RESEARCH ARTICLE
A Global Survey of Hypervirulent Aeromonas hydrophila (vAh)
Identified vAh Strains in the Lower Mekong River Basin and
Diverse Opportunistic Pathogens from Farmed Fish and Other
Environmental Sources
Tingbi Xu,a Cody R. Rasmussen-Ivey,b Francesco S. Moen,a Ana Fernández-Bravo,c Brigitte Lamy,d,e Roxana Beaz-Hidalgo,c
Chan Dara Khan,f Graciela Castro Escarpulli,g Ina Salwany M. Yasin,h Maria J. Figueras,c Mohamad Azzam-Sayuti,i
Muhammad Manjurul Karim,j K. M. Mazharul Alam,j Thao Thu Thi Le,k Ngo Huynh Phuong Thao,k Samuel Addo,l
Samuel Duodu,m Shahzad Ali,n Tooba Latif,n Sothea Mey,f Thay Somony,f Mark R. Lilesa
aDepartment of Biological Sciences, Auburn University, Alabama, USA
bDepartment of Biology, Tufts University, Medford, Massachusetts, USA
cUnit of Microbiology, Department of Basic Health Sciences, Faculty of Medicine and Health Sciences, IISPV, University Rovira i Virgili, Reus, Spain
dINSERM U1065, Laboratoire de Bactériologie, CHU Nice, Faculté de Médecine, Université Côte d’Azur, Nice, France
eCentre for Molecular Bacteriology and Infection, Imperial College of London, London, United Kingdom
fAquatic Animal Health and Disease Management Office, Department of Aquaculture Development, Fisheries Administration, Ministry of Agriculture
Forestry and Fisheries, Phnom Penh, Cambodia
gLaboratorio de Investigación Clínica y Ambiental, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Ciudad de México, Mexico
hDepartment of Aquaculture, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
iInstitute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
jDepartment of Microbiology, University of Dhaka, Dhaka, Bangladesh
kDivision of Aquacultural Biotechnology, Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Vietnam
lDepartment of Marine and Fisheries Sciences, University of Ghana, Legon, Ghana
mDepartment of Biochemistry, Cell, and Molecular Biology, University of Ghana, Legon, Ghana
nWildlife Epidemiology and Molecular Microbiology Laboratory, Department of Wildlife and Ecology, University of Veterinary and Animal Sciences,
Lahore, Pattoki, Pakistan
ABSTRACT Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent
of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of
farmed carp in China and catfish in the United States, which has caused millions of tons of
lost fish. We conducted a global survey to better understand the evolution, geographical
distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed
clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-
inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were
included in this study, including additional strains obtained from previously published
culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila
isolates, 48 of which were vAh. This collection also included five new vAh isolates from
diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthal- Editor Luke R. Iwanowicz, USGS, Eastern
mus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated Ecological Science Center
from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic Copyright © 2023 Xu et al. This is an open-
transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine access article distributed under the terms of
the Creative Commons Attribution 4.0
genes required for myo-inositol utilization revealed the close affiliation of vAh strains International license.
regardless of geographic origin and suggested lateral genetic transfer of this catabolic Address correspondence to Mark R. Liles,
pathway from an Enterobacter species. Prediction of virulence factors was conducted to lilesma@auburn.edu.
determine differences between vAh and non-vAh strains in terms of virulence and secretion The authors declare no conflict of interest.
Received 12 September 2022
systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease Accepted 5 February 2023
isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh Published 23 February 2023
and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates.
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Global Survey of Hypervirulent Aeromonas hydrophila Microbiology Spectrum
IMPORTANCE This global survey of vAh brought together scientists that study fish disease
to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other
Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four
new vAh isolates were isolated from the lower Mekong River basin in Cambodia and
Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for
improved biosecurity to prevent vAh spread.
KEYWORDS Aeromonas hydrophila, pathogen, freshwater fish, pandemic, comparative
genomics, worldwide
A eromonas species are ubiquitous in aquatic habitats and can be found in both freshand brackish water (1). Aeromonas hydrophila is one of the most well-known patho-
genic species within the genus Aeromonas and is known for its high tolerance to extremes
of temperature, pH, and salinity that enable it to flourish in a variety of environments and
to be an opportunistic pathogen in a diverse range of hosts, including fish, amphibians, birds,
reptiles, and mammals (2–6). Typically, A. hydrophila causes motile Aeromonas septicemia
(MAS) in fish that are infected with other primary pathogens, such as Flavobacterium col-
umnare, or are under stress due to harsh environmental conditions and/or high-density
farming (7, 8). MAS is associated with high mortality within a short time, and infected fish
generally show a variety of symptoms, such as hemorrhaging and lesions on the fish sur-
face (9). A. hydrophila disease isolates are known to have significant antigenic diversity,
with more than 40 O-antigen serotypes observed (10, 11).
The epidemic outbreaks among farmed fish due to hypervirulent A. hydrophila (vAh)
have been notable for their rapid emergence and high mortality (12, 13). The first isolated
strain of this deadly A. hydrophila pathotype, J-1, was obtained in Jiangsu province, China,
from an epidemic outbreak of MAS that resulted in high mortality in cultured carp and bream
in 1989 (14). Outbreaks of MAS caused by vAh were reported again in Jiangsu and in
Guangdong and Fujian provinces in 2010 (15), resulting in about 2,200 tons of fish losses
per year in China (16, 17). The isolated vAh strains J-1, NJ-35, and ZC1 were all categorized
as sequence type 251 (ST251), and were found to be clonal based on genome sequence
analyses (12).
The first report of a vAh isolate in the United States was strain S04-690, which was isolated
from channel catfish (Ictalurus punctatus) in Mississippi in 2004 (4). The first major MAS out-
break in the United States was in catfish production ponds in western Alabama in 2009, from
which vAh strain ML09-119 was isolated. The MAS outbreaks due to vAh in Alabama have
continued, causing the loss of more than 5,000 tons of farmed channel catfish each year, and
the current accumulative loss of farmed channel catfish due to vAh in the state of Alabama is
estimated to be over 40 million pounds (Anita Kelly, unpublished data) (18). Due to the lack of
effective control, vAh is a consistent threat to U.S. and Chinese aquaculture, and many more
countries could be affected. To date, there have not been any vAh global surveillance efforts.
Previous studies of vAh isolated from carp in China or from catfish in the United States
indicate that these strains share a recent common ancestor and have common features,
such as the ability to utilizemyo-inositol as the sole carbon source (19). Based on phyloge-
netic analysis of vAh-specific gene sequences, vAh isolates from carp are at the root of the
vAh tree, suggesting that the emergence of vAh in the United States was due to importa-
tion of live carp species or fish products from Asia (4). Beginning in the 1960s, silver carp
(Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) were introduced
into U.S. catfish ponds to control algal blooms. Massive flooding of the Mississippi River in
1993 resulted in the release of Asian carp into the Mississippi River basin, where these inva-
sive carp species have continued to spread and are a major threat to the Great Lakes ecosys-
tem (20). The importation of bighead carp into the United States is now prohibited based
on the Asian Carp Prevention and Control Act signed into law in 2010. The global value of
trade in exporting live carp was estimated at $164 million in 2020, with China being the
world leader in live carp export at $103 million per year (21). The lack of sufficient biosecurity
measures to prevent the spread of vAh-infected fish prompted the need for a global survey
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Global Survey of Hypervirulent Aeromonas hydrophila Microbiology Spectrum
to assess vAh dissemination among various farmed fish species in different regions of the
world.
Despite the evidence that vAh strains are clonal and have recently spread from Asia
to the United States, there are some genetic differences among vAh strains. In particular,
while vAh isolates from carp species in China typically have a complete type VI secretion
system (T6SS) (12, 22), most vAh isolates from channel catfish in the United States, and
especially from western Alabama, lack a complete T6SS and only carry hcp1, tssH, and vgrG1
(23). The carp vAh isolate NJ-35, which has a complete T6SS, has been found to express a
phospholipase that contributes to biofilm formation and virulence in zebrafish (Danio rerio)
(24). While lacking many T6SS-associated genes, the presence of hcp1 and vgrG1 have been
found to contribute to vAh ML09-119 virulence (23), but the degree to which the T6SS plays
a role in fish host specificity and virulence has yet to be defined. The evolution of vAh strains
as they infect and replicate in different fish species is of significant interest. Our lack of
knowledge regarding fish host range and geographic distribution also prompted us to con-
duct a vAh global survey. As a group of fish disease experts from around the world, we pri-
marily sampled freshwater fish with disease symptoms characteristic of MAS and obtained
pure bacterial cultures that were evaluated for growth on myo-inositol, a phenotype that
has been consistent in vAh strains isolated from China and the United States. A phylogenetic
analysis ofmyo-inositol-utilizing strains using gyrB sequences was conducted to further char-
acterize disease isolates. Finally, for representative vAh and non-vAh strains, we conducted
comparative genome analyses to provide further information on the phylogeny and pre-
dicted virulence factors of vAh strains. This study is a first step toward a better understand-
ing of vAh worldwide distribution, uniting fish disease researchers in a network that can
help track the distribution of vAh and developing methods to protect farmed fish against
this emerging pathogen.
RESULTS AND DISCUSSION
Identification ofmyo-inositol-utilizing Aeromonas sp. strains. This global vAh survey
relied upon an extensive network of microbiologists willing to participate in screening
fish disease isolates and cryopreserved collections for the presence of myo-inositol utilizing
A. hydrophila strains. There have been no previous reports of A. hydrophila strains with the
ability to use myo-inositol as the sole carbon source other than vAh strains (i.e., ST251).
