Journal of Cystic Fibrosis 14 (2015) 293 – 304 www.elsevier.com/locate/jcf

Review

Emerging bacterial pathogens and changing concepts of bacterial pathogenesis in cystic fibrosis Michael D. Parkins a,b,⁎, R. Andres Floto c,d a

b

Department of Medicine, The University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada Microbiology, Immunology and Infectious Diseases, The University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada c Cambridge Institute for Medical Research, University of Cambridge, Papworth Hospital, Cambridge CB23 3RE, UK d Cambridge Centre for Lung Infection, Papworth Hospital, Cambridge CB23 3RE, UK Received 3 December 2014; revised 21 March 2015; accepted 22 March 2015 Available online 14 April 2015

Abstract Chronic suppurative lower airway infection is a hallmark feature of cystic fibrosis (CF). Decades of experience in clinical microbiology have enabled the development of improved technologies and approaches for the cultivation and identification of microorganisms from sputum. It is increasingly apparent that the microbial constituents of the lower airways in CF exist in a dynamic state. Indeed, while changes in prevalence of various pathogens occur through ageing, differences exist in successive cohorts of patients and between clinics, regions and countries. Classical pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex and Staphylococcus aureus are increasingly being supplemented with new and emerging organisms rarely observed in other areas of medicine. Moreover, it is now recognized that common oropharyngeal organisms, previously presumed to be benign colonizers may contribute to disease progression. As infection remains the leading cause of morbidity and mortality in CF, an understanding of the epidemiology, risk factors for acquisition and natural history of infection including interactions between colonizing bacteria is required. Unified approaches to the study and determination of pathogen status are similarly needed. Furthermore, experienced and evidence-based treatment data is necessary to optimize outcomes for individuals with CF. © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Stenotrophomonas maltophilia; Achromobacter xylosoxidans; Methicillin resistant Staphylococcus aureus (MRSA); Mycobacterium abscessus; Mycobacterium avium complex; Microbiome

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . Identification of microbial agents in CF . . . . . Emerging bacterial pathogens in CF . . . . . . . 3.1. Methicillin resistant Staphylococcus aureus 3.2. Nontuberculous mycobacteria . . . . . . . 3.2.1. Achromobacter spp. . . . . . . . 3.2.2. Stenotrophomonas maltophilia . . 3.3. Other notable Gram-negatives . . . . . . . 3.4. Pathogens within the CF microbiota . . .

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⁎ Corresponding author at: Department of Medicine, The University of Calgary, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. Tel.: +1 403 220 5951; fax: +1 403 270 2772. E-mail addresses: [email protected] (M.D. Parkins), [email protected] (R.A. Floto).

http://dx.doi.org/10.1016/j.jcf.2015.03.012 1569-1993© 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

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4. Defining pathogens 5. Conclusions . . . Conflicts . . . . . . . . Acknowledgements . . References . . . . . . .

M.D. Parkins, R.A. Floto / Journal of Cystic Fibrosis 14 (2015) 293–304

in CF: looking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

to the future . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Cystic fibrosis exists as a changing disease. In the last four decades, median predicted survival has risen four-fold, with individuals born today expected to survive well into their fifth decade of life. In parallel to the changing epidemiology of patients, recent reports have highlighted the changes that are occurring within the spectrum of organisms causing infection in CF [1,2] (Fig. 1). While the driver of these changes is unknown, mechanisms postulated include: improved cultivation and identification, the selective pressure of antimicrobials, infection transmission and infection control practices, increasing prevalence of individuals with milder disease, and the survivor effect [1,3,4]. As respiratory disease continues to be the hallmark feature of CF and is primarily responsible for the attributable morbidity and mortality, understanding the spectrum and role of organisms involved in CF airways disease is of paramount importance.

