Acinetobacter variabilis sp. nov. - International Journal of Systematic

International Journal of Systematic and Evolutionary Microbiology (2015), 65, 857–863
DOI 10.1099/ijs.0.000028
Acinetobacter variabilis sp. nov. (formerly DNA
group 15 sensu Tjernberg & Ursing), isolated from
humans and animals
Lenka Krizova,1 Jana McGinnis,2 Martina Maixnerova,1 Matej Nemec,1
Laurent Poirel,3 Lisa Mingle,2 Ondrej Sedo,4 William Wolfgang2
and Alexandr Nemec1
Correspondence
Alexandr Nemec
[email protected]
1
Laboratory of Bacterial Genetics, National Institute of Public Health, Šrobárova 48,
100 42 Prague, Czech Republic
2
Wadsworth Center, Bacteriology Laboratory, New York State Dept. of Health, Albany,
NY 12201-2002, USA
3
Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science,
University of Fribourg, Rue Albert Gockel 3, CH-1700 Fribourg, Switzerland
4
Research Group Proteomics, Central European Institute of Technology and National Centre for
Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno,
Czech Republic
We aimed to define the taxonomic status of 16 strains which were phenetically congruent with
Acinetobacter DNA group 15 described by Tjernberg & Ursing in 1989. The strains were
isolated from a variety of human and animal specimens in geographically distant places over the
last three decades. Taxonomic analysis was based on an Acinetobacter-targeted, genus-wide
approach that included the comparative sequence analysis of housekeeping, protein-coding
genes, whole-cell profiling based on matrix-assisted laser desorption ionization-time-of-flight
mass spectrometry (MALDI-TOF MS), an array of in-house physiological and metabolic tests,
and whole-genome comparative analysis. Based on analyses of the rpoB and gyrB genes, the
16 strains formed respective, strongly supported clusters clearly separated from the other
species of the genus Acinetobacter. The distinctness of the group at the species level was
indicated by average nucleotide identity values of ¡82 % between the whole genome
sequences of two of the 16 strains (NIPH 2171T and NIPH 899) and those of the known
species. In addition, the coherence of the group was also supported by MALDI-TOF MS. All 16
strains were non-haemolytic and non-gelatinase-producing, grown at 41 6C and utilized a rather
limited number of carbon sources. Virtually every strain displayed a unique combination of
metabolic and physiological features. We conclude that the 16 strains represent a distinct
species of the genus Acinetobacter, for which the name Acinetobacter variabilis sp. nov. is
proposed to reflect its marked phenotypic heterogeneity. The type strain is NIPH 2171T (5CIP
110486T5CCUG 26390T5CCM 8555T).
Abbreviations: ANIb, average nucleotide identity based on BLAST; DDH,
DNA–DNA hybridization; MALDI-TOF MS, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.
The GenBank/EMBL/DDBJ accession numbers for the gyrB and rpoB
sequences of strains of Acinetobacter variabilis sp. nov. determined in this
study are KM821015–KM821030 and KM821032–KM821046, respectively; see Fig. 1 for details. The GenBank/EMBL/DDBJ accession
number for the rpoB sequence of Acinetobacter kookii 11-0202T (5 ANC
4667T) is KM821031. The GenBank/EMBL/DDBJ accession number for
the 16S rRNA sequence of Acinetobacter variabilis sp. nov. NIPH 2171T
is KP278590.
A supplementary figure and a supplementary table are available with the
online Supplementary Material.
000028 G 2015 IUMS
At the time of writing, the formal classification of the genus
Acinetobacter includes 33 distinct species with validly
published names (http://www.bacterio.net/a/acinetobacter.
html) and several species with effectively published names
awaiting validation (e.g. Krizova et al., 2014; Smet et al.,
2014). The real diversity of the genus at the species level,
however, extends far beyond this existing classification as
indicated by reports on groups of genomically related
strains that did not belong to any of the named species
(Touchon et al., 2014). Already the early taxonomic studies
of Bouvet & Grimont (1986), Bouvet & Jeanjean (1989)
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857
L. Krizova and others
and Tjernberg & Ursing (1989) have revealed several such
putative taxa based on DNA–DNA hybridization (DDH)
studies, with some of them still lacking formal classification. One of these taxa, DNA group 15, was delineated by
Tjernberg & Ursing (1989) among Acinetobacter isolates
recovered from human and environmental specimens
received from hospitals and outpatient clinics in the city
of Malmö, Sweden. This group encompassed two strains,
one isolated from urine and the other from faeces. Based
on DDH, the two strains appeared to represent a novel
genomic species (gen. sp.) clearly separated from the
validly or provisionally named species known at that time.
