.
      • Flanagan D.R.
      • Donovan M.D.
      Drug diffusion through cystic fibrotic mucus: steady-state permeation, rheologic properties, and glycoprotein morphology.
      ]. In particular liposomal antibiotics were delayed in their diffusion by mucins present in mucus [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ].
      For β-lactam antibiotics, the presence of mucin alone caused a 2-fold delay in the diffusion rate (1.1 to 0.5 μm/mm2/h) compared to the baseline rate determined in buffer. The addition of DNA resulted in a 10-fold delay in the rate of diffusion (0.1 μm/mm2/h) [
      • Bolister N.
      • Basker M.
      • Hodges N.A.
      • Marriott C.
      The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      ].
      Aminoglycoside diffusion was also delayed by mucus of CF patients compared to buffer, but no specific numbers were reported [
      • Russo P.
      • Stigliani M.
      • Prota L.
      • Auriemma G.
      • Crescenzi C.
      • Porta A.
      • et al.
      Gentamicin and leucine inhalable powder: what about antipseudomonal activity and permeation through cystic fibrosis mucus?.
      ].

      3.2.2.1 Methods to improve diffusion through the mucus layer

      Combinations of ciprofloxacin dry powder with mannitol showed enhanced diffusion and significantly higher antibacterial activity against Pa than ciprofloxacin–NaCl or ciprofloxacin–lactose particles [
      • Yang Y.
      • Tsifansky M.D.
      • Shin S.
      • Lin Q.
      • Yeo Y.
      Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
      ] (also against Pa growing in biofilm) [
      • Ong H.X.
      • Loo C.Y.
      • Lee W.H.
      • Traini D.
      • Whitchurch C.
      • Young P.
      Late-breaking abstract: sweetening antibiotic treatment for eradication of bacteria biofilm.
      ]. Ciprofloxacin–NaCl particles also showed higher antibacterial activity against Pa (albeit to a lesser extent), although NaCl alone had no effect on drug diffusion [
      • Yang Y.
      • Tsifansky M.D.
      • Shin S.
      • Lin Q.
      • Yeo Y.
      Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
      ]. Finally, ciprofloxacin–dornase alfa powder showed greater antibacterial activity than ciprofloxacin dry powder due to the better dissolution and diffusion abilities of ciprofloxacin [
      • Yang Y.
      • Tsifansky M.D.
      • Wu C.J.
      • Yang H.I.
      • Schmidt G.
      • Yeo Y.
      Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis.
      ]. Ciprofloxacin–dornase alfa powder completely diffused after 30 min while ciprofloxacin powder alone was not completely dissolved even after 2 h [
      • Yang Y.
      • Tsifansky M.D.
      • Wu C.J.
      • Yang H.I.
      • Schmidt G.
      • Yeo Y.
      Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis.
      ]. Mannitol, NaCl and dornase alfa are safe substances used for inhalation in CF. Phase I clinical trials for inhalable powder form of ciprofloxacin have been completed. The combinations have, so far, only been investigated in in vitro studies.
      In summary, the diffusion rate of aminoglycosides, β-lactam antibiotics and fluoroquinolones through CF mucus is reduced but may be increased by coadministration with mannitol or dornase alfa.