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Herein, we describe both the epidemiology and pathogenesis of those bacterial pathogens that have been considered emerging pathogens in CF for some time and/or whose clinical impact is increasingly apparent as well as those organisms/constellation of organisms that have only been recently described. We also propose a framework that may enable a better understanding of microbial pathogenesis of those organisms whose role in CF lung disease remains undefined. 2. Identification of microbial agents in CF Historically organism identification in CF has been based on semi-selective cultivation designed to enrich selection for aerobic Gram-negative organisms and Staphylococci. Initially, many uncommon Gram-negatives were misidentified as Pseudomonas aeruginosa or Burkholderia cepacia complex (Bcc), or merely reported as unidentified Gram-negatives. With the routine incorporation of technologies such as broad range 16S rRNA

Fig. 1. Prevalence data of key emerging pathogens as a function of reporting country. These data demonstrate variably increasing prevalence of key emerging organisms over time, although, this is often country dependent. Inclusion and reporting criteria vary depending on registry (hence some data point not included), and generally represent at least one culture per calendar year and do not distinguish chronic infection from transient. Data adapted where available from 2003, 2008 and 2012 Australian Cystic Fibrosis Patient Registry, Canadian Cystic Fibrosis Patient Data Registry Report, UK Cystic Fibrosis Trust Annual Data Report and Cystic Fibrosis Foundation Patient Registry Annual Data Report and [122].

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PCR and sequencing, and matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI–TOF) [5–7], the ability to rapidly and correctly identify less common organisms now exist. The widespread adoption of these technologies will further drive changes in the epidemiology of existing, emerging and novel CF pathogens. 3. Emerging bacterial pathogens in CF 3.1. Methicillin resistant Staphylococcus aureus (MRSA) S. aureus is one of more than 40 species of Staphylococci, and is distinguishable as a coccoid Gram-positive, coagulasepositive, catalase-positive species that grows on mannitol salt agar. S. aureus is one of the most notable human pathogens causing a wide range of diseases. Since the development and dissemination of penicillin resistance, methicillin and more aptly other semi-synthetic anti-Staphylococcal penicillins (such cloxacillin and flucloxacillin) have become first-line therapy [8]. Methicillin resistance, first described in 1961, two years after the introduction of methicillin is mediated through alteration of PBP2A (a transpeptidase involved in cell wall maintenance), encoded for by the mecA gene. This results in reduced affinity of this protein for penicillins and other β-lactams, thereby raising the oxacillin minimum inhibitory concentration (MIC) N 4 μg/ml. MRSA has arisen through several independent, successful MSSA clones acquiring the mobile staphylococcal chromosomal cassette (SCCmec), the genomic island containing mecA, from coagulase-negative Staphylococci. To date, up to 11 SCCmec types have been identified, which vary in their structural organisation and content. Originally, some were more commonly associated with hospital or community acquisition: SCCmec I-III are common amongst hospital-associated strains of MRSA (HA-MRSA) which have persisted at relatively modest levels for decades, community-acquired MRSA (CA-MRSA) carrying the SCCmec IV (and to a lesser extent V) have exponentially increased in the general population. However, increasingly these designations are becoming redundant, as “HA-MRSA” has become established within the community, and “CA-MRSA” outbreaks described in healthcare settings. In the United States, MRSA now accounts for ~ 60% of S. aureus skin and softtissue infections presenting to the emergency department [9]. Parallel to this, an epidemic of MRSA has been noted in US CF centres with prevalence rates increasing to 30% [10]. Despite this, the MRSA observed in American CF patients remains largely SCCmec II: (USA 100 and 800 strains), and only 30% representing SCCmec IV (predominately USA 300 [ST8] and 400 [ST1] strains) [11,12]. There has been a significant increase in prevalence of MRSA amongst CF patients in the last two decades. Notably, this phenomenon appears to be exclusive to the American CF population as levels in Europe, Canada and Australia have remained relatively low and stable suggesting other social and health-delivery factors may be involved (Fig. 1). The source of MRSA infection is better understood than other emerging agents as it is a pathogen of the general population, and transmission knowledge directly applicable. Indeed, as a

