Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis

Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis

Accepted Manuscript Title: Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis Authors: S. Stefani, S. Campana, L. C...

390KB Sizes 1 Downloads 123 Views

Accepted Manuscript Title: Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis Authors: S. Stefani, S. Campana, L. Cariani, V. Carnovale, C. Colombo, M. del Mar Lle´o, V.D. Iula, L. Minicucci, P. Morelli, G. Pizzamiglio, G. Taccetti PII: DOI: Reference:

S1438-4221(17)30023-1 http://dx.doi.org/doi:10.1016/j.ijmm.2017.07.004 IJMM 51134

To appear in: Received date: Revised date: Accepted date:

12-1-2017 13-7-2017 14-7-2017

Please cite this article as: Stefani, S., Campana, S., Cariani, L., Carnovale, V., Colombo, C., del Mar Lle´o, M., Iula, V.D., Minicucci, L., Morelli, P., Pizzamiglio, G., Taccetti, G., Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis.International Journal of Medical Microbiology http://dx.doi.org/10.1016/j.ijmm.2017.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis S. Stefania, S. Campanab, L. Carianic, V. Carnovaled, C. Colomboe, M. del Mar Lleóf, V.D. Iulag, L. Minicuccih, P. Morellii, G. Pizzamiglioj, G. Taccettib a

Department of Biomedical and Biotechnological Sciences, Division of Microbiology, University of

Catania, Catania, Italy b

Department of Paediatric Medicine, Cystic Fibrosis Centre, Anna Meyer Children's University

Hospital, Florence, Italy c

Cystic Fibrosis Microbiology Laboratory, Fondazione IRCCS Ca' Granda, Ospedale Maggiore

Policlinico, Milan, Italy d

Department of Translational Medical Sciences, Cystic Fibrosis Center, University "Federico II",

Naples, Italy e

Cystic Fibrosis Center, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan,

Italy f

Department of Diagnostics and Public Health, University of Verona, Verona, Italy

g

Department of Molecular Medicine and Medical Biotechnology, Federico II University Medical

School, Naples, Italy h

Microbiology Laboratory, Cystic Fibrosis Center, G. Gaslini Institute, Genoa, Italy

i

Department of Paediatric, Cystic Fibrosis Center, G. Gaslini Institute, Genoa, Italy

j

Respiratory Disease Department, Cystic Fibrosis Center Adult Section, Fondazione IRCCS Ca'

Granda Ospedale Maggiore Policlinico Milano, Milan, Italy.

1

Abstract Multidrug-resistant (MDR) Pseudomonas aeruginosa is an important issue for physicians who take care of patients with cystic fibrosis (CF). Here, we review the latest research on how P. aeruginosa infection causes lung function to decline and how several factors contribute to the emergence of antibiotic resistance in P. aeruginosa strains and influence the course of the infection course. However, many aspects of the practical management of patients with CF infected with MDR P. aeruginosa are still to be established. Less is known about the exact role of susceptibility testing in clinical strategies for dealing with resistant infections, and there is an urgent need to find a tool to assist in choosing the best therapeutic strategy for MDR P. aeruginosa infection. One current perception is that the selection of antibiotic therapy according to antibiogram results is an important component of the decision-making process, but other patient factors, such as previous infection history and antibiotic courses, also need to be evaluated. On the basis of the known issues and the best current data on respiratory infections caused by MDR P. aeruginosa, this review provides practical suggestions to optimize the diagnostic and therapeutic management of patients with CF who are infected with these pathogens. Keywords: Multidrug resistance; Antimicrobial susceptibility testing; Cystic fibrosis; Antibiotic therapy; Lung infection Introduction Lung infections are responsible for most of the morbidity and mortality in patients with cystic fibrosis (CF). The pathogens involved, mainly bacteria, are becoming challenging in terms of resistance and acquisition of virulence, rendering the management of these patients more difficult. In recent decades, new antibiotic therapies1-6 have emerged to treat respiratory infections that result in decline of lung function, respiratory failure and premature death of patients with CF. The quality of life and the length of survival largely depend on the success of antibiotic therapy in eradicating the initial infection7, suppression of chronic infections, and treatment of pulmonary exacerbations.

2

Despite recent progress in the treatment of CF,8 antibiotics are one of the most important components of CF management and have been responsible for an increase in median life expectancy to almost 40 years.9,10 In recent years, the microbial community resident in CF lungs has changed considerably, mainly as a result of intensive antibiotic pressure and alterations in antibiotic regimens, with the consequence that bacteria with newly acquired resistance traits or new pathogens are appearing.11,12 One of the most important issues for physicians who take care of patients with CF is the increasing problem of multidrug-resistant (MDR) isolates of Pseudomonas aeruginosa13. MDR P. aeruginosa is defined as being resistant to all antibiotics routinely evaluated in two or more of the following groups: aminoglycosides (tobramycin, gentamicin and amikacin), fluoroquinolones (ciprofloxacin), and β-lactams (ceftazidime, meropenem, imipenem, piperacillin, piperacillintazobactam, ticarcillin-clavulanate and aztreonam).14 These MDR strains result from inherent antibiotic resistance of the bacteria themselves and by their ability to acquire a plethora of adaptive mutations and diverse resistance determinants15, and they are associated with worse clinical outcomes.16 These considerations well describe the current situation. We have, nevertheless, to stress that at the moment a uniform definition of multi-drug-resistance (MDR) is lacking and would be desirable. Important initiatives in this area are being made by scientific societies but attempt to obtain a homogeneous definition in CF and non-CF contexts have not yet created a result in daily clinical practice. The attempt to find homogeneous definitions is not the objective of this work and would be premature and not based on objective data This review is based on definitions of MDRs exclusively used in the CF field and therefore uses the same definition of multiresistance in use in the North American CF Registry (www.cff.org).

3

Recent data from more than 28,000 individuals with CF included in the most recent Cystic Fibrosis Foundation Patient Registry annual report (2014)9 show that the percentage of patients with MDR P. aeruginosa has reached about 18.1%. Rates of MDR P. aeruginosa infection have increased substantially in older patients with CF. These findings likely reflect cumulative exposure to antibiotics. In addition, epidemic P. aeruginosa strains, which have an increased propensity to transmission among patients, display enhanced virulence and antimicrobial resistance, becoming an important nosocomial pathogen.17,18 In the setting of CF, a recent study has pointed out the interplay between key microbiological aspects of chronic respiratory infection by P. aeruginosa in CF: (1) the occurrence of transmissible strains and persistent strains; (2) the emergence of variants with enhanced mutation rates19; and (3) the evolution of antibiotic resistance.20 In consideration of the increasing rate of MDR P. aeruginosa, this review provides advice to support the management of patients infected with MDR P. aeruginosa and discusses whether susceptibility testing is still needed to guide clinical strategies and tackle resistant infection. P. aeruginosa adaptation mechanisms The microbiology of pulmonary infections in patients with CF is often different from similar infections in individuals who do not have CF, and the phenotypes of CF bacteria are frequently atypical.21 Microbiological and clinical studies have demonstrated that the infectious process in CF airways is a very complex phenomenon and several factors can influence the infection course. The bacterial species with established clinical relevance in CF lung disease are relatively few, but the potential pathogenicity of a large number of species that are frequently isolated remains undefined. The role of P. aeruginosa infection in the decline of lung function has been well explained. P. aeruginosa enters the lower airways by inhalation; some patients with CF (10–50%) are able to

4

clear the pathogen spontaneously or become culture-negative in subsequent specimens.22,23 However, the pathogen can persist or recur and eventually may contribute to transforming transient colonization into a chronic infection. P. aeruginosa infection is defined as chronic when it is detected in more than 50% of the cultures performed over a time span of 12 months24 (at least four airway cultures are required in different months spread throughout the year). It is well known that the long-term persistence of P. aeruginosa in CF airways is associated with sophisticated mechanisms of adaptation.21,25,26 In patients with CF, P. aeruginosa isolates display significant phenotypic variations, such as development of a mucoid phenotype and highly adherent small-colony variants (SCVs), the absence of cell motility, development of variants resistant to macrophage phagocytosis and acquisition of resistance to multiple antibiotics.27-37 In particular, SCVs exhibit different properties from the wildtype (WT) parent strains, including a slow growth rate, superior adherence, reduced motility, hyperpiliation (extra hairlike appendages), increased hydrophobicity, increased biofilm formation, reduced pyoverdin and pyocyanin production and increased resistance to antibiotics.30,37-40 An interesting property, peculiar to P. aeruginosa strains isolated from CF lung, is the unusual hypermutability.41 They possess the ability to react promptly to their environment not only by switching genes on or off but also by increasing the frequency of mutation events within the genome. In a cross-sectional study, Ciofu et al.42 detected mutator strains in 54% of Danish patients with CF who were chronically colonized with P. aeruginosa. In this study, a longitudinal (up to 25 years) evaluation of the prevalence of P. aeruginosa mutator strains in patients with CF highlighted that the proportion of hypermutable isolates increased from 0% at onset/early colonization to 65% after 20 years of chronic colonization, suggesting that the hypermutable phenotype is associated with mutations that confer adaptation of the bacteria in the lung and persistence of the infection. In a recent genomic analysis of P. aeruginosa isolated from Italian patients with CF, Marving et al.43 correlated mutations with changes in CF-relevant phenotypes such as antibiotic resistance. In 5

another recent work44 based on isolates of P. aeruginosa from the lungs of 34 children affected by CF, 52 genes were identified as more frequently mutated than what would be expected under genetic drift. These were all candidate pathoadaptive genes, where mutation optimized pathogen fitness conferring those adaptation traits already mentioned, such as antibiotic resistance (β-lactams, quinolones, chloramphenicol, macrolides, penicillin, aminoglycosides). Other authors have documented the prevalence of hypermutable P. aeruginosa isolates of approximately 5–10% at onset/early colonization in patients with CF.45,46 One of such mutation event triggers conversion to a mucoid phenotype, which is almost pathognomonic for increased severity of infection.47,48 In response to environmental triggers such as nutritional stress or hypoxia found within a CF mucus plug, exopolysaccharide (alginate)-producing mucoid mutants are selected. Another highly successful survival strategy involves the production of biofilms. This process is regulated by a “quorum sensing” system comprising networks of genes and regulators able to modulate the entire life of the microorganism.49 When these QS systems are activated, the bacterium can produce molecules such as acyl homoserine lactones that diffuse freely in and out across the bacterial membrane. As a result of this free diffusibility, the concentration within the organism reflects the concentration outside, which enables the bacteria to “sense” other bacteria in the vicinity. Once a critical mass has been achieved, the QS molecules induce expression of the genes responsible for adhesion and biofilm production. In this state, microcolonies of bacteria are surrounded by a dense matrix, which protects them against phagocytosis and prevents the penetration of antibiotic agents. A recent work19, involving 54 children and adolescents with CF, and aiming to the identification of subclonal variants of P. aeruginosa, has identified six nonsynonymous SNPs in the LasR gene, a key transcriptional regulator of QS. The coexistence of LasR isoforms reflects the diversifying selection implemented to improve the fitness of P. aeruginosa.