Human clinical isolates genotypically congruent with these
strains have been reported, although rarely, by later studies
(Nemec et al., 2000; van den Broek et al., 2009). Moreover,
Poirel et al. (2012) recently described a number of
carbapenem-resistant isolates from faeces of cattle, which
appeared to belong to DNA group 15 sensu Tjernberg &
Ursing based on rpoB gene comparative analysis.
The present study aimed to define the taxonomic status of
16 strains that were genotypically congruent with DNA
group 15 sensu Tjernberg & Ursing. For this purpose, we
used a combination of taxonomic methods, which has been
recently optimized to delineate novel species within the
genus Acinetobacter (Krizova et al., 2014). This Acinetobactertargeted, genus-wide approach includes the comparative
sequence analysis of two housekeeping, protein-coding genes
(rpoB and gyrB), whole-cell profiling based on whole-cell
matrix-assisted laser desorption ionization-time-of-flight
mass spectrometry (MALDI-TOF MS), a comprehensive
array of in-house physiological and metabolic tests, and
whole-genome comparative analysis performed on selected
strains. The obtained results revealed that the 16 strains
represented a both phenetically and phylogenetically coherent taxon, which was well-separated at the species level
from all hitherto known species of the genus Acinetobacter.
Given the relatively high number of strains isolated at
different places over a long time period, we feel appropriate
to formally name this taxon; the name Acinetobacter
variabilis sp. nov. is proposed, which is used throughout
the following text.
The 16 strains of Acinetobacter variabilis sp. nov. used in
this study are listed in Table 1. Of these, 11 strains were
recovered from a variety of human clinical specimens at
different geographical locations over the last three decades.
The remaining five strains were recently isolated from faeces
of cattle at a dairy farm in France (Poirel et al., 2012). The
diversity of the 16 isolates at the strain level was indicated
by the sequence heterogeneity of the rpoB and gyrB genes
(Fig. 1) and was confirmed by macro-restriction analysis of
genomic DNA (Fig. S1, available in the online Supplementary Material).
To determine the taxonomic position of the 16 strains
within the genus Acinetobacter, we first performed
comparative sequence analysis of the RNA polymerase bsubunit (rpoB) gene, which is currently the best-studied
858
single gene taxonomic and phylogenetic marker for the
genus Acinetobacter (Nemec et al., 2009, 2011; Krizova et al.,
2014). Similarity calculations and cluster analysis were
carried out for the region spanning nucleotide positions
2915 to 3775 of the rpoB coding region of Acinetobacter
baumannii CIP 70.34T using BioNumerics 7.1. software
(Applied-Maths) with the default parameters. The results of
cluster analysis of the rpoB sequences of the 16 strains of A.
variabilis sp. nov. and the other species of the genus
Acinetobacter are shown in Fig. 1(a). The intra-species
pairwise similarity values (expressed as the percentages of
identical nucleotides at homologous sequence positions in a
multiple alignment) for the strains of A. variabilis sp. nov.
were in the range of 96.8–100 %, whereas the similarities
between these strains and the other members of the genus
had values between 77.1 % (Acinetobacter qingfengensis ANC
4671T) and 90.0 % (Acinetobacter schindleri NIPH 1034T).
The distinctiveness of the rpoB sequences of A. variabilis
sp. nov. was also supported at the protein level. The amino
acid sequences (RpoB positions 973–1258) inferred from
nucleotide sequences were identical in all the 16 strains,
except for NIPH 2171T with a single amino acid difference
(1008E .Q), but differed from those of the other species of
the genus Acinetobacter in at least 13 amino acids (A.
schindleri NIPH 1034T).