      3.3 Other physiological issues

      3.3.1 Influence of oxygen level in mucus (Table S5, part 3)

      Thickened mucus layers in the lungs of CF patients contain areas of low oxygen tension [
      • King P.
      • Citron D.M.
      • Griffith D.C.
      • Lomovskaya O.
      • Dudley M.N.
      Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      ], with an anaerobic environment near the epithelial surface and higher oxygen levels at the top of the mucus layer [
      • Worlitzsch D.
      • Tarran R.
      • Ulrich M.
      • Schwab U.
      • Cekici A.
      • Meyer K.C.
      • et al.
      Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.
      ].
      Aminoglycosides [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ,
      • King P.
      • Citron D.M.
      • Griffith D.C.
      • Lomovskaya O.
      • Dudley M.N.
      Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      ], β-lactam antibiotics [
      • King P.
      • Citron D.M.
      • Griffith D.C.
      • Lomovskaya O.
      • Dudley M.N.
      Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      ,
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ] and chloramphenicol [
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ] showed reduced efficacies under anaerobic conditions, whereas tobramycin and ciprofloxacin were approximately twice less effective. The efficacy of antibiotics against Pa infections can be measured in different ways. The minimal inhibitory concentration (MIC) is the lowest concentration of an antibiotic that prevents visible growth of Pa and the minimal bactericidal concentration (MBC) is the concentration that results in Pa death. Both are tested using planktonic bacteria. The minimal biofilm eradication concentration (MBEC) is the concentration of an antibiotic required to kill Pa in biofilms. For tobramycin, 50% of planktonic Pa isolates were killed under aerobic conditions, 30% under anaerobic conditions [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ] and the log reduction dropped from 5.67 ± 0.00 to 2.14 ± 0.42 in 4 hour old biofilms [
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ]. For ciprofloxacin, the log reduction dropped from 5.05 ± 0.31 to 2.61 ± 0.13 in 4-h-old biofilms under anaerobic conditions [
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ]. On the contrary, levofloxacin maintained its bactericidal effect under anaerobic conditions [
      • King P.
      • Citron D.M.
      • Griffith D.C.
      • Lomovskaya O.
      • Dudley M.N.
      Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      ]. Likewise, colistin may even be more effective under anaerobic conditions. Reductions in MBECs, MIC50, MIC90, and MBC (2-fold, 8-fold, 4-fold and 2-fold reductions, respectively) against Pa were shown under anaerobic conditions [
      • Pompilio A.
      • Crocetta V.
      • Pomponio S.
      • Fiscarelli E.
      • Di Bonaventura G.
      In vitro activity of colistin against biofilm by Pseudomonas aeruginosa is significantly improved under “cystic fibrosis-like” physicochemical conditions.
      ]. However, another study found decreased killing of planktonic Pa isolates by colistin under anaerobic compared to aerobic conditions (75% vs 100%) [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ].
      In summary, low oxygen tension reduced the efficacy of aminoglycosides, β-lactam antibiotics and chloramphenicol. Colistin, however, may be more efficacious.

      3.3.2 Influence of salt content of mucus (Table S5, part 4)

      The antibacterial activity of certain antibiotics is highly dependent on the ionic environment [
      • Levy J.
      • Smith A.L.
      • Kenny M.A.
      • Ramsey B.
      • Schoenknecht F.D.
      Bioactivity of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis: comparison with activity in serum.
      ]. In CF, the ionic environment of mucus changes as a result of cell lysis; as evidenced by higher calcium levels detected in CF mucus compared to non-CF patients [
      • Bhat P.G.
      • Flanagan D.R.
      • Donovan M.D.
      Drug diffusion through cystic fibrotic mucus: steady-state permeation, rheologic properties, and glycoprotein morphology.
      ].
      Magnesium and calcium have a stabilizing influence on the cell walls of Pa, which is primarily driven by divalent cations, and thereby delay the effect of aminoglycosides [
      • Beggs W.H.
      • Andrews F.A.
      Role of ionic strength in salt antagonism of aminoglycoside action on Escherichia coli and Pseudomonas aeruginosa.
      ]. The monovalent salt sodium did not have any measurable effect on gentamicin, while the divalent magnesium salt completely shielded Pa from its activity [
      • Beggs W.H.
      • Andrews F.A.
      Role of ionic strength in salt antagonism of aminoglycoside action on Escherichia coli and Pseudomonas aeruginosa.
      ].
      For Escherichia coli, the protection by salts could be solely attributed to ionic strength and not to the type of salt. When the ionic strength was increased in vitro from 0.12 to 0.14 μ with MgCl2, NaCl or Na2SO4, gentamicin activity against E. coli ranged between 40 and 50%.
      Under anaerobic conditions, nitrate decreased the bactericidal activity of aminoglycosides and fluoroquinolones by half [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ,
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ]. Mucus of CF patients contains 250–350 μmol/l nitrate and concentrations can be as high as 1000 μmol/l [
      • King P.
      • Citron D.M.
      • Griffith D.C.
      • Lomovskaya O.
      • Dudley M.N.
      Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      ]. Nitrate had little effect on the efficacies of chloramphenicol and ceftazidime [
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ].
      In summary, the ionic environment is another important variable that can reduce the effectiveness of inhaled antibiotics against Pa.

