International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1673–1685 DOI 10.1099/ijsem.0.

000932

Taxonomy of haemolytic and/or proteolytic strains

of the genus Acinetobacter with the proposal of
Acinetobacter courvalinii sp. nov. (genomic
species 14 sensu Bouvet & Jeanjean),
Acinetobacter dispersus sp. nov. (genomic
species 17), Acinetobacter modestus sp. nov.,
Acinetobacter proteolyticus sp. nov. and
Acinetobacter vivianii sp. nov.
Alexandr Nemec,1 Lenka Radolfova-Krizova,1 Martina Maixnerova,1
Eliska Vrestiakova,1 Petr Jezek2 and Ondrej Sedo3
1
Correspondence Laboratory of Bacterial Genetics, National Institute of Public Health, Šrobárova 48, 100 42 Prague,
Alexandr Nemec Czech Republic
[email protected] 2
Department of Clinical Microbiology and Parasitology, Přı́bram Regional Hospital, 261 01 Přı́bram I,
Czech Republic
3
Research Group Proteomics, Central European Institute of Technology and National Centre for Bio-
molecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

We aimed to define the taxonomic status of 40 haemolytic and/or proteolytic strains of the genus
Acinetobacter which were previously classified into five putative species termed as genomic
species 14BJ (n59), genomic species 17 (n59), taxon 18 (n57), taxon 19 (n56) and taxon 20
(n59). The strains were recovered mostly from human clinical specimens or soil and water
ecosystems and were highly diverse in geographical origin and time of isolation. Comparative
analysis of the rpoB and gyrB gene sequences of all strains, and the whole-genome sequences of
selected strains, showed that these putative species formed five respective, well-supported clusters
within a distinct clade of the genus Acinetobacter which typically, although not exclusively,
encompasses strains with strong haemolytic activity. The whole-genome-based average nucleotide
identity (ANIb) values supported the species status of each of these clusters. Moreover, the
distinctness and coherence of the clusters were supported by whole-cell profiling based on
MALDI-TOF MS. Congruent with these findings were the results of metabolic and physiological
testing. We conclude that the five putative taxa represent respective novel species, for which the
names Acinetobacter courvalinii sp. nov. (type strain ANC 3623T5CCUG 67960T5CIP
110480T5CCM 8635T), Acinetobacter dispersus sp. nov. (type strain ANC 4105T5CCUG
67961T5CIP 110500T5CCM 8636T), Acinetobacter modestus sp. nov. (type strain NIPH
236T5CCUG 67964T5CIP 110444T5CCM 8639T), Acinetobacter proteolyticus sp. nov.
(type strain NIPH 809T5CCUG 67965T5CIP 110482T5CCM 8640T) and Acinetobacter vivianii
sp. nov. (type strain NIPH 2168T5CCUG 67967T5CIP 110483T5CCM 8642T) are proposed.

Abbreviations: ANIb, average nucleotide identity based on BLAST ; MALDI-TOF MS, matrix-assisted laser desorption/ionization-time-of-flight mass
spectrometry.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Acinetobacter courvalinii ANC 3623T, Acinetobacter dispersus
ANC 4105T, Acinetobacter modestus NIPH 236T, Acinetobacter proteolyticus NIPH 809T, Acinetobacter dispersus NIPH 1867 and Acinetobacter
vivianii NIPH 2168T are KT997472–KT997477, respectively. The GenBank/EMBL/DDBJ accession numbers for the gyrB and rpoB sequences
determined in this study are KT997478–KT997517 and KT997518–KT997552, respectively; see Fig. 1 for details.

Two supplementary figures and four supplementary tables are available with the online Supplementary Material.

000932 G 2016 IUMS Printed in Great Britain 1673

A. Nemec and others

The genus Acinetobacter is a physiologically and metaboli- focused on the diversity of acinetobacters in human clinical
cally highly diverse group of c-proteobacteria that occupy a specimens or soil and water ecosystems. Other strains were
wide spectrum of ecosystems. As shown in recent studies, isolated in a number of countries over several decades. The
this diversity is also evident at the genotypic level. One genotypic distinctness of strains belonging to the same
such study (Touchon et al., 2014) aimed to get an insight genomic species or taxon was verified by macrorestriction
into the nature and amount of genetic diversity of the analysis of genomic DNA (Fig. S1, available in the online
genus based on the comparison of whole-genome Supplementary Material). For comparative phenotypic
sequences of nearly all known species of the genus Acineto- analyses [both metabolic and physiological characterization
bacter. The authors have revealed a surprisingly high level and whole-cell matrix-assisted laser desorption/ionization-
of genomic diversification and have suggested that the time-of-flight mass spectrometry (MALDI-TOF MS)], we
evolutionary history of the genus is as ancient as that of also included 87 reference strains representing all species
the whole family Enterobacteriaceae. Furthermore, they with published names or provisional designations which
have presented a robust phylogeny of the whole genus belong to the haemolytic clade (Table S1).
based on its core genome and have provided strong evi- To approximate the taxonomic position of the 40 strains
dence for the existence of several distant phylogenetic within the genus Acinetobacter, we first performed com-
lineages. One of these lineages encompasses species which parative sequence analysis of genes encoding RNA poly-
are nearly all characterized by a strong ability to lyse merase b-subunit (rpoB) and DNA gyrase subunit B
sheep erythrocytes in ordinary agar media, and may (gyrB) as described previously (Nemec et al., 2009; Krizova
include strains with gelatinase activity. In contrast, species et al., 2014). These genes are currently the best-studied
outside this lineage are neither haemolytic nor proteolytic single-gene taxonomic and phylogenetic markers for the
except for a few which embrace a certain proportion of genus Acinetobacter, and as such, have been used in the
weakly haemolytic strains (Krizova et al., 2015a). Because most recent nomenclatural proposals for novel species of
of this strong association with the haemolytic phenotype, the genus (e.g. Krizova et al., 2014, 2015a, b; Li et al.,
the term haemolytic clade is used for this lineage hereafter. 2015). In the case of the rpoB gene, comparative analyses
According to Touchon et al. (2014), the haemolytic clade were performed for an 861 bp segment which corresponds
includes seven species with validly published names, i.e. to nucleotide positions 2915–3775 of the rpoB coding region
Acinetobacter beijerinckii, Acinetobacter gyllenbergii, Acine- of Acinetobacter baumannii CIP 70.34T (GenBank accession
tobacter haemolyticus, Acinetobacter junii, Acinetobacter no. APRG01000018.1), while in the case of the gyrB gene,
parvus (the only fully non-haemolytic species), Acinetobac- calculations were done for the region encompassing
ter tjernbergiae and Acinetobacter venetianus, and six pro- nucleotide positions 299–1258 of the gyrB coding sequence
teolytic genomic species delineated by Bouvet & Grimont of A. baumannii CIP 70.34T (APRG01000002.1). For PCR
(1986), Bouvet & Jeanjean (1989) or Tjernberg & Ursing amplification and sequencing of the gyrB gene, a novel
(1989), i.e. genomic species (gen. sp.) 6, 13BJ/14TU, pair of primers (gyrB-HP-F: 59-CCAACHGATATWCAYC-
14BJ, 15BJ, 16 and 17. Moreover, this clade was also CWGAAG-39 and gyrB-HP-R: 59- TCTTTTTCYTGACAG-
shown to encompass five novel putative species provision- TCNGCCA-39) was used, which flank the above-specified
ally termed (using a continuation of the numbering system region.
of Bouvet & Jeanjean, 1989) Acinetobacter taxon 18, taxon Comparative analysis of the partial rpoB and gyrB
19, etc. (Touchon et al., 2014). Thus, the taxonomic status sequences in the context of the whole genus Acinetobacter
of more than half of the members of the haemolytic clade showed that all 40 strains clustered with members of the
still awaits thorough investigation to decide whether they haemolytic clade, and were clearly separated from all
deserve formal recognition as distinct species. Given the species with validly published names that do not belong
close relationship of some of the clade members (Bouvet to this clade (Fig. S2). This general picture is consistent
& Jeanjean 1989; Touchon et al., 2014), the prerequisite with that based on the genus core genome (Touchon
for such investigation is the availability of multiple strains et al., 2014). Therefore, our taxonomic analysis was further
per species in order to gauge whether there is a taxonomic focused on the relationship between the 40 strains and the
discontinuity between them. Over recent years, we have
hitherto known species of the clade. Tables S2 and S3 sum-
collected a substantial number of strains with character-
marize, respectively, the intraspecies and interspecies
istics congruent with those of five of the putative species,
identity values for the rpoB and gyrB sequences of these
namely gen. sp. 14BJ, gen. sp. 17, taxon 18, taxon 19 and
organisms. The rpoB intraspecies values were higher than
taxon 20. In the present study, we summarize and discuss
97 % in all cases, which agrees with the data published
the results of the analysis of 40 strains assigned to these
for other species of the genus Acinetobacter (e.g. Krizova
species based on a set of taxonomic methods targeted at
et al., 2014, 2015a, b; Li et al., 2015; Nemec et al., 2015).
the genus Acinetobacter and a comprehensive collection
In contrast, the interspecies rpoB values were lower than
of reference strains.
97 % except for those found for few pairs of genomically
The 40 strains investigated in the present study are listed in closely related species (i.e. A. parvus, gen. sp. 14BJ, taxon
Table 1. Half of the strains were collected in the Czech 18 and/or taxon 20). The intraspecies identity values
Republic since 1993, mostly during prospective studies found for the gyrB gene were higher than 94.5 % (except

