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Emerging cystic fibrosis pathogens and the microbiome

Eshwar Mahenthiralingam *

Organisms and Environment Division, Cardiff School of Biosciences, Cardiff University, Cardiff, UK

Microbial lung infection is the major cause of morbidity andmortality in cystic fibrosis [1], and routine therapy relies on theearly detection, eradication or continuous treatment of respiratoryinfections [2]. Pseudomonas aeruginosa is the dominant pathogeninfecting more than 70% of people with CF [1,2], however, ourunderstanding of other bacterial infections associated with chroniclung disease has changed considerably in the last 20 years [2]. In

(i) Multidrug resistant non-fermenting Gram negative (MDR-NFGN) bacteria. Pathogens such as Burkholderia cepacia

complex (Bcc), Burkholderia gladioli, Achromobacter species,Ralstonia mannitolilytica, Stenotrophomonas maltophilia, andPandorea species are problematic emerging pathogens, cumu-latively infecting approximately 25% of people with CF andbeing especially resistant to therapy in older CF people with

A R T I C L E I N F O

Keywords:

Cystic fibrosis

Emerging pathogen

Burkholderia

Stenotrophomonas

Achromobacter

Pandorea

Ralstonia

Prevotella

Streptococcus milleri group

CF microbiome

S U M M A R Y

Cystic fibrosis (CF) respiratory infection is characterised by the presence of typical human bacterial

pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae and Staphylococcus aureus. Less

typical pathogens such as Burkholderia, Stenotrophomonas, Achromobacter, Pandorea and Ralstonia have

emerged as problematic infections which are largely unique to people with CF. Using molecular methods,

two groups of anaerobic bacteria Prevotella species and the Streptococcus milleri group have also recently

been shown to be highly prevalent in CF sputum. Collectively, the diversity of microorganisms present in

respiratory specimens has been designated the CF microbiome. The challenges posed by emerging CF

pathogens and a microbiome-based view of CF infection are discussed in terms of their impact on clinical

outcome, diagnosis and therapy.

� 2014 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Paediatric Respiratory Reviews

addition to P. aeruginosa, well known human pathogens such asHaemophilus influenzae and Staphylococcus aureus also cause CFinfection [2], and one could argue that because of the extensivestudy of these three bacterial species in relation to multiple humaninfections, we have a good understanding of their pathogenesisand multiple therapeutic options for their treatment. In contrast,emerging antibiotic resistant CF pathogens pose major clinicalchallenges that are largely unique to people with CF and havetypically been less studied than P. aeruginosa. In the context of thisreview an emerging pathogen will be defined as any microbialinfection that poses a challenge in terms of clinical diagnosis,therapy or outcome in CF. Non-tuberculous mycobacteria such asMycobacterium abscessus, fungal and viral infections, all posemultiple problems for people with CF [2] and hence fit thisdefinition, however, because of the unique complexity of issueseach of the latter pose, they will not be explored in detail here. Twogroups of bacteria, both multidrug resistant, will be discussed asemerging CF pathogens because they share a number of traits:

* Organisms and Environment Division, Cardiff School of Biosciences, Cardiff

University, Room 0.23 Main Building, Park Place, Cardiff, UK, CF10 3AT.

Tel.: +44 (0)29 208 75875; fax: +44 (0)29 208 74305.

E-mail address: MahenthiralingamE@cardiff.ac.uk.

Please cite this article in press as: Mahenthiralingam E. Emerging cys(2014), http://dx.doi.org/10.1016/j.prrv.2014.04.006

http://dx.doi.org/10.1016/j.prrv.2014.04.006

1526-0542/� 2014 Elsevier Ltd. All rights reserved.

severe lung disease [3–5].(ii) Novel CF microbiome players. Analyses of CF sputa, using

enhanced culture and molecular cultivation-independenttechniques, have uncovered an unprecedented diversity ofmicroorganisms, collectively known as the CF airway micro-biome [6]. Two dominant CF microbiome players that aremissed by routine culture are the Streptococcus milleri group(SMG; Gram positive, facultatively anaerobic bacteria) [7] andPrevotella species (Gram negative, obligate anaerobes) [8].These bacteria persist in most CF people receiving antibiotictherapy for chronic infection [9], but we know very little abouttheir association with lung disease and whether or how theyshould be treated.

