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Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland
JCF-01203; No of Pages 7
Journal of Cystic Fibrosis xx (2015) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland C. O'Driscoll a , J. Konjek b , B. Heym b,c , M.M. Fitzgibbon d , B.J. Plant e , M. Ní Chróinín f , D. Mullane f , M. Lynch-Healy a , G.D. Corcoran a , K. Schaffer g , T.R. Rogers d , M.B. Prentice a,h,i,⁎ a Department of Microbiology, Cork University Hospital, Cork, Ireland EA 3647, Université de Versailles Saint-Quentin-en-Yvelines, 2 avenue de la Source de la Biévre, 78180 Montigny-le-Bretonneux, France APHP Hôpitaux Universitaires Paris Ile-de-France Ouest, Service de Microbiologie, Hôpital Ambroise Paré, 9 avenue Charles de Gaulle, 92100 Boulogne-Billancourt, France d Irish Mycobacteria Reference Laboratory, St. James Hospital, Dublin, Ireland e Cork Adult Cystic Fibrosis Centre, University College Cork, Cork University Hospital, Wilton, Cork, Ireland f Department of Paediatrics, Cork University Hospital, Wilton, Cork, Ireland g Department of Microbiology, St. Vincent's University Hospital, Dublin, Ireland h Department of Pathology, University College Cork, Cork, Ireland i Department of Microbiology, University College Cork, Cork, Ireland b
c
Received 22 January 2015; revised 17 May 2015; accepted 17 May 2015
Abstract Background: The Mycobacterium abscessus complex are the rapidly growing mycobacteria (RGM) most commonly causing lung disease, especially in cystic fibrosis (CF) patients. Ireland has the world's highest CF incidence. The molecular epidemiology of M. abscessus complex in Ireland is unreported. Methods: We performed rpoB gene sequencing and multi-locus sequence typing (MLST) on M. abscessus complex strains isolated from thirty-six patients in 2006–2012 (eighteen known CF patients). Results: Twenty-eight strains (78%) were M. abscessus subsp. abscessus, eight M. abscessus subsp. massiliense, none were M. abscessus subsp. bolletii. Sequence type 1 (ST1) and ST26 (M. abscessus subsp. abscessus) were commonest. Seven M. abscessus subsp. abscessus STs (25%) were novel (two with novel alleles). Seven M. abscessus subsp. massiliense STs were previously reported (88%), including two ST23, the globally successful clone. In 2012, of 552 CF patients screened, eleven were infected with M. abscessus complex strains (2%). Conclusions: The most prevalent M. abscessus subsp. abscessus and M. abscessus subsp. massiliense strains in Ireland belong to widelydistributed STs, but there is evidence of high M. abscessus subsp. abscessus diversity. © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Mycobacterium abscessus; Epidemiology; Cystic fibrosis; Ireland; MLST
1. Introduction Clinically significant mycobacterial isolates can be divided into the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. The non-tuberculous mycobacteria are classifiable
⁎ Corresponding author at: Department of Microbiology, University College Cork, Cork, Ireland. Tel.: + 353 21 490 1420. E-mail address: [email protected] (M.B. Prentice).
as slowly growing or rapidly growing [1]. Members of the M. abscessus complex (or M. abscessus sensu lato) are the most frequent rapidly growing mycobacteria (RGM) causing lung disease in humans [2,3], and are also associated with postsurgical [4] or traumatic infections outside the respiratory tract and occasionally disseminated disease in immunocompromised patients. The taxonomy of M. abscessus complex strains has been in flux in recent years. A division into three species M. abscessus, M. massiliense [5] and M. bolletii [6] was called into question by a lack of inter-species differentiation by phenotype, DNA–DNA
http://dx.doi.org/10.1016/j.jcf.2015.05.007 1569-1993/© 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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hybridisation and individual sequences of rpoB and hsp65 genes when larger numbers of strains were evaluated [4,7]. However, a clear phylogenetic signal supporting these three groups has now been obtained by extensive Multi Locus Sequence Typing (MLST) [8] and multiple genome sequence comparisons [9,10] of widely geographically distributed strains. It is likely that three subspecies will be re-proposed based on this evidence. For the purposes of this manuscript we use the terms M. abscessus subsp. abscessus, M. abscessus subsp. massiliense and M. abscessus subsp. bolletii to describe these three taxa individually following usage in the current clinical literature on cystic fibrosis patients [9,11]. Respiratory infections with M. abscessus complex are especially associated with cystic fibrosis [12]. The largest prospective survey in western Europe (in France) cultured M. abscessus complex from 3.2% of cystic fibrosis patients [13], with a peak incidence between 11 and 15 years of age (5.8%). Case reports of fatal disseminated infection with M. abscessus complex in immunosuppressed cystic fibrosis (CF) patients following lung transplant [14,15] means carriage is viewed as a contraindication to lung transplant in some centres, but not others where longitudinal surveys have revealed no excess mortality with M. abscessus complex carriage [16]. A variety of different strains of M. abscessus complex is generally revealed on molecular typing of clinical isolates, but large outbreaks of surgical site infections with a genetically uniform (on PFGE and single gene sequencing) glutaraldehyde resistant strain of M. abscessus subsp. massiliense have occurred in Brazil associated with laparoscopy and arthroscopy [17]. Genome sequencing allowing very fine inter-isolate comparison by single nucleotide polymorphisms (SNPs) has enabled two recent reports of horizontal transmission of M. abscessus subsp. massiliense between cystic fibrosis patients within hospitals in Seattle, USA [18] and Papworth, UK [9]. The outbreak strains involved at the two centres were also very closely related on genome sequencing SNP phylogeny [9,19], despite the lack of documented inter-continental contact. Genome sequencing of the Brazilian surgical infection clone [20] showed that it was also phylogenetically very similar to the two cystic fibrosis outbreak strains [9,19]. The molecular epidemiology of M. abscessus complex strains from Ireland has not been reported. Ireland has the highest incidence of cystic fibrosis in the European Union [21] and therefore the population contains a relatively large number of individuals susceptible to M. abscessus complex infection. As the country is an island, individual strain prevalence may differ from other countries. We describe rpoB fragment typing and MLST for isolates from 36 patients from Ireland, including eighteen cystic fibrosis patients attending two different regional centres. Six patients were from one centre serving adult and paediatric patients (Cork University Hospital, Cork, CUH) and twelve from an adult centre (St Vincent's University Hospital, Dublin, SVUH).
