ORIGINAL RESEARCH
published: 17 July 2020
doi: 10.3389/fcimb.2020.00348
Identification of Antimicrobial
Resistance Determinants in
Aeromonas veronii Strain MS-17-88
Recovered From Channel Catfish
(Ictalurus punctatus)
Hasan C. Tekedar 1 , Mark A. Arick II 2 , Chuan-Yu Hsu 2 , Adam Thrash 2 , Jochen Blom 3 ,
Mark L. Lawrence 1 and Hossam Abdelhamed 1*
1
College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, United States, 2 Institute for Genomics,
Biocomputing and Biotechnology, Mississippi State University, Mississippi State, MS, United States, 3 Bioinformatics &
Systems Biology, Justus-Liebig-University Giessen, Giessen, Germany
Edited by:
Xiangmin Lin,
Fujian Agriculture and Forestry
University, China
Reviewed by:
Shengkang Li,
Shantou University, China
Xiaofeng Shan,
Jilin Agricultural University, China
*Correspondence:
Hossam Abdelhamed
abdelhamed@cvm.msstate.edu
Specialty section:
This article was submitted to
Molecular Bacterial Pathogenesis,
a section of the journal
Frontiers in Cellular and Infection
Microbiology
Received: 31 March 2020
Accepted: 08 June 2020
Published: 17 July 2020
Citation:
Tekedar HC, Arick MA II, Hsu C-Y,
Thrash A, Blom J, Lawrence ML and
Abdelhamed H (2020) Identification of
Antimicrobial Resistance Determinants
in Aeromonas veronii Strain MS-17-88
Recovered From Channel Catfish
(Ictalurus punctatus).
Front. Cell. Infect. Microbiol. 10:348.
doi: 10.3389/fcimb.2020.00348
Aeromonas veronii is a Gram-negative species ubiquitous in different aquatic
environments and capable of causing a variety of diseases to a broad host range.
Aeromonas species have the capability to carry and acquire antimicrobial resistance
(AMR) elements, and currently multi-drug resistant (MDR) Aeromonas isolates are
commonly found across the world. A. veronii strain MS-17-88 is a MDR strain isolated
from catfish in the southeastern United States. The present study was undertaken to
uncover the mechanism of resistance in MDR A. veronii strain MS-17-88 through the
detection of genomic features. To achieve this, genomic DNA was extracted, sequenced,
and assembled. The A. veronii strain MS-17-88 genome comprised 5,178,226-bp with
58.6% G+C, and it encoded several AMR elements, including imiS, ampS, mcr-7.1, mcr3, catB2, catB7, catB1, floR, vat(F), tet(34), tet(35), tet(E), dfrA3, and tetR. The phylogeny
and resistance profile of a large collection of A. veronii strains, including MS-17-88, were
evaluated. Phylogenetic analysis showed a close relationship between MS-17-88 and
strain Ae5 isolated from fish in China and ARB3 strain isolated from pond water in Japan,
indicating a common ancestor of these strains. Analysis of phage elements revealed 58
intact, 63 incomplete, and 15 questionable phage elements among the 53 A. veronii
genomes. The average phage element number is 2.56 per genome, and strain MS-1788 is one of two strains having the maximum number of identified prophage elements
(6 elements each). The profile of resistance against various antibiotics across the 53
A. veronii genomes revealed the presence of tet(34), mcr-7.1, mcr-3, and dfrA3 in all
genomes (100%). By comparison, sul1 and sul2 were detected in 7.5% and 1.8% of A.
veronii genomes. Nearly 77% of strains carried tet(E), and 7.5% of strains carried floR.
This result suggested a low abundance and prevalence of sulfonamide and florfenicol
resistance genes compared with tetracycline resistance among A. veronii strains. Overall,
the present study provides insights into the resistance patterns among 53 A. veronii
genomes, which can inform therapeutic options for fish affected by A. veronii.
Keywords: Aeromonas veronii, antibiotic resistant, phage elements, comparative genomics, phylogenetic tree
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
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July 2020 | Volume 10 | Article 348
Tekedar et al.
Antimicrobial Resistance Diversity in Aeromonas veronii
INTRODUCTION
MDR bacteria, discover potential resistance mechanisms, and
explore the mechanisms underlying resistance gene transfer (Liu
et al., 2012).
The purpose of the current work was to uncover mechanisms
of resistance in A. veronii strain MS-17-88, which was isolated
from catfish in the southeastern U.S., and to assess the diversity,
resistance profiles, and mobile elements in a large collection of
A. veronii strains. Here we present the draft genome of A. veronii
and results from a comparative analysis with 52 publicly available
A. veronii genomes with a special emphasis on patterns of AMR
genes and prophage elements distribution. To the best of our
knowledge, no study has been published reporting a core-genome
based phylogenetic relationship of sequenced A. veronii genomes,
AMR profiles, and their mobilomes. Overall, our work provides
a basis to understand the AMR profile for A. veronii in aquatic
environments, which is an important step toward curtailing AMR
spread and informing a treatment of disease caused by A. veronii.
