Journal of Cystic Fibrosis 10 (2011) 387–400www.elsevier.com/locate/jcf

Review

Novel concepts in evaluating antimicrobial therapy for bacterial lunginfections in patients with cystic fibrosis

Geraint B. Rogers a,⁎, Lucas R. Hoffman b, Gerd Döring c

a Molecular Microbiology Research Laboratory, King's College London, United Kingdomb Department of Pediatrics, University of Washington, Seattle, WA, USA

c Institute of Medical Microbiology and Hygiene, Universitätsklinikum Tübingen, Tübingen, Germany

Received 27 March 2011; received in revised form 4 June 2011; accepted 17 June 2011Available online 19 July 2011

Abstract

Cystic fibrosis (CF) patients suffer typically from bacterial infections of their airways. Whilst current antibiotic-based treatment of theseinfections has brought much benefit to patients, it has been difficult to make either direct or indirect assessments of the in vivo efficacy of anyspecific treatment used. Traditional culture-based assessment has for example been rarely used to determine the direct impact of therapy on thebacteria in the airways. Instead, the “success” of a treatment is most often gauged through measures of respiratory and general health. New culture-independent approaches though are emerging that offer much promise here however in allowing a more comprehensive evaluation ofantimicrobial efficacy. These new methods offer an opportunity to examine bacterial outcomes rather than host outcomes alone. Application ofthese novel techniques in a systematic way will lead to the rationalisation and, likely greater still individualisation, of therapy for CF patients. Thisreview discusses host and microbiological factors that may influence antibiotic efficacy. Moreover, the degree to which the inherent complexity ofCF respiratory infections complicates the process of determining treatment impact and the need to identify more robust microbiological outcomemeasures will also be reviewed.© 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Keywords: Antibiotic efficacy; Bacterial enumeration; Biofilms; Persisters; treatment design

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3882. Early infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3893. Chronic infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

3.1. Physical barriers for antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3903.2. Bacterial cell numbers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3903.3. Genetic and phenotypic diversification of pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3913.4. Efficacy of antibiotics in chronic infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392

4. Is antibiotic therapy doing more harm than good in CF patients? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3935. Novel strategies to rationalise antibiotic therapy in CF patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

5.1. Characterization of microbial pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3935.1.1. Identifying changes in viable bacterial load . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3935.1.2. Identifying changes in pathogen behaviour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 394

5.2. Selection of antibiotics for the treatment of lung infections in CF patients . . . . . . . . . . . . . . . . . . . . . . . . . . 3945.3. Treatment of acute exacerbations in CF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

⁎ Corresponding author. Tel.: +44 20 7848 4467.E-mail address: geraint.rogers@kcl.ac.uk (G.B. Rogers).

1569-1993/$ - see front matter © 2011 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.doi:10.1016/j.jcf.2011.06.014

388 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

5.4. Regulatory aspects of antibiotic therapy in CF patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3955.5. Unanswered questions regarding antibiotic treatment in CF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395

6. Antibiotic therapy beyond CF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3957. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396

1. Introduction

Despite much progress, bacterial airway infections stilldictate the course of respiratory disease in patients with cysticfibrosis (CF). The high prevalence of bacterial airway infectionsin CF patients is linked to mutations in an epithelial chloridechannel, the CF transmembrane conductance regulator (CFTR),that result in a number of defects in innate immunity and airwayclearance [1]. Antibiotic treatment of these infections remains acornerstone of therapy, with improvements in this fieldconsidered to be a major factor in the increased life expectancythat has been achieved over recent decades [2–5]. Since chroniclung disease is the main determinant of morbidity and mortalityin CF [6–8], and infections are thought to be a key driver of CFlung disease [9,10], the issues surrounding antibiotic usage haveimportant implications for the prognosis of these patients.

Pseudomonas aeruginosa is the dominant infecting organismin the majority of adult CF patients [11], and as such, is theprincipal target of antibiotic therapy. However, many bacteriaother than P. aeruginosa are frequently cultured from therespiratory secretions of CF patients, both in the absence ofP. aeruginosa and co-infecting with this pathogen, with antibiotictreatment also routinely chosen to target these bacteria. Thesepathogens include methicillin-susceptible Staphylococcus aureus(MSSA), methicillin-resistant S. aureus (MRSA), members of theBurkholderia cepacia complex (particularly B. cenocepacia,B. dolosa and B. multivorans), Haemophilus influenzae,Stenotrophomonas maltophilia, Achromobacter (formerly Alca-ligenes) xylosoxidans, and non-tuberculousmycobacteria (NTM)species (in particular M. avium-intracellulare complex,M. chelonae, and M. abscessus) [5]. In a substantial proportionof children with CF treated for respiratory exacerbations, none ofthese “standard pathogens” are cultured, necessitating theselection of antibiotics based on clinician experience or patienthistory [12]. Furthermore, antibiotic treatment approaches oftendiffer between different centres and countries [13], complicatingthe interpretation of how “current strategies” are faring, andgiving rise to a great diversity of antimicrobial strategies in currentuse. The application of culture-independent analysis has revealedthe common presence of a yet much wider group of species in thelower respiratory tract of CF patients [14–24]. However, whilstthe clinical significance of these additional species remains to bedetermined, they are usually not addressed by antibiotic treatmentstrategies.

Choices regarding antibiotic strategies in CF, including drugselection and delivery method, have traditionally been shapedby a number of integrated factors, including toxicological,pharmacokinetic, and pharmacodynamic considerations, as well

as cost; also considered are in vitro susceptibility testparameters, patients' clinical status, patient allergies, and pastsuccess rate of intervention, as determined by endpoints such aslung function, symptoms, or time to next infective exacerbation.Each of these issues is important in the decision to choose aparticular antimicrobial strategy; however, the in vivo interac-tion between host, microbe and antibiotic that is fundamental totherapeutic efficacy is difficult to predict. Current protocolsrarely use culture-based data to evaluate the bacterial impact oftreatment; success is most often gauged by subjective andobjective measures of respiratory health, such as an increase inlung function or the resolution of cough severity, which arepractical yet indirect measures of antibiotic efficacy [25].

The CF airway represents a complex system, where microbialpopulations, host defences, and antimicrobial therapy all interactin an altered physiochemical environment that results from theunderlying genetic defect but is influenced by a range of widerfactors. The reasons that these interacting factors result in severedisease in some CF patients, and more mild disease in others, arenot fully understood. Nor is it clear why some patients experienceperiods of relatively stable pulmonary status, punctuated byperiods of acute exacerbations of respiratory symptoms, or whythe frequency of these periods differs between individuals. Whendealing with such an interacting system, it becomes very difficultto accurately assess the impact of a therapy aimed at any onecomponent, particularly when the relationship between thatcomponent and the final outcome measure is poorly understood(as is often the case in CF microbiology).

The need for better insight into the effects of antimicrobialdrugs within the infected airways of CF patients is illustrated bythe fact that antibiotic treatment of these chronic bacterial lunginfections has seldom resulted in the eradication of themicrobial pathogens being targeted (usually P. aeruginosa inclinical studies, [26]), but rather achieved only a reduction in thebacterial load in the patients' airways [27,28]. Because mostcurrent, general concepts driving antibiotic treatment are basedon observations from acute infections for which antibiotics areusually curative, including pneumonia and sepsis, this relativelytepid response to antibiotic therapy is often found to besurprising. The additional observation that antibiotics caneradicate P. aeruginosa infections when initiated early afterinitial detection, but not later, is equally confusing, since thedifferences between these two infectious states are not welldefined. Moreover, the impact of antibiotic therapy on bacterianot routinely targeted or even cultured (the “bystanders”) arenot well understood. Therefore, many issues remain to beclarified regarding the microbial determinants of CF lungdisease and of response to antibiotics.

Box 1Current antibiotic therapy in chronically colonised adult CF patients.

Current antibiotic therapy in chronically infected adult CF patientsThe severity and aetiology of lower airway infections can differgreatly between CF patients [9]. As such, therapies used also varyconsiderably. Below, however, is a broad overview oftypicalantibiotic therapies in adult CF patients.Maintenance therapyInhaled antibioticsare oftenusedduring periods of quiescence inanattempt topreserve lungfunction and reduceneed for IV antibioticsto control infection. Such maintenance therapy is recommendedfor patients with chronic P. aeruginosa infection [147]. Themajority of patients receive twice daily colistin or tobramycin byinhalation (tobramycin given in one month on/one month offregimen) [5]. Courses of IV antibiotics may also be given as part ofmaintenance therapy.Treatment of acute exacerbation of pulmonary symptomsPatients chronically colonised by P. aeruginosa typically receivetwo IVanti-pseudomonal antibioticswith differentmodesof actionto reduce the development of resistance and to provide potentialfor synergy [5,42,181]. Standard treatment involves 10–14 daycourses, however, there is no rationale to support this. In patientscolonisedbyamember of theB.cepaciacomplex, thehigh levelsofresistance seen to many anti-pseudomonals [42] must beconsidered. There is currently no consensus on antibioticsusceptibility testing as a basis for selection of IV antibiotics.Whilst clinical experience indicate patients benefit from suchtherapy, the evidence base for it remains poor.Where patients are chronically infected with MSSA orH. influenzae, treatment of exacerbations is typically by orallyadministered antibiotics. Even in the absence of positive airwayculture, a 2 to 4 week course of oral antibiotics may be given inresponse to any increase in respiratory symptoms where a viralcold is suspected [5]. In such cases, antibiotics are typicallyselected to coverMSSAandH. influenzae [5].There is no evidencebase for this practice.Multi-resistant isolatesP. aeruginosa and innately resistant organisms such as Steno-trophomonas maltophilia, Achromobacter (Alcaligenes) xylosox-idans, Methicillin-resistant Staphylococcus aureus (MRSA), andnon-tuberculous mycobacteria are increasingly reported in CFairway infections [182]. The optimal treatment for these resistantbacteria, or even if treatment is always necessary, is not known.All may be associated with either a symptomatic infection, orrespiratory exacerbation.

389G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

For the first time, analytical techniques are becomingavailable that provide sufficiently detailed data about thecomplex microbiota in the CF lung, potentially affording a morecomprehensive evaluation of antimicrobial efficacy. These newmethods offer an opportunity to fundamentally re-examine andrationalise CF antimicrobial therapy using microbial outcomes,providing the means to develop a systematic basis for theindividualisation of therapy for CF patients [29]. However,because the clinical significance and utility of these novel dataare yet to be determined, they are usually not used to choose andmonitor conventional antibiotic treatments.

Here we will consider these complex issues and provide newdirections for the future study of unresolved questions. We willdiscuss host and microbiological factors that likely influenceantibiotic efficacy in this context. Further, the degree to whichthe complexity of CF respiratory infections complicates theprocess of determining treatment impact and the need to identifymore appropriate microbiological outcome measures will beexamined. Through this discussion, a rationale for the design ofmore effective antibiotic strategies against chronic bacterialrespiratory infections in CF patients will be set out.