and paediatric cystic fibrosis centre (Cork University Hospital, CUH). Thirty were anonymous isolates sent to the Irish National Mycobacterial Reference Laboratory for speciation. Twelve of these were respiratory isolates from adult cystic fibrosis patients at St Vincent's University Hospital, Dublin (SVUH), three were respiratory isolates from non-cystic fibrosis patients. Clinical details were not available for the remaining fifteen isolates. Culture and initial identification: M. abscessus complex strains from CF patients at CUH were identified by rapid growth in BacT/ ALERT 3D broth (bioMérieux) and subsequent GenoType Mycobacterium assay (Hain Lifescience) targeting the 23S rRNA gene. Twice yearly mycobacterial culture screens have been carried out at CUH on CF patients since 2008, before 2008 mycobacterial cultures were taken on clinical suspicion. At SVUH cultures were obtained by extended incubation (7–10 days) on Burkholderia cepacea selective agar [22], and B. cepacea screening was carried out 4–5 times per annum. DNA extractions were carried out on fresh cultures using Tris–EDTA, lysozyme, and proteinase as described [23]. Aspergillus culture positivity was defined as more than one isolation of Aspergillus sp. within a month before or after M. abscessus complex isolation Molecular typing: a 940-bp fragment of the rpoB gene was amplified by PCR using AmpliTaq gold polymerase (Applied Biosystems) with primers MYCOF1 and MYCOR2 [23] and trimmed to 752 bp [24]. 4–600 bp fragments from argH, cya glpK, gnd, murC, pta, and purH were amplified in 25 μl of ReddyMix PCR master mix (Thermo Fisher Inc.) with 1 μl of each primer (10 pmol) as described [8]. Minimum spanning tree created with PhyloWeb at Institut Pasteur M. abscessus MLST Database using the minimum spanning tree (MST) algorithm [25] on 118 sequence types (ST) including 22 ST from this study http://www.pasteur.fr/recherche/genopole/PF8/ mlst/Myco-abscessus.html. The neighbour-joining algorithm was used as implemented in MEGA5 [26] as described [8] using concatenated DNA sequences of each MLST type aligned with MUSCLE [27]. Codon positions were in frame and there were a total of 3576 bp in the dataset. The concatenated ST sequence of the M. massiliense type strain (ST37) was added to the alignment, strains from Ireland with the same ST as the M. abscessus type strain (ST1) were already present in the dataset. Bootstrap confidence values were based on 500 replications. Where rpoB species assignment and MLST species assignment based on localization in the minimum spanning tree or phylogenetic trees constructed with concatenated multiple sequence alignments using MLST loci were discordant, the MLST assignment was chosen because of the known horizontal transfer of rpoB in some strains [8]. An estimate of prevalence of M. abscessus complex infection in CF patients in Ireland was determined from the number of CF patients attending CUH and SVUH clinics in 2012 and the recorded number of patients from these centres from whom M. abscessus complex strains were isolated in that year.
2. Materials and methods 3. Results Thirty six isolates of the M. abscessus complex, obtained between 2006 and 2012 from 36 individuals were included in this study. Six respiratory isolates were obtained from a single adult
Twenty eight strains (78%) were assigned to M. abscessus subsp. abscessus by rpoB sequencing and eight to M. abscessus
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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subsp. massiliense, none were M. abscessus subsp. bolletii. One isolate with a novel ST identified as M. abscessus subsp. abscessus by rpoB mapped within the M. abscessus subsp. massiliense group on both minimum spanning and neighbour joining trees constructed using MLST data (isolate 27, ST156) (Tables 1 and 2, Figs. 1 and 2). The commonest ST was ST1 (N = 7), followed by ST26 (N = 5), ST126 (N = 3) and ST22 (N = 2). Two M. abscessus subsp. massiliense strains shared the same ST (ST23). The minimum spanning tree constructed with every ST represented in the international database showed no clustering of STs of strains from Ireland (Fig. 1). The neighbour joining tree constructed with the concatenated MLST sequence dataset from Ireland plus one external ST unambiguously separated M. abscessus subsp. massiliense and M. abscessus subsp. abscessus strains. Of the twenty one isolates for which patient diagnoses were available, eighteen isolates were from CF patients and 15 of these were M. abscessus subsp. abscessus (Table 3). The mean age of first isolation of Mycobacterium abscessus complex bacteria from the eighteen patients known to have cystic fibrosis was 24 years. Aspergillus sp. were isolated from 9 of these patients (50%) on multiple occasions. Five novel M. abscessus subsp. abscessus STs (combinations not previously present in the Institut Pasteur M. abscessus MLST Database) were found representing combinations of previously known alleles: ST152, ST153, ST154, ST155, and ST178 (Table 1). Two further isolates had novel alleles in ST108 (murC) and ST157 (pta). A higher proportion of M. abscessus subsp. massiliense isolates than M. abscessus subsp. abscessus isolates (75% vs 39%) were represented by single STs (Table 1). Among the 234 patients attending adult and paediatric CF clinics at CUH in 2012 (142 adult and 92 paediatric patients) and the 318 adults attending SVUH, eleven were culture positive for M. abscessus complex bacteria, a prevalence of 2%. One specimen year was used for this estimate because of changes in screening policy at CUH during the interval 2006– 12 when the isolates typed in this study were obtained. Seven typed isolates from seven patients are therefore excluded from
Table 1 M. abscessus complex isolates from Ireland (36 isolates from 36 patients). Species
Sequence type (ST)
Number of strains of each ST
Number of singleton STs (%)
M. abscessus subsp. abscessus (28 strains)
ST1 ST26 ST126 ST22 ST21, 33, 47, 125, 108 ⁎,152 ⁎, 153 ⁎, 154 ⁎, 155 ⁎, 157 ⁎,178 ⁎ ST23 ST2, 34, 48, 69, 117, 156 ⁎
7 5 3 2 1
11 (39)
2 1
6 (75)
M. abscessus subsp. massiliense (8 strains)
⁎ Novel ST in international database (http://www.pasteur.fr/recherche/ genopole/PF8/mlst/Myco-abscessus.html).
