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Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis
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Contents lists available at ScienceDirect
Journal of Cystic Fibrosis journal homepage: www.elsevier.com/locate/jcf
Short Communication
Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis Amy Coward a,1, Dervla T.D. Kenna a,∗, Neil Woodford a, Jane F. Turton a , and members of the UK CF Surveillance Working Group., The UK CF Surveillance Working Group comprised, Malcolm Armstrong b, Cressida Auckland c, Ian Bowler d, Phillipa Burns b, James Cargill e, Mary Carroll f, William Flight d, Michelle Graver g, Heather Green h, Carolyne Horner i, Andrew Jones j, Andrew M. Jones h, Graeme Jones f, Sarah Mayell e, Jeorge Orendi k, Audrey Perry l, Ali Robb l, Natasha Tucker f, David Waine m, Trevor Winstanley n, Nick Withers c a
Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, National Infection Service, Public Health England, London, United Kingdom Department of Microbiology, Royal Manchester Children’s Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom. c Microbiology Department, Royal Devon and Exeter Hospital, Exeter, United Kingdom. d Microbiology Department, John Radcliffe Hospital, Oxford, United Kingdom. e Microbiology Department, Alder Hey Children’s NHS Foundation Trust, Liverpool, United Kingdom. f Cystic Fibrosis Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom. g Department of Microbiology, King’s College Hospital NHS Foundation Trust, London, United Kingdom. h Manchester Adult Cystic Fibrosis Centre, Manchester University Hospitals NHS Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom. i Department of Microbiology, Leeds General Infirmary, Leeds Teaching Hospital NHS Trust, Leeds, United Kingdom. j Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom. k Royal Stoke University Hospital, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom. l Microbiology Department, Freeman Hospital, Newcastle-upon-Tyne, United Kingdom. m Plymouth Hospitals NHS Trust, Plymouth, United Kingdom. n Northern General Hospital, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom. b
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Article history: Received 3 July 2019 Revised 16 October 2019 Accepted 11 November 2019 Available online xxx Keywords: Survey Achromobacter Pandoraea Ralstonia CF
a b s t r a c t A structured survey of the cystic fibrosis pathogens Achromobacter, Pandoraea and Ralstonia species from thirteen sentinel hospitals throughout England was undertaken by Public Health England. One isolate per patient of these genera collected from CF patients during the seven-month survey period in 2015 was requested from participating hospitals. Species-level identification was performed using nrdA/gyrB sequence cluster analysis, and genotyping by pulsed-field gel electrophoresis. In total, 176 isolates were included in the survey; 138 Achromobacter spp. (78.4%), 29 Pandoraea spp. (16.5%) and 9 Ralstonia spp. (5.1%). Novel Achromobacter and Pandoraea clusters were identified. High levels of antimicrobial resistance were found, particularly among Pandoraea isolates. Genotyping analysis revealed considerable diversity, however one geographically-widespread cluster of A. xylosoxidans isolates from six hospitals was found, in addition to two other clusters, both comprising isolates from two hospitals, either derived from the same region (A. xylosoxidans), or from hospitals within the same city (P. apista). Crown Copyright © 2019 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. All rights reserved.
1. Introduction
∗
1
Corresponding author. E-mail address: [email protected] (D.T.D. Kenna). Present address: Broomfield Hospital, Chelmsford CM1 7ET, United Kingdom
As a UK reference laboratory for pathogens affecting cystic fibrosis (CF) patients, Public Health England’s (PHE) AMRHAI Reference Unit provides specialist identification and cross-infection investigations for CF-related organisms, including Achromobacter, Pandoraea and Ralstonia species. These genera are considered emerging CF pathogens, but their UK prevalence is relatively
https://doi.org/10.1016/j.jcf.2019.11.005 1569-1993/Crown Copyright © 2019 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. All rights reserved.
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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unknown. In 2015 our laboratory estimated the UK prevalence of different Achromobacter species by examining reference service submissions and found that 89/147 isolates from 96 patients (61%) were A. xylosoxidans [1]. A structured survey was conducted to gain a more accurate representation of Achromobacter, Pandoraea and Ralstonia species incidence in CF patients from England, and to investigate antimicrobial susceptibility patterns, cross-infection and the presence of common clones. 2. Methods Thirteen hospitals in England participated in the survey, which was conducted between 1st March and 30th September 2015. These comprised six hospitals from the North of England, one from the Midlands, two from Greater London, two from the South and two from the South–West. Each was asked to submit one isolate per CF patient of any Achromobacter, Pandoraea and Ralstonia species collected during the survey period. A case record form captured epidemiological and demographic metadata. Locally-determined susceptibility data were requested, with testing performed by disc diffusion using either European Committee on
Antimicrobial Susceptibility Testing (EUCAST) or British Society of Antimicrobial Chemotherapy (BSAC) methodologies, and interpretation using breakpoints for related genera (for example, Pseudomonas sp.). Identification was undertaken using MALDI-ToF (Bruker) analysis following ethanol and formic acid extraction, followed by sequence clustering of nrdA for Achromobacter species [1], and gyrB for Pandoraea and Ralstonia species [2]. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA, followed by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering with BioNumerics software (version 6.1, Applied Maths) using the Dice coefficient [3] (PFGE) or neighbour-joining (gyrB and nrdA clustering). 3. Results 3.1. Metadata One hundred and seventy-six isolates were included in the final analysis. Of these, 88 (50.8%) and 85 (47.5%) were isolated from male and female patients respectively, with 3 (1.7%) from an undisclosed gender. Patients’ ages ranged from 1 to 75 years (median
Fig. 1. Dendrogram showing (a) Prevalence of different Achromobacter species and (b) Pandoraea and Ralstonia species, submitted to the survey. The evolutionary history was inferred using the Neighbour-Joining method [7]. The optimal tree with the sum of branch length = 0.553 for (a) and 0.887 for (b) is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA7 [8]. † Coward et al., 2016. Examples of all nrdA/gyrB sequences for putative novel clusters/species have been submitted to Genbank under the accession numbers: MH809486, MH809487, MH809488, MH809489, MH809490, MH809491, MH809492, MH809493, MH809494, MH809495, MH809496, MH809497, MH809498, MH809499, MH809500, MH809501 and MH809502. ∗ One isolate of Pandoraea could only be identified to genus level, as it did not cluster with any currently named species or with any other isolate on our database. ‡ Recently identified as belonging to the newly described Pandoraea fibrosis [5].