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Gram-positive organism, MRSA is more resilient to desiccation, and can persist on inanimate objects and fomites making patient – intermediary – patient transmission possible, something not commonly observed with classical CF pathogens such as P. aeruginosa and Bcc [13]. Risk factors for acquisition of MRSA in CF don't substantially differ from those in the general population and include, increasing days in hospital and antibiotic exposure (in particular fluoroquinolone and cephalosporins) [14]. As MRSA is commonly isolated from asymptomatic people and companion animals, it is not surprising to find that it is found with a high frequency in family members of CF individuals residing together [15] as well as objects throughout the home [16]. Initial studies demonstrated those with MRSA infection were more likely to have advanced disease and need more antibiotics, but did not result in a disproportionate decline in lung function [17]. However, larger studies have since demonstrated the potential role of MRSA. In a ten-year study of ~ 17,500 individuals in the Cystic Fibrosis Foundation Patient Registry (CFFPR), a more rapid decline in lung function was noted in those with persistent MRSA infection relative to controls, particularly in those b 21 years [18]. Furthermore, MRSA infection has been identified as an independent risk factor for failure to recover lung function following pulmonary exacerbation (PEx) [19]. Compared to patients with chronic MSSA infection or transient MRSA infection, those with chronic MRSA have an increased risk of death [20]. Virulence of S. aureus is multi-factorial, and not substantially different in MRSA strains [21,22]. MRSA resistance to classes of antibiotics other than beta-lactams is similarly well documented in CF. In CF, SCCmecII MRSA (traditionally HA-MRSA) strains are more likely to be resistant to lincosamides, fluoroquinolones, macrolides and rifamycins than SCCmecIV strains (traditionally CA-MRSA) [11,23]. The largest multi-centre study of MRSA from CF reported resistance rates of 95% to erythromycin, 75% to clindamycin and the fluoroquinolones, 12% to rifampin, 7% to tetracyclines, 6% to gentamicin, and 4% to trimethoprim/sulfamethoxazole (SXT). While vancomycinresistant S. aureus has not been observed in CF, heteroresistance (the presence of subpopulations within a larger population of fully antimicrobial-susceptible organisms or less susceptible isolates) has been documented and is presumed commonplace [24,25]. Indeed CF derived MRSA have developed resistance to newer therapies including linezolid [11,26], ceftaroline [27] and tigecycline [11] presumably on the basis of frequent and prolonged exposures, and potentially inadequate dosing. Indeed, numerous studies have demonstrated that CF patients have an altered drug disposition (increased volume of distribution, greater total body clearance and variably increased hepatic clearance which manifest with lower drug levels and shorter half-lives) relative to the general population in which antibiotic dosing and licensing studies have been established. Accordingly, where available, dosing of antimicrobials in CF must follow data obtained from pharmacokinetic studies in this unique population [28]. Treatment strategies for MRSA in CF are being intensively pursued. Several groups have published data on eradication

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strategies. However, the natural history of infection with MRSA in CF remains poorly understood and it is not clear if chronic infections (estimated in 38–66% of cases) can be prevented with early eradication [18,20,29]. Indeed, a randomized controlled trial (RCT) comparing eradication to placebo in preventing long-term infection is underway (NCT01349192). Chronic MRSA treatment strategies are poorly described, and their impact unknown. Critically, a novel CF specific MRSA inhalational product using an airway-formulated vancomycin dry powder inhaler is currently under study (NCT01746095). Only expert opinion exists regarding MRSA treatments during PEx [30]. Indeed, vancomycin is increasingly being replaced by alternate agents owing to concerns about inferior pharmacokinetics in pulmonary infection, heteroresistant populations, reduced potency, and increased toxicity [31,32]. 3.2. Nontuberculous mycobacteria Mycobacteria are aerobic, non-motile Gram-positive rods notable for thick cell walls with high concentrations of mycolic acid resulting in poor absorption and high retention of dyes, conferring an acid-fast status. Nontuberculous mycobacteria (NTM), defined as mycobacterial species other than M. tuberculosis complex and Mycobacterium leprae, are ubiquitous environmental organisms that can cause chronic pulmonary infection associated with progressive inflammatory lung damage, termed NTM pulmonary disease (NTM-PD). NTM-PD is not unique to CF, although CF-related lung disease is a clear risk factor, presumably related to the presence of structural lung damage, impaired mucociliary clearance and inflamed airways [33]. Mutations in cftr may also have a specific role in predisposing to NTM infection as 30–50% of patients with non-CF NTM-PD are carriers of cftr mutations [34,35]. The diagnosis of NTM-PD is particularly difficult in CF. Unlike M. tuberculosis, a single positive culture for NTM does not necessarily infer disease, since NTM can transiently or permanently reside within the lungs without causing damage. It is therefore critical to accurately diagnose NTM-PD and thus identify individuals who might benefit from treatment. Consequently, the ATS/IDSA criteria have been widely adopted to help define NTM-PD for individuals with CF although not yet validated, and require the following: 1) ≥ 2 sputum samples (or a single bronchoscopic sample) to be NTM culture-positive; 2) radiological changes consistent with NTM infection; and 3) clinical features attributable to NTM [36]. Recent estimates of the prevalence of NTM-positive sputum cultures in the CF population have ranged from 6% [37] to 14% [38] with one study of CF patients ≥40 years of age reporting rates of N 30% [39]. Rates of detection of NTM from individuals with CF are increasing across the world, representing a real increase in prevalence rather than increased identification or more intensive sampling [40,41]. Potential reasons for the increased frequency of NTM in CF include: increased environmental exposure to NTM from household water, more intensive antibiotic usage creating NTM permissive lung niches [42], greater long-term administration of medications which might impair host immunity [43], and spread of NTM through person-to-person transmission [44,45].