These problematic NFGN bacteria and novel CF microbiomeplayers, Prevotella species and SMG bacteria, are emergingpathogens which constitute a health threat to people with CFbecause of the following issues.

ANTIBIOTIC RESISTANCE

NFGN bacteria comprise a pool of highly multidrug resistant CFpathogens [2]. With few effective antibiotics available, there is an

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urgent need to develop novel therapies to improve currenttreatments for these emerging CF pathogens and Gram-negativebacteria in general [10]. Despite extensive efforts to improvetherapy by performing multiple combination bactericidal antibi-otic susceptibility testing (MCBT) to aid antibiotic selection for B.

cepacia complex, P. aeruginosa, S. maltophilia, or Achromobacter

species, Aaron and colleagues [11] were not able to improve onclinical outcome in comparison to patients managed usingstandard culture and testing approaches. Little progression onhow to treat CF patients with NGFN infection has been made sincethese studies. Both Prevotella and SMG bacteria are not eradicatedby routine therapy and also demonstrate resistance to antibioticscommonly used to treat CF pulmonary infection. They are presentin the airways at levels equivalent to that of established pathogenslike P. aeruginosa [8,9] and can be considered as chronic infectionsin many patients, but are not specifically targeted for therapy.

CHALLENGING CONVENTIONAL MICROBIOLOGY,IDENTIFICATION AND TAXONOMY

All the NFGN species are often mis-identified by routine culture[2] or non-specifically reported as a ‘‘Gram Negative Rod?’’ prior tofull species identification which may take additional time toresolve. Even more concerning, Prevotella and SMG group bacteriaare not captured by routine diagnostic microbiology and to betterunderstand their role in lung disease, accurate, quantitativediagnostics are urgently required [9,12]. While the taxonomy ofBcc has become much better understood [2], all the remainingemerging CF species show genetic diversity and the presence ofnovel species groups; the pathogenic significance of this diversityhas not been fully established. For example, multiple novel speciesgroups have been recently defined within the genus Achromobacter

using a genomics-based multilocus sequence typing (MLST)scheme and polyphasic taxonomy [13,14]. The vast majority ofCF isolates were historically reported as just one species ‘‘A.

xylosoxidans’’ [15]. However, recent systematic analysis ofAchromobacter species demonstrated a very different epidemio-logical picture with prevalence rates of A. xylosoxidans 42%, A.

ruhlandii 23% and A. dolens 17% occurring in CF [15].The taxonomy of the anaerobic species found in CF respiratory

infection is also not fully defined. The SMG group comprise threespecies, S. constellatus, S. intermedius and S. aginosus, and all havebeen found in patients with CF [7]. The taxonomy of these SMGspecies was last reviewed in 1997 and it is highly likely furthertaxonomic diversity may be present in this historically definedgroup of streptococci [16]. Multiple Prevotella species have beencultivated from CF respiratory infections with P. melaninogenica, P.

histicola and an unnamed species, Prevotella oral taxon 313, beingmost commonly cultivated in a study of 12 patients [17]. Overall,both Streptococcus and Prevotella have high species diversity asbacteria genera and further systematic analysis will be required tocharacterise the species most commonly associated with CFrespiratory infection.

PATHOGENIC POTENTIAL

Bcc bacteria are established CF pathogens, with B. cenocepacia

and B. multivorans, being the dominant CF species associated withtransmission, clinical decline, loss of lung function and multiplecases of ‘‘cepacia syndrome’’ [2,18,19]. Cases of poor prognosisassociated with B. gladioli CF infection have also been reported[20,21]. Furthermore, variation in outcome post-lung transplanta-tion ranging from poor [21,22] to acceptable [23], has beenreported in B. gladioli infected CF patients. Severe lung disease [24]and bacteraemia [25] have also been observed for Pandorea speciesinfection in CF. The pathogenic potential of the remaining

Please cite this article in press as: Mahenthiralingam E. Emerging cys(2014), http://dx.doi.org/10.1016/j.prrv.2014.04.006

emerging species, Achromobacter, Stenotrophomonas, Ralstonia,

Prevotella and SMG, remains uncertain. A rise in the number ofSMG group bacteria in CF sputum has been linked with the onset ofacute pulmonary exacerbation [7]; however, there has been littlefurther follow-up of the Streptococcus species even though it is veryclear that they are frequently dominant and chronic microbiomeplayers in CF [8,9,12]. What is also clear from the literature outsideof CF is that instances of highly inflammatory and fatal infectionhave been reported for all these emerging CF pathogens.