      3.4 Mucus binding (Table S5, part 5)

      Apart from delaying the diffusion of antibiotics, macromolecules present in mucus can bind to certain antibiotics and drastically reduce their efficacy, as only free drug can be active against bacteria. In particular, the efficacy of aminoglycosides (cations) is reduced [
      • Purdy Drew K.R.
      • Sanders L.K.
      • Culumber Z.W.
      • Zribi O.
      • Wong G.C.
      Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
      ], due to their electrostatic interactions with anionic electrolytes [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ].
      Tobramycin [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ,
      • Hunt B.E.
      • Weber A.
      • Berger A.
      • Ramsey B.
      • Smith A.L.
      Macromolecular mechanisms of sputum inhibition of tobramycin activity.
      ,
      • Mendelman P.M.
      • Smith A.L.
      • Levy J.
      • Weber A.
      • Ramsey B.
      • Davis R.L.
      Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum.
      ], amikacin [
      • Bataillon V.
      • Lhermitte M.
      • Lafitte J.J.
      • Pommery J.
      • Roussel P.
      The binding of amikacin to macromolecules from the sputum of patients suffering from respiratory diseases.
      ] and gentamicin [
      • Mendelman P.M.
      • Smith A.L.
      • Levy J.
      • Weber A.
      • Ramsey B.
      • Davis R.L.
      Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum.
      ,
      • Levy J.
      • Smith A.L.
      • Kenny M.A.
      • Ramsey B.
      • Schoenknecht F.D.
      Bioactivity of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis: comparison with activity in serum.
      ] have been shown to bind to mucin and free DNA. Their affinity for mucin was higher in the presence of free DNA [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ,
      • Hunt B.E.
      • Weber A.
      • Berger A.
      • Ramsey B.
      • Smith A.L.
      Macromolecular mechanisms of sputum inhibition of tobramycin activity.
      ]. The latter is released from lysed leukocytes and bacteria during infections [
      • Hunt B.E.
      • Weber A.
      • Berger A.
      • Ramsey B.
      • Smith A.L.
      Macromolecular mechanisms of sputum inhibition of tobramycin activity.
      ]. In mucus collected from CF patients during exacerbations, a substantial fraction of inhaled tobramycin was bound to mucus [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ], which reduced its activity by a factor of approximately 30 [
      • Purdy Drew K.R.
      • Sanders L.K.
      • Culumber Z.W.
      • Zribi O.
      • Wong G.C.
      Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
      ]. Binding was dependent on tobramycin concentration [
      • Mendelman P.M.
      • Smith A.L.
      • Levy J.
      • Weber A.
      • Ramsey B.
      • Davis R.L.
      Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum.
      ] and the concentration of macromolecules [
      • Barboza M.A.
      • Brandao de Mattos C.C.
      • Barja P.R.
      • Franco de Oliveira L.V.
      • de Mattos L.C.
      Influence of secretor and non-secretor phenotypes on the solubilization of pulmonary mucus by three common medicines in cystic fibrosis patients assessed using photoacoustic analysis.
      ,
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ], in which higher tobramycin concentrations resulted in higher concentrations of free drug. Thirty percent of tobramycin was bound at tobramycin concentrations of 5–15 μg/ml while 15% was bound at concentrations of 25–50 μg/ml [
      • Mendelman P.M.
      • Smith A.L.
      • Levy J.
      • Weber A.
      • Ramsey B.
      • Davis R.L.
      Aminoglycoside penetration, inactivation, and efficacy in cystic fibrosis sputum.
      ].
      Tobramycin (15–95%) exhibited the strongest binding to mucins and DNA [
      • Purdy Drew K.R.
      • Sanders L.K.
      • Culumber Z.W.
      • Zribi O.
      • Wong G.C.
      Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
      ]. Up to 60% (range 1–60%) of amikacin [
      • Bataillon V.
      • Lhermitte M.
      • Lafitte J.J.
      • Pommery J.
      • Roussel P.
      The binding of amikacin to macromolecules from the sputum of patients suffering from respiratory diseases.
      ] and 52% (range not reported) of gentamicin were bound to mucus of patients with CF [
      • Levy J.
      • Smith A.L.
      • Kenny M.A.
      • Ramsey B.
      • Schoenknecht F.D.
      Bioactivity of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis: comparison with activity in serum.
      ].
      Aminoglycosides show lower affinity to negatively charged components in mucus in an alkalotic compared to an acidic environment [
      • Levy J.
      • Smith A.L.
      • Kenny M.A.
      • Ramsey B.
      • Schoenknecht F.D.
      Bioactivity of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis: comparison with activity in serum.
      ]. The pH in CF airways (not measured in mucus) varies from 6.5 to 7.5 and seems to be constant from central to peripheral airways. In patients with pneumonia, the pH in infected bronchi was significantly lower than that in non-infected bronchi (6.48 versus 6.69) [
      • Bodem C.R.
      • Lampton L.M.
      • Miller D.P.
      • Tarka E.F.
      • Everett E.D.
      Endobronchial pH. Relevance of aminoglycoside activity in gram-negative bacillary pneumonia.
      ].
      Polymyxin B and neomycin show strongly elevated MBCs due to binding to CF mucus [
      • Davis S.D.
      • Bruns W.T.
      Effects of sputum from patients with cystic fibrosis on the activity in vitro of 5 antimicrobial drugs on Pseudomonas aeruginosa.
      ,
      • Alipour M.
      • Suntres Z.E.
      • Halwani M.
      • Azghani A.O.
      • Omri A.
      Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      ]. Polymyxin B appeared to bind to bacterial endotoxins within CF mucus, but not to DNA or actin filaments [
      • Alipour M.
      • Suntres Z.E.
      • Halwani M.
      • Azghani A.O.
      • Omri A.
      Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      ]. No binding was observed between mucus and β-lactam antibiotics [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ,
      • Davis S.D.
      • Bruns W.T.
      Effects of sputum from patients with cystic fibrosis on the activity in vitro of 5 antimicrobial drugs on Pseudomonas aeruginosa.
      ] and the role of mucus binding in fluoroquinolones or macrolides was not studied in the selected articles.