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Fig. 1. Continued
21 years), with 42 (24%) patients aged ≤16 years, while 134 (76%) were CF adults. Most isolates were from sputum (n = 152; 86.4%), 20 (11.4%) were from cough swabs and 2 (1.1%) from bronchoalveolar lavage (BAL). For 2 (1.1%) isolates the isolation site was undisclosed. For 149 isolates where this information was provided, 119 (80%) were isolated during a routine outpatient review or home visit and 30 (20%) were isolated from inpatients. Where information was provided, CF health status was evaluated; 42/95 (44.2%) patients provided isolates during review appointments and were categorised as stable, while 18/95 (19%) isolates (from 17 adults and 1 paediatric patient) were from pulmonary exacerbations. Of these, 12 patients had co-infections and/or co-morbidities, while for the remaining 6 this information was not provided/unknown.
3.2. Species prevalence Overall, 138 isolates were identified as Achromobacter spp. (78.4%), 29 isolates as Pandoraea spp. (16.5%) and 9 isolates as Ralstonia spp. (5.1%). The most common species within the Achromobacter genus were A. xylosoxidans, A. dolens and Achromobacter sp. “cluster II” [1]. Two novel clusters were designated Achromobacter sp. clusters IV and V, one of which (IV) corresponds to the recently described A. deleyi [4]. The most common Pandoraea species were P. apista and P. sputorum. Five novel clusters were identified;
Pandoraea sp. clusters III, IV, V, VI and VII, one of which (IV) corresponds to the newly-described P. fibrosis [5]. Finally, 8 of 9 Ralstonia isolates were identified as R. mannitolilytica. The remaining Ralstonia isolate did not cluster with any currently described type strain [6] (Fig. 1). All organisms isolated during an exacerbation (n = 18) were identified as Achromobacter species. Thirteen (72%) of these were A. xylosoxidans. The organisms isolated during outpatient review (n = 42) were more diverse, with examples from all three genera, however 18 (43%) were identified as A. xylosoxidans.
3.3. Antimicrobial susceptibility Susceptibility testing was performed by the referring hospitals, with results reported for 101 isolates (57.4%). Of the Achromobacter species isolates, most (85.9–97%) were resistant to amikacin, aztreonam, ciprofloxacin, gentamicin and tobramycin, although results were submitted for an average of 51% of isolates (Table 1). Most isolates screened were susceptible to imipenem and piperacillin/tazobactam (87.5% and 88.6%, respectively). Although results were only available for an average of 28% of isolates submitted, most Pandoraea isolates (90.9–100%) were reported resistant to amikacin, aztreonam, ceftazidime, ciprofloxacin, colistin, gentamicin, meropenem and tobramycin. Results were reported for two R. mannitolilytica isolates which were resistant to amikacin, aztreonam, ceftazidime, colistin, gentamicin, meropenem, ticar-
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Table 1 Summary of the antimicrobial susceptibility data provided by the participating hospital laboratories for (a) Achromobacter species and (b) Pandoraea species.a (a) Achromobacter sp. Antimicrobial
Susceptible (%)
Intermediate (%)
Resistant (%)
Total (% of all Achromobacter spp.)
Amikacin Aztreonam Ceftazidime Ciprofloxacin Colistinc Co-trimoxazole Gentamicin Imipenem Meropenem Minocycline Pip/Tazb Ticarcillin/clavulanate Tobramycin
5 (7.8) 1 (1.5) 36 (44.4) 3 (3.7) 54 (73) 12 (66.6) 3 (4.6) 14 (87.5) 44 (54.3) 9 (60) 62 (88.6) 16 (57.1) 10 (12.5)
4 1 0 2 0 0 1 1 2 1 0 0 0
55 (85.9) 63 (97) 45 (55.6) 76 (93.8) 20 (27) 6 (33.3) 61 (93.8) 1 (6.25) 35 (43.2) 5 (33.3) 8 (11.4) 12 (42.9) 70 (87.5)
64 65 81 81 74 18 65 16 81 15 70 28 80
(b) Pandoraea sp. Antimicrobial
Susceptible (%)
Intermediate (%)
Resistant (%)
Total (% of all Pandoraea spp.)