Aeromonas species are Gram-negative rods in the family
Aeromonadaceae. They are among the most common bacteria in
aquatic environments and have been isolated from virtually all
water source types including freshwater, estuarine environments,
drinking waters, wastewaters, and sewage (Janda and Abbott,
2010). The disease caused by Aeromonas species affects
a broad host range, including freshwater fish, amphibians,
reptiles, and birds (Barony et al., 2015). Mesophilic Aeromonas
species such as A. hydrophila, A. caviae, and A. veronii, are
associated with several kinds of human infections, including
gastroenteritis, wound infections, septicemia, and respiratory
infections (Figueras, 2005; Igbinosa et al., 2012). A. veronii is
one member of the genus Aeromonas, which is known for
causing hemorrhagic septicemia in both wild and farmed fish
such as channel catfish (Ictalurus punctatus) (Liu et al., 2016;
Yang et al., 2017), snakehead (Ophiocephalus argus), whitefish
(Coregonus clupeaformis) (Loch and Faisal, 2010), obscure puffer
(Takifugu obscurus), Nile tilapia (Oreochromis niloticus) (Hassan
et al., 2017), and common carp (Cyprinus carpio) (Sun et al.,
2016). In recent years, an increasing number of Aeromonas
species have become associated with disease of predominantly
freshwater fish in most countries (Goni-Urriza et al., 2000),
which results in causing severe outbreaks in different important
aquaculture industries.
Sulfonamides
potentiated
with
trimethoprim
or
ormethoprim, oxytetracycline, florfenicol, and erythromycin
are the most commonly used antimicrobial (AM) agents for
treatment of Aeromonas-related diseases in global aquaculture
(Serrano, 2005). Although the judicious use of AM-medicated
feeds is important for treatment purposes when faced with
outbreaks of bacterial infections (Okocha et al., 2018), a
substantial number of reports suggest that indiscriminate use of
AMs can foster selection pressure and enable development of
multi-drug resistant (MDR) bacteria in aquatic environments
(Arslan and Küçüksari, 2015). MDR in bacterial pathogens can
result in therapeutic challenges for control of bacterial diseases
(Marshall and Levy, 2011).
Over the past two decades, A. veronii has gained
epidemiological and ecological importance by several research
groups due to its potential as an opportunistic and primary
pathogen for fish and the prevalence of MDR strains (SanchezCespedes et al., 2008). Previous studies have reported the
isolation of MDR A. veronii strains from different regions
of the world such as Sri Lanka (Jagoda et al., 2017), China
(Yang et al., 2017), and United States (Abdelhamed et al., 2019;
Tekedar et al., 2019). The resistance elements in Aeromonas
species are often harbored in mobile genetic elements such
as class 1 integrons, plasmids, IS elements, transposons, and
genomic islands (Piotrowska and Popowska, 2015). These mobile
elements can facilitate the spread of resistance among bacteria
via transduction and conjugation (Sanchez-Cespedes et al.,
2008; Hossain et al., 2013; Piotrowska and Popowska, 2015).
Therefore, it is important to investigate the pattern of resistance,
genetic relatedness, and mobile elements in Aeromonas species.
High-throughput sequencing provides an opportunity to detect
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
MATERIALS AND METHODS
Bacterial Strains and Data Source for
Comparative Genome Analysis
A. veronii strain MS-17-88 was recovered from a diseased channel
catfish in 2017 from the Aquatic Diagnostic Laboratory at the
College of Veterinary Medicine, Mississippi State University.
The isolate was confirmed phenotypically as A. veronii. A 20%
glycerol stock culture was stored at −80◦ C. A. veronii strain MS17-88 was cultured in brain heart infusion (BHI) agar or broth
(Difco) and incubated at 30◦ C. Fifty-two A. veronii genomes
were retrieved from the National Center for Biotechnology
Information (NCBI) genomes database on October 9, 2018,
including five complete genome sequences and forty-seven draft
genome sequences (Table 1).
Antibiotic Resistance Phenotypes of A.
