Journal of Cystic Fibrosis 12 (2013) 298–301 www.elsevier.com/locate/jcf

Short Communication Identification and distribution of Achromobacter species in cystic fibrosis ⁎ Theodore Spilker a, Peter Vandamme b, John J. LiPuma a,

a Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, MI, USA b Laboratory of Microbiology, Ghent University, Ghent, Belgium

Received 20 August 2012; received in revised form 3 October 2012; accepted 6 October 2012 Available online 7 November 2012

Abstract

Background: We recently described a multilocus sequence typing scheme for Achromobacter that identified several novel species in this genus. Methods: We assessed the ability of nrdA sequence analysis to differentiate Achromobacter species, including the seven previously named species and 14 recently described genogroups. Confirmation of distinctness between groups was confirmed using the k parameter. Using this single locus sequence to differentiate species, we analyzed Achromobacter isolates obtained from 341 CF patients in the U.S. Results: We found that Achromobacter xylosoxidans accounts for 42% of Achromobacter infections, while accounted for 23.5% of infections. Isolates from 17% of patients were members of the novel genogroup 14. The remaining 17.5% of strains belonged to 11 other species/genogroups. Conclusion: The use of nrdA sequence analysis allows differentiation of the several Achromobacter species that can infect persons with CF. Achromobacter species other than A. xylosoxidans account for the majority of Achromobacter infection in CF patients in the U.S. © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Keywords: Achromobacter; Infection; ; Clinical microbiology

1. Introduction noted that sequence analysis of a single locus in the MLST scheme, nrdA, appeared to be able to distinguish these 21 Achromobacter xylosoxidans is well recognized as an taxa. We further noted that approximately half of the opportunistic pathogen infecting persons with cystic fibrosis Achromobacter isolates included in the study, the great (CF) [1]. The remaining six currently named Achromobacter majority of which had been recovered from CF and non-CF species, including Achromobacter ruhlandii, Achromobacter clinical sources, were species other than A. xylosoxidans.In piechaudii, Achromobacter denitrificans, , the current study, we sought to validate the use of nrdA Achromobacter insolitus and Achromobacter marplatensis,are sequence as a means to differentiate Achromobacter species inhabitants of soil and have been much less frequently associated and to determine the distribution of these species among with human infection [2–5]. Achromobacter-infected CF patients in the U.S. We recently developed a multi-locus sequence typing (MLST) scheme that, in addition to clearly distinguishing 2. Methods these seven Achromobacter species, identified an additional 14 novel genogroups, each of which is genetically distinct 2.1. nrdA analysis enough to represent a novel species [6]. In that study, we Amplification and DNA sequencing of nrdA was performed ⁎ Corresponding author at: Department of Pediatrics and Communicable Diseases, as described [6]. Sequences were trimmed to 765 bp and aligned University of Michigan Medical School, 8323 MSRB III, SPC 5646, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0646, USA. Tel.: +1 734 936 9767; using MegAlign (DNASTAR, Madison, WI, USA) with Clustal fax: +1 734 764 4279. W to generate a dendrogram with 1000 bootstrap replications E-mail address: [email protected] (J.J. LiPuma). using the default parameters in MegAlign. All nrdA sequences

1569-1993/$ -see front matter © 2012 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcf.2012.10.002 T. Spilker et al. / Journal of Cystic Fibrosis 12 (2013) 298–301 299 can be found at the PubMLST site at http://pubmlst.org/ from the full MLST scheme as we described previously [6]. In that achromobacter [7]. For validation of distinctness of sequence analysis, only genogroup 5 could not be shown to be distinct; similarity clusters, the k parameter was calculated [8].This but, this may be a function of the next closest group (genogroup metric represents the distinctness of populations, expressed 7) containing only two strains. It is not clear whether or not k as a ratio (k) of the expected divergence between populations parameters would support distinct groups with the addition of to the expected divergence within a population. The expected more strains. Nevertheless, all three groups segregate into divergence within populations is determined largely by the distinct clusters in the three trees presented in the Supplemental diversity-purging effect of periodic selection, which is in turn Information. determined by the rate of recombination within populations Analysis of nrdA sequences demonstrated that A. xylosoxidans and the intensity of the periodic selection. The k parameter is accounted for only 143 (42%) of Achromobacter infection in our calculated as the ratio of the mean inter-group sequence cohort of 341 CF patients (Fig. 1). A. ruhlandii accounted for divergence to the mean intra-group sequence divergence. A k 23.5% of Achromobacter infection, while 17% of the strains parameter greater than 2 indicates that a group is distinct from were genogroup 14. We identified no individuals infected with its closest neighbor. A. marplatensis, A. piechaudii, or genogroups 1, 9, 10, 11 or 12.