3
Table 2 M. abscessus complex isolates from sputum of 18 cystic fibrosis patients at Cork University Hospital (CUH prefix) and St Vincent's University Hospital, Dublin. Year Strain
Subspecies ST
Age at first positive Aspergillus
2006 2006 2006 2008 2008 2008 2009 2009 2010 2011 2011 2011 2011 2011 2011 2012 2012 2012
massiliense abscessus massiliense abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus massiliense
16 43 24 10 30 21 29 20 26 22 5 21 33 21 35 26 19 38
CUH4619 18 21 CUH46 22 24 23 25 28 CUH2165/2011 CUH117/2011 30 19 26 20 CUH34 CUH35 29
69 26 ⁎⁎ 23 ⁎⁎ 108 ⁎ 1 ⁎⁎ 1 ⁎⁎ 178 ⁎ 1 ⁎⁎ 1 ⁎⁎ 22 ⁎⁎ 21 126 ⁎⁎ 1 ⁎⁎ 155 ⁎ 154 ⁎ 125 126 ⁎⁎ 117
− + + + − − − + − + + − − + − + − +
⁎ Novel ST in international database. ⁎⁎ Multiple strains of this type in dataset from Ireland.
the prevalence estimate. Screening coverage was 100% over 2012–2014. 4. Conclusions The data from cystic fibrosis patients and the wider culture collection of M. abscessus complex strains in Ireland shows predominance of M. abscessus subsp. abscessus, with M. abscessus subsp. massiliense forming a minority of isolates, as seen in most other reports (Tables 1 and 2) [3,13,28–31] from countries other than Brazil [32]. No M. abscessus subsp. bolletii were identified, but these form less than 5% of M. abscessus complex isolates from many countries (Table 3) and their absence could reflect inadequate sampling, as they have been reported from the neighbouring countries of Scotland [33] and England [29]. The most prevalent M. abscessus subsp. abscessus ST in our series, ST1 (Tables 1 and 2), is the most prevalent isolate in the international MLST database, with isolates recorded from France, Brazil, USA, and Switzerland from both respiratory and soft tissue infection samples [8]. ST26, the second commonest isolate, found in one CF patient at CUH and one at SVUH, has previously been reported from cystic fibrosis patients in Scotland [33], France [8] and England [11]. The other two M. abscessus subsp. abscessus STs represented by more than one isolate have also been reported from other countries: ST126 (Taiwan) and ST22 (France) [8]. The only M. abscessus subsp. massiliense ST from Ireland isolated from more than one patient was ST23 with two isolates (one from a CF patient, clinical details not available for the second). This ST is common in cystic fibrosis patients in Scotland [33], France, Germany and Switzerland [8], has been reported from a CF patient in England [11], and is also the ST of the Brazilian soft tissue infection strain. Genome sequences from ST23 strains have shown remarkably little variation in different countries
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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Fig. 1. Minimum spanning tree of STs from M. abscessus complex MLST database http://www.pasteur.fr/recherche/genopole/PF8/mlst/Myco-abscessus.html (STs from Ireland larger black circles).
[9,19]. Ireland can now be added to the list of countries where this globally successful clone is present, but further genome sequencing will be required to confirm the similarity of these strains to those isolated in other countries. M. abscessus subsp. massiliense ST34, represented by a single isolate in our series, is less common but also widely represented in Europe and Brazil [8]. The high percentage of single M. abscessus subsp. massiliense STs (75%, Table 1) is probably due to the small number of this species in this series, because five out of the six singleton STs were previously documented STs from other countries. However, the majority of the 39% of M. abscessus subsp. abscessus singleton STs (7 out of 11) were novel STs
not previously found in the database [8], suggesting the global diversity of M. abscessus subsp. abscessus strains is wider than has currently been sampled. These findings are compatible with a suggested population structure of clinical isolates based on acquisition from the environment, with superimposed loose clusters of clones that are successful in specific hospital environments (M. abscessus subsp. abscessus) and tight clusters of horizontally transmitted strains acquired from other patients (M. abscessus subsp. massiliense) [9,34]. The requirement for multiple sequence targets to correctly assign strains to M. abscessus subsp. massiliense or M. abscessus subsp. abscessus is shown by the potential misassignment of isolate 27 (ST156) to M. abscessus subsp. abscessus on rpoB
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
C. O'Driscoll et al. / Journal of Cystic Fibrosis xx (2015) xxx–xxx
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Fig. 2. Phylogenetic relationships of the 22 STs identified in this study. The tree was constructed from concatenated MLST gene sequences of the STs found in Ireland plus one type strain using the neighbour-joining method. Bootstrap support values (%) are indicated for each node. Multiple strains of the same ST (ST1*) as the type strain M. abscessus subsp. abscessus CIP 104536T were in the dataset from Ireland, ST37** corresponding to the type strain M. abscessus subsp. massiliense CIP 108297T was added from the international database.