Comparative analysis of the partial sequences of the DNA
gyrase subunit B (gyrB) gene was carried out to confirm the
genotypic relationship between the 16 strains and their
separation from the other members of the genus based on
the rpoB sequences as described (Krizova et al., 2014). To
obtain the gyrB sequences for the strains of A. variabilis sp.
nov., we used PCR amplification and sequencing primers
gyrB-F-15TU (59-CTAACCATTCATCGCGCCGG-39) and
gyrB-R-15TU (59-CGAGTGCGCTCTTACGACGG-39), which
were inferred from the whole genome sequences of strains
NIPH 2171T (GenBank accession no. APRS00000000.1) and
NIPH 899 (APPE00000000.1). These primers target the
region delimited by positions 436 and 1234 of the gyrB
coding sequence of strain NIPH 2171T. The obtained gyrB
sequences were compared to those of all species of the
genus Acinetobacter included in the rpoB analysis except
for Acinetobacter boissieri and ‘Acinetobacter gandensis’ for
which the gyrB sequence was not available (Fig. 1b). The
similarity calculations and cluster analysis were performed
for a 759 bp region corresponding to positions 456–1214.
The identity values between the A. variabilis sp. nov. strains
were 95.4–100 % in contrast to the range from 74.4 %
(Acinetobacter nectaris CIP 110549T) to 85.0 % (A. schindleri
NIPH 1034T) observed between A. variabilis sp. nov. and the
other species of the genus Acinetobacter. These values
correspond to the inter- and intra-species similarity values
found in our previous study (Krizova et al., 2014).
Genus-wide whole-genome comparative analysis was performed on the sequences of strains NIPH 2171T (GenBank
accession no. APRS00000000.1; size 3.48 Mb, no. of
contigs 23, no. of proteins 3348, DNA G+C content
40.7 %) and NIPH 899 (APPE00000000.1; size 3.77 Mb,
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D. Gopaul
D. Gopaul
Tjernberg & Ursing (1989)
Nemec et al. (2000)
Tjernberg & Ursing (1989)
M. Vaneechoutte & M. Irenge
Urine (human)
Malmö, Sweden, 1980s
Conjunctiva (human)
Sedlčany, Czech Republic, 1998
Faeces (human)
Malmö, Sweden, 1980s
Urine (human)
Bukavu, DR Congo, 2013
Blood (human)
Columbia, NY/USA, 2011
Leg (human)
Schenectady, NY/USA, 2012
Blood (human)
Queens, NY/USA, 2012
Leg wound (human)
Rensselaer, NY/USA, 2012
Toe (human)
Albany, NY/USA, 2012
Eye swab (human)
London, Canada, 1988
Peritoneal dialysis fluid (human) London, Canada, 1990
Rectal swab (cow)
France, 2010
Rectal swab (cow)
France, 2010
Rectal swab (cow)
France, 2010
Rectal swab (cow)
France, 2010
Rectal swab (cow)
France, 2010
NIPH 2171 (5CIP 110486 5CCUG 26390 5CCM 8555 5Tjernberg & Ursing 151a )
NIPH 899 (5CIP 110487)
NIPH 2026 (5CCUG 282765Tjernberg & Ursing 118)
ANC 4681
ANC 4692
ANC 4693
ANC 4694
ANC 4695
ANC 4696
ANC 4703
ANC 4723
ANC 4718
ANC 4720
ANC 4729
ANC 4750
ANC 4771
Donor or reference
Location and year of isolation
Specimen
T
T
T
T
T
Strain designation
Culture collections: CCM, Czech Collection of Microorganisms, Brno, Czech Republic; CCUG, Culture Collection, University of Göteborg, Sweden; CIP, Collection de l’Institut Pasteur, Institut
Pasteur, Paris, France. ANC and NIPH, designations used in the Laboratory of Bacterial Genetics (National Institute of Public Health, Prague, Czech Republic).
Table 1. Strains of Acinetobacter variabilis sp. nov.
Acinetobacter variabilis sp. nov.
http://ijs.sgmjournals.org
no. of contigs 88, no. of proteins 3731, DNA G+C content
39.3 %), and other species of the genus Acinetobacter,
which are all available from the NCBI website under
BioProject no. PRJNA183623 (Touchon et al., 2014).
Genome sequences were not available for six species
(A. boissieri, ‘A. gandensis’, Acinetobacter harbinensis,
Acinetobacter kookii, Acinetobacter puyangensis and A.
qingfengensis), which were clearly genotypically distinct
from A. variabilis sp. nov. at the species level, as shown by
the analysis of the rpoB and/or gyrB sequences (Fig. 1).
Average nucleotide identity based on BLAST (ANIb) was
calculated using the JSpecies web program (http://imedea.
uib-csic.es/jspecies/) with the default settings (Richter &
Rosselló-Móra, 2009). The ANIb value between the
genome sequences of strains NIPH 2171T and NIPH 899
was 96.32 %, whereas between these two sequences and
those of the other Acinetobacter taxa, it ranged from
71.12 % (A. nectaris CIP 110549T, GenBank accession no.