Amikacin Aztreonam Ceftazidime Ciprofloxacin Colistinc Co-trimoxazole Gentamicin Imipenem Meropenem Minocycline Pip/Tazb TIcarcillin/clavulanate Tobramycin
0 0 0 0 1 6 0 6 0 5 3 0 0
0 0 0 1 0 0 0 0 0 0 0 0 0
6 (100) 7 (100) 11 (100) 10 (90.9) 10 (90.9) 0 (0) 3 (100) 1 (14.3) 10 (100) 2 (28.5) 5 (62.5) 1 (100) 11 (100)
6 (20.7) 7 (24.1) 11 (37.9) 11 (37.9) 11 (37.9) 6 (20.7) 3 (10.3) 7 (24.1) 10 (34.5) 7 (24.1) 8 (27.6) 1 (3.4) 11 (37.9)
(0) (0) (0) (0) (9.1) (100) (0) (85.7) (0) (71.4) (37.5) (0) (0)
(6.3) (1.5) (0) (2.5) (0) (0) (1.5) (6.25) (2.5) (6.6) (0) (0) (0)
(0) (0) (0) (9.1) (0) (0) (0) (0) (0) (0) (0) (0) (0)
(46.4) (47.1) (58.7) (58.7) (53.6) (13) (47.1) (11.6) (58.7) (10.9) (50.7) (20.3) (58)
a Susceptibility testing methods and interpretation varied between laboratories in part due to the lack of available genus-specific European Committee on Antimicrobial Susceptibility Testing guidelines (EUCAST; http://www.eucast.org). EUCAST Pseudomonas susceptibility testing guidelines were used to interpret antibiotic sensitivities for certain antimicrobials in some hospital laboratories; i.e. for ceftazidime, aztreonam, ciprofloxacin, tobramycin, piperacillin/tazobactam, imipenem and meropenem, as available from EUCAST in 2015. b Piperacillin/Tazobactam. c Colistin resistance may be underrepresented due to the limitations of disc diffusion/gradient diffusion. This may also be reflected in the one susceptible Pandoraea isolate, as intrinsic resistance of this genus to this antibiotic has been reported [9].
cillin/clavulanate and tobramycin. One isolate was susceptible to ciprofloxacin and piperacillin/tazobactam. 3.4. PFGE Genotyping revealed considerable diversity across all species. Approximately 68% of Achromobacter species, 60% of Pandoraea species and 89% of Ralstonia species had unique profiles, clustering with less than 80% similarity to isolates in PHE’s databases (comprising profiles for Achromobacter isolates from 359 patients from 32 hospitals, Pandoraea species from 88 patients from 23 hospitals, and Ralstonia isolates from 32 patients from seven hospitals). Highly similar isolates between a maximum of three patients per centre were found for Achromobacter (6 centres), Pandoraea (2 centres) and Ralstonia (1 centre). Two novel A. xylosoxidans ‘clusters’ were found; Cluster A (n = 8 from 6 centres) had similar profiles, despite being from geographically distinct locations. Cluster B (n = 6 from 2 centres) were almost identical and comprised isolates from five patients from the same hospital, and one from a separate centre. Likewise, a ‘cluster’ of highly similar P. apista isolates from the same geographical location (Cluster C, n = 8 from 2 centres), previously identified through reference laboratory submissions [10] was also noted (Fig. 2). 4. Discussion Our survey focussed on three genera considered to be emerging pathogens in CF lung disease [11–14]. As a national CF reference laboratory, we routinely receive isolates of these genera, but only from certain centres. In this survey the inclusion of six hospitals that do not routinely submit these genera to PHE highlighted
the presence of putative novel Achromobacter and Pandoraea species and confirmed the previously reported dominance of A. xylosoxidans among UK CF patients [1]. A higher prevalence of Achromobacter with respect to Pandoraea and Ralstonia was found, broadly mirroring available CF data. Achromobacter prevalence has been found to vary widely between countries, with estimations of between 3 and 30% [14,15]. Pandoraea and Ralstonia data are limited, although one study reported a Pandoraea prevalence of 0.17 and an incidence of 0.1 for 100 CF patients [9], and another increasing Ralstonia sp. isolation from 0.6% in 2008 to 2.4% in 2016 [6]. There are currently no national guidelines for managing CF patients harbouring these organisms, and susceptibility testing guidelines are insufficient. The high level of antimicrobial resistance was therefore noteworthy, particularly across the Pandoraea genus. Indeed, of eight Achromobacter and seven Pandoraea CF isolates recently submitted to PHE, newer combinations ceftazidimeavibactam and ceftolozane-tazobactam showed limited activity, with only one Achromobacter isolate susceptible to ceftazidimeavibactam (MIC 4 mg/L), as interpreted using EUCAST non speciesspecific PK-PD breakpoints (AMRHAI, unpublished data). However, hospitals participating in this survey reported co-trimoxazole activity against limited numbers of Achromobacter and Pandoraea, as recently described by others [16]. Extensive strain diversity was found across the three genera, but three clusters of highly-related strains were noted. A. xylosoxidans cluster A comprised isolates from six hospitals, suggesting the existence of a well-adapted common clone, as recently described in a French CF study [17]. A. xylosoxidans cluster B seemed likely to represent transmission among 5 patients attending the same
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Fig. 2. Dendrograms of the XbaI-digested genomic DNA PFGE profiles of (a) Cluster A (A. xylosoxidans), (b) Cluster B (A. xylosoxidans), and (c) Cluster C (P. apista). Each isolate was from a different patient. Isolates were coded by the region of the submitting hospital, (N = North, S = South, GL = Greater London), a number within that region, and patient number within that hospital.