TRANSMISSIBILITY AND EPIDEMIOLOGY

The devastating infection control risk associated with Bccbacteria is well known [2,26]. Elucidation of Bcc epidemiology[2,27] and improved molecular species identification methods[28,29] played a major role in designing international infectioncontrol measures for this species [30]. These interventions havesuccessfully halted the large scale epidemic spread of Bcc bacteriain CF, particularly for B. cenocepacia [2,31]. However, theprevalence of certain species such as B. multivorans and thenon-Bcc species, B. gladioli, appear to be rising, with the overallincidence of Bcc infection (3 to 4%) remaining stable in CF [2].Although specific markers of transmission [32] and virulence[33,34] have been defined for the B. cenocepacia ET12 strain, nosuch markers have been identified for B. multivorans, other Bccspecies, B. gladioli, Pandorea apista and A. xylosoxidans, despitereports of patient to patient spread [24,35–37]. Finally, antibioticresistance, a known transmission risk factor for multiple patho-gens, is a trait which links all emerging CF pathogens, flaggingthem as potential infection control risks in nosocomial settings.

CONTRA-INDICATION FOR LUNG TRANSPLANT AND EXCLUSIONFROM CLINICAL TRIALS

Lung transplantation remains the only therapeutic option for CFindividuals with end-stage respiratory disease. B. cepacia complexbacteria, B. gladioli and Pandorea species bacteria are associatedwith poor outcome post-transplantation [21,22]. Access to thistherapy is specifically restricted for B. cenocepacia infectedindividuals. Additional issues associated with CF patients culturepositive for emerging CF pathogens includes the antibioticresistance of these infections which hinders clinical managementpre- and post-transplant and the frequent exclusion of infectedpatients from clinical trials.

ASSOCIATION WITH LOSS OF MICROBIAL DIVERSITY ANDSEVERE LUNG DISEASE

Recent sputum microbiome analysis of individuals with severelung disease [3,5], and sampling of explanted lungs from adultswith end-stage disease [4], has demonstrated that the microbialdiversity of these CF infections is considerably reduced. Theairways of these severely ill people with CF were dominated by asingle pathogen such as P. aeruginosa or frequently one of the MDR-NGFN emerging CF species, Burkholderia, Achromobacter orStenotrophomonas [3–5]. Furthermore, various studies havereported a positive correlation between microbial diversity andlung function [5,38,39]. Overall, these data suggest that consider-able interactions occur between members of the CF microbiome,administered anti-bacterial therapies, and the host immunesystem, which lead to either the reduced diversity during severelung disease or stable, more diverse respiratory infections inhealthy CF individuals.

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CONCLUSIONS AND PERSPECTIVE

Burkholderia, Stenotrophomonas, Achromobacter, Pandorea,Ralstonia, Streptococcus and Prevotella species can all be consideredas emerging bacterial pathogens in the context of CF respiratoryinfection. Their multi-antibiotic resistance and recalcitrance toclearance by routine therapy are shared traits which make them aclinical challenge. The linkage of MDR-NGFN species to the loss ofmicrobial diversity seen in the respiratory microbiota of individu-als with severe lung disease is a recent finding that raises manyquestions about how to optimally treat chronic lung infection.Given the cumulatively high prevalence of NFGN species and thealmost uniform presence of Prevotella and SMG bacteria inindividuals with chronic lung infection, all these emerging CFpathogens are worthy of further systematic study in the context ofCF respiratory infection and the wider role of the lung microbiome.

CONFLICT OF INTEREST

The author has no conflict of interest to report.

Acknowledgements

EM thanks Alan Brown, Thomas Connor, Aras Kadioglu, DervlaKenna, Julian Parkhill, Michael Tunney, Miguel Valvano and CraigWinstanley for their helpful comments on this text. EM acknowledgesthe European Cooperation in Science and Technology (COST) actionBM1003 for promoting networking on microbial CF pathogens.

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