The most common types of NTM infecting CF patients are the Mycobacterium avium complex (MAC), a group of slow growing species containing M. avium, Mycobacterium intracellulare and M. chimaera, and the M. abscessus complex (MABSC), comprising the subspecies M. abscessus subsp. abscessus (M. a. abscessus), Mycobacterium abscessus bolletii and Mycobacterium abscessus massiliense [46–48]. There are considerable geographical differences in both the prevalence of NTM and the frequency of different species detected in CF [37,42,49]. In North America, the most commonly isolated NTM are MAC (up to 72% of all NTM detected in sputum) followed by MABSC, while in Europe the reverse is true [49]. The different species appear to have different levels of virulence. Individuals culturing MABSC are more likely to meet ATS/IDSA criteria for diagnosing NTM-PD and have greater morbidity and mortality associated with a more rapid decline in lung function. Infection with NTM, particularly MABSC, can create considerable problems for individuals undergoing lung transplantation. Pre and/or post transplant infection with MABSC may result in increased morbidity (particularly soft tissue and surgical site infections) and, in some studies, greater post-operative mortality [50]. The management of NTM-PD in CF is challenging with limited evidence available to support clinical decision-making. As a consequence, the US CF Foundation and the European CF Society have generated joint consensus guidelines for NTM-PD due early 2015. Nevertheless advice from an NTM expert is invaluable in all but the most straightforward of cases. 3.2.1. Achromobacter spp. Achromobacter spp. are aerobic, catalase-positive, oxidasepositive, non-lactose fermenting, Gram-negative bacilli [51]. Achromobacter spp. are widely distributed in nature, but are rare opportunistic pathogens. Achromobacter spp., and in particular Achromobacter xylosoxidans and Achromobacter ruhlandii are commonly observed in CF populations [52]. Prevalence studies vary greatly with individual CF centres reporting rates ranging from 3 to 30% [53–55]. A. xylosoxidans poses diagnostic challenges and N 10% of accredited CF clinical microbiology labs incorrectly identified these isolates as Bcc or P. aeruginosa [56]. Furthermore, through new technologies the number of species within this genus is expected to increase. The development of a multi-locus sequence typing (MLST) system, and in particular the recognition that the nrdA allele correctly speciates these isolates is likely to have a dramatic impact on the epidemiology of Achromobacter spp. in CF [52]. Unfortunately, the virulence factors involved in the pathogenesis of these organisms in lung disease are poorly understood. Several single centres have demonstrated clusters of patients infected with A. xylosoxidans [55,57] isolates with the same PFGE pattern, suggesting the possibility of transmission, although this has not been universal [58]. The proportion of patients with incident infection acquiring chronic infection ranges from 11 to 30% [53,55,57]. Whether chronic infection can be prevented with eradication is unknown [59]. Moreover, risk factors for acquisition of A. xylosoxidans are not entirely clear. Indeed, older age, increasing burden of disease and chronic