1674 International Journal of Systematic and Evolutionary Microbiology 66

 Table 1. Strains of the five novel species described in this study

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, strain designation used by the Laboratory of Bacterial Genetics. Country abbreviations: BR, Brazil; CZ, Czech Republic; DE, Germany; DK, Denmark; FR, France;
GB, Great Britain; IT, Italy; NL, the Netherlands; NO, Norway; US, United States. All clinical strains were of human origin except for ANC 3623. Where known, the words in parentheses indicate
whether the specimen is from a hospitalized (in) or an ambulatory (out) patient.

Strain classification and designation Specimen/specimen from Location and/or year of isolation Donor and/or reference

Acinetobacter courvalinii sp. nov. (5genomic species 14BJ)

ANC 3623T (5CCUG 67960T5CIP 110480T5CCM 8635T) Conjunctiva (agama lizard) Přı́bram, CZ, 2008

http://ijs.microbiologyresearch.org
NIPH 1481 (5CMD0010820*) Tracheal aspirate (in) 2000 M. Vaneechoutte
NIPH 1847 (5CCUG 344355CIP 1104795382*) Conjunctiva BR, before 1990 P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
NIPH 1850 (5743*) Not known Not known P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
NIPH 1851 (5CCUG 148165513*) Wound US, before 1984 P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
ANC 3930 Wound (in) Ostrava, CZ, 2011 T. Škapová
ANC 4230 (5K50-57*) Blood (in) Tønsberg, NO, 2006 N. Karah (Karah et al., 2011)
ANC 4930 Urine (out) Petrovice (Přı́bram), CZ, 2014
ANC 5178 Shank (out) Přı́bram, CZ, 2015
Acinetobacter dispersus sp. nov. (5genomic species 17)
ANC 4105T (5CCUG 67961T5CIP 110500T5CCM 8636T) Well water (garden) Lišov, CZ, 2011D
NIPH 1867 (5CCUG 344375CIP 1104815942*) Ulcer FR, before 1990 P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
NIPH 1868 (5641*) Wound FR, before 1990 P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
NIPH 2020 (5R1-35*) Sewage water Taastrup, DK, 1997 L. Guardabassi (Guardabassi et al., 2000)
ANC 3884 Swab Ostrava, CZ, 2010 T. Škapová
ANC 4547 Well water (garden) Lišov, CZ, 2013D
ANC 4651 Soil (forest creek bank) Křivoklátsko forestland, CZ, 2013D
ANC 4933 Soil (forest) Natural reserve, Žehrov, CZ, 2014D
ANC 5122 Skin (out) Přı́bram, CZ, 2015
Acinetobacter modestus sp. nov. (5taxon 18)
NIPH 236T (5CCUG 67964T5CIP 110444T5CCM 8639T) Urine (in) Přı́bram, CZ, 1993 Nemec et al. (2000)
NIPH 972 (5RUH 175*) Blood Rotterdam, NL, 1981 L. Dijkshoorn (Dijkshoorn et al., 1998)
NIPH 2375 (5G2-79*) Sewage water Glostrup, DK, 1997 L. Guardabassi (Guardabassi et al., 2000)
ANC 3862 (5CIP 110493) Wound (out) Ostrava, CZ, 2010 T. Škapová
ANC 4109 Sludge (pond dam) Ortvı́novice, CZ, 2011D
ANC 4229 (5K50-35*) Blood (in) Drammen, NO, 2007 N. Karah (Karah et al., 2011)
ANC 5020 Throat (out) Přı́bram, CZ, 2014
Acinetobacter proteolyticus sp. nov. (5taxon 19)
NIPH 809T (5CCUG 67965T5CIP 110482T5CCM 8640T5631T*) Ear US, before 1984 P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
NIPH 1959 (5A1292*) Blood (in) Nottingham, GB, 2000 K. Towner
ANC 3839 Wound (out) Přı́bram, CZ, 2009
ANC 3849 Wound (out) Ostrava, CZ, 2010 B. Vaňková
ANC 3924 Wound (in) Ostrava, CZ, 2010 T. Škapová
ANC 3928 Wound (out) Ostrava, CZ, 2010 B. Vaňková
Five novel species of Acinetobacter

1675
A. Nemec and others

for gen. sp. 17; see below) whereas the interspecies gyrB

DGPS coordinates of sampling sites (ANC 4105 & ANC 4547: 49.0150233 N 14.6156889 E; ANC 4651: 50.1254789 N 13.8695089 E; ANC 4933: 50.5299433 N 15.0913564 E; ANC 4109:
P. J. M. Bouvet (Bouvet & Jeanjean, 1989)
identities fell, with two exceptions, within a range of

