Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively

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Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively夽 Peter Vandamme a,∗ , Edward R.B. Moore b , Margo Cnockaert a , Charlotte Peeters a , Liselott Svensson-Stadler b , Kurt Houf c , Theodore Spilker d , John J. LiPuma d a

Laboratorium voor Microbiologie, Vakgroep Biochemie en microbiologie, Faculteit Wetenschappen, Universiteit Gent, Gent, Belgium Culture Collection University of Gothenburg (CCUG), Department of Clinical Bacteriology, Sahlgrenska University Hospital, Gothenburg, Sweden c Vakgroep Veterinaire Volksgezondheid en Voedselveiligheid, Faculteit Diergeneeskunde, Universiteit Gent, Gent, Belgium d Department of Pediatrics and Communicable Disease, University of Michigan Medical School, Ann Arbor, MI 48109, USA b

a r t i c l e

i n f o

Article history: Received 4 April 2013 Received in revised form 14 June 2013 Accepted 21 June 2013 Keywords: Achromobacter Multilocus sequence analysis Cystic fibrosis

a b s t r a c t The phenotypic and genotypic characteristics of seventeen Achromobacter strains representing MLST genogroups 2, 5, 7 and 14 were examined. Although genogroup 2 and 14 strains shared a DNA–DNA hybridization level of about 70%, the type strains of both genogroups differed in numerous biochemical characteristics and all genogroup 2 and 14 strains could by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence which identified genogroups 2 and 14 as clearly distinct populations, the availability of nrdA sequence analysis as a single locus identification tool for all Achromobacter species and genogroups, and the differential phenotypic characteristics, we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845T [= CCUG 62426T ] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852T [= CCUG 62438T ] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857T [= CCUG 62444T ] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840T [= CCUG 62421T ] as the type strain). © 2013 Published by Elsevier GmbH.

Introduction Recent multilocus sequence typing (MLST) studies revealed a marked diversity among Achromobacter strains [9,11] and demonstrated the presence of 14 unnamed genogroups among 150 Achromobacter strains. Four of these genogroups were recently formally described and named as the novel species Achromobacter mucicolens, Achromobacter animicus, Achromobacter spiritinus and Achromobacter pulmonis [16]. In a study of the distribution of these novel species among Achromobacter-infected cystic fibrosis patients in the U.S. we demonstrated that Achromobacter xylosoxidans accounted for only 42% of Achromobacter infection [12]. Surprisingly, Achromobacter ruhlandii (23.5%), and isolates of the

夽 Genbank accession number: The GenBank accession numbers for the 16S rRNA gene sequence of Achromobacter strains LMG 26845T , LMG 26852T , LMG 26857T and LMG 26840T are HF586506 through HF586509, respectively. All MLST sequences are available from the Achromobacter database at http://pubmlst.org/. ∗ Corresponding author. Tel.: +32 9 264 5113; fax: +32 9 264 5092. E-mail address: [email protected] (P. Vandamme).

novel genogroups 14 (17%), 2 (4.4%) and 5 (3.8%) represented the other main fractions of Achromobacter infection in cystic fibrosis patients in the U.S. The present study describes the phenotypic and genotypic characteristics of the genogroups 2, 5 and 14, and of genogroup 7, which is the nearest phylogenetic neighbor of genogroup 5 as defined by nucleotide divergence of concatenated MLST loci [11]. Materials and methods Isolation, morphological, physiological and biochemical characteristics Achromobacter reference strains are as described in Vandamme et al. [16]; strains representing novel species, and their sources of isolation are listed in Table 1. The latter strains include three genogroup 2a and 2b strains each, three genogroup 5a and 5b strains each, two genogroup 7 strains and three genogroup 14 strains [11]. Strains were grown aerobically on trypticase soy agar (TSA) (BBL) at 28 ◦ C unless otherwise indicated. Conventional

0723-2020/$ – see front matter © 2013 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.syapm.2013.06.005

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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2 Table 1 Strains included in the present study. Strain Achromobacter insuavis Genogroup 2a LMG 26844 LMG 26845T LMG 26846 Genogroup 2b LMG 7054 LMG 26847 LMG 26848

Other strain designations

ST

Received from

Source

CCUG 62425, 8910069 CCUG 62426T , AU15210T CCUG 62427, AU18627

144 60 59

J. Baron Own isolate Own isolate

Human sputum, cystic fibrosis patient (2002, Spain) Human sputum, non-cystic fibrosis patient (2008, USA) Human sputum, non-cystic fibrosis patient (2009, USA)

CCUG 62428, CL275/75 CCUG 47223 CCUG 62429, AU18552

147 145 61

NCTC Own isolate Own isolate

Swimming pool Human sputum, cystic fibrosis patient (2002, Sweden) Human sputum, cystic fibrosis patient (2009, USA)

146 81 76

Own isolate Own isolate Own isolate

Human sputum (2002, USA) Human sputum, cystic fibrosis patient (2007, USA) Human sputum, non-cystic fibrosis patient (2003, USA)

82 86 143

Own isolate Own isolate Own isolate

Human sputum, non-cystic fibrosis patient (2004, USA) Human sputum, cystic fibrosis patient (2010, USA) Drinking water reservoir (Belgium)

100 101

Own isolate Own isolate

Human sputum, cystic fibrosis patient (2006, USA) Sludge (2002, Belgium)

49 48 54

Own isolate Own isolate Own isolate

Human sputum, non-cystic fibrosis patient (2002, USA) Human sputum, cystic fibrosis patient (2005, USA) Non-cystic fibrosis patient (2008, USA)