AYER00000000.1) to 82.09 % (Acinetobacter lwoffii NIPH
512T, AYHO00000000.1) (Table S1). These values concur
with the threshold interval (95–96 %) proposed to
discriminate between bacterial species (Richter &
Rosselló-Móra, 2009) and further support the distinctiveness of A. variabilis sp. nov. at the species level.
Whole-cell MALDI-TOF MS profiling was performed
using a standard extraction protocol based on the
extraction with acetonitrile/formic acid/water and alphacyano-4-hydroxycinnamic acid used as matrix (Krizova
et al., 2014). MALDI-TOF mass spectra measurements
were carried out using an UltrafleXtreme instrument
(Bruker Daltonics), operated in linear positive mode under
control of the FlexControl 3.4 software. Mass spectra were
processed using the Flex Analysis version 3.4 and BioTyper
version 3.1 software (Bruker Daltonics). The parameters of
methods used for calibration, acquisition and evaluation of
the obtained mass spectra were as described (Krizova et al.,
2014). Based on MALDI-TOF MS analysis, the 16 strains
formed a cohesive, although rather heterogeneous, cluster,
which was separated from the type/reference strains of the
hitherto known species of the genus Acinetobacter (Fig. 2a).
All the 16 strains shared six peaks in the range of 4.2–
7.5 kDa (Fig. 2b), but none of these peaks was unique for
A. variabilis sp. nov. The variability within the spectra of
the 16 strains resulted from the presence of intense strainspecific signals, detected mostly within the range of 8.2–
9.8 kDa.
The in-house metabolic and physiological tests were
performed as previously described (Nemec et al., 2009,
2011; Krizova et al., 2014). The assimilation and temperature growth tests were performed in fluid mineral
medium supplemented with 0.1 % (w/v) carbon source
and brain heart infusion broth (Oxoid), respectively. The
cultivation temperature was 30 uC unless indicated otherwise. All tests were performed at least twice in a strictly
standardized fashion and repeated when inconsistent
results were obtained. The properties of the 16 strains
were compared with those of nearly 800 strains deposited
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859
L. Krizova and others
(a)
93
100
99
100
73
100
97
79
71
78 86
88
100
0.05
100
100
A. qingfengensis 2BJ1T (KC631629)
A. puyangensis BQ4-1T (JX499272)
A. radioresistens CCM 3588T (EU477112)
A. soli CCUG 59023T (HQ148175)
A. baylyi ATCC 33304 (EU477155)
A. ursingii NIPH 137T (EU477105)
A. boissieri SAP 284.1T (JQ771155)
A. nectaris ANC 4385T (KJ124840)
A. junii CCM 2376T (EU477110)
Genomic sp. 6 LMG 1026 (EU477115)
A. haemolyticus CCM 2358T (EU477109)
A. towneri CCM 7201T (EU477154)
A. tandoii CCM 7199T (EU477152)
A. rudis ANC 4129T (KJ124837)
A. brisouii ANC 4119T (KJ124836)
A. bereziniae NIPH 521T (EU477116)
A. guillouiae NIPH 522T (EU477117)
A. harbinensis HITLi 7T (KF803234)
A. gerneri CCM 7197T (EU477151)
'A. gandensis' ANC 4275 (KJ569689)
'A. bohemicus' ANC 3994 (KJ124834)
A. johnsonii LMG 999T (EU477113)
A. baumannii ATCC 19606T (EU477108)
A. nosocomialis NIPH 2119T (HQ123389)
Genomic sp. 'Close to 13TU' NIPH 973 (EU477126)
A. pittii NIPH 519T (EU477114)
A. calcoaceticus ATCC 23055T (EU477149)
Genomic sp. 'Between 1 & 3' NIPH 817 (EU477122)
A. beijerinckii NIPH 838T (EU477124)
Genomic sp. 13BJ NIPH 1860 (EU477132)
Genomic sp. 14BJ NIPH 1847 (EU477147)
A. parvus NIPH 384T (EU477107)
A. venetianus NIPH 1925T (EU477136)
