NOTE

Kuncharoen et al., Int J Syst Evol Microbiol 2017;67:37–41

DOI 10.1099/ijsem.0.001566

Achromobacter aloeverae sp. nov., isolated from the root of Aloe

vera (L.) Burm.f.
Nattakorn Kuncharoen,1 Yuki Muramatsu,2 Chiyo Shibata,2 Yuki Kamakura,2 Yasuyoshi Nakagawa2 and
Somboon Tanasupawat1,*

Abstract
Two Gram-staining-negative, strictly aerobic, rod-shaped bacteria, designated strains AVA-1T and AVA-2, were isolated from
the root of Aloe vera (L.) Brum.f. derived from Chachoengsao Province, Thailand. The strains contained cytochrome oxidase
and catalase activities. They grew in 4 % (w/v) NaCl, at a pH range of 6.0–9.0 (optimally at pH 7) and at 20–42  C (optimally at
30–37  C). The major isoprenoid quinone was ubiquinone with eight isoprene units (Q-8). The major fatty acids were C16 : 0
and C17 : 0 cyclo. On the basis of 16S rRNA gene sequence analysis, the strains represent a species belonging to the genus
Achromobacter and are closely related to Achromobacter xylosoxidans NBRC 15126T (98.80 %), Achromobacter insolitus LMG
6003T (98.64 %), Achromobacter aminicus LMG 26690T (98.59 %), Achromobacter pulmonis LMG 26696T (98.58 %) and
Achromobacter insuavis LMG 26845T (98.58 %). The DNA G+C content of strain AVA-1T was 66.5 mol%. The novel strains had
low DNA–DNA relatedness values with related type strains. On the basis of the phenotypic and genotypic data obtained, the
strains clearly represent a novel species, for which the name Achromobacter aloeverae sp. nov. is proposed. The type strain
is strain AVA-1T (=LMG 29108T=NBRC 111463T=PCU 352T=TISTR 2383T).

The genus Achromobacter, of the family Alcaligenaceae in
the class b-Proteobacteria, was proposed by Yabuuchi and
Yano [1] and originally contained a single type species, Ach-
romobacter xylosoxidans. Yabuuchi et al. [2] transferred
some species of the family Alcaligenaceae, such as Alcali-
genes ruhlandii [3], Alcaligenes piechaudii [4] and Alcali-
genes denitrificans [5], to the genus Achromobacter based on
a polyphasic taxonomic study. At the time of writing, 16
species with validly published names belong to the genus
Achromobacter: Achromobacter xylosoxidans [6], Achromo-
bacter ruhlandii and Achromobacter piechaudii [2], Achro-
mobacter denitrificans [7], Achromobacter insolitus and
Achromobacter spanius [8], Achromobacter marplatensis
[9], Achromobacter animicus, Achromobacter mucicolens,
Achromobacter pulmonis and Achromobacter spiritinus [10],
Achromobacter insuavis, Achromobacter aegrifaciens, Achro-
mobacter anxifer and Achromobacter dolens [11] and Achro-
mobacter sediminum [12]. In this article two strains are
described, AVA-1T and AVA-2, which are based on a poly-
phasic taxonomic approach to their classification, represen-
tatives of a novel species, Achromobacter aloeverae sp. nov.,
a seventeenth species of the genus Achromobacter.
Strains, AVA-1T and AVA-2 were isolated from a root of
Aloe vera (L.) Burm.f. collected from Chachoengsao Prov-
ince, Thailand. The root was washed with tap water and
sonicated at 150 W. The surface sterilization method modi-
fied of Hallmann et al. [13] was used. The surface-sterilized
root was crushed with 2 ml Ringer’s solution in a sterile
mortar. The solution and the crushed root were spread on
Starch Casein Agar medium [14] supplemented with 50 mg
l 1 of cycloheximide and 25 mg l 1 of penicillin and incu-
bated at 30  C for 1 week. Cultures of strains AVA-1T and
AVA-2 (=NBRC 111779) were obtained and they were sub-
cultured on Tryptic Soy Agar (TSA, Difco) at 30  C. The cul-
tures were preserved in tryptic soy broth (TSB, Difco) with
20 % (v/v) glycerol at 20  C and in ampoules as freeze-
dried cells.
Cell morphology was examined using cells grown on TSA at
30  C for 2 days. The Hucker and Conn [15] modification
method was used for Gram staining. Flagella staining was
performed as previously described [16]. The morphology of
the cells was observed using a light microscope (Olympus
CH-2). Phenotypic characteristics (including catalase and

