Advertisement

New concepts in antimicrobial resistance in cystic fibrosis respiratory infections

  • Pavel Drevinek
    Correspondence
    Corresponding author at: V Uvalu 84, 15006 Prague, Czechia.
    Affiliations
    Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University and Motol University Hospital, Prague, Czech Republic
    Search for articles by this author
  • Rafael Canton
    Affiliations
    Servicio de Microbiología, Hospital Universitario Ramón y Cajal and Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Spain
    Search for articles by this author
  • Helle Krogh Johansen
    Affiliations
    Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Department of Clinical Medicine, University of Copenhagen, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark
    Search for articles by this author
  • Lucas Hoffman
    Affiliations
    Departments of Pediatrics and Microbiology, University of Washington/Seattle Children's Hospital, Center for Clinical and Translational Research, Seattle Children's Research Institute, Seattle, WA, USA
    Search for articles by this author
  • Tom Coenye
    Affiliations
    Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium
    Search for articles by this author
  • Pierre-Regis Burgel
    Affiliations
    National CF Reference Center and Respiratory Medicine; Cochin Hospital Assistance Publique Hôpitaux de Paris, Université Paris Cité and Institut Cochin, INSERM U1016, Paris, France
    Search for articles by this author
  • Jane C Davies
    Affiliations
    National Heart & Lung Institute, Imperial College London and Royal Brompton Hospital, Guy's & St Thomas‘ Trust, London, United Kingdom
    Search for articles by this author
Open AccessPublished:October 18, 2022DOI:https://doi.org/10.1016/j.jcf.2022.10.005

      Highlights

      • The AMR evolution in chronic infections is by large a consequence of microbial adaptation to CF lungs.
      • Both antimicrobial susceptibility testing and resistome analysis are of limited clinical value.
      • Novel strategies to fight CF infections include anti-biofilm approaches and bacteriophages.

      Abstract

      In this review, we summarize the main points that were raised and highlighted during the pre-conference meeting to the 17th European Cystic Fibrosis Society Basic Science Conference, held from 30 March to 2 April, 2022 in Albufeira, Portugal. Keynote lectures provided an update on the latest information regarding the phenomenon of antimicrobial resistance (AMR) in cystic fibrosis (CF). Traditional themes such as in vitro antibiotic susceptibility testing and its clinical value, AMR evolution in persistent Pseudomonas aeruginosa infection and the impact of biofilm on AMR were discussed. In addition, the report gives an overview on very recent AMR-related topics that include an ecological view of AMR in CF lung, referred to as resistome, and novel anti-infective approaches in preclinical or early clinical research such as antibiofilm drugs and bacteriophages.

      Keywords

      1. Introduction

      Never has there been a more exciting time to be working in the science behind cystic fibrosis (CF). The progress over the last few years in CFTR modulator therapy and the energy this has catalysed in drug development is genuinely game-changing. Improvements in diagnosis and standards of care over the last few decades have led to health and survival benefits, with a huge proportion of the CF population now reaching adulthood. However, the majority of these people have recurrent or chronic pulmonary infections and, at least to date, there is little evidence that even transformational therapies will have a major impact on these. We are still completely reliant on antimicrobials that are administered to many people with CF (pwCF) on a daily basis as a means for eradication of newly acquired infection, treatment of pulmonary exacerbations or suppressive maintenance therapy in cases of chronic infections [
      • Castellani C
      • Duff AJA
      • Bell SC
      • Heijerman HGM
      • Munck A
      • Ratjen F
      • et al.
      ECFS best practice guidelines: the 2018 revision.
      ]. Both healthcare providers and pwCF express their concerns about the inevitable increase in antimicrobial resistance (AMR), mostly perceived in an association with frequent use of inpatient (i.e., intravenous) as well as outpatient (i.e., inhaled or oral) antibiotics against traditional CF pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex or nontuberculous mycobacteria [
      • Bullington W
      • Hempstead S
      • Smyth AR
      • Drevinek P
      • Saiman L
      • Waters VJ
      • et al.
      Antimicrobial resistance: concerns of healthcare providers and people with CF.
      ]. Thus, AMR is a well-recognised and worsening problem in CF. This report aims to summarize the latest knowledge and the key aspects of the AMR in CF at the research, clinical laboratory and healthcare levels, presented by opinion leaders at the pre-conference meeting on this topic at the 2022 European CF Society Basic Science conference.

      2. Caveats of antimicrobial susceptibility testing in CF isolates

      The ultimate goal of antimicrobial susceptibility testing (AST) from a clinical standpoint is to predict the success or failure of therapy with an antimicrobial drug, based on categorization of isolates as “susceptible” or “resistant”. While the performance of AST is subject to microbiological standards of care, its results, subsequent correlation with clinical outcome and ultimately, indication and frequency of its repeated performance in chronic CF infections continue to be a matter of debate with available evidence demonstrating utility is poor [
      • Zemanick E
      • Burgel PR
      • Taccetti G
      • Holmes A
      • Ratjen F
      • Byrnes CA
      • et al.
      Antimicrobial resistance in cystic fibrosis: a delphi approach to defining best practices.
      ,
      • Waters V
      • Ratjen F.
      Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis.
      ,
      • Smith S
      • Waters V
      • Jahnke N
      • Ratjen F.
      Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis.
      ,
      • Doring G
      • Flume P
      • Heijerman H
      • Elborn JS.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ]. This most likely relates to the fact that standard AST is designed to be applied to single bacterial species, cultured in the context of acute infection, not to a community of microorganisms causing a chronic polymicrobial infection.
      A growing body of evidence raised doubts about the reliability of current microbiological tests for identifying clinically-effective antibiotics in CF. For example, multiple studies [
      • Smith S
      • Waters V
      • Jahnke N
      • Ratjen F.
      Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis.
      ,
      • Smith AL
      • Fiel SB
      • Mayer-Hamblett N
      • Ramsey B
      • Burns JL.
      Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis.
      ,
      • Waters V
      • Ratjen F.
      Combination antimicrobial susceptibility testing for acute exacerbations in chronic infection of Pseudomonas aeruginosa in cystic fibrosis.
      ,
      • Hurley MN
      • Ariff AH
      • Bertenshaw C
      • Bhatt J
      • Smyth AR.
      Results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis.
      ] found no relationship between in vitro susceptibilities of P. aeruginosa isolates and patients’ clinical responses to consequent antibiotic choices. These studies primarily focused on P. aeruginosa; however, similarly poor predictive capacities of susceptibility tests have been found for nontuberculous mycobacteria in CF [
      • van-Ingen J
      • Boeree MJ
      • van-Soolingen D
      • Mouton JW.
      Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria.
      ], as well as for a range of non-CF chronic infections [
      • Boolchandani M
      • D'Souza AW
      • Dantas G
      Sequencing-based methods and resources to study antimicrobial resistance.
      ]. Diverse potential contributors and explanations for this problem have been suggested. For example, Foweraker et al. [
      • Foweraker JE
      • Laughton CR
      • Brown DF
      • Bilton D.
      Phenotypic variability of Pseudomonas aeruginosa in sputa from patients with acute infective exacerbation of cystic fibrosis and its impact on the validity of antimicrobial susceptibility testing.
      ] demonstrated that in vitro susceptibilities of P. aeruginosa isolates in individual CF sputa vary dramatically even within a sample, raising doubts about the accuracy to be expected from testing a single isolate.
      The concept of resistance of CF lung pathogens, the potential usefulness of AST in the selection of appropriate antimicrobial therapy and the need for appropriate clinical breakpoints for the interpretation of the antibiogram have recently been reviewed [
      • Ekkelenkamp MB
      • Diez-Aguilar M
      • Tunney MM
      • Elborn JS
      • Fluit AC
      • Canton R.
      Establishing antimicrobial susceptibility testing methods and clinical breakpoints for inhaled antibiotic therapy.
      ,
      • Kidd TJ
      • Canton R
      • Ekkelenkamp M
      • Johansen HK
      • Gilligan P
      • LiPuma JJ
      • et al.
      Defining antimicrobial resistance in cystic fibrosis.
      ,
      • Waters VJ
      • Kidd TJ
      • Canton R
      • Ekkelenkamp MB
      • Johansen HK
      • LiPuma JJ
      • et al.
      Reconciling antimicrobial susceptibility testing and clinical response in antimicrobial treatment of chronic cystic fibrosis lung infections.
      ]. Neither the U.S. Clinical and Laboratory Standards Institute (CLSI) nor the European Antimicrobial Susceptibility Testing (EUCAST) Committee have included inhaled antibiotics in their proposals of defining clinical breakpoints and standardisation of AST. This decision was related to the differences between those microorganisms isolated from patients with chronic lung infection and those that cause sepsis or any other acute infection such as community or hospital-acquired pneumonia. Also, it has been challenging to apply current standards for performing AST to CF pathogens due to their characteristic growth (reduced growth rate and often in biofilms rather than in planktonic mode), great diversification to multiple morphotypes, their ability to exhibit tolerance, persistence and heteroresistance, and the high frequency of hypermutator phenotypes [
      • Waters VJ
      • Kidd TJ
      • Canton R
      • Ekkelenkamp MB
      • Johansen HK
      • LiPuma JJ
      • et al.
      Reconciling antimicrobial susceptibility testing and clinical response in antimicrobial treatment of chronic cystic fibrosis lung infections.
      ,
      • ST Clark
      • Guttman DS
      • Hwang DM.
      Diversification of Pseudomonas aeruginosa within the cystic fibrosis lung and its effects on antibiotic resistance.
      ,
      • Ciofu O
      • Tolker-Nielsen T.
      Tolerance and resistance of Pseudomonas aeruginosa biofilms to antimicrobial agents-how P. aeruginosa can escape antibiotics.
      ,
      • Ciofu O
      • Mandsberg LF
      • Wang H
      • Hoiby N.
      Phenotypes selected during chronic lung infection in cystic fibrosis patients: implications for the treatment of Pseudomonas aeruginosa biofilm infections.
      ,
      • Oliver A
      • Mena A.
      Bacterial hypermutation in cystic fibrosis, not only for antibiotic resistance.
      ].
      The CLSI does offer two brief technical recommendations: that AST of P. aeruginosa isolates from CF patients can be performed by both disc diffusion and dilution methods, and that the incubation of the tests should be extended up to 24 hours to facilitate their reading [

      Performance Standards for Antimicrobial Susceptibility Testing. 32 ed2022.