      3.4.1 Methods to reduce drug–mucus interaction

      3.4.1.1 Liposome-entrapment

      Most studies show that liposome-entrapment reduces antibiotic inhibition by macromolecules and enhances the bactericidal activity of antibiotics. Inhibition of aminoglycosides by DNA and actin filaments, and by bacterial endotoxins was reduced by 4-fold and 100-fold, respectively, when entrapped in liposomes [
      • Alipour M.
      • Suntres Z.E.
      • Halwani M.
      • Azghani A.O.
      • Omri A.
      Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.
      ]. Additionally, liposomal formulations were significantly more efficacious in reducing the Pa load in rats [
      • Meers P.
      • Neville M.
      • Malinin V.
      • Scotto A.W.
      • Sardaryan G.
      • Kurumunda R.
      • et al.
      Biofilm penetration, triggered release and in vivo activity of inhaled liposomal amikacin in chronic Pseudomonas aeruginosa lung infections.
      ] and reduced the MICs for Pa strains in vitro and Burkholderia cenocepacia strains in rats [
      • Halwani M.
      • Mugabe C.
      • Azghani A.O.
      • Lafrenie R.M.
      • Kumar A.
      • Omri A.
      Bactericidal efficacy of liposomal aminoglycosides against Burkholderia cenocepacia.
      ,
      • Mugabe C.
      • Azghani A.O.
      • Omri A.
      Liposome-mediated gentamicin delivery: development and activity against resistant strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients.
      ]. However, one study showed that liposomal aminoglycoside activity was inhibited to a greater extent by mucins than free aminoglycosides (up to 32-fold vs up to 8-fold) [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ]. Phase III trials of inhaled liposomal amikacin are currently running. To the best of our knowledge, liposomal formulations of gentamicin, tobramycin and polymyxin B are still in a pre-clinical phase.

      3.4.1.2 Co-treatment with dornase alfa

      Conflicting results were described for the co-treatment of aminoglycosides with dornase alfa. Dornase alfa was shown to increase free tobramycin in sputum by approximately 30% [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ] and enhance the bactericidal activity of free and liposomal aminoglycosides. Specifically, the higher the concentration of dornase alfa, the stronger the bactericidal activity [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ].
      Conversely, another study reported the decline in free tobramycin and its activity following dornase alfa treatment [
      • Hunt B.E.
      • Weber A.
      • Berger A.
      • Ramsey B.
      • Smith A.L.
      Macromolecular mechanisms of sputum inhibition of tobramycin activity.
      ]. A possible explanation given by the authors was that while dornase alfa did indeed cut the DNA into smaller strands, the charge of the strands remained unchanged. Therefore, the smaller strands were still able to bind positively charged antibiotics.

      3.4.1.3 Cationic amphiphiles and N-acetylcysteine

      Cationic amphiphiles are positively charged lipid solutions that have the potential to decrease binding between DNA and tobramycin and thereby enhance its antibacterial activity. They altered the binding affinity between tobramycin and DNA. By matching the cationic amphiphiles in charge and shape, the binding to DNA was enhanced and tobramycin was competitively displaced from DNA complexes by these agents. This resulted in a 15-fold increase in tobramycin activity. Whether these agents could play a role in clinical care needs to be further investigated [
      • Purdy Drew K.R.
      • Sanders L.K.
      • Culumber Z.W.
      • Zribi O.
      • Wong G.C.
      Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
      ].
      Co-treatment with 1 mg/ml of the mucolytic N-acetylcysteine (NAC) did not influence the bactericidal activity of free and liposomal aminoglycosides [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ].

      3.4.1.4 Co-treatment with NaCl

      Co-administration of NaCl (tested NaCl concentrations: 0.3%, 0.9%, 2.3% and 4.05%) had a synergistic effect with colistin at a concentration of 4.05% for the treatment of Pa and at all concentrations for E. coli [
      • Potter R.
      • Hatley R.H.M.
      Effect of saline concentration on the minimum inhibitory concentration of colistimethate sodium and tobramycin.
      ]. Studies investigating the coadministration of NaCl and tobramycin are inconclusive. Both an antagonistic effect on tobramycin [
      • Potter R.
      • Hatley R.H.M.
      Effect of saline concentration on the minimum inhibitory concentration of colistimethate sodium and tobramycin.
      ], as well as improved tobramycin efficacy against young Pa biofilms [
      • Barraud N.
      • Buson A.
      • Jarolimek W.
      • Rice S.A.
      Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      ] were shown when NaCl was added. Nevertheless, in patients with CF who use one of these antibiotic formulations and inhaled saline, the timing of inhaled saline in relation to tobramycin inhalation will need to be taken into account [
      • Potter R.
      • Hatley R.H.M.
      Effect of saline concentration on the minimum inhibitory concentration of colistimethate sodium and tobramycin.
      ].
      In summary, mucus binding appears to reduce the efficacy of aminoglycosides but not β-lactam antibiotics, and may be reduced by liposome-entrapment or coadministration of amphiphilic molecules.