Therefore, by screening bacterial isolates for growth on myo-inositol in a minimal medium,
our goal was to rapidly and cost-effectively identify putative vAh strains from diverse locales.
From this extensive survey, 43 myo-inositol-utilizing Aeromonas sp. strains were isolated from
Pabda (Ompok pabda) from Bangladesh, Pangas catfish from Cambodia, lake water from
Finland, Koi (Cyprinus rubrofuscus) from France, basa fish (Pangasius bocourti) from Malaysia,
rainbow trout (Oncorhynchus mykiss) from Mexico, crab (Brachyura spp.) from Norway, trout
(Oncorhynchus spp.) and human feces from Spain, and striped catfish from Vietnam (Table 1).
Typical vAh strains cultured on tryptic soy agar (TSA) produce smooth, rounded, opaque col-
onies that have a light yellow color with a 2- to 3-mm diameter range after 24 h of incuba-
tion (25). The strains that showed a colony morphology consistent with vAh and evident
growth on myo-inositol (i.e., increase in the optical density at 600 nm [OD600] of .0.4 over
48 h) were further validated by molecular phylogenetic analyses (26, 27).
The 43 myo-inositol-utilizing Aeromonas strains collected worldwide were subjected
to vAh-specific and/or gyrB-targeted PCR using the primer sets listed in Table 2. A phylogenetic
analysis was conducted using gyrB sequences from these isolates in addition to Aeromonas
sp. type strains and previously described vAh strains from China and the United States
(Fig. 1). The phylogeny of these strains revealed a great diversity of Aeromonas spp. that
were obtained in this survey, including A. bestiarum, A. bivalvium, A. caviae, A. dhakensis,
A. finlandensis, A. media, A. salmonicida, A. sobria, and A. veronii. Interestingly, some of
these Aeromonas spp. had not been previously shown to have the ability to use myo-
inositol as a carbon source, including A. bestiarum, A. bivalvium, A. caviae, A. dhakensis,
A. media, and A. veronii (26, 28–30). While these Aeromonas species were not the target of
this survey, this adds to our knowledge of the use of myo-inositol among diverse
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TABLE 1 Bacterial isolates used in this study
Country of GenBank species Species based on Reference
Strain ID isolation Pathotype Isolation source assignation phylogeny and ANI Accession ID or source
AL09-71 USA vAh Channel catfish A. hydrophila A. hydrophila NZ_CP007566.1 58
AL09-79 USA vAh Channel catfish A. hydrophila A. hydrophila NZ_LRRV00000000.1 47
ALG15-098 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05223361 12
IPRS15-28 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05223362 12
J-1 China (P.R.C.) vAh Crucian carp A. hydrophila A. hydrophila NZ_CP006883.1 16
JBN2301 China (P.R.C.) vAh Crucian carp A. hydrophila A. hydrophila NZ_CP013178.1 59
ML09-119 USA vAh Channel catfish A. hydrophila A. hydrophila NC_021290.1 60
ML09-121 USA vAh Channel catfish A. hydrophila A. hydrophila NZ_LRRX00000000.1 47
ML09-122 USA vAh Channel catfish A. hydrophila A. hydrophila NZ_LRRY00000000.1 47
ML10-51K USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05223363 12
NJ-35 China (P.R.C.) vAh Crucian carp A. hydrophila A. hydrophila NZ_CP006870.1 16
GYK1 China (P.R.C.) vAh Mandarin fish A. hydrophila A. hydrophila NZ_CP016392.1 61
D4 China (P.R.C.) vAh Blunt-snout bream A. hydrophila A. hydrophila NZ_CP013965.1 62
PB10-118 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN01085622 47
pc104A USA vAh Channel catfish A. hydrophila A. hydrophila NZ_CP007576.1 58
S04-690 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN02404466 4
S13-612 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05292362 12
S13-700 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05292363 12
S14-296 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05292365 12
S14-452 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05256776 12
S14-458 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05223364 12
S14-606 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05292366 12
S15-130 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05223365 12
S15-400 USA vAh Channel catfish A. hydrophila A. hydrophila SAMN05223367 12
ZC1 China (P.R.C.) vAh Grass carp A. hydrophila A. hydrophila SAMN02404465 4
AL10-121 USA vAh Channel catfish A. hydrophila A. hydrophila NZ_LRRW00000000.1 63
AL09-80 USA vAh Channel catfish A. hydrophila A. hydrophila JX275838 27
G3 China (P.R.C.) vAh Mandarin fish A. hydrophila A. hydrophila KX822741.1 64
AH11P USA vAh Catfish A. hydrophila A. hydrophila KC133524.1 65
IB102 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085433.1 66
JG102 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085448.1 66
JG103 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085449.1 66
JG101 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JN177329.1 66
DLNG201 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085458.1 66
XX-52 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JX025794.1 67
XX-22 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JX025792.1 67
4LNG202 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085443.1 66
4LNS301 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JN177325.1 66
4LNG102 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085441.1 66
PW06 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JN177338.1 66
DBHS101 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085454.1 66
2JBN302 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085474.1 66
2JBN103 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085472.1 66
DLNG102 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085457.1 66
2JBN102 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085471.1 66
2JFN201 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085476.1 66
DLNG202 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085459.1 66
LNB103 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085451.1 66
PW14 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085452.1 66
DBHS102 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JQ085455.1 66
XX-58 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JX025795.1 67
AL09-77 USA vAh Channel catfish A. hydrophila A. hydrophila JX275844.1 27
XX-14 China (P.R.C.) vAh Carp A. hydrophila A. hydrophila JX025791.1 67
AL09-138 USA vAh Channel catfish A. hydrophila A. hydrophila JX275841.1 27
AL10-13 USA vAh Channel catfish A. hydrophila A. hydrophila JX275833.1 27
ML09-139 USA vAh Channel catfish A. hydrophila A. hydrophila JX275834.1 27
AL09-74 USA vAh Channel catfish A. hydrophila A. hydrophila KF913679.1 4
CPF2-S1 Cambodia vAh Pangas catfish A. hydrophila A. hydrophila JANLOJ000000000 This study
DT-TKT-2020-677 Vietnam vAh Striped catfish A. hydrophila A. hydrophila OP198653 This study
DT-TKT-2020-680 Vietnam vAh Striped catfish A. hydrophila A. hydrophila OP198652 This study
DT-TKT-2020-681 Vietnam vAh Striped catfish A. hydrophila A. hydrophila OP198651 This study
DT-TTD-2020-734 Vietnam vAh Striped catfish A. hydrophila A. hydrophila NZ_JALRNI010000001.1 This study
VL-2013-869 Vietnam non-vAh Striped catfish A. hydrophila A. hydrophila NZ_JALRNJ000000000.1 This study
BT-2012-871 Vietnam non-vAh Striped catfish A. hydrophila A. hydrophila NZ_JALRNL000000000.1 This study
VL-2012-870 Vietnam non-vAh Striped catfish A. hydrophila A. hydrophila NZ_JALRNK000000000.1 This study
Ae34 Japan non-vAh Koi carp A. hydrophila A. hydrophila NZ_BAXY00000000.1 68
AD9 USA non-vAh Alga A. hydrophila A. hydrophila NZ_JFJO00000000.1 69
ATCC 7966 USA non-vAh Milk A. hydrophila A. hydrophila CP000462 70
ESV-357 Mexico non-vAh Rainbow trout A. hydrophila A. hydrophila KJ743520.1 71
ESV-371 Mexico non-vAh Rainbow trout A. hydrophila A. hydrophila KJ743529.1 71
ESV-381 Mexico non-vAh Rainbow trout A. hydrophila A. hydrophila KJ743537.1 71
ESV-394 Mexico non-vAh Rainbow trout A. hydrophila A. hydrophila KJ743549.1 71
ESV-399 Mexico non-vAh Rainbow trout A. hydrophila A. hydrophila KJ743514.1 71
0.14 Spain non-vAh Oscar A. hydrophila A. sobria JANLFC000000000 This study
14 Malaysia non-vAh Clinical A. hydrophila A. dhakensis NZ_AOBM00000000.1 72
D69555 Spain non-vAh Human feces A. hydrophila A. hydrophila OP198650 This study
2006 4153 Spain non-vAh Human hemoculture A. hydrophila A. hydrophila OP198649 This study
(Continued on next page)
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TABLE 1 (Continued)
Country of GenBank species Species based on Reference
Strain ID isolation Pathotype Isolation source assignation phylogeny and ANI Accession ID or source
AE210 Finland non-vAh FW lake water A. hydrophila A. hydrophila JN711784.1 73
AH10 China (P.R.C.) non-vAh Grass carp A. hydrophila A. hydrophila NZ_CP011100.1 74
TN97-08 USA non-vAh Bluegill A. hydrophila A. hydrophila NZ_LNUR00000000.1 47
AHNIH1 USA non-vAh Human tissue A. hydrophila A. hydrophila NZ_CP016380.1 75