Achromobacter aegrifaciens Genogroup 5a CCUG 62438T , AU4014T LMG 26852T CCUG 62439, AU14141 LMG 26853 CCUG 62440, AU6228 LMG 26854 Genogroup 5b CCUG 62441, AU6685 LMG 26855 CCUG 62442, AU20523 LMG 26856 CCUG 62443 LMG 11300 Achromobacter anxifer (genogroup 7) LMG 26857T CCUG 62444T , AU11412T LMG 26858 CCUG 62445, R-17672 Achromobacter dolens (genogroup 14) CCUG 62421T , AU4012T LMG 26840T CCUG 62422, AU8628 LMG 26841 LMG 26842 CCUG 62423, HI4316

Abbreviations: CCUG, Culture Collection University of Gothenburg, Göteborg, Sweden; LMG, BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Gent, Belgium; ST, sequence type.

phenotypic tests were performed as described previously [15]. API 20NE, API ZYM and Biolog GEN III microtest systems were performed according to the recommendations of the manufacturer; however, Biolog GEN III test results were scored as described before [16]. Electron microscopic analysis of strains LMG 26840T , LMG 26845T , LMG 26852T and LMG 26857T was performed as described before [16]. Cellular fatty acid analysis For cellular fatty acid methyl ester (FAME) analysis, strains were incubated for 24 h at 28 ◦ C. A loopful of well-grown cells was harvested and fatty acid methyl esters were prepared as described previously [18], and separated and identified using the Sherlock Microbial Identification System (version 3.1, MIDI Inc.). 16S rRNA gene sequencing, MLST, DNA–DNA hybridization and DNA G+C content analysis DNA for performing PCR assays was prepared by heating one or two colonies at 95 ◦ C for 15 min in 20 ␮l lysis buffer containing 0.25% (w/v) SDS and 0.05 M NaOH. Following lysis, 180 ␮l distilled water was added to the lysate. The 16S rRNA gene sequences of strains LMG 26840T , LMG 26845T , LMG 26853T and LMG 26857T were determined; 16S rRNA gene amplification, purification and sequencing were performed as described by Vandamme et al. [17]. Sequence assembly was performed using the BioNumerics 4.61 software. Sequences (1318–1508 bp) of these strains and those of type strains of all established Achromobacter species were aligned against the Silva reference database (www.arb-silva.de) using the mothur software [10]. These aligned sequences were imported into the software package MEGA (Molecular Evolutionary Genetics Analysis) version 5.0 [14] and analyzed using the neighbor-joining, maximum-likelihood and maximum-parsimony methods. All positions with less than 95% site coverage were eliminated, resulting in a total of 1275 positions in the final dataset. Uncorrected pairwise distances were calculated using pairwise.seqs() function of

the mothur software [10]. The statistical reliability of tree topologies was evaluated by bootstrapping analysis based on 1000 tree replicates. Amplification and sequencing of nusA, eno, rpoB, gltB, lepA, nuoL and nrdA gene fragments of strains LMG 7054, LMG 11300, LMG 26844, LMG 26847 and LMG 26852 was performed as described before [11]; data for all remaining strains were taken from Spilker et al. [11]. Gene number assignment to each unique allele, and assignment of sequence types to each unique allelic profile was done using the http://pubmlst.org/database tools [3]. A phylogenetic tree of the concatenated sequences (2773 bp) of seven housekeeping gene fragments [nusA (355 bp), eno (214 bp), rpoB (413 bp), gltB (241 bp), lepA (347 bp), nuoL (230 bp) and nrdA (449 bp)] was constructed using MEGA5 [14]. The tree was constructed using the Maximum Likelihood Method based on the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1650)) and allowed for some sites to be evolutionarily invariable ([+I], 31.8372% sites). Additionally, a phylogenetic tree was also constructed based on nrdA sequences (765 bp) using MEGA5. The tree was constructed using the Maximum Likelihood Method based on the General Time Reversible model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.9865)) and allowed for some sites to be evolutionarily invariable ([+I], 66.3641% sites). All sequences used are available from the PubMLST database. High-molecular weight DNA was prepared as described by Pitcher et al. [8] and DNA–DNA hybridization was performed with photobiotin-labeled probes in microplate wells as described by Ezaki et al. [1] using an HTS7000 Bio Assay Reader (Perkin-Elmer) for the fluorescence measurements. The hybridization temperature was 50 ◦ C. Analysis of the DNA base ratio of strains LMG 26840T , LMG 26845T , LMG 26852T and LMG 26857T was determined as described by Mesbah et al. [6]. DNA was enzymically degraded into nucleosides. The obtained nucleoside mixture was then separated by HPLC