2.2. Species distribution 4. Discussion

A review of isolates referred to the Burkholderia cepacia Our previous work developing a genus-level MLST scheme for Research Laboratory and Repository (University of Michigan, Achromobacter, based on loci from seven housekeeping genes USA) identified 341 CF patients from whom Achromobacter had demonstrated several novel genogroups in this genus [6]. been recovered from respiratory specimens. Isolates had been Phylogenetic analyses provided compelling evidence that these referred from 86 CF treatment centers in the U.S. between 1998 genogroups represented novel Achromobacter species; this is and 2012. A single isolate from each patient was included in this supported by on-going comprehensive taxonomic studies, includ- study. In cases where multiple Achromobacter isolates were ing DNA–DNA hybridizations (manuscripts in preparation). In a available from the same patient, the first isolate received was similar recent study, Ridderberg et al. [9] employed multilocus used. Fifty-five of the 341 isolates had previously been analyzed sequence analysis (MLSA) based on five housekeeping genes by the recently described MLST scheme [6].Fortheremaining (with only one, rpoB, in common with our MLST scheme) to study 286 isolates, only nrdA was amplified and sequenced. a collection of 77 Achromobacter strains, including type strains of the seven recognized species, as well as environmental and clinical 3. Results strains originating in Europe, Asia, and South America. They too were able to differentiate the current Achromobacter species and As expected, among the set of 55 isolates for which both nrdA identified four novel genogroups. sequence and MLST analyses had been performed, fewer nrdA In our analyses, and as reported by Ridderberg et al. [9], alleles (n=39) were detected than were MLST sequence types sequence analysis of the bacterial 16S rRNA gene was inadequate (n=55). in differentiating novel and established Achromobacter species. A dendrogram based on the full complement of concatenat- However, in developing our MLST scheme, we noted that another ed MLST loci (2249 bp) from 47 Achromobacter strains locus, nrdA, appeared capable of clustering strains into species- (representing strains from each species, genogroup, or subgroup) level groups. This was supported by the comparison of a was compared to the dendrogram based on nrdA sequence alone dendrogram based on a 765 bp internal nrdA fragment with a (765 bp) for these same 47 strains (Supplemental Fig. 1). The tree composed of the 2249 bp concatenated MLST sequences. separation of strains into distinct groups in the nrdA tree matched Although, as expected, the topology of the trees differed, since the separation of strains into the 21 species and genogroups found nrdA sequence alone is more limited in providing phylogenetic in the concatenated MLST tree. Bootstrap values supported this inferences relative to the full MLST sequence data, both trees clustering in the nrdA sequence tree. A dendrogram with clustered strains into groups representing all 21 Achromobacter bootstrap support of clusters showing all unique nrdA sequence species and genogroups. Phylogenetic analyses, including k types identified among isolates from the 341 CF patients is parameter calculation and bootstrapping analyses, supported the provided in the Supplemental Information. utility of the nrdA locus in differentiating most groups. Increasing Similar to the results we obtained previously by using the numbers of strains analyzed may increase the k parameters for concatenated MLST sequences [6], k parameter calculations the three groups with low levels of distinctness based on this single indicated that nrdA sequence alone separated most of the 21 species locus. and genogroups into genetically distinct groups; k parameters Having established that nrdA sequence analysis provides for ranged from 2.5 to 40.9 for these groups (Supplementary data species-level separation of established and novel Achromobacter Table 1). The exceptions were with genogroups 4, 5a, and species, we used this locus to determine the identity of isolates A. ruhlandii, which were not found to be distinct from their closest recovered from all individuals from whom we had previously neighbors (k= 1.3, 1.6 and 1.9, respectively). It should be noted, confirmed the recovery of Achromobacter. We limited this set to however, that both genogroup 4 and A. ruhlandii wereshowntobe individuals who were registered in the Cystic Fibrosis Founda- distinct groups based on the k parameters calculated using data tion Patient Registry [10], thereby confirming that each, in fact, 300 T. Spilker et al. / Journal of Cystic Fibrosis 12 (2013) 298–301

