micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 1
ORIGINAL RESEARCH
published: 04 Sep embe 2018
doi: 10.3389/ micb.2018.02077
Edi ed by:
Ma ha E. T ujillo,
Uni e sidad de Salamanca, Spain
Re iewed by:
John Phillip Bowman,
Uni e si y o Tasmania, Aus alia
Ja ie Pascual,
Deu sche Sammlung on
Mik oo ganismen und Zellkul u en
(DSMZ), Ge many
*Co espondence:
Jesus L. Romalde
[email p o ec ed]
Ma ía J. Figue as
ma iajose. igue as@u .ca
Special y sec ion:
This a icle was submi ed o
E olu iona y and Genomic
Mic obiology,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 15 Feb ua y 2018
Accep ed: 14 Augus 2018
Published: 04 Sep embe 2018
Ci a ion:
Pé ez-Ca aluña A, Salas-Massó N,
Diéguez AL, Balboa S, Lema A,
Romalde JL and Figue as MJ (2018)
Re isi ing he Taxonomy o he Genus
A cobac e : Ge ing O de F om
he Chaos. F on . Mic obiol. 9:2077.
doi: 10.3389/ micb.2018.02077
Re isi ing he Taxonomy o he
Genus A cobac e : Ge ing O de
F om he Chaos
Alba Pé ez-Ca aluña1, Nu ia Salas-Massó1, Ana L. Diéguez2, Sabela Balboa2,
Albe o Lema2, Jesús L. Romalde2*and Ma ia J. Figue as1*
1Depa amen de Ciències Mèdiques Bàsiques, Facul a de Medicina, Ins i u d’In es igació Sani à ia Pe e Vi gili, Uni e si a
Ro i a i Vi gili, Reus, Spain, 2Depa amen o de Mic obiología y Pa asi ología, CIBUS-Facul ad de Biología, Uni e sidade
de San iago de Compos ela, San iago de Compos ela, Spain
Since he desc ip ion o he genus A cobac e in 1991, a o al o 27 species ha e
been desc ibed, al hough some species ha e shown 16S RNA simila i ies below 95%,
which is he cu -o ha usually sepa a es species ha belong o di e en gene a.
The objec i e o he p esen s udy was o eassess he axonomy o he genus
A cobac e using in o ma ion de i ed om he co e genome (286 genes), a Mul ilocus
Sequence Analysis (MLSA) wi h 13 housekeeping genes, as well as di e en genomic
indexes like A e age Nucleo ide Iden i y (ANI), in silico DNA–DNA hyb idiza ion (isDDH),
A e age Amino-acid Iden i y (AAI), Pe cen age o Conse ed P o eins (POCPs), and
Rela i e Synonymous Codon Usage (RSCU). The s udy included a o al o 39 s ains
ha ep esen all he 27 species included in he genus A cobac e oge he wi h 13
s ains ha a e po en ially new species, and he analysis o 57 genomes. The di e en
phylogene ic analyses showed ha he A cobac e species g ouped in o ou clus e s.
In addi ion, A. leki hoch ous and he candida us species ‘A. aqua icus’ appea ed, as
did A. ni o igilis, he ype species o he genus, in sepa a e b anches. Fu he mo e,
he genomic indices ANI and isDDH no only con i med ha all he species we e well-
de ined, bu also he cohe ence o he clus e s. The AAI and POCP alues showed
in a-clus e anges abo e he espec i e cu -o alues o 60% and 50% desc ibed
o species belonging o he same genus. Pheno ypic analysis showed ha ce ain
es combina ions could allow he di e en ia ion o he ou clus e s and he h ee
o phan species es ablished by he phylogene ic and genomic analyses. The o igin
o he s ains showed ha each o he clus e s emb aced species eco e ed om
a common o ela ed en i onmen . The esul s ob ained enable he di ision o he
cu en genus A cobac e in a leas se en di e en gene a, o which he names
A cobac e ,Aliia cobac e gen. no ., Pseudoa cobac e gen. no ., Haloa cobac e gen.
no ., Malacobac e gen. no ., Poseidonibac e gen. no ., and Candida e ‘A coma inus’
gen. no . a e p oposed.
Keywo ds: A cobac e ,Aliia cobac e gen. no ., Pseudoa cobac e gen. no ., Haloa cobac e gen. no .,
Malacobac e gen. no ., Poseidonibac e gen. no ., axonomic c i e ia
F on ie s in Mic obiology | www. on ie sin.o g 1Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 2
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
INTRODUCTION
The genus A cobac e was c ea ed by Vandamme e al. (1991) o
accommoda e G am-nega i e, cu ed-shaped bac e ia belonging
o wo species Campylobac e c yae ophila (now A cobac e
c yae ophilus) and Campylobac e ni o igilis (now A. ni o igilis),
conside ed a ypical campylobac e s due o hei abili y o g ow
a lowe empe a u es (15◦C–30◦C) and wi hou mic oae ophilic
condi ions (Vandamme e al., 1991). The la e species was
selec ed as he ype species o he new genus (Vandamme e al.,
1991). One yea la e he genus was enla ged wi h he addi ion
o wo new species, A. ski owii wi h an animal o igin being
isola ed om abo ed o ine, po cine and bo ine e uses, and om
lambs wi h dia hea, and A. bu zle i, which was eco e ed om
cases o human and animal dia hea (Vandamme e al., 1992).
Ano he wo new species we e inco po a ed in o he genus in
2005. A. halophilus was isola ed om wa e om a hype saline
lagoon in Hawaii (Donachie e al., 2005), and A. ciba ius was
isola ed om b oiled ca casses in Belgium (Hou e al., 2005).
These species we e assigned o he genus A cobac e on he basis
o he 16S RNA gene simila i y (94% and 95% o A. ni o igilis
wi h A. halophilus and A. ciba ius, espec i ely). Howe e , hese
alues a e equal, o e en below, he cu -o o 95% o genus
de ini ion (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008,
2014;Tindall e al., 2010).
F om 2009 onwa d, new species we e being desc ibed yea -
by-yea , eaching a o al numbe o 27 in 2017. In some o hese
desc ip ions, he simila i y o he 16S RNA gene was he decisi e
cha ac e o axonomic assigna ion a genus le el, al hough
phylogeny based on housekeeping genes ( poB i s and hen gy B
and hsp60) was also included as addi ional, mo e disc imina o y
ools o he species (Collado e al., 2009a, 2011;De Sme e al.,
2011). Using his app oach, A. mollusco um,A. ellisii,A. de lu ii,
o A. bi al io um we e de ined, among o he s (Collado e al.,
2009a, 2011;Figue as e al., 2011a,b;Le ican e al., 2012), which
showed 16S RNA simila i ies anging om 91.1 o 94.7%, no
suppo ing hei common a ilia ion. On he o he hand, he mos
closely ela ed species, which showed a simila i y o 99.1% we e
A. ellisii and A. de lu ii (Collado e al., 2011), gi ing e idence
o he i s ime o he poo esolu ion o he 16S RNA gene
o sepa a ing closely ela ed species in he genus A cobac e .
Howe e , he phylogene ic analysis based on he conca ena ed
sequences o gy B, poB, and cpn60 genes, oge he wi h he
DNA–DNA hyb idiza ion esul s, clea ly suppo ed he exis ence
o hese wo di e en ia ed axa (Figue as e al., 2011a). Also in
2011, A. ophia um was disco e ed om he in es inal ac o
heal hy a ening pigs, which in e es ingly showed he closes
simila i ies (≥97.4%) wi h he o he species also eco e ed om
humans o animals, i.e., A. c yae ophilus,A. he eius,A. ciba ius,
o A. ski owii (De Sme e al., 2011;Figue as e al., 2014;Van den
Abeele e al., 2014).
In 2013, he species A. cloacae and A. suis we e desc ibed,
using a Mul ilocus Sequence Analysis (MLSA) app oach
including i e housekeeping genes (Le ican e al., 2013) o he
i s ime. Simul aneously, and due o he highes 16S RNA gene
simila i y wi h A. ma inus (95.5%), he species A. anae ophilus
was inco po a ed o he genus (Sasi-Jyo hsna e al., 2013).
Howe e , his species showed a ypical cha ac e is ics, including
lack o mo ili y and obliga e anae obic me abolism, which led o
he o iginal desc ip ion o he genus A cobac e being emended
(Sasi-Jyo hsna e al., 2013). The mos ecen ly desc ibed species
om shell ish a e A. leki hoch ous,A. halio is, and A. canalis
(Diéguez e al., 2017;Tanaka e al., 2017;Pé ez-Ca aluña e al.,
2018a). The i s one included se e al isola es eco e ed om
scallop la ae and om ank seawa e o a No wegian ha che y
(Diéguez e al., 2017), he second species came om an abalone
o Japan (Tanaka e al., 2017) and he hi d om oys e s
subme ged in a wa e channel con amina ed wi h was ewa e
(Pé ez-Ca aluña e al., 2018a). Howe e , Diéguez e al. (2018)
e idenced ha he species A. halio is is a la e he e o ypic
synonym o A. leki hoch ous. Addi ionally, he low 16S RNA
gene simila i y o A. leki hoch ous wi h he known A cobac e
species (91.0–94.8%) ound in he A. leki hoch ous desc ip ion
made Diéguez e al. (2017) sugges ha ce ain species migh
belong o o he gene a and ecommend ha a p o ound e ision
o he genus migh cla i y he axonomy.
On he o he hand, adding 2.5% NaCl o he en ichmen
medium and subcul u ing on ma ine aga , Salas-Massó e al.
(2016) ecognized se en po en ial new species om wa e and
shell ish (mussels and/o oys e s), and eco e ed new isola es
o A. halophilus and A. ma inus o which only he ype s ains
had been known. In addi ion, du ing he cha ac e iza ion o
he mos ecen ly desc ibed species A. canalis (Pé ez-Ca aluña
e al., 2018a) and when ying o de ine he se en men ioned
new species, we obse ed ha he A cobac e species o med
se e al di e en clus e s dis an enough o suspec hey migh
co espond o di e en gene a, in ag eemen wi h Diéguez e al.
(2017).
The e a e clea c i e ia o desc ibing new bac e ial species
(Tindall e al., 2010;Figue as e al., 2011a,b). Howe e , he
desc ip ion o a genus is usually based on a cu -o o <95%
simila i y in he 16S RNA gene sequence, and a G+C (% mol)
con en di e ing by mo e han 10% (Rosselló-Mo a and Amann,
2001;Ya za e al., 2008;Tindall e al., 2010;Ya za e al., 2014).
Nowadays, genomic da a like he A e age Nucleo ide Iden i y
(ANI) and he in silico DNA–DNA hyb idiza ion (isDDH) a e
used o de ine bac e ial species, al hough ha e no ye been ully
explo ed o delinea ing gene a (Kons an inidis and Tiedje, 2005;
Go is e al., 2007;Rich e and Rosselló-Mó a, 2009;Qin e al.,
2014;Chun e al., 2018).