Genomic sp. 17 NIPH 1867 (EU477134)
A. gyllenbergii NIPH 2150T (EU477148)
A. tjernbergiae CCM 7200T (EU477153)
Genomic sp. 16 NIPH 1872 (EU477135)
Genomic sp. 15BJ NIPH 1866 (EU477133)
A. lwoffii CCM 5581T (EU477111)
A. kookii 11-0202T (KM821031)
A. bouvetii CCM 7196T (EU477150)
A. schindleri NIPH 1034T (EU477128)
A. indicus ANC 4215T (KJ124838)
ANC 4729 (KM821042)
ANC 4771 (KM821044)
ANC 4750 (KM821043)
ANC 4681 (KM821032)
ANC 4718 (KM821039)
ANC 4720 (KM821040)
NIPH 2026 (KM821046)
ANC 4694 (KM821035)
Acinetobacter variabilis
ANC 4723 (KM821041)
ANC 4692 (KM821033)
ANC 4693 (KM821034)
ANC 4696 (KM821037)
NIPH 2171T (EU477119)
NIPH 899 (KM821045)
ANC 4695 (KM821036)
ANC 4703 (KM821038)
(b)
96
89
100
97
73
88 84
86
72
100
93
92
79
100
100
89
72
100
0.05
97
A. bouvetii CIP 107468T (APQD00000000)
A. indicus ANC 4215T (ATGH00000000)
A. kookii 11-0202T (JX844154)
A. johnsonii CIP 64.6T (APON00000000)
A. tandoii CIP 107469T (AQFM00000000)
A. gerneri CIP 107464T (APPN00000000)
A. bereziniae CIP 70.12T (APQG00000000)
A. guillouiae CIP 63.46T (APOS00000000)
A. towneri CIP 107472T (APPY00000000)
A. nectaris CIP 110549T (AYER00000000)
A. rudis CIP 110305T (ATGI00000000)
A. radioresistens CIP 103788T (APQF00000000)
A. brisouii ANC 4119T (APPR00000000)
A. qingfengensis 2BJ1T (KC686827)
A. puyangensis BQ4-1T (JQ411219)
A. lwoffii NIPH 512T (AYHO00000000)
A. harbinensis HITLi 7T (KF803235)
'A.
' bohemicus' ANC 3994 (APOH00000000)
A. ursingii CIP 107286T (APQA00000000)
A. soli CIP 110264T (APPU00000000)
A. baylyi NIPH 2280T (APPT00000000)
A. haemolyticus CIP 64.3T (APQQ00000000)
A. beijerinckii CIP 110307T (APQL00000000)
A. junii CIP 64.5T (APPX00000000)
A. gyllenbergii CIP 110306T (ATGG00000000)
A. venetianus CIP 110063T (APPO00000000)
Genomic sp. 13BJ CIP 64.2 (APRT00000000)
Genomic sp. 14BJ NIPH 1847 (APRR00000000)
Genomic sp. 'Close to 13TU' NIPH 973 (APOO00000000)
Genomic sp. 'Between1 & 3' NIPH 817 (APPF00000000)
A. pittii CIP 70.29T (APQP00000000)
A. calcoaceticus CIP 81.8T (APQI00000000)
A. baumannii CIP 70.34T (APRG00000000)
A. nosocomialis NIPH 2119T (APOP00000000)
Genomic sp. 6 CIP A165 (APOK00000000)
A. parvus CIP 108168T (APOM00000000)
A. tjernbergiae CIP 107465T (AYEV00000000)
Genomic sp. 17 NIPH 1867 (APRO00000000)
Genomic sp. 16 CIP 70.18 (APRN00000000)
Genomic sp. 15BJ CIP 110321 (AQFL00000000)
A. schindleri CIP 107287T (APPQ00000000)
ANC 4720 (KM821023)
ANC 4729 (KM821025)
ANC 4771 (KM821027)
ANC 4718 (KM821022)
ANC 4750 (KM821026)
ANC 4696 (KM821020)
ANC 4692 (KM821016)
ANC 4695 (KM821019)
Acinetobacter variabilis
NIPH 899 (KM821028)
ANC 4681 (KM821015)
ANC 4703 (KM821021)
NIPH 2171T (KM821030)
ANC 4693 (KM821017)
NIPH 2026 (KM821029)
ANC 4723 (KM821024)
ANC 4694 (KM821018)
Fig. 1. Rooted neighbour-joining trees based on partial nucleotide sequences of the (a) rpoB (861 bp) and (b) gyrB (759 bp)
genes of 16 strains of Acinetobacter variabilis sp. nov. and the type or reference strains of known species of the genus
Acinetobacter. Evolutionary distances were computed using Kimura’s two-parameter model. The sequence of Pseudomonas
aeruginosa PAO1 (derived from GenBank accession no. NC002516) was used as the outgroup. Bootstrap values (.70 %)
after 1000 simulations are shown at branch nodes. GenBank accession nos. are given in parentheses (those obtained in this
study are underlined). Bars, 5 % of change per nucleotide site. Both rpoB- and gyrB-based clusters encompassing strains of A.