hospital. Apart from small numbers of shared strains, direct transmission of Achromobacter sp. between CF patients has not been widely reported. However, two studies described the circulation of a common A. xylosoxidans clone among 21 and 22 CF patients respectively, although in both, direct patient-patient transmission was not always easy to prove [14,15]. Few CF studies have described shared Pandoraea sp. strains. One reported a shared P. pulmonicola strain between six patients, likely to be due to droplet-spread [18], while another described six patients harbouring the same strain, likely due to patientto-patient transmission either within clinics or social gatherings [19]. In this survey, recent whole genome sequencing of the P. apista cluster found it was particularly (although not exclusively)
prevalent in the north of the UK, with some evidence of patient transmission [10]. Although perhaps reflecting its prevalence, it was interesting that Achromobacter was the only genus isolated from patients with pulmonary exacerbation, particularly with respect to a recent study demonstrating an association between primary CF Achromobacter infection and exacerbation risk [20]. In this survey however, 17 of 18 affected patients were adults, of which at least 12 had other co-infections/co-morbidities, suggesting more advanced CF disease with increased likelihood of exacerbation, perhaps unrelated to Achromobacter infection. This survey has enhanced our knowledge of the population structure, antimicrobial resistance levels and prevalence of these
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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genera among CF patients in England. However, much remains to be learned about their role in CF lung disease. Funding This study used PHE reference laboratory funding and did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest The authors have no personal conflicts of interest to declare. However, PHE’s AMRHAI Reference Unit has received financial support for conference attendance, lectures, research projects or contracted evaluations from numerous sources, including: Accelerate Diagnostics, Achaogen Inc, Allecra Therapeutics, Amplex, AstraZeneca UK Ltd, Basilea Pharmaceutica, Becton Dickinson Diagnostics, BioMérieux, Bio-Rad Laboratories, The British Society for Antimicrobial Chemotherapy (BSAC), Cepheid, Check-Points B.V, Cubist Pharmaceuticals, Department of Health, Enigma Diagnostics Ltd., Food Standards Agency, Glaxo Smithkline Services Ltd, Henry Stewart Talks, IHMA Ltd, Kalidex Pharmaceuticals, Melinta Therapeutics, Merck Sharpe & Dohme Corp, Meiji Seika Pharma Co., Ltd, Mobidiag, Momentum Biosciences Ltd., Nordic Pharma Ltd., Norgine Pharmaceuticals, Rempex Pharmaceuticals Ltd, Roche, Rokitan Ltd, Smith & Nephew UK Ltd, Trius Therapeutics, VenatoRx and Wockhardt Ltd. Acknowledgments We would like to thank staff at Public Health England’s Genomic Services and Development Unit for sequencing services and Daniel Lilley for help with identification by nrdA/gyrB sequence cluster analysis. We are also grateful to Katie Hopkins and Rachel Pike of PHE’s Antimicrobial Resistance and Mechanisms service for guidance, and the use of ceftazidime-avibactam and ceftolozanetazobactam data. References [1] Coward A, Kenna DTD, Perry C, Martin K, Doumith M, Turton JF. Use of nrdA gene sequence clustering to estimate the prevalence of different Achromobacter species among cystic fibrosis patients in the UK. J Cyst Fibros 2016;15(4):479–85. [2] Kenna DTD, Lilley D, Coward A, Martin K, Perry C, Pike R, Hill R, Turton JF. Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients. J Med Microbiol 2017;66(4):490–501.
[3] Turton JF, Kaufmann ME, Mustafa N, Kawa S, Clode FE, Pitt TL. Molecular comparison of isolates of Burkholderia multivorans from patients with cystic Fibrosis in the United Kingdom. J Clin Microbiol 2013;41(12): 5750–5754. [4] Vandamme PA, Peeters C, Inganäs E, Cnockaert M, Houf K, Spilker T, et al. Taxonomic dissection of Achromobacter denitrificans coenye et al. 2003 and proposal of Achromobacter agilis sp. nov., nom. rev., Achromobacter pestifer sp. nov., nom. rev., Achromobacter kerstersii sp. nov. and Achromobacter deleyi sp. nov. Int J Syst Evol Microbiol 2016;66:3708–17. [5] See-Too W S, Ambrose M, Malley R, Ee R, Mulcahy E, Manche E, et al. Pandoraea fibrosis sp. nov., a novel Pandoraea species isolated from clinical respiratory samples. Int J Syst Evol Microbiol 2019;69(3):645–51. [6] Green HD, Bright-Thomas R, Kenna DTD, Turton JF, Woodford N, Jones AM. Ralstonia infection in cystic fibrosis. Epidemiol Infect 2017;145: 2864–2872. [7] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987;4:406–25. [8] Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016;33: 1870–1874. [9] Pugès M, Debelleix S, Fayon M, Mégraud F, Lehours P. Persistent infection because of Pandoraea sputorum in a young cystic fibrosis patient resistant to antimicrobial treatment. Pediatr Infect Dis J 2015;34:1135–7. [10] Kenna DTD, Coward A, Perry C, Pike R, Schafer U, Turton J. Investigation of a Pandoraea apista cluster common to adult and paediatric cystic fibrosis patients attending two hospitals in the same city. J Med Microbiol 2019;68:1081–95. [11] Green H, Jones AM. Emerging gram-negative bacteria: pathogenic or innocent bystanders. Curr Opin Pulm Med 2018;24(6):592–8. [12] Recio R, Brañas P, Martínez MT, Chaves F, Orellana MA. Effect of respiratory Achromobacter spp. infection on pulmonary function in patients with cystic