A pe cen age o A e age Amino-acid Iden i y (AAI) anging
om 60 o 80% be ween he compa ed genomes o species o
s ains and a Pe cen age o Conse ed P o eins (POCPs) abo e
50% has been p oposed i hey a e o belong o he same genus
(Kons an inidis and Tiedje, 2005;Qin e al., 2014). Finally, he
Rela i e Synonymous Codon Usage (RSCU) has also been used
by some au ho s o in e e olu iona y and ecological links among
bac e ial species (Ma e al., 2015;Fa ooqi e al., 2016).
Ve y ecen ly, Wai e e al. (2017) ca ied ou a compa a i e
genomic analysis o he class Epsilonp o eobac e ia. Using 16S
and 23S RNA, 120 single-copy ma ke p o eins and AAI
analysis hey p oposed i s eclassi ica ion as he new phylum
Epsilonbac e aeo a. In ha s udy, Wai e e al. (2017) also
p oposed a eclassi ica ion o he genus A cobac e as a new
F on ie s in Mic obiology | www. on ie sin.o g 2Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 3
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
Family A cobac e aceae, wi hin he class Campylobac e ia, o de
Campylobac e ales. One weakness o his s udy, speci ically
ega ding he genus A cobac e , is ha only se en alida ed
species we e included in he analysis. The new amily he e o e
comp ised only he genus A cobac e . Howe e , hese indings
also suppo he need o a cla i ica ion o he axonomy o he
cu en genus A cobac e .
The ise o genome sequencing has d ama ically changed
he landscape o sys ema ics o p oka yo es, imp o ing di e en
aspec s such as he iden i ica ion o species, he unc ional
cha ac e iza ion o esol ing axonomic g oups, and he
esolu ion o he phylogeny o highe axa (Whi man, 2015). I
seems clea ha he inco po a ion o genomics in o he axonomy
will boos i s c edibili y p o iding ep oducible, eliable, highly
in o ma i e means o in e phylogene ic ela ionships among
p oka yo es, and a oiding un eliable me hods and subjec i e
di icul - o- eplica e da a (Chun and Rainey, 2014;Chun e al.,
2018).
Wi hin his mode n axonomy con ex , he objec i e o he
p esen s udy was o eassess he axonomy o he known
and newly ecognized A cobac e species by using a MLSA o
13 housekeeping genes, he whole genome sequences and he
de i ed genomic analysis. The la e analysis included ANI,
isDDH, AAI, POCP, and RSCU o all A cobac e ype s ains. In
addi ion, phylogenies based on 16S and 23S RNA gene sequences
we e also pe o med wi h compa a i e pu poses. The new
axonomic c i e ia we e s able when including whole genome
sequences o a second s ain o each species o o unassigned
sequences ob ained om he public da abases.
MATERIALS AND METHODS
Bac e ial S ains
All 27 alid species included in he genus A cobac e ha e been
s udied. They a e ep esen ed by 39 s ains, and 13 s ains ha
a e po en ially new species (Table 1). Fu he mo e, 50 genomes
o A cobac e s ains iden i ied a species le el we e in es iga ed,
39 o which we e ob ained in ou labo a o y (27 om known
species and 13 om po en ially new species) and he o he s om
he public da abases1,2. Fi e genomes ha had been deposi ed as
A cobac e sp. in he da abases we e also included in he s udy.
I he e was mo e han one s ain o a known A cobac e species,
wo ep esen a i e genomes o each species we e included in he
analysis. The only excep ions we e: A. ac icola (Pa k e al., 2016)
and A. paci icus (Zhang e al., 2015), whose axonomic posi ions
we e only in e ed by he phylogene ic analysis o he 16S RNA
gene sequences published in hei species desc ip ions, oge he
wi h a MLSA o h ee housekeeping genes (a pA, gy B, and
poB) o A. paci icus (Zhang e al., 2015;Pa k e al., 2016). The
s ains conside ed po en ially new species, and named he ea e
as ‘candida e species,’ had been ecognized wi h an MLSA analysis
o i e housekeeping genes (a pA, gy A, gy B, hsp60, and poB)
(da a no shown).
1h ps://www.ncbi.nlm.nih.go /genome/
2h ps://gold.jgi.doe.go /
Cul u ing o genome sequencing was ca ied ou ei he on
blood aga (DIFCO, Mad id, Spain) o ma ine aga (Scha lau,
Sen mena , Spain) a 30◦C in ae obiosis o 24–72 h, depending
on he equi emen s. DNA was ex ac ed using Easy-DNATM
gDNA Pu i ica ion ki (In i ogen, Mad id, Spain) ollowing
he manu ac u e ’s ins uc ions. The in eg i y o he DNA was
e alua ed by elec opho esis o 10 µl o he sample in a 1.5%
aga ose gel. The o al amoun o DNA was quan i ied using
Qubi TM wi h he dsDNA B oad Range Assay ki (In i ogen).
Pai ed-end lib a ies we e cons uc ed wi h 50 ng o DNA
using Nex e a DNA Lib a y P epa a ion Ki (Illumina, Lisbon,
Po ugal) and sequenced wi h MiSeq pla o m (Illumina).
Sequencing gene a ed 2 ×300 bp pai ed-end eads. Clean
eads we e assembled wi h SPAdes (Nu k e al., 2013) and
he CGE assemble (La sen e al., 2012) in o de o selec
he be e assembly. Be o e deposi ing he genomes in he
NCBI da abase, FASTA iles we e sc eened o euka yo ic and
p oka yo ic sequences using BLASTn, and o adap o s wi h
VecSc een s andalone so wa e3. The i e housekeeping genes
used in he i s MLSA analysis (a pA, gy A, gy B, hsp60, and
poB) we e ex ac ed om each genome and compa ed wi h
he Sange sequences o hese genes ob ained o iginally o
he iden i ica ion o he s ain. The exis ence o a single and
iden ical copy o hese genes con i med ha he genomes we e
no con amina ed and belonged o he co ec s ain. Finally,
con igs we e dele ed i hey had less han 200 bp. The genomes
we e deposi ed in he GenBank da abase and Table 1 lis s he
accession numbe s.
The 55 genomes we e anno a ed wi h a local ins alla ion o
P okka 1.2 (Seemann, 2014) using an e- alue o 1e-06. The
anno a ion was pe o med wi h P okka, wi h he p edic ion
ools P odigal 2.6 (Hya e al., 2010) and ARAGORN 1.2
(Lasle and Canback, 2004). The p edic ion ool Ba nap
0.64included in P okka 1.2 was used o he anno a ion
o RNA genes. Coding sequences (CDS) we e anno a ed,
combining he Rapid Anno a ion Subsys ems Technology
(RAST) (O e beek e al., 2014) using he classic RAST scheme
and he Anno a ion Tools o PATRIC se e (Wa am e al.,
2017). The cha ac e is ics o each genome (i.e., N50, numbe
o con igs, numbe o CDS, G+C con en ) we e ob ained om
NCBI anno a ions.
Analysis o Housekeeping Genes,
Ribosomal Genes, and Co e Genome
Thi een housekeeping genes (a pA, a pD, dnaA, dnaJ, dnaK,
sZ, gy A, hsp60, adA, ecA, poB, poD, and s ) we e ob ained
om he genomes using BLASTn sea ch. Sequence simila i ies
o housekeeping genes we e de e mined using he MegAlign
p og am (DNASTARR
, Madison, WI, Uni ed S a es). Genes we e
aligned using Clus alW (La kin e al., 2007) and phylogenies
based on indi idual genes and on he conca ena ed sequences
was cons uc ed wi h MEGA e sion 6.0 (Tamu a e al., 2013)
using he Neighbo -Joining (NJ) and Maximum-Likelihood (ML)
algo i hms.
3 p:// p.ncbi.nlm.nih.go /blas /demo/
4h p://www. icbioin o ma ics.com/so wa e.ba nap.sh ml
F on ie s in Mic obiology | www. on ie sin.o g 3Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 4
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
The phylogene ic analysis o he co e genome was assessed
wi h he Roa y so wa e (Page e al., 2015) using 80% as cu -o o
he BLASTp sea ch. The co e genome alignmen was ex ac ed
wi h he la e so wa e and he phylogeny was in e ed using
Spli sT ee e sion 4.14.2 as desc ibed in Sawabe e al. (2007) using
Spli sT ee e sion 4.14.2, wi h a neighbo ne d awing and Jukes-
Can o co ec ion (Bandel and D ess, 1992;Huson and B yan ,
2005).
Fu he mo e, he 16S and 23S RNA genes o each genome
we e ob ained using RNamme (Lagesen e al., 2007). In some
cases, 16S RNA gene sequences we e ob ained in ou labo a o ies
by Sange sequencing o om he GenBank. The simila i y o
he 16S RNA genes was calcula ed using MegAlign e sion 7.0.0
(DNASTARR
, Madison, WI, Uni ed S a es). Phylogene ic ees
we e econs uc ed wi h MEGA e sion 6.0 (Tamu a e al., 2013)
also using he NJ and ML algo i hms. Alignmen s ob ained o
bo h genes we e isually analyzed in o de o localize signa u e
sequences o s ains o g oups o s ains.
Genomic Indices
In o de o ensu e he co ec assigna ion a species le el o
each analyzed genome, he ANI and he isDDH we e calcula ed
be ween all he genomes (Kons an inidis and Tiedje, 2005;
Rich e and Rosselló-Mó a, 2009;Qin e al., 2014). The ANIb
was calcula ed using JSpeciesWS (Rich e e al., 2016), he
esul ing ma ix was clus e ed and isualized using ggplo 2 2.2.1
package (Wickham, 2009) and he isDDH was calcula ed wi h
he GGDC so wa e using esul s ob ained wi h he o mula 2
(Meie -Kol ho e al., 2013). Two o he indices (AAI and POCP)
desc ibed o genus classi ica ion (Kons an inidis and Tiedje,
2005;Luo e al., 2014;Qin e al., 2014) we e calcula ed among
he genomes ha co esponded o he ype s ains o he accep ed
species and he e e ence s ains o he candida e species. The
AAI was calcula ed wi h he Lycoming College Newman Lab
AAI Calcula o 5using he Sequence-Based Compa ison Tools
ou pu ile om RAST (O e beek e al., 2014). The POCP was
de e mined as desc ibed by Qin e al. (2014) using he ollowing
pa ame e s o conside a pep ide as a conse ed p o ein: an
e- alue lowe han 1e-5 and an iden i y pe cen age highe han
40% om an aligned egion highe han 50%.