L. Guardabassi (Guardabassi et al., 2000)

79.6–94.3 %. Fig. 1 shows the results of cluster analysis
for the concatenated rpoB and gyrB sequences. The 40
Donor and/or reference

strains formed five strongly supported clusters encompass-

N. Karah (Karah et al., 2011)

ing, respectively, gen. sp. 14BJ, gen. sp. 17, taxon 18, taxon
19 and taxon 20. The internal sequence identities of these
clusters were $97.3 % except for gen. sp. 17 with intraclus-
ter identities of $95.5 %. This species was split into two
well-supported subclusters. However, even though these
P. G. Higgins

subclusters were consistently formed based on the individ-

H. Seifert
H. Seifert
H. Seifert

ual analysis of either gene, intracluster identities (for the

whole gen. sp. 17) were $97.3 % for the rpoB gene whereas
those for the gyrB gene were as low as $93.6 %. As shown
Natural reserve, H. Králové, CZ, 2014D

below, in the light of the results obtained by other methods,

Location and/or year of isolation

this aberration is interpreted as intraspecies heterogeneity.

The taxonomic status of the five provisional taxa was
further investigated by genome-wide comparative analysis.
Stommeln, DE, 1993–1994
Stommeln, DE, 1993–1994
Leiden, NL, before 1990

The genome sequences of selected strains of the five novel

taxa and of known species of the haemolytic clade (Table S4)
Taastrup, DK, 1997
Tosi, IT, 1993-1994

were taken from the study of Touchon et al. (2014) except

Praha, CZ, 2015

for the sequence of ANC 3849 (taxon 19) which was

obtained anew within a contract with the Genomics Core
NO, 2006
DE, 2007

Facility, EMBL, Heidelberg, Germany. Thus, each of the

five novel taxa was represented by two genome sequences.
Notably, these sequences were determined for strains
which markedly deviated from each other in the rpoB
Specimen/specimen from

and/or gyrB sequences within a given gen. sp. or taxon

(Fig. 1). To quantify the relationship between the genomes,
Soil (beetroot field)
Clinical specimen

we calculated average nucleotide identity based on BLAST

Soil (vineyard)
Soil (rye field)

Soil (wetland)

(ANIb) using the JSpecies web program (http://imedea.

Sewage water

uib-csic.es/jspecies) with the default settings (Richter &

Blood (in)
Wound

Rosselló-Móra, 2009). The obtained results are summarized

Blood

in Table S4 and depicted in Fig. 2. In all cases (including

both the putative novel species and the hitherto known
NIPH 2168T (5CCUG 67967T5CIP 110483T5CCM 8642T51240T*)

species), the intraspecies values were always higher than

48.9921289 N 14.6022636 E; ANC 4908: 50.1710233 N 15.9106683 E).

95 % and, except for gen. sp. 17, gen. sp. 16 and Acinetobac-
ter parvus (95.2 %, 95.49 % and 95.83 %, respectively), also
higher than 96 %. In contrast, the interspecies values for the
five taxa (when compared against each other or the known
species), were lower than 91 %. These values concur with
the threshold interval (95–96 %) proposed to discriminate
between bacterial species (Richter & Rosselló-Móra, 2009),
and support the distinctiveness of each of the five novel
Acinetobacter vivianii sp. nov. (5taxon 20)

taxa at the species level. Expectedly, this picture is fully

congruent with the published phylogram based on the
NIPH 758 (5CIP 1104855A110a*)
Strain classification and designation

*Strain designation used by the donor.

alignment of the 1590 protein families of the Acinetobacter

core-genome (Touchon et al., 2014) for the same set of
genome sequences.
ANC 4227 (5K50-29*)
NIPH 761 (5A114a*)
NIPH 776 (5A163a*)

The inclusion of several strains investigated by Bouvet &

Jeanjean (1989) in the present study allowed, in some
measure, to compare their results with ours. Bouvet &
Table 1. cont.

NIPH 2527

Jeanjean (1989) explored the taxonomy of 27 proteolytic

ANC 4062

ANC 4908
ANC 5387

and haemolytic strains distinct from A. haemolyticus or

gen. sp. 6. Based on DNA–DNA hybridization, they classi-
fied 20 strains into five genomic species (numbered 13–17)
whereas the other seven strains remained ungrouped.

1676 International Journal of Systematic and Evolutionary Microbiology 66

 Five novel species of Acinetobacter

100 NIPH 298 (APRM00000000.1)

Gen. sp. 6 [98.8%]
95 100 CIP A165 (APOK00000000.1)
100 CIP 64.3T (APQQ00000000.1)
A. haemolyticus [99.5%]
100 NIPH 261 (APQR00000000.1)
100 CIP 64.5T (APPX00000000.1)
100 A. junii [98.6%]
NIPH 182 (APPW00000000.1)
100 ANC 3835 (APQK00000000.1)
T A. beijerinckii [98.4%]
100 CIP 110307 (APQL00000000.1)
T
CIP 107465 (AYEV00000000.1) A. tjernbergiae
100 CIP 110321 (AQFL00000000.1) Gen. sp. 15BJ
88 100 97 CIP 56.2 (APPH00000000.1)
Gen. sp.16 [98.2%]
CIP 70.18 (APRN00000000.1)
100 ANC 4933 (KT997536/KT997496)
100
81 100 ANC 4651 (KT997533/KT997493)
NIPH 1867 (EU477134/KT997511)
2% 100 ANC 4547 (KT997532/KT997492)
T
ANC 4105 (KT997527/KT997487) Gen. sp. 17/Acinetobacter dispersus
97
ANC 3884 (KT997522/KT997482) [95.5–100%]
100 NIPH 2020 (KT997550/ KT997514)
85 ANC 5122 (KT997538/KT997498)
NIPH 1868 (KT997548/KT997512)
100 NIPH 1103 (APOL00000000.1)
T A. parvus [97.2%]
100 CIP 108168 (APOM00000000.1)
ANC 4109 (KT997528/KT997488)
100 ANC 5020 (KT997537/KT997497)
T
100 NIPH 236 (KJ124832/KT997501)
Taxon 18/Acinetobacter modestus
ANC 3862 (KT997521/KT997481)
[98.1–100%]
NIPH 972 (KT997544/KT997506)
NIPH 2375 (KT997551/KT997516)
ANC 4229 (KT997530/KT997490)
T
CIP 110063 (APPO00000000.1) A. venetianus
79 96 NIPH 1859 (APRZ00000000.1)
Gen. sp. 13BJ/14TU [96.9%]
100 NIPH 2036 (ATGK00000000.1)
95 100 NIPH 230 (AYEQ00000000.1)
A. gyllenbergii [99.8%]
93 100 CIP 110306T (ATGG00000000.1)
94
NIPH 1959 (KT997549/KT997513)
100 ANC 3849 (KT997520/KT997480)
T
100 NIPH 809 (KJ124841/KT997505) Taxon 19/Acinetobacter proteolyticus
ANC 3928 (KT997524/KT997484) [98.0–100%]
ANC 3839 (KT997519/KT997479)
79 ANC 3924 (KT997523/KT997483)
NIPH 1481 (KT997545/KT997507)
100
ANC 5178 (KT997539/KT997499)
100
NIPH 1850 (KT997546/KT997509)
ANC 4930 (KT997535/KT997495)
T Gen. sp. 14BJ/Acinetobacter courvalinii
ANC 3623 (KT997518/KT997478)
[97.3–100%]
NIPH 1851 (KT997547/KT997510)
ANC 3930 (KT997525/KT997485)
100 NIPH 1847 (EU477147/KT997508)
100 ANC 4230 (KT997531/KT997491)
ANC 4227 (KT997529/KT997489)
ANC 5387 (KT997540/KT997500)
NIPH 758 (KT997541/KT997502)
NIPH 776 (KT997543/KT997504)
Taxon 20/Acinetobacter
ANC 4908 (KT997534/KT997494)
100 vivianii [98.2–100%]
ANC 4062 (KT997526/KT997486)
97 T
NIPH 2168 (KJ124844/KT997515)
NIPH 2527 (KT997552/KT997517)
NIPH 761 (KT997542/KT997503)

