ArticlePDF Available

Taxonomy of prokaryotic viruses: update from the ICTV Bacterial and Archaeal Viruses Subcommittee

Taxonomy of prokaryotic viruses: update from the ICTV
bacterial and archaeal viruses subcommittee
Mart Krupovic
Bas E. Dutilh
Evelien M. Adriaenssens
Johannes Wittmann
Finn K. Vogensen
Mathew B. Sullivan
Janis Rumnieks
David Prangishvili
Rob Lavigne
Andrew M. Kropinski
Jochen Klumpp
Annika Gillis
Francois Enault
Rob A. Edwards
Siobain Duffy
Martha R. C. Clokie
Jakub Barylski
Hans-Wolfgang Ackermann
Jens H. Kuhn
Received: 10 December 2015 / Accepted: 12 December 2015
ÓSpringer-Verlag Wien (Outside the USA) 2016
The prokaryotic virus community is represented on the
International Committee on Taxonomy of Viruses (ICTV)
by the Bacterial and Archaeal Viruses Subcommittee. In
2008, the three caudoviral families Myoviridae, Podoviri-
dae, and Siphoviridae included only 18 genera and 36
species. Under the able chairmanship of Rob Lavigne (KU
Leuven, Belgium), major advances were made in the
classification of prokaryotic viruses and the order Cau-
dovirales was expanded dramatically, to reflect the
genome-based relationships between phages. Today, the
order includes six subfamilies, 80 genera, and 441 species.
This year, additional changes in prokaryotic virus taxon-
omy have been brought forward under the new subcom-
mittee chair, Andrew M. Kropinski (University of Guelph,
Canada). These changes are:
1. replacement of ‘phage’ with ‘virus’ in prokaryotic
virus taxon names. In recognition of the fact that
phages are first and foremost genuine viruses, and to
adhere to ICTV’s International Code of Virus Classi-
fication and Nomenclature (ICVCN), the word
‘‘phage’ will disappear from taxon names, but not
from phage names. For instance, the current taxon
Escherichia phage T4 will be renamed Escherichia
virus T4, while the name of this taxon’s member will
remain unchanged (Escherichia phage T4). It is
The content of this publication does not necessarily reflect the views
or policies of the US Department of Health and Human Services, or
the institutions and companies affiliated with the authors. The
taxonomic changes summarized here have been submitted as official
taxonomic proposals to the International Committee on Taxonomy of
Viruses (ICTV) ( and are by now accepted, but
not yet ratified. These changes therefore may differ from any new
taxonomy that is ultimately approved by the ICTV.
&Andrew M. Kropinski
Jens H. Kuhn
Unit of Molecular Biology of the Gene in Extremophiles,
Department of Microbiology, Institut Pasteur, 25 rue du Dr
Roux, 75015 Paris, France
Theoretical Biology and Bioinformatics, Utrecht University,
Utrecht, The Netherlands
Centre for Molecular and Biomolecular Informatics,
Radboud University, Medical Centre, Nijmegen,
The Netherlands
Instituto de Biologia, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
Department of Genetics, Centre for Microbial Ecology and
Genomics, University of Pretoria, Private Bag X20, Hatfield,
Pretoria 0028, South Africa
Leibniz-Institut DSMZ-Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Inhoffenstraße
7B, 38124 Braunschweig, Germany
Department of Food Science, University of Copenhagen,
Rolighedsvej 26, 1958 Frederiksberg C, Denmark
Department of Microbiology, Ohio State University,
496 W 12th Ave, Columbus, OH 43210, USA
Department of Civil, Environmental, and Geodetic
Engineering, Ohio State University, 470 Hitchcock Hall,
2070 Neil Avenue, Columbus, OH 43210, USA
Latvian Biomedical Research and Study Center, Ra¯tsupı¯tes 1,
Riga, LV 1067, Latvia
Laboratory of Gene Technology, KU Leuven, Kasteelpark
Arenberg 21-box 2462, 3001 Leuven, Belgium
Institute of Food, Nutrition and Health, ETH Zurich,
Schmelzbergstrasse 7, 8092 Zurich, Switzerland
Arch Virol
DOI 10.1007/s00705-015-2728-0
important that the community remembers the ICVCN
distinction between viral taxa (such as species, genera,
families, or orders) and their members, the actual
viruses/phages: ‘‘viruses are real physical entities
produced by biological evolution and genetics,
whereas virus species and higher taxa are abstract
concepts produced by rational thought and logic’’;
2. elimination of the infix ‘like’ from prokaryotic
virus genus names. The naming of phage taxa has
been an evolving process with genus names in the
form ‘‘P22-like virus’’, which was always considered
to be a stop-gap measure, being replaced by P22like-
virus. However, the latter convention is also prob-
lematic since it was only applied to genera included
in the order Caudovirales, and the infix ‘‘like’ was
unnecessary since the grouping of viruses in genera
implies per se that their constituent members are
alike. Consequently, the infix ‘like’ will be removed
from the names of phage genera and genus names
such as Lambdalikevirus and T4likevirus will become
Lambdavirus and T4virus, respectively. It will of
course remain correct to refer to ‘‘lambda-like
viruses’’ and ‘‘T4-like phages’’ during discussions
regarding specific groups of phages classified in these
taxa. There have also been discussions in the
Subcommittee whether all prokaryotic virus genera
should adopt the system used for some archaeal and
eukaryotic viruses, in which names of genera are
created from the root of the corresponding family
name with sequentially appended transliterated Greek
letters (e.g., Alphabaculovirus,Betabaculovirus, etc.).
However, it was decided that recognition of new
genus names is of paramount importance and that
further drastic changes in one setting might overly
confuse the community. Thus, in most cases, the infix
‘‘like’ was merely removed and the name of the
founding member of the genus was retained as a root
of the taxon name;
3. discontinuation of the use of ‘Phi’ and other
transliterated Greek letters in the naming of new
prokaryotic virus genera. Since some scientists are
under the impression that ‘Phi’ in its various forms
(phi, u,U) indicates a phage, over the years, many
phages were given names containing the prefix ‘Phi’’ .