      3.5 Barriers generated by P. aeruginosa

      3.5.1 Non-mucoid Pa, mucoid Pa and alginate formation

      Pa has the ability to grow under aerobic and anaerobic circumstances and exists in both a mucoid and non-mucoid formation. Chronic Pa infections are associated with more mucoid variants that produce the polysaccharide alginate and form biofilms within the lungs of patients with CF. Alginate is an important factor in the resistance of Pa against antibiotics, as it increases the colonization rate within the respiratory tract. Importantly, alginate seems to act as an ionic trapping agent for positively charged aminoglycosides and polymyxin B, thereby reducing the uptake and early bactericidal effect of antibiotics. Additionally, alginate inhibits the non-opsonic phagocytosis of monocytes and neutrophils, thus allowing the bacteria to avoid the phagocytic immune response [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ]. Due to these barriers, achieving an inhibitory concentration at the surface of a mucoid colony is not sufficient to eliminate Pa. To kill the bacteria, bactericidal antibiotic concentrations need to be attained at the cell surface for a sufficient period of time [
      • Bolister N.
      • Basker M.
      • Hodges N.A.
      • Marriott C.
      The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      ].

      3.5.2 Alginate and diffusion of antibiotics (Table S5, part 6)

      Alginate reduces the diffusion of antibiotics in vitro and is evidenced by the fact that aminoglycosides exhibited diffusion coefficients in alginate, which were approximately 20% of the β-lactam values (0.65 versus 3.7 × 10−6 cm2/s) [
      • Nichols W.W.
      • Dorrington S.M.
      • Slack M.P.
      • Walmsley H.L.
      Inhibition of tobramycin diffusion by binding to alginate.
      ,
      • Slack M.P.
      • Nichols W.W.
      The penetration of antibiotics through sodium alginate and through the exopolysaccharide of a mucoid strain of Pseudomonas aeruginosa.
      ,
      • Gordon C.A.
      • Hodges N.A.
      • Marriott C.
      Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
      ]. This can be attributed to the fact that not only does the alginate itself form a physical barrier to the antibiotic, but also because the positively charged aminoglycosides (in contrast to the β-lactam antibiotics) bind to the negatively charged alginate polymers [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ,
      • Nichols W.W.
      • Dorrington S.M.
      • Slack M.P.
      • Walmsley H.L.
      Inhibition of tobramycin diffusion by binding to alginate.
      ,
      • Tannenbaum C.S.
      • Hastie A.T.
      • Higgins M.L.
      • Kueppers F.
      • Weinbaum G.
      Inability of purified Pseudomonas aeruginosa exopolysaccharide to bind selected antibiotics.
      ]. This is further evidenced by the fact that 2% Pa alginate suspension completely inhibited the diffusion of gentamicin, tobramycin and polymyxin B, whereas the diffusion of the negatively charged carbenicillin was not impeded by this suspension [
      • Hatch R.A.
      • Schiller N.L.
      Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      ]. Furthermore, diffusion rates for tobramycin were consistently lower than for gentamicin [
      • Gordon C.A.
      • Hodges N.A.
      • Marriott C.
      Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
      ]. The binding of alginate to antibiotic appeared to be concentration dependent [
      • Nichols W.W.
      • Dorrington S.M.
      • Slack M.P.
      • Walmsley H.L.
      Inhibition of tobramycin diffusion by binding to alginate.
      ] as aminoglycosides formed precipitates with the alginate at a low alginate to antibiotic ratio. This phenomenon disrupted the gel structure, resulting in diffusion of aminoglycosides at a rate that was even faster than that of the β-lactams [
      • Gordon C.A.
      • Hodges N.A.
      • Marriott C.
      Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
      ].
      For β-lactam antibiotics the diffusion rate was strongly reduced by alginate as the molecular weight of these antibiotics was increased. Like mucin, free DNA further reduced the diffusion of β-lactam antibiotics through alginate gels [
      • Bolister N.
      • Basker M.
      • Hodges N.A.
      • Marriott C.
      The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.
      ].
      In summary, the diffusion of both aminoglycosides and β-lactam antibiotics is reduced through the alginate layer surrounding Pa. Ultimately, this reduced diffusion contributes significantly to the difficulty in eradicating mucoid Pa from the airways of CF patients [
      • Gordon C.A.
      • Hodges N.A.
      • Marriott C.
      Antibiotic interaction and diffusion through alginate and exopolysaccharide of cystic fibrosis-derived Pseudomonas aeruginosa.
      ].