2. Early infection

Staphylococcus aureus and Haemophilus influenzae are thebacterial species most commonly isolated from children with CF,with P. aeruginosa being detected less frequently. Therelationship between P. aeruginosa and CF lung disease hasbeen well-studied, and a wealth of epidemiological evidencesupports an association of P. aeruginosa with worse outcomes[30–32]. By comparison, the roles of S. aureus,H. influenzae, andother species often culturable from the airways of children withCF are less well-defined. However, anecdotal and epidemiolog-ical evidence indicates that bacteria other than P. aeruginosamaybe important factors in both CF lung disease and response toantibiotic therapy [27,28,33–36]. To further confuse matters, it isclear that children can have lung disease and exacerbations evenin the absence of culture detection of these “CF related bacteria”[12]. These observations have led to a lack of a standardisedapproach to early CF infections.With respect toP. aeruginosa, asdiscussed above, early antibiotic treatment for P. aeruginosaculture-positivity can be effective for eradication of this pathogen[37–44]. It has been reported that in the CF center in Copenhagen,Denmark, there are virtually no children with CF below the age of16 years who are chronically infected with P. aeruginosa (TanjaPressler, personal communication). Interestingly, the choice ofthe antibiotic to be administered for early eradication does notseem to be important. For instance, aerosolized colistin and oralciprofloxacin [38,43] or aerosolized tobramycin either alone[39,41,42] or with ciprofloxacin [45] reveal similar success rates.The determination of serum antibody titres against P. aeruginosa[41,43,46]; and genotyping of P. aeruginosa [40,43] haveprovided independent evidence of successful eradication of thepathogen.

Data from various studies suggest that the window ofopportunity to eradicate P. aeruginosa from CF airways may bearound 12 weeks from initial detection [47–49]. The fact that

intravenously or inhaled and orally administered antibiotics areeffective in early eradication suggests that the CF-specific hostimmune or clearance defects alone are not severe enough topreclude later eradication of established P. aeruginosa infection.Suggested explanations that remain for the decreasing response toantibiotics include a gradual rise in P. aeruginosa densities inairways, and a transition from planktonic cells in early infection tobiofilm-dwelling cells later [50], as well as the emergence of otherphenotypic adaptations that may decrease the response toantibiotics [51].

3. Chronic infection

Whilst treatment during early stages of disease focuses onprevention of airway infection and eradication of bacterialpathogens where detected, treatment during later infection

390 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

focuses on prevention and resolution of periodic exacerbationsof symptoms, usually without the expectation of eradication.Although the considerations required for effective treatment ofthese stages of infection differ, it can be argued that earlydisease more closely resembles the surveillance and manage-ment of acute infections found in other contexts. By contrast,such approaches are less appropriate in the management ofchronic infections, such as those that typify adult CF respiratorydisease. The key components in the management of chronicinfection are maintenance therapy, designed to preserve lungfunction and decrease the need for additional therapy, and thetreatment for acute respiratory exacerbations, designed toalleviate acute symptoms and regain levels of lung functionthat existed prior to exacerbation. In addition to these types oftherapy, prophylactic strategies may also be used at times ofincreased risk of worsening, such as where viral respiratoryinfections are suspected. These strategies are summarised inBox 1.

Until today, no antibiotic therapy strategy has beensuccessful for the routine eradication of P. aeruginosa fromthe airways of CF patients once the infection has becomechronic (commonly defined as repeated positive microbiolog-ical cultures, e.g., three out of four cultures per year, and/or thepresence of positive serum antibodies against the pathogen[52]). Multiple factors, determined by drug, bacterial, and hostcharacteristics, could impair the efficacy of antimicrobial drugsin the chronically infected CF lung, forming the subject ofseveral reviews [53–55] (summarised in Fig. 1).

3.1. Physical barriers for antibiotics

During chronic P. aeruginosa infection, individual bacterialcells are believed to commonly form large aggregates, usuallyreferred to as biofilms. Growth in biofilms, or biofilm-likestructures, may play a substantial role in the ability of bacteria toresist eradication both by the host immune response andantimicrobial therapy. In vivo data suggest that the exopoly-saccharide matrix in which P. aeruginosa cells are embeddedcan scavenge host innate immune molecules such as hypochlo-rite, reduce polymorphonuclear cell chemotaxis, inhibit activa-tion of complement, and decrease phagocytosis by neutrophils,

Fig. 1. Bacterial factors influencing antibi

macrophages and leukocytes [56–61]. Further, bacterial cells inin vitro biofilm models are able to withstand substantiallyhigher concentrations of many antibiotics, with up to 100–1000-fold higher levels often required to inhibit the growth orkill biofilm-growing bacteria compared with planktonic bacteria[62–64].

The chemical composition of CF airway secretions (in whichinfection is most often localised) can also diminish antibioticefficacy. For example, in vitro “cidal” activity of aminoglyco-sides has been shown to be completely inhibited in the presenceof sputum, permitting bacterial growth even when the antibioticconcentration is ten times the minimum inhibitory concentration[65], a phenomenon believed to be due to the binding ofaminoglycoside molecules to mucins and DNA [66]. However,repeated dosing of antibiotics during therapy may saturatebinding sites [54,66], allowing therapeutic concentrations to beachieved.

Physiochemical characteristics of CF airway secretions mayalso prevent effective antibiotic function. For example, there is asignificant decrease in oxygen tensions within mucus plugs inCF airways [67] and even in deeper layers of bacterial biofilmsthemselves (Costerton et al., 1995), with a likely reduction inthe antimicrobial activity of many antibiotics [68–74]. Inaddition, divalent cation and pyrimidine concentration, hydra-tion levels, and pH vary across the biofilm structure [55], allfactors that may affect antimicrobial efficacy.

3.2. Bacterial cell numbers

Compared to early colonisation, P. aeruginosa cell numbersare much higher during chronic infection [75], with bacterialdensities of 107 to 108 CFU/g sputum common in chronicallyinfected patients [76,77]. Such high bacterial cell densities candegrade or inactivate antibiotics through enzymatic or meta-bolic activities, potentially reducing antibiotic concentrations inthe airways. For example, P. aeruginosa has been shown tosecrete β-lactamases into the surrounding environment inmembrane bound vesicles, which may become immobilisedwithin a biofilm matrix [55,78,79] and inactivate antibiotics asthey pass through the biofilm structure [54]. The resultingdecrease in effective antibiotic concentrations in deeper layers

otic efficacy in the CF lower airways.

Fig. 2. Potential impacts of novel bacterial species in CF lung infections.

391G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

of biofilm may provide an opportunity for surviving cells to up-regulate inducible resistance mechanisms [80]. Bacterial cellsgrowing in biofilms have also been shown to produce inherentlygreater quantities of β-lactamases compared with cells of thesame strain growing planktonically [80].

Another mechanism of biofilm resistance to antibiotics is therelease of planktonic cells under specific conditions, a processthat may be mediated by a range of factors, including theactivity of bacteriophage [81] and nitric oxide concentration[82]. These actively growing cells may take up and inactivateantibiotic molecules, resulting potentially in a significantreduction in the number of antibiotic molecules available toreach biofilm embedded bacterial cells [54]. It has beensuggested that the periodic release of large numbers of cellsfrom biofilms in the CF airways may be associated with changesin clinical symptoms [83]; planktonic “blooms” could poten-tially respond to exacerbation therapy, while biofilms woulddominate in the interim, reducing the efficacy of maintenancetherapy and influencing the occurrence and resolution ofexacerbations of pulmonary symptoms.

3.3. Genetic and phenotypic diversification of pathogens

The chronic nature of CF lower airway infections, theheterogeneous airway environment, and the disruption causedby periodic antimicrobial therapy all favour the geneticdiversification of pathogens into subpopulations. For example,highly resistant, hypermutable, small colony, virulent andavirulent variants and clones have been described for a numberof CF pathogens [22,84–86]. Many of the reported adaptationsresult in altered metabolic activity [85–88], in turn, leading toan altered response to antibiotics. Such metabolic changes donot always appear to result from antibiotic exposure, anobservation that suggests that antibiotic selection is not theonly force behind these adaptive changes. Drug indifferenceresulting from a reduced metabolic rate does not however confera greater tolerance to classes of antibiotic that kill both rapidlydividing and slow- or non-growing cells, such as fluoroquino-lones or metal cations [89,90].

The observation that bacterial populations growing asbiofilms are not eradicated by high concentrations of suchantibiotics [91] is believed to be due, in a significant part, to thepresence of subpopulations of cells that are able survive lethalconcentrations of antibiotics without any specific resistancemechanisms [91,92]. These sub-populations, known as persistercells, are not drug-resistant mutants; rather, hyper-resistance inthese persisters is a transient phenotypic change and, on re-culturing, they revert to mostly wild-type cells with a new sub-population of persisters [93]. Where persisters exist they areable to survive antibiotic treatment, re-populating the airwayonce antibiotic concentrations have fallen [94]. Persisters occurboth in planktonic and aggregated bacterial populations [96,95]and their existence is not dependent on identified intercellularsignalling pathway such as quorum sensing [92], suggestingthat persisters inherently comprise a fraction of any bacterialpopulation [97]. Studies of Escherichia coli have shown thatpersister populations may be expanded by antibiotic exposure

[98,99] and high levels of persisters are known to occur amongP. aeruginosa populations in the CF airways [100].

In addition to persister cells, the CF airway may contain smallcolony variants (SCV). Whilst appearing in both biofilm andplanktonic populations, there is evidence that biofilm formationmay be linked to the development of SCVs [101]. SCVs differfrom the normal phenotype of the populations from which theyoriginate. For example, S. aureus SCVs are distinguished by theirsmall colony size, reduced growth rate, pigmentation andhaemolysis, altered expression of virulence factors and auxotro-phy for haemin, menadione, thiamine or thymidine [87,101–105]. Further, they are more resistant to the action of aminoglyco-sides and cell-wall inhibitors, as well as having a tendency topersist [102,104,105,106]. These variants may represent a stable,inheritable change or a transient colony type.

Both P. aeruginosa and S. aureus SCVs are known to bepresent in CF airway infections [100,107–111], and in addition totheir impact on antibiotic efficacy in vivo, the presence of SCVs islikely both to lead to an underestimation of bacterial numberswhen determined through culture-based methodologies, and to anunder-representation of such isolates in susceptibility testing.

As described above, in addition to those species traditionallyassociated with CF airway disease, a number of studies haveshown that many other bacterial species are also typicallypresent in the adult CF lung. Many of these are facultative orobligate anaerobes more commonly associated with the oralmicrobiota. The degree to which this wider group of speciescontributes directly to airway disease is not yet known,representing an important area of ongoing research. However,in addition to any direct role, the presence of this wider group ofspecies may contribute to disease indirectly, for instance byincreasing the virulence of key pathogens such as P. aeruginosa[112], or by “consuming” administered antibiotics and thusdiluting their effects on the intended population (usuallyP. aeruginosa) (Fig. 2). As such, this wider bacterial communitymay represent a potential target for therapy.