However, the prefix ‘Phi’ adds no informational value
when naming phage genera. Consequently, the Sub-
committee decided that, unless there was sufficient
historical precedent (e.g., U29 or UX174), Phi would
no longer be added to genus names. In addition, Greek
letters can create problems in electronic databases, as
exemplified by a PubMed search for references on
Bacillus phage U29 [1], which retrieved articles on phi
29, phi29, Phi 29, Phi29, 29 phi, {phi}29, u29, and
u29 phages. Therefore, the Subcommittee strongly
discourages phage scientists from using Phi or any
other Greek letter in virus and virus taxon names in the
4. elimination of hyphens from taxon names. The
ICVCN discourages hyphens in virus taxon names.
Accordingly, taxon names such as Yersinia phage
L-413C have been renamed (in this instance to Yersinia
virus L413C). However, hyphens are retained when
appearing in a number string: Thermus phage P2345
becomes Thermus virus P23-45 (its correct name) [2].
5. inclusion of the isolation host name in the taxon
name. On several occasions, terms such as ‘Enter-
obacteria’’ o r ‘‘ Pseudomonad’ have been used in
phage taxon names. However, such terms do not refer
to a specific bacterial host; nor do they indicate
whether the phage in question was tested upon a
variety of members of the particular host group. To
improve the situation, terms such as ‘‘Enterobacteria’’
Laboratory of Food and Environmental Microbiology,
´catholique de Louvain, Croix du Sud 2, L7.05.12,
1348 Louvain-la-Neuve, Belgium
Clermont Universite
´, Universite
´Blaise Pascal,
Laboratoire ‘‘Microorganismes: Ge
´nome et Environnement’’,
Clermont-Ferrand, France
CNRS UMR 6023, LMGE, Aubie
`re, France
Bioinformatics Lab, Department of Computer Science, San
Diego State University, 5500 Campanile Drive, San Diego,
CA 92182-7720, USA
Department of Ecology, Evolution and Natural Resources,
Rutgers University, 14 College Farm Rd, New Brunswick,
NJ 08901, USA
Department of Infection, Immunity and Inflammation,
University of Leicester, University Road,
Leicester LE1 9HN, UK
Department of Molecular Virology, Institute of Experimental
Biology, Adam Mickiewicz University, Umultowska 89,
61-614 Poznan, Poland
L’Institut de biologie inte
´grative et des systems, Universite
Laval, Pavillon Charles-Euge
`ne-Marchand, 1030, avenue de
la Me
´decine, Quebec, QC G1V 0A6, Canada
Integrated Research Facility at Fort Detrick, National
Institute of Allergy and Infectious Diseases, National
Institutes of Health, Fort Detrick, Frederick, MD 21702, USA
Departments of Food Science, Molecular and Cellular
Biology, and Pathobiology, University of Guelph, 50 Stone
Rd E, Guelph, ON N1G 2W1, Canada
M. Krupovic et al.
Table 1 Taxonomy proposals describing new taxa (genera, subfamilies, families) submitted to the ICTV in 2015
New genus Family Subfamily Type species Number of
Ap22virus Myoviridae Acinetobacter virus AP22 4
Secunda5virus Myoviridae Aeromonas virus 25 5
Biquartavirus Myoviridae Aeromonas virus 44RR2 1
Agatevirus Myoviridae Bacillus virus Agate 3
B4virus Myoviridae Bacillus virus B4 5
Bastillevirus Myoviridae Bacillus virus Bastille 2
Bv431virus Myoviridae Bacillus virus Bc431 4
Cp51virus Myoviridae Bacillus virus CP51 3
Nit1virus Myoviridae Bacillus virus NIT1 3
Wphvirus Myoviridae Bacillus virus WPh 1
Cvm10virus Myoviridae Escherichia virus CVM10 2
Kpp10virus Myoviridae Pseudomonas virus KPP10 3
Pakpunavirus Myoviridae Pseudomonas virus PAKP1 6
Rheph4virus Myoviridae Rhizobium virus RHEph4 1
Vhmlvirus Myoviridae Vibrio virus VHML 3
Tg1virus Myoviridae Yersinia virus TG1 2
P100virus Myoviridae Spounavirinae Listeria virus P100 1
Kayvirus Myoviridae Spounavirinae Staphylococcus virus K 7
Silviavirus Myoviridae Spounavirinae Staphylococcus virus Remus 2
Rb49virus Myoviridae Tevenvirinae Escherichia virus RB49 3
Rb69virus Myoviridae Tevenvirinae Escherichia virus RB69 4
Js98virus Myoviridae Tevenvirinae Escherichia virus JS98 5
Sp18virus Myoviridae Tevenvirinae Shigella virus SP18 5
S16virus Myoviridae Tevenvirinae Salmonella virus S16 2
Cc31virus Myoviridae Tevenvirinae Enterobacter virus CC31 2
Cr3virus Myoviridae Vequintavirinae (new) Cronobacter virus CR3 3
V5virus Myoviridae Vequintavirinae (new) Escherichia virus V5 4
Se1virus Myoviridae Vequintavirinae (new) Salmonella virus SE1 4
Pagevirus Podoviridae Bacillus virus Page 5
Cba41virus Podoviridae Cellulophaga virus Cba41 2
G7cvirus Podoviridae Escherichia virus G7C 8
Lit1virus Podoviridae Pseudomonas virus LIT1 3
Vp5virus Podoviridae Vibrio virus VP5 3
Kp34virus Podoviridae Autographivirinae Klebsiella virus KP34 5
Slashvirus Siphoviridae Bacillus virus Slash 4
Cba181virus Siphoviridae Cellulophaga virus Cba181 3
Cbastvirus Siphoviridae Cellulophaga virus ST 1
Nonagvirus Siphoviridae Escherichia virus 9g 4
Seuratvirus Siphoviridae Escherichia virus Seurat 2
P70virus Siphoviridae Listeria virus P70 5
Psavirus Siphoviridae Listeria virus PSA 2
Ff47virus Siphoviridae Mycobacterium virus Ff47 2
Sitaravirus Siphoviridae Paenibacillus virus Diva 5
Septima3virus Siphoviridae Pseudomonas virus 73 5
Nonanavirus Siphoviridae Salmonella virus 9NA 2
Sextaecvirus Siphoviridae Staphylococcus virus 6ec 2
Ssp2virus Siphoviridae Vibrio virus SSP002 2
Prokaryotic virus taxonomy
or ‘Pseudomonad’ in taxon names will be replaced
with the isolation host genus name: for instance,
Enterobacteria phage T7 will become Escherichia
virus T7. In addition, host species names will be
eliminated from taxon names. For example, Thermus
thermophilus phage IN93 will become Thermus virus
Further considerations
DNA-DNA relatedness is the gold standard in the classi-
fication of all prokaryotes [37], and efforts are underway
to move towards a completely genomic taxonomy in that
field [8]. The Bacterial and Archaeal Viruses Subcommit-
tee has previously used overall proteome similarity to
define genera and subfamilies, with 40 % homologous
proteins indicating membership in the same genus [911].