Finally, he RSCU was compu ed using he Codon Adap a ion
Index (CAI) de eloped by Sha p and Li (1987) h ough he
CAIcal web-se e (Puigbò e al., 2008). S a is ical di e ences in
he RSCU we e assessed by a mul inomial eg ession app oach
using he R so wa e en i onmen (R Co e Team, 2015). The
p incipal componen analysis (PCA) was pe o med by he R
so wa e en i onmen (R Co e Team, 2015, and isualized using
ggplo 2 2.2.1 and gg o i y 0.4.4 (Wickham, 2009;Ho ikoshi and
Tang, 2015;Tang e al., 2016) o pca3d 0.10 (Weine , 2017)
packages.
Pheno ypic Analysis and Me abolic
In e ence
Pheno ypic cha ac e iza ion o each desc ibed species was
ob ained om his s udy, om he o iginal desc ip ions o om
5h p://lyco s01.lycoming.edu/∼newman/AAI/
he summa y published by On e al. (2017). Fo he po en ially
new A cobac e species, he pheno ype was cha ac e ized
ollowing he ecommended minimal s anda ds desc ibed o
new axa o he amily Campylobac e aceae (U sing e al., 1994;
On e al., 2017) and wi h complemen a y es s used in he
desc ip ion o o he A cobac e species (Le ican e al., 2013).
In e ence o he me abolic ou es om he genome sequences
was pe o med wi h he so wa e package T ai a (Mic obial
T ai Analyze ) (Weimann e al., 2016), using he p o ein
coding genes iles ob ained wi h P okka 1.2 (Seemann, 2014).
T ai a so wa e is based on pheno ypic da a ex ac ed om he
Global In ec ious Disease and Epidemiology Online Ne wo k
(GIDEON) and Be gey’s Sys ema ic Bac e iology. The so wa e
uses wo p edic ion models: he phypa classi ie , which p edic s
he p esence/absence o p o eins ound in he pheno ype o 234
bac e ial species; and he phypa +PGL classi ie , which uses he
same in o ma ion as he phypa combined wi h he in o ma ion
o he acquisi ion and loss o p o ein amilies and pheno ypes
du ing e olu i e e en s. A o al o 67 ai s a ailable wi hin he
so wa e, ela ed o oxygen equi emen , enzyma ic ac i i ies,
p o eolysis, an ibio ic esis ance, mo phology and mo ili y and
he use o di e en ca bon sou ces, we e es ed and he combined
esul s o he wo p edic o s we e analyzed using a hea map.
RESULTS AND DISCUSSION
S ains and Genomes
All he 27 species cu en ly included in he genus A cobac e
and 13 candida e species ha e been in es iga ed in he p esen
s udy, which has analyzed 55 genomes, 16 o hem om he
public da abases and 39 sequenced in his s udy (Tables 1,2).
I was no possible o analyze he genomes om A. ac icola and
A. paci icus because we we e unable o ge he ype s ains o he
species. The con igs ob ained and he N50 alues complied wi h
he ecen ly p oposed minimal s anda ds o he use o genomes
in axonomic s udies (Chun e al., 2018). The genome size anged
om 1.81 Mb o A. ski owii F28 o 3.60 Mb o A. leki hoch ous
CECT 8942T(Table 2). The G+C con en anged om 26.1%
in A. mollusco um CECT 7696T o 34.9% in ‘A. aqua icus’
W112-28. The G+C alues ag ee wi h he ange om 24.6%
(which co esponded o he ype s ain o A. anae ophilus) o
31% indica ed o he genus A cobac e in he ecen emended
desc ip ion by Sasi-Jyo hsna e al. (2013). In e es ingly, 26
genomes (47.3%) showed he p esence o Clus e ed Regula ly
In e spaced Sho Palind omic Repea s (CRISPRs) and CRISPR-
associa ed genes, ela ed wi h he immune esponse o he
bac e ia.
Taxonomic and Phylogene ic Analysis
Simila i ies in he 16S RNA gene sequences among ype
and ep esen a i e s ains o he di e en A cobac e species
(all he 27 species cu en ly included in he genus and he
13 new candida e species) showed a wide ange o alues
(Supplemen a y Tables S1, S4). They anged om 90.8%
(obse ed be ween A. anae ophilus and A. aecis) o 99.9%
(be ween A. bu zle i and ‘A. lacus’). The lowe ange o
F on ie s in Mic obiology | www. on ie sin.o g 4Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 5
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
TABLE 1 | S ains used in his s udy, sou ce o isola ion and accession numbe s o he a ailable genomes.
Species S ain Sou ce Acc. No.
Genome
Species S ain Sou ce Acc. No.
Genome
A. ac icola KCTC 52212TSeawa e NAaA. my ili T234 Seawa e PDJW00b
A. anae ophilus DSM 24636TEs ua ine sedimen PDKO00bA. ni o igilis DSM7299TMa shland plan NC014166c
IR-1 U si a aqui e NZ_JXXG00cA. paci icus DSM 25018TSeawa e NAa
A. aquima inus CECT 8442TMedi e anean Sea NXIJ00bA. ski owii LMG 6621TDia heic lamb NXIC00b
A. bi al io um CECT 7835TMussels PDKM00bF28 Wild pig PDJT00b
F118-4 Mussels PDKL00bA. suis CECT 7833TPo k mea NREO00b
A. bu zle i RM4018THuman (Clinical) NC_009850cA. he eius LMG 24486TAbo ed pig oe us LLKQ01c
ED1 Mic obial uel cell NC_017187cDU22 Duck cloaca LCUJ01c
A. canalis F138-33 Oys e PNCeNWVW01bA. ophia um LMG 25534TPigle eces PDKD00b
SH-4D_Col1 Unknown FUYO00cCECT 7650 Chicken cloacal swab PDJS00b
A. ciba ius LMG 21996TB oile , skin NZ_JABW00cA. ene upis CECT 7836TClams NREP00b
A. cloacae CECT 7834TSewage NXII00bA cobac e sp. L Mic obial uel cell NC_017192c
F26 Mussels PDJZ00bAF1028 Human eces JART01c
A. c yae ophilus LMG 24291TAbo ed bo ine oe us NXGK00bCAB Ma ine Go0012496d
A. de lu ii CECT 7697TSewage NXIH00bLA11 Ma ine BDIR01c
A. eb onensis CECT 8441TMussels PDKK00bLPB0137 En i onmen al CP019070c
CECT 8993 Seawa e PDKJ00b
A. ellisii CECT 7837TMussels NXIG00b‘A. aqua icus’ W112-28 F eshwa e PNCePDKN00b
A. aecis LMG 28519THuman sep ic ank NZ_JARS00c‘A. caeni’ RW17-10 Recycled was ewa e MUXE00b
A. halophillus DSM 18005THype saline lagoon PDJY00b‘A. hispanicus’ FW-54 Was ewa e PDKI00b
F166-45 Oys e PNCePDJY00b‘A. lacus’ RW43-9 Recycled was ewa e MUXF00b
A. lan hie i LMG 28516TPig manu e JARU01c‘A. medi e aneus’ F156-34 Mussels Al acs Bay NXIE00b
LMG 28517 Dai y ca le manu e JARV01c‘A. mi oungae’ 9An Cloaca elephan seal PDKH00b
A. leki hoch ous CECT 8942TG ea scallop la ae NZ_MKCO00b‘A. nep unis’ F146-38 Mussels Al acs Bay PDKG00b
LMG 28652 Abalon PZYW00c‘A. po cinus’ LMG 24487TAbo ed pig oe us LCUH01c
A. ma inus CECT 7727TSeawa e NXAO01b‘A. pon icus’ F161-33 Cockle Al acs Bay PDKF00b
F140-37 Clams Al acs Bay NWVX01b‘A. salis’ F155-33 Oys e PNCePDKE00b
A. mollusco um CECT 7696TMussels NZ_NXFY00b‘A. iscosus’ F142-34gMussels PNCePDKC00b
F91 Mussels PDJX00b‘A. i o iensis’ FW59gWas ewa e PDKB00b
A. my ili CECT 7386TMussels NXID00bA cobac e sp. F2176 Mussels PDJV00b
aGenome no a ailable; bGenome sequenced in his s udy; cGenome ob ained om NCBI da abase; dGenome ob ained om JGI Gold a abase; ePNC means PobleNou
Channel, which is a eshwa e channel hea ily (geome ic mean o E. coli coun s 4.1 ×104c. .u./100ml) con amina ed wi h was ewa e whe e shell ish we e exposed
o 72h (Salas-Massó e al., 2016, 2018). This s ain was ob ained om F.J. Ga cía om he Labo a o io Cen al de Ve e ina ia de Alge e, MAGRAMA, Mad id, Spain;
gThese s ains we e eco e ed a he Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV-EHU), Vi o ia-Gas eiz, Spain, by R. Alonso, I. Ma inez-Malaxe xeba ia
and A. Fe nández-As o ga.
simila i y (90.8%) is due o he ac ha hose species, as
occu ed wi h o he s, we e assigned wi hin he genus based on
he p emise ha 16S RNA gene simila i y was highe wi h
any ype s ain o A cobac e han wi h o he axa. Howe e ,
in some cases being below he 95% cu -o alue o genus
delimi a ion (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008;
Tindall e al., 2010;Figue as e al., 2011a,b). I is in e es ing
o poin ou ha 16S RNA gene sequence simila i ies among
A. ni o igilis, he ype species o he genus, and he o he
desc ibed species anged om 93.2% (wi h A. he eius) o 95.9%
(wi h A. ene upis). Fu he mo e, A. ni o igilis showed highe
simila i ies han he h eshold alue o 95% wi h only se en
species (A. ac icola, ‘A. caeni,’ A. cloacae,A. de lu ii, A. ellisii,
A. suis, and A. ene upis) ou o he 27 accep ed species.
In any case, om he analysis o he simila i ies in he 16S
RNA gene sequences among he A cobac e species i is clea
ha his gene has limi ed alue and ha o he app oaches
a ailable in he genomic e a o axonomy a e needed o hei
s udy.
Phylogene ic analysis based on he co e genome made up
o 286 genes (Figu e 1 and Supplemen a y Table S5) and
also on he conca ena ed sequences o 13 housekeeping genes
o he ep esen a i e A cobac e s ains (Figu e 2) e ealed
ha he A cobac e species could be g ouped in o 4 majo
monophyle ic clus e s. Clus e 1, comp ised se en alida ed
species: A. bu zle i,A. ciba ius,A. c yae ophilus,A. lan hie i,
A. ski owii,A. he eius, and A. ophia um, oge he wi h
A. aecis (species desc ibed bu no alida ed ye ) and i e
candida e axa ‘A. hispanicus,’ ‘A. lacus,’ ‘A. mi oungae,’
‘A. po cinus,’ and ‘A. i o iensis’ (Figu e 1). Clus e 2 emb aced
he species A. aquima inus,A. cloacae,A. de lu ii,A. ellisii,
A. suis, and A. ene upis, as well as he non- alida ed
A. ac icola and he candida us ‘A. caeni.’ Clus e 3 included i e
species, A. canalis,A. halophilus,A. ma inus,A. mollusco um,
F on ie s in Mic obiology | www. on ie sin.o g 5Sep embe 2018 | Volume 9 | A icle 2077

micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 6
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
TABLE 2 | Genome cha ac e is ics and anno a ion esul s. Sou ce o whole genome sequences as indica ed in Table 1.