      3.5.3 Biofilm formation

      Within biofilms, a subpopulation of persistent Pa cells is formed and characterized by reduced metabolic activity and tolerance to antibiotics [
      • Barraud N.
      • Buson A.
      • Jarolimek W.
      • Rice S.A.
      Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      ]. It was shown that MBECs for three different Pa strains increased between 8 and 512 times for free aminoglycosides and 8 to 256 times for liposomal aminoglycosides when growing in biofilm [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ]. The efficacy of macrolides to eradicate bacteria within mature biofilms was markedly reduced relative to aminoglycosides [
      • Sommerfeld Ross S.
      • Fiegel J.
      Nutrient dispersion enhances conventional antibiotic activity against Pseudomonas aeruginosa biofilms.
      ].

      3.5.4 Therapies to overcome barriers generated by Pa

      Alginate lyase (AlgL) is an enzyme that can degrade alginate and facilitate the diffusion of aminoglycosides to the target bacteria [
      • Hatch R.A.
      • Schiller N.L.
      Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      ]. AlgL has been tested in vitro and in animal models with endocarditis. Co-treatment with AlgL increased bacterial susceptibility to antibiotics and phagocytosis, and reduced alginate levels in vitro. Additionally, AlgL was more effective at enhancing the activity of aminoglycosides than dornase alfa [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ].
      This effect differed per aminoglycoside antibiotic, where AlgL treatment alone increased the bactericidal activity of tobramycin and amikacin (free and liposomal form). Likewise, the combination of dornase alfa-AlgL enhanced the bactericidal activity of tobramycin and that of free but not liposomal amikacin [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ]. For gentamicin, one study showed enhanced bactericidal activity [
      • Hatch R.A.
      • Schiller N.L.
      Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      ], while neither AlgL nor the combination with dornase alfa demonstrated any effect on its activity in another study [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ].
      An excess of iron can induce biofilm formation by Pa and is evidenced by the fact that the iron concentration in the ELF of CF patients is 400-fold higher than in non-CF patients [
      • Moreau-Marquis S.
      • Coutermarsh B.
      • Stanton B.A.
      Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.
      ]. Drugs containing different combinations of lactoferrin (iron-binding glycoprotein) and hypothiocyanite (bactericidal agent; ALX-009 and ALX-109) had an additive effect on tobramycin and aztreonam in reducing both biofilm formation and disrupting established Pa biofilms. Both lactoferrin and hypothiocyanite are part of the normal innate immune response but their secretion by airway cells is reduced in CF [
      • Moreau-Marquis S.
      • Coutermarsh B.
      • Stanton B.A.
      Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.
      ,
      • Moreau-Marquis S.
      • Perrotto S.
      • Stanton B.A.
      ALX-009 potentiates the effect of tobramycin at killing Pseudomonas aeruginosa biofilms on human airway cells.
      ,
      • Moreau-Marquis S.
      • Drexinger J.K.
      • Perrotto S.
      • Stanton B.A.
      Treatment with ALX-009 in combination with aztreonam potentiates the killing of Pseudomonas aeruginosa biofilms on human CF airway cells.
      ].
      The addition of mannitol improved tobramycin efficacy by 99.5% against young Pa biofilms (pre-grown for 5 h) and by 77% against established biofilms (pre-grown for 20 h). However, mannitol had no effect on clinical strains with high resistance to tobramycin. The addition of glucose resulted in similar outcomes, albeit to a lesser extent. Similarly, NaCl required 2-fold higher osmolarities than mannitol to obtain a similar effect and had no effect on established biofilms [
      • Barraud N.
      • Buson A.
      • Jarolimek W.
      • Rice S.A.
      Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      ].
      Finally, co-treatment with dispersion compounds (citrate and succinic acid) has been investigated to enhance biofilm eradication. Combinations of citrate with amikacin, colistin or erythromycin and succinic acid with colistin resulted in significantly enhanced killing of bacterial populations compared with control populations. However, increased bacterial viability was seen when tobramycin and polymyxin B were combined with dispersion compounds [
      • Sommerfeld Ross S.
      • Fiegel J.
      Nutrient dispersion enhances conventional antibiotic activity against Pseudomonas aeruginosa biofilms.
      ].
      In summary, co-treatment with AlgL and dornase alfa seems to increase the efficacy of aminoglycosides in the presence of alginate. Treatment of Pa growing in biofilms can be improved by co-treatment with iron binding drugs, dispersion compounds or mannitol. All of these agents should be further investigated in order to understand the effect of combining these drugs and antibiotics for the treatment of Pa infections in vivo.