sequence type alone. This potential error would carry prognostic significance because treatment response rates to antimicrobial regimens including clarithromycin are higher for M. abscessus subsp. massiliense than M. abscessus subsp. abscessus [35]. Equally, the clear separation of M. abscessus subsp. massiliense strains from M. abscessus subsp. abscessus strains in the tree based on concatenated MLST sequences (Fig. 2) and the overall discriminatory power of MLST (22 taxa from 36 isolates) (Table 1, Fig. 2) show its utility in diagnosis. Based on this data, the suggested outbreak-screen incorporating MLST and rpoB single nucleotide polymorphism [19] would detect outbreaks in our population and is likely to be effective elsewhere. Combining centralized reference
laboratory molecular typing of this kind with frequent local culture screening using Burkholderia agar would be a powerful screening strategy. The prevalence of M. abscessus complex in the cystic fibrosis clinic population surveyed at the regional centres in Ireland at 2% is slightly less than that found in the largest European survey from France (3.2%) [13] but it is not an outlier compared with previous reports. Twenty surveys reporting M. abscessus complex prevalence in CF patients since 1980 (nineteen summarized in [36], plus the most recent survey [37]) show large geographical variations within and between countries (median prevalence 3.3%, range 0– 9.1%, IQR 1.52%–5.74%). Nine out of the eighteen CF patients infected with M. abscessus complex bacteria (50%) were also
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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Table 3 Published surveys of M. abscessus complex isolates from different countries. Year
Country
M. abscessus subsp. abscessus (%)
M. abscessus subsp. massiliense (%)
M. abscessus subsp. bolletii (%)
Other NTM/ un-speciated
Number of patients (CF patients)
Respiratory isolates (%)
Reference
2005–2007 1999–2007 2004–2010 1999–2005 2004
South Korea USA UK Netherlands France
65 (51.2) 26 (61.9) 46 (57) 25 (64) 30 (60)
59 (46.5) 12 (28.5) 20 (25) 8 (21) 11 (22)
2 (1.6) 2 (4.8) 5 (6) 6 (15) 9 (18)
1 (0.8) 2 (4.8) 10 (12) 0 0
127 (100) 34 (81) 70 (86) 30 (79) 50 (100)
(28) (3) (29) (30) (13)
2007–2010 2009–2012 2004–2012
Japan Brazil Ireland
90 (63) 6 (16.5) 28 (78)
50 (35) 0 8 (22)
3 (2) 24 (67) 0
0 6 (16.5) 0
N 99 40 (6) 40 (30) 39 (5) 50 (50) (National Cystic Fibrosis survey) 143 10 (10) 36 (18)
36 (100) 21 (58 ⁎)
(31) (32) This study
⁎ This is a minimum—most of the 15 anonymous samples are likely to be respiratory isolates.
culture positive for Aspergillus (Table 2). Aspergillosis is associated with M. abscessus complex isolation from cystic fibrosis patients in many countries [38–41]. In conclusion, this first analysis of the molecular epidemiology of M. abscessus complex strains isolated in Ireland from cystic fibrosis and other patients shows the most prevalent MLST types of M. abscessus subsp. abscessus and most M. abscessus subsp. massiliense MLST types are similar to those reported from other countries. Novel features include the absence of M. abscessus subsp. bolletii strains, and a high percentage (25%) of singleton isolates with novel M. abscessus subsp. abscessus MLST types.
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Conflict of interest [8]
The authors declare that they have no conflict of interest. [9]
Acknowledgements We thank the Strategy for the Control of Antimicrobial Resistance (SARI) Funding Committee for Cork and Kerry for funding (SARI/2010/Prentice). SARI had no role in study design, collection, analysis and interpretation of data, the writing of the report, and the decision to submit the article for publication. Ethical approval for this project was obtained from the Clinical Research Ethics Committee of the Cork Teaching Hospitals (reference ECM 4 (kk) 04/12/12). References [1] Runyon EH. Anonymous mycobacteria in pulmonary disease. Med Clin North Am 1959;43:273–90. [2] Griffith DE, Girard WM, Wallace Jr RJ. Clinical features of pulmonary disease caused by rapidly growing mycobacteria: an analysis of 154 patients. Am Rev Respir Dis 1993;147:1271–8. [3] Zelazny AM, Root JM, Shea YR, Colombo RE, Shamputa IC, Stock F, et al. Cohort study of molecular identification and typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. J Clin Microbiol 2009;47:1985–95. [4] Leao SC, Tortoli E, Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML, et al. Characterization of mycobacteria from a major Brazilian outbreak suggests that revision of the taxonomic status of members of the
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species within the Mycobacterium abscessus complex and Mycobacterium chelonae. Eur J Clin Microbiol Infect Dis 2012;31:1847–53. van Ingen J, de Zwaan R, Dekhuijzen RPN, Boeree MJ, van Soolingen D. Clinical relevance of Mycobacterium chelonae–abscessus group isolation in 95 patients. J Infect 2009;59:324–31. Yoshida S, Tsuyuguchi K, Suzuki K, Tomita M, Okada M, Hayashi S, et al. Further isolation of Mycobacterium abscessus subsp. abscessus and subsp. bolletii in different regions of Japan and susceptibility of these isolates to antimicrobial agents. Int J Antimicrob Agents 2013;42:226–31. Cândido PHC, Nunes L de S, Marques EA, Folescu TW, Coelho FS, de Moura VCN, et al. Multidrug resistant non-tuberculous mycobacteria isolated from cystic fibrosis patients. J Clin Microbiol 2014;52:2990–7. Kreutzfeldt KM, McAdam PR, Claxton P, Holmes A, Seagar AL, Laurenson IF, et al. Molecular longitudinal tracking of Mycobacterium abscessus spp. during chronic infection of the human lung. PLoS One 2013;8:e63237. Davidson RM, Hasan NA, Reynolds PR, Totten S, Garcia B, Levin A, et al. Genome sequencing of Mycobacterium abscessus isolates from patients in the United States and comparisons to globally diverse clinical strains. J Clin Microbiol 2014;52:3573–82. Koh WJ, Jeon K, Lee NY, Kim BJ, Kook YH, Lee SH, et al. Clinical significance of differentiation of Mycobacterium massiliense from Mycobacterium abscessus. Am J Respir Crit Care Med 2011;183:405–10. Qvist T, Pressler T, Høiby N, Katzenstein TL. Shifting paradigms of nontuberculous mycobacteria in cystic fibrosis. Respir Res 2014;15:41. Qvist T, Gilljam M, Jönsson B, Taylor-Robinson D, Jensen-Fangel S, Wang M, et al. Epidemiology of nontuberculous mycobacteria among patients with cystic fibrosis in Scandinavia. J Cyst Fibros 2015;14:46–52. Levy I, Grisaru-Soen G, Lerner-Geva L, Kerem E, Blau H, Bentur L, et al. Multicenter cross-sectional study of nontuberculous mycobacterial infections among cystic fibrosis patients, Israel. Emerg Infect Dis 2008;14:378. Esther Jr CR, Esserman DA, Gilligan P, Kerr A, Noone PG. Chronic Mycobacterium abscessus infection and lung function decline in cystic fibrosis. J Cyst Fibros 2010;9:117–23. Verregghen M, Heijerman HG, Reijers M, van Ingen J, van der Ent CK. Risk factors for Mycobacterium abscessus infection in cystic fibrosis patients; a case–control study. J Cyst Fibros 2012;11:340–3. Catherinot E, Roux A-L, Vibet M-A, Bellis G, Ravilly S, Lemonnier L, et al. Mycobacterium avium and Mycobacterium abscessus complex target distinct cystic fibrosis patient subpopulations. J Cyst Fibros 2013;12:74–80.