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P. aeruginosa infection appear commonly in individuals who develop chronic infection [55,57]. The impact of A. xlyosoxidans on outcomes in CF is as yet unclear. Evidence is limited to a few small cohort studies from Europe where outcomes were examined over short intervals. De Baets et al. demonstrated that patients who were chronically infected with A. xlyosoxidans were more likely to have lower lung function and worse radiographs, but did not experience a disproportionate burden of exacerbations or rate of deterioration [55]. This finding suggests that patients with increased burden of structural lung disease are predisposed to infection. Other groups have demonstrated increased rates of lung function decline following infection in a subset of patients with high antibody levels against A. xylosoxidans [59] and increased antibacterial treatment requirements [55,57]. Those patients who had infection prior to transplant have also been observed to infect the graft with the same isolates [58]. Antibiotic resistance is also common in CF Achromobacter spp. isolates. Those antibiotics most likely to have in vitro predicted activity (using P. aeruginosa breakpoints) include: imipenem/ meropenem, piperacillin/tazobactam, and minocycline (greater potency is expected with tigecycline) [53,56,60]. Very few isolates are susceptible to SXT, aztreonam or 3rd generation cephalosporins. Isolates are commonly resistant to aminoglycosides and isolation of these organisms commonly occurs in individuals receiving inhaled aminoglycosides. Most isolates are susceptible to high concentrations of colistin as might be achieved with nebulization. Multi-drug resistant (MDR) bacteria are common, and single-centre studies report MDR rates of up to 20% [57]. There are no data on optimal treatment strategies in CF for either PEx, new colonization or chronic suppression. Guidelines suggest that antibiogram directed therapy should be offered where required although this is based solely on expert opinion [30]. 3.2.2. Stenotrophomonas maltophilia Stenotrophomonas spp. are Gram-negative rod shaped, obligate aerobic, non-fermenting, oxidase-negative (mostly) organisms found in aquatic environmental reservoirs notable for their intrinsic resistance to antibiotics. While four species exist, only S. maltophilia is a human pathogen. It is an important nosocomial pathogen, and particularly notable as an agent of nosocomial or ventilator associated pneumonia [61,62]. How S. maltophilia contributes to CF lung disease is not readily understood, and few studies have sought to characterize its virulence potential. Detailed reviews have recently been published and interested readers are referred for details [63,64]. Similar to P. aeruginosa, a hypermutator phenotype in chronically colonizing strains has been identified which may give rise to significant population phenotypic heterogeneity. Whether this translates into varying antibiograms as has been observed for P. aeruginosa is as yet unknown [65]. Rates of S. maltophilia in CF vary considerably from centre to centre (0–30%) based on single-centre reports [66]. Most incident S. maltophilia isolates appear to be transient colonizers and result in persistent infection in only 13–23% of cases [67–69]. Risk factors for S. maltophilia are often debated. Several studies suggest acquisition appears to be

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linked to anti-Pseudomonal treatments including parenteral therapies and fluoroquinolones [70,71]. However, Stanojevic et al. found non-fluoroquinolone antibiotics to be protective [69]. Patients who acquire S. maltophilia have more advanced disease [68,72] and may experience an exaggerated lung function decline [69]. While debated, increasing evidence supports a pathogenic role for S. maltophilia in CF. Of N 20,000 patients followed in the CFFPR, no disproportionate decline in lung function was observed with S. maltophilia infection [68] nor any survival impact [72]. However, recent work from Toronto represents the most comprehensive assessment to date. Infection with S. maltophilia was an independent predictor of exacerbation [73]. However, patients with S. maltophilia infection were no less likely to recover baseline lung-function following an event [74]. Those patients with chronic infection had higher levels of anti-S. maltophilia flagellin antibodies, and these inversely correlated with lung function. Furthermore, chronic infection was associated with 3 × increased risk of death/transplantation [75]. Antimicrobial resistance is a characteristic of S. maltophilia. Outer membrane permeability is limited reducing potency of most antibacterial classes, in particular aminoglycosides [63,64]. A plethora of multi-drug efflux pumps, and aminoglycoside modifying enzymes similarly exist. Resistance to beta-lactams is almost universal and mediated through duplicate carbapenemases, although ticarcillin-clavulinic acid retains some potency. Unfortunately, S. maltophilia from CF display even greater reductions in antibacterial susceptibilities compared to non-CF isolates [64,76]. In vitro data of CF derived isolates suggest that the following antibiotics have the highest rates of in vitro predicted activity: tigecycline (99%), SXT (16–100%), doxycycline (80%), levofloxacin (42–90%), and ticarcillin-clavulinic acid (27–64%) [66,77,78]. Treatment data for S. maltophilia are particularly lacking in CF [79]. Eradication strategies have not been pursued, and limited data suggests that during PEx in patients with chronic infection (where typically only one anti-S. maltophilia agent was used) 25% of patients achieved organism clearance. Combination therapy for S. maltophilia is thus of great interest. For acute PEx, SXT plus an additional agent are recommended by expert panels but supporting data is lacking [30]. No agent has yet been studied in the management of chronic lung infections in CF, although inhaled colistin and inhaled levofloxacin (under development) products achieve levels within the CF airways which may overcome the MIC of many CF derived isolates. 3.3. Other notable Gram-negatives A range of other Gram-negatives, notable for their relative obscurity outside of CF have been identified in the last twenty-years. Case reports, or series of ≤ 10 patients comprise the bulk of data for organisms belonging to the genera Cupriavidus, Inquilinus, Pandorea and Ralstonia. These organisms are notable for drug/MDR resistance and are easily misidentified as P. aeruginosa or Bcc. For details on these rare pathogens, the reader is referred to the following excellent review [3]. Furthermore, it may be that a reporting bias exists in