veronii Strain MS-17-88
The AM susceptibility of A. veronii strain MS-17-88 was
determined by the Kirby-Bauer disk diffusion method (Bauer
et al., 1966). Strain MS-17-88 was streaked on MuellerHinton agar plates, and the AM disks were applied on the
streaked cultures with a Dispens-O-Disc dispenser. The AM
agents tested were florfenicol (30 µg), chloramphenicol (30
µg), tetracycline (30 µg), doxycycline (30 µg), oxytetracycline
(30 µg), sulfamethoxazole-trimethoprim (23:75; 1.25 µg),
sulfamethoxazole (25 µg), erythromycin (15 µg), gentamicin
(10 µg), streptomycin (10 µg), spectinomycin (100 µg),
amoxicillin/clavulanic acid (30 µg), ampicillin (30 µg), penicillin
(10 µg), ceftriaxone (30 µg), cefpodoxime (10 µg), ceftiofur (30
µg), ciprofloxacin (5 µg), enrofloxacin (15 µg), azithromycin
(15 µg), nalidixic acid (30 µg), bacitracin (10 µg), and
novobiocin (30 µg). These AM agents were selected based on
the World Health Organization’s list of the most common classes
of antimicrobials (aminoglycosides, tetracyclines, macrolides,
beta-lactam, phenicols, quinolones, and sulfonamides) that are
regularly used in agriculture and aquaculture and linked to
human medicine (Done et al., 2015). After 24 h of incubation
2
July 2020 | Volume 10 | Article 348
Strain names
Country
Source
Level
Size (Mb)
GC%
Scaffolds
Genes
Proteins
Accession
References
Jagoda et al., 2017
3
Sri-Lanka
Goldfish
Contig
4.56
58.7
80
–
–
BDGY00000000.1
MS-17-88
USA
Catfish
Contig
5.18
58.2
12
4,948
4,651
NZ_RAWX01000001.1
This study
ARB3
Japan
Pond water
Contig
4.54
58.8
63
4,074
3,952
NZ_JRBE00000000.1
Kenzaka et al., 2014
CIP 107763
USA
N/A
Contig
4.43
58.8
64
4,040
3,897
NZ_CDDU00000000.1
N/A
VBF557
India
Human
Contig
4.70
58.4
526
4,460
3,325
LXJN00000000.1
N/A
TTU2014-108ASC
USA
Cattle
Contig
4.53
58.7
58
4,103
3,941
NZ_LKJP00000000.1
Webb et al., 2016
TTU2014-108AME
USA
Cattle
Contig
4.53
58.7
62
4,112
3,938
NZ_LKJN00000000.1
Webb et al., 2016
TTU2014-115AME
USA
Cattle
Scaffold
4.53
58.7
53
4,108
3,943
NZ_LKJR00000000.1
Webb et al., 2016
TTU2014-115ASC
USA
Cattle
Contig
4.53
58.7
52
4,102
3,940
NZ_LKJS00000000.1
Webb et al., 2016
TTU2014-142ASC
USA
Cattle
Contig
4.68
58.6
45
4,242
4,065
NZ_LKKF00000000.1
Webb et al., 2016
TTU2014-130ASC
USA
Cattle
Scaffold
4.68
58.6
49
4,243
4,063
NZ_LKJX00000000.1
Webb et al., 2016
TTU2014-134AME
USA
Cattle
Contig
4.68
58.6
50
4,248
4,063
NZ_LKKA00000000.1
Webb et al., 2016
TTU2014-141AME
USA
Cattle
Scaffold
4.68
58.6
48
4,241
4,066
NZ_LKKD00000000.1
Webb et al., 2016
TTU2014-134ASC
USA
Cattle
Contig
4.68
58.6
59
4,242
4,060
NZ_LKKB00000000.1
Webb et al., 2016
TTU2014-143ASC
USA
Cattle
Contig
4.68
58.6
54
4,246
4,061
NZ_LKKH00000000.1
Webb et al., 2016
TTU2014-125ASC
USA
Cattle
Contig
4.68
58.6
58
4250
4,066
NZ_LKJV00000000.1
Webb et al., 2016
TTU2014-141ASC
USA
Cattle
Contig
4.68
58.6
45
4,240
4,062
NZ_LKKE00000000.1
Webb et al., 2016
TTU2014-113AME
USA
Cattle
Scaffold
4.66
58.6
122
4,239
4,037
NZ_LKJQ00000000.1
Webb et al., 2016
TTU2014-130AME
USA
Cattle
Contig
4.68
58.6
64
4,242
4,064
NZ_LKJW00000000.1
Webb et al., 2016
TTU2014-143AME
USA
Cattle
Contig
4.68
58.6
59
4,248
4,066
NZ_LKKG00000000.1
Webb et al., 2016
TTU2014-131ASC
USA
Cattle
Contig
4.68
58.6
70
4,245
4,055
NZ_LKJY00000000.1
Webb et al., 2016
TTU2014-140ASC
USA
Cattle
Contig
4.68
58.6
81
4,249
4,057
NZ_LKKC00000000.1
Webb et al., 2016
pamvotica
Greece
Sediment
Contig
4.92
58.1
21
4,581
4,317
NZ_MRUI00000000.1
N/A
AER397
USA
Human
Scaffold
4.50
58.8
5
4,014
3,888
NZ_AGWV00000000.1
*
B565
China
Pond
sediment
Complete
4.55
58.7
1
4,100
3,950
NC_015424
Li et al., 2011
CCM 4359
USA
Human
Contig
4.51
58.9
56
4,170
3,908
NZ_MRZR00000000.1
N/A
CECT 4257
USA
Human
Scaffold
4.52
58.9
52
4,101
3,955
NZ_CDDK00000000.1
Colston et al., 2014
*
July 2020 | Volume 10 | Article 348
AMC35
USA
Human
Scaffold
4.57
58.5
2
4,064
3,918
NZ_AGWW00000000.1
AVNIH1
USA
Human
Complete
4.96
58.47
2
4,551
4,321
NZ_CP014774.1
N/A
AVNIH2
USA
Human
Contig
4.52
58.9
50
4,071
3,918
NZ_LRBO00000000.1
N/A
LMG 13067
USA
N/A
Scaffold
4.74
58.4
72
4,265
4,055
NZ_CDBQ00000000.1
N/A
126-14
China
Human
Scaffold
4.37
58.6
146
4,114
3,884
NZ_PPTE00000000.1
N/A
FC951
India
Human
Contig
4.67
58.5
231
4,479
4,066
NZ_PKSR00000000.1
N/A
5.28.6
Greece
Fish
Contig
4.61
58.6
98
4,337
4,107
NZ_NNSE00000000.1
N/A
(Continued)
Antimicrobial Resistance Diversity in Aeromonas veronii
Ae52
Tekedar et al.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
TABLE 1 | The 53 A. veronii genomes used in comparative genomic analysis.