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
Journal of Cystic Fibrosis xx (2015) xxx – xxx www.elsevier.com/locate/jcf
Original Article
Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland C. O'Driscoll a , J. Konjek b , B. Heym b,c , M.M. Fitzgibbon d , B.J. Plant e , M. Ní Chróinín f , D. Mullane f , M. Lynch-Healy a , G.D. Corcoran a , K. Schaffer g , T.R. Rogers d , M.B. Prentice a,h,i,⁎ a Department of Microbiology, Cork University Hospital, Cork, Ireland EA 3647, Université de Versailles Saint-Quentin-en-Yvelines, 2 avenue de la Source de la Biévre, 78180 Montigny-le-Bretonneux, France APHP Hôpitaux Universitaires Paris Ile-de-France Ouest, Service de Microbiologie, Hôpital Ambroise Paré, 9 avenue Charles de Gaulle, 92100 Boulogne-Billancourt, France d Irish Mycobacteria Reference Laboratory, St. James Hospital, Dublin, Ireland e Cork Adult Cystic Fibrosis Centre, University College Cork, Cork University Hospital, Wilton, Cork, Ireland f Department of Paediatrics, Cork University Hospital, Wilton, Cork, Ireland g Department of Microbiology, St. Vincent's University Hospital, Dublin, Ireland h Department of Pathology, University College Cork, Cork, Ireland i Department of Microbiology, University College Cork, Cork, Ireland b
c
Received 22 January 2015; revised 17 May 2015; accepted 17 May 2015
Abstract Background: The Mycobacterium abscessus complex are the rapidly growing mycobacteria (RGM) most commonly causing lung disease, especially in cystic fibrosis (CF) patients. Ireland has the world's highest CF incidence. The molecular epidemiology of M. abscessus complex in Ireland is unreported. Methods: We performed rpoB gene sequencing and multi-locus sequence typing (MLST) on M. abscessus complex strains isolated from thirty-six patients in 2006–2012 (eighteen known CF patients). Results: Twenty-eight strains (78%) were M. abscessus subsp. abscessus, eight M. abscessus subsp. massiliense, none were M. abscessus subsp. bolletii. Sequence type 1 (ST1) and ST26 (M. abscessus subsp. abscessus) were commonest. Seven M. abscessus subsp. abscessus STs (25%) were novel (two with novel alleles). Seven M. abscessus subsp. massiliense STs were previously reported (88%), including two ST23, the globally successful clone. In 2012, of 552 CF patients screened, eleven were infected with M. abscessus complex strains (2%). Conclusions: The most prevalent M. abscessus subsp. abscessus and M. abscessus subsp. massiliense strains in Ireland belong to widelydistributed STs, but there is evidence of high M. abscessus subsp. abscessus diversity. © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Keywords: Mycobacterium abscessus; Epidemiology; Cystic fibrosis; Ireland; MLST
1. Introduction Clinically significant mycobacterial isolates can be divided into the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. The non-tuberculous mycobacteria are classifiable
⁎ Corresponding author at: Department of Microbiology, University College Cork, Cork, Ireland. Tel.: + 353 21 490 1420. E-mail address: [email protected] (M.B. Prentice).