Fig. 1. Neighbour-joining tree based on the concatenated partial sequences of the rpoB (861 bp) and gyrB (960 bp) genes
of 40 strains of five novel species and 19 reference strains representing all known species of the Acinetobacter haemolytic
clade. Evolutionary distances were computed using Kimura’s two-parameter model. The sequences of Acinetobacter calcoa-
ceticus CIP 81.8T (extracted from GenBank accession no. APQI00000000.1) were used as the outgroup. Bootstrap values
(%) based on 1000 resamplings are indicated above branches (no values are shown within clusters encompassing members
of the same species except for gen. sp. 17). Bootstrap values obtained by maximum-parsimony analysis (after 1000 resam-
plings) are given below branches. Intraspecies pairwise similarity values (percentages of identical nucleotides in equivalent
positions) are shown in square brackets. GenBank accession numbers for the rpoB/gyrB sequences or whole-genome
sequences from which the rpoB/gyrB sequences were extracted are shown in parentheses. Filled triangles and circles denote
the strains studied previously by DNA–DNA hybridization (Bouvet & Jeanjean, 1989) and those with whole-genome
sequences included in Fig. 2, respectively. The strain designations used by Bouvet & Jeanjean are shown in Table 1 or are as
follows: 79 (5CIP 110321), 78 (5CIP 70.18), 1191 (5NIPH 2036) and 1271 (5CIP 110306T). Bar, 2 % change per
nucleotide site. All calculations were done by the BioNumerics 7.5 software (Applied-Maths).

Eleven strains covered by both studies are indicated in sp. 17 (NIPH 1867 and NIPH 1868), which were separated
Fig. 1 (for their alternative designations, see Table 1). Over- into different rpoB/gyrB subclusters (Fig. 1), were
all, there was a perfect agreement between the two classifi- shown convincingly to be related at the species level by
cations. It is of particular note that two strains of gen. DNA–DNA hybridization (relative binding ratio: 83 %,

http://ijs.microbiologyresearch.org 1677
A. Nemec and others

2% NIPH 298 (APRM00000000.1)

Gen. sp. 6 [96.38%]
CIP A165 (APOK00000000.1)
CIP 64.3T (APQQ00000000.1)
A. haemolyticus [97.63%]
NIPH 261 (APQR00000000.1)
CIP 64.5T (APPX00000000.1)
A. junii [97.39%]
NIPH 182 (APPW00000000.1)
ANC 3835 (APQK00000000.1)
A. beijerinckii [96.48%]
CIP 110307T (APQL00000000.1)
CIP 110063T (APPO00000000.1) A. venetianus
NIPH 1103 (APOL00000000.1)
A. parvus [95.83%]
CIP 108168T (APOM00000000.1)
CIP 107465T (AYEV00000000.1) A. tjernbergiae
NIPH 236T (APOJ00000000.1)
Taxon 18/Acinetobacter modestus [96.85%]
ANC 3862 (APRP00000000.1)
NIPH 1859 (APRZ00000000.1)
Gen. sp. 13BJ/14TU [96.45%]
NIPH 2036 (ATGK00000000.1)
CIP 110321 (AQFL00000000.1) Gen. sp. 15BJ
CIP 56.2 (APPH00000000.1)
Gen. sp.16 [95.49%]
CIP 70.18 (APRN00000000.1)
NIPH 1867 (APRO00000000.1)
Gen. sp. 17/Acinetobacter dispersus [95.2%]
ANC 4105T (APRL00000000.1)
NIPH 230 (AYEQ00000000.1)
A. gyllenbergii [98.53%]
CIP 110306T (ATGG00000000.1)
ANC 3849 (This study)
Taxon 19/Acinetobacter proteolyticus [97.48%]
NIPH 809T (APOI00000000.1)
ANC 3623T (APSA00000000.1)
Gen. sp. 14BJ/Acinetobacter courvalinii [96.23%]
NIPH 1847 (APRR00000000.1))
NIPH 758 (APPC00000000.1)
Taxon 20/Acinetobacter vivianii [96.78%]
NIPH 2168T (APRW00000000.1)

Fig. 2. Dendrogram of cluster analysis of ANIb values between the whole-genome sequences of the representatives of the
five novel species and other species of the haemolytic clade. The tree was reconstructed based on the similarity matrix
shown in Table S1 using the UPGMA algorithm. Intraspecies ANIb values are given in parentheses.