fibrosis. J Med Microbiol 2018;67(7):952–6. [13] Somayaji R, Stanojevic S, Tullis DE, Stephenson AL, Ratjen F, Waters V. Clinical outcomes associated with Achromobacter species infection in patients with cystic fibrosis. Ann Am Thorac Soc 2017;14(9):1412–18. [14] Cools P, Ho E, Vranckx K, Schelstraete P, Wurth B, Franckx H, et al. Epidemic Achromobacter xylosoxidans strain among Belgian cystic fibrosis patients and review of literature. BMC Microbiol 2016;16(1):122 24. [15] Pereira RH, Carvalho-Assef AP, Albano RM, Folescu TW, Jones MC, Leao RS, et al. Achromobacter xylosoxidans: characterisation of strains in Brazilian cystic fibrosis patients. J Clin Microbiol 2011;49(10):3649–51. [16] Díez-Aguilar M, Ekkelenkamp M, Morosini MI, Merino I, de Dios Caballero J, Jones M, et al. Antimicrobial susceptibility of non-fermenting gram-negative pathogens isolated from cystic fibrosis patients. Int J Antimicrob Agents 2019;53:84–8. [17] Amoureux L, Sauge J, Sarret B, Lhoumeau M, Bajard A, Tetu J, Bador J, Neuwirth C. MucoMicrobes group. Study of 109 Achromobacter spp. isolates from 9 French CF centres reveals the circulation of a multiresistant clone of A. xylosoxidans belonging to ST 137. J Cyst Fibros 2019. doi:10.1016/j.jcf.2019. 04.005. [18] Degand N, Lotte R, Decondé Le Butor C, Segonds C, Thouverez M, Ferroni A, et al. Epidemic spread of Pandoraea pulmonicola in a cystic fibrosis center. BMC Infect Dis 2015;15:583. [19] Jørgensen IM, Johansen HK, Frederiksen B, Pressler T, Hansen A, Vandamme P, et al. Epidemic spread of Pandoraea apista, a new pathogen causing severe lung disease in cystic fibrosis patients. Pediatr Pulmonol 2003;36: 439–446. [20] Edwards BD, Greysson-Wong J, Somayaji R, Waddell B, Whelan FJ, Storey DG, et al. Prevalence and outcomes of Achromobacter species infections in adults with cystic fibrosis: a North American cohort study. J Clin Microbiol 2017;55(7):2074–85.
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Contents lists available at ScienceDirect
Journal of Cystic Fibrosis journal homepage: www.elsevier.com/locate/jcf
Short Communication
Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis Amy Coward a,1, Dervla T.D. Kenna a,∗, Neil Woodford a, Jane F. Turton a , and members of the UK CF Surveillance Working Group., The UK CF Surveillance Working Group comprised, Malcolm Armstrong b, Cressida Auckland c, Ian Bowler d, Phillipa Burns b, James Cargill e, Mary Carroll f, William Flight d, Michelle Graver g, Heather Green h, Carolyne Horner i, Andrew Jones j, Andrew M. Jones h, Graeme Jones f, Sarah Mayell e, Jeorge Orendi k, Audrey Perry l, Ali Robb l, Natasha Tucker f, David Waine m, Trevor Winstanley n, Nick Withers c a
Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit, National Infection Service, Public Health England, London, United Kingdom Department of Microbiology, Royal Manchester Children’s Hospital, Manchester University NHS Foundation Trust, Manchester, United Kingdom. c Microbiology Department, Royal Devon and Exeter Hospital, Exeter, United Kingdom. d Microbiology Department, John Radcliffe Hospital, Oxford, United Kingdom. e Microbiology Department, Alder Hey Children’s NHS Foundation Trust, Liverpool, United Kingdom. f Cystic Fibrosis Unit, University Hospital Southampton NHS Foundation Trust, Southampton, United Kingdom. g Department of Microbiology, King’s College Hospital NHS Foundation Trust, London, United Kingdom. h Manchester Adult Cystic Fibrosis Centre, Manchester University Hospitals NHS Foundation Trust, Wythenshawe Hospital, Manchester, United Kingdom. i Department of Microbiology, Leeds General Infirmary, Leeds Teaching Hospital NHS Trust, Leeds, United Kingdom. j Respiratory Medicine, Royal Brompton and Harefield NHS Foundation Trust, London, United Kingdom. k Royal Stoke University Hospital, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom. l Microbiology Department, Freeman Hospital, Newcastle-upon-Tyne, United Kingdom. m Plymouth Hospitals NHS Trust, Plymouth, United Kingdom. n Northern General Hospital, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom. b
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Article history: Received 3 July 2019 Revised 16 October 2019 Accepted 11 November 2019 Available online xxx Keywords: Survey Achromobacter Pandoraea Ralstonia CF
a b s t r a c t A structured survey of the cystic fibrosis pathogens Achromobacter, Pandoraea and Ralstonia species from thirteen sentinel hospitals throughout England was undertaken by Public Health England. One isolate per patient of these genera collected from CF patients during the seven-month survey period in 2015 was requested from participating hospitals. Species-level identification was performed using nrdA/gyrB sequence cluster analysis, and genotyping by pulsed-field gel electrophoresis. In total, 176 isolates were included in the survey; 138 Achromobacter spp. (78.4%), 29 Pandoraea spp. (16.5%) and 9 Ralstonia spp. (5.1%). Novel Achromobacter and Pandoraea clusters were identified. High levels of antimicrobial resistance were found, particularly among Pandoraea isolates. Genotyping analysis revealed considerable diversity, however one geographically-widespread cluster of A. xylosoxidans isolates from six hospitals was found, in addition to two other clusters, both comprising isolates from two hospitals, either derived from the same region (A. xylosoxidans), or from hospitals within the same city (P. apista). Crown Copyright © 2019 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. All rights reserved.