Box 2Current outcome measures. Categories reflect the use of theseoutcomes measures in a number of previous studies ([36,115,128–130,142,160], and reviewed in [12]).

Short-term outcomesPrimary measures• Lung function (absolute change or percentage change

compared to baseline)• Resolution of clinical signs and symptoms• Changes in sputum bacteriology (quantitative or qualitative)• Adverse effects (e.g. allergic reactions, candidal infections)

Secondary measures• Quality of life• Change in nutritional status (weight, height, BMI)• Time to exacerbation• Change in inflammatory status (blood or sputum markers)• Cost• Treatment failure (switching to another treatment due to

clinical deterioration)

Long-term outcomesPrimary measures• Frequency of exacerbations• Lung function (absolute change or percentage change

compared to baseline)• Development of antibiotic resistant strains

Secondary measures• Adverse effects (decline in renal function)• Number of courses of IV antibiotics• Quality of life• Cost

392 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

3.4. Efficacy of antibiotics in chronic infections

Despite these barriers to effective action, antibiotic treatmentis associated with a decrease in bacterial load and improvementin symptoms when given for the treatment of CF respiratoryexacerbations [28,113–115]. Although eradication is rarelyachieved, many trials have reported a decrease in bacterialnumbers of one to two log10 orders. What are the reasons forthis efficacy, albeit limited, given the factors described above?

Physical barriers such as mucins or DNA may be saturable,and sufficient antimicrobial molecules may then reach bacterialcells, with the degree to which the extracellular matricesassociated with biofilms restrict the penetration of antimicrobialagents differing significantly between antibiotics [116–120].Furthermore, as above, antibiotics may selectively kill aplanktonic subpopulation of cells, while sparing many of theco-infecting biofilm-dwelling cells [83]; if these planktoniccells are major contributors to the signs and symptoms ofexacerbations, this antibiotic effect could be sufficient to speedrecovery.

Whilst targeting specific routinely-cultured pathogens (such asP. aeruginosa) antibiotics may also impact other bacteria in theCF lung, resulting in a decreased total bacterial burden. Forexample, using standard, selective cultures, anti-pseudomonalantibiotics have been shown to have more significant antimicro-bial effects in vivo on H. influenzae [27] than on P. aeruginosa,raising the question of whether anti-pseudomonal antibioticsameliorate exacerbations through their targeted species, orthrough another mechanism. Similarly, antibiotics such asazithromycin are likely to have an impact on the bacterialcommunity present in the CF airways in ways not yet welldefined. This possibility may be reflected by the finding thatazithromycin reduces exacerbations in people culture-negativefor P. aeruginosa [36].

Finally, antibiotics may impact host cells directly, forexample by decreasing inflammation, in turn, resulting in anincrease of lung function. For instance, azithromycin is knownto have a wide range of anti-inflammatory effects ([121] andothers — reviewed by [122]) that may influence clinical signs.In addition, azithromycin is known to aid sputum expectoration,prevent airway remodelling, limit tissue damage, and correctairways surface liquid composition ([123–127], reviewed by[122]). The multiple non-antibiotic effects of azithromycin mayexplain why administration of this drug is associated withpositive outcomes, including improvement in lung function andin the frequency of episodes of infective exacerbation, despitethe lack of any observed, significant changes in bacterialdensities [128–130]. At sub-inhibitory concentrations, anumber of macrolide antibiotics, including azithromycin, caninfluence the gene expression of P. aeruginosa ([131–133] andothers), and interfere with quorum sensing [134,135]. Disrup-tion of quorum sensing pathways in turn affects the ability ofP. aeruginosa to express a range of virulence traits, includingthe formation of biofilms [136].

Guidelines governing antibiotic selection in CF care havehistorically supported the use of in vitro sensitivity testing toinform antibiotic selection [137,138]. However, the usefulness

of such analysis is now being questioned [139]. This isprimarily due to the observation of wide variability in the resultsof susceptibility testing of multiple isolates of P. aeruginosafrom individual sputum samples by more than one laboratory ortechnician [140]. Foweraker et al. reported substantial differ-ences in antibiotic sensitivities between isolates with the samecolony morphology [86], as well as differences in results fromthe same isolates among different observers [140]. Further,these problems do not appear to be resolved by the ongoingrefinement of the way in which such analysis is performed[140–141]. Given these findings, the poor correlation betweenin vitro susceptibility data and clinical outcome is unsurprising[142,143].

It must also be considered that antibiotics are usually givenas part of a complex, multimodal regimen, that includesmucolytic drugs, physiotherapy, and nutritional supplements,all of which may affect the same clinical outcome measures andpatient reported outcomes used to determine antibiotic efficacy(Box 2). Further, in a proportion of cases, the decline inrespiratory health that precipitates acute antibiotic therapymight result from a non-bacterial trigger, such as respiratoryviral infection [144–146]. In such cases, the role of antibioticsin recovery is difficult to predict.

393G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

The example of azithromycin highlighted here is an extremecase, being an antibiotic most often given for its theoretical non-antibiotic impact. However, these same issues of indirect orpoorly defined therapeutic impact affecting conventionaloutcome measures will exist to some degree for all antibiotics.Taken together, a range of factors are likely to contribute to theeffect of an administered antibiotic on lung function inchronically infected CF patients. As such, determing theindividual contribution of antibiotics to clinical improvement,and the mechanism of any such effect, is very difficult.

4. Is antibiotic therapy doing more harm than good in CFpatients?

Typically, treatment strategies are designed to achieve as higha concentration of antibiotics at the site of infection as possible,with appropriate upper limits determined primarily by toxicolog-ical and pharmacokinetic considerations. With continuing in-creases in patient longevity, and the concomitant rise in the totalnumber of courses of antibiotics recieved, there is growingconcern about cumulative damage, particularly to renal and oticfunction, that can result from such drug exposure [148–151].Furthermore, antibiotic resistance rates are known to increasewith multiple cycles of therapy [27], yielding multi-resistantclones which have been associated with both worse outcomes[152], and with epidemic infection among CF patients [153,154].Increases in lung function observed in CF antibiotic treatmenttrials tend to decrease when repeated. For example, while in the1999 tobramycin trial [155] an increase of FEV1 of more than10% was observed, such increases are currently rarely seen withthis drug [156]. Furthermore, bacteria that survive antibiotictreatment may adapt phenotypically by becoming even moreresistant to killing, such as by antibiotic-mediated up-regulationof cyclic-di-GMP levels byP. aeruginosa [157] and an increase inalginate synthesis [48,79].

On the other hand, there is evidence of an overall beneficialeffect of antibiotics on long-term respiratory outcomes in CF:lung function declines less rapidly with chronic suppressivetherapy, as does the frequency of acute exacerbations andinflammation [128–130,149,158–161].

5. Novel strategies to rationalise antibiotic therapy inCF patients

There are many factors that must be considered in the designof any antibiotic treatment strategy. These include, but are notlimited to, the drug or drug combination used, formulation,mode of delivery, frequency of delivery, dose, the need forallergic desensitisation, duration of treatment, potential side-effects, potential antibiotic synergy, the use of non-antibioticco-therapies, and inter-treatment interval. Given the complexityof CF respiratory infections, making decisions based on ourcurrent microbiological understanding strays close to guess-work. Indeed, it could be argued that there is insufficientevidence to support any tailoring of antibiotic treatment basedon current bacterial surveillance strategies in individual CFpatients, as opposed to empirically chosen antibiotics. Never-

theless, our expanding understanding of CF microbiology offersnew opportunities to rationally design and test novel treatmentstrategies based on the best current data.

5.1. Characterization of microbial pathogens

Currently, CF antibiotic therapies are usually informed bytwo types of microbiological data: the semi-quantitative,selective culture-based detection of the defined group ofpathogens set out above, and the determination of theirantibiotic susceptibility profiles in vitro [139]. The detectionof pathogens through culture-based microbiology provides areasonably robust indication of the presence of a limited groupof key pathogens. However, the presence of bacteria in formsthat grow poorly in vitro, such as small colony variants andauxotrophic strains [162–166], can lead both to false negativeresults and inaccuracy in enumeration. Further, there isincreasing recognition that species refractory to culture understandard diagnostic conditions may be clinically significant[21,33,109]. As such, in order to obtain a more substantial basisfor the design of drug trials or treatment strategies, it isnecessary to compile a body of empirical data, based on directmicrobiological measurements that accurately represent in vivoscenarios.

Bacteria in CF airways could influence the progression of lungdisease in at least two ways: They could have a passive impact,through the host detecting their presence and an immune responsebeing triggered, or they could have an active impact, through theexpression of genes that code for proteins involved inpathogenicity. Following this logic, the key questions indescribing the success of antimicrobial therapy could be:

1) Has there been a reduction in viable bacterial load, either fortotal bacteria or for specific species?

2) Has there been a change in the expression of genesassociated with pathogenicity?

5.1.1. Identifying changes in viable bacterial loadDue to diversity of growth phenotypes of bacteria isolated

from CF airways (as described above), traditional culture-basedmicrobiology is unable to provide sufficiently representativedata when applied to bacterial enumeration in CF respiratorysamples. However, culture-independent approaches to bacterialenumeration can avoid many of the problems associated withculture-based strategies. Nucleic acids can be extracted directlyfrom clinical samples and quantitative PCR (Q-PCR) used toenumerate either total bacteria or bacteria belonging to aparticular species of interest that are present. Further, nucleicacid extraction and Q-PCR can be, to a large degree, automated,with each extract being used for multiple species-specificassays, thus reducing associated costs.

However, whilst such culture-independent strategies have theadvantage of not requiring in vitro cultivation to be performed,this is also one of their key flaws. Standard PCR-baseddiagnostics are unable to differentiate between DNA present inviable bacterial cells, in non-viable cells, and in the extracellularmatrix. A failure to exclude non-viable bacteria from analysis is

394 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

particularly important when analysing the impact of therapy,given that DNA may persist in the CF airways for extendedperiods of time without being cleared or degraded [167–169].

The contribution of DNA other than that present in viablebacteria can be effectively excluded by pre-treating samples withpropidium monoazide photo-crosslinking. Whilst a relativelysimple additional step, this method has been shown to be effectivein preventing DNA from non-viable bacterial cells present in CFsputum from acting as PCR template [170]. The use of Q-PCR, incombination with PMA treatment, has been used to show adecrease in density of P. aeruginosa in CF sputum samples as aresult of IV antibiotic therapy [171].Whilst such a reduction is notevident in all patients, and comparison with results from standard,culture-based studies (in the absence of a gold standard measureof microbial content) will be required before they can beemployed diagnostically, such a strategy may allow accurateenumeration of bacteria in CF respiratory samples through theaddition of a relatively minor step in sample processing.

