Microbiol Immunol 2016; 60: 303–311

doi: 10.1111/1348-0421.12374

ORIGINAL ARTICLE

Phylogenetics of family Enterobacteriaceae and proposal

to reclassify Escherichia hermannii and Salmonella
subterranea as Atlantibacter hermannii and Atlantibacter
subterranea gen. nov., comb. nov.
Hiroyuki Hata1, Tatsuya Natori1, Takuya Mizuno1, Izumi Kanazawa1, Ibrahim Eldesouky4,
Masahiro Hayashi2, Machiko Miyata1, Hajime Fukunaga1, Shoko Ohji3, Akira Hosoyama3, Eiji Aono3,
Atsushi Yamazoe3, Keiko Tsuchikane3, Nobuyuki Fujita3 and Takayuki Ezaki1
1
Department of Microbiology, Gifu University Graduate School of Medicine, 2Division of Anaerobe Research, Life Science Research
Center, Gifu University, 1-1 Yanagido, Gifu 501-1194, 3Biological Resource Center, National Institute of Technology and Evaluation,
2-5-8, Kazusakamatari, Kisarazu-shi, Chiba 292-0818, Japan and 4Department of Bacteriology, Mycology and Immunology, Faculty
of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt

ABSTRACT
Multilocus sequence analysis based on hypervariable housekeeping proteins was utilized to
differentiate closely related species in the family Enterobacteriaceae. Of 150 housekeeping proteins,
the top 10 hypervariable proteins were selected and concatenated to obtain distance data. Distances
between concatenated proteins within the family were 0.9–41.2%, whereas the 16S rRNA and
atpD-gyrB-infB-rpoB concatenated sequence (4MLSA) distances were 0.8–6.0% and 0.9–22.1%,
respectively. These data indicate that phylogenetic analysis by concatenation of hypervariable
proteins is a powerful tool for discriminating species in the family Enterobacteriaceae. To confirm
the discriminatory power of the 10 chosen concatenated hypervariable proteins (C10HKP),
phylogenetic trees based on C10HKP, 4MLSA, and the 16S rRNA gene were constructed.
Comparison of average bootstrap values among C10HKP, 4MLSA and 16S rRNA genes indicated
that the C10HKP tree was the most reliable. Location via the C10HKP tree was consistent with
existing assignments for almost all species in the family Enterobacteriaceae. However, the C10HKP
tree suggested that several species (including Enterobacter massiliensis, Escherichia vulneris,
Escherichia hermannii, and Salmonella subterranea) should be reassigned to different clusters than
those defined in previous analyses. Furthermore, E. hermannii and S. subterranea appeared to fall
onto a branch independent from those occupied by the other Enterobacteriaceae. Therefore, we
propose Atlantibacter gen. nov., such that E. hermannii and S. subterranea would be transferred to
genus Atlantibacter as Atlantibacter hermannii, comb. nov. and Atlantibacter subterranea. comb.
nov., respectively.

Key words Enterobacteriaceae, genes, essential, multilocus sequencing typing, phylogeny.

Correspondence
Hiroyuki Hata, Department of Microbiology, Gifu University Graduate School of Medicine, Gifu University, Yanagido 1-1, Gifu, 501-1194, Japan.
Tel: þ81 58 230 6488; Fax: þ81 58 267 0156; email: h-hatabou@feb.email.ne.jp
Received 21 December 2015; revised 25 February 2016; accepted 7 March 2016.

List of Abbreviations: 4MLSA, atpD-gyrB-infB-rpoB concatenated sequences; C10HKP, 10 concatenated hypervariable proteins; CDC, Centers for
Disease Control and Prevention; DSM, Deutsche Sammlung von Mikroorganismen (German Collection of Microorganisms); JNBP, Japan National
Bioresource of Bacterial Pathogen; KCN, potassium cyanide; LIM, lysine indole motility; MLSA, multilocus sequence analysis; NBRC, National Biological
Resource Center; TSI, triple sugar iron.