1. Introduction
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Corresponding author. E-mail address: [email protected] (D.T.D. Kenna). Present address: Broomfield Hospital, Chelmsford CM1 7ET, United Kingdom
As a UK reference laboratory for pathogens affecting cystic fibrosis (CF) patients, Public Health England’s (PHE) AMRHAI Reference Unit provides specialist identification and cross-infection investigations for CF-related organisms, including Achromobacter, Pandoraea and Ralstonia species. These genera are considered emerging CF pathogens, but their UK prevalence is relatively
https://doi.org/10.1016/j.jcf.2019.11.005 1569-1993/Crown Copyright © 2019 Published by Elsevier B.V. on behalf of European Cystic Fibrosis Society. All rights reserved.
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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unknown. In 2015 our laboratory estimated the UK prevalence of different Achromobacter species by examining reference service submissions and found that 89/147 isolates from 96 patients (61%) were A. xylosoxidans [1]. A structured survey was conducted to gain a more accurate representation of Achromobacter, Pandoraea and Ralstonia species incidence in CF patients from England, and to investigate antimicrobial susceptibility patterns, cross-infection and the presence of common clones. 2. Methods Thirteen hospitals in England participated in the survey, which was conducted between 1st March and 30th September 2015. These comprised six hospitals from the North of England, one from the Midlands, two from Greater London, two from the South and two from the South–West. Each was asked to submit one isolate per CF patient of any Achromobacter, Pandoraea and Ralstonia species collected during the survey period. A case record form captured epidemiological and demographic metadata. Locally-determined susceptibility data were requested, with testing performed by disc diffusion using either European Committee on
Antimicrobial Susceptibility Testing (EUCAST) or British Society of Antimicrobial Chemotherapy (BSAC) methodologies, and interpretation using breakpoints for related genera (for example, Pseudomonas sp.). Identification was undertaken using MALDI-ToF (Bruker) analysis following ethanol and formic acid extraction, followed by sequence clustering of nrdA for Achromobacter species [1], and gyrB for Pandoraea and Ralstonia species [2]. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA, followed by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering with BioNumerics software (version 6.1, Applied Maths) using the Dice coefficient [3] (PFGE) or neighbour-joining (gyrB and nrdA clustering). 3. Results 3.1. Metadata One hundred and seventy-six isolates were included in the final analysis. Of these, 88 (50.8%) and 85 (47.5%) were isolated from male and female patients respectively, with 3 (1.7%) from an undisclosed gender. Patients’ ages ranged from 1 to 75 years (median
Fig. 1. Dendrogram showing (a) Prevalence of different Achromobacter species and (b) Pandoraea and Ralstonia species, submitted to the survey. The evolutionary history was inferred using the Neighbour-Joining method [7]. The optimal tree with the sum of branch length = 0.553 for (a) and 0.887 for (b) is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA7 [8]. † Coward et al., 2016. Examples of all nrdA/gyrB sequences for putative novel clusters/species have been submitted to Genbank under the accession numbers: MH809486, MH809487, MH809488, MH809489, MH809490, MH809491, MH809492, MH809493, MH809494, MH809495, MH809496, MH809497, MH809498, MH809499, MH809500, MH809501 and MH809502. ∗ One isolate of Pandoraea could only be identified to genus level, as it did not cluster with any currently named species or with any other isolate on our database. ‡ Recently identified as belonging to the newly described Pandoraea fibrosis [5].
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Fig. 1. Continued
21 years), with 42 (24%) patients aged ≤16 years, while 134 (76%) were CF adults. Most isolates were from sputum (n = 152; 86.4%), 20 (11.4%) were from cough swabs and 2 (1.1%) from bronchoalveolar lavage (BAL). For 2 (1.1%) isolates the isolation site was undisclosed. For 149 isolates where this information was provided, 119 (80%) were isolated during a routine outpatient review or home visit and 30 (20%) were isolated from inpatients. Where information was provided, CF health status was evaluated; 42/95 (44.2%) patients provided isolates during review appointments and were categorised as stable, while 18/95 (19%) isolates (from 17 adults and 1 paediatric patient) were from pulmonary exacerbations. Of these, 12 patients had co-infections and/or co-morbidities, while for the remaining 6 this information was not provided/unknown.
3.2. Species prevalence Overall, 138 isolates were identified as Achromobacter spp. (78.4%), 29 isolates as Pandoraea spp. (16.5%) and 9 isolates as Ralstonia spp. (5.1%). The most common species within the Achromobacter genus were A. xylosoxidans, A. dolens and Achromobacter sp. “cluster II” [1]. Two novel clusters were designated Achromobacter sp. clusters IV and V, one of which (IV) corresponds to the recently described A. deleyi [4]. The most common Pandoraea species were P. apista and P. sputorum. Five novel clusters were identified;
Pandoraea sp. clusters III, IV, V, VI and VII, one of which (IV) corresponds to the newly-described P. fibrosis [5]. Finally, 8 of 9 Ralstonia isolates were identified as R. mannitolilytica. The remaining Ralstonia isolate did not cluster with any currently described type strain [6] (Fig. 1). All organisms isolated during an exacerbation (n = 18) were identified as Achromobacter species. Thirteen (72%) of these were A. xylosoxidans. The organisms isolated during outpatient review (n = 42) were more diverse, with examples from all three genera, however 18 (43%) were identified as A. xylosoxidans.