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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using a Waters Symmetry Shield C8 column thermostated at 37 ◦ C. The solvent was 0.02 M NH4 H2 PO4 (pH 4.0) with 1.5% acetonitrile. Non-methylated Lambda phage (Sigma, Saint Louis, MO, USA) was used as the calibration reference. Results and discussion MLST data were available for 12 genogroup 2, 5, 7 or 14 isolates examined previously [11] and were generated for strains LMG 7054, LMG 11300, LMG 26844, LMG 26847 and LMG 26852 in the present study. The results demonstrated that each of them represented a novel sequence type and allowed us to assign them to their respective geno(sub)groups [11] (cfr. Table 1 and http://pubmlst.org/). In a previous MLST study of 150 Achromobacter strains the k parameter, which is the ratio of the intergroup divergence to the mean of the intragroup divergence, was calculated to validate the distinctness of MLST sequence similarity clusters [11]; ratios greater than 2 are considered distinct sequence similarity clusters [7]. Applying a maximum intraspecies divergence of 2.1% as a cutoff in the concatenated MLST dendrogram identified 14 unnamed genogroups, most of which had distinctness of population ratio (k) parameters ≥2. Achromobacter xylosoxidans and Achromobacter marplatensis, two validly named species, could not be considered separate sequence similarity clusters, with k values of 1.68; in addition, genogroup 5 failed to meet the distinctness of population ratio (k = 1.58) but this might have been caused by the small number of isolates of its nearest neighbor, genogroup 7, which thus far comprises only 2 isolates from different sources on different continents but representing single locus variants of one strain (Table 1). The concatenated MLST tree revealed that Achromobacter genogroups 2 and 5 contained distinct subgroups, each labeled a and b ([11] and Fig. 1). Using bootstrapping with a 1000 replicates genogroups 2a and 2b showed 100% support that distinct nodes would be maintained; similarly, genogroups 5a and 5b showed 93.8% support that distinct nodes would be maintained [11]. Both intradivergence and interdivergence levels, as well as k parameters, indicated that genogroups 2a and 2b and genogroups 5a and 5b could be considered as separate clusters, with k parameters of 3.27 and 2.62, respectively, thus providing evidence that these two genogroups contain distinct diverging subgroups. Yet, the validated use [11,16] of 2.1% concatenated nusA, eno, rpoB, gltB, lepA, nuoL and nrdA sequence divergence as a tool to discriminate Achromobacter species, indicated that genogroups 2 (including both 2a and 2b), 5 (including both 5a and 5b), 7 and 14 represented four distinct Achromobacter species. Therefore, we examined the taxonomic relationships of these bacteria further by DNA–DNA hybridization experiments. In a first experiment, reciprocal DNA–DNA hybridization experiments were performed between DNAs of strains LMG 26845T (representative of genogroup 2a), LMG 26848 (representative of genogroup 2b), LMG 26840T (representative of genogroup 14), A. xylosoxidans LMG 1863T , A. ruhlandii LMG 1866T and A. marplatensis LMG 26219T , as they are nearest neighbors as defined by divergence in concatenated MLSA sequences [11] (Table S1, Fig. 1); in a second experiment, reciprocal DNA–DNA hybridization experiments were performed between DNAs of strains LMG 26853 (representative of genogroup 5a), LMG 26856 (representative of genogroup 5b) and LMG 26857T (representative of genogroup 7), again, because they are nearest neighbors as defined by divergence in concatenated MLSA sequences [11] (Table S2, Fig. 1). The level of DNA–DNA hybridization between genogroup 2a LMG 26845T and 2b LMG 26848 was 78% indicating that they indeed represent a single species. The average of reciprocal DNA–DNA hybridization values between the genogroup 2 strains and LMG 26840T (representative of genogroup 14, its nearest phylogenetic neighbor; [11]) was 69.5% which corresponds to the threshold level for species delineation. The final classification of genogroups 2 and

3

14 should therefore be based on polyphasic taxonomic information. The averages of reciprocal DNA–DNA hybridization levels observed between the genogroup 2 strains, genogroup 14 strain LMG 26840T , A. xylosoxidans LMG 1863T and A. ruhlandii LMG 1866T were all in the range of 59–66%, i.e. just below the threshold level for species delineation. The average value of 59% between A. xylosoxidans LMG 1863T and A. ruhlandii LMG 1866T was similar to a value previously reported by Gomila et al. [2] and somewhat higher than the value reported by Yabuuchi et al. [19] (32%); also the average values of 33% and 36% between A. marplatensis LMG 26219T and A. xylosoxidans LMG 1863T and A. ruhlandii LMG 1866T , respectively, were similar to those reported by Gomila et al. [2] (38% and 40%, respectively). These high interspecies DNA–DNA hybridization levels all point to a very close genotypic relatedness of these Achromobacter species. The interpretation of DNA–DNA hybridization levels in the second experiment was more straightforward: the representatives of the genogroups 5a and 5b (LMG 26853 and LMG 26856, respectively) shared a high level of average reciprocal DNA–DNA hybridization (80%) and a clearly lower value (56%) was observed between these strains and LMG 26857T (representative of genogroup 7, its nearest phylogenetic neighbor [11]. The 16S rRNA gene sequences of strains LMG 26840T (genogroup 14), LMG 26845T (genogroup 2a), LMG 26852T (genogroup 5a), and LMG 26857T (genogroup 7) were more than 99% similar to those of the current Achromobacter species. The highest percentage similarity values were determined toward the A. xylosoxidans (99.9), A. xylosoxidans (99.7), A. denitrificans (99.6), and A. denitrificans (99.8) type strains, respectively. Fig. 2 shows a phylogenetic tree of these bacteria based on maximum-likelihood analysis of the 16S rRNA gene sequences. The bootstrap consensus tree, inferred from 1000 replicates, was constructed using the Maximum Likelihood Method based on the Tamura-Nei model [13]. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1000)) and allowed for some sites to be evolutionarily invariable ([+I], 27.5085% sites). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches if greater than 50%. The sequence of Kerstersia gyiorum LMG 5906T was used as outgroup. The scale bar indicates the number of substitutions per site. The DNA base ratio of genogroup 14 strain LMG 26840T (67.5 mol%), genogroup 2a strain LMG 26845T (68.0 mol%), genogroup 5a strain LMG 26852T (66.0 mol%) and genogroup 7 strain LMG 26857T (66.5 mol%) proved very similar. Average cellular fatty acid profiles for all Achromobacter strains are shown in Table 2. Summed features 2 (iso-C16:1 I or C14:0 3-OH, or both) and 3 (iso-C15:0 2-OH or C16:1 w7c, or both) most likely correspond to C14:0 3-OH and C16:1 w7c, respectively [16]. Achromobacter genogroup 14 strains have a distinctive fatty acid profile characterized by the presence of C14:0 2-OH and very low amounts of C18:1 w7c, which discriminate this taxon from all other Achromobacter species (Table 2 and [16]). The remaining genogroups all have very similar average fatty acid profiles (Table 2). Subsequently, cell and colony morphological and 191 biochemical characteristics were recorded for all 17 Achromobacter strains as described before [16]. The general conclusions with regard to phenotyping achromobacters, which we reported previously were further substantiated: these bacteria are biochemically poorly reactive, and there is a general paucity of species differential characteristics and a considerable intraspecies variability [4,5,16]. Most of the 119 phenotypic characteristics that we reported previously as genus specific were confirmed as such in the present study. The few exceptions were mainly observed in the tests included in the Biolog GEN III microtest system (see below). In the present study