Species No. Con igs N50 (Kb) CDS (To al) CDS (Coding) RNA Genes RNAs ncRNAs CRISPR A ays G+C (%) Size (Mb)
A. anae ophilus DSM 24636T40 186 2,938 2,922 45 40 2 1 29.9 2.98
A. anae ophilus IR1 7 1,179 3,360 3,024 61 47 2 3 30.2 3.25
‘A. aqua icus’ W112-28T20 370 2,500 2,487 55 45 3 0 34.9 2.53
A. aquima inus CECT 8442T68 75 2,473 2,463 46 42 2 0 26.6 2.46
A. bi al io um CECT 7835T179 461 2,786 2,728 50 41 3 0 28.2 2.75
A. bi al io um F118-4 26 209 2,652 2,652 47 38 3 0 28.1 2.71
A. bu zle i RM4018T1 – 2,261 2,256 71 54 2 0 27.0 2.34
A. bu zle i ED1 1 – 2,151 2,145 71 54 2 0 27.1 2.26
‘A. caeni RW17-10T59 123 2,357 2,337 58 51 3 0 27.1 2.42
A. canalis CECT8984T50 166 2,733 2,720 53 48 2 1 27.3 2.78
A. canalis SH-4D_Col1 69 72 2,716 2,663 63 52 2 1 27.1 2.82
A. ciba ius LMG 21996T44 119 2,156 2,110 68 46 2 0 27.1 2.20
A. cloacae CECT 7834T135 135 2,826 2,795 58 51 2 3 26.8 2.78
A. cloacae F26 40 218 2,470 2,459 53 44 2 1 26.9 2.51
A. c yae ophilus LMG 24291T91 54 2,092 2,081 49 40 3 0 27.2 2.06
A. de lu ii CECT 7697T80 166 2,921 2,894 57 49 2 2 26.3 2.94
A. eb onensis CECT 8441T103 188 3,089 3,072 47 39 3 1 29.2 3.15
A. eb onensis W129-34 126 217 3,206 3,171 46 40 3 2 29.2 3.23
A. ellisii CECT 7837T135 177 2,875 2,840 64 52 2 1 26.9 2.80
A. aecis LMG 28519T55 127 2,429 2,376 76 53 2 1 27.2 2.50
A. halophilus DSM 18005T111 56 2,677 2,660 54 46 3 2 27.4 2.75
A. halophilus F166-45 90 56 2,879 2,864 59 51 2 2 27.0 2.96
‘A. hispanicus’ FW54T76 148 2,228 2,207 46 40 3 1 26.4 2.21
‘A. lacus’ RW43-9T24 295 2,194 2,182 47 40 2 0 26.8 2.22
A. lan hie i LMG 28516T29 466 2,223 2,190 73 52 3 1 26.7 2.29
A. lan hie i AF1581 24 353 2,199 2,186 88 57 3 0 26.8 2.26
A. leki hoch ous CECT 8942T436 343 3,628 3,316 88 75 3 0 28.6 3.61
A. leki hoch ous LMG 28652 82 343 3,499 3,330 61 55 3 0 28.2 3.50
A. ma inus CECT 7727T162 54 2,809 2,781 55 50 2 0 27.0 2.87
A. ma inus F140-37 76 67 2,725 2,652 59 48 2 0 27.0 2.78
‘A. medi e aneus’ F156-34T29 689 2,769 2,750 47 41 3 1 27.3 2.83
‘A. mi oungae’ 9An T35 363 1,868 1,847 46 41 2 1 28.1 1.84
A. mollusco um CECT 7696T117 121 2,746 2,736 58 49 3 6 26.1 2.76
(Con inued)
F on ie s in Mic obiology | www. on ie sin.o g 6Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 7
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
TABLE 2 | Con inued
Species No. Con igs N50 (Kb) CDS (To al) CDS (Coding) RNA Genes RNAs ncRNAs CRISPR A ays G+C (%) Size (Mb)
A. mollusco um F91 240 150 2,951 2,889 71 58 3 2 26.3 2.89
A. my ili CECT 7386T126 70 2,950 2,934 58 48 3 1 26.3 2.97
A. my ili T234 145 37 2,735 2,723 54 48 3 0 26.4 2.77
‘A. nep unis’ F146-38T36 267 2,627 2,614 57 45 3 0 27.1 2.65
A. ni o igilis DSM 7299T1 – 3,101 3,086 69 55 2 1 28.4 3.19
‘A. pon icus’ F161-33 24 597 2,632 2,621 46 36 3 0 28.1 2.74
‘A. po cinus’ LMG 24487T70 123 2,186 2,112 47 41 2 0 27.0 2.14
‘A. salis’ F155-33T153 169 2,932 2,904 50 43 3 0 29.0 2.93
A. ski owii LMG 6621T62 306 2,029 2,006 48 42 2 2 27.7 1.97
A. ski owii F28 110 40 1,911 1,897 46 41 2 0 27.8 1.81
A. suis CECT 7833T122 142 2,646 2,613 57 52 2 0 27.3 2.62
A. he eius LMG 24486T2 1,039 1,896 1,883 57 46 2 3 27.0 1.91
A. he eius DU22 19 252 2,006 1,983 47 42 2 1 26.8 2.01
A. ophia um CECT 7650 37 152 1,911 1,894 48 37 3 0 28.0 1.90
A. ophia um LMG 25534T266 86 2,167 2,071 49 41 3 0 29.4 2.00
A. ene upis CECT 7836T234 182 3,319 3,267 64 52 2 0 28.0 3.28
‘A. iscosus’ F142-34T82 65 2,772 2,756 55 48 3 1 26.6 2.79
‘A. i o iensis’ FW59T144 179 2,617 2,570 53 46 2 0 27.4 2.58
A cobac e sp. CAB 367 20 3,596 3,392 NA 31 NA NA 28.2 3.48
A cobac e sp. F2176 99 178 3,212 3,186 67 57 2 0 28.1 3.27
A cobac e sp. LA11 53 229 3,006 2,961 49 43 3 0 27.9 3.10
A cobac e sp. LPB0137 1 – 2,731 2,698 85 64 2 0 27.7 2.87
A cobac e sp. La1 – 2,847 2,834 73 56 2 1 26.6 2.95
A cobac e sp. AF1028b46 148 2,336 2,285 71 51 2 1 27.2 2.41
aGenome sequenced in his s udy; bGenome ob ained om NCBI da abase; cGenome ob ained om JGI Gold da abase. Ou esul s show ha hese s ains belong o he species. dA. de lu ii and eA. aecis.
F on ie s in Mic obiology | www. on ie sin.o g 7Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 8
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
and A. my ili, oge he wi h wo candida es, ‘A. nep unis’
and ‘A. iscosus.’ Finally, Clus e 4 included he species
A. anae ophilus,A. bi al io um, and A. eb onensis, as well as he
candida es ‘A. medi e aneus,’ ‘A. pon icus,’ and ‘A. salis.’ The
spli decomposi ion ne wo k analysis o he co e genome showed
ha he species A. leki hoch ous CECT 8942Tand A. ni o igilis
DSM 7299Tappea ed as o phan species. Fu he mo e, wi h his
analysis he candida us ‘A. aqua icus’ W112-28 also appea ed
in a sepa a e b anch nea o A. ni o igilis DSM 7299T. On he
o he hand, bo h analyses, MLSA and co e genome, con i med
he exis ence o wo sub-clus e s in Clus e 1 (again A. bu zle i
and ‘A. lacus’ we e loca ed in he mos dis an b anch wi hin he
clus e ), and also wo subg oups could be obse ed in Clus e 4,
one comp ising he species A. anae ophilus and A. eb onensis,
and he o he including he es o species wi hin his clus e
(Figu es 1,2). All he clus e s and sub-clus e s showed a
simila i y in he conca ena ed sequences o he 13 housekeeping
genes highe han 85% (Figu e 2).
Phylogenies based on he 16S and 23S RNA gene sequences,
unde aken wi h he NJ and ML app oachese ealso cons uc ed
wi h compa a i e pu poses. 16S RNA based ee showed also
he ou majo clus e s al hough less de ined (Supplemen a y
Figu e S1A). Species wi hin Clus e 1, showed 16S RNA gene
sequence simila i ies anging om 96.1 o 99.9%. Clus e 2
yielded simila i ies among species o he 16S RNA gene be ween
96.7 and 99.6%, whe eas wi hin Clus e 3 anged be ween 93.0
and 99.1%. Finally, Clus e 4 included species wi h a ange o 16S
RNA sequence simila i y om 94.0 o 99.5%. Wi h he excep ion
o Clus e 3, simila i y alues wi hin he clus e s (>94–95%) we e
wi hin he classical bounda ies o genus assigna ion in bac e ial
axonomy (Rosselló-Mo a and Amann, 2001;Ya za e al., 2008,
2014;Tindall e al., 2010;Figue as e al., 2011a,b). Ou esul s
ag ee wi h hose om a ecen s udy by Ya za e al. (2014), who
in es iga ed 568 axa and desc ibed a h eshold in 16S RNA
sequence iden i y o 94.5% o genus delinea ion.
Simila g oups and opology, wi h only mino di e ences,
we e ob ained when he 23S RNA gene sequences we e used o
analyze he phylogeny o he genus (Supplemen a y Figu e S2).
In his analysis, he ecen ly desc ibed species A. ac icola, and
A. paci icus could no be included because o he una ailabili y o
he ype s ains and/o whole genome sequences. The same ou
majo clus e s o med in he 23S RNA gene phylogene ic ee,
and he species A. leki hoch ous and A. ni o igilis appea ed also
as o phan species (Supplemen a y Figu e S2). Wi hin Clus e 1
wo subg oups could also be ob ained, di e en ia ing he species
A. bu zle i and ‘A. lacus’ om he es o he species. Simila ly, he
species A. anae ophilus and A. eb onensis o med a di e en ia ed
subg oup in Clus e 4.