variabilis sp. nov. were supported by bootstrap values of 100 % using maximum-parsimony analysis. All calculations were done
by the BioNumerics 7.1 software (Applied-Maths).
in the Acinetobacter collection of the Laboratory of
Bacterial Genetics, which represent all species with validly
published names and a number of tentative species or
taxonomically unique strains. This comparison revealed
two characteristic metabolic features of A. variabilis sp.
nov. First, each of the 16 strains used a rather limited
number of carbon sources; individual strains utilized 4–10
out of 36 different substrates tested (mean, 7.5 substrates).
Thus, they could be easily differentiated from more
biochemically active species such as those of the Acinetobacter calcoaceticus–Acinetobacter baumannii complex,
Acinetobacter baylyi, Acinetobacter soli, Acinetobacter tandoii,
Acinetobacter rudis, Acinetobacter gerneri and Acinetobacter
860
brisouii, and from most species encompassing proteolytic
and/or haemolytic strains [Acinetobacter beijerinckii, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter
venetianus, gen. sp. 6 and gen. sp. 13–17 sensu Bouvet &
Jeanjean (1989)]. Second, each strain displayed a unique
combination of metabolic and physiological features. The
name A. variabilis sp. nov. is proposed to reflect this unusual
intra-species variability (as well as that of MALDI-TOF mass
spectra). A consequence of this variability is the absence of
unequivocal diagnostic traits enabling to differentiate A.
variabilis sp. nov. from some other relatively inactive species
including Acinetobacter indicus, Acinetobacter junii, A. kookii,
A. lwoffii, or Acinetobacter towneri. The properties of A.
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Acinetobacter variabilis sp. nov.
(a)
1000
900
800
Distance level
600
500
400
700
300
200
100
x105
1.50
1.25
4266
0.25
0.00
2000
6091
0.50
3000
4000
5000
6000
7000
7452
0.75
6832
4844
1.00
5176
(b)
Intensity (a.u.)
A. nosocomialis LMG 10619T
Gen. sp. 'Close to 13TU' NIPH 973
Gen. sp. 'Between 1 & 3' NIPH 817
Gen. sp. 14BJ NIPH 1847
A. harbinensis ANC 4817T
A. nectaris LMG 26958T
A. indicus CCM 7832T
A. schindleri NIPH 1034T
A. lwoffii CCM 5581T
A. brisouii ANC 4119T
‘A. gandensis’ ANC 4275
A. tandoii CCM 7199T
A. johnsonnii LMG 999T
‘A. bohemicus’ ANC 3994
A. kookii 11-0202T
A. bouvetii CCM 7196T
A. towneri CCM 7201T
A. boisseri LMG 26959T
A. venetianus ATCC 31012T
A. radioresistens CCM 3588T
A. rudis CIP 110305T
A. pyuangensis BQ4-1T
A. gerneri CCM 7197T
A. guillouiae LMG 988T
A. bereziniae LMG 1003T
Gen. sp. 6 LMG 1026
A. qingfengensis 2BJ1T
A. tjernbergiae CCM 7200T
A. parvus NIPH 384T
A. junii CCM 2376T
Gen. sp. 17 NIPH 1867
Gen. sp. 15BJ CIP 110321
A. haemolyticus CCM 2358T
A. gyllenbergii NIPH 2150T
Gen. sp. 16 ATCC 17988
Gen. sp. 13BJ/14TU ATCC 17905
A. beijerinckii NIPH 838T
A. soli CCUG 59023T
A. baylyi CIP 107474T
A. ursingii NIPH 137T
A. pittii LMG 1035T
A. calcoaceticus ATCC 23055T
A. baumannii ATCC 19606T
NIPH 2171T
ANC 4720
ANC 4692
ANC 4771
ANC 4694
NIPH 899
ANC 4723
ANC 4729
Acinetobacter variabilis
ANC 4750
ANC 4718
ANC 4703
ANC 4693
NIPH 2026
ANC 4681
ANC 4696
ANC 4695
8000
9000
10 000
11 000
m/z
Fig. 2. (a) Dendrogram based on the MALDI-TOF mass spectra of 16 strains of Acinetobacter variabilis sp. nov. and the type/
reference strains of other species of the genus Acinetobacter. The dendrogram was constructed using UPGMA. (b) Combined
spectra of the strains of A. variabilis sp. nov. with the peaks shared by all the strains indicated by molecular mass.