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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Fig. 1. Maximum-likelihood phylogenetic tree based on concatenated MLSA sequences (2249 bp alignment) of Achromobacter type strains and genogroup 2, 5, 7 and 14 strains. Strains LMG 26844 through LMG 26846 are genogroup 2a, strains LMG 7054, LMG 26847 and LMG 26848 are genogroup 2b, strains LMG 26852T through LMG 26854 are genogroup 5a, and strains LMG 11300, LMG 26855 and LMG 26856 are genogroup 2b (Table 1). Bootstrap values based on 1000 replications are indicated at the nodes; values of less than 50% are not shown. The scale bar indicates the number of substitutions per site. All sequences are available from the PubMLST database.

some additional tests were uniformly present or absent: assimilation of maltose (API 20NE and classical test), l-arabinose (API 20NE) and dl-norleucine, and liquefaction of gelatine were uniformly absent, whereas assimilation of l-malate and phenylacetate, and oxidase activity (all API 20NE) and were uniformly present. Also, valine arylamidase activity was uniformly absent (API ZYM). In the Biolog GEN III microtest system, mannose was not oxidized, whereas l-lactic acid and ␣-keto-glutaric acid were uniformly

oxidized and, for the sensitivity tests, the tetrazolium redox dye was uniformly reduced in the presence of fusidic acid, troleandomycin, tetrazolium bleu, nalidixic acid and niaproof 4, but oxidation was inhibited in the presence of sodium bromate. All species or strain dependent phenotypic characteristics are shown in Table 3; these included 21 traditional biochemical tests, 8 from the API 20NE microtest system, 5 from the API ZYM microtest system, and 25 and 7 substrate oxidation and sensitivity

Table 2 Average fatty acid composition of all Achromobacter speciesa

C12:0 C12:0 2-OH C14:0 C14:0 2-OH C14:1 w5c C16:0 Cyclo-C17:0 C18:0 C18:1 w7c Summed feature 2c Summed feature 3d

A. aegrifaciens (genogroup 5) (6)b

A. anxifer (genogroup 7) (2)

A. dolens (genogroup 14) (3)

A. insuavis (genogroup 2) (6)

1.18 ± 0.88 2.94 ± 1.60 5.52 ± 1.10 ND T 32.08 ± 1.68 6.75 ± 4.15 1.22 ± 0.62 6.16 ± 3.38 9.94 ± 1.59 31.69 ± 4.54

T 3.71 ± 0.42 4.79 ± 0.23 ND ND 31.59 ± 1.15 6.70 ± 1.35 1.93 ± 0.59 7.59 ± 0.39 11.18 ± 0.40 30.37 ± 0.79

T 3.93 ± 0.85 3.43 ± 1.18 4.03 ± 1.01 1.00 ± 0.50 33.31 ± 1.71 8.49 ± 2.53 T 2.11 ± 1.56 10.43 ± 1.83 30.29 ± 1.67

T 3.42 ± 0.60 2.95 ± 1.28 1.86 ± 1.11 ND 32.19 ± 2.53 8.12 ± 5.04 3.15 ± 3.05 8.47 ± 1.41 9.73 ± 1.37 28.02 ± 4.60

a Those fatty acids for which the average amount for all taxa was less than 1% are not included. Therefore, the percentages may not add up to 100%. T, trace amount (less than 1%); ND, not detected. b Number of strains examined. c Summed feature 2 comprises iso-C16:1 I or C14:0 3-OH or both. d Summed feature 3 comprises iso-C15:0 2-OH or C16:1 w7c or both.

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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Table 3 Differential biochemical characteristics of all Achromobacter strains examined.