© 2016 The Societies and John Wiley & Sons Australia, Ltd 303
 H. Hata et al.
Members of the family Enterobacteriaceae are classified
into 52 genera and approximately 290 species. These
bacteria have been classified into several systems
according to different standards during their taxonomic
history. Some pathogenic species of the family were
misclassified historically based solely on their pathogenic
characteristics or the results of 16S rRNA–based
phylogenetic analyses. Because of the low discriminatory
power of 16S rRNA gene analysis, several housekeeping
genes have instead been used for phylogenetic analyses;
these housekeeping loci have included rpoB (1, 2), gyrB
(3), dnaJ (4) and recA (5). Moreover, MLSA has become
a powerful alternative to 16S rRNA gene or single
housekeeping gene phylogenetic analysis (5–8).
Approximately 10 years have passed since the
emergence of next-generation sequencing techniques;
these methods have made it relatively easy and inexpen-
sive to obtain draft genome sequences for bacterial strains.
In addition, many housekeeping protein sequences now
can be readily identified using high-performance auto-
mated protein sequence annotation programs. These
bioinformatics improvements have enabled the selection
of a greater variety of housekeeping proteins and therefore
might provide more reliable and precise MLSA data.
The family Enterobacteriaceae contains 52 historically
well-characterized genera, including Escherichia, Citro-
bacter and Salmonella. The results of 16S rRNA gene
analyses have indicated that these three genera are
closely related. However, Escherichia vulneris, Eschrichia
hermannii, and Salmonella subterranea do not form a
clade with the respective genus type species. To
determine the proper phylogenetic positions of these
species, we determined the draft genome sequences of
several strains of species in the family Entero-
bacteriaceae, and then compared these genomes by
MLSA using hypervariable housekeeping proteins.
MATERIALS AND METHODS
Bacterial strains and sequence information
All bacterial strains subjected to draft genome analysis
were obtained from the stocks of the JNBP and NBRC
collections. Nucleotide and protein sequence data were
obtained from the NCBI genome database. The strains
and information used in this study are listed in
Supplemental Table 1.
Draft genome sequences
Draft genome sequences for the bacterial strains were
determined using HiSeq 1000, 2500 or Miseq (Illumina,
San Diego, CA, USA) or 454 GS-FLX Titanium (Roche
Diagnostics, Pleasanton, CA, USA) sequencing systems.
304
Raw sequence data were assembled using Newbler
version 2.6 (Roche Diagnostics) and ABySS version 1.3.2
(9) software. After assembly, more than 90% of the
sequences were auto-annotated using the Microbial
Genome Annotation Pipeline (MiGAP) program suite
version 1.0.50 (10).
Selection of housekeeping proteins
One hundred and fifty housekeeping proteins, including
ribosomal proteins; proteins involved in tRNA synthesis,
DNA replication and modification, and cell wall
synthesis; and molecular chaperones were selected
from the clusters of orthologous groups database (11)
for Escherichia coli strain K-12 (substrain MG1655),
Salmonella enterica subsp. enterica, serovar Typhimu-
rium strain LT2 and Yersinia pestis strain CO92
(Supplemental Table 2). The numbers of amino acid
sequence differences between strains for each protein
were determined using MEGA version 6 software (12).
Distance and phylogenetic analysis
After concatenation of the 10 selected protein sequences,
the concatenated sequences together with 16S rRNA
gene sequences were aligned using MEGA6 software.
MLSA using the atpD, gyrB, infB and rpoB genes was
performed as previously described (7). Distances among
the strains were calculated by MGEA6 software and
correlation analysis was performed by Microsoft Excel
2007 software. Phylogenetic trees for the 16S rRNA
genes and MLSA using the atpD, gyrB, infB and rpoB
genes were prepared using the maximum likelihood
method based on the Tamura–Nei model (13). Phylo-
genetic trees of concatenated protein sequences were
prepared using the maximum likelihood method based
on the JTT matrix-based model (14).
Biochemical tests
Strains subject to biochemical tests are listed in
Supplemental Table 3. TSI agar, LIM medium, Simmons
citrate agar and Voges–Proskauer medium were pur-
chased from Kyokuto Pharmaceutical Industrial (Tokyo,
Japan). The KCN test was performed according to the
Sakazaki–Yamada modified test (15). Other biochemical
tests were conducted by ID 32 E, API 20 E and oxidase
tests (bioMerieux, Lyon, France).
RESULTS
Selecting the top 10 hypervariable
housekeeping proteins
To select hypervariable proteins, we calculated amino
acid distances for 150 housekeeping proteins. The
© 2016 The Societies and John Wiley & Sons Australia, Ltd
 Atlantibacter hermannii gen. nov., comb. nov.