Strain names
Country
Source
Level
Size (Mb)
GC%
Scaffolds
Genes
Proteins
Accession
References
VCK
Greece
Fish
Contig
4.63
58.6
120
4,366
4,133
NZ_NNSF00000000.1
N/A
NS
Greece
European
bass
Contig
4.71
58.5
140
4,503
4,244
NZ_NMUR00000000.1
N/A
PDB
Greece
Fish
Contig
4.72
58.5
141
4,542
4,285
NZ_NMUS00000000.1
N/A
AER39
USA
Human
Scaffold
4.42
58.8
4
3,987
3,832
NZ_AGWT00000000.1
N/A
X12
China
Wuchang
bream
Complete
4.77
58.3
1
4,440
4,183
NZ_CP024933
N/A
A29
South Africa
Surface water
Scaffold
4.48
58.8
54
4,142
3,979
NJGB00000000.1
N/A
X11
China
Wuchang
bream
Complete
4.28
58.8
1
3,901
3,716
NZ_CP024930
N/A
CCM 7244
Germany
Surface water
Contig
4.42
58.9
74
4,069
3,807
NZ_MRZQ00000000.1
N/A
CECT 4486
USA
Surface water
Scaffold
4.41
58.9
66
4,022
3,831
NZ_CDBU00000000.1
Colston et al., 2014
Hm21
Turkey
Digestive tract
Contig
4.68
58.7
50
4,252
4,116
NZ_ATFB00000000.1
Bomar et al., 2013
CB51
China
Fish
Complete
4.58
58.6
1
4,152
3,623
CP015448
N/A
ZWY-AV1
China
Liver
Contig
4.62
58.6
31
4,317
4,153
NZ_PXYZ00000000.1
N/A
4
Z2-7
China
N/A
Scaffold
4.41
58.7
48
4,092
3,915
NZ_UETI00000000.1
N/A
ZJ12-3
China
Human
Scaffold
4.70
58.4
124
4,380
4,122
NZ_UETM00000000.1
N/A
ML09-123
USA
Fish
Contig
4.75
58.4
32
4,422
4,204
PPUW01000001
N/A
TH0426
China
Catfish
Complete
4.92
58.3
1
4,528
4,282
NZ_CP012504.1
Kang et al., 2016
XH.VA.1
China
Catfish
Contig
5.36
58.5
62
5,207
4,912
NZ_PZKL00000000.1
N/A
XH.VA.2
China
Catfish
Scaffold
4.91
58.1
48
4,637
4,389
NZ_QQOQ00000000.1
N/A
RU31B
N/A
N/A
Scaffold
4.53
58.7
93
4,203
3,976
NZ_FTMU00000000.1
N/A
Tekedar et al.
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
TABLE 1 | Continued
*Human Microbiome U54 initiative, Broad Institute (broadinstitute.org).
N/A, Not available.
Antimicrobial Resistance Diversity in Aeromonas veronii
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Tekedar et al.
Antimicrobial Resistance Diversity in Aeromonas veronii
at 30◦ C, the zones of inhibition diameter were measured and
compared to the criteria of the National Committee for Clinical
Laboratory Standards. The assay was performed in triplicate and
repeated as two independent experiments.
90 was deemed questionable; and score < 70 was considered
incomplete phage region (Arndt et al., 2019).
DNA Extraction, Whole-Genome
Sequencing, Assembly, and Annotation
The potential AMR genes and related elements for each
genome were identified using ResFinder 3.1 (Zankari et al.,
2012). ResFinder database was downloaded and used in CLC
Workbench version 11.0.1 (CLC Bio) for the BLAST search.
The contig files for each genome were concatenated, and
concatenated nucleotide files were uploaded to CLC Workbench.
A BLAST search was run with the following settings: 40%
minimum identity and 40% minimum matching length.
Comparative Analysis of Putative AMR
Elements
Genomic DNA of A. veronii strain MS-17-88 was extracted
using the DNeasy Blood & Tissue Kit (Qiagen., USA) according
to the manufacturer’s instructions. Genome sequencing was
conducted using HiSeq X Ten (Illumina, San Diego, CA,
USA) and MinION (Oxford Nanopore Technologies, Oxford,
UK), producing approximately 848.86X and 229.35X genome
coverages, respectively. Together, the genome coverage is 1077X.
Trimmomatic (Bolger et al., 2014) was used to trim Illumina
reads, Nanopore reads were corrected with Canu (version 1.6)
(Koren et al., 2017), and contig errors were corrected using Pilon
(version 1.21) (Walker et al., 2014). Assembly of the Illumina
and Nanopore reads into contigs was done using MaSuRCA
(version 3.2.4 (Zimin et al., 2013). Average nucleotide identity
(ANI) was calculated based on whole genome sequencing using
BLAST alignments (Richter and Rossello-Mora, 2009).