◦

Growth at 42 C Motility Growth in O/F medium with d-xylosec Growth on cetrimide agar Growth in the presence of in 3% NaCl Growth in the presence of in 4.5% NaCl Growth in the presence of in 6% NaCl Nitrate reduction Nitrate reduction (API 20NE) Nitrite reductionb Denitrification Growth on acetamide Assimilation of glucose Assimilation of d-glucose (API 20NE) Assimilation of d-gluconate Assimilation of d-gluconate (API 20NE) Assimilation of caprate Assimilation of caprate (API 20NE) Assimilation of adipate Assimilation of adipate (API 20NE) Assimilation of l-malate Assimilation of citrate Assimilation of phenylacetate Assimilation of sucrose Assimilation of d-mannose (API 20NE) Assimilation of d-lactate Assimilation of lactate + methionine Acid production from glucose (API 20NE) Activity of alkaline phosphatase (API ZYM) Activity of C4 -lipase (API ZYM) Activity of C8 -lipase (API ZYM) Activity of acid phosphatase (API ZYM) Activity of phosphoamidase (API ZYM) Oxidation of substrates (Biolog GEN III): ␣-d-Glucose d-Aspartic acid d-Serine Glycyl-l-proline l-Alanine l-Aspartic acid l-Glutamic acid l-Histidine l-Pyroglutamic acid l-Serine d-Gluconic acid d-Glucuronic acid Glucuronamide Muric acid d-Saccharic acid Methyl pyruvate d-Malic acid Bromo-succinic acid ␥-Amino-butyric acid ␣-Hydroxy-butyric acid ␤-Hydroxy-d,l-butyric acid ␣-Keto butyric acid Propionic acid Acetic acid Formic acid Sensitivity tests (Biolog GEN III): pH5 4% NaCl 8% NaCl d-Serine Minocycline Potassium tellurite Sodium butyrate

A. aegrifaciensa (5a 5b)b

A. anxifer (7)

A. dolens (14)

A. insuavisa (2a 2b)

−−+ −++ +++ +++ −−− −−− −−+ +++ +−w −w− +−− −−− −−− −−− +−+ +++ +−+ +++ +++ +++ −−− −−− +−+ +++ −−− −−− +−w −−− +−+ +++ +−+ +++ +−+ +++ +−+ +++ +−+ +++ +−+ +++ +−+ +++ +−+ +++ +−+ +++ −−− −−− −−− −−− +−+ +++ +−+ +++ −−− −−− +−+ w−− +++ ++w −−− −−− +++ +w+ −−− −−w

++ ++ −− ++ ++ −− −− ++ ++ ++ −− ++ −− −− ++ ++ ++ ++ ++ ++ ++ ++ ++ −− −− ++ ++ −− −− ++ −− +− −−

+++ +++ ++− +++ ++w +w− +−− +++ +++ −−− −−− −−− +++ ++w ++w ++w −++ −+− +++ ++w +++ +++ +++ −−− ++− +++ +++ −−− +++ +w+ −−− www www

+−− −+− −−+ +++ +++ +−+ +++ +++ +−w +++ −−− w−+ −−− −−w +++ +++ +++ +++ +++ +−+ ++− +++ +++ +−+ +++ +++ +++ +++ +++ +++ ++− +++ +++ +++ +++ +++ +++ +++ +w+ w++ +++ +++ +++ +++ −++ +++ −−− −−+ ww− −−w +++ +++ +++ +++ −−− −−+ +++ +ww −++ ++w −−− −w− +++ +ww w−+ +w−

−−− −−− −−− −−− −−+ −−− −−− −−− +−+ +++ +w+ ++w +w+ +++ −−− −−− +−+ +++ +−+ +++ +−+ +++ −−− −−− −−− −−− −−− −−− +−+ +++ +−− ww− +−− +ww −−− ww− −−− −−− −−− −−− +−− +++ −−− ww− +−+ +++ +−+ +++ −+− −−−

+− −− −− ++ ++ ++ ++ ++ ++ ++ ++ −− −− +− ++ +− +− w− ++ w− ++ w− ++ ++ −−

+−+ −−− −−− −−− ++− +++ +++ −−− +++ +w+ +w+ −−− −−− −+− +++ −−+ +++ −−− −−− −−− +−+ −−− +++ +++ −−−

+−+ +++ w−− −−− −−− −−−−− −−+−+ +++ +−+ +++ +−+ +++ −−− −−− +−+ +++ +−+ +++ +−+ +++ −+− −−− −+− −−− +−− −++ +w+ −++ +−+ w+w +++ +++ −−w +−w −−− −−− −−− −−− +−+ +++ −−− −−− +++ +++ +++ +++ −−− −−−

+−+ +++ +−+ +++ −−− −+− w−w +++ −−− −−− +−+ +++ +−+ +++

++ ++ −− ++ +− ++ ++

+++ +++ w+− +w− w++ +++ +++

+++ +++ +++ +++ +−+ w−+ +++ +−+ +−+ −++ +−+ +++ +++ +++

a Test results are given for each strain individually. Test results of the type strain is given first, followed by the remaining strains in order as presented in Table 1. +, characteristic is present; −, characteristic is absent; w, weak reaction. b Numbers refer to genogroup designations. c Characteristics marked in bold character type are particularly useful for distinguishing Achromobacter species.