Table 1. Amino acid sequence distances of housekeeping proteins

Category Number of proteins E. coli–S. enterica E. coli–Y. pestis S. enterica–Y. pestis

†
Cell wall synthesis 31 4.8% 16.9% 16.7%
Chaperonins 9 8.3% 23.1% 24.5%
Modification 17 3.8% 10.4% 10.3%
Replication 12 7.0% 21.1% 20.7%
Ribosomal proteins 52 1.6% 7.9% 7.9%
tRNA synthesis 29 4.9% 14.8% 15.1%
Total 150 4.0% 13.3% 13.4%
16S rRNA gene 3.2% 5.2% 6.0%

†, percentages indicate the average amino acid distance for each protein category.

average amino acid distances between E. coli and
S. enterica subsp. enterica, E. coli and Y. pestis, and
S. enterica subsp. enterica and Y. pestis were 4.0, 13.3
and 13.4%, respectively. Relatively high average amino
acid distances were observed for three of six protein
categories: chaperonins, DNA replication proteins and
cell wall–synthesis proteins (Table 1). We then selected
10 hypervariable proteins from these categories (Table 2).
We did not include some proteins exhibiting higher
distances among those ultimately selected because there
were too many deletion/insertion sites (e.g., FtsY) or
protein was absent in some species (e.g., HslJ and HscB).
Discriminatory power of concatenated
hypervariable proteins
To confirm the power of the selected proteins for
discriminating among species within the family Entero-
bacteriaceae, we calculated distances among E. coli
K12 MG1655 and several other members of the
Enterobacteriaceae using the 10 concatenated hypervar-
iable proteins (designated as C10HKP), the 16rRNA
sequences, and the atpD-gyrB-infB-rpoB concatenated
sequences (designated as 4MLSA) (Fig. 1). Distances
within the family using C10HKP were 0.9–41.2%,
whereas the distances using 16S rRNA and 4MLSA
were 0.8–6.0% and 0.9–22.1%, respectively. In addition,
we implemented correlation analysis to determine
whether C10HKP has the same divergence as 4MLSA.
The coefficient of correlation between these two models
was 0.902 (Fig. 2).
Phylogenetic analysis of the family
Enterobacteriaceae using C10HKP
We separately used the C10HKP, 4MLSA and 16S rRNA
sequences to construct phylogenetic trees for the family
Enterobacteriaceae (Fig. 3; Supplemental Figs 1–3).
Average bootstrap values were 89.2% for the C10HKP-
based tree, 77.0% for the 4MLSA-based tree, and 49.8%