5.1.2. Identifying changes in pathogen behaviourThe expression of particular behavioural traits by CF

pathogens is likely to contribute to lung disease pathogenesis[172,173]. Treatments that are able tomodify pathogen behaviourso that virulence traits are not exhibited may therefore bebeneficial, even where the number of bacteria present remainsunaltered.

In many cases, such changes in behaviour will be reflected ingene and protein expression patterns. A meta-transcriptomic or-proteomic analysis could therefore be used to identify genes ormolecules whose up- or down-regulation correlates with clinicalsymptoms, with such data used to inform the development ofbiomarkers and outcomes measures [174].

Analysis based on gene expression or protein productioneach have both advantages and disadvantages. The ability ofprotein-based analysis to report short-term changes is likely tobe reduced by poor clearance of material from the CF lowerairways [167–179] and low rates of protein turnover. Further,potential biomarker proteins may be of very low abundancewithin the meta-proteome, making their initial identificationthrough global profiling techniques difficult. However, onceuseful protein biomarkers have been identified, routinequantification can be performed through relatively simplyimmuno-assays.

In comparison to protein, mRNA has a very short half-life,making transcriptomic analysis better suited for characterisingrapid changes that occur as a result of acute therapy. Broad meta-transcriptomic analysis can be achieved through the reversetranscription of total mRNA extracts and their subsequentcharacterisation, either by microarray analysis or through highthroughput next generation sequencing. Again, identification ofgenes whose transcription correlates with clinical data throughsuch a process would allow specific assays to be developed. Therelative instability of mRNA presents a significant challenge inthe search for biomarkers, with the recovery of sufficient highintegrity mRNA from clinical samples a key issue. Sometechnological advance is therefore likely to be required beforesuch an approach can be applied to the large patients groups and

temporal sample sets needed to identify useful biomarkers.Further, with deployment of such technology in a diagnosticsetting currently a distant proposition.

Novel protein or mRNA biomarkers may be most useful aspredictors of exacerbations, and/or to provide an indication fortreatment. For example, the identification of a bacterial gene orprotein whose upregulation precedes the worsening of pulmo-nary symptoms could provide a cue to initiate IV antibiotictherapy before clinical signs and symptoms become apparent.

However, to limit the use of such insight to an indication forthe initiation of treatment might be a missed opportunity. Whilstbactericidal treatments ultimately prevent gene expression ofbacterial cells, such approaches are associated with a number ofsignificant drawbacks due to the high concentrations of antibioticsoften required. Since sub-inhibitory antibiotics have been showntomodify bacterial gene expression in ways that are likely to haveclinical significance [132,157], the potential for bacterial markersto be used to re-design antimicrobial therapy for CF airwayinfections (for example, to study the effects of less toxic drugsand/or doses in clinically meaningful ways) should not beignored. The rationale for antibiotic therapy, both in themanagement of chronic infection and in the treatment of acuteperiods of exacerbation, could be shifted from achieving areduction in the bacterial load in the airways (an outcome that hasnot been definitively related with clinical improvement [27]), to areduction of the expression of genes associated with poor clinicaloutcomes, or the up-regulation of genes associated with clinicalstabilty. For example, recent data suggest that the production ofpyocyanin byP. aeruginosa cells can cause significant changes ingene expression profiles of bronchiolar epithelial cells [173]. Ifthese epithelial expression changes can be correlated with clinicaloutcomes, intervening to prevent the production of pyocyaninmay confer clinical benefits. The use of antibiotics as “behaviourmodifers” in this way has been demonstrated in the treatment ofmethicillin resistant S. aureus (MRSA), where protein synthesisinhibitory antibiotics such as aminoglycosides and macrolidescan be used to inhibit exotoxin production [175]. Such strategiesare potentially applicable more widely.

5.2. Selection of antibiotics for the treatment of lung infectionsin CF patients

As described above, evidence indicates that the traditionalstrategy of using susceptibilities to guide antibiotic selection forCF treatment is of limited value [143]. As a result of this lack ofinformative data, antibiotics are frequently chosen for thetreatment of bacterial lung infections based on factors such aswhether they have worked well for a given patient previously,their cost, availability, or toxicity. A reduction in pulmonarysymptoms is the primary aim of therapy in this context, and assuch, should be the ultimate measure of success. However, failingto achieve a better understanding of the direct impact ofantibiotics on their target – populations of bacterial pathogensin the airways – confounds a better understanding of when, why,and how antibiotics result in clinical outcomes, precludingimprovement of current therapeutic strategies. Thus, a betterunderstanding of the overall impact of antibiotics on CF

395G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

microbiology, and correlation of these changes with clinicaloutcomes, is needed.

5.3. Treatment of acute exacerbations in CF

The antibiotic strategy most commonly used for acute CFpulmonary exacerbations is similar to that used to eradicatebacteria in acute infection contexts elsewhere, typically involvinga ten to fourteen day course of IV antibiotics [5,11]. This is despitethe wide acceptance that eradication of chronically infectingpathogens is not a realistic aim in the majority of cases [176].Whilst the ‘14 day’ schedule is an historically accepted approach,supported by the weight of clinical experience [177], there is littleevidence to indicate whether this is the most appropriate format.For example, there is yet to even be a sufficiently poweredcomparison of IV antibiotic benefit versus placebo [5]. Whilstretrospective analysis of FEV1 response to IV antibiotic therapydata in the CFF Patient Registry suggests that treatment periods of1–2 weeks are associated with better lung function outcomescompared with shorter or longer treatment periods [178], the factthat patients treated for longer periods do worse is difficult toexplain from amicrobiological standpoint (and could conceivablybe attributable to confounding — patients who are sicker mayoften be treated for longer, rather than the reverse). Further, if aninability to achieve eradication is accepted, there is no consensusrationale for dose determination from a microbiological perspec-tive. In the absence of compelling evidence about the effective-ness of the 14 day duration of therapy, such high dose strategiesfor acute exacerbations persist. The generation of detailedmicrobiological data, in parallel with clinical trials using standardclinical outcome measures, would therefore provide a comple-mentary basis for assessing novel strategies empirically.

5.4. Regulatory aspects of antibiotic therapy in CF patients

Current systems for determining antibiotic efficacy, such asthose employed by the Food and Drug Administration (FDA)[179], rightly rely on validated clinical outcomes. FDA guidelineson the development of antimicrobials for respiratory infectionsstate “Although microbiological outcome may provide usefulinformation regarding the biological activity of antimicrobials,microbiological outcome is not a direct measure of benefit topatients and, therefore, should be viewed as being supportiveinformation but not as a substitute for clinical outcome in aspecific trial” [180].

Whilst sophisticated bacteriological measures cannot beapplied in such a role until they have been further validated andapplied to large patient groups and longitudinal sample sets,antibiotic treatment studies offer an excellent opportunity to dothis. Despite not directly contributing to drug licencing, thewidespread application of such measures during drug trialswould result in the generation of large and detailed data sets, inturn providing a potential basis for the more formal inclusion ofsuch assays as routine outcome measures (the inclusion of suchsecondary and additional endpoints is broadly supported byFDA guidelines, where sufficiently powered [180]). Further, the

data generated would be invaluable in helping us determine themicrobial determinants of CF lung disease.

5.5. Unanswered questions regarding antibiotic treatment in CF

Despite a rich literature, with respect to antibiotic choicesand delivery modes, many more specific questions remain. Forexample, does dry powder inhalation result in altered drugdistribution in vivo compared to nebulization? Do liposomaldrug formulations result in better biofilm penetration? Doinhaled antibiotics differ from IV and oral therapies in thepersistence time within the lung compartment? Is combinationtherapy more effective than monotherapy in killing targetorganisms or other microbes? What are the effects ofantimicrobial drugs on inflammation? Is tailored or empiricaltreatment more effective? Does cycling different maintenancetreatments offer advantages over single-agent maintenance?Should IV treatments be administered prophylactically, or onlyas indicated by clinical or laboratory parameters? These andmany more questions remain to be answered, and until they are,guidelines will be difficult to compose and defend, andtherapeutic strategies are likely to continue to differ betweentreatment sites.

6. Antibiotic therapy beyond CF

In many ways, CF lung disease represents a model system,providing insight into the mechanisms involved in, and potentialtreatments for, a wide range of acute and chronic infections.Techniques and concepts developed through the study of CFrespiratory infections can help develop our understanding of anarray of other respiratory infections, including those associatedwithCOPD, non-CF bronchiectasis, chronic intubation, aswell aschronic, polymicrobial infections more widely.

7. Summary

Sincewidespread antibiotic therapy for CF lung infectionswasintroduced in the 1960s, it has developed into a central componentof care. A process of refinement of antibiotic strategy, includingprogress in the drugs available, and the manner in which they aredelivered, has resulted in a significant beneficial impact on thecourse of CF airway disease. However, as antibiotic resistancedevelops in step with drug development, and the cumulativelevels of antibiotics recieved by patients increases with improvinglife expectancy, the need to develop more effective treatmentstrategies is as urgent as ever. The inclusion of sophisticatedbacteriology in development and assessment of antibiotictreatment strategies is a vital next step in the process of improvingthe effectiveness of CF antibiotic therapy.

Acknowledgment

The authors wish to thank Kenneth Bruce for his help andsupport in the preparation of this manuscript.

396 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

References

[1] Döring G, Gulbins E. Cystic fibrosis and innate immunity: how chloridechannel mutations provoke lung disease. Cell Microbiol 2009 Feb;11(2):208–16.

[2] Döring G, Conway SP, Heijerman HG, Hodson ME, Hoiby N, Smyth A,et al. Antibiotic therapy against Pseudomonas aeruginosa in cysticfibrosis: a European consensus. Eur Respir J 2000 Oct;16(4):749–67.

[3] Döring G, Hoiby N, Consensus Study Group. Early intervention andprevention of lung disease in cystic fibrosis: a European consensus. J CystFibros 2004 Jun;3(2):67–91.

[4] Ratjen F. Changes in strategies for optimal antibacterial therapy in cysticfibrosis. Int J Antimicrob Agents 2001 Feb;17(2):93–6.

[5] Cystic Fibrosis Trust. Antibiotic treatment for cystic fibrosis— report ofthe UK cystic fibrosis antibiotic working group. 3rd; 2009. http://www.cftrust.org.uk/aboutcf/publications/consensusdoc/Antibiotic_treatment_for_Cystic_Fibrosis.pdf.

[6] Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL.Pseudomonas aeruginosa and other predictors of mortality and morbidityin young children with cystic fibrosis. Pediatr Pulmonol 2002 Aug;34(2):91–100.

[7] Hudson VL, Wielinski CL, Regelmann WE. Prognostic implications ofinitial oropharyngeal bacterial flora in patients with cystic fibrosisdiagnosed before the age of two years. J Pediatr 1993 Jun;122(6):854–60.

[8] Rosenfeld M, Emerson J, Williams-Warren J, Pepe M, Smith A,Montgomery AB, et al. Defining a pulmonary exacerbation in cysticfibrosis. J Pediatr 2001 Sep;139(3):359–65.