3.3. Antimicrobial susceptibility Susceptibility testing was performed by the referring hospitals, with results reported for 101 isolates (57.4%). Of the Achromobacter species isolates, most (85.9–97%) were resistant to amikacin, aztreonam, ciprofloxacin, gentamicin and tobramycin, although results were submitted for an average of 51% of isolates (Table 1). Most isolates screened were susceptible to imipenem and piperacillin/tazobactam (87.5% and 88.6%, respectively). Although results were only available for an average of 28% of isolates submitted, most Pandoraea isolates (90.9–100%) were reported resistant to amikacin, aztreonam, ceftazidime, ciprofloxacin, colistin, gentamicin, meropenem and tobramycin. Results were reported for two R. mannitolilytica isolates which were resistant to amikacin, aztreonam, ceftazidime, colistin, gentamicin, meropenem, ticar-
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Table 1 Summary of the antimicrobial susceptibility data provided by the participating hospital laboratories for (a) Achromobacter species and (b) Pandoraea species.a (a) Achromobacter sp. Antimicrobial
Susceptible (%)
Intermediate (%)
Resistant (%)
Total (% of all Achromobacter spp.)
Amikacin Aztreonam Ceftazidime Ciprofloxacin Colistinc Co-trimoxazole Gentamicin Imipenem Meropenem Minocycline Pip/Tazb Ticarcillin/clavulanate Tobramycin
5 (7.8) 1 (1.5) 36 (44.4) 3 (3.7) 54 (73) 12 (66.6) 3 (4.6) 14 (87.5) 44 (54.3) 9 (60) 62 (88.6) 16 (57.1) 10 (12.5)
4 1 0 2 0 0 1 1 2 1 0 0 0
55 (85.9) 63 (97) 45 (55.6) 76 (93.8) 20 (27) 6 (33.3) 61 (93.8) 1 (6.25) 35 (43.2) 5 (33.3) 8 (11.4) 12 (42.9) 70 (87.5)
64 65 81 81 74 18 65 16 81 15 70 28 80
(b) Pandoraea sp. Antimicrobial
Susceptible (%)
Intermediate (%)
Resistant (%)
Total (% of all Pandoraea spp.)
Amikacin Aztreonam Ceftazidime Ciprofloxacin Colistinc Co-trimoxazole Gentamicin Imipenem Meropenem Minocycline Pip/Tazb TIcarcillin/clavulanate Tobramycin
0 0 0 0 1 6 0 6 0 5 3 0 0
0 0 0 1 0 0 0 0 0 0 0 0 0
6 (100) 7 (100) 11 (100) 10 (90.9) 10 (90.9) 0 (0) 3 (100) 1 (14.3) 10 (100) 2 (28.5) 5 (62.5) 1 (100) 11 (100)
6 (20.7) 7 (24.1) 11 (37.9) 11 (37.9) 11 (37.9) 6 (20.7) 3 (10.3) 7 (24.1) 10 (34.5) 7 (24.1) 8 (27.6) 1 (3.4) 11 (37.9)
(0) (0) (0) (0) (9.1) (100) (0) (85.7) (0) (71.4) (37.5) (0) (0)
(6.3) (1.5) (0) (2.5) (0) (0) (1.5) (6.25) (2.5) (6.6) (0) (0) (0)
(0) (0) (0) (9.1) (0) (0) (0) (0) (0) (0) (0) (0) (0)
(46.4) (47.1) (58.7) (58.7) (53.6) (13) (47.1) (11.6) (58.7) (10.9) (50.7) (20.3) (58)
a Susceptibility testing methods and interpretation varied between laboratories in part due to the lack of available genus-specific European Committee on Antimicrobial Susceptibility Testing guidelines (EUCAST; http://www.eucast.org). EUCAST Pseudomonas susceptibility testing guidelines were used to interpret antibiotic sensitivities for certain antimicrobials in some hospital laboratories; i.e. for ceftazidime, aztreonam, ciprofloxacin, tobramycin, piperacillin/tazobactam, imipenem and meropenem, as available from EUCAST in 2015. b Piperacillin/Tazobactam. c Colistin resistance may be underrepresented due to the limitations of disc diffusion/gradient diffusion. This may also be reflected in the one susceptible Pandoraea isolate, as intrinsic resistance of this genus to this antibiotic has been reported [9].
cillin/clavulanate and tobramycin. One isolate was susceptible to ciprofloxacin and piperacillin/tazobactam. 3.4. PFGE Genotyping revealed considerable diversity across all species. Approximately 68% of Achromobacter species, 60% of Pandoraea species and 89% of Ralstonia species had unique profiles, clustering with less than 80% similarity to isolates in PHE’s databases (comprising profiles for Achromobacter isolates from 359 patients from 32 hospitals, Pandoraea species from 88 patients from 23 hospitals, and Ralstonia isolates from 32 patients from seven hospitals). Highly similar isolates between a maximum of three patients per centre were found for Achromobacter (6 centres), Pandoraea (2 centres) and Ralstonia (1 centre). Two novel A. xylosoxidans ‘clusters’ were found; Cluster A (n = 8 from 6 centres) had similar profiles, despite being from geographically distinct locations. Cluster B (n = 6 from 2 centres) were almost identical and comprised isolates from five patients from the same hospital, and one from a separate centre. Likewise, a ‘cluster’ of highly similar P. apista isolates from the same geographical location (Cluster C, n = 8 from 2 centres), previously identified through reference laboratory submissions [10] was also noted (Fig. 2). 4. Discussion Our survey focussed on three genera considered to be emerging pathogens in CF lung disease [11–14]. As a national CF reference laboratory, we routinely receive isolates of these genera, but only from certain centres. In this survey the inclusion of six hospitals that do not routinely submit these genera to PHE highlighted