MN98-04 USA non-vAh Tilapia A. hydrophila A. hydrophila SAMN04967900 47
AL97-91 USA non-vAh Channel catfish A. hydrophila A. hydrophila SAMN04967787 47
AL06-06 USA non-vAh Goldfish A. hydrophila A. hydrophila NZ_CP010947.1 23
AL10-121 USA non-vAh Channel catfish A. hydrophila A. hydrophila NZ_LRRW00000000.1 47
116 Malaysia non-vAh Clinical A. hydrophila A. dhakensis NZ_ANPN00000000.1 72
173 Malaysia non-vAh Clinical A. hydrophila A. dhakensis NZ_AOBN00000000.1 72
187 Malaysia non-vAh Clinical A. hydrophila A. dhakensis NZ_AOBO00000000.1 72
226 Malaysia non-vAh Clinical A. hydrophila A. hydrophila NZ_JEML00000000.1 72
259 Malaysia non-vAh Clinical A. hydrophila A. dhakensis NZ_AOBP00000000.1 72
277 Malaysia non-vAh Clinical A. hydrophila A. dhakensis NZ_AOBQ00000000.1 72
RB-AH Canada non-vAh Soil A. hydrophila A. hydrophila NZ_JPEH00000000.1 76
CIP 107985 Thailand non-vAh Frog A. hydrophila subsp. ranae A. hydrophila NZ_CDDC00000000.1 77
YL17 Malaysia non-vAh Compost A. hydrophila A. dhakensis NZ_CP007518.2 78
SSU USA non-vAh Human A. hydrophila A. dhakensis NZ_AGWR00000000.1 79
665N Spain non-vAh Seafood A. bivalvium A. bivalvium DQ504430 80
ESV-353 Mexico non-vAh Rainbow trout A. bestiarum A. bestiarum KJ743516.1 71
ESV-364 Mexico non-vAh Rainbow trout A. bestiarum A. bestiarum KJ743524.1 71
ESV-367 Mexico non-vAh Rainbow trout A. bestiarum A. bestiarum KJ743526.1 71
0.2 Spain non-vAh Oscar A. caviae A. caviae OP198648 This study
1P11S3 Malaysia non-vAh Basa fish A. dhakensis A. dhakensis NZ_JADPIC000000000 81
KOR1 China (P.R.C.) non-vAh Mangrove A. dhakensis A. dhakensis NZ_LJOE00000000.1 82
P1S3 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP198647 This study
P2L2 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP198646 This study
P3I3 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP222574 This study
P3L1 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP222573 This study
P3L2 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP222572 This study
P3L3 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP222571 This study
P3S1 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP222570 This study
P3S3 Bangladesh non-vAh Pabda A. dhakensis A. dhakensis OP222569 This study
HE40 Finland non-vAh FW lake water A. finlandensis A. finlandensis HG970924.1 83
4287D Finland non-vAh FW lake water A. finlandensis A. finlandensis NZ_JRGK00000000.1 29
4AK4 China non-vAh Carp A. hydrophila A. media NZ_CP006579.1 84
ESV-360 Mexico non-vAh Rainbow trout A. media A. media KJ743508.1 71
ESV-383 Mexico non-vAh Rainbow trout A. media A. media KJ743513.1 71
R100 Spain non-vAh Trout A. media A. hydrophila KP400944.1 85
AH31 Norway non-vAh Crab A. media A. media KP400946.1 86
BWH65 USA non-vAh Perch A. caviae A. media NZ_LESK00000000.1 12
0890 France non-vAh Koi carp A. media A. media OP222568 This study
18900 USA non-vAh Canadian perch A. salmonicida A. salmonicida JANLFD000000000 This study
ESV-355 Mexico non-vAh Rainbow trout A. sobria A. sobria KJ743518.1 71
ESV-396 Mexico non-vAh Rainbow trout A. salmonicida A. salmonicida KJ743550.1 71
ESV-400 Mexico non-vAh Rainbow trout A. veronii A. veronii KJ743553.1 71
0.15 Spain non-vAh Oscar A. veronii A. veronii OP222567 This study
D47366 Spain non-vAh Human feces A. veronii A. veronii OP222566 This study
ESV-393 Mexico non-vAh Rainbow trout A. veronii A. sobria KJ743548.1 71
ESV-397 Mexico non-vAh Rainbow trout A. veronii A. veronii KJ743551.1 71
EN3600 China (P.R.C.) non-vAh Human tissue E. cloacae E. cloacae NZ_CP035633.1 87
GGT036 Korea non-vAh Soil E. cloacae E. cloacae NZ_CP009756.1 88
M12X01451 USA non-vAh Human tissue E. cloacae E. cloacae NZ_CP017475.1 89
B1 Ghana non-vAh Tilapia Plesiomonas shigelloides P. shigelloides OP222552 This study
D1 Ghana non-vAh Tilapia Enterobacter spp. Enterobacter spp. OP222553 This study
E1 Ghana non-vAh Tilapia P. shigelloides P. shigelloides OP222554 This study
G1 Ghana non-vAh Tilapia P. shigelloides P. shigelloides OP222555 This study
B2 Ghana non-vAh Tilapia A. veronii A. veronii OP222556 This study
F2 Ghana non-vAh Tilapia P. shigelloides P. shigelloides OP222557 This study
8 Pakistan non-vAh Rohu P. aeruginosa P. aeruginosa OP222558 This study
21 Pakistan non-vAh Rohu P. aeruginosa P. aeruginosa OP222559 This study
37 Pakistan non-vAh Gulfam P. aeruginosa P. aeruginosa OP222560 This study
38 Pakistan non-vAh Rohu P. aeruginosa P. aeruginosa OP222561 This study
53A Pakistan non-vAh Rainbow trout Serratia liquefaciens S. liquefaciens OP222562 This study
62 Pakistan non-vAh Silver carp P. aeruginosa P. aeruginosa OP222563 This study
63 Pakistan non-vAh Rohu Eneterobacter cancerogenus E. cancerogenus OP222564 This study
PB USA non-vAh Rainbow trout A. sobria A. sobria OP222565 This study
Aeromonas species. Additionally, it suggests that this ability may contribute to the per-
sistence of these bacteria in aquatic habitats and the virulence of these opportunistic
pathogens in diverse warm-water fish species.
Based on the gyrB phylogeny, A. hydrophila strains isolated from Spain, Mexico, Finland,
Cambodia, and Vietnam grouped together and formed well-supported clades. Furthermore,
the gyrB phylogeny indicated that all previously described vAh strains (i.e., ST251) grouped to-
gether within a monophyletic clade with bootstrap support, clearly distinct from other myo-
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TABLE 2 Primer sets for PCR to amplify gyrB or vAh-specific genetic locia
Primer set Direction Sequence Amplicon size (bp)
2986F Forward 59-CTATTACTGCCCCCTCGTTC-39 167
2986R Reverse 59-ATTGAGCGGTATGCTGTCG-39
vAh-SerF Forward 59-AG9CATCACCAGCGTTGGCCC-39 502
vAh-SerR Reverse 59-GCCGGGCTGAACTTCCGCAT-39
gyrB3F Forward 59-TCCGGCGGTCTGCACGGCGT-39 680
gyrB9R Reverse 59-ACCTTGACGGAGATAACGGC-39
gyrB7F Forward 59-GGGGTCTACTGCTTCACCAA-39 680
gyrB14R Reverse 59-TTGTCCGGGTTGTACTCGTC-39
aPrimer sets 2986F/R and vAh-SerF/R were used for vAh identification, and primer sets gyrB3F/9R and 7F/14R
were used for gyrB amplification.
inositol utilizing Aeromonas sp. strains (Fig. 1). Interestingly, the vAh clade included strains
from Cambodia (CPF2-S1) and Vietnam (DT-TKT-2020-677, DT-TKT-2020-680, DT-TKT-
2020-681, and DT-TTD-2020-734). The Cambodian vAh strain CPF2-S1 was one of five
myo-inositol utilizing bacterial isolates that were positive for vAh-specific PCR and isolated
from Pangas catfish in the Mekong River basin. The four vAh isolates from Vietnam were
all obtained from diseased striped catfish in the Mekong River delta, and three of
them (DT-TKT-2020-677, DT-TKT-2020-680, DT-TKT-2020-681) are closely related to a
recently reported vAh strain, DT-TTD-2020-734, obtained from striped catfish in the
Mekong River delta (31). These newly described vAh strains indicate that additional fish
species are susceptible to vAh and that the Mekong River basin is an active region of vAh
disease transmission.
Inositol catabolism phylogeny. The evolutionary history of the inositol catabolism
pathway among myo-inositol-utilizing Aeromonas spp. was inferred based on the amino
acid sequences of IolA, IolC, IolD, IolE, IolG, InoE, InoF, and InoL, which were obtained from
representative vAh and other Aeromonas sp. genomes (Fig. S1A to H). The variability in inositol
gene content among these strains precluded a concatenated phylogenetic analysis. Among
vAh strains, there were no differences observed in the evolutionary history of gene products
required for myo-inositol transport (InoE, InoF, and InoL) or catabolism (IolA, IolC, IolD, IolE,
and IolG), with all vAh strains present in the same monophyletic clade and having strong
bootstrap support (Fig. S1A to H). This is consistent with the observation that vAh strains are
clonal, including the newly isolated vAh strains from Cambodia (CPF2-S1) and Vietnam (e.g.
DT-TTD-2020-734). Another consistent observation was that the inositol-related gene products
from vAh strains share a close relationship with orthologous sequences from Enterobacter cloa-
cae, which has been hypothesized to be the origin of the inositol catabolism pathway present
in vAh strains (27). In contrast, the sequences obtained from other Aeromonas species were
distantly related to those from vAh strains and E. cloacae, including A. dhakensis 1P11S3,
A. dhakensis P3I3, A. dhakensis P3L3, A. media R100, A. sobria ESV-355, and A. sobria ESV-393.
The evolutionary history of inositol utilization among Aeromonas sp. therefore appears to be
complex, with horizontal gene transfer of inositol transport and catabolism postulated to
play an important role. This survey revealed a large diversity of other Aeromonas species
that can utilize myo-inositol. Future research should explore the role ofmyo-inositol utilization
in the persistence and virulence of opportunistic Aeromonas sp. pathogens.