This has resulted in spurious taxonomic lumping [1214].
Furthermore, EMBOSS Stretcher [15,16], which has been
used for calculating nucleotide similarities between related
phages (e.g., [17]), suffers from certain limitations (in
particular the requirement for the genomes to be collinear).
Problems with EMBOSS Stretcher are highlighted when an
alignment of the phage T7 genome with a randomly
shuffled T7 DNA sequence (http://www.bioinformatics.
org/sms2/shuffle_dna.html) is attempted. The resulting
value, 47.6 % identity, demonstrates that EMBOSS
Stretcher values below a certain threshold are meaningless.
Accordingly, more recent phage classification efforts have
explored alternative approaches. Specifically, BLASTN
[19] was found to be superior to EMBOSS Stretcher for
identification and quantitative comparison of closely rela-
ted phages [16]. Indeed, a BLASTN search seeded with the
shuffled sequence of phage T7 specifically against ‘‘En-
terobacteria phage T7’’ results in no detectable similarity,
as expected from a randomized sequence with 48.4 % GC
content. Moreover, BLASTN has also been used to deter-
mine relationships between phages at larger phylogenetic
distances [17,18], although the meaning of a similarity
search hit in the absence of a true-shared ancestry remains
unclear. Most of the newer programs that calculate phy-
logenetic relationships between genome sequences,
including CLANS [20], GEGENEES [21], and mVISTA
[22], are based upon sequence similarity analyses such as
provided by BLASTN [19]. Complete and near-complete
viral genome and protein homologies will be the focus of
the Bacterial and Archaeal Viruses Subcommittee’s atten-
tion in 2016 to develop clearer parameters for the molec-
ular definition of genera, subfamilies, and families.
The changes described here were formalized and sub-
mitted in more than 40 ICTV taxonomic proposals (Tax-
oProps) for consideration by the ICTV Executive
Committee ( One new archaeal
virus family (Pleolipoviridae), four new bacterial sub-
families (Guernseyvirinae [Salmonella phage Jersey], Ve-
quintavirinae [Escherichia phage rV5], Tunavirinae
[Escherichia phage T1], and Bullavirinae [Escherichia
phage UX174]), and 59 new genera including 232 species
are covered in these proposals (summarized in Table 1).
While the Bacterial and Archaeal Viruses Subcommittee
is delighted with the progress described here, some
400–600 new genomes of novel phages are deposited to
Table 1 continued
New genus Family Subfamily Type species Number of
K1gvirus Siphoviridae Guernseyvirinae (new) Escherichia virus K1G 4
Jerseyvirus (existing) Siphoviridae Guernseyvirinae (new) Salmonella virus Jersey 6
Sp31virus Siphoviridae Guernseyvirinae (new) Salmonella virus SP31 1
T1virus (existing) Siphoviridae Tunavirinae (new) Escherichia virus T1 4
Tlsvirus Siphoviridae Tunavirinae (new) Escherichia virus TLS 3
Rtpvirus Siphoviridae Tunavirinae (new) Escherichia virus Rtp 2
Kp36virus Siphoviridae Tunavirinae (new) Klebsiella virus KP36 3
Rogue1virus Siphoviridae Tunavirinae (new) Escherichia virus Rogue1 8
Alpha3microvirus Microviridae Bullavirinae (new) Escherichia virus alpha3 8
G4microvirus Microviridae Bullavirinae (new) Escherichia virus G4 3
Phix174microvirus Microviridae Bullavirinae (new) Escherichia virus phiX174 1
Alphapleolipovirus Pleolipoviridae (new) Halorubrum virus HRPV-1 5
Betapleolipovirus Pleolipoviridae (new) Halorubrum virus HRPV-3 2
Gammapleolipovirus Pleolipoviridae (new) Haloarcula virus His2 1
M. Krupovic et al.
GenBank annually. Many of these may have to be assigned
to novel species or higher taxa via the ICTV TaxoProp
process. Phage classification will therefore remain a highly
demanding and daunting process, unless a genomic tax-
onomy for viruses is embraced (see [8]). Although a tax-
onomy that is based on the genome sequence alone might
be incorrect due to rampant genomic rearrangements in
viruses [23], such an approach may turn out to be the only
scalable solution.
Compliance with ethical standards
Funding This work was funded in part through Battelle Memorial
Institute’s prime contract with the US National Institute of Allergy
and Infectious Diseases (NIAID) under Contract No.
HHSN272200700016I. A subcontractor to Battelle Memorial Institute
who performed this work is: J.H.K., an employee of Tunnell
Government Services, Inc. B.E.D. was supported by the Netherlands
Organization for Scientific Research (NWO) Vidi Grant 864.14.004
Conflict of interest The authors declare that they have no conflict
of interest.
Ethical approval This article does not contain any studies with
human participants or animals performed by any of the authors.
1. Salas M (2012) My life with bacteriophage phi29. J Biol Chem
287(53):44568–44579. doi:10.1074/jbc.X112.433458
2. Yu MX, Slater MR, Ackermann HW (2006) Isolation and charac-
terization of Thermus bacteriophages. Arch Virol 151(4):663–679
3. Auch AF, Klenk HP, Go
¨ker M (2010) Standard operating pro-
cedure for calculating genome-to-genome distances based on
high-scoring segment pairs. Stand Genomic Sci 2(1):142–148.