Author affiliations: 1Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330,
Thailand; 2NITE Biological Resource Center (NBRC), National Institute of Technology and Evaluation (NITE), 2-5-8 Kazusakamatari, Kisarazu, Chiba
292-0818, Japan.
*Correspondence: Somboon Tanasupawat, somboon.t@chula.ac.th
Keywords: Alcaligenaceae; b-proteobacteria; Achromobacter; plant root.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of strains AVA-1T and AVA-2 are LC094463 and LC147657,
respectively.
Two supplementary figures are available with the online Supplementary Material.

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oxidase activities, and the ability to hydrolyse starch, casein,
gelatin and Tweens 20, 40 and 80) were determined accord-
ing to standard methods [17]. DNase activity was tested by
using DNase agar (Difco). Anaerobic growth was tested by
incubating cultures on TSA plates in anaerobic jars with an
AnaeroPack-Anaero (Mitsubishi Gas Chemical). The pH
range for growth was evaluated in buffers at pH 4.0–9.0 (at
intervals of 1 pH unit) using the following buffer system:
acetate buffer (4.0–5.0), phosphate buffer (pH 6.0–7.0) and
Tris-HCl (pH 8.0–9.0). The temperature range for growth
was tested on TSA plates at 20, 30, 37, 40, 42 and 45  C. The
effects of NaCl were investigated in TSB with different con-
centrations of NaCl present (1, 2, 3, 4 and 5 %, w/v). The
activity of constitutive enzymes and other physiological
properties were determined after growth on TSA (Difco) at
30  C for 2 days using the API 20NE and API ZYM systems
(BioMerieux), according to the manufacturer’s instructions.
Freeze-dried cells for the chemotaxonomic analysis of
strains AVA-1T, AVA-2 and related type strains were
obtained from cultures grown in TSB on an incubating
shaker at 30  C for 2 days. Isoprenoid quinones were
extracted by the method of Collins et al. [18] and were ana-
lyzed by HPLC. For cellular fatty acid analysis, strains
AVA-1T, AVA-2 and related type strains were cultivated on
NBRC medium no. 802 (Wako Pure Chemical Industries)
agar plates [composed of (l 1) 10 g polypeptone, 2 g yeast
extract, 1 g MgSO4.7H2O and 15 g agar; pH 7.0], at 25  C
for 24 h. The quantitative analysis of cellular fatty acid com-
position was carried out by using gas chromatography,
according to the instructions of the Microbial Identification
System (MIDI) Sherlock system version 6.0 [19, 20].
The genomic DNA of strains AVA-1T and AVA-2 was
extracted and purified from cells grown on TSA plates
according to the method of Saito and Miura [21]. The DNA
G+C content was determined by HPLC [22]. DNA–DNA
relatedness was determined in microplates according to the
method of Ezaki et al. [23]. The amplification of 16S rRNA
genes was carried out with two primers (20F: 5¢-AGTTT-
GATCCTGGCTC-3¢ and 1530R: 5¢-AAGGAGGTGATC-
CAGCC-3¢) [24] and the PCR products were sequenced

Table 1. Differential phenotypic characteristics of strains AVA-1T, AVA-2 and closely related type strains
Strains: 1, AVA-1T; 2, AVA-2; 3, Achromobacter xylosoxidans NBRC 15126T; 4, Achromobacter insolitus LMG 6003T; 5, Achromobacter animicus LMG
26690T; 6, Achromobacter pulmonis LMG 26696T; 7, Achromobacter insuavis LMG 26845T. All data are from this study. +, Positive reaction; w, weakly
positive reaction; , negative reaction.