DTm: 2.5 %). This is in line with the ANIb value found for results of cluster analysis of the MALDI-TOF spectra of
NIPH 1867 and ANC 4105T (95.2 %), each of which also the 40 novel strains and 87 strains representing all known
represents a different rpoB/gyrB subcluster, and further species of the haemolytic clade. The strains of each of the
supports the genomic coherence of gen. sp. 17. It is also five novel species formed a cohesive cluster that was separ-
worthy to mention that strains NIPH 809T and NIPH ated from the strains of all other species or taxa. The spectra
2168T (taxon 19 and taxon 20, respectively) were found of the 40 strains were also compared with those derived from
to be distinct from each of the genomic species 13–17 the type strains of species of the genus Acinetobacter that are
based both on DNA–DNA hybridization and ANIb. not the members of the haemolytic clade. Cluster analysis of
these spectra confirmed a clear separation of the five novel
The 40 strains were further studied by whole-cell
species from all known species of the genus Acinetobacter
MALDI-TOF MS profiling. To improve the differentiation
(data not shown).
of taxonomically closely related taxa, a new method pro-
posed by Šedo et al. (2013) was used, with a matrix solution Finally, the metabolic and physiological properties of the
consisting of ferulic acid 12.5 mg ml21 in water/acetonitrile/ 40 strains were assessed using an array of strictly standar-
formic acid (50:33:17, by vol.). Compared with the standard dized, in-house tests targeting the genus Acinetobacter as
method of Freiwald & Sauer (2009), this new approach described previously (Nemec et al., 2009; Krizova et al.,
allowed the detection of a wider mass range of proteins 2014). All the strains were haemolytic except for NIPH
and more species-specific signals (data not shown). 1481 (gen. sp. 14BJ). In all cases, the zones of haemolysis
MALDI-TOF mass spectrum measurements and processing were seen on Columbia agar plates containing 5 % sheep
were carried out as described by Krizova et al. (2014) with blood (bioMérieux) or on other agar media with sheep
changes in the calibration strategy (Protein Calibration erythrocytes within one day of culture at 30 8C. Gelatinase
Standard I from Bruker Daltonics was used) and peak activity was expressed by all strains of gen. sp. 14BJ, gen.
frequency threshold limit (25 %). The determinations sp. 17 and taxon 19, whereas it was completely absent in
were performed using an Ultraflextreme instrument the strains of taxon 18 and taxon 20. These results were
operated in linear positive mode under control of the obtained concordantly with both the Frazier gelatinase
FlexControl 3.4, Flex Analysis (version 3.4) and BioTyper method (Nemec et al., 2009) and the API 20 NE system
(version 3.1) software (Bruker Daltonics). Fig. 3 shows the (bioMérieux) after culture at 30 8C for 3 days. Based on

1678 International Journal of Systematic and Evolutionary Microbiology 66

 Five novel species of Acinetobacter

A. parvus (n=10)

A. venetianus (n=5)

A. junii (n=14)

ANC 4109
ANC 5020
NIPH 972
NIPH 2375 Taxon 18/ Acinetobacter modestus
ANC 4229
NIPH 236T
ANC 3862

A. haemolyticus (n=16)

Genomic sp. 6 (n=3)

A tjernbergiae (n=2)
A. gyllenbergii
NIPH 1481
ANC 3930
NIPH 1851
ANC 3623 T
NIPH 1850 Gen. sp. 14BJ/Acinetobacter courvalinii
NIPH 1847
ANC 4230
ANC 5178
ANC 4930
ANC 4908
NIPH 776
ANC 4062
ANC 5387
NIPH 2527 Taxon 20/Acinetobacter vivianii
NIPH 758
NIPH 2168T
NIPH 761
ANC 4227
Genomic sp. 16 (n=4)
Genomic sp. 15BJ (n=2)
NIPH 1868
ANC 5122
NIPH 1867
ANC 4547
ANC 4933 Gen. sp. 17/Acinetobacter dispersus
ANC 4651
ANC 4105 T
NIPH 2020
ANC 3884

Genomic sp. 13BJ/14TU (n=7)

A. gyllenbergii (n=2)
NIPH 809T
ANC 3924
NIPH 1959
ANC 3928 Taxon 19/ Acinetobacter proteolyticus
ANC 3849
ANC 3839

A. gyllenbergii (n=6)

A. beijerinckii (n=15)

1000 900 800 700 600 500 400 300 200 100 0
Distance Level

Fig. 3. Dendrogram based on MALDI-TOF mass spectra of the 40 strains of five novel species and 87 reference strains
representing all known species of the Acinetobacter haemolytic clade. The reference strains are listed in Table S1. The den-
drogram was reconstructed using UPGMA.

http://ijs.microbiologyresearch.org 1679
 Table 2. Metabolic and physiological features of the five novel species of the genus Acinetobacter and other members of the haemolytic clade

1680
Species: 1, gen. sp. 14BJ/A. courvalinii sp. nov. (n59 where n is the number of strains); 2, gen. sp. 17/A. dispersus sp. nov. (n59); 3, taxon 18/A. modestus sp. nov. (n57); 4, taxon 19/A. proteolyticus
sp. nov. (n56); 5, taxon 20/A. vivianii sp. nov. (n59); 6, A. beijerinckii (n515); 7, A. gyllenbergii (n59); 8, A. haemolyticus (n516); 9, A. junii (n514); 10, A. parvus (n510); 11, A. tjernbergiae
(n52); 12, A. venetianus (n55); 13, gen. sp. 6 (n53); 14, gen. sp. 13BJ/14TU (n57); 15, gen. sp. 15BJ (n52); 16, gen. sp. 16 (n54). All species with validly published names include the respective
type strains. Results were obtained either in this study or have been presented previously (Krizova et al., 2015b). Tests were performed as described by Nemec et al. (2009). Bacteria were cultured at
30 8C except for in temperature growth tests. Assimilation tests were interpreted after culture for 6 days in fluid mineral medium supplemented with 0.1 % (w/v) carbon source. Other tests were
A. Nemec and others

assessed after 3 days (haemolytic and gelatinase activities) or 2 days (D -glucose acidification, temperature growth tests in Brain-Heart Infusion Broth). +, All strains positive; 2, all strains
negative; D , (mostly) doubtful or irreproducible reactions; W , (mostly) weak positive reactions. Numbers are percentages of strains with clearly positive reactions. For strain-dependent reactions,
results for type strains are given in parentheses.

Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Growth at:
44 8C 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
41 8C 50W (D ) 2 2 2 2 2 2 94 (+) 93 (+) 2 2 2 2 2 2 2
37 8C + + + (W ) + + + + + + 90 (+) 2 + + 86 50 +
35 8C + + + + + + + + + + 2 + + + + +
Acidification of D -glucose + 2 2 2 89 (+) 2 2 75 (+) 2 2 2 2 + + 2 2
Haemolysis of sheep blood 89 (+) + + + + + + + 50 (2) 2 + + + + + +
Liquefaction of gelatin + + 2 + 2 13 (2) + 94 (+) 2 2 2 80 (+) + + + +
Assimilation of:
Acetate + + + + + + + + + + + + + + + +
trans-Aconitate 44 (2) 11 (2) 2 + 56 (2) 2 2 63 (+) 2 2 2 2 2 2 2 2
Adipate + 22 (2) 14 (2) 67 (2) 89 (+) 2 + 2 2 2 2 20 (2) 2 2 2 25
b-Alanine 89 (+) + 2 + + 2 + 2 2 2 2 2 2 29 2 +
4-Aminobutyrate + + 2 D (2) + + D + 86 (+) 2 2 + 2 2 2 D
L -Arabinose 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
L -Arginine + + + + + 2 + 94 (+) 93 (+) 2 50 (+) + + + + +
L -Aspartate 11 (2) 2 2 2 11 (D ) + 2 31 (2) 21 (+) 2 2 2 67 2 2 2
Azelate + 22 (2) 2 67 (2) 89 (+) 2 + 2 2 2 2 20 (2) 2 2 2 25
Benzoate + + + + + 2 + 2 79 (+) 2 2 + 2 29 + +
2,3-Butanediol 2 2 14 (2) 2 2 2 2 2 2 2 2 2 2 2 2 2
Citraconate 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
Citrate (Simmons) + + 2 + + + + 75 (+) 79 (+) 2 2 + + + 50 +
Ethanol 2 11 (2) + 2 2 + 22 (2) 94 (+) 93 (+) + + + + 14 2 2
Gentisate 2 33 (2) 2 + 89 (+) 2 11 (+) 81 (+) 2 2 2 2 2 + + +
D -Gluconate 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
D -Glucose 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
L -Glutamate + + + + + + + + + 2 + + + + + +
Glutarate + + 2 33 (2) 89 (+) 2 D (2) 2 2 2 2 2 2 2 2 2
Histamine 11 (2) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
L -Histidine + + + + + + + + 93 (+) 2 + + + + + +
4-Hydroxybenzoate + + 2 + + 2 89 (+) 81 (+) 2 2 2 2 33 71 + +
DL -Lactate + + 2 + + 2 + 2 93 (+) 2 2 2 2 + + +