      ]. The EUCAST includes epidemiological cut-off (ECOFF) values for topical use, but explicitly excludes their use for inhaled antibiotics []. ECOFF refers to inhibitory concentration values that discriminate wild-type bacterial populations from those with acquired resistance mechanisms [
      • Kahlmeter G
      • Turnidge J.
      How to: ECOFFs-the why, the how, and the don'ts of EUCAST epidemiological cutoff values.
      ,
      • Giske CG
      • Turnidge J
      • Canton R
      • Kahlmeter G
      • Committee ES.
      Update from the european committee on antimicrobial susceptibility testing (EUCAST).
      ]. However, they are not applicable to scenarios where much higher concentrations of antibiotic are reached at the infection site when compared with those obtained with the oral or intravenous route of administration [
      • Ekkelenkamp MB
      • Diez-Aguilar M
      • Tunney MM
      • Elborn JS
      • Fluit AC
      • Canton R.
      Establishing antimicrobial susceptibility testing methods and clinical breakpoints for inhaled antibiotic therapy.
      ].
      Consensus documents recommend the performance of AST for isolates from pwCF for varying reasons; specifically, for understanding the potential impact of antimicrobial use on pathogens and their evolution of AMR, selection of treatment for current or next exacerbation, and to explain treatment failure [
      • Doring G
      • Flume P
      • Heijerman H
      • Elborn JS.
      Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
      ,
      • Waters VJ
      • Kidd TJ
      • Canton R
      • Ekkelenkamp MB
      • Johansen HK
      • LiPuma JJ
      • et al.
      Reconciling antimicrobial susceptibility testing and clinical response in antimicrobial treatment of chronic cystic fibrosis lung infections.
      ,
      • Doring G
      • Conway SP
      • Heijerman HG
      • Hodson ME
      • Hoiby N
      • Smyth A
      • et al.
      Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.
      ,
      • Canton R
      • Maiz L
      • Escribano A
      • Olveira C
      • Oliver A
      • Asensio O
      • et al.
      Spanish consensus on the prevention and treatment of Pseudomonas aeruginosa bronchial infections in cystic fibrosis patients.
      ]. It is routinely recommended to study different isolate morphotypes separately, avoiding the pooling of multiple colony types, and to incubate with antimicrobials for 24 hours. Diffusion techniques, either with discs or gradient strips, also allow the phenotypic detection of potential hypermutator strains, which can lead to a closer monitoring of the possible failure due to selection of populations with lower antimicrobial sensitivity [
      • Macia MD
      • Borrell N
      • Perez JL
      • Oliver A.
      Detection and susceptibility testing of hypermutable Pseudomonas aeruginosa strains with the Etest and disk diffusion.
      ]. The study of CF isolates’ susceptibilities as biofilms has also been proposed, in general applying a methodology similar to the determination of minimal inhibitory concentration (MIC) (by using the Calgary biofilm device), in which the proposed value is the biofilm inhibitory concentration (BIC), i.e. the lowest concentration preventing the growth in biofilms [
      • Macia MD
      • Rojo-Molinero E
      • Oliver A.
      Antimicrobial susceptibility testing in biofilm-growing bacteria.
      ] (Table 1). In this case, the inoculum used is an already formed biofilm. Arguably more representative AST value would be the concentration that eliminates biofilm (biofilm bactericidal concentration; BBC) or the concentration that prevents biofilm formation (BPC) [
      • Macia MD
      • Rojo-Molinero E
      • Oliver A.
      Antimicrobial susceptibility testing in biofilm-growing bacteria.
      ,
      • Fernandez-Olmos A
      • Garcia-Castillo M
      • Maiz L
      • Lamas A
      • Baquero F
      • Canton R.
      In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.
      ] where the antimicrobial interacts with the biofilm at the time as it is formed (Figure 1).
      Table 1Different antimicrobial susceptibility testing parameters and inoculum used.
      ParameterDefinitionInoculum
      Minimal inhibitory concentration (MIC)Lowest antibiotic concentration that inhibits the visible bacterial (planktonic) growth after overnight incubationPlanktonic (105 CFU/ml)
      Minimal bactericidal concentration (MBC)Lowest antibiotic concentration that reduces an initial bacterial (planktonic) inoculum with 99.9% (≥3 log)Planktonic (105 CFU/ml)
      Biofilm inhibitory concentration (BIC)Lowest antibiotic concentration that results in an OD650 nm difference of ≤10% (1 log difference in growth after 6 h of incubation) of the mean of two positive control well readings when a biofilm is used as inoculumSessile (biofilm previously developed)
      Biofilm prevention concentration (BPC)Same definition as the BIC, but bacterial (planktonic) inoculation and antibiotic exposure occur simultaneously to avoid biofilm developmentPlanktonic (105 CFU/ml)
      Biofilm bactericidal concentration (BBC)Lowest antibiotic concentration that reduces an initial biofilm inoculum with 99.9% (≥3 log) as compared to the growth controlSessile (biofilm)
      Fig 1
      Fig. 1Concentration over time at the site of infection of four hypothetical antibiotics. The dashed lines indicate the concentrations required for various effects against planktonic cells and biofilms as defined by MIC, MBC, BPC, BIC, and BBC ().
      However, current evidence does not support the use of biofilm AST to guide antimicrobial treatment of P. aeruginosa pulmonary infections in pwCF. Neither microbiological (e.g., a change in P. aeruginosa density in sputum), nor clinical outcomes (see below) demonstrated that biofilm AST was superior to conventional AST [
      • Yau YC
      • Ratjen F
      • Tullis E
      • Wilcox P
      • Freitag A
      • Chilvers M
      • et al.
      Randomized controlled trial of biofilm antimicrobial susceptibility testing in cystic fibrosis patients.
      ,
      • Moskowitz SM
      • Emerson JC
      • McNamara S
      • Shell RD
      • Orenstein DM
      • Rosenbluth D
      • et al.
      Randomized trial of biofilm testing to select antibiotics for cystic fibrosis airway infection.
      ].
      In addition to the inherent methodological problems with AST mentioned above, there are also problems of defining microbiological or clinical endpoints to evaluate the efficacy of the therapy, either empirical or driven by the AST results [
      • Smith S
      • Rowbotham NJ
      • Charbek E.
      Inhaled antibiotics for pulmonary exacerbations in cystic fibrosis.
      ]. Eradication, once chronic infection is established, is difficult to achieve, so other parameters such as the decrease in bacterial load, reduced antibody responses, reduction in exacerbation frequency, time to the next exacerbation, improvement in lung function or even improvement in the quality of life have to be assessed [
      • Ekkelenkamp MB
      • Diez-Aguilar M
      • Tunney MM
      • Elborn JS
      • Fluit AC
      • Canton R.
      Establishing antimicrobial susceptibility testing methods and clinical breakpoints for inhaled antibiotic therapy.
      ,
      • Tiddens H
      • Puderbach M
      • Venegas JG
      • Ratjen F
      • Donaldson SH
      • Davis SD
      • et al.
      Novel outcome measures for clinical trials in cystic fibrosis.
      ,
      • Karampitsakos T
      • Papaioannou O
      • Kaponi M
      • Kozanidou A
      • Hillas G
      • Stavropoulou E
      • et al.
      Low penetrance of antibiotics in the epithelial lining fluid. The role of inhaled antibiotics in patients with bronchiectasis.
      ,
      • Munzenberger PJ
      • Van Wagnen CA
      • Abdulhamid I
      • Walker PC
      Quality of life as a treatment outcome in patients with cystic fibrosis.
      ]. As a consequence, efforts to define the clinical breakpoints for inhaled antibiotics and both microbiological and clinical outcomes should continue, to better understand benefits of antimicrobial treatment. Without overcoming technical issues and finding meaningful clinical correlates, reservations about utility and clinical value of AST are appropriate and shared by the authors of this review.