as slowly growing or rapidly growing [1]. Members of the M. abscessus complex (or M. abscessus sensu lato) are the most frequent rapidly growing mycobacteria (RGM) causing lung disease in humans [2,3], and are also associated with postsurgical [4] or traumatic infections outside the respiratory tract and occasionally disseminated disease in immunocompromised patients. The taxonomy of M. abscessus complex strains has been in flux in recent years. A division into three species M. abscessus, M. massiliense [5] and M. bolletii [6] was called into question by a lack of inter-species differentiation by phenotype, DNA–DNA
http://dx.doi.org/10.1016/j.jcf.2015.05.007 1569-1993/© 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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hybridisation and individual sequences of rpoB and hsp65 genes when larger numbers of strains were evaluated [4,7]. However, a clear phylogenetic signal supporting these three groups has now been obtained by extensive Multi Locus Sequence Typing (MLST) [8] and multiple genome sequence comparisons [9,10] of widely geographically distributed strains. It is likely that three subspecies will be re-proposed based on this evidence. For the purposes of this manuscript we use the terms M. abscessus subsp. abscessus, M. abscessus subsp. massiliense and M. abscessus subsp. bolletii to describe these three taxa individually following usage in the current clinical literature on cystic fibrosis patients [9,11]. Respiratory infections with M. abscessus complex are especially associated with cystic fibrosis [12]. The largest prospective survey in western Europe (in France) cultured M. abscessus complex from 3.2% of cystic fibrosis patients [13], with a peak incidence between 11 and 15 years of age (5.8%). Case reports of fatal disseminated infection with M. abscessus complex in immunosuppressed cystic fibrosis (CF) patients following lung transplant [14,15] means carriage is viewed as a contraindication to lung transplant in some centres, but not others where longitudinal surveys have revealed no excess mortality with M. abscessus complex carriage [16]. A variety of different strains of M. abscessus complex is generally revealed on molecular typing of clinical isolates, but large outbreaks of surgical site infections with a genetically uniform (on PFGE and single gene sequencing) glutaraldehyde resistant strain of M. abscessus subsp. massiliense have occurred in Brazil associated with laparoscopy and arthroscopy [17]. Genome sequencing allowing very fine inter-isolate comparison by single nucleotide polymorphisms (SNPs) has enabled two recent reports of horizontal transmission of M. abscessus subsp. massiliense between cystic fibrosis patients within hospitals in Seattle, USA [18] and Papworth, UK [9]. The outbreak strains involved at the two centres were also very closely related on genome sequencing SNP phylogeny [9,19], despite the lack of documented inter-continental contact. Genome sequencing of the Brazilian surgical infection clone [20] showed that it was also phylogenetically very similar to the two cystic fibrosis outbreak strains [9,19]. The molecular epidemiology of M. abscessus complex strains from Ireland has not been reported. Ireland has the highest incidence of cystic fibrosis in the European Union [21] and therefore the population contains a relatively large number of individuals susceptible to M. abscessus complex infection. As the country is an island, individual strain prevalence may differ from other countries. We describe rpoB fragment typing and MLST for isolates from 36 patients from Ireland, including eighteen cystic fibrosis patients attending two different regional centres. Six patients were from one centre serving adult and paediatric patients (Cork University Hospital, Cork, CUH) and twelve from an adult centre (St Vincent's University Hospital, Dublin, SVUH).
and paediatric cystic fibrosis centre (Cork University Hospital, CUH). Thirty were anonymous isolates sent to the Irish National Mycobacterial Reference Laboratory for speciation. Twelve of these were respiratory isolates from adult cystic fibrosis patients at St Vincent's University Hospital, Dublin (SVUH), three were respiratory isolates from non-cystic fibrosis patients. Clinical details were not available for the remaining fifteen isolates. Culture and initial identification: M. abscessus complex strains from CF patients at CUH were identified by rapid growth in BacT/ ALERT 3D broth (bioMérieux) and subsequent GenoType Mycobacterium assay (Hain Lifescience) targeting the 23S rRNA gene. Twice yearly mycobacterial culture screens have been carried out at CUH on CF patients since 2008, before 2008 mycobacterial cultures were taken on clinical suspicion. At SVUH cultures were obtained by extended incubation (7–10 days) on Burkholderia cepacea selective agar [22], and B. cepacea screening was carried out 4–5 times per annum. DNA extractions were carried out on fresh cultures using Tris–EDTA, lysozyme, and proteinase as described [23]. Aspergillus culture positivity was defined as more than one isolation of Aspergillus sp. within a month before or after M. abscessus complex isolation Molecular typing: a 940-bp fragment of the rpoB gene was amplified by PCR using AmpliTaq gold polymerase (Applied Biosystems) with primers MYCOF1 and MYCOR2 [23] and trimmed to 752 bp [24]. 4–600 bp fragments from argH, cya glpK, gnd, murC, pta, and purH were amplified in 25 μl of ReddyMix PCR master mix (Thermo Fisher Inc.) with 1 μl of each primer (10 pmol) as described [8]. Minimum spanning tree created with PhyloWeb at Institut Pasteur M. abscessus MLST Database using the minimum spanning tree (MST) algorithm [25] on 118 sequence types (ST) including 22 ST from this study http://www.pasteur.fr/recherche/genopole/PF8/ mlst/Myco-abscessus.html. The neighbour-joining algorithm was used as implemented in MEGA5 [26] as described [8] using concatenated DNA sequences of each MLST type aligned with MUSCLE [27]. Codon positions were in frame and there were a total of 3576 bp in the dataset. The concatenated ST sequence of the M. massiliense type strain (ST37) was added to the alignment, strains from Ireland with the same ST as the M. abscessus type strain (ST1) were already present in the dataset. Bootstrap confidence values were based on 500 replications. Where rpoB species assignment and MLST species assignment based on localization in the minimum spanning tree or phylogenetic trees constructed with concatenated multiple sequence alignments using MLST loci were discordant, the MLST assignment was chosen because of the known horizontal transfer of rpoB in some strains [8]. An estimate of prevalence of M. abscessus complex infection in CF patients in Ireland was determined from the number of CF patients attending CUH and SVUH clinics in 2012 and the recorded number of patients from these centres from whom M. abscessus complex strains were isolated in that year.