6

By switching to the biofilm mode of growth, P. aeruginosa acquires tolerance towards the inflammatory defence mechanism and to antibiotic therapy,50,51 becoming 100- to 1000-fold more tolerant to antimicrobial agents than planktonic bacteria.52,53 Biofilm-associated antimicrobial tolerance is fundamentally different from antimicrobial resistance, which can be displayed by bacteria in planktonic culture, and is not connected to the biofilm mode of growth per se.54,55 Biofilm antimicrobial tolerance results from a combination of mechanisms, including restricted penetration of antimicrobials through the exopolysaccharide matrix, differential physiological activity caused by limited oxygen and nutrient penetration through the biofilm as a result of bacterial consumption and differential expression of specific genes.26 The biofilm mode of growth leads to oxidative stress, which causes enhanced mutability in biofilms.56,57 These phenotypical changes are likely to play a major role in the persistence of P. aeruginosa infection in most patients with CF despite best medical attempts at eradication. P. aeruginosa multidrug-resistance development The adaptation mechanisms described provide a fertile ground for the emergence of antibiotic resistance in P. aeruginosa strains. High percentages of hypermutable P. aeruginosa isolates associated with antibiotic resistance have been found in patients with CF.41,42,58 In CF isolates from France, Ferroni et al.59 analysed the effect of hypermutation over the time required for the appearance of antibiotic resistance and demonstrated that mutator strains acquired additional resistance mechanisms much more quickly than non-mutator strains. Biofilm-growing bacteria easily become multidrug resistant by means of traditional resistance mechanisms against β-lactam antibiotics, aminoglycosides and fluoroquinolones, including production of enzymes that degrade antibiotics, the presence of low-affinity antibiotic targets and overexpression of efflux pumps that have a broad range of substrates.

7

Surprisingly, an in vitro evaluation of P. aeruginosa phenotypes collected among the baseline respiratory isolates of 194 newly identified infections60, evidenced the prevalence of phenotypes traditionally associated with adaptive changes during chronic infections, suggesting that the P. aeruginosa infection could be silent for a long time and undetected in the upper airways or in the lower respiratory tract. These phenotypes (associated with mucoid, wrinkly colony surface, irregular colony edges) are significantly associated with antibiotic failure. If these results are validated, these colonies may be used as markers of eradication failure and lead to an early identification and prediction of those patients where a more aggressive eradication strategy should be followed. Resistance to β-lactams The pathogenesis of P. aeruginosa resistance to β-lactam antibiotics is multifactorial but is mediated mainly by inactivating enzymes called β-lactamases. Resistance to β-lactams occurs due to mutations in the regulatory genes of β-lactamase production, leading to the occurrence of isolates with stable or partially stable derepressed production of AmpC β-lactamase. Overproduction of chromosomally encoded AmpC cephalosporinase is considered the main mechanism of resistance to β-lactam antibiotics in CF P. aeruginosa isolates. The most common β-lactamase production phenotype in CF isolates is the partially derepressed phenotype with high basal levels of βlactamase, which can be further increased when β-lactam antibiotics are present.61 Different types of acquired β-lactamases have been found in P. aeruginosa isolates around the world. Among them, metallo-β-lactamases (MBLs) are emerging as resistance determinants of increasing clinical importance. MBLs can confer a very broad spectrum of resistance to β-lactams (including the expanded-spectrum cephalosporins and carbapenems), which is not reversible by the available β-lactamase inhibitors. Due to linkage of MBL genes with other resistance genes, MBLproducing strains usually exhibit a complex MDR phenotype.62 MBL-producing P. aeruginosa has been reported worldwide in nosocomial settings.63-68 Its presence in patients with CF has been 8

reported in Portugal69 and, more recently, an epidemic Pseudomonas aeruginosa clone producing IMP-13 metallo-β-lactamase, causing infections and even large outbreaks in Italian critical care settings, was detected in a young patient with CF.70 Several chromosomally encoded efflux systems play an important role, together with other mechanisms, in the development of multidrug resistance in P. aeruginosa. Because the bacteria in the CF lung are often exposed to oxygen radicals and some of the efflux systems of P. aeruginosa are upregulated in response to oxygen radicals, mutants overexpressing these pumps might be selected. Among the various efflux systems of P. aeruginosa, mexX and mexA overexpression are the main contributing factors to carbapenem resistance.71 In a recent study72 the old antibiotic, recently revived, temocillin has been investigated to verify its activity on a large collection of P. aeruginosa from CF patients. Temocillin susceptibility was observed for isolates resistant to other β-lactams or in isolates considered as MDR. Resistance to aminoglycosides Resistance to aminoglycosides is mediated by acquisition of plasmid- and/or integron-borne genes encoding various transferable aminoglycoside-modifying enzymes (AMEs), rRNA methylases and derepression of endogenous efflux systems. In addition, overexpression of efflux pumps (MexXY)73 is also an important mechanism. MexXY-mediated aminoglycoside resistance is disproportionately represented among strains of P. aeruginosa recovered from CF lungs.71,74 Resistance to fluoroquinolones Resistance to ciprofloxacin in CF P. aeruginosa isolates was shown to be mediated by overexpression of multidrug efflux pumps as a result of mutations in the genes involved in the regulation of pump expression75 and/or mutational changes within the target’s DNA gyrase (gyrA, gyrB) and/or topoisomerase IV (parC and parE).76 Mutants overexpressing MexCD-OprJ may be selected by ciprofloxacin treatment of hypermutable P. aeruginosa mutT and mutY strains77. 9

Azithromycin, routinely prescribed for CF as an anti-inflammatory drug, might select in P. aeruginosa biofilms for mutants overexpressing MexCD-OprJ, leading to cross-resistance to ciprofloxacin.78 Resistance to colistin (polymyxin E) Several studies have shown that P. aeruginosa can develop resistance to polymyxins via constitutive modification of its lipopolysaccharides (LPSs). which have overall negative charges and are the initial targets of polymyxins. The activation of two-component systems (TCSs) involving PhoP/PhoQ and PmrA/PmrB is triggered by environmental stimuli and specific mutations within the TCSs that result in their constitutive activation and subsequent overexpression of LPSmodifying genes.79 Only one study has reported a mutation in PmrA that may be responsible for P. aeruginosa resistance to date,80 while the rest of the mutations have been mainly localized to PmrB. Moskowitz et al.81 showed that PmrB mutations promote polymyxin resistance of P. aeruginosa isolated from colistin-treated patients with CF. The PmrB mutants were cultured from patients with CF attending clinical centres in Denmark and the UK, where inhaled colistin is often used as a longterm treatment for chronic airway infection.82,83 In addition, epidemic spread of colistin-resistant P. aeruginosa strains among patients with CF has been documented in Denmark and the UK.84,85 Epidemiology of resistance Development of antibiotic resistance in P. aeruginosa during chronic lung infection in CF has been demonstrated in Denmark,86 UK,87 Italy88 and USA.89 Retrospective analysis of microbiological data of patients with CF, who attended two CF centres in Germany from 2001 to 2011, revealed a substantial increase in MDR CF-specific isolates. An increasing rate of resistance for P. aeruginosa during chronic infection was documented, which is most likely due to repeated intravenous (IV) courses of antibiotic treatment, leading to the selection of resistant isolates.90

10

In a recent study91, 17 P aeruginosa antimicrobial susceptibility loci, candidate as hot spots of mutations in bacteria colonizing CF lungs, were sequenced. Different SNP patterns in P. aeruginosa isolates from the initial and the chronic stages of infection support the idea that the adaptation to the CF lung habitat and the regular anti-pseudomonal chemotherapy are major drivers for the emergence of amino acid sequence variants at the antimicrobial targets. The observed amino acid changes either are harmless or contribute to immune evasion, improved fitness, drug resistance or a combination of the previous in the CF lungs. A 5-year cohort study of individuals enrolled in the Cystic Fibrosis Foundation Registry from 1998 to 2002 evaluated the risk factors for multiple antibiotic-resistant P. aeruginosa acquisition in patients with CF.92 Diabetes, long-term inhaled tobramycin usage and frequent acute pulmonary exacerbations requiring hospitalization and IV antibiotic treatments were found to increase the risk for MDR P. aeruginosa. A slight increase in tobramycin-resistant P. aeruginosa had been reported also during the pivotal trial in the group of patients treated with tobramycin compared with placebo.93 However, the association between long-term use of inhaled tobramycin and the emergence of MDR P. aeruginosa is more complex and deserves further evaluation.92 Antibiotic susceptibility testing in CF: standard versus novel methods Optimal clinical care of patients with CF requires access to a well-equipped microbiology laboratory where diagnostic testing relevant to CF disease management can be undertaken. For many years, concerns regarding slow growth, auxotrophy and the impact of mucoid exopolysaccharide had led to questions about the accuracy of antibiotic susceptibility testing for P. aeruginosa isolates. Subsequently, agar diffusion methods including disc-diffusion susceptibility testing and agar-based stable gradient methods (Etest) were shown to compare favourably with reference broth microdilution methods, whereas correlation to reference methods was unacceptably low for several automated commercial systems.94 A study that compared the accuracy of