[9] Kosorok MR, Jalaluddin M, Farrell PM, Shen G, Colby CE, Laxova A,et al. Comprehensive analysis of risk factors for acquisition ofPseudomonas aeruginosa in young children with cystic fibrosis. PediatrPulmonol 1998 Aug;26(2):81–8.

[10] KosorokMR, Zeng L,West SE, RockMJ, SplaingardML, Laxova A, et al.Acceleration of lung disease in children with cystic fibrosis afterPseudomonas aeruginosa acquisition. Pediatr Pulmonol 2001 Oct;32(4):277–87.

[11] CF Foundation. Patient registry annual data report 2007. http://cff.org/UploadedFiles/research/ClinicalResearch/2007-Patient-Registry-Report.pdf.

[12] Zemanick ET, Wagner BD, Harris JK, Wagener JS, Accurso FJ, SagelSD. Pulmonary exacerbations in cystic fibrosis with negative bacterialcultures. Pediatr Pulmonol 2010 Jun;45(6):569–77.

[13] Smyth A. Prophylactic antibiotics in cystic fibrosis: a conviction withoutevidence? Pediatr Pulmonol 2005 Dec;40(6):471–6.

[14] 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 rDNAterminal restriction fragment length polymorphism profiling. J ClinMicrobiol 2003 Aug;41(8):3548–58.

[15] Rogers GB, Carroll MP, Serisier DJ, Hockey PM, Jones G, Bruce KD.characterization of bacterial community diversity in cystic fibrosis lunginfections by use of 16 s ribosomal DNA terminal restriction fragment lengthpolymorphism profiling. J Clin Microbiol 2004 Nov;42(11):5176–83.

[16] Rogers GB, Carroll MP, Serisier DJ, Hockey PM, Jones G, Kehagia V,et al. Use of 16S rRNA gene profiling by terminal restriction fragmentlength polymorphism analysis to compare bacterial communities insputum and mouthwash samples from patients with cystic fibrosis. J ClinMicrobiol 2006 Jul;44(7):2601–4.

[17] Sibley CD, Parkins MD, Rabin HR, Duan K, Norgaard JC, Surette MG. Apolymicrobial perspective of pulmonary infections exposes an enigmaticpathogen in cystic fibrosis patients. Proc Natl Acad Sci USA 2008 Sep30;105(39):15070–5.

[18] Stressmann FA, Rogers GB, Klem ER, Lilley AK, Donaldson SH,Daniels TW, et al. Analysis of the bacterial communities present in lungsof patients with cystic fibrosis from American and British centers. J ClinMicrobiol 2011 Jan;49(1):281–91.

[19] TunneyMM, Klem ER, Fodor AA, Gilpin DF, Moriarty TF, McGrath SJ,et al. Use of culture and molecular analysis to determine the effect ofantibiotic treatment on microbial community diversity and abundance

during exacerbation in patients with cystic fibrosis. Thorax 2011,doi:10.1136/thx.2010.137281.

[20] Tunney MM, Field TR, Moriarty TF, Patrick S, Doering G, MuhlebachMS, et al. Detection of anaerobic bacteria in high numbers in sputumfrom patients with cystic fibrosis. Am J Respir Crit Care Med 2008 May1;177(9):995–1001.

[21] van der Gast CJ, Walker AW, Stressmann FA, Rogers GB, Scott P, DanielsTW, et al. Partitioning core and satellite taxa fromwithin cystic fibrosis lungbacterial communities. ISME J 2010 Dec 9, doi:10.1038/ismej.2010.175.

[22] Worlitzsch D, Rintelen C, Bohm K, Wollschlager B, Merkel N, Borneff-Lipp M, et al. Antibiotic-resistant obligate anaerobes during exacerbationsof cystic fibrosis patients. Clin Microbiol Infect 2009 May;15(5):454–60.

[23] Bittar F, Richet H, Dubus JC, Reynaud-Gaubert M, Stremler N, Sarles J,et al. Molecular detection of multiple emerging pathogens in sputa fromcystic fibrosis patients. PLoS One 2008 Aug 6;3(8):e2908.

[24] Guss AM, Roeselers G, Newton IL, Young CR, Klepac-Ceraj V, Lory S,et al. Phylogenetic and metabolic diversity of bacteria associated withcystic fibrosis. ISME J 2010 Jan;5(1):20–9.

[25] Flume PA, Mogayzel PJ, Robinson KA, Goss CH, Rosenblatt RL, KuhnRJ, et al. Cystic fibrosis pulmonary guidelines: treatment of pulmonaryexacerbations. Am J Respir Crit Care Med 2009 Nov 1;180(9):802–8.

[26] Gibson RL, Emerson J, Mayer-Hamblett N, Burns JL, McNamara S,Accurso FJ, et al. Duration of treatment effect after tobramycin solutionfor inhalation in young children with cystic fibrosis. Pediatr Pulmonol2007 Jul;42(7):610–23.

[27] Smith AL, Doershuk C, Goldmann D, Gore E, Hilman B, Marks M, et al.Comparison of a beta-lactam alone versus beta-lactam and anaminoglycoside for pulmonary exacerbation in cystic fibrosis. J Pediatr1999 Apr;134(4):413–21.

[28] Ordonez CL, Henig NR, Mayer-Hamblett N, Accurso FJ, Burns JL,Chmiel JF, et al. Inflammatory and microbiologic markers in inducedsputum after intravenous antibiotics in cystic fibrosis. Am J Respir CritCare Med 2003 Dec 15;168(12):1471–5.

[29] Zemanick ET, Harris JK, Conway S, Konstan MW, Marshall B, QuittnerAL, et al. Measuring and improving respiratory outcomes in cysticfibrosis lung disease: opportunities and challenges to therapy. J CystFibros 2010 Jan;9(1):1–16.

[30] Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cysticfibrosis. Clin Microbiol Rev 2002 Apr;15(2):194–222.

[31] Konstan MW, Morgan WJ, Butler SM, Pasta DJ, Craib ML, Silva SJ,et al. Risk factors for rate of decline in forced expiratory volume in onesecond in children and adolescents with cystic fibrosis. J Pediatr 2007Aug;15(2):134–9 139.e1.

[32] McPhail GL, Acton JD, Fenchel MC, Amin RS, Seid M. Improvementsin lung function outcomes in children with cystic fibrosis are associatedwith better nutrition, fewer chronic pseudomonas aeruginosa infections,and dornase alfa use. J Pediatr 2008 Dec;153(6):752–7.

[33] Ulrich M, Beer I, Braitmaier P, Dierkes M, Kummer F, Krismer B, et al.Relative contribution of Prevotella intermedia and Pseudomonasaeruginosa to lung pathology in airways of patients with cystic fibrosis.Thorax 2010 Nov;65(11):978–84.

[34] Sagel U, Schulte B, Heeg P, Borgmann S. Vancomycin-resistantenterococci outbreak, Germany, and calculation of outbreak start.Emerg Infect Dis 2008 Feb;14(2):317–9.

[35] Dasenbrook EC, Merlo CA, Diener-West M, Lechtzin N, Boyle MP.Persistent methicillin-resistant staphylococcus aureus and rate of FEV1decline in cystic fibrosis. Am J Respir Crit Care Med 2008 Oct 15;178(8):814–21.

[36] Saiman L, Anstead M, Mayer-Hamblett N, Lands LC, Kloster M,Hocevar-Trnka J, et al. Effect of azithromycin on pulmonary function inpatients with cystic fibrosis uninfected with Pseudomonas aeruginosa: arandomized controlled trial. JAMA 2010 May 5;303(17):1707–15.

[37] Littlewood JM, Miller MG, Ghoneim AT, Ramsden CH. Nebulisedcolomycin for early Pseudomonas colonisation in cystic fibrosis. Lancet1985 Apr 13;1(8433):865.

[38] Valerius NH, Koch C, Hoiby N. Prevention of chronic Pseudomonasaeruginosa colonisation in cystic fibrosis by early treatment. Lancet 1991Sep 21;338(8769):725–6.

397G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

[39] Wiesemann HG, Steinkamp G, Ratjen F, Bauernfeind A, Przyklenk B,Döring G, et al. Placebo-controlled, double-blind, randomized study ofaerosolized tobramycin for early treatment of Pseudomonas aeruginosacolonization in cystic fibrosis. Pediatr Pulmonol 1998 Feb;25(2):88–92.

[40] Munck A, Bonacorsi S, Mariani-Kurkdjian P, Lebourgeois M, GerardinM, Brahimi N, et al. Genotypic characterization of Pseudomonasaeruginosa strains recovered from patients with cystic fibrosis afterinitial and subsequent colonization. Pediatr Pulmonol 2001 Oct;32(4):288–92.

[41] Ratjen F, Döring G, Nikolaizik WH. Effect of inhaled tobramycin onearly Pseudomonas aeruginosa colonisation in patients with cysticfibrosis. Lancet 2001 Sep 22;358(9286):983–4.

[42] Gibson RL, Emerson J, McNamara S, Burns JL, Rosenfeld M, Yunker A,et al. Significant microbiological effect of inhaled tobramycin in youngchildren with cystic fibrosis. Am J Respir Crit Care Med 2003 Mar15;167(6):841–9.

[43] Taccetti G, Campana S, Festini F, Mascherini M, Döring G. Earlyeradication therapy against Pseudomonas aeruginosa in cystic fibrosispatients. Eur Respir J 2005 Sep;26(3):458–61.

[44] Ratjen F, Munck A, Kho P, Angyalosi G, ELITE Study Group. Treatmentof early Pseudomonas aeruginosa infection in patients with cysticfibrosis: the ELITE trial. Thorax 2010 Apr;65(4):286–91.

[45] Mayer-Hamblett N, Burns JL, Khan U, Retsch-Bogart G, Treggiari M,Ramsey BW. Predictors of Pseudomonas aeruginosa recurrence in cysticfibrosis: results from the EPIC trial. Published Abstract PediatricPulmonology; 2010. supplement 33.

[46] Ratjen F, Walter H, Haug M, Meisner C, Grasemann H, Döring G.Diagnostic value of serum antibodies in early Pseudomonas aeruginosainfection in cystic fibrosis patients. Pediatr Pulmonol 2007 Mar;42(3):249–55.

[47] Hansen CR, Pressler T, Hoiby N. Early aggressive eradication therapy forintermittent Pseudomonas aeruginosa airway colonization in cystic fibrosispatients: 15 years experience. J Cyst Fibros 2008 Nov;7(6):523–30.

[48] Wood LF, Leech AJ, Ohman DE. Cell wall-inhibitory antibiotics activatethe alginate biosynthesis operon in Pseudomonas aeruginosa: roles ofsigma (AlgT) and the AlgW and Prc proteases. Mol Microbiol 2006Oct;62(2):412–26.