2. Materials and methods 3. Results Thirty six isolates of the M. abscessus complex, obtained between 2006 and 2012 from 36 individuals were included in this study. Six respiratory isolates were obtained from a single adult
Twenty eight strains (78%) were assigned to M. abscessus subsp. abscessus by rpoB sequencing and eight to M. abscessus
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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subsp. massiliense, none were M. abscessus subsp. bolletii. One isolate with a novel ST identified as M. abscessus subsp. abscessus by rpoB mapped within the M. abscessus subsp. massiliense group on both minimum spanning and neighbour joining trees constructed using MLST data (isolate 27, ST156) (Tables 1 and 2, Figs. 1 and 2). The commonest ST was ST1 (N = 7), followed by ST26 (N = 5), ST126 (N = 3) and ST22 (N = 2). Two M. abscessus subsp. massiliense strains shared the same ST (ST23). The minimum spanning tree constructed with every ST represented in the international database showed no clustering of STs of strains from Ireland (Fig. 1). The neighbour joining tree constructed with the concatenated MLST sequence dataset from Ireland plus one external ST unambiguously separated M. abscessus subsp. massiliense and M. abscessus subsp. abscessus strains. Of the twenty one isolates for which patient diagnoses were available, eighteen isolates were from CF patients and 15 of these were M. abscessus subsp. abscessus (Table 3). The mean age of first isolation of Mycobacterium abscessus complex bacteria from the eighteen patients known to have cystic fibrosis was 24 years. Aspergillus sp. were isolated from 9 of these patients (50%) on multiple occasions. Five novel M. abscessus subsp. abscessus STs (combinations not previously present in the Institut Pasteur M. abscessus MLST Database) were found representing combinations of previously known alleles: ST152, ST153, ST154, ST155, and ST178 (Table 1). Two further isolates had novel alleles in ST108 (murC) and ST157 (pta). A higher proportion of M. abscessus subsp. massiliense isolates than M. abscessus subsp. abscessus isolates (75% vs 39%) were represented by single STs (Table 1). Among the 234 patients attending adult and paediatric CF clinics at CUH in 2012 (142 adult and 92 paediatric patients) and the 318 adults attending SVUH, eleven were culture positive for M. abscessus complex bacteria, a prevalence of 2%. One specimen year was used for this estimate because of changes in screening policy at CUH during the interval 2006– 12 when the isolates typed in this study were obtained. Seven typed isolates from seven patients are therefore excluded from
Table 1 M. abscessus complex isolates from Ireland (36 isolates from 36 patients). Species
Sequence type (ST)
Number of strains of each ST
Number of singleton STs (%)
M. abscessus subsp. abscessus (28 strains)
ST1 ST26 ST126 ST22 ST21, 33, 47, 125, 108 ⁎,152 ⁎, 153 ⁎, 154 ⁎, 155 ⁎, 157 ⁎,178 ⁎ ST23 ST2, 34, 48, 69, 117, 156 ⁎
7 5 3 2 1
11 (39)
2 1
6 (75)
M. abscessus subsp. massiliense (8 strains)
⁎ Novel ST in international database (http://www.pasteur.fr/recherche/ genopole/PF8/mlst/Myco-abscessus.html).
3
Table 2 M. abscessus complex isolates from sputum of 18 cystic fibrosis patients at Cork University Hospital (CUH prefix) and St Vincent's University Hospital, Dublin. Year Strain
Subspecies ST
Age at first positive Aspergillus
2006 2006 2006 2008 2008 2008 2009 2009 2010 2011 2011 2011 2011 2011 2011 2012 2012 2012
massiliense abscessus massiliense abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus abscessus massiliense
16 43 24 10 30 21 29 20 26 22 5 21 33 21 35 26 19 38
CUH4619 18 21 CUH46 22 24 23 25 28 CUH2165/2011 CUH117/2011 30 19 26 20 CUH34 CUH35 29
69 26 ⁎⁎ 23 ⁎⁎ 108 ⁎ 1 ⁎⁎ 1 ⁎⁎ 178 ⁎ 1 ⁎⁎ 1 ⁎⁎ 22 ⁎⁎ 21 126 ⁎⁎ 1 ⁎⁎ 155 ⁎ 154 ⁎ 125 126 ⁎⁎ 117
− + + + − − − + − + + − − + − + − +
⁎ Novel ST in international database. ⁎⁎ Multiple strains of this type in dataset from Ireland.
the prevalence estimate. Screening coverage was 100% over 2012–2014. 4. Conclusions The data from cystic fibrosis patients and the wider culture collection of M. abscessus complex strains in Ireland shows predominance of M. abscessus subsp. abscessus, with M. abscessus subsp. massiliense forming a minority of isolates, as seen in most other reports (Tables 1 and 2) [3,13,28–31] from countries other than Brazil [32]. No M. abscessus subsp. bolletii were identified, but these form less than 5% of M. abscessus complex isolates from many countries (Table 3) and their absence could reflect inadequate sampling, as they have been reported from the neighbouring countries of Scotland [33] and England [29]. The most prevalent M. abscessus subsp. abscessus ST in our series, ST1 (Tables 1 and 2), is the most prevalent isolate in the international MLST database, with isolates recorded from France, Brazil, USA, and Switzerland from both respiratory and soft tissue infection samples [8]. ST26, the second commonest isolate, found in one CF patient at CUH and one at SVUH, has previously been reported from cystic fibrosis patients in Scotland [33], France [8] and England [11]. The other two M. abscessus subsp. abscessus STs represented by more than one isolate have also been reported from other countries: ST126 (Taiwan) and ST22 (France) [8]. The only M. abscessus subsp. massiliense ST from Ireland isolated from more than one patient was ST23 with two isolates (one from a CF patient, clinical details not available for the second). This ST is common in cystic fibrosis patients in Scotland [33], France, Germany and Switzerland [8], has been reported from a CF patient in England [11], and is also the ST of the Brazilian soft tissue infection strain. Genome sequences from ST23 strains have shown remarkably little variation in different countries
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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Fig. 1. Minimum spanning tree of STs from M. abscessus complex MLST database http://www.pasteur.fr/recherche/genopole/PF8/mlst/Myco-abscessus.html (STs from Ireland larger black circles).