The isual analysis o he alignmen s ob ained wi h he
sequences o he 16S and 23S RNA genes allowed he
localiza ion o signa u e mo i s, especially in he 16S RNA
gene, o he di e en clus e s es ablished in he phylogene ic
analysis. In hese sequences, a o al o 16 loca ions we e ound,
p esen ing nucleo ide combina ions cha ac e is ic o he clus e s
(Supplemen a y Figu e S3). Some o hese mo i s we e loca ed
in helix egions as in e ac ions wi h p o eins o he ibosomal
30S subuni , such as helix 21 ( egion V4) o helix 28/44 ( egion
V9), and he e o e had a conside able le el o p o ec ion agains
mu a ions (Adilakshmi e al., 2008;Ki aha a e al., 2012). The e
a e some s udies on he p esence o signa u e egions wi h
axonomic/phylogene ic implica ions in he ibosomal genes
(Ma ínez-Mu cia e al., 1992, 2007;Ue e al., 2011;ˇ
Reháko á
e al., 2014;Ma ínez-Mu cia and Lamy, 2015). Some egions
wi h signa u e mo i s de ec ed in he p esen s udy ha e also
shown implica ions o phylogene ic analysis in cyanobac e ia,
including egions H15, H17, H21, H22-H23, H41, and H44
(ˇ
Reháko á e al., 2014). A ee was also cons uc ed weigh ing
such posi ions (Supplemen a y Figu e S1B), which allowed a
be e de ini ion o he main clus e s obse ed wi h he whole 16S
RNA sequences al hough, as expec ed, di e en ia ion among
species wi hin each clus e was lowe . Two sub-clus e s we e
obse ed in Clus e 1, whe e he species A. bu zle i and ‘A. lacus’
g ouped in o a well-di e en ia ed b anch wi h espec o he
o he species in he clus e (Supplemen a y Figu e S1B). In
his analysis, A. paci icus was clea ly loca ed in he Clus e 3,
whe eas in Clus e 4, A anae ophilus was he bo de line species,
while A. eb onensis and ‘A. medi e aneus’ we e loca ed in an
independen b anch (Supplemen a y Figu e S1B). The e o e,
he signa u e mo i s desc ibed he e migh be a new ool o
iden i ica ion o he di e en clus e s and/o genus.
Genomic Indices
The esul s o he calcula ions o he ANI and he isDDH
among he 36 s udied genomes a e gi en in he Supplemen a y
Table S2 and Supplemen a y Figu e S4. The esul s o he
ANI and isDDH calcula ions showed ha he genomes g ouped
in o he same clus e s obse ed by he analyses o he MLSA
o he 13 housekeeping and co e genes (Figu es 1,2). Ranges
o ANI wi hin each clus e we e om 75.2 o 95.4%, whe eas
isDDH alues we e be ween 19.5 and 65.4% (Figu e 2 and
Table 3). These esul s con i m he phylogene ic analysis o
he 13 new candida e species because all o hem showed ANI
and isDDH alues o <96% and <70%, espec i ely, which
a e he cu -o alues p oposed o he delinea ion o new
species (Kons an inidis and Tiedje, 2005;Go is e al., 2007;
Rich e and Rosselló-Mó a, 2009;Figue as e al., 2017). As
discussed in o he s udies, he ANI and isDDH indices p o ided
eliable in o ma ion o he delinea ion o A cobac e species and
a e also included in he minimal guidelines o de ine species
using genomes (Whi educk-Lé eillée e al., 2015, 2016;Figue as
e al., 2017;Chun e al., 2018). Al hough hose indices a e no
conside ed use ul o delimi ing gene a, each o he ou clus e s
showed alues ha anged be ween 75.2 and 81.8% as hei lowes
ANI, which migh be he sui able ange o sepa a ing di e en ,
closely ela ed gene a. These alues a e ela i ely simila o hose
epo ed by Qin e al. (2014) ha ound 68–82% in e species
ANI alues among he gene a ha hey s udied. Values o ANI
ob ained o he candida e species ‘A. aqua icus’ we e lowe han
he o he esul s, om 70.0% wi h A. c yae ophilus LMG 24291T
o 71.9% wi h A. bi al io um CECT 7835Tand mo e in line wi h
he Qin e al. (2014) esul s o 68% (Supplemen a y Table S2). In
he case o he isDDH he lowe alues among species in he same
clus e anged be ween 19.5 and 24.8%, and again hese migh be
he le els associa ed o di e en gene a.
F on ie s in Mic obiology | www. on ie sin.o g 8Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 9
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
FIGURE 1 | Spli decomposi ion ne wo k cons uc ed wi h he conca ena ed sequences o 284 co e genes om he genomes o 36 ype and ep esen a i e s ains
o A cobac e . Scale ba , base subs i u ions pe si e.
Wi h he aim o con i ming i he clus e s obse ed migh
ep esen di e en gene a, as sugges ed by he phylogene ic
analyses, he simila i y indices AAI and POCP we e also
calcula ed (Supplemen a y Table S3). In ag eemen wi h he
60–80% AAI ha ha e been desc ibed o species belonging o
he same genus (Kons an inidis and Tiedje, 2005) all ou clus e s
showed lowe anges o be ween 67.6 o 80.3% (Table 3). All
he clus e s also complied wi h he POCP p oposed o genus
sepa a ion abo e 50% (Luo e al., 2014;Qin e al., 2014) because
as shown in Table 3 all clus e s showed he lowes alues om
67.0 o 75.4%.
I is widely known ha synonymous codon usage a ies
among o ganisms and ha i is ela ed o di e ences in G+C
con en , eplica ion s and skew, o gene exp ession (Suzuki e al.,
2008;Fa ooqi e al., 2016). The in e ac ion o hese ac o s may
a y among species depending on hei e olu iona y p ocess
(Ma e al., 2015). I has also been sugges ed ha he ex en o
codon usage bias plays a ole in he adap a ion o p oka yo ic
o ganisms o hei en i onmen s and li es yles (Bo zman and
Ma gali , 2011). To analyze he o e all codon usage ends o
he A cobac e species, he equencies o he di e en codons
we e ob ained om he whole genomes and he RSCU was
compu ed using he CAI, which is a use ul ool o es ima ing
codon usage bias (Ma e al., 2015;Fa ooqi e al., 2016). A i s
inding was ha all he A cobac e species p esen ed a p e e en ial
use o he codons inishing in A o T (Supplemen a y Figu e
S5), which migh be expec ed due o hei low G+C% con en .
The cha ac e is ic pa e n showed by A. aqua icus is no ewo hy
(Supplemen a y Figu e S5), which suppo s i s di e en ia ion
om he o he species in Clus e 3 as well as i s unique axonomy.
Such di e ence was he only s a is ically signi ican (p<0.05) in
he mul inomial eg ession analysis ca ied ou .
Nex , he codon usage ends we e analyzed by PCA
o e eal possible e olu iona y ela ionships. In e es ingly,
di e en g oups o s ains could be obse ed in he h ee-
dimensional g aphic (Figu e 3), which co ela ed wi h hose
clus e s es ablished in he di e en phylogene ic analyses, as
shown abo e. As epo ed p e iously o di e en species o
Mycoplasma (Ma enda e al., 2005;Ma e al., 2015), PCA
p o ides an addi ional pa hway o in es iga e he e olu iona y
di ec ion o he A cobac e species. In addi ion, simila i ies in
he synonymous codon usage pa e ns migh e lec simila
li es yles (pa hogenic s. non-pa hogenic) and adap a ion o
ce ain en i onmen s (ma ine wa e , shell ish, e c.).
Me abolic In e ence and Pheno ypic
Analysis
Phylogene ic and genomic analysis con i med he exis ence o
ou clus e s among he alida ed and candida e A cobac e
F on ie s in Mic obiology | www. on ie sin.o g 9Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 16
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
Desc ip ion o Malacobac e canalis
comb. no .
Basonym: A cobac e canalis Pé ez-Ca aluña e al., 2018b.
The desc ip ion is he same gi en by Pé ez-Ca aluña e al.
(2018b). The ype s ain is F138-33T(= CECT 8984T= LMG
29148T).
Desc ip ion o Malacobac e
mollusco um comb. no .
Basonym: A cobac e mollusco um Figue as e al., 2011a.
The desc ip ion is he same gi en by Figue as e al. (2011a).
The ype s ain is F98-3T(= CECT 7696T= LMG 25693T).
Desc ip ion o Malacobac e paci icus
comb. no .
Basonym: A cobac e paci icus Zhang e al., 2015.
The desc ip ion is he same gi en by Zhang e al. (2015). The
ype s ain is SW028T(= DSM 25018T = JCM 17857T= LMG
26638T).
Desc ip ion o Haloa cobac e gen. no .
Haloa cobac e (Ha.lo.a .co.bac’ e , G . n. halo, sal ; N.L.
masc. n. A cobac e , a bac e ial gene ic name; N.L. masc. n.
Haloa cobac e , A cobac e sal lo ing).
G am-nega i e, cells a e od shaped and mo ile. Cell size 0.1–
0.5 µm in diame e and 0.9–2.5 µm in leng h. Oxidase posi i e
and ca alase a iable among species. Halophilic, g ow h can be
ob ained wi hin he ange o 0.5% ( a iable among species)
and up o 4% NaCl. G ow h occu s a 15–42◦C. G ow h a
37◦C in mic oae ophilic condi ions o a 42◦C in anae obiosis
a iable among species. Ca bohyd a es a e no e men ed. Some
species may educe ni a e o ni i e. Nega i e o he hyd olysis
o u ea (wi h he excep ion o H. eb onensis). Some species
may hyd olyze indoxyl ace a e. G ow h does no occu in
he p esence o oxgall (1% w / ol) (wi h he excep ion o
H. mollusco um) o 2,3,5- iphenyl e azolium chlo ide (0.04%,
w / ol). No g ow h on CCDA. Some species may g ow in he
p esence o glycine (1% w / ol) o sa anin (0.05% w / ol).
Sensi i e o ce ope azone (64 mg/l). Range o DNA G+C con en
is 27.3–29.9 mol%.
The ype species is Haloa cobac e bi al io um.
Desc ip ion o Haloa cobac e
bi al io um comb. no .
Basonym: A cobac e bi al io um Le ican e al., 2012.
The desc ip ion is he same gi en by Le ican e al. (2012). The
ype s ain is F4T(= CECT 7835T= LMG 26154T).
Desc ip ion o Haloa cobac e
anae ophilus comb. no .
Basonym: A cobac e anae ophilus Sasi-Jyo hsna e al., 2013.
The desc ip ion is he same gi en by Sasi-Jyo hsna e al.
(2013). The ype s ain is JC84T(= KCTC 15071T= MTCC
10956T= DSM 24636T).
Desc ip ion o Haloa cobac e
eb onensis comb. no .
Basonym: A cobac e eb onensis Le ican e al., 2015.
The desc ip ion is he same gi en by Le ican e al. (2015). The
ype s ain is F128-2T(= CECT 8441T= LMG 27922T).
Desc ip ion o Poseidonibac e gen. no .
Poseidonibac e (Po.se.i.do.ni.bac’ e , G . n. Poseidon, God o he
sea; G . n. bac e , od; N.L. masc. n. Poseidonibac e e e ing o
he ma ine habi a o his bac e ia).