RESULTS AND DISCUSSION
General Genome Features of A. veronii
Strain MS-17-88
The present study reported the draft genome of A. veronii strain
MS-17-88 isolated from diseased catfish in the southeastern
U.S. The draft genome of A. veronii strain MS-17-88 consisted
of 5,178,226 bp with 58.6% G+C content and encoded 4,944
predicted coding sequences (CDSs). A total of 181 RNA genes
were predicted in the genome including 139 tRNAs, 4 ncRNAs,
and 38 rRNAs (12, 13, 13 for 5, 16, and 23 s, respectively). The
final assembly contained 13 contigs. The largest contig assembled
was 1,457,362-bp length, and the smallest contig was 7,082-bp.
The genome has been deposited in GenBank (accession number
NZ_RAWX01000000). A. veronii contains two biovars (A. veronii
biovar veronii and A. veronii biovar sobria) (Janda and Abbott,
2010). ANI and phylogenetic tree calculation confirmed that
strain MS-17-88 belongs to A. veronii biovar veronii (ANI score
higher than 95%).
Phylogenetic Tree
A phylogenetic tree was constructed based on the complete
core genome of A. veronii strain MS-17-88 and 52 A. veronii
genomes to evaluate taxonomic positions. All publicly available
A. veronii genome sequences (52 genomes) were downloaded
from NCBI. Gene sets of the core genome were aligned
using MUSCLE (Edgar, 2004) and concatenated. Concatenated
alignment files were used as an input to compute a Kimura
distance matrix, which was followed by using the concatenated
files for the Neighbor-Joining algorithm as implemented in
PHYLP (Felsenstein, 1989).
Genotypic and Phenotypic
Characterization of A. veronii Strain
MS-17-88
Subsystem Coverages and Genomes
Structure Variation
A. veronii strain MS-17-88 and 52 A. veronii genomes
were submitted to Rapid Annotations using Subsystems
Technology (RAST) for annotation, subsystem categorization,
and comparison purposes (Aziz et al., 2008). The following
criteria were used for annotation pipeline: classic RAST for
annotation, RAST gene caller for open reading frame (ORF)
identification, and Figfam (version release70 with automatic fix
errors and fix frameshifts options). A. veronii strain MS-17-88
genome was compared against 52 A. veronii using BRIG (BLAST
Ring Image Generator) (Alikhan et al., 2011).
The Rasfinder and CARD analysis revealed 14 resistance
elements in the A. veronii strain MS-17-88 genome (Table 2),
including beta-lactamase resistance genes (imiS and ampS),
chloramphenicol and florfenicol resistance gene (floR), macrolide
resistance genes (mcr-3 and mcr-7.1), streptogramin B resistance
vat(F), tetracycline resistance genes [tet(34), tet(35), and tet(E)],
and acetyltransferase genes conferring resistance to phenicol
compounds. The disk diffusion results (Table 3) demonstrated
that A. veronii strain MS-17-88 strain is resistant to phenicol class
(florfenicol and chloramphenicol), tetracyclines (tetracycline,
doxycycline, and oxytetracycline), macrolides (erythromycin,
azithromycin,), aminoglycoside (gentamicin), sulfamethoxazole,
beta-lactam class (amoxicillin/clavulanic acid, ampicillin, and
penicillin), spectinomycin, bacitracin, and novobiocin.
Resistance to β-lactam antibiotics in Aeromonas species is
primarily mediated by β-lactamases, whose mode of action
involves hydrolyzing the β-lactam ring. Many different βlactamases have been detected in Aeromonas species, such as
TEM-, SHV-, OXA-, CMY-, and CTX-M-type β-lactamases. In
Prophages
The presence of prophages in the 53 A. veronii genomes was
determined using PHASTER (PHAge Search Tool Enhanced
Release) (Arndt et al., 2016, 2019). Nucleotide sequences from 53
genomes were concatenated using Sequencher 5.4.5 to serve as
an input file prior to submission to the PHASTER server. Results
from PHASTER were arranged into three categories: score > 90
was considered intact phage element; a score between 70 and
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Tekedar et al.
Antimicrobial Resistance Diversity in Aeromonas veronii
TABLE 2 | Predicted antibiotic resistance genes in A. veronii strain MS-17-88.
Protein name
Protein ID
Gene
% Identity
Query/HSP Length
Predicted phenotype
CphA family subclass B2 metallo-beta-lactamase
RKJ87494.1
imiS
89.7
767/768
Beta-lactam resistance
Class D beta-lactamase
RKJ86357.1
ampS
93.84
795/795
Beta-lactam resistance
Phosphoethanolamine-lipid A transferase
RKJ90059.1
mcr-3
67.19
756/1,626
Phosphoethanolamine–lipid A transferase
RKJ90059.1
mcr-7.1
73.2
1,601/1,620
Colistin resistance
Antibiotic acetyltransferase
D6R50_13775
catB2
65.85
201/633
Phenicol resistance
Antibiotic acetyltransferase
D6R50_13775
catB7
68.78
235/639
Phenicol resistance
Antibiotic acetyltransferase
RKJ86442.1
catB7
66.61
565/639
Phenicol resistance
Vat family streptogramin A O-acetyltransferase
RKJ85500.1
catB1
70.79
265/630
Phenicol resistance
Chloramphenicol/florfenicol efflux MFS
RKJ86396.1
floR
98.19
1,214/1,215
Vat family streptogramin A O-acetyltransferase
RKJ85500.1
vat(F)
69.52
581/666
Streptogramin B resistance
Xanthine phosphoribosyltransferase
RKJ89311.1
tet(34)
66.95
340/465
Tetracycline resistance
Na+/H+ antiporter NhaC family protein
RKJ91399.1
tet(35)
70.13
231/1,110
Tetracycline resistance
Tetracycline efflux MFS transporter Tet(E)
RKJ91234.1
tet(E)
99.92
1218/1,218
Tetracycline resistance
Type 3 dihydrofolate reductase
RKJ87621.1
dfrA3
68.93
348/489
Trimethoprim resistance
Phenicol resistance
(strains 5.28.6, VCK, NS, and PDB) were clustered together.