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L. Krizova and others
variabilis sp. nov. and phenotypically most similar species are
summarized in Table 2.
aerobic, oxidase-negative and catalase-positive coccobacilli
typically occurring in pairs, incapable of swimming
motility, capable of growing in mineral media with acetate
as the sole carbon source and ammonia as the sole source
of nitrogen, and incapable of dissimilative denitrification.
Positive in the transformation assay (Juni, 1972).
Description of Acinetobacter variabilis sp. nov.
Acinetobacter variabilis (va.ri.a9bi.lis. L. masc. adj. variabilis
variable, referring to the heterogeneous, strain-dependent
phenotypic properties of the species).
Colonies on tryptic soy agar (Oxoid) after 24 h incubation at 30 uC are 1.0–2.5 mm in diameter, grey–white,
slightly opaque, circular, convex and smooth, with entire
The phenotypic characteristics correspond to those of the
genus (Baumann et al., 1968), i.e. Gram-negative, strictly
Table 2. Phenotypic characteristics of Acinetobacter variabilis sp. nov. and phenotypically most similar species of the genus
Acinetobacter
Species: 1, A. variabilis sp. nov. (n516, where n is no. of strains); 2, A. bouvetii (n51); 3, A. gandensis (n56); 4, A. harbinensis (n51); 5, A. indicus
(n52); 6, A. johnsonii (n520); 7, A. junii (n514); 8, A. kookii (n51); 9, A. lwoffii (n516); 10, A. parvus (n510); 11, A. radioresistens (n512); 12, A.
schindleri (n523); 13, A. towneri (n52); 14, A. ursingii (n529). All species with validly published names include the type strain. Results were
obtained either in this study or have been published previously (Krizova et al., 2014; Smet et al., 2014). Tests were evaluated after six (assimilation
tests), three (haemolytic and gelatinase activities), or two (D-glucose acidification, temperature growth tests) days. All strains grew on acetate,
whereas no strains liquefied gelatin or grew on b-alanine, citraconate, D-gluconate, D-glucose, levulinate, trigonelline instead of putrescine. +, All
strains positive; 2, all strains negative; numbers, percentages of strains giving a positive reaction; D, mostly doubtful reactions; W, mostly weak
positive reactions.
Characteristic
Growth at:
44 uC
41 uC
37 uC
Acidification of D-glucose
Haemolysis of sheep blood
Utilization of:
trans-Aconitate
Adipate
4-Aminobutyrate
L-Arabinose
L-Arginine
L-Aspartate
Azelate
Benzoate
2,3-Butanediol
Citrate (Simmons)
Ethanol
Gentisate
L-Glutamate
Glutarate
L-Histidine
4-Hydroxybenzoate
DL-Lactate
L-Leucine
D-Malate
Malonate
L-Ornithine
Phenylacetate
L-Phenylalanine
Putrescine
D-Ribose
L-Tartrate
Tricarballylate
862
1
2
3
4
5
6
7
8
9
10
11
12
13
14
31
+
+
13
2
2
2
2
2
2
2
+
2
2
2
2
2
2
2
2
+
+
2
2
2
2
25W
2
70W
2
93
+
2
50
2
+
+
2
2
2
6
+
19
2
2
2
90
2
2
2
+
+
2
2
2
+
+
2
2
2
+W
+
2
2
2
2
+
2
2
6
69
19
19
19
2
81
88
81
25
+
2
25
19
2
2
6
2
13
2
2
75
38
2
13
2
2
2
2
2
2
2
2
2
+
2
+
+
2
+
+
+
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
33
50
+
2
33
83
2
2
+
2
2
17
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
2
+
2
2
2
2
2
+
2
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
50
50
2
2
+
2
50
2
2
2
+
2
2
2
2
+
2
2
2
2
2
2
2
+
2
+
95
2
+
80
90
+
2
+
2
2
30
+
2
15W
90
2
2
2
2
2
45
2
2
2
86
2
93
21
2
79
2
79
93
2
+
2
93
2
93
14
79
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
+
+
2
+
2
2
2
2
2
+
2
2
2
2
+
2
2
2
2
2
6
81
88
2
2
2
+
88
6
13
+
2
6
2
2
2
89
2
19W
6
2
69
2
2
2
2
6
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
2
2
2
2
2
20
2
2
2
2
2
2
2
92
+
2
83
2
+
+
+
2
+
2
92
+
2
2
+
92
2
+
2
+
92
92
2
2
2
2
30
2
2
2
2
64
75
32
59
95
30
2
95
2
64
+
2
95
2
2
2
2
2
2
18
25
2
2
2
2
2
2
2
+
50
2
+
2
50
2
2
2
+
2
50
2
2
2
2
2
2
2
2
2
+
2
2
2
97W
+
50
2
+
+
2
+
97
2
97
+
2
+
2
2
2
2
2
2
2
2
D
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International Journal of Systematic and Evolutionary Microbiology 65
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Acinetobacter variabilis sp. nov.