G am-nega i e, cells a e od shaped and mo ile. Oxidase and
ca alase posi i e. Halophilic, no g ow h can be ob ained wi hou
seawa e o he addi ion o combined ma ine sal s o he medium.
G ow h occu s a 15◦C–25◦C, bu no a 37◦C o 42◦C. Range
o pH o g ow h is 6–8. Ca bohyd a es a e no e men ed.
Reduce ni a e o ni i e. Nega i e o he hyd olysis o indoxyl
ace a e and u ea. G ow h occu s in he p esence o sa anin
(0.05% w / ol), and 2,3,5- iphenyl e azolium chlo ide (0.04%,
w / ol), bu no in he p esence o glycine (1% w / ol) sensi i e o
ce ope azone (30 µg). Possess ubiquinone MK-6 as a espi a o y
quinone. DNA G+C con en is 28.7 mol%.
The ype species is Poseidonibac e leki hoch ous.
Desc ip ion o Poseidonibac e
leki hoch ous comb. no .
Basonym: A cobac e leki hoch ous Diéguez e al., 2017.
The desc ip ion is he same gi en by Diéguez e al. (2017). The
ype s ain is LFT1.7T(= CECT 8942T= DSM 100870T).
AUTHOR CONTRIBUTIONS
MF and JR designed he wo k. AP-C, NS-M, and AD pe o med
he pheno ypic and phylogene ic expe imen s. AP-C and SB
ca ied ou he genome sequencing and analysis. AP-C, AL, and
JR pe o med he bioin o ma ic wo k. JR, MF, AP-C, and AD
w o e he pape .
FUNDING
This wo k was suppo ed in pa by G an s JPIW2013-69095-
C03-03 om he Minis e io de Economía y Compe i i idad
(MINECO), AQUAVALENS o he Se en h F amewo k
P og am (FP7/2007-2013) g an ag eemen 311846 om he
Eu opean Union and AGL2013-42628-R and AGL2016-77539-R
(AEI/FEDER UE) om he Agencia Es a al de In es igación
(Spain).
ACKNOWLEDGMENTS
The au ho s hank D . F. J. Ga cía (Labo a o io Cen al de
Ve e ina ia de Alge e, MAGRAMA, Mad id, Spain) and D s. R.
Alonso, I. Ma inez-Malaxe xeba ia, and A. Fe nandez-As o ga
[Facul y o Pha macy, Uni e si y o he Basque Coun y (UPV-
EHU), Vi o ia-Gas eiz, Spain], o kindly p o iding some o he
F on ie s in Mic obiology | www. on ie sin.o g 16 Sep embe 2018 | Volume 9 | A icle 2077

micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 17
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
A cobac e s ains. AP-C hanks Ins i u d’In es igació Sani à ia
Pe e Vi gili (IISPV) o he Ph.D. ellowship and NS-M hanks
he Uni e si a Ro i a i Vi gili (URV), he Ins i u de Rece ca i
Tecnologia Ag oalimen à ia (IRTA) and he Banco San ande o
he Ph.D. ellowship.
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h ps://www. on ie sin.o g/a icles/10.3389/ micb.
2018.02077/ ull#supplemen a y-ma e ial
REFERENCES
Adilakshmi, T., Bellu , D. L., and Woodson, S. A. (2008). Concu en nuclea ion o
16S olding and induced i in 30S ibosome assembly. Na u e 455, 1268–1272.
doi: 10.1038/na u e07298
Bandel , H. J., and D ess, A. W. M. (1992). Spli decomposi ion: a new and use ul
app oach o phylogene ic analysis o dis ance da a. Mol. Phylogene . E ol. 1,
242–252. doi: 10.1016/1055-7903(92)90021-8
Bo zman, M., and Ma gali , H. (2011). Va ia ion in global codon usage bias among
p oka yo ic o ganisms is associa ed wi h hei li es yles. Genome Biol. 12:R109.
doi: 10.1186/gb-2011-12-10- 109
Chun, J., O en, A., Ven osa, A., Ch is ensen, H., A ahal, D. R., da Cos a,
M. S., e al. (2018). P oposed minimal s anda ds o he use o genome da a
o he axonomy o p oka yo es. In . J. Sys . E ol. Mic obiol. 68, 461–466.
doi: 10.1099/ijsem.0.002516
Chun, J., and Rainey, F. A. (2014). In eg a ing genomics in o he axonomy and
sys ema ics o he Bac e ia and A chaea. In . J. Sys . E ol. Mic obiol. 64, 316–324.
doi: 10.1099/ijs.0.054171-0
Collado, L., Cleenwe ck, I., Van T appen, S., De Vos, P., and Figue as, M. J.
(2009a). A cobac e my ili sp. no ., an indoxyl ace a e –hyd olysis- nega i e
bac e ium isola ed om mussels. In . J. Sys . E ol. Mic obiol. 59, 1391–1396.
doi: 10.1099/ijs.0.003749-0
Collado, L., Gua o, J., and Figue as, M. J. (2009b). P e alence o A cobac e in
mea and shell ish. J. Food P o . 72, 1102–1106.
Collado, L., and Figue as, M. J. (2011). Taxonomy, epidemiology, and clinical
ele ance o he genus A cobac e .Clin. Mic obiol. Re . 24, 174–192.
doi: 10.1128/CMR.00034-10
Collado, L., Inza, I., Gua o, J., and Figue as, M. J. (2008). P esence o A cobac e
spp. in en i onmen al wa e s co ela es wi h high le els o ecal pollu ion.
En i on. Mic obiol. 10, 1635–1640. doi: 10.1111/j.1462-2920.2007.01555.x
Collado, L., Kasimi , G., Pe ez, U., Bosch, A., Pin o, R., Saucedo, G., e al.
(2010). Occu ence and di e si y o A cobac e spp. along he Llob ega Ri e
ca chmen , a sewage e luen s and in a d inking wa e ea men plan . Wa e
Res. 44, 3696–3702. doi: 10.1016/j.wa es.2010.04.002
Collado, L., Le ican, A., Pé ez, J., and Figue as, M. J. (2011). A cobac e de lu ii sp.
no ., isola ed om sewage samples. In . J. Sys . E ol. Mic obiol. 61, 2155–2161.
doi: 10.1099/ijs.0.025668-0
De Sme , S., Vandamme, P., De Zu e , L., On, S. L. W., Douidah, L., and Hou , K.
(2011). A cobac e ophia um sp. no ., isola ed om a ening pigs. In . J. Sys .
E ol. Mic obiol. 61, 356–361. doi: 10.1099/ijs.0.022665-0
Diéguez, A. L., Balboa, S., Magnesen, T., and Romalde, J. L. (2017). A cobac e
leki hoch ous sp. no ., isola ed om a molluscan ha che y. In . J. Sys . E ol.
Mic obiol. 67, 1327–1332. doi: 10.1099/ijsem.0.001809
Diéguez, A. L., Pé ez-Ca aluña, A., Figue as, M. J., and Romalde, J. L. (2018).
A cobac e halio is Tanaka e al., 2017 is a la e he e o ypic synonym o
A cobac e leki hoch ous Diéguez e al., 2017.In . J. Sys . E ol. Mic obiol.
doi: 10.1099/ijsem.0.002909 [Epub ahead o p in ].
Donachie, S. P., Bowman, J. P., On, S. L. W., and Alam, M. (2005). A cobac e
halophilus sp. no ., he i s obliga e halophile in he genus A cobac e . In . J.
Sys . E ol. Mic obiol. 55, 1271–1277. doi: 10.1099/ijs.0.63581-0
Fa ooqi, M. S., Mish a, D., Rai, N., Singh, D., Rai, A., Cha u edi, K., e al. (2016).
Genome-wide ela i e analysis o codon usage bias and codon con ex pa e n
in he bac e ia Salinibac e ube ,Ch omohalobac e salexigens and Rhizobium
e li.Biochem. Anal. Biochem. 5:257. doi: 10.4172/2161-1009.1000257
Figue as, M. J., Collado, L., and Gua o, J. (2008). A new 16S DNA-RFLP me hod
o he disc imina ion o he accep ed species o A cobac e .Diagn. Mic obiol.
In ec . Dis. 62, 11–15. doi: 10.1016/j.diagmic obio.2007.09.019
Figue as, M. J., Collado, L., Le ican, A., Pé ez, J., Solsona, M. J., and Yus es, C.
(2011a). A cobac e mollusco um sp. no ., a new species isola ed om shell ish.
Sys . Appl. Mic obiol. 34, 105–109. doi: 10.1016/j.syapm.2010.10.001
Figue as, M. J., Le ican, A., Collado, L., Inza, M. I., and Yus es, C. (2011b).
A cobac e ellisii sp. no ., isola ed om mussels. Sys . Appl. Mic obiol. 34,
414–418. doi: 10.1016/j.syapm.2011.04.004
Figue as, M. J., Le ican, A., Pujol, I., Balles e , F., Rabada Quilez, M. J., and Gomez-
Be omeu, F. (2014). A se e e case o pe sis en dia hoea associa ed wi h
A cobac e c yae ophilus bu a ibu ed o Campylobac e sp. and a e iew o
he clinical incidence o A cobac e spp. New Mic obes New In ec . 2, 31–37.
doi: 10.1002/2052-2975.35
Figue as, M. J., Pé ez-Ca aluña, A., Salas-Massó, N., Le ican, A., and Collado, L.
(2017). ‘A cobac e po cinus’ sp. no ., a no el A cobac e species unco e ed by
A cobac e he eius.New Mic obe New In ec . 15, 104–106. doi: 10.1016/j.nmni.
2016.11.014
Fishe , J. C., Le ican, A., Figue as, M. J., and McLellan, S. L. (2014). Popula ion
dynamics and ecology o A cobac e in sewage. F on . Mic obiol. 5:525.
doi: 10.3389/ micb.2014.00525
Giacome i, F., Salas-Massó, N., Se aino, A., and Figue as, M. J. (2015).
Cha ac e iza ion o A cobac e suis isola ed om wa e bu alo (Bubalus
bubalis) milk. Food Mic obiol. 51, 186–191. doi: 10.1016/j. m.2015.06.004
Go is, J., Kons an inidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P.,
and Tiedje, J. M. (2007). DNA-DNA hyb idiza ion alues and hei ela ionship
o whole-genome sequence simila i ies. In . J. Sys . E ol. Mic obiol. 57, 81–91.
doi: 10.1099/ijs.0.64483 -0
Ho ikoshi, M., and Tang, Y. (2015). gg o i y: Da a Visualiza ion Tools o S a is ical
Analysis Resul s. A ailable a : h ps://CRAN.R-p ojec .o g/package=gg o i y
Hou , K., On, S. L. W., Coenye, T., Deb uyne, L., De Sme , S., and Vandamme, P.