Characteristics 1 2 3 4 5 6 7

Motility + + + + + +
Growth at 40  C + + + + +
Growth at 42  C + + + + +
Growth in 5 % (w/v) NaCl +
Anaerobic growth + + +
Hydrolysis of:
Tween 20 + +
Tween 80 + +
API 20NE
Nitrate reduction + + + + +
Glucose fermentation W

Assimilation of:
L-Arabinose + +
Capric acid + +
D-Glucose + + + +
D-Mannose +
D-Mannitol + +
Phenylacetic acid + + + + +
API ZYM
Alkaline phosphatase + + + + +
a-Chymotrypsin W W

Esterase (C 4) + + + + + +
Esterase Lipase (C 8) + +
Lipase (C 14) + +
Naphthol-AS-BI-phosphohydrolase + +
Trypsin + +
Valine arylamidase + + W W

DNA G+C content (mol%) 66.5 66.5 66.8 66.6 65.7 66.8 68.5

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Table 2. Cellular fatty acid compositions (% of totals) of strain AVA-1T, AVA-2 and closely related type strains
Strains: 1, AVA-1T; 2, AVA-2; 3, Achromobacter xylosoxidans NBRC 15126T; 4, Achromobacter insolitus LMG 6003T; 5, Achromobacter animicus LMG
26690T; 6, Achromobacter pulmonis LMG 26696T; 7, Achromobacter insuavis LMG 26845T. All data are from this study. Values are percentages of total
fatty acids. –, Not detected.

Fatty acid 1 2 3 4 5 6 7

C12 : 0 0.21 0.23 0.37 0.61 0.58 0.44 0.64

C14 : 0 0.52 0.42 2.12 4.22 4.63 5.41 2.87
C16 : 0 33.73 33.99 33.71 30.14 33.09 37.45 33.07
C17 : 0 0.38 0.34 0.7 1.21 0.46 – 1.44
C18 : 0 4.48 4.53 2.70 2.66 1.52 0.52 2.33
C12 : 0 2OH 2.54 2.56 2.52 2.10 2.23 2.47 2.27
C14 : 0 2OH – – 1.79 – – – 1.36
C16 : 0 2OH 3.53 3.60 0.36 – – – –
C16 : 0 3OH 0.23 0.22 0.38 0.51 – 0.27 0.40
C17 : 0 cyclo 10.52 9.27 7.05 7.08 8.26 2.48 6.99
Summed Feature 2* 7.28 7.58 8.04 7.92 7.55 7.09 8.01
Summed Feature 3† 27.28 28.65 28.50 28.91 31.69 36.58 29.03
Summed Feature 8‡ 8.11 8.12 10.77 13.43 9.46 6.60 10.54

*Summed Feature 2 contained unknown 10.928, C14 : 0 3OH/C16 : 1 iso I.

†Summed Feature 3 contained C16 : 1!7c/C16 : 1!6c.
‡Summed Feature 8 contained C18 : 1!7c or C18 : 1!6c.

(Macrogen, Seoul, Korea) by using universal primers [25]. DDBJ databases employing CLUSTAL_X [26]. Phylogenetic
The 16S rRNA gene sequence was multiply aligned with trees were reconstructed by using the neighbour-joining
selected sequences obtained from the GenBank/EMBL/ [27], maximum-parsimony [28] and maximum-likelihood

T
90 Achromobacter marplatensis B2 (EU150134)
89 Achromobacter spiritinus LMG 26692T (HE613447)
Achromobacter piechaudii ATCC 43552T (AB010841)
97 Achromobacter spanius LMG 5911T (AY170848)
Achromobacter animicus LMG 26690T (HE613448)
0.005
59 Achromobacter mucicolens LMG 26685T (HE613446)
Achromobacter aegrifaciens LMG 26852T (HF586507)
Achromobacter insuavis LMG 26845T (HF586506)
Achromobacter insolitus LMG 6003T (AY170847)
62 Achromobacter anxifer LMG 26857T (HF586508)
Achromobacter dolens LMG 26840T (HF586509)
93
Achromobacter denitrificans DSM 30026T (Y14907)
65
Achromobacter ruhlandii ATCC 15749T (AB010840)
95 Achromobacter pulmonis LMG 26696T (HE798552)
Achromobacter xylosoxidans NBRC 15126T (CP006958)
AVA-1T (LC094463)
100 AVA-2 (LC147657)
Achromobacter sediminum DSM 27279T (KC986352)
Taylorella equigenitalis ATCC 35865T (X68645)