      4. Discussion

      To our knowledge, this is the first systematic review on what happens to inhaled antibiotics after deposition in the airways of patients with CF. All results were primarily drawn from in vitro studies, from which we can conclude that the clinical efficacy of antibiotics is negatively affected by many factors after deposition in the airways (Fig. 2). Aminoglycosides, which were the most intensively studied relative to other inhaled antibiotics, seemed to be most adversely affected by these factors.
      Fig. 2
      Fig. 2Pathway of the inhaled antibiotic after deposition on the mucus layer.
      After depositing in the airways, the aerosol particle needs to dissolve in the airway surface layer or mucus layer. Next, the antibiotic needs to diffuse to the site where the bacteria are located. During the diffusion process through the mucus layer the aerosol particle can bind to molecules in the mucus. Also, the oxygen level, salt content and pH of the mucus are of influence on the antibiotic efficacy. Finally, the antibiotic has to overcome barriers generated by the microorganisms.
      Following dissolution, the drug needs to diffuse through the mucus layer to reach the site of the bacteria. The high concentration of macromolecules in mucus of patients with CF increases its viscosity, thereby impeding antibiotic diffusion [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ]. Slow diffusion across CF mucus layers may play an important role in reduced pulmonary bioavailability as antibiotic molecules may be cleared by alveolar macrophages before reaching the bacteria. Therefore, coadministration of mannitol and dornase alfa may improve the diffusion of antibiotic molecules through mucus [
      • Yang Y.
      • Tsifansky M.D.
      • Shin S.
      • Lin Q.
      • Yeo Y.
      Mannitol-guided delivery of Ciprofloxacin in artificial cystic fibrosis mucus model.
      ,
      • Yang Y.
      • Tsifansky M.D.
      • Wu C.J.
      • Yang H.I.
      • Schmidt G.
      • Yeo Y.
      Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis.
      ].
      During the diffusion process the inhaled antibiotic may bind to mucus, thereby limiting the amount of free drug available to be efficacious against bacteria. Aminoglycosides showed substantial binding to mucus from CF patients [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ] while this was not observed in β-lactam antibiotics. This difference in binding can be attributed to the fact that aminoglycosides are positively charged, whereas β-lactam antibiotics are neutrally or negatively charged. As mucus macromolecules are negatively charged they show a high affinity for positively charged antibiotics. This level of binding may be reduced by the coadministration of drugs such as cationic amphiphiles, which competitively bind to the macromolecules within the mucus. This resulted in saturation of the binding sites and a higher aminoglycoside bioavailability [
      • Purdy Drew K.R.
      • Sanders L.K.
      • Culumber Z.W.
      • Zribi O.
      • Wong G.C.
      Cationic amphiphiles increase activity of aminoglycoside antibiotic tobramycin in the presence of airway polyelectrolytes.
      ]. Conflicting results on the coadministration of dornase alfa have been observed by different authors; one study found a decrease in binding [
      • Ramphal R.
      • Lhermitte M.
      • Filliat M.
      • Roussel P.
      The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.
      ], while another found an increase in binding [
      • Hunt B.E.
      • Weber A.
      • Berger A.
      • Ramsey B.
      • Smith A.L.
      Macromolecular mechanisms of sputum inhibition of tobramycin activity.
      ]. To the best of our knowledge, the effect of mucus on the efficacy of fluoroquinolones or macrolides has not been studied.
      Low oxygen levels and high salt concentrations within the mucus were shown to reduce the effectiveness of antibiotics. Specifically, aminoglycosides, β-lactam antibiotics and chloramphenicol were rendered less efficacious against Pa under anaerobic conditions. Low oxygen levels within mucus may increase colistin activity [
      • Hill D.
      • Rose B.
      • Pajkos A.
      • Robinson M.
      • Bye P.
      • Bell S.
      • et al.
      Antibiotic susceptibilities of Pseudomonas aeruginosa isolates derived from patients with cystic fibrosis under aerobic, anaerobic, and biofilm conditions.
      ,
      • King P.
      • Citron D.M.
      • Griffith D.C.
      • Lomovskaya O.
      • Dudley M.N.
      Effect of oxygen limitation on the in vitro activity of levofloxacin and other antibiotics administered by the aerosol route against Pseudomonas aeruginosa from cystic fibrosis patients.
      ,
      • Pompilio A.
      • Crocetta V.
      • Pomponio S.
      • Fiscarelli E.
      • Di Bonaventura G.
      In vitro activity of colistin against biofilm by Pseudomonas aeruginosa is significantly improved under “cystic fibrosis-like” physicochemical conditions.
      ]. With regard to salt concentrations, Pa was rendered less susceptible to killing by aminoglycosides in the presence of magnesium and calcium [
      • Beggs W.H.
      • Andrews F.A.