[49] Treggiari MM, Rosenfeld M, Retsch-Bogart G, Gibson R, Ramsey B.Approach to eradication of initial Pseudomonas aeruginosa infection inchildren with cystic fibrosis. Pediatr Pulmonol 2007 Sep;42(9):751–6.

[50] Döring G, Parameswaran GI, Murphy TF. Differential adaptation ofmicrobial pathogens to airways of patients with cystic fibrosis andchronic obstructive pulmonary disease. FEMS Microbiol Rev 28 MAY2010, doi:10.1111/j.1574-6976.2010.00237.

[51] Oliver A, Canton R, Campo P, Baquero F, Blazquez J. High frequency ofhypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection.Science 2000 May 19;288(5469):1251–4.

[52] Döring G, Worlitzsch D. Inflammation in cystic fibrosis and itsmanagement. Paediatr Respir Rev 2000 Jun;1(2):101–6.

[53] Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibioticresistance of bacterial biofilms. Int J Antimicrob Agents 2010 Apr;35(4):322–32.

[54] Anwar H, Strap JL, Costerton JW. Establishment of aging biofilms:possible mechanism of bacterial resistance to antimicrobial therapy.Antimicrob Agents Chemother 1992 Jul;36(7):1347–51.

[55] del Pozo JL, Patel R. The challenge of treating biofilm-associatedbacterial infections. Clin Pharmacol Ther 2007 Aug;82(2):204–9.

[56] Oliver AM,Weir DM. The effect of Pseudomonas alginate on rat alveolarmacrophage phagocytosis and bacterial opsonization. Clin Exp Immunol1985 Jan;59(1):190–6.

[57] Meshulam T, Obedeanu N, Merzbach D, Sobel JD. Phagocytosis ofmucoid and nonmucoid strains of Pseudomonas aeruginosa. ClinImmunol Immunopathol 1984 Aug;32(2):151–65.

[58] Learn DB, Brestel EP, Seetharama S. Hypochlorite scavenging byPseudomonas aeruginosa alginate. Infect Immun 1987 Aug;55(8):1813–8.

[59] Krieg DP, Helmke RJ, German VF, Mangos JA. Resistance of mucoidPseudomonas aeruginosa to nonopsonic phagocytosis by alveolarmacrophages in vitro. Infect Immun 1988 Dec;56(12):3173–9.

[60] Pedersen SS, Kharazmi A, Espersen F, Hoiby N. Pseudomonasaeruginosa alginate in cystic fibrosis sputum and the inflammatoryresponse. Infect Immun 1990 Oct;58(10):3363–8.

[61] Leid JG, Willson CJ, Shirtliff ME, Hassett DJ, Parsek MR, Jeffers AK.The exopolysaccharide alginate protects Pseudomonas aeruginosabiofilm bacteria from IFN-gamma-mediated macrophage killing.J Immunol 2005 Dec 1;175(11):7512–8.

[62] Anwar H, Costerton JW. Enhanced activity of combination of tobramycinand piperacillin for eradication of sessile biofilm cells of Pseudomonasaeruginosa. Antimicrob Agents Chemother 1990 Sep;34(9):1666–71.

[63] Moskowitz SM, Foster JM, Emerson J, Burns JL. Clinically feasiblebiofilm susceptibility assay for isolates of Pseudomonas aeruginosa frompatients with cystic fibrosis. J Clin Microbiol 2004 May;42(5):1915–22.

[64] Drenkard E. Antimicrobial resistance of Pseudomonas aeruginosabiofilms. Microbes Infect 2003 Nov;5(13):1213–9.

[65] Levy J. Antibiotic activity in sputum. J Pediatr 1986 May;108(5 Pt 2):841–6.

[66] Hunt BE, Weber A, Berger A, Ramsey B, Smith AL. Macromolecularmechanisms of sputum inhibition of tobramycin activity. AntimicrobAgents Chemother 1995 Jan;39(1):34–9.

[67] Worlitzsch D, Tarran R, Ulrich M, Schwab U, Cekici A, Meyer KC, et al.Effects of reduced mucus oxygen concentration in airway Pseudomonasinfections of cystic fibrosis patients. J Clin Invest 2002 Feb;109(3):317–25.

[68] Tack KJ, Sabath LD. Increased minimum inhibitory concentrations withanaerobiasis for tobramycin, gentamicin, and amikacin, compared tolatamoxef, piperacillin, chloramphenicol, and clindamycin. Chemother-apy 1985;31(3):204–10.

[69] Folsom JP, Richards L, Pitts B, Roe F, Ehrlich GD, Parker A, et al.Physiology of Pseudomonas aeruginosa in Biofilms as Revealed byTranscriptome Analysis. BMC Microbiol 2010 Nov 17;10(1):294.

[70] Walters III MC, Roe F, Bugnicourt A, Franklin MJ, Stewart PS.Contributions of antibiotic penetration, oxygen limitation, and lowmetabolic activity to tolerance of Pseudomonas aeruginosa biofilms tociprofloxacin and tobramycin. Antimicrob Agents Chemother 2003Jan;47(1):317–23.

[71] King P, Citron DM, Griffith DC, Lomovskaya O, Dudley MN. Effect ofoxygen limitation on the in vitro activity of levofloxacin and otherantibiotics administered by the aerosol route against Pseudomonasaeruginosa from cystic fibrosis patients. Diagn Microbiol Infect Dis 2010Feb;66(2):181–6.

[72] King P, Lomovskaya O, Griffith DC, Burns JL, Dudley MN. In vitropharmacodynamics of levofloxacin and other aerosolized antibioticsunder multiple conditions relevant to chronic pulmonary infection incystic fibrosis. Antimicrob Agents Chemother 2010 Jan;54(1):143–8.

[73] Park MK, Myers RA, Marzella L. Oxygen tensions and infections:modulation of microbial growth, activity of antimicrobial agents, andimmunologic responses. Clin Infect Dis 1992 Mar;14(3):720–40.

[74] Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS, O'Toole GA. Agenetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.Nature 2003 Nov 20;426(6964):306–10.

[75] Billard-Pomares T, Herwegh S, Wizla-Derambure N, Turck D, CourcolR, Husson MO. Application of quantitative PCR to the diagnosis andmonitoring of Pseudomonas aeruginosa colonization in 5-18-year-oldcystic fibrosis patients. J Med Microbiol 2011 Feb;60(Pt 2):157–61.

[76] Regelmann WE, Elliott GR, Warwick WJ, Clawson CC. Reduction ofsputum Pseudomonas aeruginosa density by antibiotics improveslung function in cystic fibrosis more than do bronchodilators andchest physiotherapy alone. Am Rev Respir Dis 1990 Apr;141(4 Pt 1):914–21.

[77] Stressmann FA, Rogers GB, Marsh P, Lilley AK, Daniels TWV, CarrollMP, et al. Does bacterial density in cystic fibrosis sputum increaseprior to pulmonary exacerbation? J Cyst Fibros 2011, doi:10.1016/j.jcf.2011.05.002.

[78] Ciofu O, Beveridge TJ, Kadurugamuwa J, Walther-Rasmussen J, HoibyN. Chromosomal beta-lactamase is packaged into membrane vesicles andsecreted from Pseudomonas aeruginosa. J Antimicrob Chemother 2000Jan;45(1):9–13.

398 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

[79] Bagge N, Hentzer M, Andersen JB, Ciofu O, Givskov M, Hoiby N.Dynamics and spatial distribution of beta-lactamase expression inPseudomonas aeruginosa biofilms. Antimicrob Agents Chemother2004 Apr;48(4):1168–74.

[80] Giwercman B, Jensen ET, Hoiby N, Kharazmi A, Costerton JW.Induction of beta-lactamase production in Pseudomonas aeruginosabiofilm. Antimicrob Agents Chemother 1991 May;35(5):1008–10.

[81] Webb JS, Thompson LS, James S, Charlton T, Tolker-Nielsen T, Koch B,et al. Cell death in Pseudomonas aeruginosa biofilm development.J Bacteriol 2003 Aug;185(15):4585–92.

[82] Kjelleberg S, Molin S. Is there a role for quorum sensing signals inbacterial biofilms? Curr Opin Microbiol 2002 Jun;5(3):254–8.

[83] VanDevanter DR, Van Dalfsen JM. Howmuch do Pseudomonas biofilmscontribute to symptoms of pulmonary exacerbation in cystic fibrosis?Pediatr Pulmonol 2005 Jun;39(6):504–6.

[84] Ciofu O, Riis B, Pressler T, Poulsen HE, Hoiby N. Occurrence ofhypermutable Pseudomonas aeruginosa in cystic fibrosis patients isassociated with the oxidative stress caused by chronic lung inflammation.Antimicrob Agents Chemother 2005 Jun;49(6):2276–82.

[85] Haussler S, Tummler B, Weissbrodt H, Rohde M, Steinmetz I. Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin InfectDis 1999 Sep;29(3):621–5.

[86] Foweraker JE, Laughton CR, Brown DF, Bilton D. Comparison ofmethods to test antibiotic combinations against heterogeneous popula-tions of multiresistant Pseudomonas aeruginosa from patients with acuteinfective exacerbations in cystic fibrosis. Antimicrob Agents Chemother2009 Nov;53(11):4809–15.

[87] Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M,et al. Small colony variants: a pathogenic form of bacteria that facilitatespersistent and recurrent infections. Nat Rev Microbiol 2006 Apr;4(4):295–305.

[88] Yoon SS, Hassett DJ. Chronic Pseudomonas aeruginosa infection incystic fibrosis airway disease: metabolic changes that unravel novel drugtargets. Expert Rev Anti Infect Ther 2004 Aug;2(4):611–23.

[89] Spoering AL, Lewis K. Biofilms and planktonic cells of Pseudomonasaeruginosa have similar resistance to killing by antimicrobials.J Bacteriol 2001 Dec;183(23):6746–51.

[90] Harrison JJ, Turner RJ, Ceri H. Persister cells, the biofilm matrix andtolerance to metal cations in biofilm and planktonic Pseudomonasaeruginosa. Environ Microbiol 2005 Jul;7(7):981–94.

[91] Lewis K. Persister cells, dormancy and infectious disease. Nat RevMicrobiol 2007 Jan;5(1):48–56.

[92] Lewis DD, Ruane ML, Scheeline A. Biofilm effects on theperoxidase-oxidase reaction. J Phys Chem B 2006 Apr 20;110(15):8100–4.

[93] Brooun A, Liu S, Lewis K. A dose-response study of antibiotic resistancein Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother2000 Mar;44(3):640–6.

[94] Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother2001 Apr;45(4):999–1007.

[95] Desai M, Buhler T, Weller PH, Brown MR. Increasing resistance ofplanktonic and biofilm cultures of Burkholderia cepacia to ciprofloxacinand ceftazidime during exponential growth. J Antimicrob Chemother1998 Aug;42(2):153–60.