margins. Growth occurs in brain heart infusion (Oxoid) at
temperatures ranging from 25 uC to 41 uC and to a certain
extent at 44 uC (about one-third of strains weakly positive).
Acid is produced from D-glucose by the minority of strains.
Gelatin is not hydrolysed. Haemolysis is not observed
on agar media supplemented with sheep erythrocytes,
although some strains display a greenish discoloration of
the agar. Acetate and ethanol are utilized as sole sources
of carbon with growth visible in six (mostly two) days of
incubation. No growth occurs on b-alanine, L-aspartate,
citraconate, gentisate, D-gluconate, D-glucose, histamine, Lhistidine, 4-hydroxybenzoate, L-leucine, levulinate, malonate, L-ornithine, putrescine, L-tartrate, tricarballylate,
trigonelline, or tryptamine in 10 days. Various numbers of
strains utilize trans-aconitate, adipate, 4-aminobutyrate, Larabinose, L-arginine, azelate, benzoate, 2,3-butanediol,
citrate (Simmons), L-glutamate, glutarate, DL-lactate, Dmalate, phenylacetate, L-phenylalanine or D-ribose within
6 days (Table 2).
The type strain is NIPH 2171T (5CIP 110486T5CCUG
26390T5NIPH 546T5CCM 8555T5Tjernberg & Ursing
151aT), isolated from urine of a human patient in Malmö ,
Sweden in the 1980s. It was used as the reference strain of
DNA group 15 by Tjernberg & Ursing (1989). The type
strain (NIPH 2171T) grows weakly at 44 uC and assimilates
azelate, benzoate, 2,3-butanediol, D-malate and phenylacetate. It produces whitish colonies 2.0 mm in diameter on
tryptic soy agar after 24 h at 30 uC. Strain NIPH 2171T
neither produces acid from D-glucose nor utilizes transaconitate, 4-aminobutyrate, L-arabinose, L-arginine, citrate
(Simmons), L-glutamate, glutarate, DL-lactate, L-phenylalanine or D-ribose. Growth on adipate is visible between
the sixth and tenth day of incubation. The fatty acid
pattern of this strain was published by Kämpfer (1993).
The whole genome sequence of is available from NCBI
under accession no. APRS00000000. The GenBank accession nos. for the partial rpoB and 16S rRNA gene sequences
of strain NIPH 2171T are EU477119 and KP278590,
respectively.
Acknowledgements
We thank all the donors listed in Table 1 for generous provision of
strains and Eva Kodytkova (National Institute of Public Health,
Prague) for linguistic revision of the manuscript. The authors also
thank the Wadsworth Center Applied Genomic Technologies Core
for Sanger and next-generation sequencing, and the Wadsworth
Center Bacteriology Laboratory for maintaining the novel strains
described in this study. The study benefited from the public
availability of Acinetobacter genome sequences generated by the
Broad Institute Genome Sequencing Platform within the collaborative project (NCBI BioProject no. PRJNA183623). This work was
supported by a grant from the Czech Science Foundation (13-26693S)
and by MH CZ - DRO [National Institute of Public Health (NIPH)
75010330].
http://ijs.sgmjournals.org
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