[9,19]. Ireland can now be added to the list of countries where this globally successful clone is present, but further genome sequencing will be required to confirm the similarity of these strains to those isolated in other countries. M. abscessus subsp. massiliense ST34, represented by a single isolate in our series, is less common but also widely represented in Europe and Brazil [8]. The high percentage of single M. abscessus subsp. massiliense STs (75%, Table 1) is probably due to the small number of this species in this series, because five out of the six singleton STs were previously documented STs from other countries. However, the majority of the 39% of M. abscessus subsp. abscessus singleton STs (7 out of 11) were novel STs
not previously found in the database [8], suggesting the global diversity of M. abscessus subsp. abscessus strains is wider than has currently been sampled. These findings are compatible with a suggested population structure of clinical isolates based on acquisition from the environment, with superimposed loose clusters of clones that are successful in specific hospital environments (M. abscessus subsp. abscessus) and tight clusters of horizontally transmitted strains acquired from other patients (M. abscessus subsp. massiliense) [9,34]. The requirement for multiple sequence targets to correctly assign strains to M. abscessus subsp. massiliense or M. abscessus subsp. abscessus is shown by the potential misassignment of isolate 27 (ST156) to M. abscessus subsp. abscessus on rpoB
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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Fig. 2. Phylogenetic relationships of the 22 STs identified in this study. The tree was constructed from concatenated MLST gene sequences of the STs found in Ireland plus one type strain using the neighbour-joining method. Bootstrap support values (%) are indicated for each node. Multiple strains of the same ST (ST1*) as the type strain M. abscessus subsp. abscessus CIP 104536T were in the dataset from Ireland, ST37** corresponding to the type strain M. abscessus subsp. massiliense CIP 108297T was added from the international database.
sequence type alone. This potential error would carry prognostic significance because treatment response rates to antimicrobial regimens including clarithromycin are higher for M. abscessus subsp. massiliense than M. abscessus subsp. abscessus [35]. Equally, the clear separation of M. abscessus subsp. massiliense strains from M. abscessus subsp. abscessus strains in the tree based on concatenated MLST sequences (Fig. 2) and the overall discriminatory power of MLST (22 taxa from 36 isolates) (Table 1, Fig. 2) show its utility in diagnosis. Based on this data, the suggested outbreak-screen incorporating MLST and rpoB single nucleotide polymorphism [19] would detect outbreaks in our population and is likely to be effective elsewhere. Combining centralized reference
laboratory molecular typing of this kind with frequent local culture screening using Burkholderia agar would be a powerful screening strategy. The prevalence of M. abscessus complex in the cystic fibrosis clinic population surveyed at the regional centres in Ireland at 2% is slightly less than that found in the largest European survey from France (3.2%) [13] but it is not an outlier compared with previous reports. Twenty surveys reporting M. abscessus complex prevalence in CF patients since 1980 (nineteen summarized in [36], plus the most recent survey [37]) show large geographical variations within and between countries (median prevalence 3.3%, range 0– 9.1%, IQR 1.52%–5.74%). Nine out of the eighteen CF patients infected with M. abscessus complex bacteria (50%) were also
Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007
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Table 3 Published surveys of M. abscessus complex isolates from different countries. Year
Country
M. abscessus subsp. abscessus (%)
M. abscessus subsp. massiliense (%)
M. abscessus subsp. bolletii (%)
Other NTM/ un-speciated
Number of patients (CF patients)
Respiratory isolates (%)
Reference
2005–2007 1999–2007 2004–2010 1999–2005 2004
South Korea USA UK Netherlands France
65 (51.2) 26 (61.9) 46 (57) 25 (64) 30 (60)
59 (46.5) 12 (28.5) 20 (25) 8 (21) 11 (22)
2 (1.6) 2 (4.8) 5 (6) 6 (15) 9 (18)
1 (0.8) 2 (4.8) 10 (12) 0 0
127 (100) 34 (81) 70 (86) 30 (79) 50 (100)
(28) (3) (29) (30) (13)
2007–2010 2009–2012 2004–2012
Japan Brazil Ireland
90 (63) 6 (16.5) 28 (78)
50 (35) 0 8 (22)
3 (2) 24 (67) 0
0 6 (16.5) 0
N 99 40 (6) 40 (30) 39 (5) 50 (50) (National Cystic Fibrosis survey) 143 10 (10) 36 (18)
36 (100) 21 (58 ⁎)
(31) (32) This study
⁎ This is a minimum—most of the 15 anonymous samples are likely to be respiratory isolates.
culture positive for Aspergillus (Table 2). Aspergillosis is associated with M. abscessus complex isolation from cystic fibrosis patients in many countries [38–41]. In conclusion, this first analysis of the molecular epidemiology of M. abscessus complex strains isolated in Ireland from cystic fibrosis and other patients shows the most prevalent MLST types of M. abscessus subsp. abscessus and most M. abscessus subsp. massiliense MLST types are similar to those reported from other countries. Novel features include the absence of M. abscessus subsp. bolletii strains, and a high percentage (25%) of singleton isolates with novel M. abscessus subsp. abscessus MLST types.
[5]
[6]
[7]
Conflict of interest [8]
The authors declare that they have no conflict of interest. [9]
Acknowledgements We thank the Strategy for the Control of Antimicrobial Resistance (SARI) Funding Committee for Cork and Kerry for funding (SARI/2010/Prentice). SARI had no role in study design, collection, analysis and interpretation of data, the writing of the report, and the decision to submit the article for publication. Ethical approval for this project was obtained from the Clinical Research Ethics Committee of the Cork Teaching Hospitals (reference ECM 4 (kk) 04/12/12). References [1] Runyon EH. Anonymous mycobacteria in pulmonary disease. Med Clin North Am 1959;43:273–90. [2] Griffith DE, Girard WM, Wallace Jr RJ. Clinical features of pulmonary disease caused by rapidly growing mycobacteria: an analysis of 154 patients. Am Rev Respir Dis 1993;147:1271–8. [3] Zelazny AM, Root JM, Shea YR, Colombo RE, Shamputa IC, Stock F, et al. Cohort study of molecular identification and typing of Mycobacterium abscessus, Mycobacterium massiliense, and Mycobacterium bolletii. J Clin Microbiol 2009;47:1985–95. [4] Leao SC, Tortoli E, Viana-Niero C, Ueki SYM, Lima KVB, Lopes ML, et al. Characterization of mycobacteria from a major Brazilian outbreak suggests that revision of the taxonomic status of members of the
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Please cite this article as: O'Driscoll C, et al, Molecular epidemiology of Mycobacterium abscessus complex isolates in Ireland, J Cyst Fibros (2015), http://dx.doi.org/ 10.1016/j.jcf.2015.05.007