Table 2. Selected hypervariable housekeeping genes

Amino acid E. coli– E. coli– S. enterica–

Category Symbol† Gene product† length‡ S. enterica Y. pestis Y. pestis

Replication holB DNA polymerase III, delta prime subunit 334 21.0% 48.2% 47.6%
Cell wall synthesis murB UDP-N-acetylenolpyruvoylglucosamine reductase 342 17.8% 38.6% 38.0%
Chaperonin grpE Molecular chaperone GrpE (heat shock protein) 191 7.3% 38.2% 39.3%
Cell wall synthesis ftsQ Cell division septal protein FtsQ 266 6.4% 30.8% 29.3%
Chaperonin lolA Outer membrane lipoprotein-sorting protein 202 5.9% 29.7% 30.7%
Cell wall synthesis murF UDP-N-acetylmuramyl pentapeptide synthase 452 12.4% 29.4% 31.4%
Cell wall synthesis ftsX Cell division protein FtsX 316 5.4% 28.5% 26.9%
Replication holA DNA polymerase III, delta subunit 343 10.2% 28.3% 28.6%
Cell wall synthesis pssA Phosphatidylserine synthase 451 6.2% 25.9% 26.2%
Cell wall synthesis mreD Cell shape-determining protein MreD 162 5.6% 24.7% 24.1%
Chaperonin hslJ§ Heat shock protein HslJ 136 29.4% 51.5% 58.8%
Cell wall synthesis ftsY§ Signal recognition particle GTPase 487 15.0% 32.0% 32.6%
Chaperonin hscB§ DnaJ-domain-containing proteins 1 171 8.8% 32.7% 33.9%

†, symbols and gene names used for orthologous sequences are consistently those used in the E. coli genome annotation; ‡, amino acid length
indicates the average amino acid length for E. coli, S. enterica and Y. pestis; §, hslJ, ftsY and hscB had large distances but were not included among the
concatenated proteins.

© 2016 The Societies and John Wiley & Sons Australia, Ltd 305
 H. Hata et al.

Fig. 1. 16S rRNA gene, 4MLSA, and C10HKP sequence distances for several species in the family Enterobacteriaceae. The percentage of
nucleotide base differences (16S rRNA gene and 4MLSA) and amino acid sequence differences (C10HKP) relative to Escherichia coli K-12
MG1655 are shown. All positions containing gaps and missing data were eliminated. There were a total of 1154 (16S rRNA gene), 2633
(4MLSA), and 2907 (C10HKP) positions in the final datasets.

for the 16S rRNA sequence-based tree. In addition, more

than 70% of the branches in the C10HKP-based tree
exhibited bootstrap values >95%.

Phenotypic characteristics of Genus

Fig. 2. Relationship between 4MLSA and C10HKP distances.
Each dot represents a pairwise comparison between 4MLSA and
C10HKP distances. Both distances were calculated round-robin using
92 nucleotides (4MLSA) or 92 proteins (C10HKP).
Escherichia and Salmonella
To identify specific phenotypic characteristics for
Escherichia vulneris, Escherichia hermannii and Salmo-
nella subterranea, we conducted several biological tests
using type strains and reference strains of Genuses
Escherichia and Salmonella (Supplemental Table 3 and
4). We found that almost all the phenotypic character-
istics of E. hermannii and S. subterraneaare are identical.
Only beta-glucosidase and D-arabitol fermentation/
oxidation differed. We further found that the character-
istics of Escherichia fergusonii are similar to those of
E. hermanii and S. subterranea. However, we could
distinguish E. fergusonii from those species by lysine
decarboxylation, adonitol fermentation/oxidation, yellow

306 © 2016 The Societies and John Wiley & Sons Australia, Ltd
 Atlantibacter hermannii gen. nov., comb. nov.

Fig. 3. (a) 4MLSA- and (b) C10HKP-based phylogenetic trees of members of the family Enterobacteriaceae. The evolutionary history was
inferred by using the maximum likelihood method based on the Tamura–Nei model (13) for 4MLSA and JTT matrix-based model (14) for
C10HKP. The tree with the highest log likelihood (56653.1344 for 4MLSA and 104327.7197 for C10HKP) is shown. The percentages of trees
in which the associated taxa clustered together are shown next to the branches. Initial tree(s) for the heuristic search were obtained by applying
the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood approach. The tree is drawn to
scale, with branch lengths measured in the number of substitutions per site. The analysis involved 92 nucleotide sequences for 4MSLA and
92 amino acid sequences for C10HKP. All positions containing gaps and missing data were eliminated. There were a total of 2633 positions for
4MLSA and 2907 positions for C10HKP in the final dataset.