the presence of putative novel Achromobacter and Pandoraea species and confirmed the previously reported dominance of A. xylosoxidans among UK CF patients [1]. A higher prevalence of Achromobacter with respect to Pandoraea and Ralstonia was found, broadly mirroring available CF data. Achromobacter prevalence has been found to vary widely between countries, with estimations of between 3 and 30% [14,15]. Pandoraea and Ralstonia data are limited, although one study reported a Pandoraea prevalence of 0.17 and an incidence of 0.1 for 100 CF patients [9], and another increasing Ralstonia sp. isolation from 0.6% in 2008 to 2.4% in 2016 [6]. There are currently no national guidelines for managing CF patients harbouring these organisms, and susceptibility testing guidelines are insufficient. The high level of antimicrobial resistance was therefore noteworthy, particularly across the Pandoraea genus. Indeed, of eight Achromobacter and seven Pandoraea CF isolates recently submitted to PHE, newer combinations ceftazidimeavibactam and ceftolozane-tazobactam showed limited activity, with only one Achromobacter isolate susceptible to ceftazidimeavibactam (MIC 4 mg/L), as interpreted using EUCAST non speciesspecific PK-PD breakpoints (AMRHAI, unpublished data). However, hospitals participating in this survey reported co-trimoxazole activity against limited numbers of Achromobacter and Pandoraea, as recently described by others [16]. Extensive strain diversity was found across the three genera, but three clusters of highly-related strains were noted. A. xylosoxidans cluster A comprised isolates from six hospitals, suggesting the existence of a well-adapted common clone, as recently described in a French CF study [17]. A. xylosoxidans cluster B seemed likely to represent transmission among 5 patients attending the same
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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Fig. 2. Dendrograms of the XbaI-digested genomic DNA PFGE profiles of (a) Cluster A (A. xylosoxidans), (b) Cluster B (A. xylosoxidans), and (c) Cluster C (P. apista). Each isolate was from a different patient. Isolates were coded by the region of the submitting hospital, (N = North, S = South, GL = Greater London), a number within that region, and patient number within that hospital.
hospital. Apart from small numbers of shared strains, direct transmission of Achromobacter sp. between CF patients has not been widely reported. However, two studies described the circulation of a common A. xylosoxidans clone among 21 and 22 CF patients respectively, although in both, direct patient-patient transmission was not always easy to prove [14,15]. Few CF studies have described shared Pandoraea sp. strains. One reported a shared P. pulmonicola strain between six patients, likely to be due to droplet-spread [18], while another described six patients harbouring the same strain, likely due to patientto-patient transmission either within clinics or social gatherings [19]. In this survey, recent whole genome sequencing of the P. apista cluster found it was particularly (although not exclusively)
prevalent in the north of the UK, with some evidence of patient transmission [10]. Although perhaps reflecting its prevalence, it was interesting that Achromobacter was the only genus isolated from patients with pulmonary exacerbation, particularly with respect to a recent study demonstrating an association between primary CF Achromobacter infection and exacerbation risk [20]. In this survey however, 17 of 18 affected patients were adults, of which at least 12 had other co-infections/co-morbidities, suggesting more advanced CF disease with increased likelihood of exacerbation, perhaps unrelated to Achromobacter infection. This survey has enhanced our knowledge of the population structure, antimicrobial resistance levels and prevalence of these
Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005
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genera among CF patients in England. However, much remains to be learned about their role in CF lung disease. Funding This study used PHE reference laboratory funding and did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest The authors have no personal conflicts of interest to declare. However, PHE’s AMRHAI Reference Unit has received financial support for conference attendance, lectures, research projects or contracted evaluations from numerous sources, including: Accelerate Diagnostics, Achaogen Inc, Allecra Therapeutics, Amplex, AstraZeneca UK Ltd, Basilea Pharmaceutica, Becton Dickinson Diagnostics, BioMérieux, Bio-Rad Laboratories, The British Society for Antimicrobial Chemotherapy (BSAC), Cepheid, Check-Points B.V, Cubist Pharmaceuticals, Department of Health, Enigma Diagnostics Ltd., Food Standards Agency, Glaxo Smithkline Services Ltd, Henry Stewart Talks, IHMA Ltd, Kalidex Pharmaceuticals, Melinta Therapeutics, Merck Sharpe & Dohme Corp, Meiji Seika Pharma Co., Ltd, Mobidiag, Momentum Biosciences Ltd., Nordic Pharma Ltd., Norgine Pharmaceuticals, Rempex Pharmaceuticals Ltd, Roche, Rokitan Ltd, Smith & Nephew UK Ltd, Trius Therapeutics, VenatoRx and Wockhardt Ltd. Acknowledgments We would like to thank staff at Public Health England’s Genomic Services and Development Unit for sequencing services and Daniel Lilley for help with identification by nrdA/gyrB sequence cluster analysis. We are also grateful to Katie Hopkins and Rachel Pike of PHE’s Antimicrobial Resistance and Mechanisms service for guidance, and the use of ceftazidime-avibactam and ceftolozanetazobactam data. References [1] Coward A, Kenna DTD, Perry C, Martin K, Doumith M, Turton JF. Use of nrdA gene sequence clustering to estimate the prevalence of different Achromobacter species among cystic fibrosis patients in the UK. J Cyst Fibros 2016;15(4):479–85. [2] Kenna DTD, Lilley D, Coward A, Martin K, Perry C, Pike R, Hill R, Turton JF. Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients. J Med Microbiol 2017;66(4):490–501.
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Please cite this article as: A. Coward, D.T.D. Kenna and N. Woodford et al., Structured surveillance of Achromobacter, Pandoraea and Ralstonia species from patients in England with cystic fibrosis, Journal of Cystic Fibrosis, https://doi.org/10.1016/j.jcf.2019.11.005