[96] Silo-Suh L, Suh SJ, Phibbs PV, Ohman DE. Adaptations of Pseudomonasaeruginosa to the cystic fibrosis lung environment can includederegulation of zwf, encoding glucose-6-phosphate dehydrogenase.J Bacteriol 2005 Nov;187(22):7561–8.

[97] Lewis K. Persister cells. Annu Rev Microbiol 2010 Oct 13;64:357–72.[98] Dorr T, Vulic M, Lewis K. Ciprofloxacin causes persister formation by

inducing the TisB toxin in Escherichia coli. PLoS Biol 2010 Feb 23;8(2):e1000317.

[99] Dorr T, Lewis K, Vulic M. SOS response induces persistence tofluoroquinolones in Escherichia coli. PLoS Genet 2009 Dec;5(12):e1000760.

[100] Mulcahy LR, Burns JL, Lory S, Lewis K. Emergence of Pseudomonasaeruginosa strains producing high levels of persister cells in patients withcystic fibrosis. J Bacteriol 2010 Dec;192(23):6191–9.

[101] Williams I, Venables WA, Lloyd D, Paul F, Critchley I. The effects ofadherence to silicone surfaces on antibiotic susceptibility in Staphylo-coccus aureus. Microbiology 1997 Jul;143(Pt 7):2407–13.

[102] Acar JF, Goldstein FW, Lagrange P. Human infections caused bythiamine- or menadione-requiring Staphylococcus aureus. J ClinMicrobiol 1978 Aug;8(2):142–7.

[103] Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD.Persistent and relapsing infections associated with small-colony variantsof Staphylococcus aureus. Clin Infect Dis 1995 Jan;20(1):95–102.

[104] Proctor RA, Peters G. Small colony variants in staphylococcal infections:diagnostic and therapeutic implications. Clin Infect Dis 1998 Sep;27(3):419–22.

[105] Proctor RA, Kahl B, von Eiff C, Vaudaux PE, Lew DP, Peters G.Staphylococcal small colony variants have novel mechanisms forantibiotic resistance. Clin Infect Dis 1998 Aug;27(Suppl 1):S68–74.

[106] Chuard C, Vaudaux PE, Proctor RA, Lew DP. Decreased susceptibility toantibiotic killing of a stable small colony variant of Staphylococcusaureus in fluid phase and on fibronectin-coated surfaces. J AntimicrobChemother 1997 May;39(5):603–8.

[107] Starkey M, Hickman JH, Ma L, Zhang N, De Long S, Hinz A, et al.Pseudomonas aeruginosa rugose small-colony variants have adaptationsthat likely promote persistence in the cystic fibrosis lung. J Bacteriol 2009Jun;191(11):3492–503.

[108] Nelson LK, Stanton MM, Elphinstone RE, Helwerda J, Turner RJ, CeriH. Phenotypic diversification in vivo: Pseudomonas aeruginosa gacS-strains generate small colony variants in vivo that are distinct from invitro variants. Microbiology 2010 Dec;156(Pt 12):3699–709.

[109] Goerke C, Wolz C. Adaptation of Staphylococcus aureus to the cysticfibrosis lung. Int J Med Microbiol 2010 Dec;300(8):520–5.

[110] Schneider M, Muhlemann K, Droz S, Couzinet S, Casaulta C, ZimmerliS. Clinical characteristics associated with isolation of small-colonyvariants of Staphylococcus aureus and Pseudomonas aeruginosa fromrespiratory secretions of patients with cystic fibrosis. J Clin Microbiol2008 May;46(5):1832–4.

[111] Kahl BC, Duebbers A, Lubritz G, Haeberle J, Koch HG, Ritzerfeld B,et al. Population dynamics of persistent Staphylococcus aureus isolatedfrom the airways of cystic fibrosis patients during a 6-year prospectivestudy. J Clin Microbiol 2003 Sep;41(9):4424–7.

[112] Sibley CD, Duan K, Fischer C, Parkins MD, Storey DG, Rabin HR, et al.Discerning the complexity of community interactions using a Drosophilamodel of polymicrobial infections. PLoS Pathog 2008 Oct;4(10):e1000184.

[113] Smith AL, Redding G, Doershuk C, Goldmann D, Gore E, Hilman B,et al. Sputum changes associated with therapy for endobronchialexacerbation in cystic fibrosis. J Pediatr 1988 Apr;112(4):547–54.

[114] McLaughlin FJ, Matthews Jr WJ, Strieder DJ, Sullivan B, Taneja A,Murphy P, et al. Clinical and bacteriological responses to three antibioticregimens for acute exacerbations of cystic fibrosis: ticarcillin-tobramycin,azlocillin-tobramycin, and azlocillin-placebo. J Infect Dis 1983 Mar;147(3):559–67.

[115] Gold R, Overmeyer A, Knie B, Fleming PC, Levison H. Controlled trialof ceftazidime vs. ticarcillin and tobramycin in the treatment of acuterespiratory exacerbations in patients with cystic fibrosis. Pediatr InfectDis 1985 Mar-Apr;4(2):172–7.

[116] Vrany JD, Stewart PS, Suci PA. Comparison of recalcitrance tociprofloxacin and levofloxacin exhibited by Pseudomonas aeruginosabofilms displaying rapid-transport characteristics. Antimicrob AgentsChemother 1997 Jun;41(6):1352–8.

[117] Anderl JN, Franklin MJ, Stewart PS. Role of antibiotic penetrationlimitation in Klebsiella pneumoniae biofilm resistance to ampicillinand ciprofloxacin. Antimicrob Agents Chemother 2000 Jul;44(7):1818–24.

[118] Stone G, Wood P, Dixon L, Keyhan M, Matin A. Tetracycline rapidlyreaches all the constituent cells of uropathogenic Escherichia colibiofilms. Antimicrob Agents Chemother 2002 Aug;46(8):2458–61.

[119] Darouiche RO, Dhir A, Miller AJ, Landon GC, Raad II, Musher DM.Vancomycin penetration into biofilm covering infected prostheses andeffect on bacteria. J Infect Dis 1994 Sep;170(3):720–3.

399G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

[120] Shigeta M, Tanaka G, Komatsuzawa H, Sugai M, Suginaka H, Usui T.Permeation of antimicrobial agents through Pseudomonas aeruginosabiofilms: a simple method. Chemotherapy 1997 Sep-Oct;43(5):340–5.

[121] Jaffe A, Bush A. Anti-inflammatory effects of macrolides in lung disease.Pediatr Pulmonol 2001 Jun;31(6):464–73.

[122] Yousef AA, Jaffe A. The role of azithromycin in patients with cysticfibrosis. Paediatr Respir Rev 2010 Jun;11(2):108–14.

[123] Tagaya E, Tamaoki J, Kondo M, Nagai A. Effect of a short course ofclarithromycin therapy on sputum production in patients with chronicairway hypersecretion. Chest 2002 Jul;122(1):213–8.

[124] Takizawa H, Desaki M, Ohtoshi T, Kawasaki S, Kohyama T, Sato M,et al. Erythromycin and clarithromycin attenuate cytokine-inducedendothelin-1 expression in human bronchial epithelial cells. Eur RespirJ 1998 Jul;12(1):57–63.

[125] Yatsunami J, Fukuno Y, Nagata M, Tsuruta N, Aoki S, Tominaga M,et al. Roxithromycin and clarithromycin, 14-membered ring macrolides,potentiate the antitumor activity of cytotoxic agents against mouse B16melanoma cells. Cancer Lett 1999 Dec 1;147(1–2):17–24.

[126] Feldman C, Anderson R, Theron A, Mokgobu I, Cole PJ, Wilson R.The effects of ketolides on bioactive phospholipid-induced injury tohuman respiratory epithelium in vitro. Eur Respir J 1999 May;13(5):1022–8.

[127] BergaminiG,CiganaC, Sorio C,Della PerutaM, Pompella A, Corti A, et al.Effects of azithromycin on glutathione S-transferases in cystic fibrosisairway cells. Am J Respir Cell Mol Biol 2009 Aug;41(2):199–206.

[128] Equi A, Balfour-Lynn IM, Bush A, Rosenthal M. Long termazithromycin in children with cystic fibrosis: a randomised, placebo-controlled crossover trial. Lancet 2002 Sep 28;360(9338):978–84.

[129] Wolter J, Seeney S, Bell S, Bowler S, Masel P, McCormack J. Effect oflong term treatment with azithromycin on disease parameters in cysticfibrosis: a randomised trial. Thorax 2002 Mar;57(3):212–6.

[130] Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL,Cibene DA, et al. Azithromycin in patients with cystic fibrosischronically infected with Pseudomonas aeruginosa: a randomizedcontrolled trial. JAMA 2003 Oct 1;290(13):1749–56.

[131] Kobayashi H. Biofilm disease: its clinical manifestation and therapeuticpossibilities of macrolides. Am J Med 1995 Dec 29;99(6A):26S–30S.

[132] Mitsuya Y, Kawai S, Kobayashi H. Influence of macrolides on guanosinediphospho-D-mannose dehydrogenase activity in Pseudomonas biofilm.J Infect Chemother 2000 Mar;6(1):45–50.

[133] Sofer D, Gilboa-Garber N, Belz A, Garber NC. ‘Subinhibitory’erythromycin represses production of Pseudomonas aeruginosa lectins,autoinducer and virulence factors. Chemotherapy 1999;45:335–41.

[134] Tateda K, Comte R, Pechere JC, Kohler T, Yamaguchi K, Van Delden C.Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa.Antimicrob Agents Chemother 2001 Jun;45(6):1930–3.

[135] Skindersoe ME, Alhede M, Phipps R, Yang L, Jensen PO, RasmussenTB, et al. Effects of antibiotics on quorum sensing in Pseudomonasaeruginosa. Antimicrob Agents Chemother 2008 Oct;52(10):3648–63.

[136] Bala A, Kumar R, Harjai K. Inhibition of quorum sensing inPseudomonas aeruginosa by azithromycin and its effectiveness inurinary tract infections. J Med Microbiol 2011 Mar;60(Pt 3):300–6.

[137] Razvi S, Saiman L. Microbiology of cystic fibrosis: role of the clinicalmicrobiology laboratory, susceptibility and synergy studies and infectioncontrol. In: Hodson M, Geddes D, Bush A, editors. Cystic fibrosis. 3rded. London: Hodder Arnold; 2007. p. 123–33.

[138] Standards Unit Department for Evaluations Standards and TrainingCentre for Infections. Investigation of bronchoalveolar lavage, sputumand associated specimens. (www.evaluations-standards.org.uk) BSOP2008;57.

[139] Cystic Fibrosis Trust. Laboratory standards for processing microbiolog-ical samples from people with cystic fibrosis — report of the UK cysticfibrosis trust microbiology standards working group; 2010.

[140] Foweraker JE, Laughton CR, Brown DF, Bilton D. Phenotypic variabilityof Pseudomonas aeruginosa in sputa from patients with acute infectiveexacerbation of cystic fibrosis and its impact on the validity ofantimicrobial susceptibility testing. J Antimicrob Chemother 2005Jun;55(6):921–7.