4. Colston SM, Fullmer MS, Beka L, Lamy B, Gogarten JP, Graf J
(2014) Bioinformatic genome comparisons for taxonomic and
phylogenetic assignments using Aeromonas as a test case. MBio
5(6):e02136. doi:10.1128/mBio.02136-14
5. Lee I, Kim YO, Park SC, Chun J (2015) OrthoANI: An improved
algorithm and software for calculating average nucleotide iden-
tity. Int J Syst Evol Microbiol. doi:10.1099/ijsem.0.000760
6. Richter M, Rossello
´ra R, Glo
¨ckner FO, Peplies J (2015)
JSpeciesWS: a web server for prokaryotic species circumscrip-
tion based on pairwise genome comparison. Bioinformatics pii:
btv681 [Epub ahead of print]
7. Caputo A, Merhej V, Georgiades K, Fournier PE, Croce O,
Robert C, Raoult D (2015) Pan-genomic analysis to redefine
species and subspecies based on quantum discontinuous varia-
tion: the Klebsiella paradigm. Biol Direct 10:55. doi:10.1186/
8. Thompson CC, Amaral GR, Campea
˜o M, Edwards RA, Polz MF,
Dutilh BE, Ussery DW, Sawabe T, Swings J, Thompson FL
(2015) Microbial taxonomy in the post-genomic era: rebuilding
from scratch? Arch Microbiol 197(3):359–370. doi:10.1007/
9. Lavigne R, Seto D, Mahadevan P, Ackermann HW, Kropinski
AM (2008) Unifying classical and molecular taxonomic classi-
fication: analysis of the Podoviridae using BLASTP-based tools.
Res Microbiol 159(5):406–414. doi:10.1016/j.resmic.2008.03.
10. Lavigne R, Darius P, Summer EJ, Seto D, Mahadevan P, Nilsson
AS, Ackermann HW, Kropinski AM (2009) Classification of
Myoviridae bacteriophages using protein sequence similarity.
BMC Microbiol 9:224. doi:10.1186/1471-2180-9-224
11. Adriaenssens EM, Edwards R, Nash JH, Mahadevan P, Seto D,
Ackermann HW, Lavigne R, Kropinski AM (2015) Integration of
genomic and proteomic analyses in the classification of the
Siphoviridae family. Virology 477:144–154. doi:10.1016/j.virol.
12. Eriksson H, Maciejewska B, Latka A, Majkowska-Skrobek G,
Hellstrand M, Melefors O
¨, Wang JT, Kropinski AM, Drulis-
Kawa Z, Nilsson AS (2015) A suggested new bacteriophage
genus, ‘‘Kp34likevirus’’, within the Autographivirinae subfamily
of Podoviridae. Viruses 7(4):1804–1822. doi:10.3390/v7041804
13. Niu YD, McAllister TA, Nash JH, Kropinski AM, Stanford K
(2014) Four Escherichia coli O157:H7 phages: a new bacterio-
phage genus and taxonomic classification of T1-like phages.
PLoS One 9(6):e100426. doi:10.1371/journal.pone.0100426
14. Wittmann J, Klumpp J, Moreno Switt AI, Yagubi A, Ackermann
HW, Wiedmann M, Svircev A, Nash JH, Kropinski AM (2015)
Taxonomic reassessment of N4-like viruses using comparative
genomics and proteomics suggests a new subfamily—’’Enquar-
tavirinae’’. Arch Virol. 160(12):3053–3062. doi:10.1007/s00705-
15. Rice P, Longden I, Bleasby A (2000) EMBOSS: the European
Molecular Biology Open Software Suite. Trends Genet
16. Ceyssens PJ, Glonti T, Kropinski NM, Lavigne R, Chanishvili N,
Kulakov L, Lashkhi N, Tediashvili M, Merabishvili M (2011)
Phenotypic and genotypic variations within a single bacterio-
phage species. Virol J 8:134. doi:10.1186/1743-422X-8-134
17. Rohwer F, Edwards R (2002) The Phage Proteomic Tree: a
genome-based taxonomy for phage. J Bacteriol 184(16):
18. Mizuno CM, Rodriguez-Valera F, Kimes NE, Ghai R (2013)
Expanding the marine virosphere using metagenomics. PLoS
Genet 9(12):e1003987. doi:10.1371/journal.pgen.1003987
19. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)
Basic local alignment search tool. J Mol Biol 215(3):403–410
20. Asare PT, Jeong TY, Ryu S, Klumpp J, Loessner MJ, Merrill BD,
Kim KP (2015) Putative type 1 thymidylate synthase and dihy-
drofolate reductase as signature genes of a novel Bastille-like
group of phages in the subfamily Spounavirinae. BMC Genomics
16:582. doi:10.1186/s12864-015-1757-0
21. Agren J, Sundstro
¨m T, Segerman B (2012) Gege-
nees: fragmented alignment of multiple genomes for determining
phylogenomic distances and genetic signatures unique for spec-
ified target groups. PLoS One 7(6):e39107. doi:10.1371/journal.
22. Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I (2004)
VISTA: computational tools for comparative genomics. Nucleic
Acids Res; 32(Web Server issue): W273–W279
23. Krupovic M, Prangishvili D, Hendrix RW, Bamford DH (2011)
Genomics of bacterial and archaeal viruses: dynamics within the
prokaryotic virosphere. Microbiol Mol Biol Rev 75(4):610–635.
Prokaryotic virus taxonomy
... As the age of genomics dawned in the early 2000s, the sequencing of phage genomes revealed a much higher genomic diversity than had previously been considered, especially in bacteriophages belonging to the order Caudovirales, leading to the creation of the first subfamilies within the existing three families Podoviridae [7], Myoviridae [8], and later on Siphoviridae [9]. As the number of phage genomes in databases rose, it quickly became apparent that these three families were not monophyletic and cohesive within a monophyletic order. ...
... For the filamentous, ssDNA phages, the family Inoviridae has been split into two families, Inoviridae and Plectroviridae which are grouped together in the order Tubulavirales [21], with a potential further increase with five new families based on the analysis of cryptic inoviruses from bacterial genome datasets [32]. In a similar vein, many additional subfamilies have been proposed in the ssDNA family Microviridae, beyond the existing subfamilies Bullavirinae [9] and Gokushovirinae based on the detection in virome data, i.e. the subfamilies "Alpavirinae" [33], "Pichovirinae" [34], "Stokavirinae" [35], and "Aravirinae" [35]. Recently, computational approaches identified a massive expansion in the number of ssRNA phage genomes of the Leviviridae family, first with 158 [36] then with a further 1k complete and 15k partial genomes [37]. ...