      3. Is measuring CF lung resistome clinically useful?

      The term resistome was coined in 2006 by Gerry Wright at the University of Michigan [
      • D'Costa VM
      • McGrann KM
      • Hughes DW
      • Wright GD.
      Sampling the antibiotic resistome.
      ,
      • Wright GD.
      The antibiotic resistome: the nexus of chemical and genetic diversity.
      ], referring to an ecological, rather than a clinical, concept [
      • Surette MD
      • Wright GD.
      Lessons from the environmental antibiotic resistome.
      ]. His definition was “a collection of all the antibiotic resistance genes and their precursors in pathogenic and non-pathogenic bacteria”, i.e., specifically including bacteria that are both identified and not identified as pathogens, and also “precursor” genes that could confer resistance only if adapted or upregulated. This focus of the term resistome on the presence or absence of genes within an entire, diverse population of microbes highlights a key difference from what the clinical microbiology laboratory usually measures from CF respiratory samples: the expression of resistance in individual microbial isolates during in vitro monoculture.
      Current methods used in clinical CF microbiology are intentionally selective [
      • Saiman L
      • Siegel JD
      • LiPuma JJ
      • Brown RF
      • Bryson EA
      • Chambers MJ
      • et al.
      Infection prevention and control guideline for cystic fibrosis: 2013 update.
      ]. Respiratory samples are cultured using a battery of media formulated to identify pathogens most associated with CF lung disease, while selecting against common microbes without a known role in disease. Cultured isolates are then individually tested for AST without defining mechanisms of resistance. These features of clinical laboratory results - defining phenotypes of specific pathogens - contrast sharply with those of genomics-based resistome analyses that focus on the presence or absence (but not activity) of canonical resistance mechanisms among all bacteria in a sample without considering whether those bacteria are pathogens [
      • Surette MD
      • Wright GD.
      Lessons from the environmental antibiotic resistome.
      ,
      • Sherrard LJ
      • Tunney MM
      • Elborn JS.
      Antimicrobial resistance in the respiratory microbiota of people with cystic fibrosis.
      ]. For these reasons, CF resistome results can be expected to differ substantially from conventional CF laboratory phenotypic test results. In addition, sequencing-based (microbiome) methods often identify many bacteria in CF respiratory samples at abundances resembling those of conventional, cultured CF pathogens [
      • Zhao J
      • Schloss PD
      • Kalikin LM
      • Carmody LA
      • Foster BK
      • Petrosino JF
      • et al.
      Decade-long bacterial community dynamics in cystic fibrosis airways.
      ,
      • Rogers GB
      • Hart CA
      • Mason JR
      • Hughes M
      • Walshaw MJ
      • Bruce KD.
      Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling.
      ]. While the roles of these nonconventional bacteria in pathogenesis or response to therapy remain unknown, it has been suggested that interspecies interactions and other influences common in the CF airway [
      • Van den Bossche S
      • De Broe E
      • Coenye T
      • Van Braeckel E
      • Crabbe A.
      The cystic fibrosis lung microenvironment alters antibiotic activity: causes and effects.
      ] can alter the effects of antibiotics on pathogens [
      • O'Brien TJ
      • Figueroa W
      • Welch M
      Decreased efficacy of antimicrobial agents in a polymicrobial environment.
      ].
      Recent studies demonstrated the power of genomic methods for identifying the dominant contributors to in vitro susceptibilities for individual pathogens, such as Mycobacterium tuberculosis and Staphylococcus aureus [
      • Bradley P
      • Gordon NC
      • Walker TM
      • Dunn L
      • Heys S
      • Huang B
      • et al.
      Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.
      ], with the capacity to be faster and cheaper than culture-based methods. Notably, these pathogens are well-represented in genomic databases and are therefore ideal test organisms for molecular methods. By comparison, many CF pathogens have relatively few complete genomes available for computational comparison; for example, genomics seem more likely to predict resistance for P. aeruginosa, given the numerous genomes available for computational comparison, than for Achromobacter spp. [
      • Gabrielaite M
      • Bartell JA
      • Norskov-Lauritsen N
      • Pressler T
      • Nielsen FC
      • Johansen HK
      • et al.
      Transmission and antibiotic resistance of Achromobacter in cystic fibrosis.
      ]. In addition, given the limited clinical utility of in vitro AST [
      • Smith AL
      • Fiel SB
      • Mayer-Hamblett N
      • Ramsey B
      • Burns JL.
      Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis.
      ,
      • Hurley MN
      • Ariff AH
      • Bertenshaw C
      • Bhatt J
      • Smyth AR.
      Results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis.
      ,
      • van-Ingen J
      • Boeree MJ
      • van-Soolingen D
      • Mouton JW.
      Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria.
      ,
      • Van den Bossche S
      • De Broe E
      • Coenye T
      • Van Braeckel E
      • Crabbe A.
      The cystic fibrosis lung microenvironment alters antibiotic activity: causes and effects.
      ], and because genomic methods are generally optimized to predict in vitro resistance of individual isolates of specific, well-studied species, it is unclear whether a pathogen-focused genomic predictor will be any more useful for clinical care than culture-based predictors.
      Published reviews have detailed the enormous promise resistomics holds for improving cost and efficiency of predicting resistance among pathogens such as M. tuberculosis [
      • Cox H
      • Mizrahi V.
      The coming of age of drug-susceptibility testing for tuberculosis.
      ]. However, CF respiratory infections are often polymicrobial, with additional genomic diversity among populations of traditional pathogens such as P. aeruginosa. The roles in clinical responses to antibiotics of each microbe identified using untargeted genomics of CF respiratory samples is a topic of controversy and the focus of ongoing studies [
      • Nelson MT
      • Wolter DJ
      • Eng A
      • Weiss EJ
      • Vo AT
      • Brittnacher MJ
      • et al.
      Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.
      ,
      • Heirali A
      • Thornton C
      • Acosta N
      • Somayaji R
      • Laforest-Lapointe I
      • Storey D
      • et al.
      Sputum microbiota in adults with CF associates with response to inhaled tobramycin.
      ,
      • Heirali AA
      • Workentine ML
      • Acosta N
      • Poonja A
      • Storey DG
      • Somayaji R
      • et al.
      The effects of inhaled aztreonam on the cystic fibrosis lung microbiome.
      ]. Resistome analyses would not easily determine which specific species carries a given resistance determinant, whether that species is important for clinical response, or whether that gene is active in vivo. Therefore, there are many challenges inherent in developing genomics-based CF resistomics measures for clinical use, including therapy guidance. However, the growing efficiency and power of genomic methodology provide hope for a future role for these approaches in directing CF care. This future will require a great deal of research, data validation and methodologic refinement.

      4. Development AMR in bacteria: P. aeruginosa as an exemplar

      Pathoadaptation to the environment of CF airways has been most extensively studied in P. aeruginosa, the pathogen that still causes chronic infections in over 40% of the European adult CF population [

      Orenti A, Zolin A, Jung A, van Rens J. ECFSPR Annual Report 2020. 2022.

      ]. The following section focuses specifically on findings in P. aeruginosa, recovered from young pwCF. Almost half of these pwCF were persistently infected with a single P. aeruginosa clone type [
      • Marvig RL
      • Sommer LM
      • Molin S
      • Johansen HK.
      Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis.
      ,
      • Bartell JA
      • Sommer LM
      • Marvig RL
      • Skov M
      • Pressler T
      • Molin S
      • et al.
      Omics-based tracking of Pseudomonas aeruginosa persistence in "eradicated" cystic fibrosis patients.
      ] and the AST on a collection of early and subsequent P. aeruginosa isolates showed that during the first 5 to 10 years of infection, most of them remained susceptible to all anti-pseudomonas antibiotics, except for quinolones towards which resistance had developed in about 10 to 20% of the isolates. If we assume that AST provides clinically meaningful information (despite all the concerns related to the AST mentioned earlier), then these P. aeruginosa isolates should remain susceptible to antimicrobial therapy. However, they survive antibiotic exposure in vivo. Thus, their ability to establish chronic infection is likely a consequence of several other mechanisms beyond those conventionally involved in the development of resistance [
      • La-Rosa R
      • Johansen HK
      • Molin S
      Persistent bacterial infections, antibiotic treatment failure, and microbial adaptive evolution.
      ].
      P. aeruginosa is known to develop various tolerance traits during infection in CF lungs (Figure 2). Slow growth is one of its adaptive phenotypes, and the metabolic footprint for amino acids, organic acids, and sugars also changes over time. In association with slow growth, antibiotic resistance towards ceftazidime, carbapenems, quinolones and aminoglycosides has been observed [
      • Bartell JA
      • Sommer LM
      • Haagensen JAJ
      • Loch A
      • Espinosa R
      • Molin S
      • et al.
      Evolutionary highways to persistent bacterial infection.
      ]. Persister cells, tolerant to antibiotics, are found in all bacterial populations. Although the persister phenotype per se is not associated with genetic changes, a fraction as high as 20% with a high-persister phenotype has been found among early CF isolates [
      • Bartell JA
      • Cameron DR
      • Mojsoska B
      • Haagensen JAJ
      • Pressler T
      • Sommer LM
      • et al.
      Bacterial persisters in long-term infection: emergence and fitness in a complex host environment.
      ].
      Fig 2
      Fig. 2P. aeruginosa infection timeline. Environmental P. aeruginosa strains colonize the airways of people with cystic fibrosis persisting for decades. To escape the immune system and resist antibiotic treatment, bacteria have to survive natural selection due to their pre-existing variants of resistant phenotype; furthermore, they modify their phenotype and adapt their physiology through accumulation of adaptive mutations and changes in gene expression profiles. Unconventional mechanisms such as heteroresistance development, metabolic specialization, growth rate reduction, persister phenotype and biofilm associated lifestyle strengthen further their persistence in the host.
      As an example of a less expected AMR mechanism, it was found that P. aeruginosa isolates from patients receiving tobramycin therapy developed L6 ribosomal protein mutations and associated with aminoglycoside resistance. The L6 mutations had additional impacts on the bacterial phenotype such as decreased growth rate, and development of collateral sensitivity to chloramphenicol. The L6 mutants were eliminated from the patient airways after cessation of tobramycin treatment [
      • Halfon Y
      • Jimenez-Fernandez A
      • La-Rosa R
      • Espinosa-Portero R
      • Krogh-Johansen H
      • Matzov D
      • et al.
      Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate.
      ]. Another common mechanism of aminoglycoside resistance in P. aeruginosa CF isolates is associated with mutations in the mexZ gene encoding a negative regulator protein, resulting in over-expression of the efflux pump proteins MexY and MexX. In the collection of nearly 500 whole genome sequenced P. aeruginosa clinical isolates, almost 40% carried a mutation in mexZ. However, only a minority showed clinical aminoglycoside resistance. Instead, they showed subtle, no more than 2-fold increased aminoglycoside and fluoroquinolone resistance relative to the wild type [
      • Frimodt-Moller J
      • Rossi E
      • Haagensen JAJ
      • Falcone M
      • Molin S
      • Johansen HK.
      Mutations causing low level antibiotic resistance ensure bacterial survival in antibiotic-treated hosts.
      ]. The link between mexZ mutations and the level of AMR as well as the reasons for the high frequency of these mutations among P. aeruginosa isolates needs to be further studied.
      The use of azithromycin was adopted for treatment of pwCF in the 1990s to take advantage of its immunomodulatory and anti-alginate effects [
      • Goltermann L
      • Andersen KL
      • Johansen HK
      • Molin S
      • La-Rosa R
      Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.
      ]. It was assumed that, as P. aeruginosa is inherently resistant to macrolides (high MIC values in standard AST), azithromycin resistance should not be induced in P. aeruginosa infecting CF airways. However, macrolide therapy in fact does select for AMR development in P. aeruginosa, related to mutations in the ribosomal protein gene L4 when assessed in alternative substrates [
      • Goltermann L
      • Andersen KL
      • Johansen HK
      • Molin S
      • La-Rosa R
      Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.
      ]. When azithromycin resistance did develop, both the immunomodulatory effect and the inhibition of mucoidy were severely impaired. It is therefore important to reconsider this long-term therapy in pwCF; specifically, that azithromycin may lose efficacy after one to two years from the start of therapy [
      • Goltermann L
      • Andersen KL
      • Johansen HK
      • Molin S
      • La-Rosa R
      Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.
      ].
      As stated above, the successful survival of P. aeruginosa exposed to frequent intensive antibiotic treatment in CF airways arises from a combination of bacterial features. Initial infection usually involves antibiotic-sensitive and fast-growing environmental P. aeruginosa isolates. Over the course of infection, the bacteria adapt to the CF lung environment via the accumulation of pathoadaptive mutations and changes in their metabolism. A substantial fraction of the bacterial population enters a state of dormancy, becoming persisters. Eventual development of a reduced growth rate markedly contributes to the development of phenotypic resistance [
      • La-Rosa R
      • Johansen HK
      • Molin S
      Persistent bacterial infections, antibiotic treatment failure, and microbial adaptive evolution.
      ,
      • Rossi E
      • La-Rosa R
      • Bartell JA
      • Marvig RL
      • Haagensen JAJ
      • Sommer LM
      • et al.
      Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis.
      ].