Fig. 1. Neighbour-joining phylogenetic tree, based on 16S rRNA gene sequences, showing the relationships between AVA-1T, AVA-2
and closely related type strains of species of the genus Achromobacter. Bootstrap values (>50 %), based on 1000 replications, are given
at the branching nodes. Taylorella equigenitalis ATCC 35865T was used as an out group. Bar, 0.005 substitutions per nucleotide
position.

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 Kuncharoen et al., Int J Syst Evol Microbiol 2017;67:37–41
[29] methods in the program MEGA 6.0 [30]. The confidence
values of the nodes were evaluated by using the bootstrap
resampling method with 1000 replicates [31].
Cells of strains AVA-1T and AVA-2 were Gram-staining-
negative, aerobic, motile rods with peritrichous flagella.
Colonies were cream, circular (1.0–2.0 mm in diameter),
convex and opaque with entire margins after growth on
TSA for 2 days at 30  C. Results for oxidase and catalase
were positive. Tween 20 and Tween 40 were hydrolysed,
but not starch, casein, gelatin, DNA or Tween 40. The
novel isolates did not grow under anaerobic conditions, but
they did grow in the presence of 4 % (w/v) NaCl. The pH
range for growth was 6.0–9.0, with an optimum pH for
growth of 7.0. The optimum temperature for growth was
30–37  C. Detailed physiological and biochemical properties
are shown in Table 1 and given in the species description.
Analysis of the chemotaxonomic characteristics showed
that strains AVA-1T and AVA-2 contained Q-8 as the major
ubiquinone. The predominant fatty acids were C16 : 0
(33.73–33.99 % of the totals), C17 : 0 cyclo (9.27–10.52 % of
totals) and summed feature 3 of C16 : 1!7c/C16 : 1!6c (27.28–
28.65 %). Strains AVA-1T, AVA-2 and all type strains
showed the same fatty acid profiles, whereas distinct differ-
ences in the relative amounts of C14 : 0, C17 : 0., C18 : 0, C14 : 0
2OH and C16 : 0 2OH could be observed. They were different
to the most closely related strain, Achromobacter xylosoxi-
dans NBRC 15126T, with respect to the presence of C14 : 0,
C14 : 0 2OH and C16 : 0 2OH (Table 2). The DNA G+C con-
tent of strains AVA-1T and AVA-2 was 66.5 mol%, which
was in the range of species of the genus Achromobacter with
validly published names (Table 1).
Almost complete 16S rRNA gene sequences of strain AVA-
1T (1419 nt) and AVA-2 (1409 nt) were compared with the
16S rRNA gene sequences of all members of the genus Ach-
romobacter. The highest 16S rRNA gene sequence similari-
ties were with Achromobacter xylosoxidans NBRC 15126T
(98.80 %), Achromobacter insolitus LMG 6003T (98.64 %),
Achromobacter aminicus LMG 26690T (98.59 %), Achromo-
bacter pulmonis LMG 26696T (98.58 %) and Achromobacter
insuavis LMG 26845T (98.58 %). The phylogenetic trees
based on 16S rRNA gene sequences using neighbour-joining
(Fig. 1), maximum-likelihood (Fig. S1, available in the
online Supplementary Material) and maximum-parsimony
(Fig. S2) algorithms show that strains AVA-1Tand AVA-2
form a tight cluster with type strains of species of the genus
Achromobacter. Strains AVA-1T and AVA-2 exhibited
86.1 % DNA–DNA relatedness with each other. Strain
AVA-1T showed low levels of DNA–DNA relatedness to
Achromobacter xylosoxidans NBRC 15126T (33.6 %), Achro-
mobacter insolitus LMG 6003T (19.7 %), Achromobacter
aminicus LMG 26690T (22.1 %), Achromobacter pulmonis
LMG 26696T (29.9 %) and Achromobacter insuavis LMG
26845T (30.7 %). These values were lower than the 70 % cut