11

susceptibility results generated by the disc-diffusion and Etest methods against agar dilution as the reference method showed an unacceptable number of relevant errors for various antimicrobials when using the Etest or disc-diffusion methods.95 Other studies have shown variable accuracy for different automated systems.96,97 Finally, compared with agar dilution, other susceptibility testing methods (broth microdilution, Etest and disc diffusion) have shown high rates of apparent false polymyxin susceptibility for CF isolates of P. aeruginosa.98 The clinical validity of antimicrobial susceptibility testing by any method has been extensively investigated, and there is a widespread perception that “antibiotic resistance” primarily based on breakpoint concentrations achieved by parenteral administration is inappropriate in the context of CF pulmonary infections.95 Antibiotic resistance and the clinical utility of susceptibility testing have to be defined according to the pathogens under consideration, their resistance mechanisms, the complex pathophysiology of CF pulmonary infection, the high antibiotic concentrations that can occur in the infected airways by inhaled antibiotic therapies and the therapeutic endpoints to be achieved. The antibiotic susceptibilities of the microorganisms growing in planktonic and biofilm states vary significantly. In particular, biofilm-growing bacteria are much more resistant to antibiotics than those growing planktonically.52,53 The biofilm mode of growth of P. aeruginosa is considered one of the main explanations for the poor association between in vitro antibiotic susceptibility and clinical response.100 A 5-year retrospective review of antibiotic treatment for chronic P. aeruginosa lung infection showed no association between change in clinical endpoints (forced expiratory volume in first second of expiration [FEV1], body mass index or time to next exacerbation) and the in vitro antibiotic susceptibility (disc diffusion, Etest, microtitre broth assay) to the antibiotics administered.101 12

However, Foweraker et al.102 have argued that the debate on the clinical validity of susceptibility testing must distinguish between the treatment of P. aeruginosa causing early and chronic infections. Another reason for the lack of concordance between clinical outcome and susceptibility testing of P. aeruginosa isolates from patients with chronic CF is the variable susceptibility patterns within the same or different colonial morphotypes from the same sputum, the clinical impact of subinhibitory concentrations of antibiotic and the issue of testing a representative bacterial sample from a variable population of P. aeruginosa, which can exceed 108 colony forming units/ml of sputum.103,104 In contrast, P. aeruginosa isolates from patients with an initial infection showing a nonmucoid phenotype are not associated with alginate production and do not have the variable susceptibility patterns associated with chronic infection. However, although most environmental and early clinical P. aeruginosa isolates are susceptible, some are resistant, even using the higher breakpoints proposed for inhaled antibiotics.105 Therefore, it may be prudent to maintain susceptibility testing of P. aeruginosa isolates responsible for the initial infection.102 There is growing interest in the development of susceptibility tests specific for biofilmgrowing microorganisms to improve clinical practice. By analysing randomized controlled trials of antibiotic therapy based on biofilm antimicrobial susceptibility testing compared with antibiotic therapy based on conventional antimicrobial susceptibility testing in the treatment of P. aeruginosa infection in patients with CF, a Cochrane review concluded that the data did not provide evidence that biofilm susceptibility testing was superior to conventional susceptibility testing.106 Although over the last decade several in vitro biofilm models and new pharmacodynamic parameters (minimal biofilm inhibitory concentration, minimal biofilm-eradication concentration, biofilm bactericidal concentration, and biofilm-prevention concentration) have been defined to quantify antibiotic activity in biofilms, lack of standardization of the methods, parameters and

13

breakpoints by official agencies does not allow their use in microbiology laboratories for routine susceptibility testing.107 With regard to selection of appropriate antibiotics to treat multiresistant bacterial strains, drug combination testing has been developed, including both synergy testing and multiple combination bactericidal testing. In a prospective randomized, double-blind, controlled trial in patients with CF infected with MDR bacteria with acute pulmonary exacerbations, antibiotic therapy directed by combination antibiotic susceptibility testing did not result in better clinical and bacteriological outcomes compared with therapy directed by standard culture and sensitivity techniques.108 Poor correlation among the results obtained by the different methods of synergy testing was recently demonstrated and none of the methods was shown to be able to predict the response to treatment in patients with CF infected with MDR P. aeruginosa with acute infective exacerbations, suggesting that the in vitro effects of antibiotic combinations against different isolates from the same sputum sample may vary according to the methodology used.109 Based on these findings, a Cochrane review scrutinized the available evidence on combination antimicrobial susceptibility testing in CF and concluded that there is insufficient evidence to determine the effect of choosing antimicrobial therapy based on combination testing.110 Despite potential methodological flaws, in vitro susceptibility testing continues to be requested by clinicians. If properly interpreted and used, determination of the minimum inhibitory concentration represents a valuable tool for choosing the best therapeutic strategy for MDR P. aeruginosa infection. Whenever inhaled tobramycin is considered for therapy, Morosini et al.111 suggested that the P. aeruginosa strain from patients with CF should be tested with high-range Etest strips and categorized using MENSURA interpretive criteria (susceptible, 64

g/ml; resistant, 128

g ml).

Using the EUCAST criteria, the results of resistance would be much higher. In practice, Etest strips

14

with a wider antibiotic concentration range are not available for other antibiotics. Finally, there is an urgent need to redefine breakpoints appropriate for inhaled antibiotic formulations. The selection of antibiotic therapy according to antibiogram results is only one component of the decision-making process and other patient factors, such as previous infection history and antibiotic courses, also need to be considered. It has been recommended9 that P. aeruginosa susceptibility testing should be considered for surveillance of resistant or MDR P. aeruginosa strains when a new isolate is isolated in an individual patient and when a modification of therapy (IV, nebulized or oral) is proposed because of a lack of therapeutic response. Determination of antibiotic susceptibilities in P. aeruginosa isolates remains an effective tool for monitoring the development of resistance in a specific population. It may also be of some epidemiological support in disclosing the emergence of MDR CF clonal complexes of P. aeruginosa from an otherwise sensitive population. Therapeutic strategies for P. aeruginosa pulmonary infections Advancements in antibiotic therapies have certainly contributed to improvement in the survival and quality of life of patients with CF. Over the last several years, an increasing number of antimicrobial agents, of different classes and formulations, have become available for the management of pulmonary infections in patients with CF (including antibiotic combinations). A recent retrospective cohort study112 has highlighted that an aggressive antibiotic eradication therapy of new P. aeruginosa infections is effective in the majority of adults and paediatric patients. Vaccination against P. aeruginosa could also be a second line therapy. As stated previously, once P. aeruginosa has colonized CF lungs it is almost impossible to eradicate the infection. For this reason, vaccination strategy could be effective in reducing the infection. Vaccines dedicated to patients, not yet, or intermittently colonised have been developed. Despite two large and one small

15

trial113 no effectiveness in the tested vaccines was demonstrated. Thus, antibiotic seems to be the only strategy available at the moment. Improvement in our understanding of the CF microbiology can offer new opportunities to rationally design and test novel treatment strategies based on the best current data13,114,115. Suppression of chronic infection Chronic mucoid P. aeruginosa infection is associated with an accelerated decline in FEV1 in both adults and children and has been shown to be a strong predictor of pulmonary decline in comparison with non-mucoid P. aeruginosa.116 Long-term inhaled antibiotic therapy is the standard of care for chronic maintenance treatment in patients with stable CF.9,10,117 Aerosolized colistin inhalation had long been used off label for several years when Tobramycin was introduced in clinical practice for CF after the results of a rigorous clinical trial had shown lung function improvement and reduction of pulmonary exacerbations in patients with CF. For individuals with CF aged 6 years and older who have moderate to severe lung disease (the severity of lung disease, defined by FEV1 percentage of predicted as follows: moderately impaired, 40–69% predicted; and severely impaired, less than 40% predicted) and with P. aeruginosa persistently present in cultures of the airways, the Cystic Fibrosis Foundation strongly recommended the chronic use of inhaled tobramycin to improve lung function and reduce exacerbations.118,119 Tobramycin dry powder displayed similar tolerability and efficacy to tobramycin inhalation solution117 and significantly improved FEV1 compared with placebo.120 The most common adverse event was cough; the frequency of cough was higher in patients receiving placebo (26.5%) versus tobramycin inhalation powder (13.0%) in cycle 1 and appeared to be increased in the tobramycin inhalation group (26.1%) in cycles 2 and 3.120

16

In a phase III open-label trial, colistimethate sodium inhalation powder was shown to be not inferior in efficacy to tobramycin inhaler solution, based on change in FEV1 % predicted after 24 weeks of treatment.121 In several clinical studies, aztreonam lysine resulted in safe and efficacious suppression of chronic P. aeruginosa lung infection in patients with CF.122-124 In a 6-month active comparator trial, aztreonam lysine was superior to tobramycin inhalation solution with regard to lung function improvements.125 One of the most common therapeutic approaches for inhaled antibiotic therapy in patients with chronic P. aeruginosa infection is an intermittent 1 month on/1 month off regime with tobramycin.10 However, other therapeutic strategies, including continuous therapy with colistimethate sodium or continuous alternating therapy with tobramycin and aztreonam lysine, could be considered in patients who frequently experience acute exacerbations or whose lung function deteriorates rapidly.10 Starting from these recommendations,10 the right balance between the well-demonstrated clinical benefits of inhaled antibiotic usage and the increased risk of resistance development should be considered. The long-term microbiological effects of continuous suppressive therapy on P. aeruginosa antibiotic susceptibility are of crucial importance in CF clinical practice and decreases in antibiotic susceptibility are probably inevitable. Therefore, clinicians may need to devise novel treatment paradigms to preserve antibiotic efficacy in their patients. In patients infected with MDR P. aeruginosa, three alternative antibiotic strategies may be considered: 

1. Use of tobramycin even with evidence of resistance. It has been shown that patients with CF with highly resistant pathogens detected in sputum cultures may still derive clinical benefit from aerosolized tobramycin.126 This may be due to the substantial pharmacodynamic benefits of aerosolized antibiotic: high concentrations of drug (tobramycin concentration in the lung is 17

900–1000

g/ml) can be delivered to the site of infection with low risk of toxicity.127 For other

inhaled antibiotics (colistin, aztreonam), the concentration that can be achieved in the lung has not yet been established. 2. Use of colistin in the case of resistance to tobramycin. According to Valenza et al.,128 resistance to tobramycin may be present in P. aeruginosa isolates from patients with chronically colonized CF under long-term antimicrobial therapy, even when the highest breakpoints suggested for inhaled tobramycin are considered. In contrast, colistin resistance remains uncommon even after many years of inhaled therapy. Schuster et al.121 investigated, in addition to the efficacy and safety of inhaled colistimethate sodium, any changes in microbiological susceptibility over time and did not detect emergence of resistance of P. aeruginosa to colistin. 