      5. Biofilm, the inherent defence mechanism of CF pathogens against antibiotics

      In vivo, microorganisms behave in a very different way from how they behave under laboratory conditions, forming multicellular aggregates embedded in a host-derived and/or self-produced extracellular matrix. These aggregates are designated as biofilms, and can be surface-attached (e.g. on the surface of a medical device), suspended (e.g. in synovial fluid) or embedded in host tissue (e.g. in a chronically infected wound). Cells in a biofilm are much less susceptible to antimicrobial agents compared with planktonic cells, and treatment of biofilm-related infections is often difficult (Figure 1). There is convincing evidence that important pathogens like P. aeruginosa occur as biofilms in the lungs of pwCF, which might help to explain (along with other bacterial adaptation processes mentioned earlier) why eradication of these infections is so difficult [
      • Kolpen M
      • Kragh KN
      • Enciso JB
      • Faurholt-Jepsen D
      • Lindegaard B
      • Egelund GB
      • et al.
      Bacterial biofilms predominate in both acute and chronic human lung infections.
      ,
      • Schwab U
      • Abdullah LH
      • Perlmutt OS
      • Albert D
      • Davis CW
      • Arnold RR
      • et al.
      Localization of Burkholderia cepacia complex bacteria in cystic fibrosis lungs and interactions with Pseudomonas aeruginosa in hypoxic mucus.
      ,
      • Bjarnsholt T
      • Alhede M
      • Alhede M
      • Eickhardt-Sorensen SR
      • Moser C
      • Kuhl M
      • et al.
      The in vivo biofilm.
      ,
      • Bjarnsholt T
      • Jensen PO
      • Fiandaca MJ
      • Pedersen J
      • Hansen CR
      • Andersen CB
      • et al.
      Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
      ]. Multiple mechanisms are involved in reduced antibiotic susceptibility of bacteria in biofilms [
      • Van Acker H
      • Van Dijck P
      • Coenye T.
      Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms.
      ,
      • Ciofu O
      • Moser C
      • Jensen PO
      • Hoiby N.
      Tolerance and resistance of microbial biofilms.
      ] and the micro-environmental conditions in the lung play an important role in this [
      • Van den Bossche S
      • De Broe E
      • Coenye T
      • Van Braeckel E
      • Crabbe A.
      The cystic fibrosis lung microenvironment alters antibiotic activity: causes and effects.
      ,
      • Bjarnsholt T
      • Whiteley M
      • Rumbaugh KP
      • Stewart PS
      • Jensen PO
      Frimodt-Moller N. The importance of understanding the infectious microenvironment.
      ]. Indeed, changes in microbial metabolism, at least partly related to gradients in oxygen and nutrient levels, can have a profound effect on antimicrobial susceptibility [
      • Van Acker H
      • Coenye T.
      The role of reactive oxygen species in antibiotic-mediated killing of bacteria.
      ,
      • Crabbe A
      • Ostyn L
      • Staelens S
      • Rigauts C
      • Risseeuw M
      • Dhaenens M
      • et al.
      Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.
      ,
      • Crabbe A
      • Jensen PO
      • Bjarnsholt T
      • Coenye T.
      Antimicrobial tolerance and metabolic adaptations in microbial biofilms.
      ]. The exact metabolomic adaptations vary between different microorganisms [
      • Crabbe A
      • Jensen PO
      • Bjarnsholt T
      • Coenye T.
      Antimicrobial tolerance and metabolic adaptations in microbial biofilms.
      ], and much remains to be learned about microbial metabolism in the infectious micro-environment (e.g. how the presence of multiple species affects metabolism and susceptibility [
      • Armbruster CR
      • Coenye T
      • Touqui L
      • Bomberger JM.
      Interplay between host-microbe and microbe-microbe interactions in cystic fibrosis.
      ]), but a common theme nevertheless starts to emerge. Biofilm-associated bacteria typically downregulate their central metabolism (e.g. the tricarboxylic acid, TCA cycle) with a concomitant upregulation of alternative pathways (e.g. the glyoxylate shunt); by doing so they produce fewer reducing equivalents (NADH, FADH2) which slows down the electron transport chain and reduces the production of toxic reactive oxygen species [
      • Van Acker H
      • Coenye T.
      The role of reactive oxygen species in antibiotic-mediated killing of bacteria.
      ,
      • Van Acker H
      • Sass A
      • Bazzini S
      • De Roy K
      • Udine C
      • Messiaen T
      • et al.
      Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.
      ,
      • Kohanski MA
      • Dwyer DJ
      • Hayete B
      • Lawrence CA
      • Collins JJ.
      A common mechanism of cellular death induced by bactericidal antibiotics.
      ]. The important contribution of the microenvironment and metabolism to biofilm susceptibility also has implications for in vitro evaluation of antimicrobial strategies and strongly suggests that the model systems to be used should closely mimic the in vivo micro-environmental conditions.
      The observation that microbial metabolism plays a crucial role in reduced susceptibility during biofilm-associated infections also opens the door for novel treatment approaches: what if we could counteract the metabolic changes in vivo? Would this allow us to overcome bacterial defence mechanisms and increase antibiotic susceptibility? In this context, carbon sources can likely work as potentiators of antimicrobial activity. While their use to increase activity of antibiotics is not new (see for example [
      • Barraud N
      • Buson A
      • Jarolimek W
      • Rice SA.
      Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
      ,
      • Meylan S
      • Porter CBM
      • Yang JH
      • Belenky P
      • Gutierrez A
      • Lobritz MA
      • et al.
      Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control.
      ]), this strategy has not been systematically explored for biofilms. A recent study using P. aeruginosa biofilms formed in an artificial CF sputum medium [
      • Bao X
      • Bove M
      • Coenye T.
      Organic acids and their salts potentiate the activity of selected antibiotics against Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium.
      ] demonstrated that, by activating the TCA cycle, it is feasible to potentiate the anti-biofilm activity of ciprofloxacin (using D,L-malic acid) and ceftazidime (using sodium acetate). While the observed anti-biofilm effects appeared to be antibiotic and strain dependent, and while much more work is needed (including validation in in vivo models), this study can be considered as a proof-of-concept that direct interference with biofilm metabolism can increase antibiotic susceptibility. Another intriguing alternative approach to overcome the biofilm barrier is hyperbaric oxygen therapy (HBOT). In CF, infected endobronchial mucus quickly becomes anoxic due to O2 consumption by activated polymorphonuclear leukocytes that are recruited to kill the infecting bacteria. The resulting very low levels of oxygen force P. aeruginosa to generate energy in a different way (e.g. using nitrate as terminal electron acceptor), but this switch results in lower metabolic activity and growth, which in turn reduces the susceptibility to antibiotics [
      • Jensen PO
      • Moller SA
      • Lerche CJ
      • Moser C
      • Bjarnsholt T
      • Ciofu O
      • et al.
      Improving antibiotic treatment of bacterial biofilm by hyperbaric oxygen therapy: Not just hot air.
      ]. The idea behind using HBOT is that reoxygenation of the anoxic zones (by exposure to 100% O2 at 2.8 bar for 90 min) will activate microbial aerobic metabolism and will increase antibiotic susceptibility. Indeed, in vitro it has been shown that HBOT dramatically increased killing of P. aeruginosa biofilms by ciprofloxacin [
      • Kolpen M
      • Mousavi N
      • Sams T
      • Bjarnsholt T
      • Ciofu O
      • Moser C
      • et al.
      Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.
      ,
      • Kolpen M
      • Lerche CJ
      • Kragh KN
      • Sams T
      • Koren K
      • Jensen AS
      • et al.
      Hyperbaric oxygen sensitizes anoxic Pseudomonas aeruginosa biofilm to ciprofloxacin.
      ] and tobramycin [
      • Moller SA
      • Jensen PO
      • Hoiby N
      • Ciofu O
      • Kragh KN
      • Bjarnsholt T
      • et al.
      Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients.
      ]. In addition, HBOT lowered the tobramycin concentration required to achieve a 3-log (99.9%) reduction in the number of colony forming units by over 50% (i.e., the same killing could be achieved with much lower antibiotic concentrations) [
      • Moller SA
      • Jensen PO
      • Hoiby N
      • Ciofu O
      • Kragh KN
      • Bjarnsholt T
      • et al.
      Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients.
      ]. While HBOT has been used to treat various infections, mostly wounds with anaerobic bacteria, more evidence is needed that it will be clinically beneficial as adjuvant therapy for antibiotics in the treatment of respiratory tract infections in CF [
      • Jensen PO
      • Moller SA
      • Lerche CJ
      • Moser C
      • Bjarnsholt T
      • Ciofu O
      • et al.
      Improving antibiotic treatment of bacterial biofilm by hyperbaric oxygen therapy: Not just hot air.
      ].
      Finally, while development of resistance against these alternative (combination) treatments seems less likely than with current strategies, it cannot be ruled out. For example, while several quorum sensing inhibitors drastically increased the antimicrobial activity of conventional antibiotics against different bacterial biofilms [
      • Brackman G
      • Cos P
      • Maes L
      • Nelis HJ
      • Coenye T.
      Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo.
      ,
      • Christensen LD
      • van Gennip M
      • Jakobsen TH
      • Alhede M
      • Hougen HP
      • Hoiby N
      • et al.
      Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model.
      ], resistance towards this potentiating activity rapidly develops in vitro [
      • Sass A
      • Slachmuylders L
      • Van Acker H
      • Vandenbussche I
      • Ostyn L
      • Bove M
      • et al.
      Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum-sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms.
      ,
      • Bove M
      • Bao X
      • Sass A
      • Crabbe A
      • Coenye T.
      The quorum-sensing inhibitor furanone C-30 rapidly loses its tobramycin-potentiating activity against pseudomonas aeruginosa biofilms during experimental evolution.
      ]. Moreover, resistance against these antibiotic-potentiating quorum sensing inhibitors was observed in clinical P. aeruginosa isolates that were never exposed to them before, illustrating the difficulties of finding anti-biofilm therapies that are “evolution-proof” [
      • Garcia-Contreras R
      • Perez-Eretza B
      • Jasso-Chavez R
      • Lira-Silva E
      • Roldan-Sanchez JA
      • Gonzalez-Valdez A
      • et al.
      High variability in quorum quenching and growth inhibition by furanone C-30 in Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients.
      ,
      • Garcia-Contreras R
      • Martinez-Vazquez M
      • Velazquez Guadarrama N
      • Villegas Paneda AG
      • Hashimoto T
      • Maeda T
      • et al.
      Resistance to the quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil in Pseudomonas aeruginosa clinical isolates.
      ].