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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Fig. 2. Maximum-likelihood phylogenetic tree based on 16S rRNA gene sequences (1275 bp alignment) of Achromobacter type strains. Bootstrap values based on 1000 replications are indicated at the nodes; values of less than 50% are not shown. The scale bar indicates the number of substitutions per site.

tests, respectively, from the Biolog GEN III microtest system. The four taxa could be distinguished phenotypically from each other (Table 3) and from other Achromobacter species (Table S3 [data for Achromobacter reference species were taken from reference 16]). Genogroup 5a strain LMG 26853 proved particularly unreactive, a finding observed in strains of some other Achromobacter species as well [16]. Genogroup 2a strains could not be differentiated from genogroup 2b strain, nor could genogroup 5a strains be differentiated from genogroup 5b strains. Finally, the cellular morphology of the four type strains proved very similar (Fig. S1). Biochemical tests that are particularly useful for distinguishing all 15 Achromobacter species are nitrite reduction, denitrification, growth on acetamide, assimilation of glucose, d-gluconate, caprate, and d-lactate, alkaline and acid phosphatase activity, oxidation of the following Biolog GEN III substrates: glycyl-l-proline, l-alanine, l-histidine, l-pyroglutamic acid, d-gluconic acid, muric acid, bromo-succinic acid, ␥-amino-butyric acid and ␤-hydroxy-d,l-butyric acid, and sensitivity tests of Biolog GEN III substrate oxidation toward 8% NaCl, d-serine, troleandomycin, minocycline, and potassium tellurite. In conclusion, although k parameters indicated that genogroups 2 and 5 each consist of two diverging subgroups, these subgroups could not be distinguished by phenotypic characteristics and DNA–DNA hybridizations confirmed that they represented a single genospecies each. Genogroups 2 and 14 strains share a DNA–DNA hybridization level of about 70% (Table S1), which corresponds to the threshold level for species delineation. The type strains of both genogroups differ in numerous biochemical characteristics and in spite of considerable phenotypic variability,

especially within genogroup 2, both sets of strains can by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence, which identifies genogroups 2 and 14 as clearly distinct populations, the potential of nrdA sequence analysis as a single locus tool to identify all of these Achromobacter species and genogroups ([12] and Fig. 3) and the differential phenotypic characteristics we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 [11] as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845T [= CCUG 62426T ] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852T [= CCUG 62438T ] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857T [= CCUG 62444T ] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840T [= CCUG 62421T ] as the type strain). Description of Achromobacter insuavis sp. nov. (in.su.a’vis, L. adj. insuavis unpleasant, disagreeable) A. insuavis cells are Gram-negative, small bacilli (about 0.4 to 0.7 ␮m wide and 1.4 to 2.8 ␮m long) with rounded ends that occur as single units or in pairs and with strain dependent motility. After 48 h of incubation on trypticase soy agar at 28 ◦ C, colonies of all strains except LMG 7054 and LMG 26848 are slightly convex, translucent and non-pigmented, with smooth margins and 1–1.5 mm in diameter; colonies of strains LMG 7054 and LMG 26848 were 2–3 mm in diameter but otherwise similar to those of other A. insuavis strains.

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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Fig. 3. Maximum-likelihood phylogenetic tree based on nrdA gene sequences (765 bp alignment) of Achromobacter type strains and genogroup 2, 5, 7 and 14 strains. Strains LMG 26844 through LMG 26846 are genogroup 2a, strains LMG 7054, LMG 26847 and LMG 26848 are genogroup 2b, strains LMG 26852T through LMG 26854 are genogroup 5a, and strains LMG 11300, LMG 26855 and LMG 26856 are genogroup 2b (Table 1). Bootstrap values based on 1000 replications are indicated at the nodes; values of less than 50% are not shown. The scale bar indicates the number of substitutions per site. All sequences are available from the PubMLST database.

Biochemical characteristics are as described above for all Achromobacter strains. In addition, A. insuavis strains grow on cetrimide agar, reduce nitrate, assimilate glucose, d-gluconate (classical test), caprate, adipate (although sometimes weakly in API 20NE), lmalate, citrate, d-lactate and lactate + methionine, exhibit alkaline and acid phosphatase activity (although some strains only weakly), and oxidize the following Biolog GEN III substrates: d-malic acid, propionic acid and acetic acid. They do not oxidize d-serine, glycyll-proline, l-histidine, ␥-amino-butyric acid, ␣-hydroxy-butyric acid, ␣-keto butyric acid or formic acid as Biolog GEN III substrates. Finally, in Biolog GEN III sensitivity tests, oxidation is not inhibited at pH5, in the presence of 4% NaCl or by sodium butyrate. Growth at 42 ◦ C, in O/F medium with d-xylose, in the presence of 3, 4.5 and 6% NaCl, and on acetamide, reduction of nitrite, denitrification, assimilation of d-gluconate (API 20NE), phenylacetate, d-mannose and sucrose, acid production from glucose, activity of C4 -lipase, C8 -lipase and phosphoamidase, oxidation of the following Biolog GEN III substrates: ␣-d-glucose, d-aspartic acid, l-alanine, l-aspartic acid, l-glutamic acid, l-pyroglutamic acid, lserine, d-gluconic acid, d-glucuronic acid, d-glucuronamide, muric

acid, d-saccharic acid, methyl pyruvate, bromo-succinic acid and ␤hydroxy-dl-butyric acid, and inhibition of oxidation in Biolog GEN III sensitivity tests by 8% NaCl, d-serine, minocycline and potassium tellurite are all strain dependent. The following fatty acid components were present in major amounts (28–33%): C16:0 and summed feature 3 (most likely C16:1 w7c); cyclo-C17:0 , C18:1 w7c and summed feature 2 (most likely C14:0 3-OH) were present in moderate amounts (8–10%); other fatty acids are present in minor amounts (less than 4%). The G+C content is 68.0 mol%. The type strain is LMG 26845T (= CCUG 62426T ) and was isolated from human sputum of a non-cystic fibrosis patient in the USA in 2008. Additional strains have been isolated from human respiratory samples in different countries and from swimming pool water (Table 1, [11]). of Achromobacter aegrifaciens sp. nov. Description (ae.gri.fa’ciens, L. adj. aeger unwell, sick, ill; L. v. facere to make; L. pres. part. faciens making; N. L. part. adj. aegrifaciens sick making)