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the CF literature against presumed common human pathogens. Escherichia coli, a single member of the Enterobacteriaceae, has been identified in 25% of patients yet the CF literature has a paucity of information and no outcome data [80].

3.4. Pathogens within the CF microbiota The traditional means by which microbes are identified in CF rely on cultivation using differential culture, designed specifically to increase the yield of classical pathogens. Intrinsic to these protocols is the selection against organisms that exist within the oropharynx. Increasingly researchers have questioned the basis of this fundamental assumption. With the revolution of low-cost sequencing, our ability to understand complex polymicrobial populations through studying 16s rRNA diversity in mixed populations has exploded. The term ‘CF microbiota’ is intended to represent all organisms present in the lower airways. This consists of a wide range of anaerobic and aerobic organisms, with studies identifying 20–1000 different bacterial taxa depending on the method of assessment and sampling depth [81]. Indeed the vast majority of these organisms are biologically active [82]. While classical CF pathogens such as P. aeruginosa and Bcc dominate in some patients, others have tremendous diversity. The most commonly identified non-classical genera include; Streptococcus, Prevotella, Veillonella, Rothia, Actinomyces, Gamella, Graulicatella and Fusobacterium [83–85]. The relative abundance and prevalence of these organisms vary considerably within different areas of the lung, and between individual patients and datasets. While contamination issues are inherent for respiratory samples taken through the mouth, those communities colonizing the lungs are more than just sputum contaminated by oral flora [86]. Concordance has been demonstrated between expectorated sputum, induced sputum and protected brochoalveolar lavage [87–90] and there is also a high degree of similarity between serially produced sputum samples [89]. Furthermore, sputum community profiles are significantly different from those of the oropharynx in CF [86]. Cross-sectional studies have been performed to understand the make-up and diversity of the microbiota in different populations. There is universal consensus that patients with less advanced disease have a greater diversity and those with chronic P. aeruginosa and/or Bcc have less diversity [81,87,91,92]. Indeed some studies have reported that specific communities may be associated with certain genotypes [81,87] and that females have lower diversity [93]. To understand both the stability and how the microbiota changes in time, few longitudinal studies have been conducted. These studies have generally identified stability within each patient but considerable differences between patients. The exception is during antibacterial pressure exerted during PEx, where a temporary disruption is induced only to later normalize [94]. In those individuals followed for longer periods of time diversity disproportionally decreases in those with advancing disease and is strongly associated with antibiotic usage [93,95].

The microbiota of PEx has been extensively studied [91,92,96–99] as these events are tremendously important, resulting in reduced quality of life, cost, permanent loss of lung function and increased risk of mortality [19,100–104]. Moreover, their frequency, short-duration and the ability to assess serial samples, makes understanding population dynamics possible. In general, there is consensus that the CF microbiota is relatively resilient, changing little before and during PEx [91,92,97]. However, the emergence of members of the Streptococcus anginosus group also known as the Streptococcus milleri group, (SMG), as a numerically dominant pathogen has been observed preceding PEx in a subset of patients; treatments targeting SMG were effective at resolving PEx and reducing future events [98,99,105]. If, and how, the constituents of the microbiome impact CF is unknown. Some of these may have direct virulence potential [106,107]. Other seemingly benign commensals may interact with established pathogens and modulate virulence indirectly [108,109]. Indeed, data suggests that even low abundance community members can have dramatic effects on the community structure and behaviour [110]. It may be that the CF microbiota influences antibacterial treatment response. Relative to isolates from individuals without CF, several members of the OF including the Viridans Streptococci (SVG) and in particular SMG, and Prevotella spp. have markedly increased rates of antibiotic resistance to multiple classes of drugs [111–113]. Most notably, antibiotic resistance correlates with prior drug exposure demonstrating that both the effects and consequences of antibiotic treatment extend into other constituents of the CF microbiome [112]. Of particular note, reduced susceptibility to macrolides is commonly seen in SMG, SVG and Prevotella isolates from patients receiving chronic azithromycin therapy [111,113]. Given that one postulated mechanism by which azithromycin exerts its clinical benefit is through its effect on components of the oropharyngeal flora (OF), the development of resistance may serve to reduce efficacy [114]. Indeed, in a study assessing long-term efficacy, the clinical benefit of azithromycin was not observed to extend beyond the first year which could be consistent with an antimicrobial effect mechanism (and subsequent resistance) as opposed to the more commonly proposed anti-inflammatory mechanism [115]. Furthermore, through the production and excretion of antibiotic modifying enzymes, members of the OF may degrade or deactivate antibiotics used to target traditional pathogens such as P. aeruginosa thereby extending protection. Beta-lactamases including TEM type extended-spectrum beta-lactamases (ESBLs) capable of hydrolyzing several anti-Pseudomonal beta-lactams including ceftazidime have been identified in Prevotella isolates from CF [111,116]. Indeed, precedent exists for OF flora producing beta-lactamases reducing efficacy of beta-lactam treatments in multiple infectious syndromes [117]. Therefore, researchers are now considering the concept of the resistome — the sum of all the antibiotic resistance genes and their determinants in pathogenic and non-pathogenic bacteria within the airways [118]. It may be that the CF resistome influences treatment outcomes, and in part explains why antibiogram directed therapies against