The role of myo-inositol utilization in vAh persistence and virulence should also be
further explored. Channel catfish have been shown to synthesize myo-inositol in brain,
kidney, and liver tissues, and soy-based fish feed containing a high concentration of
phytic acid (inositol hexaphosphate) (32, 33). The inositol derived from fish tissues and
dietary sources may provide both a carbon source and an environmental signal that
induces expression of vAh virulence factors. The transcriptional regulator IolR is responsible
for the regulation of iol genes as well as other virulence factors in bacterial pathogens, such
as Salmonella enterica (34, 35). IolR has also been found to regulate autoaggregation and
biofilm formation in the vAh strain NJ-35 (36). Furthermore, the presence of myo-ino-
sitol that accumulates in sediment from fish feed may help vAh to persist within the
environment.
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FIG 1 Phylogeny of Aeromonas species isolates based on the gyrB gene. The evolutionary relationships of vAh and other
Aeromonas sp. isolates were inferred using the maximum likelihood method based on gyrB gene sequences. A total of 1,000
iterations were performed for determination of bootstrap support, with bootstrap values indicated by the size of the circle at
each supported node.
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FIG 2 Pairwise comparison of average nucleotide identity (ANI) of vAh, non-vAh, and myo-inositol-utilizing Aeromonas sp. isolates. Genome sequences of
vAh, non-vAh, and myo-inositol-utilizing Aeromonas sp. isolates were pairwise compared using JSpeciesWS. ANI values of .95% indicate that two strains belong to
the same species.
Average nucleotide identity (ANI). The pairwise ANI comparisons for 63 Aeromonas
sp. genomes, including representative vAh strains from China, Cambodia, the United States,
and Vietnam showed high ANI values (.99%) for all vAh strains (Fig. 2), which was consist-
ent with the previous core genome-based phylogeny indicating the clonality of all known
vAh strains (12). In contrast, only a few non-vAh A. hydrophila strains showed high ANI values
compared with vAh strains, and most ANI values ranged from 96% to 97%. The exceptions to
this were strains that had been putatively indicated as A. media, A. sobria, and A. veronii based
on phylogenetic analyses, all of which had discrepancies between the species affiliation indi-
cated by ANI values and their species affiliation indicated in GenBank as previously described
(37). Based on these ANI data and a core genome-based phylogeny, the phylogenetic affilia-
tions of several strains were revised (see below and Table 1). This survey also included diverse
Aeromonas sp. isolated from diseased fish and other environments, as revealed by the
A. hydrophila-Aeromonas sp. pairwise ANI comparisons that ranged from 67% to 93%.
Aeromonas core genome phylogenetic analysis. The phylogenetic relationships
among the representative vAh and diverse Aeromonas sp. strains included in this survey
were inferred based on a set of core genome sequences totaling 3.8 Mb (Fig. 3). A subset
of vAh strains was included in the core genome phylogeny due to some of the strains
lacking high-quality genome sequences (e.g., Vietnamese vAh strains DT-TKT-2020-681
DT-TKT-2020-677, DT-TKT-2020-680). Consistent with the gyrB phylogeny, the Aeromonas
core genome phylogeny indicated that all vAh strains, including the newly identified
strains from Cambodia and Vietnam, form a monophyletic clade with strong bootstrap
support that is distinct from other A. hydrophila or other Aeromonas sp. strains (Fig. 3). While
the clonal vAh clade showed little variation among its members for the core genome phy-
logeny, there was significant intraspecies genetic variability observed among the other
Aeromonas sp. Strains, including within A. hydrophila, A. dhakensis, A. media, and A. sobria.
Based on this core genome phylogeny (and ANI values), there were many bacterial isolates
described as A. hydrophila that were affiliated with A. dhakensis, A. media, or A. sobria, and
these revised phylogenetic affiliations have been indicated (Table 1). In this analysis, the
exclusion of small fragments was set to 10 kbp because these fragments were found to be
flanked by highly repetitive sequences, which were previously demonstrated to contribute
less to the production of core genomes. This removal was chosen as a blanket approach
to increase computational efficiency and decrease the noise generated from repetitive
sequences, as this study is solely based on sequence-based comparisons. However, with the
growing body of knowledge that shows repetitive regions as significant in regulation, future
studies should focus on these noncoding regions.
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FIG 3 Phylogeny of Aeromonas sp. isolates based on the core genome sequences. The evolutionary relationships of vAh and other Aeromonas sp. isolates were
inferred using the maximum likelihood method, based on core genome sequences. A total of 1,000 bootstrap replications were conducted, and bootstrap values are
represented by the size of the circle for each supported node.
Virulence factors encoded in Aeromonas sp. genomes. Representative vAh and
non-vAh genomes were evaluated for their encoded potential to secrete virulence factors
(Fig. 4). In agreement with previous studies, vAh strains were universally found to encode
complete type 2 secretion systems, which have been found to be essential to the virulence
of a vAh strain isolated from a channel catfish in the United States (38). In contrast, type 3
secretion systems were only identified in non-vAh strains. Interestingly, the type 6 secre-
tion systems (T6SS) were complete only in a subset of vAh strains as has been previously
described (23). Most of the vAh isolates from China, with the one exception of strain GYK1,
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FIG 4 Predicted virulence factors for vAh and other Aeromonas sp. strains. Aeromonas genomes were annotated using RAST and submitted to MacSyFinder
for secretion system analysis. Maximum independent E value and minimal profile coverage were set as the default, while the maximum E value was set as
1.0. Virulence factors include type 1 secretion system (T1SS), type 2 secretion system (T2SS), type 4 pili (T4P), tight adherence system (TAD), type 3 secretion system
(T3SS), flagellum, a phylogenetic subtype of type 6 secretion system (T6SSi), and type 9 secretion system (T9SS).
were predicted to possess the complete T6SS, which has been shown to contribute to bio-
film formation and virulence in fish (24). The two new vAh isolates from Cambodia and
Vietnam (CPF2-S1, DT-TTD-2020-734) possessed the entire T6SS, which further demonstrates
their close relationship to vAh strains isolated from carp in China. In contrast, many of the
vAh strains isolated from channel catfish in the United States lacked a complete T6SS, with
the notable exception of S14-452 and other strains isolated from the Mississippi delta (23).
VAh core genome phylogenetic analysis. The phylogenetic relationships among
the representative vAh strains included in this survey were inferred based on a set of core ge-
nome sequences present in all sequenced vAh strains (Fig. 5). The vAh core genome phylogeny
FIG 5 Phylogeny of vAh isolates based on the core genome sequences. The evolutionary relationships of vAh isolates were inferred using the maximum
likelihood method, based on core genome sequences. A total of 1,000 bootstrap replications were conducted, and bootstrap values are represented by the
size of the circle for each supported node.
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indicated that the newly identified strains obtained from diseased fish in Cambodia and
Vietnam form a monophyletic clade with strong bootstrap support with vAh strains isolated
from crucian carp (Carassius carassius) and mandarin fish (Siniperca chuatsi) in China.
Moreover, these vAh isolates from Cambodia and Vietnam share a close relationship,
indicating that they originated from a common ancestor. In contrast, two other strains isolated
from carp in China, ZC1 and JBN2301, form a well-supported clade with vAh strains isolated
from catfish in the United States (4).
The successful isolation of vAh from farmed Pangas catfish in Cambodia and from
farmed striped catfish in Vietnam broadens the knowledge of the geographical distribution
of vAh and the fish species in which this emerging pathogen can cause disease. Due to the
rapid growth of the live fish trade in Asia and beyond, this pathogen could be transmitted
to more countries and infect more fish species without sufficient biosafety (39). This calls for
future development of rapid and inexpensive diagnostic assays to identify vAh strains and
aid in biosecurity precautions to prevent further dissemination of this virulent pathogen.
MATERIALS ANDMETHODS
Bacterial isolates. Fish that demonstrated the typical symptoms of MAS, especially with external
hemorrhaging and in farms experiencing high fish mortality, were collected for diagnosis and autopsy at the local
institution. Aeromonas sp. isolates were recovered from diseased fish from aquaculture ponds in Bangladesh,
Cambodia, Finland, France, Ghana, Malaysia, Mexico, Norway, Pakistan, Spain, Thailand, and Vietnam (Table 1).
The fish species sampled were tilapia (Oreochromis niloticus), striped catfish, pabda, Pangas catfish, basa catfish,
rainbow trout, carp (Cyprinidae spp.), and perch (Perca spp.), while in some cases isolates were obtained from
other environmental samples such as lake water, crab, seafood, and human feces (Table 1). Organs with the high-
est concentration of vAh, including liver, spleen, and kidney, were used to inoculate tryptic soy agar (TSA) plates
(Beckton Dickinson, New Jersey, USA) or other bacteriologic growth medium appropriate for A. hydrophila cultiva-
tion, and these cultures were incubated at 30°C for 24 to 48 h. The vAh strain ML09-119 served as a control for
comparison.