3. Use of aztreonam. Assael et al.125 did not observe any change in the percentage of patients with CF with MDR P. aeruginosa after treatment with inhaled aztreonam lysine. In addition, a recent open label study129 enrolling CF paediatric patients, 3 months to <18 years of age, with P. aeruginosa confirmed the efficacy of azetreonam inhalation solution in the treatment of early infections. Eradication rates were, also in this case, consistent with those reported in literature. Furthermore, few, if any, decreases in aztreonam susceptibility were observed in patients

with CF receiving inhaled aztreonam lysine thrice daily (28 days on/28 days off) for up to 18 months, in addition to other antibiotics, mainly tobramycin. The long-term use of inhaled aztreonam lysine not only did not change the percentage of patients with isolates resistant to β-lactams or quinolones but also increased the susceptibility to tobramycin.130 It can be assumed that alternating tobramycin and aztreonam does not confer crossresistance to each other, because the main mechanisms of resistance to aminoglycosides differ from those to aztreonam in P. aeruginosa 61,73,74,131-133 and may suppress the development of resistance. Furthermore, it should be remembered that whereas the activity of tobramycin is concentration 18

dependent (i.e. related to the peak drug concentration achieved in the airway), the antimicrobial activity of aztreonam is time dependent (i.e. related to the elapsed time above a specific drug concentration in the airway). A recent multinational, randomized, double blind study134 has evaluated the efficacy and safety profile of levofloxacin inhaled solution (LIS) as an antibiotic treatment in stable cystic fibrosis patients. Results have evidenced an improvement in lung function (measured as FEV1 improvement) and the reduction of bacterial density in sputum. The study results were consistent with others on Tobramycin inhaled solution (TIS) and the safety and tolerability record endorse LIS as a promising therapy for patients with CF and chronic P. aeruginosa infection. Thus, new therapeutic strategies based on continuous alternating use of inhaled antibiotics with different mechanisms of action are perhaps feasible and advantageous in terms of efficacy and decreased risk of resistance development in patients infected with P. aeruginosa strains. The conclusions of a recent review135, identifying 45 trials comparing single antipseudomonal agent to a combination of the same antibiotic and other administered intravenously stressed and evidenced the lack and the need of well-designed RCT, where results are standardised to a consistent method of reporting, in order to validate pooling of results from multiple trials. This lack is an important bias affecting the formulation of proper statements on therapeutic strategies. Treatment of pulmonary exacerbations Patients with CF chronically infected by P. aeruginosa undergo a progressive decline of lung function with pulmonary exacerbations characterized by acute worsening of respiratory symptoms. After a severe pulmonary exacerbation,136 a quarter of adult and paediatric patients with CF do not recover their baseline FEV1. There is no agreed definition of a pulmonary exacerbation137 but it is essential that these episodes are diagnosed and treated promptly138. Indeed, optimal 19

therapeutic strategies for pulmonary exacerbations could significantly improve the quality of life and survival of patients with CF. The Cystic Fibrosis Foundation (CFF) guidelines recommend treating an acute pulmonary exacerbation with two IV anti-pseudomonal antibiotics with different mechanisms of action to reduce the chance of the development of resistance and enhance antibiotic efficacy.10,139 Several surveys support the CFF recommendations by showing β-lactam (98%), ciprofloxacin (1.5%), and aminoglycoside (61–84.3%) use in acute pulmonary exacerbations.140-142 One of the most used treatment options is the combination of aminoglycoside administered once daily with an antipseudomonal β-lactam antibiotic.143 There is no evidence to support the use of inhaled antibiotics in addition to intravenously administered antibiotics.144-146 Even so, inhaled antibiotics have been used in a quarter of pulmonary exacerbations in North America between 2003 and 2005.147 A consensus document produced by the European Cystic Fibrosis Society suggested10,148 that exacerbations should be treated until respiratory symptoms resolve and an improvement in lung function is observed. However, therapeutic treatments should not be extended more than 3 weeks, except under very special circumstances. Patients with MDR P. aeruginosa infection may require longer therapy with careful evaluation of their clinical status. Factors predictive of pulmonary exacerbations in CF patients infected with multiresistant bacteria were younger age, female sex, lower FEV1, a previous history of multiple pulmonary exacerbations and infection with Stenotrophomonas maltophilia and Achromobacter xylosoxidans bacteria.149 In one study, failure to recover lung function after CF pulmonary exacerbation was more likely to occur in female patients and patients with allergic bronchopulmonary aspergillosis, pancreatic insufficiency or malnutrition, persistent infection with P. aeruginosa, Burkholderia cepacia complex, or methicillin-resistant Staphylococcus aureus.150 Thus, establishing the local 20

epidemiology of pulmonary exacerbations is definitely the first step in their management. Then, physicians should assess the severity of exacerbation and start antibiogram-oriented therapy. The continued use of conventional susceptibility testing can play a role in the management of pulmonary exacerbations. Although pulmonary exacerbation should be treated with combination antibiotic therapy after susceptibility test results, the finding of in vitro antibiotic resistance does not necessarily indicate that treatment should be changed if the patient is responding to the current therapy. In a study on the incidence and risk factors of pulmonary exacerbation treatment failures in patients chronically infected with P. aeruginosa, 57% of exacerbations were successfully treated even though P. aeruginosa was resistant to the antibiotics used for treatment. In that study, failure rates decreased with increasing number of active antimicrobial agents used based on in vitro susceptibility,151 suggesting that the one-host, one-pathogen, one-treatment model of acute infection where the most important predictor of outcome is the time of delivery of antibiotic therapy with in vitro-predicted activity, may have limited applicability to CF and that an event-specific approach should be adopted. In this frame, it is interesting to underline the action of azithromycin, determined by various mechanisms such as anti-inflammatory action or the effect on the biofilm of P. aeruginosa. As previously stated, azithromycin, might select in P. aeruginosa biofilms for mutants overexpressing MexCD-OprJ, leading to cross-resistance to ciprofloxacin. Nevertheless, in RCT studies, azithromycin resulted in a substantial reduction in the number of pulmonary exacerbations in chronic P. pneumonia infected patients. The effect on FEV1 on the use of azithromycin is usually less apparent. 152,153 If anti-pseudomonal antibiotics are an integral component of PEx management, a recent study154 verified that a reduction in total P. aeruginosa burden (mucoid or non-mucoid isolates) does not predict clinical outcomes. Decline in pulmonary function and exacerbations were not 21

associated with P. aeruginosa sputum density. The results of this study mirror other recent works challenging the common opinion that an increase in P. aeruginosa triggers PEx. Factors (other than changes in P. aeruginosa CF lung population) that should be taken into account might be: environmental (pollution, allergens); patient characteristics (medication compliance, comorbidities, lung microbiome). It is likely that there are multiple convergent pathways leading to a PEx, and these vary in manifestation and potential outcomes. In conclusion, we would emphasize that the evaluation of patients with exacerbations should be based on demographic and episode-specific factors to determine the severity of each event and the potential for failure of clinical management, with treatments tailored accordingly. Only through such an event-specific tailored approach can outcomes be optimized and the potential for emergence of resistance avoided. Eradication of transmissible P. aeruginosa strains There is growing evidence suggesting that adaptation of P. aeruginosa to the CF lung environment may escape from the scale of the individual patients.21 The existence of epidemic strains capable of infecting patients with CF in different geographical locations has been well documented for over two decades. Microbiological surveillance using molecular typing (genotyping) has provided compelling evidence for P. aeruginosa cross-infection at many European, Australian and Canadian CF centers.17,155,156 The transmissible strains responsible for this cross-infection pose an increased risk for infection acquisition for patients currently free of P. aeruginosa. As transmissible strains are often multiresistant to antibiotics, they may also be more difficult to eradicate. Gilchrist et al.157 reported the efficacy of eradication therapy in a cohort of six patients who acquired a transmissible strain as their first isolate of P. aeruginosa. Despite aggressive treatment, only one (17%) achieved successfully eradicated.157

22

Therefore, MDR transmissible strains of P. aeruginosa pose a double risk to patients who are free of P. aeruginosa, that of developing chronic infection as a result of increased risk of acquisition by cross-infection and failure of early eradication treatment. The exact reason why transmissible strains of P. aeruginosa are more difficult to eradicate is unclear.157 Prevention can only be achieved by the implementation of infection control measures, and the success of these measures should be monitored by continued microbiological surveillance.157 Conclusions Resistance to antimicrobial agents is currently one of the most relevant problems in the treatment of CF respiratory infections, the major cause of morbidity and mortality in this patient population, and the increasing incidence of MDR P. aeruginosa strains poses significant challenges for clinicians who take care of patients with CF. The present work offers practical suggestions to optimize the diagnostic and therapeutic management of patients with CF with respiratory infections caused by MDR P. aeruginosa. The first step towards successful management of these patients is the appropriate use of an antibiogram138 for deciding antimicrobial therapy. In particular, physicians should be guided by, but be also aware of the limitations of, laboratory-based antimicrobial susceptibility testing when considering treatment for MDR P. aeruginosa infection. The second step is the adoption of a decision-making process that starts from the antibiogram results and includes other patient factors, such as previous infection history and antibiotic courses. Because there is evidence that antimicrobial resistance is potentiated in patients with CF due to the extensive use of antimicrobial agents from a young age, both for prophylaxis and treatment of respiratory infection, the clinician must pay particular attention to achieving the right balance

23

between the well-demonstrated clinical benefits of antibiotic use and the increased risk of resistance development. Starting from international guidelines10 and from the data considered in this review that support long-term inhaled antibiotic therapy as the standard of care for chronic maintenance treatment in patients with stable CF, novel treatment paradigms in the management of chronic pulmonary infection with MDR P. aeruginosa in CF are needed. In particular, a new strategy based on continuous alternating use of inhaled antibiotics from different classes, especially if these antibiotics have a different mechanism of action, could be ideal for patients with CF. Patients with CF with acute pulmonary exacerbations who are infected with MDR P. aeruginosa require careful evaluation of their clinical status throughout the course of combination antibiotic therapy. Before starting therapy, an assessment of episode-specific factors to determine the severity of each event and a tailored approach should be adopted to optimize clinical outcomes. Finally, the emergence of transmissible epidemic P. aeruginosa strains, which are often multiresistant to antibiotics and more difficult to eradicate, also significantly contributes to the scenario in the management of patients with CF infected with P. aeruginosa. Strategies to prevent the occurrence of mutational resistance, promote the development of novel antimicrobial agents against MDR P. aeruginosa strains and implement rigorous infection control measures are thus needed. References 1. Narasimhan, M., Cohen, R. New and investigational treatments in cystic fibrosis. Ther. Adv. Respir. Dis. 2011, 5, 275–282. 2. Jain, K., Smyth, A.R. Current dilemmas in antimicrobial therapy in cystic fibrosis. Exp. Rev. Respir. Med. 2012, 6, 407–422.