      6. Expanding the therapeutic arsenal against CF pathogens?

      The emergence of AMR and the increased prevalence of difficult-to-treat pathogens highlight the need for novel antimicrobial molecules and/or strategies in pwCF. The novel molecules currently under evaluation as anti-infective drugs in the U.S. CF Foundation and the European CF Society drug development pipelines are mostly in early (phase 1 and 2) stages of development (Figure 3). These investigational products include gallium, nitric oxide and other antimicrobial substances (e.g., lactoferrin-hypothiocyanite or substances active against biofilm), and bacteriophages.
      Fig3
      Fig.3The antimicrobial compounds in the CF therapeutic development pipeline as of October 2022 (adapted from CF Foundation website).
      Gallium is a metal, nearly identical to iron, that disrupts iron metabolism in bacteria and exhibits therapeutic effects in mice and humans with lung infections [
      • Goss CH
      • Kaneko Y
      • Khuu L
      • Anderson GD
      • Ravishankar S
      • Aitken ML
      • et al.
      Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections.
      ]. Intravenous gallium is approved by the Food and Drug Administration for intravenous use in humans and is being studied in phase 1 or 2 trials in pwCF using intravenous or inhaled formulations for targeting P. aeruginosa or Mycobacterium abscessus infections. Novel formulations of gallium are being studied and may show improved antimicrobial effects against Gram-positive and Gram-negative bacteria, and nontuberculous mycobacteria [
      • Visaggio D
      • Frangipani E
      • Hijazi S
      • Pirolo M
      • Leoni L
      • Rampioni G
      • et al.
      Variable susceptibility to gallium compounds of major cystic fibrosis pathogens.
      ].
      Nitric oxide is a gas that exerts natural antimicrobial effects. One hypothesis that has been suggested for many years regarding severe infections is that increasing levels of nitric oxide could help kill bacteria and eliminate their biofilms in the lungs of pwCF [
      • Howlin RP
      • Cathie K
      • Hall-Stoodley L
      • Cornelius V
      • Duignan C
      • Allan RN
      • et al.
      Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic Pseudomonas aeruginosa Infection in cystic fibrosis.
      ]. Phase 1 and 2 studies are being conducted in pwCF. A new inhaled glycopolymer SNSP113 that may disrupt bacterial biofilms and target the mucus layer in the lung has been recently developed and will be tested in pwCF.
      A combination of lactoferrin and hypothiocyanite, two natural substances with antimicrobial activities, has been proposed to be a potentially useful strategy for treating bacterial infection in pwCF. In vitro studies have revealed promising antibacterial effects on CF pathogens, including P. aeruginosa [
      • Tunney MM
      • Payne JE
      • McGrath SJ
      • Einarsson GG
      • Ingram RJ
      • Gilpin DF
      • et al.
      Activity of hypothiocyanite and lactoferrin (ALX-009) against respiratory cystic fibrosis pathogens in sputum.
      ]. However, the first in man clinical study has been ongoing for several years and has been terminated due to financial issues; it is unknown whether this compound will be further developed in pwCF.
      Great hope has emerged with the revival of research into bacteriophages, which had been put on hold or overlooked for many years during past periods of full confidence in success of antibiotics. Bacteriophages are viruses that exclusively infect bacteria and can act as potent bactericidal agents [
      • Kortright KE
      • Chan BK
      • Koff JL
      • Turner PE.
      Phage therapy: a renewed approach to combat antibiotic-resistant bacteria.
      ] thanks to their advantageous features such as self-amplification at the site of infection or the capacity to disrupt biofilm matrix. Their high host specificity makes them very promising tools for targeted and personalised anti-infective therapy. Anecdotal reports described interesting effects of inhaled or intravenous bacteriophages in pwCF who developed infections with untreatable M. abscessus [
      • Dedrick RM
      • Guerrero-Bustamante CA
      • Garlena RA
      • Russell DA
      • Ford K
      • Harris K
      • et al.
      Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus.
      ,
      • Nick JA
      • Dedrick RM
      • Gray AL
      • Vladar EK
      • Smith BE
      • Freeman KG
      • et al.
      Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.
      ], pan-drug resistant A. xylosoxidans[
      • Lebeaux D
      • Merabishvili M
      • Caudron E
      • Lannoy D
      • Van Simaey L
      • Duyvejonck H
      • et al.
      A case of phage therapy against pandrug-resistant Achromobacter xylosoxidans in a 12-year-old lung-transplanted cystic fibrosis patient.
      ] or P. aeruginosa[
      • Chan BK
      • Stanley G
      • Modak M
      • Koff JL
      • Turner PE.
      Bacteriophage therapy for infections in CF.
      ]. Phage cocktails (ready-to-use, or “magistral”, customized preparations [
      • Verbeken G
      • Pirnay JP.
      European regulatory aspects of phage therapy: magistral phage preparations.
      ]) have been produced by several laboratories worldwide, and early phase clinical trials on the phage therapy of P. aeruginosa in CF have been designed. Compassionate use of bacteriophages is also ongoing in multiple countries. Yet many questions remain unanswered, including how to test the efficacy of phage therapy for pwCF both in vitro and in vivo, how to select phage cocktails, how to combine phages with antibiotics, and how best to deliver phages to CF airways [
      • Trend S
      • Fonceca AM
      • Ditcham WG
      • Kicic A
      • Cf A.
      The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and delivery considerations for use in Pseudomonas aeruginosa-infected airways.
      ,
      • Rossitto M
      • Fiscarelli EV
      • Rosati P.
      Challenges and promises for planning future clinical research into bacteriophage therapy Against Pseudomonas aeruginosa in cystic fibrosis.
      ].
      Of note, the CF pipelines mentioned above are not the only routes for approval of new antimicrobial compounds for pwCF. Additional novel antibiotics, applicable also to CF infections, have been commercialized in the past few years, although they have not been subject to clinical trials in pwCF. These novel broad spectrum antibiotics, mostly beta-lactams in combination with beta-lactamase inhibitor (including ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-cilastatin-relebactam, meropenem-vaborbactam and cefidorocol) may be useful in the treatment of Gram-negative bacteria (e.g., P. aeruginosa, A. xylosoxidans, Stenotrophomonas maltophilia, and B. cepacia complex) and nontuberculous mycobacteria and could therefore be considered in pwCF with difficult-to-treat airway infections [
      • Belcher R
      • Zobell JT.
      Optimization of antibiotics for cystic fibrosis pulmonary exacerbations due to highly resistant nonlactose fermenting Gram negative bacilli: Meropenem-vaborbactam and cefiderocol.
      ,
      • Zobell JT
      • Epps KL
      • Young DC.
      Optimization of anti-pseudomonal antibiotics for cystic fibrosis pulmonary exacerbations: II. Cephalosporins and penicillins update.
      ]. These novel antibiotics have shown interesting in vitro activity in several studies using Gram-negative bacterial strains isolated from CF sputum [
      • Beauruelle C
      • Lamoureux C
      • Mashi A
      • Ramel S
      • Le Bihan J
      • Ropars T
      • et al.
      In vitro activity of 22 antibiotics against Achromobacter isolates from people with cystic fibrosis.
      ,
      • Papp-Wallace KM
      • Becka SA
      • Zeiser ET
      • Ohuchi N
      • Mojica MF
      • Gatta JA
      • et al.
      Overcoming an extremely drug resistant (XDR) pathogen: avibactam restores susceptibility to ceftazidime for Burkholderia cepacia complex isolates from cystic fibrosis patients.
      ,
      • Grohs P
      • Taieb G
      • Morand P
      • Kaibi I
      • Podglajen I
      • Lavollay M
      • et al.
      In vitro activity of ceftolozane-tazobactam against multidrug-resistant nonfermenting gram-negative bacilli isolated from patients with cystic fibrosis.
      ] and are being increasingly used in pwCF, as reported in short case series [
      • Warner NC
      • Bartelt LA
      • Lachiewicz AM
      • Tompkins KM
      • Miller MB
      • Alby K
      • et al.
      Cefiderocol for the treatment of adult and pediatric patients with cystic fibrosis and Achromobacter xylosoxidans infections.
      ,
      • Ottino L
      • Bartalesi F
      • Borchi B
      • Bresci S
      • Cavallo A
      • Baccani I
      • et al.
      Ceftolozane/tazobactam for Pseudomonas aeruginosa pulmonary exacerbations in cystic fibrosis adult patients: a case series.
      ]. Imamovic et al. have further suggested novel strategies of cycling approaches using available antibiotics, as the evolution of AMR to P. aeruginosa confers predictable sensitivities to other classes of antibiotics [
      • Imamovic L
      • Ellabaan MMH
      • Dantas-Machado AM
      • Citterio L
      • Wulff T
      • Molin S
      • et al.
      Drug-driven phenotypic convergence supports rational treatment strategies of chronic infections.
      ]. To the best of our knowledge, this recently-presented approach is not currently being tested in clinical trials.
      At this time, antibiotics remain the main approach to fight airway infection in pwCF and their wise use, with the aim to maximize therapeutic effect and to minimize adverse events, should be guided by professionals from antimicrobial stewardship teams who are knowledgeable of specifics of CF microbiology [
      • Cogen JD
      • Kahl BC
      • Maples H
      • McColley SA
      • Roberts JA
      • Winthrop KL
      • et al.
      Finding the relevance of antimicrobial stewardship for cystic fibrosis.
      ]. Other approaches are still in early stages of drug development and there will be major challenges in designing clinical trials, especially at the upcoming time when highly effective CFTR modulators reduce both exacerbation rates and sputum expectoration in pwCF. Nonetheless, current CFTR modulators have limited effects on established bacterial infection [
      • Heltshe SL
      • Mayer-Hamblett N
      • Burns JL
      • Khan U
      • Baines A
      • Ramsey BW
      • et al.
      Pseudomonas aeruginosa in cystic fibrosis patients with G551D-CFTR treated with ivacaftor.
      ], and developing novel anti-infective strategies for pwCF is of utmost importance.