These data clearly suggest that the ecological niche is a more
important factor contributing to relatedness among A. veronii
isolates than geographical location. However, two isolates (CIP
107763 and VBF557) showed a distinct genetic relationship to the
other isolates.
It has been postulated that a fish pathogenic Aeromonas
hydrophila clonal group was transferred to the U.S. channel
catfish aquaculture industry from China (Hossain et al., 2014).
In the current study, we observed two different clonal groups
of A. veronii that contain isolates from both U.S. and China.
One clade has MDR A. veronii strain MS-17-88 from U.S. catfish
aquaculture and MDR isolate Ae5 from goldfish in China, and
another clade has U.S. channel catfish isolate ML09-123 and
China catfish strain TH0426 (Figure 1).
the present study, ampS and imiS were detected in A. veronii
stain 17-88. AmpS is a class 2d penicillinase and ImiS is a
class 3 metallo-β-lactamase. The imiS gene has been detected
in clinical isolates of A. veronii biovar sobria (Wu et al., 2012).
MS-17-88 also harbors floR, which encodes a major facilitator
superfamily efflux pump that exports florfenicol (Schwarz et al.,
2004). Florfenicol is one among the three approved AMs for use
in catfish aquaculture in the U.S (Bowker et al., 2010). Most
fish pathogenic bacteria mediate florfenicol resistance through
FloR (Dang et al., 2007; Gordon et al., 2008). Dissemination of
florfenicol resistance among bacterial pathogens isolated from
aquaculture can limit the efficacy of this agent as an important
treatment option.
Phylogenetic Tree
The phylogenetic relationship between the A. veronii MS-1788 genome and 52 other A. veronii genomes was assessed. The
strains used in this analysis are from different countries and
hosts, and they had distinct resistance profiles (Table 1). The
phylogenetic tree for the 53 A. veronii genomes was built from a
core genome of 2,563 genes per genome (135,839 genes in total).
The core has 2,538,377 bp per genome (134,533,981 bp in total).
The phylogenetic tree showed that there are multiple highly
conserved branches that are separated from the other A. veronii
genomes. MDR A. veronii strain MS-17-88 from U.S. channel
catfish, goldfish (Carassius auratus) MDR strain Ae5 from China,
and A. veronii ARB3 from pond water in Japan were clustered
together, which may indicate a common origin. Similarly, U.S.
channel catfish isolate ML09-123 and China catfish isolate
TH0426 were closely related, which also may suggest derivation
from the same monophyletic origin despite their geographic
disparity. Moreover, dairy cattle isolates and Greece surface
sediment isolates (strain pamvotica) formed another closely
related group. U.S. human isolates (strains AER 397, CECT
4257, and CCM 4359) and China pond sediment isolate B565
formed another clade. Lastly, U.S. surface water and Germany
surface water isolates were closely related, and Greece fish isolates
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Colistin resistance
Subsystems Coverage
The subsystems categorization based on RAST annotation is
shown in Figure 2. SEED subsystem categorization analysis
predicted 26 different categories for the evaluated A. veronii
genomes. The most abundant systems are “amino acid and
derivatives,” followed by “carbohydrates” and “protein
metabolism.” These subsystems are essential for bacteria to
perform basic cellular processes and may indicate the potential
ability of A. veronii to utilize different kinds of sugars and
amino acids available in the environment (Liang et al., 2019).
On the other hand, A. veronii genomes show remarkably
low numbers of mobile genetic elements including phages,
prophages, transposable elements, and plasmids. These mobile
elements can mediate alteration of genotypes, and these findings
may suggest that horizontal gene exchange may not contribute
to A. veronii genomic variation as much as other species.
Interestingly, U.S. catfish isolate strain MS-17-88 carries the
most abundant subsystems (63 elements) associated with phages,
prophages, transposable elements, and plasmids. Chinese
catfish isolate strain TH0426 has the second largest number
(52 elements) of phage and transposable elements. Therefore,
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Antimicrobial Resistance Diversity in Aeromonas veronii
aquatic bacteria (about 70%) are infected with prophages (Chen
et al., 2006). Of particular interest, bacteriophages can mediate
horizontal gene transfer, including genes encoding virulence
factors and antibiotic resistance (Colomer-Lluch et al., 2011). A
higher number of phages may represent a concern because they
can expand the pathogenicity of a bacterial strain or convert an
avirulent strain into a virulent one (Canchaya et al., 2004). Even
though phage elements may not be the main spreading factors
of resistance elements, recent studies indicate that they can
infrequently contribute to the dissemination of these elements
(Allen et al., 2011; Enault et al., 2017). In Aeromonas, the transfer
of resistance gene by phage elements has never been observed
previously (Piotrowska and Popowska, 2015).