International Journal of Systematic and Evolutionary Microbiology 66

 Table 2. cont.

http://ijs.microbiologyresearch.org
Characteristic 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

L -Leucine + + 2 83 (2) + 93 (+) + 88 (+) 14 (2) 2 2 + + 14 + +

Levulinate 11 (2) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
D -Malate 89 (+) + D + + + + 88 (+) 79 (D ) 2 2 + 67 + 50 +
Malonate + 22 (2) 2 67 (+) + + 78 (+) 2 2 2 2 + 2 29 + 50
L -Ornithine 89 (+) 89 (D ) 2 + + 2 56 (+) 2 2 20 (2) 2 2 2 D 2 50
Phenylacetate + 89 (+) 2 83 (2) + 2 + 2 2 2 2 2 2 + + +
L -Phenylalanine + 89 (+) 2 + + 2 89 (+) 2 2 2 2 2 2 + + +
Putrescine + + 2 2 78 (+) 2 2 2 2 2 2 2 2 2 2 2
D -Ribose 2 2 2 2 22 (+) 2 2 2 2 2 2 2 2 2 2 2
L -Tartrate 22 (2) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
Tricarballylate 44 (2) 11 (2) 2 + 56 (2) 2 2 2 2 2 2 2 2 2 2 2
Trigonelline 67 (+) 2 + 2 + 2 2 2 2 2 50 (+) 2 2 57 + 25
Tryptamine 22 (D ) 11 (+) 2 50 (+) D 2 2 2 2 2 2 2 2 2 2 50
No. of differences from:*
Gen. sp. 14BJ/ 1 (+3) 14 (+3) 3 1 (+1) 12 (+3) 2 9 (+4) 10 (+4) 19 (+3) 17 (+3) 8 (+4) 11 (+2) 6 (+1) 7 (+2) 4
A. courvalinii sp. nov.
Gen. sp. 17/ 10 (+3) 1 (+2) 2 (+1) 8 (+6) 1 (+2) 5 (+5) 5 (+5) 15 (+3) 12 (+4) 5 (+5) 7 (+4) 4 (+2) 5 (+2) 2
A. dispersus sp. nov.
Taxon 18/ 12 (+2) 11 (+5) 7 (+1) 8 (+5) 3 (+6) 1 (+3) 6 2 5 (+1) 6 7 9 10
A. modestus sp. nov.
Taxon 19/ 2 (+2) 12 (+1) 2 (+1) 6 (+3) 8 (+2) 16 (+3) 14 (+2) 9 (+1) 10 (+1) 3 5 2
A. proteolyticus sp. nov.
Taxon 20/ 10 (+7) 2 (+2) 8 (+6) 7 (+7) 17 (+6) 14 (+6) 8 (+5) 11 (+5) 2 (+5) 4 (+5) 1 (+3)
A. vivianii sp. nov.

*Number of differences in individual characters between the given novel species and another species. Only the homogeneous characters (i.e. those that were either positive or negative in all strains
of a species) are considered. In addition, the number of characters for which the difference in the percentages of positivity between two species is $75 % is indicated in parentheses. Of the
temperature growth tests, only the test at 37 8C was taken into account.
Five novel species of Acinetobacter