      7. Conclusions

      Adaptation of pathogens to the CF lung environment results in the development of persistent infections. One, but not the only, adaptive mechanism is the evolution towards the AMR phenotype, which is not a simple correlate of mutational changes in their known resistance genes. Bacteria tend to switch to a metabolically less active state with slower growth rate, characteristic of the biofilm associated mode of growth; existing subpopulations of persisters also survive exposure to antibiotics. Standard AST is not designed to consider these bacterial properties, and for these reasons, a broader concept of resistome testing may currently be also of limited clinical value. For more reliable AST, concentration values related to biofilm may be further investigated and clinical breakpoints for antibiotics when administered via inhalation need to be defined. The drug development pipeline for anti-infective therapeutics is rather limited but includes a number of relatively unconventional approaches, such as the use of bacteriophages and antibiotic potentiating drugs.

      Declaration of Competing Interest

      P.D. reports personal fees from Vertex Pharmaceuticals, Chiesi CZ and I.T.A.-Intertact outside the submitted work. P-R. B. reports grants and/or personal fees from Vertex Pharmaceuticals, GlaxoSmithKline, AstraZeneca, Chiesi, Insmed, Viatris, Zambon, Pari, Pfizer, Boehringer Ingelheim outside the submitted work. J.C.D. reports grants and/or personal fees from UK CF Trust, CF Foundation, CF Ireland, EPSRC, Vertex Pharmaceuticals, Boehringer Ingelheim, Eloxx, Algipharma, Abbvie, Arcturus, Enterprise Therapeutics, Recode, LifeArc, Genentech outside the submitted work. All other authors have no conflicts of interest.

      Acknowledgements

      We thank the CF patient organisations from France (Vaincre la Mucoviscidose), The Netherlands (Nederlandse Cystic Fibrosis Stichting), Belgium (Mucovereniging), Italy (Lega Italiana Fibrosi Cistica), United Kingdom (CF Trust) and Germany (Mukoviszidoze e.V.) and to the European CF Society for organizing the pre-conference meeting. We are most grateful to Sylvia Hafkemeyer, David Debisshop and Christine Dubois for their excellent assistance in preparation of the symposium. We acknowledge the help from Dr. R. La Rosa at the Novo Nordisk Foundation Center for Biosustainability (Kgs. Lyngby, Denmark) for designing and creating the Figure 2. PD is supported by the National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103 ) - Funded by the European Union - Next Generation EU. RC is supported by Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades (Ref. CIBERINFEC CB21/13/00084 and PI19-01043). JCD is supported by the National Institutes of Health Research through the Imperial Biomedical Research Centre, the Royal Brompton Clinical Research Facility and a Senior Investigator Award. LRH is supported by the National Institutes of Health ( K24HL141669 ) and the Cystic Fibrosis Foundation ( HOFFMA20Y2-SVC ). HKJ is supported by a Challenge grant ( NNF19OC0056411 ) and a research grant ( NNF18OC0052776 ) from the Novo Nordisk Foundation .

      References

        • Castellani C
        • Duff AJA
        • Bell SC
        • Heijerman HGM
        • Munck A
        • Ratjen F
        • et al.
        ECFS best practice guidelines: the 2018 revision.
        J Cyst Fibros. 2018; 17: 153-178
        • Bullington W
        • Hempstead S
        • Smyth AR
        • Drevinek P
        • Saiman L
        • Waters VJ
        • et al.
        Antimicrobial resistance: concerns of healthcare providers and people with CF.
        J Cyst Fibros. 2021; 20: 407-412
        • Zemanick E
        • Burgel PR
        • Taccetti G
        • Holmes A
        • Ratjen F
        • Byrnes CA
        • et al.
        Antimicrobial resistance in cystic fibrosis: a delphi approach to defining best practices.
        J Cyst Fibros. 2020; 19: 370-375
        • Waters V
        • Ratjen F.
        Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis.
        Cochrane Database Syst Rev. 2017; 10CD009528
        • Smith S
        • Waters V
        • Jahnke N
        • Ratjen F.
        Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis.
        Cochrane Database Syst Rev. 2020; 6CD009528
        • Doring G
        • Flume P
        • Heijerman H
        • Elborn JS.
        Treatment of lung infection in patients with cystic fibrosis: current and future strategies.
        J Cyst Fibros. 2012; 11: 461-479
        • Smith AL
        • Fiel SB
        • Mayer-Hamblett N
        • Ramsey B
        • Burns JL.
        Susceptibility testing of Pseudomonas aeruginosa isolates and clinical response to parenteral antibiotic administration: lack of association in cystic fibrosis.
        Chest. 2003; 123: 1495-1502
        • Waters V
        • Ratjen F.
        Combination antimicrobial susceptibility testing for acute exacerbations in chronic infection of Pseudomonas aeruginosa in cystic fibrosis.
        Cochrane Database Syst Rev. 2017; 6CD006961
        • Hurley MN
        • Ariff AH
        • Bertenshaw C
        • Bhatt J
        • Smyth AR.
        Results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis.
        J Cyst Fibros. 2012; 11: 288-292
        • van-Ingen J
        • Boeree MJ
        • van-Soolingen D
        • Mouton JW.
        Resistance mechanisms and drug susceptibility testing of nontuberculous mycobacteria.
        Drug Resist Updat. 2012; 15: 149-161
        • Boolchandani M
        • D'Souza AW
        • Dantas G
        Sequencing-based methods and resources to study antimicrobial resistance.
        Nat Rev Genet. 2019; 20: 356-370
        • Foweraker JE
        • Laughton CR
        • Brown DF
        • Bilton D.
        Phenotypic variability of Pseudomonas aeruginosa in sputa from patients with acute infective exacerbation of cystic fibrosis and its impact on the validity of antimicrobial susceptibility testing.
        J Antimicrob Chemother. 2005; 55: 921-927
        • Ekkelenkamp MB
        • Diez-Aguilar M
        • Tunney MM
        • Elborn JS
        • Fluit AC
        • Canton R.
        Establishing antimicrobial susceptibility testing methods and clinical breakpoints for inhaled antibiotic therapy.
        Open Forum Infect Dis. 2022; 9: ofac082
        • Kidd TJ
        • Canton R
        • Ekkelenkamp M
        • Johansen HK
        • Gilligan P
        • LiPuma JJ
        • et al.
        Defining antimicrobial resistance in cystic fibrosis.
        J Cyst Fibros. 2018; 17: 696-704
        • Waters VJ
        • Kidd TJ
        • Canton R
        • Ekkelenkamp MB
        • Johansen HK
        • LiPuma JJ
        • et al.
        Reconciling antimicrobial susceptibility testing and clinical response in antimicrobial treatment of chronic cystic fibrosis lung infections.
        Clin Infect Dis. 2019; 69: 1812-1816
        • ST Clark
        • Guttman DS
        • Hwang DM.
        Diversification of Pseudomonas aeruginosa within the cystic fibrosis lung and its effects on antibiotic resistance.
        FEMS Microbiol Lett. 2018; 365
        • Ciofu O
        • Tolker-Nielsen T.
        Tolerance and resistance of Pseudomonas aeruginosa biofilms to antimicrobial agents-how P. aeruginosa can escape antibiotics.
        Front Microbiol. 2019; 10: 913
        • Ciofu O
        • Mandsberg LF
        • Wang H
        • Hoiby N.
        Phenotypes selected during chronic lung infection in cystic fibrosis patients: implications for the treatment of Pseudomonas aeruginosa biofilm infections.
        FEMS Immunol Med Microbiol. 2012; 65: 215-225
        • Oliver A
        • Mena A.
        Bacterial hypermutation in cystic fibrosis, not only for antibiotic resistance.
        Clin Microbiol Infect. 2010; 16: 798-808
      1. Performance Standards for Antimicrobial Susceptibility Testing. 32 ed2022.

      2. EUCAST breakpoints for topical agents. 2022. Available from: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Guidance_documents/Topicals_guidance_note_220412.pdf.