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A. aegrifaciens cells are motile Gram-negative, small bacilli (about 0.4–0.7 ␮m wide and 1.4–2.8 ␮m long) with rounded ends that occur as single units or in pairs. After 48 h of incubation on trypticase soy agar at 28 ◦ C, colonies are slightly convex, translucent and non-pigmented, with smooth margins and 1–1.5 mm in diameter. Biochemical characteristics are as described above for all Achromobacter strains. In addition, A. aegrifaciens strains reduce nitrite, exhibit C4 -lipase and acid phosphatase activity (although some strains only weakly) and oxidize l-aspartic acid and l-glutamic acid as Biolog GEN III substrates (although some strains only weakly). Except for one biochemically unreactive strain (LMG 26853–see above), A. aegrifaciens strains also reduce nitrate, grow on acetamide, assimilate d-gluconate, caprate, adipate, lmalate, citrate, phenylacetate, d-lactate and lactate + methionine, and oxidize the following Biolog GEN III substrates: l-alanine, l-pyroglutamic acid, l-serine, d-gluconic acid, d-saccharic acid, propionic acid and acetic acid. A. aegrifaciens strains do not grow in O/F medium with d-xylose or in the presence of 6% NaCl, denitrify, assimilate glucose (classical test), sucrose or d-mannose, acidify glucose or exhibit C8 -lipase activity, and the following Biolog GEN III substrates are not oxidized: ␣-d-glucose, d-aspartic acid, glycyl-l-proline, l-histidine, d-glucuronic acid, d-glucuronamide, muric acid, ␥-amino-butyric acid and ␣-hydroxy-butyric acid. In Biolog GEN III sensitivity tests, oxidation is inhibited by minocycline, but not at pH5 or in the presence of 4% NaCl, potassium tellurite or sodium butyrate (except for strain LMG 26853). Growth at 42 ◦ C, on cetrimide agar, in the presence of 3 and 4.5% NaCl, assimilation of glucose (API 20NE), alkaline phosphatase and phosphoamidase activity, and oxidation of the following Biolog GEN III substrates: d-serine, methyl pyruvate, d-malic acid, bromosuccinic acid, ␤-hydroxy-dl-butyric acid, ␣-keto butyric acid and formic acid, and inhibition of oxidation in Biolog GEN III sensitivity tests by 8% NaCl and d-serine are all strain dependent. The following fatty acid components were present in major amounts (30–35%): C16:0 and summed feature 3 (most likely C16:1 w7c); C14:0 , cyclo-C17:0 , C18:1 w7c and summed feature 2 (most likely C14:0 3-OH) were present in moderate amounts (5–10%); other fatty acids are present in minor amounts (less than 3%). The G+C content is 66.0 mol%. The type strain is LMG 26852T (= CCUG 62438T ) and was isolated from human sputum in the USA in 2002. Additional strains have been isolated from human respiratory samples in different countries and from a drinking water reservoir (Table 1, [11]). Description of Achromobacter dolens sp. nov. (do’lens, L. v. dolere to hurt, L. pres. part. dolens hurting, causing pain, distressing) A. dolens cells are motile, Gram-negative, small bacilli (about 0.4–0.7 ␮m wide and 1.4 to 2.8 ␮m long) with rounded ends that occur as single units or in pairs. After 48 h of incubation on trypticase soy agar at 28 ◦ C, colonies are slightly convex, translucent and non-pigmented, with smooth margins and 1–1.5 mm in diameter. Biochemical characteristics are as described above for all Achromobacter strains. In addition, A. dolens strains grow at 42 ◦ C, on cetrimide agar, in the presence of 3% NaCl (although some strains only weakly), reduce nitrate, assimilate glucose, d-gluconate, adipate (although sometimes weakly in API 20NE), l-malate, citrate, phenylacetate, d-lactate and lactate + methionine, exhibit alkaline and acid (weakly) phosphatase, phosphoamidase (weakly) and C4 lipase (although some strains only weakly) activity and oxidize the following Biolog GEN III substrates: l-aspartic acid, l-glutamic acid, l-pyroglutamic acid, l-serine (although some strains only weakly), d-gluconic acid (although some strains only weakly), dsaccharic acid, d-malic acid, propionic acid and acetic acid. A.

dolens strains do not reduce nitrite, denitrify, grow on acetamide, assimilate sucrose, produce acid from glucose or exhibit C8 -lipase activity, do not oxidize the following Biolog GEN III substrates: daspartic acid, d-serine, glycyl-l-proline, l-histidine, d-glucuronic acid, d-glucuronamide, bromo-succinic acid, ␥-amino-butyric acid, ␣-hydroxy-butyric acid, ␣-keto butyric acid or formic acid. Finally, in Biolog GEN III sensitivity tests, oxidation is not inhibited at pH5 or in the presence of 4% NaCl, minocycline (although for some strains only weak reactions are observed), potassium tellurite or sodium butyrate. Growth in O/F medium with d-xylose, in the presence of 4.5 and 6% NaCl, assimilation of caprate and d-mannose, oxidation of ␣-dglucose, l-alanine, muric acid, methyl pyruvate and ␤-hydroxy-dlbutyric acid as Biolog GEN III substrates and inhibition of oxidation in Biolog GEN III sensitivity tests by 8% NaCl and d-serine are all strain dependent. The following fatty acid components were present in major amounts (30–35%): C16:0 and summed feature 3 (most likely C16:1 w7c); cyclo-C17:0 and summed feature 2 (most likely C14:0 3-OH) were present in moderate amounts (8–11%); other fatty acids are present in minor amounts (less than 4%). The G+C content is 67.5% mol%. The type strain is LMG 26840T (= CCUG 62421T ) and was isolated from human sputum of a non-cystic fibrosis patient in the USA in 2002. Additional strains have been isolated from human respiratory samples in the U.S.A. (Table 1, [11]).