© 2016 The Societies and John Wiley & Sons Australia, Ltd 307
 H. Hata et al.
pigmented colonies and growth in KCN. Although we
found that the characteristics of E. vulneris are similar to
those of E. albertii by TSI agar, LIM medium, Simmons
citrate agar and Voges–Proskauer medium, we easily
differentiated these two strains by motility.
DISCUSSION
We selected 10 hypervariable proteins from 150
housekeeping proteins commonly possessed by mem-
bers of the family Enterobacteriaceae. In distance
calculations, C10HKP yielded distance values more
than twofold those obtained using 4MLSA (Fig. 1).
Moreover, distances derived using C10HKP strongly
correlated with those derived using 4MLSA (Fig. 2). In
addition, the phylogenetic tree obtained using C10HKP
had higher bootstrap values than the trees obtained using
either 4MLSA or 16S rRNA sequences (Supplemental
Figs 1–3). These data indicate that the C10HKP-based
tool is more reliable than 16S rRNA and 4MLSA tools for
phylogenetic analysis of the family Enterobacteriaceae.
Almost all genera were grouped into a single cluster in
the C10HKP-based tree. However, some species were
grouped as parts of clusters distinct from those suggested
by previous classifications. For example, the type strains
of Enterobacter aerogenes and Enterobacter massiliensis
were not classified with the Enterobacter cloacae cluster
when analyzed using C10HKP. E. aerogenes (16) and
Klebsiella mobilis (17) have the same type strain and are
therefore homotypic synonyms (Rules 24a and 24b) (18).
Our results led us to conclude that E. aerogenes belongs
in the genus Klebsiella. In addition, given that strain CHS
78 was assigned to the E. cloacae cluster by C10HKP
analysis, it appears that Lelliottia amnigena should be
classified in the genus Enterobacter. However, because
there is thus far no type strain genome information for
L. amnigena, further information is needed before this
species can be reclassified with confidence.
The 16S rRNA gene–based phylogenetic tree indi-
cated that species of the genus Citrobacter are closely
related to species of both the genus Escherichia and
the genus Salmonella. However, the C10HKP-based tree
divided the Citrobacter species into two groups. The
animal-derived species Citrobacter rodenticum and
Citrobacter farmeri (19) segregated with members of
the genus Escherichia, whereas Citrobacter freundii,
isolated from humans (20), segregated with members of
the genus Salmonella. Although these results support
division of the genus Citrobacter into two distinct
genera, we do not propose any taxonomic changes for
Citrobacter both because no clear genus definition is
currently available and we did not (in the present study)
analyze all of the Citrobacter species' genomes.
308
E. fergusonii and E. albertii are independent and
established species (21, 22). However, these species were
difficult to distinguish from E. coli by 16S rRNA gene–
based phylogenetic analysis (Supplemental Fig. 1). In
contrast, these two species were clearly differentiated
from E. coli in the C10HKP-based tree, which provided
100% bootstrap values (Supplemental Fig. 3). However,
species of the genus Shigella could not be differentiated
from E. coli, even via the C10HKP-based tree.
The 16S rRNA gene–based phylogenetic tree suggests
that E. vulneris has been misclassified in the genus
Escherichia (Supplemental Fig. 1). 4MLSA analysis also
indicated that E. vulneris does not belong to the genus
Escherichia. Consistent with those analyses, data from
the C10HKP-based tree strongly suggested that
E. vulneris is independent from the genus Escherichia,
and is instead related to Kosakonia sacchari and
E. massiliensis. Phenotypic characteristics of E. vulneris
are clearly differentiated from those of E. coli, E. albertii
and E. fergusonii by ornithine decarboxylase, arginine
dihydrolase and lysine decarboxylase activities (Supple-
mental Table 4). These data indicate that E. vulneris
belongs to a genus distinct from Escherichia. Further
investigation is needed to clarify the correct taxonomic
position of E. vulneris.
E. hermannii and S. subterranea also sorted separately
from their respective genera according to data from the
16S rRNA gene–based tree. Both the 4MLSA- and
C10HKP-based trees indicated that E. hermannii and
S. subterranea should be assigned to a shared genus.
Analyses using either of these concatenation tools
suggested that these two species are closely related to
members of the genus Cronobacter. Indeed, the
phenotypic characteristics of these two species are quite
similar, while clearly different from those of other
Escherichia and Salmonella species (Table 3). These data
strongly suggest that E. hermannii and S. subterranea
represent a new genus. Hence, we propose Atlantibacter
gen. nov. for both of these species.
Description of Atlantibacter gen. nov.
Atlantibacter (Atlan.ti.bac'ter. N.L. gen. fem. n. Atlanta,
the city of Atlanta, Georgia, USA, where the U.S. Centers
for Disease Control and many researchers investigating
the family Enterobacteriaceae are located; N.L. masc.
n. bacter a rod; N.L. masc. n. Atlantibacter a rod named
in memory of Atlanta).
This description is based on those of Brenner et al.
(23) and Shelobolina et al. (24).
Gram-negative non-spore-forming rods that are facul-
tatively anaerobic and motile. After 24 hr aerobic
incubation at 37°C on trypticase soy agar, colonies are
© 2016 The Societies and John Wiley & Sons Australia, Ltd
 Atlantibacter hermannii gen. nov., comb. nov.