Single colonies that showed A. hydrophila morphology were cultured on TSA (30°C, 24 h) to obtain
isolated colonies. Three colonies of each strain were cultured separately in 2 mL of M9 broth medium
supplemented with 5.5 mMmyo-inositol as previously described (27). The vAh strain ML09-119 and the non-vAh
strain AL06-06 served as positive and negative controls, respectively. Cultures were grown at 30°C for 48 h to re-
cord their growth as measured by the optical density at 600 nm (OD600). The utilization of myo-inositol of an
unknown isolate was monitored by turbidity and CFU counts (as previously described). An increase in turbidity
(change in OD600 of.0.4) was observed formyo-inositol-utilizing strains over 48 h. Pure cultures ofmyo-inositol-
utilizing strains were subsequently identified as vAh by phylogenetic analysis of gyrB sequences following previ-
ously described methods (12, 40). Validated vAh strains were cryopreserved in tryptic soy broth (TSB) containing
20% glycerol at280°C.
Phylogenetic analysis based on gyrB from myo-inositol-utilizing strains. Genomic DNA of the
myo-inositol-utilizing isolates was isolated using the E.Z.N.A. bacterial DNA isolation kit according to the
manufacturer’s protocol (Omega Bio-Tek, Norcross, GA, USA). Bacterial DNA was quantified with a NanoDrop
instrument (Thermo Fisher Scientific, Waltham, MA, USA) and used as a template for PCR amplify gyrB gene
sequences using Aeromonas genus-level primer sets (Table 2) (41). To avoid the potential off-target priming
and increase PCR specificity (42), touchdown PCR was conducted to generate gyrB products and performed on
a Mastercycler Nexus thermo cycler (Eppendorf, Hamburg, Germany) with 50 ng of genomic DNA (gDNA) iso-
lated from each strain, 25 mL of EconoTaq Plus green 2X master mix (Lucigen Corp., Middleton, WI, USA), and
0.5 mL of 20 mM reverse and forward primers. The thermal cycling parameters were 94°C for 3 min, 10 cycles
of 94°C for 30 sec, 68°C for 30 sec (21°C per cycle), and 72°C for 1 min, and then 25 cycles of 94°C for 30 sec,
58°C for 30 sec, and 72°C for 1 min and a final extension at 72°C for 5 min.
The gyrB gene amplicons were Sanger sequenced as described previously (27, 40) and assembled
into consensus sequences using CLC Genomics Workbench (Qiagen, Inc., Aarhus, Denmark). The gyrB sequence
reads were trimmed for quality, assembled into consensus sequences, and aligned with an existing gyrB
sequence database obtained from Chinese and U.S. vAh and non-vAh strains (12), using ClustalW in MEGA X
(43). The gyrB sequence database included sequences varying from 422 to 1,068 bp and included Aeromonas
sp. type strains to confirm species affiliations. Phylogenetic relationships of the inositol-utilizing Aeromonas sp.
strains and appropriate type strains were determined by the construction of a phylogenetic tree using MEGA X
(43). In total, 100 strains were included in the tree, including 5 new vAh strains and 38 new non-vAh strains.
Among the 66 A. hydrophila strains, some were removed due to poor sequence quality and/or the lack of an
available viable culture from which to recover a better-quality gyrB sequence. The evolutionary history of the
strains in the gyrB database was inferred using the maximum likelihood (ML) method (44). The ML analysis was
conducted with 1,000 iterations for bootstrap support, with bootstrap values shown on each branch of the
gyrB tree as a circle proportional to bootstrap support. The gyrB tree was annotated and visualized using iTOL
v6 (45). The gyrB tree was rooted using the A. sobria type strain.
Genome sequencing based on the gyrB phylogeny. Representative isolates of different inositol-
utilizing Aeromonas lineages were selected for Illumina sequencing based on the results of the gyrB phylo-
genetic tree. The sequenced strains were selected to represent vAh and inositol-utilizing Aeromonas from
multiple geographical locations, including Spain, Mexico, Cambodia, Vietnam, Bangladesh, and Malaysia. The
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fragment libraries were constructed using a Nextera XT DNA library prep kit (Illumina, San Diego, CA, USA)
based on the manufacturer’s protocol, followed by paired-end sequencing conducted on an Illumina MiSeq
platform (46). Sequence reads were imported into CLC Genomics Workbench, which was used to trim
sequences for quality, followed by de novo assembly using default settings. Draft genome contig sequences
were generated for strains CPF2-S1 (Cambodia), 14 (Malaysia), 1P11S3 (Malaysia), ESV-393 (Mexico), ESV-399
(Mexico), R100 (Spain), VL-2013-869 (Vietnam), BT-2012-871 (Vietnam), VL-2012-870 (Vietnam), and DT-TTD-
2020-734 (Vietnam). For subsequent phylogenomic analyses and prediction of virulence factors, a database
was constructed that also included the existing sequences of vAh strains isolated from carp species in China
and from catfish in the United States, along with other Aeromonas sp. strains isolated in China, the United
States, Malaysia, and Japan.
Inositol catabolism phylogeny. A genomic database was generated that included draft genome
sequences of strains isolated in Mexico, Spain, Cambodia, Bangladesh, Malaysia, and Vietnam that were
supplemented with genome sequences of vAh strains from China and the United States, as well as from
Enterobacter spp. which are predicted to be the origin of the inositol catabolism pathway present in vAh
strains (22, 47). A total of 15 open reading frames (ORFs) were identified in the myo-inositol catabolism
pathway in vAh strains, from which we selected proteins shown to be required for inositol utilization
(IolA, IolC, IolD, IolE, and IolG) for phylogenetic analysis (48). The proteins predicted to be involved in
myo-inositol transport, InoE, InoF, and InoL, were also included in the phylogenetic analyses (49). Given
the unique evolutionary histories associated with these proteins involved in inositol catabolism, a sepa-
rate phylogenetic analysis was conducted for each of these amino acid sequences. An ML tree with
1,000 iterations for bootstrap support was conducted on MEGA X as described above and visualized
using ITOL v6.
Average nucleotide identity. To evaluate the overall genetic similarity between vAh, non-vAh, and
myo-inositol-utilizing Aeromonas spp., the ANI values of the 63 Aeromonas genomes were compared using
JSpeciesWS (50) and visualized with Daniel’s XL Toolbox v7.3.4 (51). According to the criteria for taxonomic affilia-
tion of new genomes, an ANI value of.95% indicated that two strains belong to the same species (52, 53).
Aeromonas core genome analysis. Both noncoding and coding sequences of vAh and non-vAh
strains, including A. sobria, A. media, A. dhakensis, and A. salmonicida strains, were used for a core genome phy-
logenetic analysis. In general, small fragments do not influence the overall quality of core genomes due to
these small genomic regions being flanked by highly repetitive sequences. Removing small fragments helped
to improve overall accuracy by decreasing the noise generated from repetitive sequences; therefore, any con-
tigs less than 10 kb were filtered from Aeromonas genomes by limiting them from mapping to multiple
regions. FASTA files of the filtered sequences were submitted to Mugsy v1.2.3, a multiple whole genome align-
ment tool, using default parameters (54). The alignment was processed by GBLOCK v0.91b for the identifica-
tion of highly conserved regions across all Aeromonas spp. strains as previously described (55). The parameters
used in GBLOCK for retention were dependent upon the input alignment as previously described (12). Briefly,
a maximum of 8 contiguous regions, a minimum of 30 sequences for conserved regions, and 51 sequences for
flanked positions were used for the gapped positions within a block. A ML tree based on the final alignment
was generated using MEGAX with default parameters for the 60 Aeromonas spp. strains, including 28 vAh
strains. The ML tree was further visualized using iTOL v6.
VAh core genome phylogenetic analysis. To assess the phylogeny of vAh strains isolated from
Cambodia, China, the US, and Vietnam, a vAh core genome was created using both coding and noncoding
sequences of representative vAh. Contigs less than 10 kb in size were removed to increase computational effi-
ciency, and filtered data were submitted as FASTA files to the multiple whole-genome alignment tool Mugsy
v1.2.3 (54), under default parameters. The resulting alignment was subsequently processed with GBLOCK
v0.91b (55) in order to identify regions of high conservation across all isolates. Parameters for retention by
GBLOCK are dictated by the input alignment and were the following: a minimum of 31 and 51 sequences for
conserved and flanked positions, respectively, a maximum of 8 contiguous, but nonconserved positions, a min-
imum block length of 10, and one-half of the sequences allowed to possess gapped positions within a block.
From the final alignment, a maximum likelihood (ML) phylogeny for the vAh isolates was inferred using
RAxML v8.2.8 (56) under the general time reversible model of evolution with estimated proportions of invaria-
ble sites and rate variation among sites (i.e., GTR1 I1 G) and 1,000 bootstraps to determine branch supports,
as described previously (22). Trees were visualized using iTOL v6.
Virulence factor prediction. Virulence factor prediction and identification of secretion systems fol-
lowed previously described methods (23, 57). Briefly, the secretion systems of Aeromonas strains were
identified with the program MacSyFinder. The data set option was set as “unordered” to evaluate the draft
genome of each strain. The minimal profile coverage was set to 0.5, the maximum E value was set to 1.0,
and the maximum independent E value was 0.001. Secretion systems of vAh, non-vAh, and myo-inositol-
utilizing Aeromonas strains were identified and indicated with mandatory and accessory genes, and the
corresponding copy numbers were determined.
Data availability. The sequences were deposited in the GenBank database under accession no. OP198646
to OP198653 for the gyrB sequences, and the genome sequences were deposited as JANLOJ000000000.1,
NZ_JALRNI010000001.1, NZ_JALRNJ000000000.1, NZ_JALRNL000000000.1, NZ_JALRNK000000000.1,
JANLFC000000000.1, and JANLFD000000000.1 (Table 1).