Description of Achromobacter anxifer sp. nov. (an’xi.fer L. adj. anxifer distressing, bringing anxiety) A. anxifer cells are motile, Gram-negative, small bacilli (about 0.4–0.7 ␮m wide and 1.4–2.8 ␮m long) with rounded ends that occur as single units or in pairs. After 48 h of incubation on trypticase soy agar at 28 ◦ C, colonies are slightly convex, translucent and non-pigmented, with smooth margins and 1–1.5 mm in diameter. Biochemical characteristics are as described above for all Achromobacter strains. In addition, A. anxifer strains grow at 42 ◦ C, on cetrimide agar, in the presence of 3% NaCl, reduce nitrate and nitrate, grow on acetamide, assimilate d-gluconate, caprate, adipate, l-malate, citrate, phenylacetate, d-lactate and lactate + methionine, exhibit C4 -lipase activity and oxidize the following Biolog GEN III substrates: glycyl-l-proline, l-alanine, l-aspartic acid, l-glutamic acid, l-histidine, l-pyroglutamic acid, l-serine, d-gluconic acid, d-saccharic acid, ␥-amino-butyric acid, ␤hydroxy-dl-butyric acid, propionic acid and acetic acid. A. anxifer strains do not grow in O/F medium with d-xylose, in the presence of 4.5 and 6% NaCl, denitrify, assimilate glucose, sucrose, d-mannose, produce acid from glucose or exhibit alkaline phosphatase, C8 lipase or phosphoamidase activity. The following Biolog GEN III substrates are not oxidized: d-aspartic acid, d-serine, d-glucuronic acid, d-glucuronamide and formic acid. Finally, in Biolog GEN III sensitivity tests, oxidation is inhibited in the presence of 8% NaCl but not at pH5, or in the presence of 4% NaCl, d-serine, potassium tellurite or sodium butyrate. Finally, acid phosphatase activity, oxidation of ␣-d-glucose, muric acid, methyl pyruvate, d-malic acid, bromo-succinic acid, ␣-hydroxy-butyric acid and ␣-keto butyric as Biolog GEN III substrates, and inhibition of oxidation in Biolog GEN III sensitivity tests by minocycline are all strain dependent. The following fatty acid components were present in major amounts (30–33%): C16:0 and summed feature 3 (most likely C16:1 w7c); cyclo-C17:0 , C18:1 w7c and summed feature 2 (most likely C14:0 3-OH) were present in moderate amounts (6–12%); other fatty acids are present in minor amounts (less than 5%). The G + C content is 66.5 mol%.

Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005

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The type strain is LMG 26857T (= CCUG 62444T ) and was isolated from human sputum of a cystic fibrosis patient in the USA in 2006. One additional strain was isolated from a drinking water reservoir in Belgium (Table 1, [11]). Acknowledgements P.V. is indebted to the Fund for Scientific Research – Flanders (Belgium) for research grants. J.J.L. and T.S. are supported by the Cystic Fibrosis Foundation (US). We are indebted to J. Euzéby for helpful comments on nomenclatural issues. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.syapm.2013.06.005. References [1] Ezaki, T., Hashimoto, Y., Yabuuchi, E. (1989) Fluorometric deoxyribonucleic acid deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane-filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39, 224–229. [2] Gomila, M., Tvrzova, L., Teshim, A., Sedlacek, I., Gonzalez-Escalona, N., Zdrahal, Z., Sedo, O., Gonzalez, J.F., Bennasar, A., Moore, E.R.B., Lalucat, J., Murialdo, S.E. (2011) Achromobacter marplatensis sp nov., isolated from a pentachlorophenolcontaminated soil. Int. J. Syst. Evol. Microbiol. 61, 2231–2237. [3] Jolley, K.A., Maiden, M.C.J. (2010) BIGSdb: Scalable analysis of bacterial genome variation at the population level. Bmc Bioinformatics 11. [4] Kersters, K., Hinz, K.H., Hertle, A., Segers, P., Lievens, A., Siegmann, O., Deley, J. (1984) Bordetella avium sp. nov., isolated from the respiratory tracts of turkeys and other birds. Int. J. Syst. Bacteriol. 34, 56–70. [5] Kiredjian, M., Popoff, M., Coynault, C., Lefevre, M., Lemelin, M. (1981) Taxonomy of the genus Alcaligenes. Ann. Microbiol. (Paris) B132, 337–374. [6] Mesbah, M., Whitman, W.B. (1989) Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance liquid chromatography for determination of the mole percentage guanine + cytosine of DNA. J. Chromatogr. 479, 297–306. [7] Palys, T., Nakamura, L.K., Cohan, F.M. (1997) Discovery and classification of ecological diversity in the bacterial world: the role of DNA sequence data. Int. J. Syst. Bacteriol. 47, 1145–1156.

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Please cite this article in press as: P. Vandamme, et al., Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively, Syst. Appl. Microbiol. (2013), http://dx.doi.org/10.1016/j.syapm.2013.06.005