Full-text available
Bacteriophage (phage) taxonomy has been in flux since its inception over four decades ago. Genome sequencing has put pressure on the classification system and recent years have seen significant changes to phage taxonomy. Here, we reflect on the state of phage taxonomy and provide a roadmap for the future, including the abolition of the order Caudovirales and the families Myoviridae, Podoviridae, and Siphoviridae. Furthermore, we specify guidelines for the demarcation of species, genus, subfamily and family-level ranks of tailed phage taxonomy.
... M. aeruginosa cyanophage MinS1 (MinS1) [27] was isolated from the surface freshwater samples obtained from the Mayang Stream, located in Fujian, China, which had an outbreak of cyanobacterial blooms (more specific information is shown in Table S1). The plaques resulting from MinS1 lysis appeared clear and circular, with diameters of 3-4 mm, following a 4-day incubation (25 • C, 2000 Lux, 12 h:12 h light-dark cycle) on host algal plates at a multiplicity of infection (MOI) of 0.1 (Figure 1a). ...
Full-text available
Microcystis aeruginosa, as one of the major players in algal bloom, produces microcystins, which are strongly hepatotoxic, endangering human health and damaging the ecological environment. Biological control of the overgrowth of Microcystis with cyanophage has been proposed to be a promising solution for algal bloom. In this study, a novel strain of Microcystis cyanophage, MinS1, was isolated. MinS1 contains an icosahedral head approximately 54 nm in diameter and a 260 nm-long non-contractile tail. The phage genome consists of a linear, double-stranded 49,966 bp DNA molecule, which shares very low homology with known phages in the NCBI database (only 1% of the genome showed weak homology with known phages when analyzed by megablast). The phage contains 75 ORFs, of which 23 ORFs were predicted to code for proteins of known function, 39 ORFs were predicted to code for proteins of unknown function, and 13 ORFs showed no similarity to any protein sequences. Transmission electron microscopy and phylogenetic analysis showed that MinS1 belongs to the family Siphoviridae. Various experiments confirmed that the phage could infect several different orders of cyanobacteria, including Chroococcales, Nostocales, Oscillatoriales, Hormogonales, and Synechococcales, indicating that it has a very broad host range. In addition, MinS1 has no known antibiotic tolerance genes, virulence genes, and tRNAs, and it is tolerant to temperature, pH, UV, and salinity, suggesting that MinS1 has good potential for application as a biological control agent against cyanobacterial blooms. This study expands the diversity and knowledge of cyanophages, and it provides useful information for the development of novel prevention and control measures against cyanobacterial blooms.
... Visualization and alignment of the phage genomes in circular and linear form were carried out using the program DNA-Plotter [27]. The percent nucleotide sequence identity for the complete bacteriophage Athena1 and VBM1 genomes was determined using BLASTn as recommended previously [28]. The common genes and open reading frames (ORFs), presented in Supplementary Table S1, were identified using the online platform CoreGenes3.5 [29] with a BLASTp threshold score of 65. ...
Full-text available
Tailed bacteriophages have been at the center of attention, not only for their ability to infect and kill pathogenic bacteria but also due to their peculiar and intriguing complex contractile tail structure. Tailed bacteriophages with contractile tails are known to have a Myoviridae morphotype and are members of the order Caudovirales. Large bacteriophages with a genome larger than 150 kbp have been studied for their ability to use multiple infection and lysis strategies to replicate more efficiently. On the other hand, smaller bacteriophages with fewer genes are represented in the GenBank database in greater numbers, and have several genes with unknown function. Isolation and molecular characterization of a newly reported bacteriophage named Athena1 revealed that it is a strongly lytic bacteriophage with a genome size of 39,826 bp. This prompted us to perform a comparative genomic analysis of Vibrio myoviruses with a genome size of no more than 50 kbp. The results revealed a pattern of genomic organization that includes sets of genes responsible for virion morphogenesis, replication/recombination of DNA, and lysis/lysogeny switching. By studying phylogenetic gene markers, we were able to draw conclusions about evolutionary events that shaped the genomic mosaicism of these phages, pinpointing the importance of a conserved organization of the genomic region encoding the baseplate protein for successful infection of Gram-negative bacteria. In addition, we propose the creation of new genera for dwarf Vibrio myoviruses. Comparative genomics of phages infecting aquatic bacteria could provide information that is useful for combating fish pathogens in aquaculture, using novel strategies.
... However, for the commonly used clustering techniques with these features that were generated from phage genomes, they were difficult to classify the different phage families [5]. Moreover, because of the lack of corresponding biological and experimental data, clustering techniques with these genomes into the ICTV(the International Committee on Taxonomy of Viruses) scheme were difficult also [5,6,7]. Furthermore, some approaches of phage classifications used the singly selected marker molecules to define sequence alignment and similarities, but these approaches were restricted to closely related phage taxa only [8,9], such as these comparative sequence analysis [10,11,12,13,14]. ...
Full-text available
Background: Phages are the most abundant biological entities, but the commonly used clustering techniques are difficult to separate them from other virus families and classify the different phage families together. Results: This work uses GI-clusters to separate phages from other virus families and classify the different phage families, where GI-clusters are constructed by GI-features, GI-features are constructed by the togetherness with F-features, training data, MG-Euclidean and Icc-cluster algorithms, F-features are the frequencies of multiple-nucleotides that are generated from genomes of viruses, MG-Euclidean algorithm is able to put the nearest neighbors in the same mini-groups, and Icc-cluster algorithm put the distant samples to the different mini-clusters. For these viruses that the maximum element of their GI-features are in the same locations, they are put to the same GI-clusters, where the families of viruses in test data are identified by GI-clusters, and the families of GI-clusters are defined by viruses of training data. Conclusions: From analysis of 4 data sets that are constructed by the different family viruses, we demonstrate that GI-clusters are able to separate phages from other virus families, correctly classify the different phage families, and correctly predict the families of these unknown phages also.
... Thereafter, the prepared sample was observed with a TEM (Philips EM 208S) at 100 KV. The latest changes in the International virus classification committee (ICTV) reports were used to further identify the possible family of the isolated phages (40,41). ...