      Role of ionic strength in salt antagonism of aminoglycoside action on Escherichia coli and Pseudomonas aeruginosa.
      ]. Additionally, nitrate decreased the efficacy of aminoglycosides and fluoroquinolones by half under anaerobic conditions [
      • Borriello G.
      • Werner E.
      • Roe F.
      • Kim A.M.
      • Ehrlich G.D.
      • Stewart P.S.
      Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms.
      ].
      Ultimately, when antibiotic molecules make it to the vicinity of the bacteria they still need to overcome multiple barriers generated by the microorganisms. The alginate layer surrounding Pa is an important contributing factor to its resistance of Pa against antibiotics and the patient's innate immune response. In general aminoglycosides bind to alginate while β-lactam antibiotics do not. Diffusion through alginate was impaired for all tested antibiotics, but most strongly for aminoglycosides. Coadministration of AlgL showed promising results in vitro, with improved diffusion rates and enhanced bactericidal activity [
      • Alipour M.
      • Suntres Z.E.
      • Omri A.
      Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.
      ,
      • Hatch R.A.
      • Schiller N.L.
      Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
      ]. Another important barrier generated by Pa is biofilm formation, which drastically reduced effective killing. Treatment of Pa growing in biofilms can be further improved by co-treatment with iron binding glycoproteins [
      • Moreau-Marquis S.
      • Coutermarsh B.
      • Stanton B.A.
      Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.
      ,
      • Moreau-Marquis S.
      • Perrotto S.
      • Stanton B.A.
      ALX-009 potentiates the effect of tobramycin at killing Pseudomonas aeruginosa biofilms on human airway cells.
      ,
      • Moreau-Marquis S.
      • Drexinger J.K.
      • Perrotto S.
      • Stanton B.A.
      Treatment with ALX-009 in combination with aztreonam potentiates the killing of Pseudomonas aeruginosa biofilms on human CF airway cells.
      ], mannitol or dispersion compounds [
      • Barraud N.
      • Buson A.
      • Jarolimek W.
      • Rice S.A.
      Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      ,
      • Sommerfeld Ross S.
      • Fiegel J.
      Nutrient dispersion enhances conventional antibiotic activity against Pseudomonas aeruginosa biofilms.
      ].
      The limitations of this systematic review are the following; firstly, out of the 35 publications selected for analysis, 9 publications were selected by screening the reference lists of the included articles. The search term “INHALED” was obligated in the title or abstract, while not all studies specified the route of administration.
      Secondly, a high level of inter-publication variability was observed for the following aspects of the studies; concentrations of antibiotic, types of alginate or exopolysaccharide, types of buffer or medium. These differences prevented an accurate comparison between the results.
      Thirdly, the majority of the studies were performed in vitro and hence, caution is required when extrapolating these results to in vivo conditions. Clearly, in vivo studies are needed to investigate the relevance of the in vitro observations for the effectiveness of inhaled antibiotics in patients. In addition, most publications did not distinguish between intravenous, nebulized or dry powder antibiotics. This is important as aerosol particles are first required to dissolve before diffusion through the mucus and alginate can occur. Finally, there is a clear disbalance in the antibiotics studied in the literature; aminoglycosides were most extensively studied, followed by β-lactam antibiotics. Although, these antibiotic classes are frequently used to treat pulmonary infections in CF, little is known about other frequently used antibiotics such as colistin.
      Based on in vitro studies we conclude that aminoglycosides can be strongly affected after deposition in the airways. The composition of CF mucus is an important determinant of the in vitro efficacy of aminoglycosides, as well as the alginate layer surrounding Pa. In order to eradicate a microorganism, an antibiotic needs to overcome all the aforementioned barriers before reaching the outer membrane of the microorganism and ultimately bind to specific target sites. Importantly, higher concentrations allow more antibiotic molecules to reach these target sites. For future research, both advanced modelling and in vivo studies are required to further establish the role of encapsulated antibiotic formulations and coadministration with other drugs in improving the local efficacy of inhaled antibiotics.
      The following are the supplementary data related to this article.

      Acknowledgements

      The authors thank Wichor Bramer, Medical Library Erasmus MC, Rotterdam, for performing the search, Prof. dr. de Jongste, department of Paediatric Pulmonology, Rotterdam, for drawing the image of the antibiotic pathway after deposition and Clara Mok, Telethon Kids Institute, Australia, for checking the grammar and spelling of the manuscript.

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