543 2 2 1 A. xylosoxidans 1 1 15 13 13 A. ruhlandii 58 A. genogroup 14 A. genogroup 2 A. genogroup 5 A. insolitus A. genogroup 3 143 80 A. genogroup 4 A. genogroup 6 A. genogroup 7 A. genogroup 8 A. denitrificans A. genogroup 13 A. spanius

Fig. 1. Distribution of Achromobacter species/genogroups isolated from 341 CF patients. Achromobacter species/genogroups are listed top to bottom in order from most to least abundant. The numbers in each wedge indicate the number of patients infected with the species/genogroup.

had CF. Essentially all isolates from these patients had been Acknowledgments identified as A. xylosoxidans by commercial test systems in the referring laboratories. We found, however, that this species This work was supported by the Cystic Fibrosis Foundation. accounted for the minority (42%) of Achromobacter infection in U.S. CF patients. A. xylosoxidans accounted for 64% of infection Appendix A. Supplementary data in the 56 CF patients studied by Ridderberg et al. [9]. A. ruhlandii was found in nearly one-quarter of our patients. Supplementary data to this article can be found online at It is clear that commercial test systems do not identify this http://dx.doi.org/10.1016/j.jcf.2012.10.002. species, which was formally described in 1998 [11]. Of interest, Ridderberg et al. [9] found that the Danish epidemic strain (DES), an exceptionally resistant Achromobacter clone causing References infection in Copenhagen [12] and Aarhus [13], is identified as A. ruhlandii based on their MLSA scheme. [1] LiPuma JJ. The changing microbial epidemiology in cystic fibrosis. Clin We found that 17% of patients were infected by a novel Microbiol Rev 2010;23:299-323. Achromobacter genogroup (genogroup 14), while Ridderberg [2] Coenye T, Vancanneyt M, Cnockaert MC, Falsen E, Swings J, Vandamme P. Kerstersia gyiorum gen. nov., sp. nov., a novel Alcaligenes noted 18% of patients were infected with a novel species (their faecalis-like organism isolated from human clinical samples, and MLSA cluster III). Our preliminary analysis suggests that these reclassification of Alcaligenes denitrificans Ruger and Tan 1983 as two groups do not represent the same species. We further found Achromobacter denitrificans comb. nov. Int J Syst Evol Microbiol that only six of the 21 Achromobacter species/genogroups 2003;53:1825-31. (A. xylosoxidans, A. ruhlandii, A. insolitus, and genogroups 14, [3] Coenye T, Vancanneyt M, Falsen E, Swings J, Vandamme P. Achromobacter insolitus sp. nov. and Achromobacter spanius sp. nov., 2a, 2b, 5a and 5b) account for 94% of Achromobacter infection from human clinical samples. Int J Syst Evol Microbiol 2003;53:1819-24. in CF patients in the U.S. Of note, we identified no patients [4] Gomila M, Tvrzová L, Teshim A, Sedlácek I, González-Escalona N, infected with A. marplatensis, a recently described species that is Zdráhal Z, et al. Achromobacter marplatensis sp. nov., isolated from a phylogenetically very closely related to A. xylosoxidans [4,6]. pentachlorophenol-contaminated soil. Int J Syst Evol Microbiol 2011;61: This observation suggests that despite their very close genetic 2231-7. [5] Kiredjian M, Holmes B, Kersters K, Guilvout I De, Ley J. Alcaligenes relatedness these two species differ in their capacity for human piechaudii, a new species from human clinical specimens and the infection (and/or their biogeography), suggesting that a compar- environment. Int J Syst Evol Microbiol 1986;36:282-7. ative study of these two taxa may prove useful in elucidating [6] Spilker T, Vandamme P, LiPuma JJ. An Achromobacter multilocus factors involved in A. xylosoxidans pathogenicity. sequence typing scheme infers population structure and reveals several Most studies addressing the impact of Achromobacter putative novel Achromobacter species. J Clin Microbiol Jul. 11 2012;50: 3010-5 [Epub ahead of print]. infection on clinical outcomes in CF have concluded that [7] Jolley KA, Maiden MCJ. BIGSdb: scalable analysis of bacterial genome infection does not appreciably seem to affect clinical course variation at the population level. BMC Bioinformatics 2010;11:595. [14–16]. Nevertheless, it appears that some specific clones are [8] Palys T, Nakamura LK, Cohan FM. Discovery and classification of associated with increased rates of pulmonary decline [12,17,18]. ecological diversity in the bacterial world: the role of DNA sequence data. The recognition that several distinct Achromobacter species are Int J Syst Bacteriol 1997;47:1145-56. [9] Ridderberg W, Wang M, Nørskov-Lauritsen N. Multilocus sequence responsible for infection in CF patients now provides an analysis of isolates of Achromobacter from patients with cystic fibrosis opportunity to determine whether certain species are more likely reveals infecting species other than Achromobacter xylosoxidans. J Clin associated with poor outcome than others. Microbiol 2012;50:2688-94. T. Spilker et al. / Journal of Cystic Fibrosis 12 (2013) 298–301 301