24

3. Ryan, G., Singh, M., Dwan, K. Inhaled antibiotics for long-term therapy in cystic fibrosis. Cochrane Database Syst. Rev. 2011, 16, CD001021. 4. Sawicki, G.S., Signorovitch, J.E., Zhang, J., Latremouille-Viau, D., von Wartburg, M., Wu, E.Q., et al. Reduced mortality in cystic fibrosis patients treated with tobramycin inhalation solution. Pediatr. Pulmonol. 2012, 47, 44–52. 5. Zobell, J.T., Waters, C.D., Young, D.C., Stockmann, C., Ampofo, K., Sherwin, C.M., et al. Optimization of anti-pseudomonal antibiotics for cystic fibrosis pulmonary exacerbations: II. cephalosporins and penicillins. Pediatr. Pulmonol. 2013, 48, 107–122. 6. Waters, V., Smyth, A. Cystic fibrosis microbiology: advances in antimicrobial therapy. J Cyst Fibros. 2015, 14, 551–560. 7. Mayer-Hamblett, N., Kloster, M., Rosenfeld, M., Gibson, R.L., Retsch-Bogart G.Z., Emerson, J., et al. Impact of Sustained Eradication of New Pseudomonas aeruginosa Infection on Longterm Outcomes in Cystic Fibrosis. Clin Infect Dis. 2015 Sep 1;61(5):707-15. 8. Wainwright, C.E., Elborn, J.S., Ramsey, B.W., Marigowda, G., Huang, X., Cipolli, M., et al.; TRAFFIC Study Group; TRANSPORT Study Group. Lumacaftor-ivacaftor in patients with cystic fibrosis homozygous for Phe508del CFTR. N. Engl. J. Med. 2015, 373, 220–231. 9. Cystic Fibrosis Foundation. Cystic Fibrosis Foundation Patient Registry. 2014 Annual Data Report. Available at https://www.cff.org/2014_CFF_Annual_Data_Report_to_the_Center_Directors.pdf/ 10. Döring, G., Flume, P., Heijerman, H., Elborn, J.S.; Consensus Study Group. Treatment of lung infection in patients with cystic fibrosis: current and future strategies. J. Cyst. Fibros. 2012, 11, 461–479. 11. Nguyen, D., Singh, P.K. Evolving stealth: genetic adaptation of Pseudomonas aeruginosa during cystic fibrosis infections. Proc. Natl. Acad. Sci. USA 2006, 103, 8305–8306. 25

12. Bittar, F., Richet, H., Dubus, J.C., Reynaud-Gaubert, M., Stremler, N., Sarles, J., et al. Molecular detection of multiple emerging pathogens in sputa from cystic fibrosis patients. PLoS One 2008, 3, e2908. 13. Lund-Palau, H., Turnbull, A.R., Bush, A., Bardin, E., Cameron, L., Soren, O., et al. Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches. Expert Rev Respir Med. 2016 Jun;10(6):685-97. 14. Saiman, L., Schidlow, D., Smith, A., editors. Concepts in Care: Microbiology and Infectious Disease in Cystic Fibrosis. V. Bethesda, MD: Cystic Fibrosis Foundation; 1994. 15. Winstanley, C., O'Brien, S., Brockhurst, M.A. Pseudomonas aeruginosa Evolutionary Adaptation and Diversification in Cystic Fibrosis Chronic Lung Infections. Trends Microbiol. 2016 May;24(5):327-37. 16. Conway, S.P., Brownlee, K.G., Denton, M., Peckham, D.G. Antibiotic treatment of multidrugresistant organisms in cystic fibrosis. Am. J. Respir. Med. 2003, 2, 321–332. 17. Jones, A.M., Govan, J.R., Doherty, C.J., Dodd, M.E., Isalska, B.J., Stanbridge, T.N., et al. Spread of a multiresistant strain of Pseudomonas aeruginosa in an adult cystic fibrosis clinic. Lancet 2001, 358, 557–558.n 18. Salunkhe, P., Smart, C.H.M., Morgan, J.A., Panagea, S., Walshaw, M.J., Hart, C.A., et al. A cystic fibrosis epidemic strain of Pseudomonas aeruginosa displays enhanced virulence and antimicrobial resistance. J. Bacteriol. 2005, 187, 4908–4920. 19. Fischera, S., Greipela, L., Klockgethera, J., Dordaa, M., Wiehlmanna, L., Cramera, N., et al. Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy. J Cyst Fibros. 2017 May;16(3):346-352.

26

20. López-Causapé, C., Rojo-Molinero, E., Mulet, X., Cabot, G., Moyà, B., Figuerola, J., et al. Clonal dissemination., emergence of mutator lineages and antibiotic resistance evolution in Pseudomonas aeruginosa cystic fibrosis chronic lung infection. PLoS One 2013, 8, e71001. 21. Folkesson, A., Jelsbak, L., Yang, L., Johansen, H.K., Ciofu, O., Høiby, N., et al. Adaptation of Pseudomonas aeruginosa to the cystic fibrosis airway: an evolutionary perspective. Nat. Rev. Microbiol. 2012, 10, 841–851. 22. Burns, J.L., Gibson, R.L., McNamara, S., Yim, D., Emerson, J., Rosenfeld, M., et al. Longitudinal assessment of Pseudomonas aeruginosa in young children with cystic fibrosis. J. Infect. Dis. 2001, 183, 444–452. 23. Van Ewijk, B.E., Wolfs, T.F.W., Fleer, A., Kimpen, J.L.L., Van der Ent, C.K. High Pseudomonas aeruginosa acquisition rate during acute respiratory infection in healthy and cystic fibrosis children. Thorax 2006, 61, 641–642. 24. Lee, T.W.R., Brownlee, K.G., Conway, S.P., Denton, M., Littlewood, J.M. Evaluation of a new definition for chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J. Cyst. Fibros. 2003, 2, 29–34. 25. Hogardt, M., Heesemann, J. Microevolution of Pseudomonas aeruginosa to a chronic pathogen of the cystic fibrosis lung. Curr. Top. Microbiol. Immunol. 2013, 358, 91–118. 26. Sousa, A.M., Pereira, M.O. Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs-a review. Pathogens 2014, 3, 680–703. 27. Martin, D.W., Schurr, M.J., Mudd, M.H., Govan, J.R., Holloway, B.W., Deretic, V. Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc. Natl. Acad. Sci. USA 1993, 90, 8377–8381. 28. Govan, J.R., Deretic, V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol. Rev. 1996, 60, 539–574. 27

29. Häussler, S., Tümmler, B., Weissbrodt, H., Rohde, M., Steinmetz, I. Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin. Infect. Dis. 1999, 29, 621–625. 30. Déziel, E., Comeau, Y., Villemur, R.., Comeau, Y., Villemur, R. Initiation of biofilm formation by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming, swarming, and twitching motilities. J. Bacteriol. 2001, 183, 1195–1204. 31. Häussler, S., Ziegler, I., Löttel, A., von Götz, F., Rohde, M., Wehmhöhner, D., et al. Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J. Med. Microbiol. 2003, 52(Pt 4), 295–301. 32. Webb, J.S., Lau, M., Kjelleberg, S. Bacteriophage and phenotypic variation in Pseudomonas aeruginosa biofilm development. J. Bacteriol. 2004, 186, 8066–8073. 33. Oliver, A. Mutators in cystic fibrosis chronic lung infection: prevalence, mechanisms, and consequences for antimicrobial therapy. Int. J. Med. Microbiol. 2010, 300, 563–572. 34. Oliver, A., Mena, A. Bacterial hypermutation in cystic fibrosis., not only for antibiotic resistance. Clin. Microbiol. Infect. 2010, 16, 798–808. 35. Mulcahy, L.R., Burns, J.L., Lory, S., Lewis, K. Emergence of Pseudomonas aeruginosa strains producing high levels of persister cells in patients with cystic fibrosis. J. Bacteriol. 2010, 192, 6191–6199. 36. Wei, Q., Tarighi, S., Dötsch, A., Häussler, S., Müsken, M., Wright, V.J., et al. Phenotypic and genome-wide analysis of an antibiotic-resistant small colony variant (SCV) of Pseudomonas aeruginosa. PLoS One 2011, 6, e29276. 37. Kirisits, M.J., Prost, L., Starkey, M., Parsek, M.R. Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 2005, 71, 4809–4821. 28