diarizonae JNBP 4573T, S. enterica subsp. houtenae JNBP 4858T and S. enterica subsp. salamae JNBP 4556T; ‡, S. enterica subsp houtenae JNBP 4858T is negative for D-sorbitol fermentation and positive for
S. bongori

†, this column shows common characteristics of the following strains: S. enterica subsp. enterica serovar Typhimurium JNBP 7710 (¼ATCC 13311), S. enterica subsp. arizonae JNBP 4588T, S. enterica subsp.
NBP 4536
yellow-pigmented and convex. Catalase-positive and

þ
þ

þ
þ
þ
þ
þ
þ

Genus Salmonella negative for oxidase. Positive for indole reaction and
nitrate reduction, growth in KCN, ornithine decarboxylase
and beta- galactosidase activities. Negative results in tests
S. enterica†
for urease, ornithine decarboxylase, arginine dihydrolase,
lysine decarboxylase and L-asparagine arylamidase, lipase,

þ‡
‡
þ
þ

þ
þ
þ
þ


alpha-glucosidase, alpha-galactosidase, alpha-maltosidase
and beta- glucuronidase. N-acetyl-beta-D-glucosamini-
dase activities, H2S production and the Voges–Proskauer
NBRC 102420T

reaction. Produce acid from the following compounds:

E. vulneris

D-amygdalin, L-arabinose, L-rhamnose, D-cellobiose,


þ

þ

þ
þ



D-galacturonic acid, D-glucose, D-mannitol, D-maltose
and trehalose. No acid production is observed for
L-arabitol, 5-ketogluconate, sodium pyruvate, adonitol,
E. fergusonii
JNBP 2381T

inositol, D-melibiose, palatinose or D-sorbitol.

The type species is Atlantibacter hermannii.

þ
þ
þ






Genus Escherichia

Description of Atlantibacter hermannii

comb. nov.
NBRC 107761T

Atlantibacter hermannii (her.man'ni.i. N.L. gen. n.

E. albertii

hermannii of Hermann, in honor of George J. Hermann,


þ









former chief of the Enteric Section at the CDC, for his

many contributions to enteric bacteriology, and Lloyd G.
Herman, formerly of the Environmental Services Branch,
National Institutes of Health, Bethesda, MD, for his
JNBP 2020T

contributions to the study of yellow-pigmented bacteria).

E. coli


þ
þ
þ


þ
þ



Basonym: Escherichia hermannii Brenner et al. 1983.

The description of this taxon is the same as that given
by Brenner et al. (23).
The type strain is NBRC 105704T ¼ ATCC 33650T ¼
S. subterranea
JNBP 5029T

DSM 4560T. Sequence accession numbers: JN175345

Atlantibacter gen. nov.