(2009). A cobac e he eius sp. no ., isola ed om pigs and ducks. In . J. Sys .
E ol. Mic obiol. 59, 2599–2604. doi: 10.1099/ijs.0.006650-0
Hou , K., On, S. L. W., Coenye, T., Mas , J., Van Hoo , J., and Vandamme, P. (2005).
A cobac e ciba ius sp. no ., isola ed om boiled ca casses. In . J. Sys . E ol.
Mic obiol. 55, 713–717. doi: 10.1099/ijs.0.63103-0
Hsu, T. T. D., and Lee, J. (2015). Global dis ibu ion and p e alence o A cobac e
in ood and wa e . Zoonoses Public Heal h 62, 579–589. doi: 10.1111/zph.12215
Huson, D. H., and B yan , D. (2005). Applica ion o phylogene ic ne wo ks in
e olu iona y s udies. Mol. Biol. E ol. 23, 254–267. doi: 10.1093/molbe /msj030
Hya , D., Chen, G. L., Locascio, P. F., Land, M. L., La ime , F. W., and Hause ,
L. J. (2010). P odigal: p oka yo ic gene ecogni ion and ansla ion ini ia ion
si e iden i ica ion. BMC Bioin o ma ics 11:119. doi: 10.1186/1471-2105-11-119
Kiehlbauch, A., B enne , D. J., Nicholson, M. A., Bake , C. N., Pa on, C. M.,
S eige wal , A. G., e al. (1991). Campylobac e bu zle i sp. no ., isola ed om
humans and dia heal illness. J. Clin. Mic obiol. 29, 376–385.
Kim, H. M., Hwang, C. Y., and Cho, B. C. (2010). A cobac e ma inus sp. no . In .
J. Sys . E ol. Mic obiol. 60, 531–536. doi: 10.1099/ijs.0.007740-0
Ki aha a, K., Yasu ake, Y., and Miyazaki, K. (2012). Mu a ional obus ness o 16S
ibosomal RNA, shown by expe imen al ho izon al gene ans e in Esche ichia
coli.PNAS 109, 19220–19225. doi: 10.1073/pnas.1213609109
Kons an inidis, K. T., and Tiedje, J. M. (2005). Genomic insigh s ha ad ance he
species de ini ion o p oka yo es. P oc. Na l. Acad. Sci. U.S.A. 102, 2567–2572.
doi: 10.1073/pnas.0409727102
Lagesen, K., Hallin, P., Rødland, E. A., S æ eld , H.-H., Rognes, T., and Usse y,
D. W. (2007). RNAmme : consis en and apid anno a ion o ibosomal RNA
genes. Nucleic Acids Res. 35, 3100–3108. doi: 10.1093/na /gkm160
La kin, M. A., Blackshields, G., B own, N. P., Chenna, R., McGe igan, P. A.,
McWilliam, H., e al. (2007). Clus al W and Clus al X e sion 2.0. Bioin o ma ics
23, 2947–2948. doi: 10.1093/bioin o ma ics/b m404
La sen, M. V., Cosen ino, S., Rasmussen, S., F iis, C., Hasman, H., Ma ig, R. L.,
e al. (2012). Mul ilocus sequence yping o o al-genome-sequenced bac e ia.
J. Clin. Mic obiol. 50, 1355–1361. doi: 10.1128/JCM.06094-11
Lasle , D., and Canback, B. (2004). ARAGORN, a p og am o de ec RNA
genes and mRNA genes in nucleo ide sequences. Nucleic Acids Res. 32, 11–16.
doi: 10.1093/na /gkh152
F on ie s in Mic obiology | www. on ie sin.o g 17 Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 18
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
Le ican, A., Collado, L., Aguila , C., Yus es, C., Diéguez, A. L., Romalde, J. L.,
e al. (2012). A cobac e bi al io um sp. no . and A cobac e ene upis sp.
no ., new species isola ed om shell ish. Sys . Appl. Mic obiol. 35, 133–138.
doi: 10.1016/j.syapm.2012.01.002
Le ican, A., Collado, L., and Figue as, M. J. (2013). A cobac e cloacae sp. no . and
A cobac e suis sp. no ., wo new species isola ed om ood and sewage. Sys .
Appl. Mic obiol. 36, 22–27. doi: 10.1016/j.syapm.2012.11.003
Le ican, A., Collado, L., and Figue as, M. J. (2016). The use o wo cul u ing
me hods in pa allel e eals a high p e alence and di e si y o A cobac e spp.
in a was ewa e ea men plan . Biomed Res. In . 2016:8132058. doi: 10.1155/
2016/8132058
Le ican, A., Collado, L., Yus es, C., Aguila , C., and Figue as, M. J. (2014). Highe
wa e empe a u e and incuba ion unde ae obic and mic oae obic condi ions
inc ease he eco e y and di e si y o A cobac e spp. om shell ish. Appl.
En i on. Mic obiol. 80, 385–391. doi: 10.1128/AEM.03014-13
Le ican, A., Rubio-A cos, S., Ma ínez-Mu cia, A., Collado, L., and Figue as, M. J.
(2015). A cobac e eb onensis sp. no . and A cobac e aquima inus sp. no ., wo
new species isola ed om ma ine en i onmen . Sys . Appl. Mic obiol. 38, 30–35.
doi: 10.1016/j.syapm.2014.10.011
Luo, C., Rod iguez-R, L. M., and Kons an inidis, K. T. (2014). MyTaxa: an
ad anced axonomic classi ie o genomic and me agenomic sequences.
Nucleic Acids Res. 42:e73. doi: 10.1093/na /gku169
Ma, X. X., Feng, Y. P., Bai, J. L., Zhang, D. R., Lin, X. S., and Ma, Z. R. (2015).
Nucleo ide composi ion bias and codon usage ends o gene popula ions in
Mycoplasma cap iolum subsp. cap iolum and M. agalac iae.J. Gene ics 94,
251–260. doi: 10.1007/s12041-015-0512-2
Ma enda, M. S., Sagne, E., Pouma a , F., and Ci i, C. (2005). Supp ession
sub ac i e hyb idiza ion as a basis o assess Mycoplasma agalac iae
and Mycoplasma bo is genomic di e si y and species-speci ic sequences.
Mic obiology 151, 475–489. doi: 10.1099/mic.0.27590-0
Ma ínez-Mu cia, A., and Lamy, B. (2015). “Molecula diagnos ics by gene ic
me hods,” in Ae omonas, ed. J. G a (Poole: Cais e Academic P ess),
155–200.
Ma ínez-Mu cia, A. J., Benlloch, S., and Collins, M. D. (1992). Phylogene ic
in e ela ionships o membe s o he gene a Ae omonas and Plesiomonas as
de e mined by 16S ibosomal DNA sequencing: lack o cong uence wi h esul s
o DNA-DNA hyb idiza ions. In . J. Sys . Bac e iol. 42, 412–421. doi: 10.1099/
00207713-42-3-412
Ma ínez-Mu cia, A. J., Figue as, M. J., Saa ed a, M. J., and S ackeb and , E.
(2007). The ecen ly p oposed species Ae omonas sha mana sp. no ., isola e
GPTSA-6T, is no a membe o he genus Ae omonas. In . Mic obiol. 10, 61–64.
Meie -Kol ho , J. P., Auch, A. F., Klenk, H. P., and Göke , M. (2013). Genome
sequence-based species delimi a ion wi h con idence in e als and imp o ed
dis ance unc ions. BMC Bioin o ma ics 14:60. doi: 10.1186/1471-2105-14-60
Neill, S. D., Campbell, J. N., O’B ien, J. J., Wea he up, S. T. C., and Ellis, W. A.
(1985). Taxonomic posi ion o Campylobac e c yae ophila sp. no .In . J. Sys .
Bac e iol. 35, 342–356. doi: 10.1099/00207713-35-3-342
Nu k, S., Banke ich, A., An ipo , D., Gu e ich, A. A., Ko obeyniko , A.,
Lapidus, A., e al. (2013). Assembling single-cell genomes and mini-
me agenomes om chime ic MDA p oduc s. J. Compu . Biol. 20, 714–737.
doi: 10.1089/cmb.2013.0084
On, S. L. W., Mille , W. G., Hou , K., Fox, J. G., and Vandamme, P. (2017).
Minimal s anda ds o desc ibing new species belonging o he amilies
Campylobac e aceae and Helicobac e aceae: campylobac e , A cobac e ,
Helicobac e and Wolinella spp. In . J. Sys . E ol. Mic obiol. 67, 5296–5311.
doi: 10.1099/ijsem.0.002255
O e beek, R., Olson, R., Pusch, G. D., Olsen, G. J., Da is, J. J., Disz, T., e al. (2014).
The SEED and he apid anno a ion o mic obial genomes using subsys ems
echnology ( RAST ). Nucleic Acids Res 42, D206–D214. doi: 10.1093/na /
gk 1226
Page, A. J., Cummins, C. A., Hun , M., Wong, V. K., Reu e , S., Holden,
M. T. G., e al. (2015). Roa y: apid la ge-scale p oka yo e pan genome analysis.
Bioin o ma ics 31, 3691–3693. doi: 10.1093/bioin o ma ics/b 421
Pa k, S., Jung, Y. T., Kim, S., and Yoon, J. H. (2016). A cobac e ac icola sp. no .,
isola ed om seawa e o he Eas Sea in Sou h Ko ea. J. Mic obiol. 54, 655–659.
doi: 10.1007/s12275-016-6268-4
Pé ez-Ca aluña, A., Collado, L., Salgado, O., Le iñanco, V., and Figue as, M. J.
(2018a). A polyphasic and axogenomic e alua ion unco e s A cobac e
c yae ophilus as a species complex ha emb aces ou genomo a s. F on .
Mic obiol 9:805. doi: 10.3389/ micb.2018.00805
Pé ez-Ca aluña, A., Salas-Massó, N., and Figue as, M. J. (2018b). A cobac e canalis
sp. no ., isola ed om a wa e canal con amina ed wi h u ban sewage. In . J.
Sys . E ol. Mic obiol. 68, 1258–1264. doi: 10.1099/ijsem.0.002662
Puigbò, P., B a o, I. G., and Ga cia-Vall e, S. (2008). CAIcal: a combined se o ools
o assess codon usage adap a ion. Biol. Di . 3:38. doi: 10.1186/1745-6150-3-38
Qin, Q.-L., Xie, B. B., Zhang, X. Y., Chen, X. L., Zhou, B. C., Zhou, J., e al. (2014).
A p oposed genus bounda y o he p oka yo es based on genomic insigh s.
J. Bac e iol. 196, 2210–2215. doi: 10.1128/JB.01688-14
R Co e Team. (2015). R: A Language and En i onmen o S a is ical Compu ing.
Vienna: R Founda ion o S a is ical Compu ing.