Full-text available
Background and Objectives: Prevalence of extended spectrum β-lactamase (ESBL) leads to the development of antibiotic resistance and mortality in burn patients. One of the alternative strategies for controlling ESBL bacterial infections is clinical trials of bacteriophage therapy. The aim of this study was to isolate and characterize specific bacteriophages against ESBL-producing Klebsiella pneumoniae in patients with burn ulcers. Materials and Methods: Clinical samples were isolated from the hospitalized patient in burn medical centers, Iran. Biochemical screenings and 16S rRNA gene sequencing were determined. The phages were isolated from municipal sewerage treatment plants, Isfahan, Iran. TEM and FESEM, adsorption velocity, growth curve, host range, and the viability of the phage particles as well as proteomics and enzyme digestion patterns were examined. Results: The results showed that Klebsiella pneumoniae Iaufa_lad2 (GenBank accession number: MW836954) was confirmed as an ESBL-producing strain using combined disk method. This bacterium showed significant sensitivity to three phages including PɸBw-Kp1, PɸBw-Kp2, and PɸBw-Kp3. Morphological characterization demonstrated that the phage PɸBw-Kp3 to the Siphoviridae family (lambda-like phages) and both phages PɸBw-Kp1 and ɸBw-Kp2 to the Podoviridae family (T1-like phages). The isolated bacteriophages had a large burst size, thermal and pH viability and efficient adsorption rate to the host cells. Conclusion: In present study, the efficacy of bacteriophages against ESBL pathogenic bacterium promises a remarkable achievement for phage therapy. It seems that, these isolated bacteriophages, in the form of phage cocktails, had a strong antibacterial impacts and a broad-spectrum strategy against ESBL-producing Klebsiella pneumoniae isolated from burn ulcers.
... All Microviridae phages encoded two relatively conserved proteins, a capsid protein and a replication initiator protein (Cherwa and Fane, 2011). Currently, Microviridae is mainly classified into two subfamilies, Bullavirinae and Gokushovirinae, and an unclassified group (Cherwa and Fane, 2011;Krupovic et al., 2016). Bullavirinae contain 11 genes with genome size from 5.4 to 6 kb, typified by the bacteriophage phiX174 (Sanger et al., 1977). ...
Full-text available
“Candidatus Liberibacter asiaticus” (CLas) is an unculturable phloem-limited α-proteobacterium associated with citrus Huanglongbing (HLB; yellow shoot disease). HLB is currently threatening citrus production worldwide. Understanding the CLas biology is critical for HLB management. In this study, a novel single-stranded DNA (ssDNA) phage, CLasMV1, was identified in a CLas strain GDHZ11 from Guangdong Province of China through a metagenomic analysis. The CLasMV1 phage had a circular genome of 8,869 bp with eight open reading frames (ORFs). While six ORFs remain uncharacterized, ORF6 encoded a replication initiation protein (RIP), and ORF8 encoded a major capsid protein (MCP). Based on BLASTp search against GenBank database, amino acid sequences of both MCP and RIP shared similarities (coverage > 50% and identity > 25%) to those of phages in Microviridae, an ssDNA phage family. Phylogenetic analysis revealed that CLasMV1 MCP and RIP sequences were clustered with genes from CLas and “Ca. L. solanacearum” (CLso) genomes and formed a unique phylogenetic lineage, designated as a new subfamily Libervirinae, distinct to other members in Microviridae family. No complete integration form but partial sequence (∼1.9 kb) of CLasMV1 was found in the chromosome of strain GDHZ11. Read-mapping analyses on additional 15 HiSeq data sets of CLas strains showed that eight strains harbored complete CLasMV1 sequence with variations in single-nucleotide polymorphisms (SNPs) and small sequence insertions/deletions (In/Dels). PCR tests using CLasMV1-specific primer sets detected CLasMV1 in 577 out of 1,006 CLas strains (57%) from southern China. This is the first report of Microviridae phage associated with CLas, which expands our understanding of phage diversity in CLas and facilitates current research in HLB.
... After drying, phage particles were observed in the sample with TEM (EM 208S 100 Kv, Philips). Detection of the phage family according to the morphological features was performed by using the latest changes in the International Virus Classification Committee (ICTV) reports (46)(47)(48)43). ...
Full-text available
Objectives: With emergence of drug resistance, novel approaches such as phage therapy for treatment of bacterial infections have received significant attention. The purpose of this study was to isolate and identify effective bacteriophages on extremely drug-resistant (XDR) bacteria isolated from burn wounds. Materials and methods: Pathogenic bacteria were isolated from hospitalized patient wounds in specialized burn hospitals in Iran, and their identification was performed based on biochemical testing and sequencing of the gene encoding 16S rRNA. Bacteriophages were isolated from municipal sewage, Isfahan, Iran. The phage morphology was observed by TEM. After detection of the host range, adsorption rate, and one-step growth curve, the phage proteomics pattern and restriction enzyme digestion pattern were analyzed. Results: All isolates of bacteria were highly resistant to antibiotics. Among isolates, Acinetobacter baumannii strain IAU_FAL101 (GenBank accession number: MW845680), which was an XDR bacterium, showed significant sensitivity to phage Pɸ-Bw-Ab. TEM determined the phage belongs to Siphoviridae. They had double-stranded DNA. This phage showed the highest antibacterial effect at 15 °C and pH 7. Analysis of the restriction enzyme digestion pattern showed Pɸ-Bw-Ab phage was sensitive to most of the used enzymes and based on SDS-PAGE, protein profiles were revealed 43 to 90 kDa. Conclusion: Considering the potential ability of the isolated phage, it had an antibacterial impact on other used bacterial spp and also strong antibacterial effects on XDR A. baumannii. Also, it had long latency and low burst size. This phage can be a suitable candidate for phage therapy.
... The first 12 BLASTN results sorted by E-value were collected in Table 2. Phage LPSTLL showed significant similarity to Salmonella phage E1, Salmonella phage LPST10, Salmonella phage IME207, Salmonella phage 64795_sal3, Escherichia phage vB_EcoS-Sa179w3YLVW and Shigella phage Sf11 (more than 86% nucleotide identity with at least 43% coverage). Interestingly, none of the stated phages has been assigned to any genus by International Committee on Taxonomy of Viruses (ICTV) (Adriaenssens & Wittmann, 2018;Krupovic & Dutilh, 2016). Therefore, we hypothesized that these phages, together with phage LPSTLL, might constitute a new genus. ...