        • Kahlmeter G
        • Turnidge J.
        How to: ECOFFs-the why, the how, and the don'ts of EUCAST epidemiological cutoff values.
        Clin Microbiol Infect. 2022; 28: 952-954
        • Giske CG
        • Turnidge J
        • Canton R
        • Kahlmeter G
        • Committee ES.
        Update from the european committee on antimicrobial susceptibility testing (EUCAST).
        J Clin Microbiol. 2022; 60e0027621
        • Doring G
        • Conway SP
        • Heijerman HG
        • Hodson ME
        • Hoiby N
        • Smyth A
        • et al.
        Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European consensus.
        Eur Respir J. 2000; 16: 749-767
        • Canton R
        • Maiz L
        • Escribano A
        • Olveira C
        • Oliver A
        • Asensio O
        • et al.
        Spanish consensus on the prevention and treatment of Pseudomonas aeruginosa bronchial infections in cystic fibrosis patients.
        Arch Bronconeumol. 2015; 51: 140-150
        • Macia MD
        • Borrell N
        • Perez JL
        • Oliver A.
        Detection and susceptibility testing of hypermutable Pseudomonas aeruginosa strains with the Etest and disk diffusion.
        Antimicrob Agents Chemother. 2004; 48: 2665-2672
        • Macia MD
        • Rojo-Molinero E
        • Oliver A.
        Antimicrobial susceptibility testing in biofilm-growing bacteria.
        Clin Microbiol Infect. 2014; 20: 981-990
        • Fernandez-Olmos A
        • Garcia-Castillo M
        • Maiz L
        • Lamas A
        • Baquero F
        • Canton R.
        In vitro prevention of Pseudomonas aeruginosa early biofilm formation with antibiotics used in cystic fibrosis patients.
        Int J Antimicrob Agents. 2012; 40: 173-176
        • Yau YC
        • Ratjen F
        • Tullis E
        • Wilcox P
        • Freitag A
        • Chilvers M
        • et al.
        Randomized controlled trial of biofilm antimicrobial susceptibility testing in cystic fibrosis patients.
        J Cyst Fibros. 2015; 14: 262-266
        • Moskowitz SM
        • Emerson JC
        • McNamara S
        • Shell RD
        • Orenstein DM
        • Rosenbluth D
        • et al.
        Randomized trial of biofilm testing to select antibiotics for cystic fibrosis airway infection.
        Pediatr Pulmonol. 2011; 46: 184-192
        • Smith S
        • Rowbotham NJ
        • Charbek E.
        Inhaled antibiotics for pulmonary exacerbations in cystic fibrosis.
        Cochrane Database Syst Rev. 2018; 10CD008319
        • Tiddens H
        • Puderbach M
        • Venegas JG
        • Ratjen F
        • Donaldson SH
        • Davis SD
        • et al.
        Novel outcome measures for clinical trials in cystic fibrosis.
        Pediatr Pulmonol. 2015; 50: 302-315
        • Karampitsakos T
        • Papaioannou O
        • Kaponi M
        • Kozanidou A
        • Hillas G
        • Stavropoulou E
        • et al.
        Low penetrance of antibiotics in the epithelial lining fluid. The role of inhaled antibiotics in patients with bronchiectasis.
        Pulm Pharmacol Ther. 2020; 60101885
        • Munzenberger PJ
        • Van Wagnen CA
        • Abdulhamid I
        • Walker PC
        Quality of life as a treatment outcome in patients with cystic fibrosis.
        Pharmacotherapy. 1999; 19: 393-398
        • D'Costa VM
        • McGrann KM
        • Hughes DW
        • Wright GD.
        Sampling the antibiotic resistome.
        Science. 2006; 311: 374-377
        • Wright GD.
        The antibiotic resistome: the nexus of chemical and genetic diversity.
        Nat Rev Microbiol. 2007; 5: 175-186
        • Surette MD
        • Wright GD.
        Lessons from the environmental antibiotic resistome.
        Annu Rev Microbiol. 2017; 71: 309-329
        • Saiman L
        • Siegel JD
        • LiPuma JJ
        • Brown RF
        • Bryson EA
        • Chambers MJ
        • et al.
        Infection prevention and control guideline for cystic fibrosis: 2013 update.
        Infect Control Hosp Epidemiol. 2014; 35 (Suppl): S1-S67
        • Sherrard LJ
        • Tunney MM
        • Elborn JS.
        Antimicrobial resistance in the respiratory microbiota of people with cystic fibrosis.
        Lancet. 2014; 384: 703-713
        • Zhao J
        • Schloss PD
        • Kalikin LM
        • Carmody LA
        • Foster BK
        • Petrosino JF
        • et al.
        Decade-long bacterial community dynamics in cystic fibrosis airways.
        Proc Natl Acad Sci U S A. 2012; 109: 5809-5814
        • Rogers GB
        • Hart CA
        • Mason JR
        • Hughes M
        • Walshaw MJ
        • Bruce KD.
        Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling.
        J Clin Microbiol. 2003; 41: 3548-3558
        • Van den Bossche S
        • De Broe E
        • Coenye T
        • Van Braeckel E
        • Crabbe A.
        The cystic fibrosis lung microenvironment alters antibiotic activity: causes and effects.
        Eur Respir Rev. 2021; 30
        • O'Brien TJ
        • Figueroa W
        • Welch M
        Decreased efficacy of antimicrobial agents in a polymicrobial environment.
        ISME J. 2022; 16: 1694-1704
        • Bradley P
        • Gordon NC
        • Walker TM
        • Dunn L
        • Heys S
        • Huang B
        • et al.
        Rapid antibiotic-resistance predictions from genome sequence data for Staphylococcus aureus and Mycobacterium tuberculosis.
        Nat Commun. 2015; 6: 10063
        • Gabrielaite M
        • Bartell JA
        • Norskov-Lauritsen N
        • Pressler T
        • Nielsen FC
        • Johansen HK
        • et al.
        Transmission and antibiotic resistance of Achromobacter in cystic fibrosis.
        J Clin Microbiol. 2021; 59
        • Cox H
        • Mizrahi V.
        The coming of age of drug-susceptibility testing for tuberculosis.
        N Engl J Med. 2018; 379: 1474-1475
        • Nelson MT
        • Wolter DJ
        • Eng A
        • Weiss EJ
        • Vo AT
        • Brittnacher MJ
        • et al.
        Maintenance tobramycin primarily affects untargeted bacteria in the CF sputum microbiome.
        Thorax. 2020; 75: 780-790
        • Heirali A
        • Thornton C
        • Acosta N
        • Somayaji R
        • Laforest-Lapointe I
        • Storey D
        • et al.
        Sputum microbiota in adults with CF associates with response to inhaled tobramycin.
        Thorax. 2020; 75: 1058-1064
        • Heirali AA
        • Workentine ML
        • Acosta N
        • Poonja A
        • Storey DG
        • Somayaji R
        • et al.
        The effects of inhaled aztreonam on the cystic fibrosis lung microbiome.
        Microbiome. 2017; 5: 51
      3. Orenti A, Zolin A, Jung A, van Rens J. ECFSPR Annual Report 2020. 2022.