ˇ
Reháko á, K., Johansen, J. R., Bowen, M. B., Ma in, M. P., and Sheil, C. A. (2014).
Va ia ion in seconda y s uc u e o he 16S RNA molecule in Cyanobac e ia
wi h implica ions o phylogene ic analysis. Fo ea 14, 161–178. doi: 10.5507/
o .2014.013
Rich e , M., and Rosselló-Mó a, R. (2009). Shi ing he genomic gold s anda d
o he p oka yo ic species de ini ion. P oc. Na l. Acad. Sci. U.S.A. 106,
19126–19131. doi: 10.1073/pnas.0906412106
Rich e , M., Rosselló-Mó a, R., Oli e Glöckne , F., and Peplies, J. (2016).
JSpeciesWS: a web se e o p oka yo ic species ci cumsc ip ion based
on pai wise genome compa ison. Bioin o ma ics 32, 929–931. doi: 10.1093/
bioin o ma ics/b 681
Rosselló-Mo a, R., and Amann, R. (2001). The species concep o
p oka yo es. FEMS Mic obiol. Re . 25, 39–67. doi: 10.1111/j.1574-6976.2001.
b00571.x
Salas-Massó, N., And ee, K. B., Fu ones, M. D., and Figue as, M. J. (2016).
Enhanced eco e y o A cobac e spp. using NaCl in cul u e media and e-
assessmen o he ai s o A cobac e ma inus and A cobac e halophilus
isola ed om ma ine wa e and shell ish. Sci. To al En i on. 56, 1355–1361.
doi: 10.1016/j.sci o en .2016.05.197
Salas-Massó, N., Pé ez-Ca aluña, A., Collado, L., Le ican, A., and Figue as, M.
J. (2018). “A cobac e ”, in Handbook o Foodbo ne Diseases, ed. D. Liu (Boca
Ra on, FL: CRC P ess, Taylo & F ancis G oup).
Sasi-Jyo hsna, T. S., Rahul, K., Ramap asad, E. V. V., Sasikala, C., and Ramana,
C. V. (2013). A cobac e anae ophilus sp. no ., isola ed om an es ua ine
sedimen and emended desc ip ion o he genus A cobac e . In . J. Sys . E ol.
Mic obiol. 63, 4619–4625. doi: 10.1099/ijs.0.054155-0
Sawabe, T., Ki a-Tsukamo o, K., and Thompson, F. L. (2007). In e ing he
e olu iona y his o y o ib ios by means o mul ilocus sequence analysis.
J. Bac e iol. 189, 7932–7936. doi: 10.1128/JB.00693-07
Seemann, T. (2014). P okka: apid p oka yo ic genome anno a ion. Bioin o ma ics
30, 2068–2069. doi: 10.1093/bioin o ma ics/b u153
Sha p, P. M., and Li, W. H. (1987). The codon adap a ion index – a mesu e
o di ec ional synonymous codon usage bias, and i s po en ial applica ions.
Nucleic Acid Res. 15, 1281–1295. doi: 10.1093/na /15.3.1281
Suzuki, H., B own, C. J., Fo nay, L. J., and Top, E. M. (2008). Compa ison o
co espondence analysis me hods o synonymous codon usage in bac e ia.
DNA Res. 15, 357–365. doi: 10.1093/dna es/dsn028
Tamu a, K., S eche , G., Pe e son, D., Filipski, A., and Kuma , S. (2013). MEGA6:
molecula e olu iona y gene ics analysis e sion 6.0. Mol. Biol. E ol. 30,
2725–2729. doi: 10.1093/molbe /ms 197
Tanaka, R., Cleenwe ck, I., Mizu ani, Y., Ieha a, S., Bossie , P., and Vandamme, P.
(2017). A cobac e halio is sp. no ., isola ed om abalone species Halio is
gigan ea. In . J. Sys . E ol. Mic obiol 67, 3050–3056. doi: 10.1099/ijsem.0.00
2080
Tang, Y., Ho ikoshi, M., and Li, W. (2016). Gg o i y: uni ied in e ace o isualize
s a is ical esul o popula R Packages. The R J. 8, 474–485.
Tindall, B. J., Rosselló-Mo a, R., Busse, H. J., Ludwig, W., and Kämp e , P. (2010).
No es on he cha ac e iza ion o p oka yo e s ains o axonomic pu poses. In .
J. Sys . E ol. Mic obiol. 60, 249–266. doi: 10.1099/ijs.0.016949-0
Toh, H., Sha ma, V. K., Oshima, K., Kondo, S., Ha o i, M., Wa d, F. B.,
e al. (2011). Comple e genome sequences o A cobac e bu zle i ED-1 and
A cobac e sp. s ain L, bo h isola ed om a mic obial uel cell. J. Bac e iol. 193,
6411–6412. doi: 10.1128/JB.06084-11
Ue, H., Ma suo, Y., Kasai, H., and Yoko a, A. (2011). Demequina globuli o mis
sp. no ., Demequina oxidasica sp. no . and Demequina au an iaca sp.
no ., ac inobac e ia isola ed om ma ine en i onmen s, and p oposal o
F on ie s in Mic obiology | www. on ie sin.o g 18 Sep embe 2018 | Volume 9 | A icle 2077
micb-09-02077 Sep embe 1, 2018 Time: 10:25 # 19
Pé ez-Ca aluña e al. Re isi ing he Taxonomy o he Genus A cobac e
Demequinaceae am. no . In . J. Sys . E ol. Mic obiol. 61, 1322–1329. doi: 10.
1099/ijs.0.024299-0
U sing, J. B., Lio , H., and Owen, R. J. (1994). P oposal o minimal s anda ds o
desc ibing new species o he amily Campylobac e aceae. In . J. Sys . Bac e iol.
44, 842–845. doi: 10.1099/00207713-44-4-842
Van den Abeele, A. M., Vogelae s, D., Van Hende, J., and Hou , K.
(2014). P e alence o A cobac e species among humans, Belgium,
2008-2013. Eme g. In ec . Dis. 20, 1731–1734. doi: 10.3201/eid2010.
140433
Vandamme, P., Falsen, E., Rossau, R., Hos e, B., Sege s, P., Ty ga , R.,
e al. (1991). Re ision o Campylobac e , Helicobac e and Wolinella
axonomy: emenda ion o gene ic desc ip ions and p oposal o A cobac e
gen. no .In . J. Sys . Bac e iol. 41, 88–103. doi: 10.1099/00207713-
41-1-88
Vandamme, P., Vancanney , M., Po , B., Mels, L., Hos e, B., Dewe inck, D., e al.
(1992). Polyphasic axonomic s udy o he emended genus A cobac e wi h
A cobac e bu zle i comb. no . and A cobac e ski owii sp. no ., an ae o ole an
bac e ium isola ed om e e ina y specimens. In . J. Sys . Bac e iol. 42,
344–356. doi: 10.1099/00207713-42-3-344
Wai e, D. W., Vanwon e ghem, I., Rinke, C., Pa ks, D. H., Zhang, Y., Takai, K., e al.
(2017). Compa a i e genomic analysis o he class Epsilonp o eobac e ia and
p oposed eclassi ica ion o Epsilonbac e aeo a (phyl. no .).F on . Mic obiol.
8:682. doi: 10.3389/ micb.2017.00682
Wa am, A. R., Da is, J. J., Assa , R., Bois e , S., B e in, T., Bun, C., e al.
(2017). Imp o emen s o PATRIC, he all-bac e ial bioin o ma ics da abase and
analysis esou ce cen e . Nucleic Acids Res. 45, D535–D542. doi: 10.1093/na /
gkw1017
Weimann, A., Moo en, K., F ank, J., Pope, P. B., B emges, A., and McHa dy,
A. C. (2016). F om genomes o pheno ypes: ai a , he mic obial ai analyze .
mSys ems 1, e101–e116. doi: 10.1128/mSys ems.00101-16
Weine , J. (2017). Pca3d: Th ee Dimensional PCA Plo s. A ailable a : h ps://CRAN.
R-p ojec .o g/package=pca3d.
Whi educk-Lé eillée, K., Whi educk-Lé eillée, J., Clou ie , M., Tambong, J. T.,
Xu, R., Topp, E., e al. (2015). A cobac e lan hie i sp. no ., isola ed om
pig and dai y ca le manu e. In . J. Sys . E ol. Mic obiol. 65, 2709–2716.
doi: 10.1099/ijs.0.000318
Whi educk-Lé eillée, K., Whi educk-Lé eillée, J., Clou ie , M., Tambong, J. T.,
Xu, R., Topp, E., e al. (2016). Iden i ica ion, cha ac e iza ion and desc ip ion o
A cobac e aecis sp. no ., isola ed om a human was e sep ic ank. Sys . Appl.
Mic obiol. 39, 93–99. doi: 10.1016/j.syapm.2015.12.002
Whi man, W. B. (2015). Genome sequences as he ype ma e ial o axonomic
desc ip ions o p oka yo es. Sys . Appl. Mic obiol. 38, 217–222. doi: 10.1016/j.
syapm.2015.02.003
Wickham, H. (2009). Ggplo 2: Elegan G aphics o Da a Analysis. New Yo k, NY:
Sp inge -Ve lag. doi: 10.1007/978-0-387-98141-3
Ya za, P., Rich e , M., Peplies, J., Euzeby, J., Amann, R., Schlei e , K. H., e al. (2008).
The All-Species Li ing T ee p ojec : a 16S RNA-based phylogene ic ee o all
sequenced ype s ains. Sys . Appl. Mic obiol. 31, 241–250. doi: 10.1016/j.syapm.
2008.07.001
Ya za, P., Yilmaz, P., P uesse, E., Glöckne , F. O., Ludwig, W., Schlei e , K. H.,
e al. (2014). Uni ing he classi ica ion o cul u ed and uncul u ed bac e ia
and a chaea using 16S RNA gene sequences. Na . Re . Mic obiol. 12, 635–645.
doi: 10.1038/n mic o3330
Zhang, Z., Yu, C., Wang, X., Yu, S., and Zhang, X. H. (2015). A cobac e paci icus
sp. no ., isola ed om seawa e o he Sou h Paci ic Gy e. In . J. Sys . E ol.
Mic obiol. 66, 542–547. doi: 10.1099/ijsem.0.000751
Con lic o In e es S a emen : The au ho s decla e ha he esea ch was
conduc ed in he absence o any comme cial o inancial ela ionships ha could
be cons ued as a po en ial con lic o in e es .
Copy igh © 2018 Pé ez-Ca aluña, Salas-Massó, Diéguez, Balboa, Lema, Romalde
and Figue as. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in
o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s)
a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance
wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed
which does no comply wi h hese e ms.
F on ie s in Mic obiology | www. on ie sin.o g 19 Sep embe 2018 | Volume 9 | A icle 2077