Salmonella is one of the most common foodborne pathogens around the world. Phages are envisioned as a new strategy to control foodborne pathogenic bacteria and food safety. A Salmonella specific lytic phage vB_SalS-LPSTLL (LPSTLL) was selected for food applications on the basis of lytic range, lytic efficiency, functional stability and characteristics. Phage LPSTLL was able to lyse 11 Salmonella serotypes, which represents the broadest range reported Salmonella phages, and was able to suppress the growth of Salmonella enterica in liquid culture over nine hours. LPSTLL exhibited rapid reproductive activity with a short latent period and a large burst size in one-step growth experiment. LPSTLL remained active over a pH range of 3.0 to 12.0, and at incubation temperatures up to 60 °C for 60 minutes, indicating wide applicability for food processing and storage. Significant reductions of viable Salmonella were observed in diverse foods (milk, apple juice, chicken and lettuce) with reductions up to 2.8 log CFU/mL recorded for milk. Sensory evaluation indicated that treatment with phage LPSTLL did not alter the visual or tactile quality of food matrices. Genome analysis of LPSTLL indicated the absence of any virulence or antimicrobial resistance genes. Genomic comparisons suggest phage LPSTLL constitutes a novel member of a new genus, the LPSTLLvirus with the potential for Salmonella biocontrol in the food industry.
... taxonomy/p/taxonomy_releases). Recent advances in next-generation sequencing (NGS) technologies unveiled the 'hidden' genomic and metagenomic sequence of unknown phages, but unfortunately, a systematic classification of these phage genomes into the ICTV scheme is not available due to lack of related biological properties [112][113][114]. Therefore, taxonomical revision based on the genomic information of phages has become indispensable, and modernized comprehensive guidelines for phage classification have been recently suggested, which is expected to cause a substantial increase in the list of virus taxa in the coming years [115,116]. ...
Acinetobacter strains are widely present in the environment. Some antimicrobial-resistant strains of this genus have been implicated in infections acquired in hospitals. Genetic similarities have been reported between Acinetobacter strains in nosocomial infections and those isolated from foods. However, the antimicrobial resistance of Acinetobacter strains in foods, such as meat, remains unclear. This study initially aimed to isolate Campylobacter strains; instead, strains of the genus Acinetobacter were isolated from meat products, and their antimicrobial resistance was investigated. In total, 58 Acinetobacter strains were isolated from 381 meat samples. Of these, 32 strains (38.6%) were from beef, 22 (26.5%) from pork, and 4 (4.8%) from duck meat. Antimicrobial susceptibility tests revealed that 12 strains were resistant to more than one antimicrobial agent, whereas two strains were multidrug-resistant; both strains were resistant to colistin. Cephalosporin antimicrobials showed high minimal inhibitory concentration against Acinetobacter strains. Resfinder analysis showed that one colistin-resistant strain carried mcr-4.3; this plasmid type was not confirmed, even when analyzed with PlasmidFinder. Analysis of the contig harboring mcr-4.3 using BLAST confirmed that this contig was related to mcr-4.3 of Acinetobacter baumannii. The increase in antimicrobial resistance in food production environments increases the resistance rate of Acinetobacter strains present in meat, inhibits the isolation of Campylobacter strains, and acts as a medium for the transmission of antimicrobial resistance in the environment. Therefore, further investigations are warranted to prevent the spread of antimicrobial resistance in food products.
The prevalence of multidrug-resistant (MDR) strains has caused serious problems in the treatment of burn infections. MDR Enterobactercloacae and Enterobacterhormaechei have been defined as the causative agents of nosocomial infections in burn patients. In this situation, examination of phages side effects on human cell lines before any investigation on human or animal that can provide beneficial information about the safety of isolated phages. The aim of this study was to isolate and identify the specific bacteriophages on MDR E. cloacae and E. hormaechei isolated from burn wounds and to analyze the efficacy, cell viability and cell cytotoxicity of phages on A-375 and HFSF-PI cell lines by MTT (3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide) colorimetric assay and lactate dehydrogenase (LDH) release assay. Phages were isolated from urban sewage Isfahan, Iran. Enterobactercloacae strain Iau-EC100 (GenBank accession number: MZ314381) and E. hormaechei strain Iau-EHO100 (GenBank accession number: MZ348826) were sensitive to the isolated phages. Transmission electron microscopy (TEM) results revealed that PɸEn-CL and PɸEn-HO that were described had the morphologies of Myovirus and Inovirus, respectively. Overall, MTT and LDH assays showed moderate to excellent correlation in the evaluation of cytotoxicity of isolated phages. The results of MTT and LDH assays showed that, phages PɸEn-CL and PɸEn-HO had no significant toxicity effect on A375 and HFSF-PI 3 cells. Phage PɸEn-HO had a better efficacy on the two tested cell lines than other phage. Our results indicated that, there were significant differences between the two cytotoxicity assays in phage treatment compared to control.
Full-text available
Species demarcation in Bacteria and Archaea is mainly based on overall genome relatedness, which serves a framework for modern microbiology. Current practice of obtaining these measures between two strains is shifting from experimentally determined similarity obtained by DNA-DNA hybridization (DDH) to genome sequence-based similarity. Average nucleotide identity (ANI) is a simple algorithm that mimics DDH. Like DDH, ANI values between two genome sequences may be different from each other when reciprocal calculations are compared. We compared 63,690 pairs of genome sequences and found that the differences in reciprocal ANI values are significantly high, showing over 1% in some cases. To resolve this problem of not being symmetrical, new algorithm, named OrthoANI, was developed to accommodate the concept of orthology for which both genome sequences were fragmented and only orthologous fragment pairs taken into consideration for calculating nucleotide identities. OrthoANI is highly correlated with ANI (using BLASTn) and the former showed ~0.1% higher values than the latter. In conclusion, OrthoANI provides a more robust and faster means of calculating average nucleotide identity for the taxonomic purposes. The standalone software tools are freely available at
Full-text available
Unlabelled: JSpecies Web Server (JSpeciesWS) is a user-friendly online service for in silico calculating the extent of identity between two genomes, a parameter routinely used in the process of polyphasic microbial species circumscription. The service measures the average nucleotide identity (ANI) based on BLAST+ (ANIb) and MUMmer (ANIm), as well as correlation indexes of tetra-nucleotide signatures (Tetra). In addition, it provides a Tetra Correlation Search function, which allows to rapidly compare selected genomes against a continuously updated reference database with currently about 32 000 published whole and draft genome sequences. For comparison, own genomes can be uploaded and references can be selected from the JSpeciesWS reference database. The service indicates whether two genomes share genomic identities above or below the species embracing thresholds, and serves as a fast way to allocate unknown genomes in the frame of the hitherto sequenced species. Availability and implementation: JSpeciesWS is available at Supplementary information: Supplementary data are available at Bioinformatics online. Contact:
Full-text available
Background Various methods are currently used to define species and are based on the phylogenetic marker 16S ribosomal RNA gene sequence, DNA-DNA hybridization and DNA GC content. However, these are restricted genetic tools and showed significant limitations. Results In this work, we describe an alternative method to build taxonomy by analyzing the pan-genome composition of different species of the Klebsiella genus. Klebsiella species are Gram-negative bacilli belonging to the large Enterobacteriaceae family. Interestingly, when comparing the core/pan-genome ratio; we found a clear discontinuous variation that can define a new species. Conclusions Using this pan-genomic approach, we showed that Klebsiella pneumoniae subsp. ozaenae and Klebsiella pneumoniae subsp. rhinoscleromatis are species of the Klebsiella genus, rather than subspecies of Klebsiella pneumoniae. This pan-genomic analysis, helped to develop a new tool for defining species introducing a quantic perspective for taxonomy. Reviewers This article was reviewed by William Martin, Pierre Pontarotti and Pere Puigbo (nominated by Dr Yuri Wolf).