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Table 1 Factors affecting chronic lung infections involving Emerging CF pathogens benchmarked against Pseudomonas aeruginosa the archetypical CF pathogen [11,12,55,57,123–131]. Factor

Epidemic strains

Chronically infecting organisms

Existence of Patient-patient clonal strains spread, postulated in CF patients mechanism

Worse outcomes Stability of with clonal chronically strains infecting strains

Pseudomonas aeruginosa

Documented

Documented, Droplet spread, aerosol spread

Marked heterogeneity Commonplace Chronically infecting but not universal strains persist a, including in phenotypes following transplantation including antibiograms

Stenotrophomonas maltophilia Achromobacter xylosoxidans Methicillin resistant Staphylococcus aureus M. avium complex M. abscessus complex CF Microbiota

Documented

Unknown b

Unknown

Variable

Unknown

Conversion to mucoidy associated with accelerated clinical decline Unknown

Documented

Unknown b

Unknown

Demonstrated

Unknown

Unknown

Documented

Suspected, direct contact, Unknown +/− droplet spread, also indirect via Fomites, HCW

Unknown

Unknown Documented

Unknown Unknown

Unknown Unknown

Limited supporting data c Methicillin resistance phenotype increases S. aureus clinical impact* Unknown Unknown Unknown Unknown

N/A

Demonstrated

Unknown

Unknown, not suspected Documented, droplet/airborne No supporting N/A evidence d

Heterogeneity of chronically infecting strains

Phenotype influences clinical outcomes

N/A d

Definitions: HCW = health care workers, N/A not applicable. a Except in situations of super-infection with epidemic strains. b While clonal complexes have been documented in small patient series, common source acquisition or dominant environmental clones have not been excluded. c Documented in methicillin sensitive S. aureus. d Few groups have attempted to cultivate those other members of the CF microbiota to explore this.

traditional pathogens such as P. aeruginosa correlate poorly with clinical response [119]. To explore this concept an international multi-centre randomized controlled trial comparing antimicrobial treatment regimens targeting known constituents of the CF microbiota through PEx versus standard of care is currently underway [4].

Whether components of the CF microbiota may in fact be protective are unknown. It may be that specific resident flora in the CF lung serve as a means against colonization by exogenous microbes either through competition for local nutrients and/or the production of bacteriocins. This protective function, known as the barrier

Fig. 2. A framework for investigating novel CF pathogens. Importantly, an identified agent may have some but not all of these factors, and may have a role some, but not all of the time.