1681
A. Nemec and others

the presence of the haemolytic and/or proteolytic activity, Description of Acinetobacter courvalinii sp. nov.
the five putative species can be easily distinguished from
Acinetobacter courvalinii (cour.va.li9ni.i. N.L. gen. masc. n.
any species that is not a member of the haemolytic clade
courvalinii of Courvalin, named after Patrice Courvalin,
except for Acinetobacter bohemicus, Acinetobacter johnsonii
a French microbiologist).
and Acinetobacter soli, each of which includes a certain pro-
portion of weakly haemolytic strains (Krizova et al., 2014, The phenotypic characteristics correspond to those of the
2015a). Nevertheless, these three species can be genus (Baumann et al., 1968), i.e. Gram-negative, strictly
differentiated from the non-proteolytic strains of taxon aerobic, oxidase-negative and catalase-positive coccobacilli
18 and taxon 20 based on a number of other metabolic typically occurring in pairs, incapable of swimming moti-
characters (Krizova et al., 2015a). Table 2 summarizes the lity, capable of growing in mineral media with acetate as
phenotypic properties of the clade members and indicates the sole carbon source and ammonia as the sole source
the amount of characters that can be used for the differen- of nitrogen, and incapable of dissimilative denitrification.
tiation of the five putative species from each other and Positive in the transformation assay of Juni (1972).
from the other species. The number of these characters
Colonies on tryptic soy agar (Oxoid) after incubation for
ranges from 1 to 18 (if only those which are either positive
24 h at 30 uC are 1.5–2.0 mm in diameter, grey–white,
or negative in all strains of a given species are considered)
slightly opaque, circular, convex and smooth, with entire
or from 2 to 23 (if considering also those for which the
margins. Growth occurs in Brain-Heart Infusion (Oxoid)
difference in the percentages of positivity between two
at temperatures ranging from 15 uC to 37 uC, but not at
species is $75 %). Our results were largely congruent
44 uC. Some strains show weak growth at 41 uC. Acid is
with those of Bouvet & Jeanjean (1989) for the 11 strains
produced from D -glucose. Gelatin is hydrolysed. The vast
covered by both studies. A notable exception is NIPH
majority of strains produce wide zones of haemolysis
2168T (taxon 20) which was reported to be proteolytic by
around colonies on agar media supplemented with sheep
Bouvet & Jeanjean (1989) but was negative in our hands
erythrocytes. Adipate, 4-aminobutyrate, L -arginine, azelate,
after repeated testing. Our finding is supported by the
benzoate, citrate (Simmons), L -glutamate, glutarate, L -his-
fact that all other strains of taxon 20 consistently showed
tidine, 4-hydroxybenzoate, DL -lactate, L -leucine, malonate,
the absence of gelatinase activity.
phenylacetate, L -phenylalanine and putrescine are utilized
In conclusion, the data presented and discussed above as sole sources of carbon, with growth visible within six
support the species status of each of the five putative (mostly two) days of culture at 30 uC. No growth occurs
species. This also holds true for those that appeared to on L -arabinose, 2,3-butanediol, citraconate, ethanol, genti-
be closely related at both the genotypic and the phenoty- sate, D -gluconate, D -glucose or D -ribose in 10 days. Utiliz-
pic levels such as gen. sp. 14BJ and taxon 20. These two ation of trans-aconitate, b-alanine, L -aspartate, histamine,
taxa share a shallow branch in the core-genome-based levulinate, D -malate, L -ornithine, L -tartrate, tricarballylate,
phylogram published by Touchon et al. (2014) and as trigonelline and tryptamine is strain-dependent.
shown in the present study, they are also closely related The type strain is ANC 3623T (5CCUG 67960T5CIP
based on the analyses of several independent taxonomic 110480T5CCM 8635T), isolated from the conjunctiva of
markers (Figs 1, 3 and Table 2). Nevertheless, all these an agama lizard (Pogona vitticeps) in the Czech Republic in
analyses also congruently revealed that the strains of February 2008. This strain haemolyses sheep erythrocytes
gen. sp. 14BJ and taxon 20 formed two respective, and assimilates b-alanine, D -malate, L -ornithine and trigo-
internally cohesive and mutually exclusive groups. Their nelline, but does not grow on trans-aconitate, L -aspartate,
separation at the species level is evidenced by both the histamine, levulinate, L -tartrate or tricarballylate. Growth
ANIb values of 89.01–89.31 % and the digital DNA– on tryptamine is visible between the sixth and tenth day
DNA hybridization values of 37.8–38.7 % for the same of culture. The whole genome sequence of the type strain
two pairs of whole-genome sequences (the latter values is available under GenBank/EMBL/DDBJ accession no.
were calculated using the GGDC 2.0 web server at APSA00000000.1 (size: 3.94 Mb, number of contigs: 18,
http://ggdc.dsmz.de/distcalc2.php; Meier-Kolthoff et al., number of proteins: 3617, DNA G+C content: 45.34 %).
2013). Finally, despite their overall phenotypic similarity, The GenBank/EMBL/DDBJ accession numbers for the partial
gen sp. 14BJ and taxon 20 can be distinguished based on 16S rRNA gene, gyrB and rpoB sequences of the type strain
the differences in the production of gelatinase and growth are KT997472, KT997478 and KT997518, respectively.
on gentisate (Table 2). Similar results were found for
another pair of closely related taxa, taxon 19 and
A. gyllenbergii. Based on these findings, we propose the Description of Acinetobacter dispersus sp. nov.
formal names Acinetobacter courvalinii sp. nov., Acinetobac-
ter dispersus sp. nov., Acinetobacter modestus sp. nov., Acine- Acinetobacter dispersus (dis.per9sus. L. masc. adj. dispersus
dispersed, pertaining to the occurrence of the species in
tobacter proteolyticus sp. nov. and Acinetobacter vivianii
diverse ecosystems).
sp. nov. for the five putative species, i.e. genomic species
14BJ, genomic species 17, taxon 18, taxon 19 and taxon The phenotypic characteristics correspond to those of the
20, respectively. genus (Baumann et al., 1968), i.e. Gram-negative, strictly

1682 International Journal of Systematic and Evolutionary Microbiology 66

 Five novel species of Acinetobacter

aerobic, oxidase-negative and catalase-positive coccobacilli slightly opaque, circular, convex and smooth, with entire
typically occurring in pairs, incapable of swimming moti- margins. Growth occurs in Brain-Heart Infusion (Oxoid)
lity, capable of growing in mineral media with acetate as at temperatures ranging from 15 uC to 37 uC, but not at
the sole carbon source and ammonia as the sole source 41 uC. Acid is not produced from D -glucose. Gelatin is
of nitrogen, and incapable of dissimilative denitrification. not hydrolysed. Colonies on agar media supplemented
Positive in the transformation assay of Juni (1972). with sheep erythrocytes are surrounded by wide zones of
haemolysis. L -Arginine, benzoate, ethanol, L -glutamate,
Colonies on tryptic soy agar (Oxoid) after incubation for
L -histidine and trigonelline are utilized as sole sources of
24 h at 30 uC are 1.0–1.5 mm in diameter, grey–white,
carbon, with growth visible in six (mostly two) days of
slightly opaque, circular, convex and smooth, with entire
culture at 30 uC. No growth occurs on trans-aconitate,
margins. Growth occurs in Brain-Heart Infusion (Oxoid)
b-alanine, 4-aminobutyrate, L -arabinose, L -aspartate,
at temperatures ranging from 15 uC to 37 uC, but not at
azelate, citraconate, citrate (Simmons), gentisate, D -gluco-
41 uC. Acid is not produced from D -glucose. Gelatin is hydro-
nate, D -glucose, glutarate, histamine, 4-hydroxybenzoate,
lysed. Colonies on agar media supplemented with sheep
DL -lactate, L -leucine, levulinate, malonate, L -ornithine, pheny-
erythrocytes are surrounded by wide zones of haemolysis.
lacetate, L -phenylalanine, putrescine, D -ribose, L -tartrate,
b-Alanine, 4-aminobutyrate, L -arginine, benzoate, citrate
tricarballylate or tryptamine in 10 days. The vast majority of
(Simmons), L -glutamate, glutarate, L -histidine, 4-hydroxy-
strains do not utilize adipate or 2,3-butanediol. Growth on
benzoate, DL -lactate, L -leucine, D -malate and putrescine are
D -malate is unclear in most strains.
utilized as sole sources of carbon, with growth visible in six
(mostly two) days of culture at 30 uC. No growth occurs The type strain is NIPH 236T (5CCUG 67964T5CIP
on L -arabinose, L -aspartate, 2,3-butanediol, citraconate, 110444T5CCM 8639T), isolated from the urine of a
D -gluconate, D -glucose, histamine, levulinate, D -ribose, female injured in a traffic accident (Přı́bram Regional Hos-
L -tartrate or trigonelline in 10 days. Utilization of trans- pital, Czech Republic) in 1993. This strain does not grow
aconitate, adipate, azelate, ethanol, gentisate, malonate, on 2,3-butanediol or adipate. The whole genome sequence
L -ornithine, phenylacetate, L -phenylalanine, tricarballylate of the type strain is available under GenBank/EMBL/DDBJ
and tryptamine is strain-dependent. accession no. APOJ00000000.1 (size: 3.54 Mb, number of
The type strain is ANC 4105T (5CCUG 67961T5CIP contigs: 32, number of proteins: 3347, DNA G+C content
110500T5CCM 8636T), isolated from well water in the 41.24 %). The GenBank/EMBL/DDBJ accession numbers
Czech Republic (Lišov, GPS coordinates: 49.0150233 N for the partial 16S rRNA gene, gyrB and rpoB sequences
14.6156889 E) in July 2011. This strain assimilates phenyl- of the type strain are KT997474, KT997501 and
acetate, L -phenylalanine and tryptamine, but not trans- KJ124832, respectively.
aconitate, adipate, azelate, ethanol, gentisate, malonate or
tricarballylate. Growth on L -ornithine is visible between
the sixth and tenth day of culture. The whole genome Description of Acinetobacter proteolyticus sp. nov.
sequence of the type strain is available under GenBank/ Acinetobacter proteolyticus (pro.te.o.ly9ti.cus. N.L. masc.
EMBL/DDBJ accession no. APRL00000000.1 (size: 4.10 Mb, adj. proteolyticus proteolytic, pertaining to the ability of
number of contigs: 15, number of proteins: 3835, DNA the species to hydrolyse gelatin).
G+C content: 42.41 %). The GenBank/EMBL/DDBJ acces-
sion numbers for the partial 16S rRNA gene, gyrB and rpoB The phenotypic characteristics correspond to those of the
sequences of the type strain are KT997473, KT997487 and genus (Baumann et al., 1968), i.e. Gram-negative, strictly
KT997527, respectively. aerobic, oxidase-negative and catalase-positive coccobacilli
typically occurring in pairs, incapable of swimming moti-
lity, capable of growing in mineral media with acetate as
Description of Acinetobacter modestus sp. nov. the sole carbon source and ammonia as the sole source
of nitrogen, and incapable of dissimilative denitrification.
Acinetobacter modestus (mo.des9tus. L. masc. adj. modestus
Positive in the transformation assay of Juni (1972).
moderate, pertaining to the limited capacity of the species
to utilize different carbon sources). Colonies on tryptic soy agar (Oxoid) after incubation for
24 h at 30 uC are 1.0–1.5 mm in diameter, grey–white,
The phenotypic characteristics correspond to those of the
slightly opaque, circular, convex and smooth, with entire
genus (Baumann et al., 1968), i.e. Gram-negative, strictly
margins. Growth occurs in Brain-Heart Infusion (Oxoid)
aerobic, oxidase-negative and catalase-positive coccobacilli
at temperatures ranging from 15 uC to 37 uC, but not at
typically occurring in pairs, incapable of swimming moti-
41 uC. Acid is not produced from D -glucose. Gelatin is
lity, capable of growing in mineral media with acetate as
hydrolysed. Colonies on agar media supplemented with
the sole carbon source and ammonia as the sole source
sheep erythrocytes are surrounded by wide zones of
of nitrogen, and incapable of dissimilative denitrification.
haemolysis. trans-Aconitate, b-alanine, L -arginine, benzo-
Positive in the transformation assay of Juni (1972).
ate, citrate (Simmons), gentisate, L -glutamate, L -histidine,
Colonies on tryptic soy agar (Oxoid) after incubation for 4-hydroxybenzoate, DL -lactate, D -malate, L -ornithine,
24 h at 30 uC are 1.0–1.5 mm in diameter, grey–white, L -phenylalanine and tricarballylate are utilized as sole