off level for assigning strains to the same species [32] and
indicated that strains AVA-1T and AVA-2 are members of a
novel species.
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In addition, the novel strains could be differentiated from
Achromobacter xylosoxidans NBRC 15126T, Achromobacter
aminicus LMG 26690T, Achromobacter pulmonis LMG
26696T, Achromobacter insuavis LMG 26845T and Achro-
mobacter insolitus LMG 6003T in terms of their biochemi-
cal and growth characteristics as well as their motility
(Table 1). On the basis of the phenotypic and chemotaxo-
nomic characteristics as well as the genotypic data
described above, strains AVA-1T and AVA-2 are distinct
from species of the genus Achromobacter with validly pub-
lished names. Therefore, these two strains should be viewed
as representatives of a novel species of the genus Achromo-
bacter, for which the name Achromobacter aloeverae sp.
nov. is proposed.
DESCRIPTION OF ACHROMOBACTER
ALOEVERAE SP. NOV.
Achromobacter aloeverae (a.lo.e.ve¢rae. N. L. gen. n. of Aloe
vera (L.) Burm.f., from which the type strain was isolated).
Cells are Gram-staining-negative, aerobic, non-spore form-
ing rods (0.8–1.22.5–3.0 µm), which are motile with perit-
richous flagella. Colonies are cream, circular (1.0–2.0 mm in
diameter), convex and opaque with entire margins after
incubation at 30  C on Tryptic Soy agar medium for 2 days.
Positive for oxidase and catalase enzymes. Growth is
observed in 4 % (w/v) NaCl, at a pH range of 6.0–9.0 (opti-
mum pH 7.0) and at 20–42  C (optimum 30–37  C). No
growth is seen at 45  C and in 5 % (w/v) NaCl. In API 20
NE tests, urease, indole production and assimilation of cit-
rate are positive. Nitrate reduction, glucose fermentation, b-
galactosidase, hydrolysis of arginine, esculin and gelatin,
and the assimilation of D-glucose, L-arabinose, D-mannose,
D-mannitol, N-acetylglucosamine, D-maltose, potassium
gluconate, carpric acid, adipic acid, L-malate and phenylace-
tic acid are negative. In the API ZYM system, alkaline phos-
phatase, esterase lipase (C4), esterase lipase (C8), esterase
lipase (C14), leucine arylamidase, valine arylamidase, tryp-
sin, acid phosphatase and naphthol-AS-BI-phosphohydro-
lase are positive. Cysteine arylamidase, a-chymotrypsin,
a-galactosidase, b-galactosidase, b-glucuronidase, a-gluco-
sidase, b-glucosidase, N-acetyl-b-glucosamidase, a-manno-
sidase and a-fucosidase are negative. The predominant
ubiquinone is Q-8. The predominant fatty acids are C16 : 0,
C17 : 0 cyclo and summed feature 3 of C16 : 1!7c/C16 : 1!6c.
The type strain, AVA-1T (=LMG 29108T=NBRC 111463T
=PCU 352T=TISTR 2383T), was isolated from Aloe vera
from Thailand. The DNA G+C content of the type strain is
66.5 mol%.
Funding information
This research was supported by Grant for International Research Inte-
gration: Research Pyramid, Ratchadaphiseksomphot Endowment Fund
(GCURP_58_01_33_01), Chulalonglorn University.
Conflicts of interest
The authors declare that there are no conflicts of interest.
 Kuncharoen et al., Int J Syst Evol Microbiol 2017;67:37–41
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3. Our journals have a global readership with subscriptions
held in research institutions around the world.
4. 80% of our authors rate our submission process as
‘excellent’ or ‘very good’.
5. Your article will be published on an interactive journal
platform with advanced metrics.
Find out more and submit your article
at microbiologyresearch.org.