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 2.1 MB.
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Global Survey of Hypervirulent Aeromonas hydrophila Microbiology Spectrum
ACKNOWLEDGMENTS
This work has been supported by the Vietnam National Foundation for Science and
Technology Development (NAFOSTED) under grant number 106.05.2019.340 and by Auburn
University for funding the professional improvement leave for Mark R. Liles.
REFERENCES
1. Isonhood JH, Drake M. 2002. Aeromonas species in foods. J Food Prot 65: 20. Changnon S. 2019. The great flood of 1993: causes, impacts, and responses.
575–582. https://doi.org/10.4315/0362-028x-65.3.575. Routledge, New York, NY.
2. Palumbo S, Williams A, Buchanan R, Phillips J. 1987. Thermal resistance of 21. Simoes AJG, Hidalgo CA. The economic complexity observatory: an ana-
Aeromonas hydrophila. J Food Prot 50:761–764. https://doi.org/10.4315/ lytical tool for understanding the dynamics of economic development. In
0362-028X-50.9.761. Workshops at the twenty-fifth AAAI conference on artificial intelligence,
3. Huys G, Pearson M, Kämpfer P, Denys R, Cnockaert M, Inglis V, Swings J. San Francisco, CA.
2003. Aeromonas hydrophila subsp. ranae subsp. nov., isolated from sep- 22. Wang N, Liu J, Pang M, Wu Y, Awan F, Liles MR, Lu C, Liu Y. 2018. Diverse
ticaemic farmed frogs in Thailand. Int J Syst Evol Microbiol 53:885–891. https:// roles of Hcp family proteins in the environmental fitness and pathogenicity of
doi.org/10.1099/ijs.0.02357-0. Aeromonas hydrophila Chinese epidemic strain NJ-35. Appl Microbiol Biotech-
4. Hossain MJ, Sun D, McGarey DJ, Wrenn S, Alexander LM, Martino ME, Xing nol 102:7083–7095. https://doi.org/10.1007/s00253-018-9116-0.
Y, Terhune JS, Liles MR. 2014. An Asian origin of virulent Aeromonas hydro- 23. Tekedar HC, Abdelhamed H, Kumru S, Blom J, Karsi A, Lawrence ML. 2018.
phila responsible for disease epidemics in United States-farmed catfish. mBio Comparative genomics of Aeromonas hydrophila secretion systems and
5:e00848-14. https://doi.org/10.1128/mBio.00848-14. mutational analysis of hcp1 and vgrG1 genes from T6SS. Front Microbiol
5. Liu J, Xie L, Zhao D, Yang T, Hu Y, Sun Z, Yu X. 2019. A fatal diarrhoea out- 9:3216. https://doi.org/10.3389/fmicb.2018.03216.
break in farm-raised Deinagkistrodon acutus in China is newly linked to 24. Ma S, Dong Y, Wang N, Liu J, Lu C, Liu Y. 2020. Identification of a new effector-
potentially zoonotic Aeromonas hydrophila. Transbound Emerg Dis 66:287–298. immunity pair of Aeromonas hydrophila type VI secretion system. Vet Res 51:
https://doi.org/10.1111/tbed.13020. 71. https://doi.org/10.1186/s13567-020-00794-w.
6. Guerra IM, Fadanelli R, Figueiró M, Schreiner F, Delamare APL, Wollheim C, 25. Al-Fatlawy HNK, Al-Hadrawy HA. 2014. Isolation and characterization of A.
Costa SOP, Echeverrigaray S. 2007. Aeromonas associated diarrhoeal disease hydrophila from the Al-Jadryia River in Baghdad (Iraq). Am J Educ Res 2:
in south Brazil: prevalence, virulence factors and antimicrobial resistance. Braz 658–662. https://doi.org/10.12691/education-2-8-14.
J Microbiol 38:638–643. https://doi.org/10.1590/S1517-83822007000400011. 26. Azzam-Sayuti M, Ina-Salwany MY, Zamri-Saad M, Annas S, Yusof MT, Monir MS,
7. Grizzle JM, Kiryu Y. 1993. Histopathology of gill, liver, and pancreas, and Mohamad A, Muhamad-Sofie MHN, Lee JY, Chin YK, Amir-Danial Z, Asyiqin A,
serum enzyme levels of channel catfish infected with Aeromonas hydro- Lukman B, Liles MR, Amal MNA. 2021. Comparative pathogenicity of Aeromo-
phila complex. J Aquat Anim Health 5:36–50. https://doi.org/10.1577/ nas spp. in cultured red hybrid tilapia (Oreochromis niloticus
 O. mossambi-
1548-8667(1993)005%3C0036:HOGLAP%3E2.3.CO;2. cus). Biology 10:1192. https://doi.org/10.3390/biology10111192.
8. El-Son MA, Nofal MI, Abdel-Latif HM. 2021. Co-infection of Aeromonas 27. Hossain MJ, Waldbieser GC, Sun D, Capps NK, Hemstreet WB, Carlisle K,
hydrophila and Vibrio parahaemolyticus isolated from diseased farmed Griffin MJ, Khoo L, Goodwin AE, Sonstegard TS, Schroeder S, Hayden K,
striped mullet (Mugil cephalus) in Manzala, Egypt: a case report. Aquacul- Newton JC, Terhune JS, Liles MR. 2013. Implication of lateral genetic
ture 530:735738. https://doi.org/10.1016/j.aquaculture.2020.735738. transfer in the emergence of Aeromonas hydrophila isolates of epidemic
9. Plumb JA, Hanson LA. 2010. Health maintenance and principal microbial
outbreaks in channel catfish. PLoS One 8:e80943. https://doi.org/10.1371/
diseases of cultured fishes. John Wiley & Sons, New York, NY.
journal.pone.0080943.
10. Sakazaki R, Shimada T. 1984. O-serogrouping scheme for mesophilic
28. von Graevenitz A, Bucher C. 1983. Evaluation of differential and selective
Aeromonas strains. Jpn J Med Sci Biol 37:247–255. https://doi.org/10.7883/
media for isolation of Aeromonas and Plesiomonas spp. from human feces. J
yoken1952.37.247.
11. Wise AL, LaFrentz BR, Kelly AM, Khoo LH, Xu T, Liles MR, Bruce TJ. 2021. A Clin Microbiol 17:16–21. https://doi.org/10.1128/jcm.17.1.16-21.1983.
review of bacterial co-infections in farmed cat sh: components, diagnos- 29. Beaz-Hidalgo R, Latif-Eugenín F, Hossain M, Berg K, Niemi R, Rapala J, Lyrafi
tics, and treatment directions. Animals 11:3240. https://doi.org/10.3390/ C, Liles M, Figueras M. 2015. Aeromonas aquatica sp. nov., Aeromonas fin-
ani11113240. landiensis sp. nov. and Aeromonas lacus sp. nov. isolated from Finnish
12. Rasmussen-Ivey CR, Hossain MJ, Odom SE, Terhune JS, Hemstreet WG, waters associated with cyanobacterial blooms. Syst Appl Microbiol 38:
Shoemaker CA, Zhang D, Xu D-H, Grif n MJ, Liu Y-J, Figueras MJ, Santos SR, 161–168. https://doi.org/10.1016/j.syapm.2015.02.005.fi
Newton JC, Liles MR. 2016. Classi cation of a hypervirulent Aeromonas hydro- 30. Dallaire-Dufresne S, Barbeau X, Sarty D, Tanaka KH, Denoncourt AM, Lagüefi
phila pathotype responsible for epidemic outbreaks in warm-water shes. P, Reith ME, Charette SJ. 2013. Aeromonas salmonicida Ati2 is an effector pro-fi
Front Microbiol 7:1615. https://doi.org/10.3389/fmicb.2016.01615. tein of the type three secretion system. Microbiology (Reading) 159:1937–1945.
13. Rasmussen-Ivey CR, Figueras MJ, McGarey D, Liles MR. 2016. Virulence https://doi.org/10.1099/mic.0.067959-0.
factors of Aeromonas hydrophila: in the wake of reclassi cation. Front 31. Ngo TP, Vu HT, Le TT, Bui HC, Liles MR, Rodkhum C. 2022. Comparative ge-fi
Microbiol 7:1337. https://doi.org/10.3389/fmicb.2016.01337. nomic analysis of hypervirulent Aeromonas hydrophila strains from striped
14. Huaiqing C, Chengping L. 1991. Study on the pathogen of epidemic septi- catfish (Pangasianodon hypophthalmus) in Vietnam. Aquaculture 558:738364.
cemia occurred in cultured cyprinoid fishes in southeastern China. Bio- https://doi.org/10.1016/j.aquaculture.2022.738364.
divers Sci 6:31–36. 32. Burtle G, Lovell R. 1989. Lack of response of channel catfish (Ictalurus puncta-
15. Deng G, Jiang X, Ye X, Liu M, Xu S, Liu L, Bai Y, Luo X. 2009. Isolation, iden- tus) to dietary myo-inositol. Can J Fish Aquat Sci 46:218–222. https://doi.org/
tification and characterization of Aeromonas hydrophila from hemor- 10.1139/f89-030.
rhagic grass carp. Microbiol China 36:1170–1177. 33. Jackson LS, Li MH, Robinson EH. 1996. Use of microbial phytase in channel
16. Pang M, Jiang J, Xie X, Wu Y, Dong Y, Kwok AHY, Zhang W, Yao H, Lu C, catfish Ictalurus punctatus diets to improve utilization of phytate phos-
Leung FC, Liu Y. 2015. Novel insights into the pathogenicity of epidemic phorus 1. J World Aquacult Soc 27:309–313. https://doi.org/10.1111/j
Aeromonas hydrophila ST251 clones from comparative genomics. Sci .1749-7345.1996.tb00613.x.