Full-text available
The GenBank database currently contains sequence data for 33 N4-like viruses, with only one, Escherichia phage N4, being formally recognized by the ICTV. The genus N4likevirus is uniquely characterized by that fact that its members possess an extremely large, virion-associated RNA polymerase. Using a variety of proteomic, genomic and phylogenetic tools, we have demonstrated that the N4-like phages are not monophyletic and that N4 is actually a genomic orphan. We propose to create four new genera: "G7cvirus" (consisting of phages G7C, IME11, KBNP21, vB_EcoP_PhAPEC5, vB_EcoP_PhAPEC7, Bp4, EC1-UPM and pSb-1), "Lit1virus" (LIT1, PA26 and vB_PaeP_C2-10_Ab09), "Sp58virus" (SP058 and SP076), and "Dss3virus" (DSS3φ2 and EE36φ1). We propose that coliphage N4, the members of "G7cvirus", Erwinia phage Ea9-2, and Achromobacter phage JWAlpha should be considered members of the same subfamily, which we tentatively call the "Enquartavirinae".
Full-text available
Spounavirinae viruses have received an increasing interest as tools for the control of harmful bacteria due to their relatively broad host range and strictly virulent phenotype. In this study, we collected and analyzed the complete genome sequences of 61 published phages, either ICTV-classified or candidate members of the Spounavirinae subfamily of the Myoviridae. A set of comparative analyses identified a distinct, recently proposed Bastille-like phage group within the Spounavirinae. More importantly, type 1 thymidylate synthase (TS1) and dihydrofolate reductase (DHFR) genes were shown to be unique for the members of the proposed Bastille-like phage group, and are suitable as molecular markers. We also show that the members of this group encode beta-lactamase and/or sporulation-related SpoIIIE homologs, possibly questioning their suitability as biocontrol agents. We confirm the creation of a new genus-the "Bastille-like group"-in Spounavirinae, and propose that the presence of TS1- and DHFR-encoding genes could serve as signatures for the new Bastille-like group. In addition, the presence of metallo-beta-lactamase and/or SpoIIIE homologs in all members of Bastille-like group phages makes questionable their suitability for use in biocontrol.
Full-text available
Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 (NC_013649), F19 (NC_023567) and NTUH-K2044-K1-1 (NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called "Kp34likevirus" after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.
Full-text available
Microbial taxonomy should provide adequate descriptions of bacterial, archaeal, and eukaryotic microbial diversity in ecological, clinical, and industrial environments. Its cornerstone, the prokaryote species has been re-evaluated twice. It is time to revisit polyphasic taxonomy, its principles, and its practice, including its underlying pragmatic species concept. Ultimately, we will be able to realize an old dream of our predecessor taxonomists and build a genomic-based microbial taxonomy, using standardized and automated curation of high-quality complete genome sequences as the new gold standard.
Full-text available
Unlabelled: Prokaryotic taxonomy is the underpinning of microbiology, as it provides a framework for the proper identification and naming of organisms. The "gold standard" of bacterial species delineation is the overall genome similarity determined by DNA-DNA hybridization (DDH), a technically rigorous yet sometimes variable method that may produce inconsistent results. Improvements in next-generation sequencing have resulted in an upsurge of bacterial genome sequences and bioinformatic tools that compare genomic data, such as average nucleotide identity (ANI), correlation of tetranucleotide frequencies, and the genome-to-genome distance calculator, or in silico DDH (isDDH). Here, we evaluate ANI and isDDH in combination with phylogenetic studies using Aeromonas, a taxonomically challenging genus with many described species and several strains that were reassigned to different species as a test case. We generated improved, high-quality draft genome sequences for 33 Aeromonas strains and combined them with 23 publicly available genomes. ANI and isDDH distances were determined and compared to phylogenies from multilocus sequence analysis of housekeeping genes, ribosomal proteins, and expanded core genes. The expanded core phylogenetic analysis suggested relationships between distant Aeromonas clades that were inconsistent with studies using fewer genes. ANI values of ≥ 96% and isDDH values of ≥ 70% consistently grouped genomes originating from strains of the same species together. Our study confirmed known misidentifications, validated the recent revisions in the nomenclature, and revealed that a number of genomes deposited in GenBank are misnamed. In addition, two strains were identified that may represent novel Aeromonas species. Importance: Improvements in DNA sequencing technologies have resulted in the ability to generate large numbers of high-quality draft genomes and led to a dramatic increase in the number of publically available genomes. This has allowed researchers to characterize microorganisms using genome data. Advantages of genome sequence-based classification include data and computing programs that can be readily shared, facilitating the standardization of taxonomic methodology and resolving conflicting identifications by providing greater uniformity in an overall analysis. Using Aeromonas as a test case, we compared and validated different approaches. Based on our analyses, we recommend cutoff values for distance measures for identifying species. Accurate species classification is critical not only to obviate the perpetuation of errors in public databases but also to ensure the validity of inferences made on the relationships among species within a genus and proper identification in clinical and veterinary diagnostic laboratories.