[141] Milne KE, Gould IM. Combination testing of multidrug-resistant cysticfibrosis isolates of Pseudomonas aeruginosa: use of a new parameter, thesusceptible breakpoint index. J Antimicrob Chemother 2010 Jan;65(1):82–90.

[142] Aaron SD, Vandemheen KL, Ferris W, Fergusson D, Tullis E, Haase D,et al. Combination antibiotic susceptibility testing to treat exacerbationsof cystic fibrosis associated with multiresistant bacteria: a randomised,double-blind, controlled clinical trial. Lancet 2005 Aug 6–12;366(9484):463–71.

[143] Smith AL, Fiel SB, Mayer-Hamblett N, Ramsey B, Burns JL.Susceptibility testing of Pseudomonas aeruginosa isolates and clinicalresponse to parenteral antibiotic administration: lack of association incystic fibrosis. Chest 2003 May;123(5):1495–502.

[144] Wang EE, Prober CG, Manson B, Corey M, Levison H. Association ofrespiratory viral infections with pulmonary deterioration in patients withcystic fibrosis. N Engl J Med 1984 Dec 27;311(26):1653–8.

[145] Abman SH, Ogle JW, Butler-Simon N, Rumack CM, Accurso FJ. Roleof respiratory syncytial virus in early hospitalizations for respiratory distressof young infants with cystic fibrosis. J Pediatr 1988 Nov;113(5):826–30.

[146] Smyth AR, Smyth RL, Tong CY, Hart CA, Heaf DP. Effect of respiratoryvirus infections including rhinovirus on clinical status in cystic fibrosis.Arch Dis Child 1995 Aug;73(2):117–20.

[147] UK Cystic Fibrosis Trust Antibiotic Group. Antibiotic treatment forcystic fibrosis: report of the UK Cystic Fibrosis Trust Antibiotic Group.2nd; 2002.

[148] Ring E, Eber E, Erwa W, Zach MS. Urinary N-acetyl-beta-D-glucosaminidase activity in patients with cystic fibrosis on long-termgentamicin inhalation. Arch Dis Child 1998 Jun;78(6):540–3.

[149] Moss RB. Administration of aerosolized antibiotics in cystic fibrosispatients. Chest 2001 Sep;120(3 Suppl):107S–13S.

[150] Turvey SE, Cronin B, Arnold AD, Dioun AF. Antibiotic desensitizationfor the allergic patient: 5 years of experience and practice. Ann AllergyAsthma Immunol 2004 Apr;92(4):426–32.

[151] Steinkamp G, Wiedemann B, Rietschel E, Krahl A, Gielen J, Barmeier H,et al. Prospective evaluation of emerging bacteria in cystic fibrosis. J CystFibros 2005 Mar;4(1):41–8.

[152] Lechtzin N, John M, Irizarry R, Merlo C, Diette GB, Boyle MP.Outcomes of adults with cystic fibrosis infected with antibiotic-resistantPseudomonas aeruginosa. Respiration 2006;73(1):27–33.

[153] Aaron SD, Vandemheen KL, Ramotar K, Giesbrecht-Lewis T, Tullis E,Freitag A, et al. Infection with transmissible strains of Pseudomonasaeruginosa and clinical outcomes in adults with cystic fibrosis. JAMA2010 Nov 17;304(19):2145–53.

[154] Salunkhe P, Smart CH, Morgan JA, Panagea S, Walshaw MJ, Hart CA,et al. A cystic fibrosis epidemic strain of Pseudomonas aeruginosadisplays enhanced virulence and antimicrobial resistance. J Bacteriol2005 Jul;187(14):4908–20.

[155] Ramsey BW, Pepe MS, Quan JM, Otto KL, Montgomery AB, Williams-Warren J, et al. Intermittent administration of inhaled tobramycin inpatients with cystic fibrosis. Cystic Fibrosis Inhaled Tobramycin StudyGroup. N Engl J Med 1999 Jan 7;34(1):23–30.

[156] Oermann CM, Assael B, Makamura C, Boas SR, Bresnik M,Montgomery AB, et al. Aztreonam for inhalation solution (AZLI) vs.tobramycin inhalation solution (TIS), a 6-month comparative trial incystic fibrosis patients with Pseudomonas aeruginosa. PublishedAbstract Pediatric Pulmonology; 2010. supplement 33.

[157] Hoffman LR, D'Argenio DA, MacCoss MJ, Zhang Z, Jones RA, MillerSI. Aminoglycoside antibiotics induce bacterial biofilm formation.Nature 2005 Aug 25;436(7054):1171–5.

[158] Retsch-Bogart GZ, Quittner AL, Gibson RL, Oermann CM, McCoy KS,Montgomery AB, et al. Efficacy and safety of inhaled aztreonam lysine forairway Pseudomonas in cystic fibrosis. Chest 2009May;135(5):1223–32.

[159] Stelmach I, Korzeniewska A, Stelmach W. Long-term benefits of inhaledtobramycin in children with cystic fibrosis: first clinical observationsfrom Poland. Respiration 2008;75(2):178–81.

[160] Clement A, Tamalet A, Leroux E, Ravilly S, Fauroux B, Jais JP. Longterm effects of azithromycin in patients with cystic fibrosis: a doubleblind, placebo controlled trial. Thorax 2006 Oct;61(10):895–902.

400 G.B. Rogers et al. / Journal of Cystic Fibrosis 10 (2011) 387–400

[161] McCoy KS, Quittner AL, Oermann CM, Gibson RL, Retsch-Bogart GZ,Montgomery AB. Inhaled aztreonam lysine for chronic airwayPseudomonas aeruginosa in cystic fibrosis. Am J Respir Crit CareMed 2008 Nov 1;178(9):921–8.

[162] Kaplan ML, Dye W. Growth requirements of some small-colony-formingvariants of Staphylococcus aureus. J Clin Microbiol 1976 Oct;4(4):343–8.

[163] Barth AL, Pitt TL. Auxotrophic variants of Pseudomonas aeruginosa areselected from prototrophic wild-type strains in respiratory infections inpatients with cystic fibrosis. J Clin Microbiol 1995 Jan;33(1):37–40.

[164] Barth AL, Pitt TL. Auxotrophy of Burkholderia (Pseudomonas) cepaciafrom cystic fibrosis patients. J Clin Microbiol 1995 Aug;33(8):2192–4.

[165] Taylor RF, Hodson ME, Pitt TL. Auxotrophy of Pseudomonas aeruginosain cystic fibrosis. FEMS Microbiol Lett 1992 May 1;71(3):243–6.

[166] Taylor RF, Hodson ME, Pitt TL. Adult cystic fibrosis: association ofacute pulmonary exacerbations and increasing severity of lung diseasewith auxotrophic mutants of Pseudomonas aeruginosa. Thorax 1993Oct;48(10):1002–5.

[167] Ranasinha C, Assoufi B, Shak S, Christiansen D, Fuchs H, Empey D,et al. Efficacy and safety of short-term administration of aerosolisedrecombinant human DNase I in adults with stable stage cystic fibrosis.Lancet 1993 Jul 24;342(8865):199–202.

[168] Brandt T, Breitenstein S, von der Hardt H, Tummler B. DNA concentrationand length in sputum of patients with cystic fibrosis during inhalation withrecombinant human DNase. Thorax 1995 Aug;50(8):880–2.

[169] Ulmer JS, Herzka A, Toy KJ, Baker DL, Dodge AH, Sinicropi D, et al.Engineering actin-resistant human DNase I for treatment of cysticfibrosis. Proc Natl Acad Sci USA 1996 Aug 6;93(16):8225–9.

[170] Rogers GB, Stressmann FA, Koller G, Daniels T, Carroll MP, Bruce KD.Assessing the diagnostic importance of nonviable bacterial cells inrespiratory infections. Diagn Microbiol Infect Dis 2008 Oct;62(2):133–41.

[171] Rogers GB, Marsh P, Stressmann AF, Allen CE, Daniels TV, Carroll MP,et al. The exclusion of dead bacterial cells is essential for accuratemolecular analysis of clinical samples. Clin Microbiol Infect 2010Nov;16(11):1656–8.

[172] Caldwell CC, Chen Y, Goetzmann HS, Hao Y, Borchers MT, Hassett DJ,et al. Pseudomonas aeruginosa exotoxin pyocyanin causes cystic fibrosisairway pathogenesis. Am J Pathol 2009 Dec;175(6):2473–88.

[173] Cobb LM, Mychaleckyj JC, Wozniak DJ, Lopez-Boado YS. Pseudomo-nas aeruginosa flagellin and alginate elicit very distinct gene expressionpatterns in airway epithelial cells: implications for cystic fibrosis disease.J Immunol 2004 Nov 1;173(9):5659–70.

[174] Rogers GB, Hoffman LR, Johnson MW, Mayer-Hamblett N, Schwarze J,Carroll MP, et al. Using bacterial biomarkers to identify early indicatorsof cystic fibrosis pulmonary exacerbation onset. Expert Rev Mol Diagn2011, doi:10.1586/ERM.10.117.

[175] Stevens DL, Ma Y, Salmi DB, McIndoo E, Wallace RJ, Bryant AE.Impact of antibiotics on expression of virulence-associated exotoxingenes in methicillin-sensitive and methicillin-resistant Staphylococcusaureus. J Infect Dis 2007 Jan 15;195(2):202–11.

[176] Fernandes B, Plummer A, WildmanM. Duration of intravenous antibiotictherapy in people with cystic fibrosis. Cochrane Database Syst Rev 2008Apr;16(2):CD006682.

[177] Smyth A, Elborn JS. Exacerbations in cystic fibrosis: 3–Management.Thorax 2008 Feb;63(2):180–4.

[178] VanDevanter DR, O'Riordan MA, Blumer JL, Konstan MW. Assessingtime to pulmonary function benefit following antibiotic treatment of acutecystic fibrosis exacerbations. Respir Res 2010 Oct 6;11:137.

[179] U.S. Department of Health and Human Services Food and DrugAdministration. Guidance for industry: developing antimicrobial drugs —general considerations for clinical trials (draft guidance). 1998. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070983.pdf.

[180] U.S. Department of Health and Human Services Food and DrugAdministration. Guidance for industry: acute bacterial exacerbations ofchronic bronchitis in patients with chronic obstructive pulmonary disease:developing antimicrobial drugs for treatment (draft guidance). 2008.http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070935.pdf.

[181] Chernish RN, Aaron SD. Approach to resistant gram-negative bacterialpulmonary infections in patients with cystic fibrosis. Curr Opin PulmMed 2003 Nov;9(6):509–15.

[182] Radhakrishnan DK, Yau Y, Corey M, Richardson S, Chedore P,Jamieson F, et al. Non-tuberculous mycobacteria in children with cysticfibrosis: isolation, prevalence, and predictors. Pediatr Pulmonol 2009Nov;44(11):1100–6.