Rep 5:9833. https://doi.org/10.1038/srep09833. 34. Zhang D, Pridgeon JW, Klesius PH. 2013. Expression and activity of recombi-
17. Zhang Y, Arakawa E, Leung K. 2002. Novel Aeromonas hydrophila PPD134/ nant proaerolysin derived fromAeromonas hydrophila cultured from diseased
91 genes involved in O-antigen and capsule biosynthesis. Infect Immun 70: channel catfish. Vet Microbiol 165:478–482. https://doi.org/10.1016/j.vetmic
2326–2335. https://doi.org/10.1128/IAI.70.5.2326-2335.2002. .2013.04.023.
18. Hemstreet B. 2010. An update on Aeromonas hydrophila from a fish health 35. Cordero-Alba M, Bernal-Bayard J, Ramos-Morales F. 2012. SrfJ, a Salmo-
specialist for summer 2010. Catfish J 24:4. nella type III secretion system effector regulated by PhoP, RcsB, and IolR. J
19. Hanson L, Liles M, Hossain M, Griffin M, Hemstreet W. 2014. Motile aero- Bacteriol 194:4226–4236. https://doi.org/10.1128/JB.00173-12.
monas septicemia. In Fish health section blue book. American Fisheries 36. Dong Y, Li S, Zhao D, Liu J, Ma S, Geng J, Lu C, Liu Y. 2020. IolR, a negative
Society, Bethesda, MD. regulator of themyo-inositol metabolic pathway, inhibits cell autoaggregation
March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03705-22 13
Downloaded from https://journals.asm.org/journal/spectrum on 01 June 2023 by 197.255.68.131.
Global Survey of Hypervirulent Aeromonas hydrophila Microbiology Spectrum
and biofilm formation by downregulating RpmA in Aeromonas hydrophila. 58. Pridgeon JW, Zhang D, Zhang L. 2014. Complete genome sequence of a
NPJ Biofilms Microbiomes 6:1–12. https://doi.org/10.1038/s41522-020-0132-3. moderately virulent Aeromonas hydrophila strain, pc104A, isolated from
37. Beaz-Hidalgo R, Hossain MJ, Liles MR, Figueras M-J. 2015. Strategies to soil of a catfish pond in West Alabama. Genome Announc 2:e00554-14. https://
avoid wrongly labelled genomes using as example the detected wrong doi.org/10.1128/genomeA.00554-14.
taxonomic affiliation for Aeromonas genomes in the GenBank database. 59. Yang W, Li N, Li M, Zhang D, An G. 2016. Complete genome sequence of fish
PLoS One 10:e0115813. https://doi.org/10.1371/journal.pone.0115813. pathogen Aeromonas hydrophila JBN2301. Genome Announc 4:e01615-15.
38. Barger PC, Liles MR, Newton JC. 2020. Type II secretion is essential for viru- https://doi.org/10.1128/genomeA.01615-15.
lence of the emerging fish pathogen, hypervirulent Aeromonas hydro- 60. Liles M, Hemstreet W, Waldbieser G, Griffin M, Khoo L, Bebak J, Garcia J,
phila. Front Vet Sci 7:574113. https://doi.org/10.3389/fvets.2020.574113. Goodwin A, Capps N, Hayden K. 2011. Comparative genomics of Aeromonas
39. Kobayashi M, Msangi S, Batka M, Vannuccini S, Dey MM, Anderson JL. hydrophila isolates from an epidemic in channel catfish. Poster 1489. American
2015. Fish to 2030: the role and opportunity for aquaculture. Aquacult Econ Society for MicrobiologyMeeting.
Manage 19:282–300. https://doi.org/10.1080/13657305.2015.994240. 61. Pan H, Wu S, Dong C, Shi C, Ye M, Lin T, Huang Z. 2004. Identification, viru-
40. Griffin MJ, Goodwin AE, Merry GE, Liles MR, Williams MA, Ware C, Waldbieser lence, hemolytic activity of GYK1, a strain of pathogenic Aeromonas
GC. 2013. Rapid quantitative detection of Aeromonas hydrophila strains asso- hydrophila isolated frommandarinfish. J Shanghai Fish Univ 13:e29.
ciated with disease outbreaks in catfish aquaculture. J Vet Diagn Invest 25: 62. Zhu L, Zheng J-S, Wang W-M, Luo Y. 2019. Complete genome sequence
473–481. https://doi.org/10.1177/1040638713494210. of highly virulent Aeromonas hydrophila strain D4, isolated from a dis-
41. Yanez M, Catalán V, Apraiz D, Figueras M, Martinez-Murcia A. 2003. Phylo- eased blunt-snout bream in China. Microbiol Resour Announc 8:e01035-
genetic analysis of members of the genus Aeromonas based on gyrB gene 18. https://doi.org/10.1128/MRA.01035-18.
sequences. Int J Syst Evol Microbiol 53:875–883. https://doi.org/10.1099/ijs.0 63. Tekedar HC, Kumru S, Karsi A, Waldbieser GC, Sonstegard T, Schroeder SG,
.02443-0. Liles MR, Griffin MJ, Lawrence ML. 2016. Draft genome sequences of four viru-
42. Green MR, Sambrook J. 2018. Touchdown polymerase chain reaction lent Aeromonas hydrophila strains from catfish aquaculture. GenomeAnnounce-
(PCR). Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot095133. ments 4:e00860-16. https://doi.org/10.1128/genomeA.00860-16.
43. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: molecular ev- 64. Zhang Y, Gao X, Ye J, Chen N, Zhang X, Bing X. 2017. Molecular characteri-
olutionary genetics analysis across computing platforms. Mol Biol Evol zation and establishment of LAMP detection method of pathogenic Aero-
35:1547–1549. https://doi.org/10.1093/molbev/msy096. monas hydrophila isolated from Siniperca chuatsi. Acta Hydrobiologica
44. Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. Sinica 41:1225–1231.
New algorithms and methods to estimate maximum-likelihood phylogenies: 65. Pridgeon J, Yildirim-Aksoy M, Klesius P, Kojima K, Mobley J, Srivastava K,
assessing the performance of PhyML 3.0. Syst Biol 59:307–321. https://doi.org/ Reddy P. 2013. Identification of gyrB and rpoB gene mutations and differ-
10.1093/sysbio/syq010. entially expressed proteins between a novobiocin-resistant Aeromonas
45. Letunic I, Bork P. 2021. Interactive Tree of Life (iTOL) v5: an online tool for hydrophila catfish vaccine strain and its virulent parent strain. Vet Micro-
phylogenetic tree display and annotation. Nucleic Acids Res 49:W293–W296. biol 166:624–630. https://doi.org/10.1016/j.vetmic.2013.07.025.
https://doi.org/10.1093/nar/gkab301. 66. Zhang X, Yang W, Li T, Li A. 2013. The genetic diversity and virulence char-
46. Sato MP, Ogura Y, Nakamura K, Nishida R, Gotoh Y, Hayashi M, Hisatsune acteristics of Aeromonas hydrophila isolated from fishponds with disease
J, Sugai M, Takehiko I, Hayashi T. 2019. Comparison of the sequencing outbreaks in Hubei province. Acta Hydrobiologica Sinica 37:458–466.
67. Hu M, Wang N, Pan Z, Lu C, Liu Y. 2012. Identity and virulence properties
bias of currently available library preparation kits for Illumina sequencing
of Aeromonas isolates from diseased fish, healthy controls and water
of bacterial genomes and metagenomes. DNA Res 26:391–398. https://
environment in China. Lett Appl Microbiol 55:224–233. https://doi.org/10
doi.org/10.1093/dnares/dsz017.
.1111/j.1472-765X.2012.03281.x.
47. Hossain M. 2012. Molecular Interactions between phage and the catfish
68. Jagoda SDS, Tan E, Arulkanthan A, Kinoshita S, Watabe S, Asakawa S.
pathogen Edwardsiella ictaluri and comparative genomics of epidemic strains
2014. Draft genome sequence of Aeromonas hydrophila strain Ae34, iso-
of Aeromonas hydrophila. PhD thesis. Auburn University, Auburn, AL. lated from a septicemic and moribund koi carp (Cyprinus carpio koi), a
48. Yoshida K, Yamaguchi M, Morinaga T, Kinehara M, Ikeuchi M, Ashida H, freshwater aquarium fish. Genome Announcements 2:e00572-14. https://
Fujita Y. 2008. myo-Inositol catabolism in Bacillus subtilis. J Biol Chem doi.org/10.1128/genomeA.00572-14.
283:10415–10424. https://doi.org/10.1074/jbc.M708043200. 69. Lenneman EM, Barney BM. 2014. Draft genome sequences of the alga-
49. Rodionova IA, Leyn SA, Burkart MD, Boucher N, Noll KM, Osterman AL, degrading bacteria Aeromonas hydrophila strain AD9 and Pseudomonas
Rodionov DA. 2013. Novel inositol catabolic pathway in Thermotogamaritima. pseudoalcaligenes strain AD6. Genome Announcements 2:e00709-14.