In the present study, we identified 58 intact, 63 incomplete,
and 15 questionable phage elements among the 53 A. veronii
genomes (Figure 4). The average phage element number is 2.56
per genome (136 identified phage elements/53 A. veronii strains).
In a previous study, A. hydrophila genomes were found to
harbor an average of 2.91 phage elements per genome (143
identified phage elements/49 A. hydrophila strains) (Awan et al.,
2018). Almost all the strains carry one or more prophage
elements, with the exceptions being strains FC951, CECT4486,
and CCM7244, which do not carry any type of prophage. Strains
MS-17-88 and TH0426 had the maximum number of identified
prophage elements (6 elements per strain). The maximum
number of complete prophage elements (5 complete phages)
was present in strain MS-17-88 along with one incomplete
phage element (Figure 4). The maximum number of incomplete
prophage elements (4 incomplete phages) was present in strains
TTU2014-108ASC and TTU2014-115AME. Strain MS-17-88
carries three different types of phage elements: vB_AbaM_ME3,
phi018P, and RSA1. Interestingly, two of these phage elements
(vB_AbaM_ME3 and RSA1) are not carried by any other
evaluated A. veronii genomes, suggesting that this strain has been
exposed to different environments and acquired unique phage
elements. Phage vB_AbaM_ME3 was previously isolated from
wastewater effluent using the propagating host Acinetobacter
baumannii DSM 30007 (Kropinski et al., 2009). A. baumannii is
a known nosocomial pathogen that causes pneumonia, urinary
tract infection, and septicemia (Buttimer et al., 2016). Phage
vB_AbaM_ME3 of A. baumannii has a size of 234,900 bp and 326
ORFs (Buttimer et al., 2016). The Myovirus-type phage RSA1 is
relatively small (39 to 40 kb) and has lytic activity. It has restricted
host range, mainly Ralstonia solanacearum, a soil-borne species
pathogenic to many important crops (Yamada et al., 2007; Addy
et al., 2019).
Interestingly, the MS-17-88 genome has four prophages
sharing structural similarities with temperate Aeromo_phiO18P
elements found in Aeromonas media isolated from a pond in
Germany (Beilstein and Dreiseikelmann, 2008). The phiO18P
phage type belongs to the Myoviridae phage family and consists
of 33 kb. The phiO18P phage elements typically have 46 ORFs
encoding proteins responsible for integration and regulation,
replication, packaging, head and tail, and lysis (Beilstein
and Dreiseikelmann, 2008). Unlike other prophage elements,
Aeromo_phiO18P does not have a lytic phase; it replicates
lysogenically by integrating its genome into the bacterial
TABLE 3 | Antimicrobial resistance phenotype of A. veronii strain MS-17-88.
Antimicrobial agents
Disk content
(µg)
Diameter of
inhibition zone (mm)
Sensitivity
Florfenicol FFC30
30
0
R
Chloramphenicol C30
30
0
R
Tetracycline TE30
30
6.16 ± 0.44
R
Doxycycline D30
30
10.8 ± 0.41
R
Oxytetracycline T30
30
0
R
Sulfamethoxazole
25
0
R
Sulphamethoxazole
trimethoprim SXT
25
17.9 ± 0.20
S
Erythromycin E15
15
11.20 ± 0.11
R
Gentamicin GM10
10
16.24 ± 0.40
R
Streptomycin S10
10
11.4 ± 0.23
S
Spectinomycin SPT100
100
12.7 ± 0.14
R
Amoxicillin/clavulanic
acid AMC30
30
9.2 ± 0.11
R
Ampicillin AM10
10
0
R
Penicillin P10
10
0
R
Ceftriaxone CRO30
30
29.43 ± 0.31
S
Cefpodoxime CPD10
10
19.63 ± 0.18
S
Ceftiofur XNL30
30
19.76 ± 0.12
S
Ciprofloxacin CIP5
25
24.76 ± 0.14
S
Enrofloxacin E15
15
24.76 ± 0.14
S
Azithromycin AZM15
15
16.3 ± 0.17
R
Nalidixic acid NA30
30
24.8 ± 0.10
S
Bacitracin B10
10
0
R
Novobiocin NB30
30
9.3 ± 0.15
R
R, resistant; S, sensitive.
Data represented as means diameter of inhibition zones (mm) ± SD of two independent
experiments in triplicates.
these two strains (MS-17-88 and TH0426) may have acquired
significant genome structure changes by gene acquisitions
from mobile elements. It is notable that both strains were
isolated from aquatic environments, and we speculate that the
aquatic environment may be favorable for genetic exchange and
horizontal gene acquisition.
Genome Structure Variation
Visualization of the alignment between A. veronii MS-17-88
and 52 other A. veronii genomes revealed that phage elements,
transposons, and genomic islands comprise many of the MS17-88-specific regions (Figure 3). This suggests that mobile
elements, especially phages and transposons, play an important
role in A. veronii genome variation. These mobile elements
can result in genomic rearrangements and evolution through
acquisition of novel virulence or antibiotic resistance genes which
may result in emergence of new phenotypes (Brown-Jaque et al.,
2015).