38. Schneider, M., Mühlemann, K., Droz, S., Couzinet, S., Casaulta, C., Zimmerli, S. Clinical characteristics associated with isolation of small-colony variants of Staphylococcus aureus and Pseudomonas aeruginosa from respiratory secretions of patients with cystic fibrosis. J. Clin. Microbiol. 2008, 46, 1832–1834. 39. Starkey, M., Hickman, J.H., Ma, L., Zhang, N., De Long, S., Hinz, A., et al. Pseudomonas aeruginosa rugose small-colony variants have adaptations that likely promote persistence in the cystic fibrosis lung. J. Bacteriol. 2009, 191, 3492–3503. 40. Nelson, A., De Soyza, A., Bourke, S.J., Perry, J.D., Cummings, S.P. Assessment of sample handling practices on microbial activity in sputum samples from patients with cystic fibrosis. Lett. Appl. Microbiol. 2010, 51, 272–277. 41. Oliver, A., Cantón, R., Campo, P., Baquero, F., Blázquez, J. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 2000, 288, 1251–1254. 42. Ciofu, O., Riis, B., Pressler, T., Poulsen, H.E., Høiby, N. Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation. Antimicrob. Agents Chemother. 2005, 49, 2276– 2282. 43. Marvig, R.L., Dolce, D., Sommer, L.M., Petersen, B., Ciofu, O., Campana, S., et al. Within-host microevolution of Pseudomonas aeruginosa in Italian cystic fibrosis patients. BMC Microbiol. 2015, 15, 218. 44. Marvig, R.L., Sommer, L.M., Molin, S., Johansen, H.K. Convergent evolution and adaptation of Pseudomonas aeruginosa within patients with cystic fibrosis. Nat Genet. 2015 Jan;47(1):5764. 45. Kenna, D.T., Doherty, C.J., Foweraker, J., Macaskill, L., Barcus, V.A., Govan, J.R.W. Hypermutability in environmental Pseudomonas aeruginosa and in populations causing

29

pulmonary infection in individuals with cystic fibrosis. Microbiology 2007, 153, 1852– 1859. 46. Mena, A., Smith, E.E., Burns, J.L., Speert, D.P., Moskowitz, S.M., Perez, J.L., et al. Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation. J. Bacteriol. 2008, 190, 7910–7917. 47. Pedersen, S.S., Lung infection with alginate-producing, mucoid Pseudomonas aeruginosa in cystic fibrosis APMIS Suppl. 1992, 28, 1–79. 48. Schurr, M.J., Martin, D.W., Mudd, M.H., Hibler, N.S., Boucher, J.C., Deretic, V. The algD promoter: regulation of alginate production by Pseudomonas aeruginosa in cystic fibrosis. Cell. Mol. Biol. Res. 1993, 39, 371–376. 49. Singh, P.K., Schaefer, A.L., Parsek, M.R., Moninger, T.O., Welsh, M.J., Greenberg, E.P. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature 2000, 407, 762–764. 50. Bjarnsholt, T., Jensen, P.Ø., Fiandacam, M.J., Pedersenm, J., Hansenm, C.R., Andersenm, C.B., et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr. Pulmonol. 2009, 44, 547–558. 51. Høiby, N., Ciofu, O., Bjarnsholt, T. Pseudomonas aeruginosa biofilms in cystic fibrosis. Future Microbiol. 2010, 5, 1663–1674. 52. Sriramulu, D.D., Lunsdorf, H., Lam, J.S., Romling, U. Microcolony formation: a novel biofilm model of Pseudomonas aeruginosa for the cystic fibrosis lung. J. Med. Microbiol. 2005, 54(Pt 7), 667–676. 53. Mah, T.F., O’Toole, G,A. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001, 9, 34–39.

30

54. Høiby, N., Bjarnsholt, T., Givskov, M., Molin, S., Ciofu, O. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Agents 2010, 35, 322–332. 55. Ciofu, O., Tolker-Nielsen, T., Jensen, P.Ø., Wang, H., Høiby, N. Antimicrobial resistance., respiratory tract infections and role of biofilms in lung infections in cystic fibrosis patients. Adv. Drug Deliv. Rev. 2015, 85, 7–23. 56. Driffield, K., Miller, K., Bostock, J.M., O'Neill, A.J., Chopra, I. Increased mutability of Pseudomonas aeruginosa in biofilms. J. Antimicrob. Chemother. 2008, 61, 1053–1056. 57. Conibear, T.C., Collins, S.L., Webb, J.S. Role of mutation in Pseudomonas aeruginosa biofilm development. PLoS One 2009, 4, e6289. 58. Rodríguez-Rojas A., Oliver A., Blázquez J. Intrinsic and environmental mutagenesis drive diversification and persistence of Pseudomonas aeruginosa in chronic lung infections. J. Infect. Dis. 2012; 205, 121–127. 59. Ferroni, A., Guillemont, D., Moumile, K., Bernede, C., Le Bourgeois, M., Waernessyckle, S., et al. Effect of mutator P. aeruginosa on antibiotic resistance acquisition and respiratory function in cystic fibrosis. Pediatr. Pulmonol. 2009, 44, 820–825. 60. Mayer-Hamblett, N., Ramsey, B.W., Kulasekara, H.D., Wolter, D.J., Houston L.S., Pope C.E., et al. Pseudomonas aeruginosa phenotypes associated with eradication failure in children with cystic fibrosis. Clin Infect Dis. 2014 Sep 1;59(5):624-31. 61. Ciofu, O., Pseudomonas aeruginosa chromosomal beta-lactamase in patients with cystic fibrosis and chronic lung infection. Mechanism of antibiotic resistance and target of the humoral immune response. APMIS Suppl. 2003, 1–47. 62. Queenan, A.M., Bush, K. Carbapenemases: the versatile b-lactamases. Clin. Microbiol. Rev. 2007, 20, 440–458.

31

63. Watanabe, M., Iyobe, S., Inoue, M., Mitsuhashi, S. Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 1991, 35, 147–151. 64. Lauretti, L., Riccio, M.L., Mazzariol, A., Cornaglia, G., Amicosante, G., Fontana, R., et al. Cloning and characterization of blaVIM., a new integron-borne metallo-β-lactamase gene from a Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother. 1999, 43, 1584–1590. 65. Salabi, A.E., Toleman, M.A., Weeks, J., Bruderer, T., Frei, R., Walsh, T.R. First report of the metallo-beta-lactamase SPM-1 in Europe. Antimicrob. Agents Chemother. 2010, 54, 582. 66.Yong, D., Bell, J.M., Ritchie, B., Pratt, R., Toleman, M.A., Walsh, T.R. A novel sub-group metallo-b-lactamase (MBL) , AIM-1, emerges in Pseudomonas aeruginosa (PSA) from Australia. 47th Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, IL, USA. 2007, Abstract C1-593. 67. Jovcic, B., Lepsanovic, Z., Suljagic, V., Rackov, G., Begovic, J., Topisirovic, L., et al. Emergence of NDM-1 metallo-{beta}-lactamase in Pseudomonas aeruginosa clinical isolates from Serbia. Antimicrob. Agents Chemother. 2011, 55, 3929–3931. 68. Pollini, S., Maradei, S., Pecile, P., Olivo, G., Luzzaro, F., Docquier, J.D., et al. FIM-1, a new acquired metallo-β-lactamase from a Pseudomonas aeruginosa clinical isolate from Italy. Antimicrob. Agents Chemother. 2013, 57, 410–416. 69. Cardoso, O., Alves, A.F., Leitao, R. Metallo-b-lactamase VIM-2 in Pseudomonas aeruginosa isolates from a cystic fibrosis patient. Int. J. Antimicrob. Agents 2008, 31, 375–379. 70. Pollini, S., Fiscarelli, E., Mugnaioli, C., Di Pilato, V., Ricciotti, G., Neri, A.S., et al. Pseudomonas aeruginosa infection in cystic fibrosis caused by an epidemic metallo-βlactamase-producing clone with a heterogeneous carbapenem resistance phenotype. Clin. Microbiol. Infect. 2011, 17, 1272–1275.

32

71. Aghazadeh, M., Hojabri, Z., Mahdian, R., Nahaei, M.R., Rahmati, M., Hojabri, T., Pirzadeh, T., Pajand, O. Role of efflux pumps: MexAB-OprM and MexXY(-OprA), AmpC cephalosporinase and OprD porin in non-metallo-b-lactamase producing Pseudomonas aeruginosa isolated from cystic fibrosis and burn patients. Infection. Genet. Evol. 2014, 24, 187–192. 72. Chalhoub, H., Pletzer, D., Weingart, H., Braun, Y., Tunney, M.M., Elborn, J.S., et al. Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic. Sci Rep. 2017 Jan 16;7:40208. 73. Sobel, M.L., McKay, G.A., Poole, K. Contribution of the MexXY multidrug transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. Antimicrob. Agents Chemother. 2003, 47, 3202–3207. 74. Islam, S., Oh, H., Jalal, S., Karpati, F., Ciofu, O., Hoiby, N., et al. Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients., Clin. Microbiol. Infect. 2009, 15, 60–66. 75. Jalal, S., Ciofu, O., Hoiby, N., Gotoh, N., Wretlind, B. Molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob. Agents Chemother. 2000, 44, 710–712. 76. Lister, P.D., Wolter, D.J., Hanson, N.D. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin. Microbiol. Rev. 2009, 22, 582–610. 77. Mandsberg, L.F., Ciofu, O., Kirkby, N., Christiansen, L.E., Poulsen, H.E., Hoiby, N. Antibiotic resistance in Pseudomonas aeruginosa strains with increased mutation frequency due to inactivation of the DNA oxidative repair system. Antimicrob. Agents Chemother. 2009, 53, 2483–2491. 33

78. Mulet, X., Macia, M.D., Mena, A., Juan, C., Perez, J.L., Oliver, A. Azithromycin in Pseudomonas aeruginosa biofilms: bactericidal activity and selection of nfxB mutants. Antimicrob. Agents Chemother. 2009, 53, 1552–1560. 79. Olaitan, A.O., Morand, S., Rolain, J.M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front. Microbiol. 2014, 5, 643. 80. Lee., J.Y., Ko, K.S. Mutations and expression of PmrAB and PhoPQ related with colistin resistance in Pseudomonas aeruginosa clinical isolates. Diagn. Microbiol. Infect. Dis. 2014, 78 , 271–276. 81. Moskowitz, S.M., Brannon, M.K., Dasgupta, N., Pier, M., Sgambati, N., Miller, A.K., et al. PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin-treated cystic fibrosis patients. Antimicrob. Agents Chemother. 2012, 56, 1019– 1030. 82. Frederiksen, B., Koch, C., Høiby, N. Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr. Pulmonol. 1997, 23, 330–335. 83. Frederiksen, B., Lanng, S., Koch, C., Høiby, N. Improved survival in the Danish center-treated cystic fibrosis patients: results of aggressive treatment. Pediatr. Pulmonol. 1996, 21, 153– 158. 84. Johansen, H.K., Moskowitz, S.M., Ciofu, O., Pressler, T., Hoiby, N. Spread of colistin resistant non-mucoid Pseudomonas aeruginosa among chronically infected Danish cystic fibrosis patients. J. Cyst. Fibros. 2008, 7, 391–397. 85. Denton, M., Kerr, K., Mooney, L., Keer, V., Rajgopal, A., Brownlee, K., et al. Transmission of colistin-resistant Pseudomonas aeruginosa between patients attending a paediatric cystic fibrosis center. Pediatr. Pulmonol. 2002, 34, 257–261.