EnvironMicrobiol 15:2254–2266. https://doi.org/10.1111/1462-2920.12096. https://doi.org/10.1128/genomeA.00709-14.
50. Richter M, Rosselló-Móra R, Oliver Glöckner F, Peplies J. 2016. JSpeciesWS: 70. Seshadri R, Joseph SW, Chopra AK, Sha J, Shaw J, Graf J, Haft D, Wu M,
a web server for prokaryotic species circumscription based on pairwise Ren Q, Rosovitz MJ, Madupu R, Tallon L, Kim M, Jin S, Vuong H, Stine OC,
genome comparison. Bioinformatics 32:929–931. https://doi.org/10.1093/ Ali A, Horneman AJ, Heidelberg JF. 2006. Genome sequence of Aeromo-
bioinformatics/btv681. nas hydrophila ATCC 7966T: jack of all trades. J Bacteriol 188:8272–8282.
51. Kraus D. 2014. Consolidated data analysis and presentation using an https://doi.org/10.1128/JB.00621-06.
open-source add-in for the Microsoft Excel spreadsheet software. Med 71. Vega-Sánchez V, Acosta-Dibarrat J, Vega-Castillo F, Castro-Escarpulli G,
Writing 23:25–28. https://doi.org/10.1179/2047480613Z.000000000181. Aguilera-Arreola MG, Soriano-Vargas E. 2014. Phenotypical characteristics,
52. Figueras MJ, Beaz-Hidalgo R, Hossain MJ, Liles MR. 2014. Taxonomic affili- genetic identification, and antimicrobial sensitivity of Aeromonas species iso-
ation of new genomes should be verified using average nucleotide iden- lated from farmed rainbow trout (Onchorynchus mykiss) in Mexico. Acta Trop
tity and multilocus phylogenetic analysis. Genome Announcements 2: 130:76–79. https://doi.org/10.1016/j.actatropica.2013.10.021.
e00927-14. https://doi.org/10.1128/genomeA.00927-14. 72. Chan K-G, Puthucheary SD, Chan X-Y, YinW-F, Wong C-S, TooW-SS, Chua K-H.
53. Konstantinidis KT, Tiedje JM. 2005. Genomic insights that advance the 2011. Quorum sensing in Aeromonas species isolated from patients in Malay-
species definition for prokaryotes. Proc Natl Acad Sci U S A 102: sia. Curr Microbiol 62:167–172. https://doi.org/10.1007/s00284-010-9689-z.
2567–2572. https://doi.org/10.1073/pnas.0409727102. 73. Berg KA, Lyra C, Sivonen K, Paulin L, Suomalainen S, Tuomi P, Rapala J. 2009.
54. Angiuoli SV, Salzberg SL. 2011. Mugsy: fast multiple alignment of closely High diversity of cultivable heterotrophic bacteria in association with cyanobac-
related whole genomes. Bioinformatics 27:334–342. https://doi.org/10 terial water blooms. ISME J 3:314–325. https://doi.org/10.1038/ismej.2008.110.
.1093/bioinformatics/btq665. 74. Xu L, Wang H, Yang X, Lu L. 2013. Integrated pharmacokinetics/pharma-
55. Castresana J. 2000. Selection of conserved blocks from multiple align- codynamics parameters-based dosing guidelines of enrofloxacin in grass
ments for their use in phylogenetic analysis. Mol Biol Evol 17:540–552. carp Ctenopharyngodon idella to minimize selection of drug resistance.
https://doi.org/10.1093/oxfordjournals.molbev.a026334. BMC Veterinary Res 9:126. https://doi.org/10.1186/1746-6148-9-126.
56. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis and 75. Hughes HY, Conlan SP, Lau AF, Dekker JP, Michelin AV, Youn J-H, Henderson
post-analysis of large phylogenies. Bioinformatics 30:1312–1313. https:// DK, Frank KM, Segre JA, Palmore TN. 2016. Detection and whole-genome
doi.org/10.1093/bioinformatics/btu033. sequencing of carbapenemase-producing Aeromonas hydrophila isolates
57. Abby SS, Néron B, Ménager H, Touchon M, Rocha EP. 2014. MacSyFinder: from routine perirectal surveillance culture. J Clin Microbiol 54:1167–1170.
a program to mine genomes for molecular systems with an application to https://doi.org/10.1128/JCM.03229-15.
CRISPR-Cas systems. PLoS One 9:e110726. https://doi.org/10.1371/journal 76. Emond-Rheault J-G, Vincent AT, Trudel MV, Brochu F, Boyle B, Tanaka KH,
.pone.0110726. Attéré SA, Jubinville E
, Loch TP, Winters AD, Faisal M, Frenette M, Derome N,
March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03705-22 14
Downloaded from https://journals.asm.org/journal/spectrum on 01 June 2023 by 197.255.68.131.
Global Survey of Hypervirulent Aeromonas hydrophila Microbiology Spectrum
Charette SJ. 2015. Variants of a genomic island in Aeromonas salmonicida 83. Berg A, Rødseth OM, Hansen T. 2007. Fish size at vaccination influence
subsp. salmonicida link isolates with their geographical origins. Vet Microbiol the development of side-effects in Atlantic salmon (Salmo salar L.). Aqua-
175:68–76. https://doi.org/10.1016/j.vetmic.2014.11.014. culture 265:9–15. https://doi.org/10.1016/j.aquaculture.2007.02.014.
77. Colston SM, Fullmer MS, Beka L, Lamy B, Gogarten JP, Graf J. 2014. Bioin- 84. Gao X, Jian J, Li W-J, Yang Y-C, Shen X-W, Sun Z-R, Wu Q, Chen G-Q. 2013.
formatic genome comparisons for taxonomic and phylogenetic assign- Genomic study of polyhydroxyalkanoates producing Aeromonas hydro-
ments using Aeromonas as a test case. mBio 5:e02136-14. https://doi.org/ phila 4AK4. Appl Microbiol Biotechnol 97:9099–9109. https://doi.org/10
10.1128/mBio.02136-14. .1007/s00253-013-5189-y.
78. Lim Y-L, Roberts RJ, Ee R, Yin W-F, Chan K-G. 2016. Complete genome 85. Talagrand-Reboul E, Roger F, Kimper J-L, Colston SM, Graf J, Latif-Eugenín
sequence and methylome analysis of Aeromonas hydrophila strain YL17, F, Figueras MJ, Petit F, Marchandin H, Jumas-Bilak E, Lamy B. 2017. Delinea-
isolated from a compost pile. Genome Announcements 4:e00060-16. https:// tion of taxonomic species within complex of species: Aeromonas media and
doi.org/10.1128/genomeA.00060-16. related species as a test case. Front Microbiol 8:621. https://doi.org/10.3389/
79. Erova TE, Sha J, Horneman AJ, Borchardt MA, Khajanchi BK, Fadl AA, fmicb.2017.00621.
Chopra AK. 2007. Identification of a new hemolysin from diarrheal isolate 86. Granum PE, O’Sullivan K, Tomás JM, Ørmen Ø. 1998. Possible virulence factors
SSU of Aeromonas hydrophila. FEMS Microbiol Lett 275:301–311. https://
of Aeromonas spp. from food and water. FEMS Immunol Med Microbiol 21:
doi.org/10.1111/j.1574-6968.2007.00895.x.
80. Minana-Galbis D, Farfan M, Fusté MC, Lorén JG. 2007. Aeromonas bival- 131–137. https://doi.org/10.1111/j.1574-695X.1998.tb01158.x.
vium sp. nov., isolated from bivalve molluscs. Int J Syst Evol Microbiol 57: 87. Yuan S, Wu G, Zheng B. 2019. Complete genome sequence of an IMP-8,
582–587. https://doi.org/10.1099/ijs.0.64497-0. CTX-M-14, CTX-M-3 and QnrS1 co-producing Enterobacter asburiae iso-
81. Azzam-Sayuti M, Ina-Salwany MY, Zamri-Saad M, Annas S, Liles MR, Xu T, late from a patient with wound infection. J Glob Antimicrob Resist 18:
Amal MNA, Yusof MT. 2022. Draft genome sequence of myo-inositol uti- 52–54. https://doi.org/10.1016/j.jgar.2019.05.029.
lizing Aeromonas dhakensis 1P11S3 isolated from striped catfish (Panga- 88. Gong G, Um Y, Park TH, Woo HM. 2015. Complete genome sequence of
sianodon hypopthalmus) in a local fish farm in Malaysia. Data in Brief 41: Enterobacter cloacae GGT036: A furfural tolerant soil bacterium. J Biotechnol
107974. https://doi.org/10.1016/j.dib.2022.107974. 193:43–44. https://doi.org/10.1016/j.jbiotec.2014.11.012.
82. Yin M, Ma Z, Cai Z, Lin G, Zhou J. 2015. Genome sequence analysis reveals 89. Carter MQ, Pham A, Huynh S, He X. 2017. Complete genome sequence
evidence of quorum-sensing genes present in Aeromonas hydrophila strain of a Shiga toxin-producing Enterobacter cloacae clinical isolate. Ge-
KOR1, isolated from a mangrove plant (Kandelia obovata). Genome Announce- nome Announcements 5:e00883-17. https://doi.org/10.1128/genomeA
ments 3:e01461-15. https://doi.org/10.1128/genomeA.01461-15. .00883-17.
March/April 2023 Volume 11 Issue 2 10.1128/spectrum.03705-22 15
Downloaded from https://journals.asm.org/journal/spectrum on 01 June 2023 by 197.255.68.131.