http://ijs.microbiologyresearch.org 1683
A. Nemec and others

sources of carbon, with growth visible in six (mostly two) Leiden (the Netherlands) before 1990. This strain produces
days of culture at 30 uC. No growth occurs on L -arabinose, acid from D -glucose, and assimilates adipate, azelate,
L -aspartate, 2,3-butanediol, citraconate, ethanol, D - gentisate, glutarate, putrescine and D -ribose, but not trans-
gluconate, D -glucose, histamine, levulinate, putrescine, aconitate or tricarballylate. Growth on L -aspartate is
D -ribose, L -tartrate or trigonelline in 10 days. Utilization doubtful. The whole genome sequence of the type strain
of adipate, azelate, glutarate, L -leucine, malonate, phenyla- is available under GenBank/EMBL/DDBJ accession no.
cetate and tryptamine is strain-dependent. No or doubtful APRW00000000.1 (size: 4.03 Mb, number of contigs: 16,
growth on 4-aminobutyrate. number of proteins: 3695, DNA G+C content: 44.68 %).
The GenBank/EMBL/DDBJ accession numbers for the
The type strain is NIPH 809T (5CCUG 67965T5CIP 110482T
partial 16S rRNA gene, gyrB and rpoB sequences of the
5CCM 8640T5Bouvet & Jeanjean 631T5Gilardi GLG
type strain are KT997477, KT997515 and KJ124844,
4638T), isolated from a human ear in the USA before 1984.
respectively.
This strain grows on malonate and tryptamine, but not on
adipate, 4-aminobutyrate, azelate, glutarate, L -leucine or
phenylacetate. The whole genome sequence of the type
strain is available under GenBank/EMBL/DDBJ accession Acknowledgements
no. APOI00000000.1 (size: 4.25 Mb, number of contigs: 32, Addendum: Most of the 40 strains analysed in the present study have
number of proteins: 3929, DNA G+C content: 42.72 %). now been deposited in the Czech National Collection of Type Cul-
The GenBank/EMBL/DDBJ accession numbers for the partial tures (http://apps.szu.cz/cnctc). Our special thanks go to all the
16S rRNA gene, gyrB and rpoB sequences of the type strain are donors listed in Table 1 for generous provision of strains. We are
KT997475, KT997505 and KJ124841, respectively. also indebted to Eva Kodytková (National Institute of Public
Health, Prague) for linguistic revision of the manuscript and to
Šimon Nemec (Waldorf Lyceum, Prague) and Martin Španěl (Charles
University in Prague) for their help with comparative genome anal-
Description of Acinetobacter vivianii sp. nov.
ysis. The study benefited from the public availability of whole-
Acinetobacter vivianii (vi.vi.a9ni.i. N.L. gen. masc. n. vivianii of genome sequences generated by the Broad Institute Genome Sequen-
Vivian, named after Alan Vivian, a British microbiologist). cing Platform within NCBI BioProject no. PRJNA183623. The present
work was supported by a grant from the Czech Science Foundation
The phenotypic characteristics correspond to those of the (13-26693S).
genus (Baumann et al., 1968), i.e. Gram-negative, strictly
aerobic, oxidase-negative and catalase-positive coccobacilli
typically occurring in pairs, incapable of swimming moti- References
lity, capable of growing in mineral media with acetate as
the sole carbon source and ammonia as the sole source Baumann, P., Doudoroff, M. & Stanier, R. Y. (1968). A study of the
of nitrogen, and incapable of dissimilative denitrification. Moraxella group. II. Oxidative-negative species (genus
Acinetobacter). J Bacteriol 95, 1520–1541.
Positive in the transformation assay of Juni (1972).
Bouvet, P. J. M. & Grimont, P. A. D. (1986). Taxonomy of the genus
Colonies on tryptic soy agar (Oxoid) after incubation for Acinetobacter with the recognition of Acinetobacter baumannii
24 h at 30 uC are 1.5–2.0 mm in diameter, grey–white, sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii
slightly opaque, circular, convex and smooth, with entire sp. nov., and Acinetobacter junii sp. nov. and emended descriptions
margins. Growth occurs in Brain-Heart Infusion (Oxoid) of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst
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