[10] Cystic Fibrosis Foundation. Cystic Fibrosis Foundation patient registry. [14] De Baets F, Schelstraete P, Van Daele S, Haerynck F, Vaneechoutte M. Annual data report. Bethesda, Maryland: Cystic Fibrosis Foundation; Achromobacter xylosoxidans in cystic fibrosis: prevalence and clinical 2010. relevance. J Cyst Fibros 2007;6:75-8. [11] Yabuuchi E, Kawamura Y, Kosako Y, Ezaki T. Emendation of genus [15] Raso T, Bianco O, Grosso B, Zucca M, Savoia D. Achromobacter Achromobacter and Achromobacter xylosoxidans (Yabuuchi and Yano) xylosoxidans respiratory tract infections in cystic fibrosis patients. APMIS and proposal of Achromobacter ruhlandii (Packer and Vishniac) comb. 2008;116:837-41. nov., (Kiredjian et al.) comb. nov., and [16] Lambiase A, Catania MR, Del Pezzo M, Rossano F, Terlizzi V, Sepe A, Achromobacter xylosoxidans subsp. denitrificans (Rüger and Tan) comb. et al. Achromobacter xylosoxidans respiratory tract infection in cystic nov. Microbiol Immunol 1998;42:429-38. fibrosis patients. Eur J Clin Microbiol Infect Dis 2011;30:973-80. [12] Rønne Hansen C, Pressler T, Høiby N, Gormsen M. Chronic infection [17] McPhail GL, VanDyke R, Fenchel M, LiPuma JJ, Joseph PM. An update with Achromobacter xylosoxidans in cystic fibrosis patients; a retrospec- on clinical outcomes associated with a clonal strain of Achromobacter tive case control study. J Cyst Fibros 2006;5:245-51. (Alcaligenes) xylosoxidans. Pediatr Pulm 2009(Suppl. 32):310. [13] Ridderberg W, Bendstrup KE, Olesen HV, Jensen-Fangel S, Nørskov- [18] Magni A, Trancassini M, Varesi P, Iebba V, Curci A, Pecoraro C, et al. Lauritsen N. Marked increase in incidence of Achromobacter xylosoxidans Achromobacter xylosoxidans genomic characterization and correlation of infections caused by sporadic acquisition from the environment. J Cyst randomly amplified polymorphic DNA profiles with relevant clinical Fibros 2011;10:466-9. features of cystic fibrosis patients. J Clin Microbiol 2010;48:1035-9.