34

86. Ciofu, O., Giwercman, B., Pedersen, S.S., Høiby, N. Development of antibiotic resistance in Pseudomonas aeruginosa during two decades of antipseudomonal treatment at the Danish CF Center. APMIS 1994, 102, 674–680. 87. Pitt, T.L., Sparrow, M., Warner, M., Stefanidou, M. Survey of resistance of Pseudomonas aeruginosa from UK patients with cystic fibrosis to six commonly prescribed antimicrobial agents. Thorax 2003, 58, 794–796. 88. Taccetti, G., Campana, S., Marianelli, L. Multiresistant non-fermentative gram-negative bacteria in cystic fibrosis patients: the results of an Italian multicenter study. Italian Group for Cystic Fibrosis microbiology. Eur. J. Epidemiol. 1999, 15, 85–88. 89. Emerson, J. McNamara, S., Buccat, A.M., Worrell, K., Burns, J.L. Changes in cystic fibrosis sputum microbiology in the United States between 1995 and 2008. Pediatr Pulmonol. 2010, 45, 363–370. 90. Raidt, L., Idelevich, E.A., Dübbers, A., Küster, P., Drevinek, P., Peters, G., et al. Increased prevalence and resistance of important pathogens recovered from respiratory specimens of cystic fibrosis patients during a decade. Pediatr. Infect. Dis. J. 2015, 34, 700–705. 91. Greipel, L., Fischer, S., Klockgether, J., Dorda, M., Mielke, S., Wiehlmann, L. Molecular Epidemiology of Mutations in Antimicrobial Resistance Loci of Pseudomonas aeruginosa Isolates from Airways of Cystic Fibrosis Patients. Antimicrob Agents Chemother. 2016 Oct 21;60(11):6726-6734 92. Merlo, C.A., Boyle, M.P., Diener-West, M., Marshall, B.C., Goss, C.H., Lechtzin, N. Incidence and risk factors for multiple antibiotic-resistant Pseudomonas aeruginosa in cystic fibrosis. Chest 2007, 132, 562–568.

35

93. Ramsey, B.W., Pepe, M.S., Quan, J.M., Otto, K.L., Montgomery, A.B., Williams-Warren, J., et al. Intermittent administration of inhaled tobramycin in patients with cystic fibrosis: Cystic Fibrosis Inhaled Tobramycin Study Group. N. Engl. J. Med. 1999, 340, 23–30. 94. Burns, J.L., Saiman, L., Whittier, S., Larone, D., Krzewinski, J., Liu, Z., et al. Comparison of agar diffusion methodologies for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients. J. Clin. Microbiol. 2000, 38, 1818–1822. 95. Bradbury, R.S., Tristram, S.G., Roddam, L.F., Reid, D.W., Inglis, T.J., Champion, A.C. Antimicrobial susceptibility testing of cystic fibrosis and non-cystic fibrosis clinical isolates of Pseudomonas aeruginosa: a comparison of three methods. Br. J. Biomed. Sci. 2011, 68, 1–4. 96. Sader, H., Fritsche, T.R., Jones, R.N. Accuracy of three automated systems (MicroScan Walkaway, VITEK, and VITEK2) for susceptibility testing of Pseudomonas aeruginosa against five broad spectrum β-lactam agents. J. Clin. Microbiol. 2006, 44, 1101–1104. 97. Burns, J.L., Saimon, L., Whittier, S., Krzewinski, J., Liu, Z., Larone, D., et al. Comparison of two commercial systems (Vitek and MicroScan WalkAway) for antimicrobial susceptibility testing of Pseudomonas aeruginosa isolates from cystic fibrosis patients. Diagn. Microbiol. Infect. Dis. 2001, 39, 257–260. 98. Moskowitz, S.M., Garber, E., Chen, Y., Clock, S.A., Tabibi, S., Miller, A.K., et al. Colistin susceptibility testing: evaluation of reliability for cystic fibrosis isolates of Pseudomonas aeruginosa and Stenotrophomonas maltophilia. J. Antimicrob. Chemother. 2010, 65, 1416– 1423. 99. Hurley, M.E., Amin Ariff, A.H., Bertenshaw, C., Bhatt, J., Smyth, A.R. Results of antibiotic susceptibility testing do not influence clinical outcome in children with cystic fibrosis. J. Cyst. Fibros. 2012, 11, 288–292.

36

100. Smyth, A. Multiresistant pulmonary infection in cystic fibrosis – prevention is better than cure. Lancet 2005, 366, 433–435. 101. Moskowitz, S.M., Foster, J.M., Emerson, J.C., Gibson, R.L., Burns, J.L. Use of Pseudomonas biofilm susceptibilities to assign simulated antibiotic regimens for cystic fibrosis airway infection. J. Antimicrob. Chemother. 2005, 56, 879–886. 102. Foweraker, J.E., Govan, J.R. Antibiotic susceptibility testing in early and chronic respiratory infections with Pseudomonas aeruginosa. J. Cyst. Fibros. 2013, 12, 302. 103. Foweraker, J.E., Laughton, C.R., Brown 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. 104. Govan, J.R.W. Multiresistant pulmonary infection in cystic fibrosis — what does resistant mean? J. Med. Microbiol. 2006, 55, 1615–1617. 105. Macdonald, D., Cuthbertson, L., Doherty, C., Campana, S., Revenni, N., Taccetti, G., et al. Early Pseudomonas aeruginosa infection in individuals with cystic fibrosis: is susceptibility testing justified? J. Antimicrob. Chemother. 2010, 65, 2373–2375. 106. Waters, V., Ratjen, F. Standard versus biofilm antimicrobial susceptibility testing to guide antibiotic therapy in cystic fibrosis. Cochrane Database Syst. Rev. 2012, 11, CD009528. 107. Macia, M.D., Rojo-Molinero, E., Oliver, A. Antimicrobial susceptibility testing in biofilmgrowing bacteria. Clin. Microbiol. Infect. 2014, 20, 981–990. 108. Aaron, S.D., Vandemheen, K.L., Ferris, W., Fergusson, D., Tullis, E., Haase, D., et al. Combination antibiotic susceptibility testing to treat exacerbations of cystic fibrosis associated with multiresistant bacteria: a randomised., double-blind., controlled clinical trial. Lancet 2005, 366, 463–471. 37

109. Foweraker, J.E., Laughton, C.R., Brown, D.F., Bilton, D. Comparison of methods to test antibiotic combinations against heterogeneous populations of multiresistant Pseudomonas aeruginosa from patients with acute infective exacerbations in cystic fibrosis. Antimicrob. Agents Chemother. 2009, 53, 4809–15. 110. Waters, V., Ratjen, F. Combination antimicrobial susceptibility testing for acute exacerbations in chronic infection of Pseudomonas aeruginosa in cystic fibrosis. Cochrane Database Syst. Rev. 2008, CD006961. 111. Morosini, M.I., García-Castillo, M., Loza, E., Pérez-Vázquez, M., Baquero, F., Cantón, R. Breakpoints for predicting Pseudomonas aeruginosa susceptibility to inhaled tobramycin in cystic fibrosis patients: use of high-range Etest strips. J. Clin. Microbiol. 2005, 43, 4480– 4485. 112. Kenny SL, et al. Eradication of Pseudomonas aeruginosa in adults with cystic fibrosis. BMJ Open Respir Res. 2014 Apr 23;1(1):e000021. 113. Johansen, H.K., Gøtzsche, P.C. Vaccines for preventing infection with Pseudomonas aeruginosa in cystic fibrosis. Cochrane Database Syst Rev. 2015 Aug 23;(8):CD001399. 114. Langan, K.M., Kotsimbos, T., Peleg, A.Y. Managing Pseudomonas aeruginosa respiratory infections in cystic fibrosis. Curr Opin Infect Dis. 2015 Dec;28(6):547-56. 115. Talwalkar, J.S., Murray, T.S. The Approach to Pseudomonas aeruginosa in Cystic Fibrosis. Clin Chest Med. 2016 Mar;37(1):69-81. 116. Parad, R., Gerard, C., Zurakowski, D., Nichols, D., Pier, G. Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype. Infect. Immun. 1999, 67, 4744–4750.

38

117. Smyth, A.R., Bell, S.C., Bojcin, S., Bryon, M., Duff, A., Flume, P., et al.; European Cystic Fibrosis Society. European Cystic Fibrosis Society Standards of Care: Best Practice guidelines. J. Cyst. Fibros. 2014, 13, S23–S42. 118. Flume, P.A., O'Sullivan, B.P., Robinson, K.A., Goss, C.H., Mogayzel, P.J., Jr., WilleyCourand, D.B., et al.; Cystic Fibrosis Foundation., Pulmonary Therapies Committee. Cystic fibrosis pulmonary guidelines: chronic medications for maintenance of lung health. Am. J. Respir. Crit. Care Med. 2007, 176, 957–969. 119. Konstan, M.W., Flume, P.A., Kappler, M., Chirond, R., Higgins, M., Brockhaus, F., et al. Safety., efficacy and convenience of tobramycin inhalation powder in cystic fibrosis patients: the EAGER trial. J. Cyst. Fibros. 2011, 10, 54–61. 120. Konstan, M.W., Geller, D.E., Minić, P., Brockhaus, F., Zhang, J., Angyalosi, G. Tobramycin inhalation powder for P. aeruginosa infection in cystic fibrosis: the EVOLVE trial. Pediatr. Pulmonol. 2011, 46, 230–238. 121. Schuster, A., Haliburn, C., Döring, G., Goldman, M.H.; Freedom Study Group. Safety, efficacy and convenience of colistimethate sodium dry powder for inhalation (Colobreathe DPI) in patients with cystic fibrosis: a randomised study. Thorax 2013, 68, 344–350. 122. McCoy, K.S., Quittner, A.L., Oermann, C.M., Gibson, R.L., Retsch-Bogart, G.Z., Montgomery, A.B. Inhaled aztreonam lysine for chronic airway Pseudomonas aeruginosa in cystic fibrosis. Am. J. Respir. Crit. Care Med. 2008, 178, 921–928. 123. Retsch-Bogart, G.Z., Quittner, A.L., Gibson, R.L., Oermann, C.M., McCoy, K.S., Montgomery, A.B., et al. Efficacy and safety of inhaled aztreonam lysine for airway pseudomonas in cystic fibrosis. Chest 2009, 135, 1223–1232.