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effect or colonization resistance, may be disrupted through the inappropriate or excessive administration of antibiotics [120]. Furthermore, certain microbiota constituents may interact with the host immune system in a similar fashion to those organisms in the gastrointestinal tract to modify host inflammatory response in the lung. 4. Defining pathogens in CF: looking to the future With the emergence of technologies allowing for the exquisite characterization of the nature and spectrum of organism within the lower airways of CF and our ability to better understand outcomes associated with lung disease, the need to reassess some of the fundamental assumptions of CF airway pathogenesis exists. Most importantly, we must be able to define what constitutes a CF pathogen. A CF pathogen simply put is an organism capable of causing lung damage in CF. However, multiple models and caveats exist even in a normal host without chronic airway inflammation to describe pathogens based on variables including organism, host and host immune-response, situation, and putative mechanism [121]. In the past the isolation of an organism in CF that was not a component of the OF was interpreted as being a pathogen, particularly if this organism was uncommon in other diseases and persisted for prolonged periods. However, this is not always the case as reports of patients with positive lower airway cultures without clinical deterioration abound suggesting colonization is possible and perhaps even common. Similarly, members of the long presumed benign normal OF have evidence supporting a role in disease pathogenesis [98,108]. Furthermore, we must acknowledge that organisms may oscillate between roles as commensals, synergins and pathogens whereby their potential for inciting harm may be influenced by factors such as inter and intra bacterial species signalling, exogenous viruses, environmental triggers and medical therapies. Strategies to better delineate these distinctions are required. Detailed assessment of these organisms must also be done within the context of what has been learned/ understood for the principle CF pathogen: P. aeruginosa through four decades of study (Table 1). Finally, the nature of the host must be considered. Host related factors including genetic background, functional immune defects, and structural lung abnormalities may render a benign commensal a pathogen in some individuals. To understand pathogenicity in the context of these potential confounders we must be able to adequately power future studies — especially for low prevalence organisms. This means complementing those initial reports from single-centres that have often used a variety of definitions of infection with large scale, longitudinal studies. Collaborative efforts using uniform diagnostic and treatment criteria are required. Similarly, a more inclusive reporting structure is required of national registries. In a time where P. aeruginosa and S. aureus were the principle pathogens, and chronic infection the presumed natural consequence of exposure, once yearly prevalence reporting of pathogens provided a reasonable assessment of airway microbiology. However, in the new age of CF, where the natural history of infection with many of these organisms is one that may not culminate in

chronic infection, and early eradication regimens with high success exist to prevent chronic infection, this is no longer the case. Each identified organism from each encounter should be prospectively reported in order to get an accurate reflection of the epidemiology of lower airway infection. Finally, the focus of these studies must move beyond merely reporting the epidemiology of infection and in vitro predicted antimicrobial resistance of these agents to studies assessing the natural history of infection, and the affect of acute and chronic interventions on patient and bacteriological response (Fig. 2). 5. Conclusions CF lower airway infection exists in a dynamic state. Owing to changes in patient survival, the impact of strategies to preserve lung function and prevent deterioration/new infections, tremendous selective pressures will continue to affect microorganisms recovered from the lower airways. Indeed with the introduction of new and novel antibacterial treatment strategies, combined with enhanced ability to recognize organisms, a new generation of emerging pathogens can be expected. Some of these may even include previously considered benign oropharynx colonizers. However, it is clear that a coordinated approach to the identification and analysis of the pathogenic potential of these organisms is required. Conflicts MDP has accepted research grants from Cystic Fibrosis Canada and Gilead Sciences. He has served on advisory boards for Gilead, Novartis, Roche but does not accept honoraria. RAF has served on advisory boards for and accepted honoraria/research funding from Vertex, Gilead and Forest. Acknowledgements No funding was required for this work. References [1] Millar FA, Simmonds NJ, Hodson ME. Trends in pathogens colonising the respiratory tract of adult patients with cystic fibrosis, 1985–2005. J Cyst Fibros Dec 2009;8(6):386–91. [2] Emerson J, McNamara S, Buccat AM, Worrell K, Burns JL. Changes in cystic fibrosis sputum microbiology in the United States between 1995 and 2008. Pediatr Pulmonol Apr 2010;45(4):363–70. [3] Lipuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin Microbiol Rev Apr 2010;23(2):299–323. [4] Ingram R. CF matters. [cited 2014 Sept, 14]; Available from: www. cfmatters.ue; 2014. [5] Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res Jan 2009;37:D141–5 [Database issue]. [6] Fernandez-Olmos A, Garcia-Castillo M, Morosini MI, Lamas A, Maiz L, Canton R. MALDI-TOF MS improves routine identification of nonfermenting Gram negative isolates from cystic fibrosis patients. J Cyst Fibros Jan 2012;11(1):59–62. [7] Marko DC, Saffert RT, Cunningham SA, Hyman J, Walsh J, Arbefeville S, et al. Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for

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