        • Marvig RL
        • Sommer LM
        • Molin S
        • Johansen HK.
        Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis.
        Nat Genet. 2015; 47: 57-64
        • Bartell JA
        • Sommer LM
        • Marvig RL
        • Skov M
        • Pressler T
        • Molin S
        • et al.
        Omics-based tracking of Pseudomonas aeruginosa persistence in "eradicated" cystic fibrosis patients.
        Eur Respir J. 2021; 57
        • La-Rosa R
        • Johansen HK
        • Molin S
        Persistent bacterial infections, antibiotic treatment failure, and microbial adaptive evolution.
        Antibiotics (Basel). 2022; 11
        • Bartell JA
        • Sommer LM
        • Haagensen JAJ
        • Loch A
        • Espinosa R
        • Molin S
        • et al.
        Evolutionary highways to persistent bacterial infection.
        Nat Commun. 2019; 10: 629
        • Bartell JA
        • Cameron DR
        • Mojsoska B
        • Haagensen JAJ
        • Pressler T
        • Sommer LM
        • et al.
        Bacterial persisters in long-term infection: emergence and fitness in a complex host environment.
        PLoS Pathog. 2020; 16e1009112
        • Halfon Y
        • Jimenez-Fernandez A
        • La-Rosa R
        • Espinosa-Portero R
        • Krogh-Johansen H
        • Matzov D
        • et al.
        Structure of Pseudomonas aeruginosa ribosomes from an aminoglycoside-resistant clinical isolate.
        Proc Natl Acad Sci U S A. 2019; 116: 22275-22281
        • Frimodt-Moller J
        • Rossi E
        • Haagensen JAJ
        • Falcone M
        • Molin S
        • Johansen HK.
        Mutations causing low level antibiotic resistance ensure bacterial survival in antibiotic-treated hosts.
        Sci Rep. 2018; 8: 12512
        • Goltermann L
        • Andersen KL
        • Johansen HK
        • Molin S
        • La-Rosa R
        Macrolide therapy in Pseudomonas aeruginosa infections causes uL4 ribosomal protein mutations leading to high-level resistance.
        Clin Microbiol Infect. 2022;
        • Rossi E
        • La-Rosa R
        • Bartell JA
        • Marvig RL
        • Haagensen JAJ
        • Sommer LM
        • et al.
        Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis.
        Nat Rev Microbiol. 2021; 19: 331-342
        • Kolpen M
        • Kragh KN
        • Enciso JB
        • Faurholt-Jepsen D
        • Lindegaard B
        • Egelund GB
        • et al.
        Bacterial biofilms predominate in both acute and chronic human lung infections.
        Thorax. 2022;
        • Schwab U
        • Abdullah LH
        • Perlmutt OS
        • Albert D
        • Davis CW
        • Arnold RR
        • et al.
        Localization of Burkholderia cepacia complex bacteria in cystic fibrosis lungs and interactions with Pseudomonas aeruginosa in hypoxic mucus.
        Infect Immun. 2014; 82: 4729-4745
        • Bjarnsholt T
        • Alhede M
        • Alhede M
        • Eickhardt-Sorensen SR
        • Moser C
        • Kuhl M
        • et al.
        The in vivo biofilm.
        Trends Microbiol. 2013; 21: 466-474
        • Bjarnsholt T
        • Jensen PO
        • Fiandaca MJ
        • Pedersen J
        • Hansen CR
        • Andersen CB
        • et al.
        Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients.
        Pediatr Pulmonol. 2009; 44: 547-558
        • Van Acker H
        • Van Dijck P
        • Coenye T.
        Molecular mechanisms of antimicrobial tolerance and resistance in bacterial and fungal biofilms.
        Trends Microbiol. 2014; 22: 326-333
        • Ciofu O
        • Moser C
        • Jensen PO
        • Hoiby N.
        Tolerance and resistance of microbial biofilms.
        Nat Rev Microbiol. 2022;
        • Bjarnsholt T
        • Whiteley M
        • Rumbaugh KP
        • Stewart PS
        • Jensen PO
        Frimodt-Moller N. The importance of understanding the infectious microenvironment.
        Lancet Infect Dis. 2022; 22: e88-e92
        • Van Acker H
        • Coenye T.
        The role of reactive oxygen species in antibiotic-mediated killing of bacteria.
        Trends Microbiol. 2017; 25: 456-466
        • Crabbe A
        • Ostyn L
        • Staelens S
        • Rigauts C
        • Risseeuw M
        • Dhaenens M
        • et al.
        Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics.
        PLoS Pathog. 2019; 15e1007697
        • Crabbe A
        • Jensen PO
        • Bjarnsholt T
        • Coenye T.
        Antimicrobial tolerance and metabolic adaptations in microbial biofilms.
        Trends Microbiol. 2019; 27: 850-863
        • Armbruster CR
        • Coenye T
        • Touqui L
        • Bomberger JM.
        Interplay between host-microbe and microbe-microbe interactions in cystic fibrosis.
        J Cyst Fibros. 2020; 19 (Suppl): S47-S53
        • Van Acker H
        • Sass A
        • Bazzini S
        • De Roy K
        • Udine C
        • Messiaen T
        • et al.
        Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.
        PLoS One. 2013; 8: e58943
        • Kohanski MA
        • Dwyer DJ
        • Hayete B
        • Lawrence CA
        • Collins JJ.
        A common mechanism of cellular death induced by bactericidal antibiotics.
        Cell. 2007; 130: 797-810
        • Barraud N
        • Buson A
        • Jarolimek W
        • Rice SA.
        Mannitol enhances antibiotic sensitivity of persister bacteria in Pseudomonas aeruginosa biofilms.
        PLoS One. 2013; 8: e84220
        • Meylan S
        • Porter CBM
        • Yang JH
        • Belenky P
        • Gutierrez A
        • Lobritz MA
        • et al.
        Carbon sources tune antibiotic susceptibility in Pseudomonas aeruginosa via tricarboxylic acid cycle control.
        Cell Chem Biol. 2017; 24: 195-206
        • Bao X
        • Bove M
        • Coenye T.
        Organic acids and their salts potentiate the activity of selected antibiotics against Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium.
        Antimicrob Agents Chemother. 2022; 66e0187521
        • Jensen PO
        • Moller SA
        • Lerche CJ
        • Moser C
        • Bjarnsholt T
        • Ciofu O
        • et al.
        Improving antibiotic treatment of bacterial biofilm by hyperbaric oxygen therapy: Not just hot air.
        Biofilm. 2019; 1100008
        • Kolpen M
        • Mousavi N
        • Sams T
        • Bjarnsholt T
        • Ciofu O
        • Moser C
        • et al.
        Reinforcement of the bactericidal effect of ciprofloxacin on Pseudomonas aeruginosa biofilm by hyperbaric oxygen treatment.
        Int J Antimicrob Agents. 2016; 47: 163-167
        • Kolpen M
        • Lerche CJ
        • Kragh KN
        • Sams T
        • Koren K
        • Jensen AS
        • et al.
        Hyperbaric oxygen sensitizes anoxic Pseudomonas aeruginosa biofilm to ciprofloxacin.
        Antimicrob Agents Chemother. 2017; 61
        • Moller SA
        • Jensen PO
        • Hoiby N
        • Ciofu O
        • Kragh KN
        • Bjarnsholt T
        • et al.
        Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients.
        J Cyst Fibros. 2019; 18: 657-664
        • Brackman G
        • Cos P
        • Maes L
        • Nelis HJ
        • Coenye T.
        Quorum sensing inhibitors increase the susceptibility of bacterial biofilms to antibiotics in vitro and in vivo.
        Antimicrob Agents Chemother. 2011; 55: 2655-2661
        • Christensen LD
        • van Gennip M
        • Jakobsen TH
        • Alhede M
        • Hougen HP
        • Hoiby N
        • et al.
        Synergistic antibacterial efficacy of early combination treatment with tobramycin and quorum-sensing inhibitors against Pseudomonas aeruginosa in an intraperitoneal foreign-body infection mouse model.
        J Antimicrob Chemother. 2012; 67: 1198-1206
        • Sass A
        • Slachmuylders L
        • Van Acker H
        • Vandenbussche I
        • Ostyn L
        • Bove M
        • et al.
        Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum-sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms.
        Antimicrob Agents Chemother. 2019; 63
        • Bove M
        • Bao X
        • Sass A
        • Crabbe A
        • Coenye T.
        The quorum-sensing inhibitor furanone C-30 rapidly loses its tobramycin-potentiating activity against pseudomonas aeruginosa biofilms during experimental evolution.
        Antimicrob Agents Chemother. 2021; 65e0041321
        • Garcia-Contreras R
        • Perez-Eretza B
        • Jasso-Chavez R
        • Lira-Silva E
        • Roldan-Sanchez JA
        • Gonzalez-Valdez A
        • et al.
        High variability in quorum quenching and growth inhibition by furanone C-30 in Pseudomonas aeruginosa clinical isolates from cystic fibrosis patients.
        Pathog Dis. 2015; 73: ftv040
        • Garcia-Contreras R
        • Martinez-Vazquez M
        • Velazquez Guadarrama N
        • Villegas Paneda AG
        • Hashimoto T
        • Maeda T
        • et al.
        Resistance to the quorum-quenching compounds brominated furanone C-30 and 5-fluorouracil in Pseudomonas aeruginosa clinical isolates.
        Pathog Dis. 2013; 68: 8-11
        • Goss CH
        • Kaneko Y
        • Khuu L
        • Anderson GD
        • Ravishankar S
        • Aitken ML
        • et al.
        Gallium disrupts bacterial iron metabolism and has therapeutic effects in mice and humans with lung infections.
        Sci Transl Med. 2018; 10
        • Visaggio D
        • Frangipani E
        • Hijazi S
        • Pirolo M
        • Leoni L
        • Rampioni G
        • et al.
        Variable susceptibility to gallium compounds of major cystic fibrosis pathogens.
        ACS Infect Dis. 2022; 8: 78-85
        • Howlin RP
        • Cathie K
        • Hall-Stoodley L
        • Cornelius V
        • Duignan C
        • Allan RN
        • et al.
        Low-dose nitric oxide as targeted anti-biofilm adjunctive therapy to treat chronic Pseudomonas aeruginosa Infection in cystic fibrosis.
        Mol Ther. 2017; 25: 2104-2116
        • Tunney MM
        • Payne JE
        • McGrath SJ
        • Einarsson GG
        • Ingram RJ
        • Gilpin DF
        • et al.
        Activity of hypothiocyanite and lactoferrin (ALX-009) against respiratory cystic fibrosis pathogens in sputum.
        J Antimicrob Chemother. 2018; 73: 3391-3397
        • Kortright KE
        • Chan BK
        • Koff JL
        • Turner PE.
        Phage therapy: a renewed approach to combat antibiotic-resistant bacteria.
        Cell Host Microbe. 2019; 25: 219-232
        • Dedrick RM
        • Guerrero-Bustamante CA
        • Garlena RA
        • Russell DA
        • Ford K
        • Harris K
        • et al.
        Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus.
        Nat Med. 2019; 25: 730-733
        • Nick JA
        • Dedrick RM
        • Gray AL
        • Vladar EK
        • Smith BE
        • Freeman KG
        • et al.
        Host and pathogen response to bacteriophage engineered against Mycobacterium abscessus lung infection.
        Cell. 2022; 185 (e12): 1860-1874
        • Lebeaux D
        • Merabishvili M
        • Caudron E
        • Lannoy D
        • Van Simaey L
        • Duyvejonck H
        • et al.
        A case of phage therapy against pandrug-resistant Achromobacter xylosoxidans in a 12-year-old lung-transplanted cystic fibrosis patient.
        Viruses. 2021; 13
        • Chan BK
        • Stanley G
        • Modak M
        • Koff JL
        • Turner PE.
        Bacteriophage therapy for infections in CF.
        Pediatr Pulmonol. 2021; 56 (Suppl): S4-S9
        • Verbeken G
        • Pirnay JP.
        European regulatory aspects of phage therapy: magistral phage preparations.
        Curr Opin Virol. 2022; 52: 24-29
        • Trend S
        • Fonceca AM
        • Ditcham WG
        • Kicic A
        • Cf A.
        The potential of phage therapy in cystic fibrosis: Essential human-bacterial-phage interactions and delivery considerations for use in Pseudomonas aeruginosa-infected airways.
        J Cyst Fibros. 2017; 16: 663-670
        • Rossitto M
        • Fiscarelli EV
        • Rosati P.
        Challenges and promises for planning future clinical research into bacteriophage therapy Against Pseudomonas aeruginosa in cystic fibrosis.
        An Argumentative Rev Front Microbiol. 2018; 9: 775
        • Belcher R
        • Zobell JT.
        Optimization of antibiotics for cystic fibrosis pulmonary exacerbations due to highly resistant nonlactose fermenting Gram negative bacilli: Meropenem-vaborbactam and cefiderocol.
        Pediatr Pulmonol. 2021; 56: 3059-3061
        • Zobell JT
        • Epps KL
        • Young DC.
        Optimization of anti-pseudomonal antibiotics for cystic fibrosis pulmonary exacerbations: II. Cephalosporins and penicillins update.
        Pediatr Pulmonol. 2017; 52: 863-865
        • Beauruelle C
        • Lamoureux C
        • Mashi A
        • Ramel S
        • Le Bihan J
        • Ropars T
        • et al.
        In vitro activity of 22 antibiotics against Achromobacter isolates from people with cystic fibrosis.
        Are There New Therapeutic Options? Microorg. 2021; 9
        • Papp-Wallace KM
        • Becka SA
        • Zeiser ET
        • Ohuchi N
        • Mojica MF
        • Gatta JA
        • et al.
        Overcoming an extremely drug resistant (XDR) pathogen: avibactam restores susceptibility to ceftazidime for Burkholderia cepacia complex isolates from cystic fibrosis patients.
        ACS Infect Dis. 2017; 3: 502-511
        • Grohs P
        • Taieb G
        • Morand P
        • Kaibi I
        • Podglajen I
        • Lavollay M
        • et al.
        In vitro activity of ceftolozane-tazobactam against multidrug-resistant nonfermenting gram-negative bacilli isolated from patients with cystic fibrosis.
        Antimicrob Agents Chemother. 2017; 61
        • Warner NC
        • Bartelt LA
        • Lachiewicz AM
        • Tompkins KM
        • Miller MB
        • Alby K
        • et al.
        Cefiderocol for the treatment of adult and pediatric patients with cystic fibrosis and Achromobacter xylosoxidans infections.
        Clin Infect Dis. 2021; 73 (-e7): e1754
        • Ottino L
        • Bartalesi F
        • Borchi B
        • Bresci S
        • Cavallo A
        • Baccani I
        • et al.
        Ceftolozane/tazobactam for Pseudomonas aeruginosa pulmonary exacerbations in cystic fibrosis adult patients: a case series.
        Eur J Clin Microbiol Infect Dis. 2021; 40: 2211-2215
        • Imamovic L
        • Ellabaan MMH
        • Dantas-Machado AM
        • Citterio L
        • Wulff T
        • Molin S
        • et al.
        Drug-driven phenotypic convergence supports rational treatment strategies of chronic infections.
        Cell. 2018; 172 (-2e14): 121-134
        • Cogen JD
        • Kahl BC
        • Maples H
        • McColley SA
        • Roberts JA
        • Winthrop KL
        • et al.
        Finding the relevance of antimicrobial stewardship for cystic fibrosis.
        J Cyst Fibros. 2020; 19: 511-520
        • Heltshe SL
        • Mayer-Hamblett N
        • Burns JL
        • Khan U
        • Baines A
        • Ramsey BW
        • et al.
        Pseudomonas aeruginosa in cystic fibrosis patients with G551D-CFTR treated with ivacaftor.
        Clin Infect Dis. 2015; 60: 703-712