þ
þ




þ
þ

(16S rRNA gene) and GCA_000248015.2 (whole

genome) for the type strain. Atlantibacter hermannii
Table 3. Key tests for phenotypic characteristics of Atlantibacter gen. nov.

was previously known as CDC Enteric Group 11.

NBRC105704T

Description of Atlantibacter subterranea

E. hermannii

comb. nov.


þ
þ




þ
þ

Altantibacter subterranea (sub.ter.ra'ne.a. L. adj. subter-

ranea, -us, underground, subterranean).
Basonym:Salmonella subterranea Shelobolina et al.
Lysine decarboxylase (LIM, API 20E and ID 32E)

2005.
D-sorbitol fermentation (API 20E and ID 32E)
Arginine dihydrolase (API 20E and ID 32E)

The description of this taxon is the same as that given

by Shelobolina et al. (24).
The type strain is FRClT ¼ ATCC BAA-836T ¼ DSM
16208T. Sequence accession numbers: AY373829 (16S
Alpha-galactosidase (ID 32E)

rRNA gene) and DRR015979 (whole genome, raw

Phenotypic characteristic

Indole production (LIM)

sequence data) for the type strain.

Hydrogen sulfide (TSI)

Citrate (API 20E)

ACKNOWLEDGEMENT
Yellow pigment

growth in KCN.
Growth in KCN
Motility (LIM)

This work was performed as part of a project supported

by the Ministry of Economy, Trade and Industry of
Japan.

© 2016 The Societies and John Wiley & Sons Australia, Ltd 309
 H. Hata et al.
DISCLOSURE
None of the authors have any conflicts of interest
associated with this study.
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SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article at the publisher's web-site.
Fig S1. 16S rRNA gene–based phylogenetic tree of
members of the family Enterobacteriaceae. The evolu-
tionary history was inferred using the maximum
© 2016 The Societies and John Wiley & Sons Australia, Ltd
 Atlantibacter hermannii gen. nov., comb. nov.
likelihood method based on the Tamura–Nei model
(13). The tree with the highest log likelihood
(9295.6582) is shown. The percentages of trees in
which the associated taxa clustered together are shown
next to the branches. Initial tree(s) for the heuristic
search were obtained by applying the neighbor-joining
method to a matrix of pairwise distances estimated using
the maximum composite likelihood approach. The tree
is drawn to scale, with branch lengths measured in the
number of substitutions per site. The analysis involved
92 nucleotide sequences. All positions containing gaps
and missing data were eliminated. There were a total of
1154 positions in the final dataset.
Fig S2. 4MLSA-based phylogenetic tree of members of
the family Enterobacteriaceae. The multilocus sequence
analysis method has been reported previously (7). The
phylogenetic tree was constructed in the same manner
as the tree shown in Supplemental Figure 1. The tree
with the highest log likelihood (56653.1344) is
shown. There were a total of 2633 positions in the final
dataset.
© 2016 The Societies and John Wiley & Sons Australia, Ltd
Fig S3. C10HKP-based phylogenetic tree of members of
the family Enterobacteriaceae. The evolutionary history
was inferred by using the maximum likelihood method
based on the JTT matrix-based model (14). The tree with
the highest log likelihood (104327.7197) is shown. The
percentages of trees in which the associated taxa clustered
together are shown next to the branches. Initial tree(s) for
the heuristic search were obtained by applying the
neighbor-joining method to a matrix of pairwise distances
estimated using a JTT model. The tree is drawn to scale,
with branch lengths measured in the number of
substitutions per site. The analysis involved 92 amino
acid sequences. All positions containing gaps and missing
data were eliminated. There were a total of 2907 positions
in the final dataset.
Table S1. Genome sequence data used in this study.
Table S2. Amino acid distances of housekeeping proteins.
Table S3. Phenotypic characteristics of Genus Escherichia
and Salmonella (Part 1).
Table S4. Phenotypic characteristics of Genus Escherichia
and Salmonella (Part 2).
311