39

124. Oermann, C.M., Retsch-Bogart, G.Z., Quittner, A.L., Gibson, R.L., McCoy, K.S., Montgomery, A.B., et al. An 18-month study of the safety and efficacy of repeated courses of inhaled aztreonam lysine in cystic fibrosis. Pediatr. Pulmonol. 2010, 45, 1121–1134. 125. Assael, B.M., Pressler, T., Bilton, D., Fayond, M., Fischer, R., Chiron, R., et al. Inhaled aztreonam lysine vs. inhaled tobramycin in cystic fibrosis: a comparative efficacy trial. J. Cyst. Fibros. 2013, 12, 130–140. 126. LiPuma, J.J. Microbiological and immunologic considerations with aerosolized drug delivery. Chest 2001, 120(3 Suppl), 118S–123S. 127. Lipworth, B.J. Pharmacokinetics of inhaled drugs. Br. J. Clin. Pharmacol. 1996, 42, 697–705. 128. Valenza, G., Radike, K., Schoen, C., Horn, S., Oesterlein, A., Frosch, M., et al. Resistance to tobramycin and colistin in isolates of Pseudomonas aeruginosa from chronically colonized patients with cystic fibrosis under antimicrobial treatment. Scand. J. Infect. Dis. 2010, 42, 885–889. 129. Tiddens, H.A., De Boeck, K., Clancy, J.P., Fayon, M., H G M A, Bresnik, M., et al. Open label study of inhaled aztreonam for Pseudomonas eradication in children with cystic fibrosis: The ALPINE study. J Cyst Fibros. 2015 Jan;14(1):111-9. 130. Oermann, C.M., McCoy, K.S., Retsch-Bogart, G.Z., Gibson, R.L., McKevitt, M., Montgomery, A.B. Pseudomonas aeruginosa antibiotic susceptibility during long-term use of aztreonam for inhalation solution (AZLI). J. Antimicrob. Chemother. 2011, 66, 2398– 2404. 131. Poole, K. Resistance to β-lactam antibiotics. Cell. Mol. Life Sci. 2004, 61, 2200–2223. 132. Masuda, N., Sakagawa, E., Ohya, S., Gotoh, N., Tsujimoto, H., Nishino, T. Substrate specificities of MexAB-OprM., MexCD-OprJ., and MexXY-OprM efflux pumps in Pseudomonas aeruginosa. J. Antimicrob. Chemother. 2000, 44, 3322–3327. 40

133. Maseda, H., Yoneyama, H., Nakae, T. Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa. J. Antimicrob. Chemother. 2000, 44, 658–664. 134. Flume, P.A., VanDevanter, D.R., Morgan, E.E., Dudley, M.N., Loutit, J.S., Bell, S.C., et al. A phase 3, multi-center, multinational, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of levofloxacin inhalation solution (APT-1026) in stable cystic fibrosis patients. J Cyst Fibros. 2016 Jul;15(4):495-502. 135. Elphick, H.E., Scott, A. Single versus combination intravenous anti-pseudomonal antibiotic therapy for people with cystic fibrosis. Cochrane Database Syst Rev. 2016 Dec 1;12:CD002007. 136. Waters, V., Stanojevic, S., Atenafu, E.G., Lu, A., Yau, Y., Tullis, E., et al. Effect of pulmonary exacerbations on long-term lung function decline in cystic fibrosis. Eur. Respir. J. 2012, 40, 61–66. 137. Bhatt, J.M. Treatment of pulmonary exacerbations in cystic fibrosis. Eur. Respir. Rev. 2013, 22, 205–216. 138. Cohen-Cymberknoh, M., Gilead, N., Gartner, S., Rovira, S., Blau, H., Mussaffi, H., et al. Eradication failure of newly acquired Pseudomonas aeruginosa isolates in cystic fibrosis. J Cyst Fibros. 2016 Nov;15(6):776-782. 139. Flume, P.A., Mogayzel, P.J., Jr., Robinson, K.A., Goss, C.H., Rosenblatt, R.L., Kuhn, R.J., et al.; Clinical Practice Guidelines for Pulmonary Therapies Committee. Cystic fibrosis pulmonary guidelines: treatment of pulmonary exacerbations. Am. J. Respir. Crit. Care Med. 2009, 180, 802–808.

41

140. Zobell, J.T., Young, D.C., Waters, C.D., Ampofo, K., Cash, J., Marshall, B.C., et al. A survey of the utilization of antipseudomonal beta-lactam therapy in cystic fibrosis patients. Pediatr. Pulmonol. 2011, 46, 987–990. 141. Van Meter, D.J., Corriveau, M., Ahern, J.W., Lahiri, T. A survey of once-daily dosage tobramycin therapy in patients with cystic fibrosis. Pediatr. Pulmonol. 2009, 44, 325–329. 142. Prescott, W.A., Jr. National survey of extended-interval aminoglycoside dosing in pediatric cystic fibrosis pulmonary exacerbations. J. Pediatr. Pharmacol. Ther. 2011, 16, 262–269. 143. Smyth, A., Tan, K.H., Hyman-Taylor, P., Mulheran, M., Lewis, S., Stableforth, D., et al.; TOPIC Study Group. Once versus three-times daily regimens of tobramycin treatment for pulmonary exacerbations of cystic fibrosis--the TOPIC study: a randomised controlled trial. Lancet 2005, 365, 573–578. 144. Cooper, D.M., Harris, M., Mitchell, I. Comparison of intravenous and inhalation antibiotic therapy in acute pulmonary deterioration in cystic fibrosis. Am. Rev. Respir. Dis. 1985, 131, A242. 145. Ryan, G., Jahnke, N., Remmington, T. Inhaled antibiotics for pulmonary exacerbations in cystic fibrosis. Cochrane Database Syst. Rev. 2012, 12, CD008319. 146. Schaad, U.B., Wedgwood-Krucko, J., Suter, S., Kraemer, R. Efficacy of inhaled amikacin as adjunct to intravenous combination therapy (ceftazidime and amikacin) in cystic fibrosis. J. Pediatr. 1987, 111, 599–605. 147. Moskowitz, S.M., Silva, S.J., Mayer-Hamblett, N., Pasta, D.J., Mink, D.R., Mabie, J.A., et al.; Investigators and Coordinators of the Epidemiologic Study of Cystic Fibrosis (ESCF). Shifting patterns of inhaled antibiotic use in cystic fibrosis. Pediatr. Pulmonol. 2008, 43, 874–881.

42

148. Saiman, L., Siegel, J.D., LiPuma, J.J., Brown, R.F., Bryson, E.A., Chambers, M.J., et al. Infection Prevention and Control Guideline for Cystic Fibrosis: 2013 Update. Infect Control Hosp Epidemiol. 2014 Aug;35 Suppl 1:S1-S67. 149. Block, J.K., Vandemheen, K.L., Tullis, E., Fergusson, D., Doucette, S., Haase, D., et al. Predictors of pulmonary exacerbations in patients with cystic fibrosis infected with multiresistant bacteria. Thorax 2006, 61, 969–974. 150. Sanders, D.B., Bittner, R.C.L., Rosenfeld, M., Hoffman, L.R., Redding, G.J., Goss, C.H. Failure to recover to baseline pulmonary function after cystic fibrosis pulmonary exacerbation. Am. J. Respir. Crit. Care Med. 2010, 182, 627–632. 151. Parkins, M.D., Rendall, J.C., Elborn, J.S. Incidence and risk factors for pulmonary exacerbation treatment failures in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa. Chest 2012, 141, 485–493. 152. Saiman, L., Marshall, B.C., Mayer-Hamblett, N., Burns, J.L., Quittner, A.L., Cibene, D.A., et al. Azithromycin in patients with cystic fibrosis chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA. 2003 Oct 1;290(13):1749-56. 153. Equi, A., Balfour-Lynn, I.M., Bush, A., Rosenthal, M. Long term azithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial. Lancet. 2002 Sep 28;360(9338):978-84. 154. Lam, J.C., Somayaji, R., Surette, M.G., Rabin, H.R., Parkins, M.D. Reduction in Pseudomonas aeruginosa sputum density during a cystic fibrosis pulmonary exacerbation does not predict clinical response. BMC Infect Dis. 2015 Mar 22;15:145.

43

155. Cheng, K., Smyth, R.L., Govan, J.R., Doherty, C., Winstanley, C., Denning, N., et al. Spread of b-lactam-resistant Pseudomonas aeruginosa in a cystic fibrosis clinic. Lancet 1996, 348, 639–642. 156. Armstrong, D.S., Nixon, G.M., Carzino, R., Bigham A, Carlin JB, Robins-Browne RM, et al. Detection of a widespread clone of Pseudomonas aeruginosa in a pediatric cystic fibrosis clinic. Am. J. Respir. Crit. Care Med. 2002, 166, 983–987. 157. Gilchrist, F.J., France, M., Bright-Thomas, R., Doherty, C.J., Govan, J.R., Webb, A.K., et al., Can transmissible strains of Pseudomonas aeruginosa be successfully eradicated? Eur. Respir. J. 2011, 38, 1483–1486.

44