Prophages
Bacteriophages are responsible for loci rearrangements and
deletions and are recognized as an important element in
bacterial evolution (Tinsley et al., 2006). The vast majority of
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Antimicrobial Resistance Diversity in Aeromonas veronii
FIGURE 1 | Phylogenetic tree analysis based on the core genomes of A. veronii.
the MS-17-88 genome using PHASTER, we identified that the
strain carries the floR gene inside incomplete phage element
PHAGE_Staphy_SPbeta_like_NC_029119 (genome position
2631302-2644102), but later analysis with PHASTER showed
that this may not be a true phage element. Further investigation
of this region is warranted to determine whether a phage element
mediated dissemination of the floR resistance gene (Garriss et al.,
2009).
chromosome (Vincent et al., 2017). In some instances, the phageencoded genes are advantageous to the host bacteria (Dziewit and
Radlinska, 2016). Aeromo_phiO18P shows significant similarity
to the P2 phage family in Aeromonas salmonicida and Vibrio
cholerae K139 genomes (Beilstein and Dreiseikelmann, 2008).
The present study documented 40 different types of phage
elements across the 53 A. veronii genomes (Figure 5). Among
these 40 phage elements, phage type “Aeromo_phiO18P” is the
most abundant type in all the evaluated A. veronii genomes
as well as the most abundant in strain MS-17-88. Strains
CCM 4359 and XH.VA.1 carry five different phage elements.
Incomplete Staphy_SPbeta_like phage element was detected in
two strains (MS-17-88 and AVNIH1). In our initial analysis of
Frontiers in Cellular and Infection Microbiology | www.frontiersin.org
Comparative Analysis of Antibiotic
Resistance Determinants
A comparative analysis of 87 AMR determinants and
components was conducted to determine their distribution
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Antimicrobial Resistance Diversity in Aeromonas veronii
FIGURE 2 | Comparison of functional categories in 53 A. veronii genomes based on SEED. Functional categorization is based on roles of annotated and assigned
genes. Each colored bar represents the number of genes assigned to each category.
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Antimicrobial Resistance Diversity in Aeromonas veronii
FIGURE 3 | Comparative circular map of the A. veronii MS-17-88 genome. Phage regions are highlighted with red color: phage region-1 encodes 41 proteins
(31.8 Kb), phage region-2 encodes 12 proteins (12.6 Kb), phage region-3 encodes 39 proteins (37.7 Kb), phage region-4 encodes 31 proteins (22.9 Kb), phage
region-5 encodes 45 proteins (48.6 Kb), and phage region-6 encodes 30 proteins (24.1 Kb).
2009; Lagana et al., 2011; Tamminen et al., 2011). In addition
to tet(34), tet(E), and dfrA3, A. veronii MS-17-88 genome
carries colistin resistance genes (mcr-7.1 and mcr-3). There have
been an increasing number of reports on the identification of
mcr genes in many bacterial species globally (Stoesser et al.,
2016; Elbediwi et al., 2019). A recent study reported that
mcr-3 variants are more common in Aeromonas than in other
bacterial species but aeromonads do not inherently carry the
mcr-3 gene (Shen et al., 2018). However, it is speculated that
Aeromonas isolates from the aquatic environment may be the
major reservoir for the dissemination of mcr-3 genes to other
among the 53 A. veronii strains. A. veronii MS-17-88 shared a
common AMR gene composition with the other A. veronii
genomes. Figure 6 shows the distribution of antibiotic
resistance genes in each strain. All the A. veronii genomes
carry tetracycline [tet(34) and tet(E)], and trimethoprim
(dfrA3) resistance genes. Oxytetracycline, tetracycline, and
trimethoprim/sulfamethoxazole have been extensively used
in human clinical, veterinary, and agricultural sectors for
decades. The linkage between AM use and resistance has been
demonstrated for other bacteria in aquaculture ecosystems
and other animal husbandry facilities (Verner-Jeffreys et al.,
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Antimicrobial Resistance Diversity in Aeromonas veronii
FIGURE 4 | Number of prophages with their completeness profiles in A. veronii genomes. Strains FC91, CCM7244, and CECT4486 did not have any prophage
elements.
FIGURE 5 | Type of prophage elements present in the A. veronii genomes. Red color represents presence of the gene.
strain was isolated from human stool, and the genome exhibits
clear evidence of horizontal gene transfer (Hughes et al.,
2016). In our analyses, we did not observe any pattern of
antimicrobial resistance in specific bacterial host or sources.
However, there was one exception: the highest number of
AMR elements were observed in two human isolates (strain
AVNIH from U.S.A; (Hughes et al., 2016) and ZJ12-3 from
China; Shen et al., 2018), and one isolate from septicemic
goldfish (strain Ae52 from Sri-Lanka; Jagoda et al., 2017).
The use of antimicrobial agents in human medicine may
be associated with these nosocomial trends (Hughes et al.,
2016).
bacteria (Ling et al., 2017; Eichhorn et al., 2018). Furthermore,
A. veronii MS-17-88 genome harbors a macrolide resistance
gene vat(F) that encodes an acetyltransferase that acetylates class
A streptogramins (Seoane and García Lobo, 2000). Six genes
encoding resistance to β-lactamases were identified in A. veronii
MS-17-88 genome including ampH and blaOXA-427 belong to
class D beta-lactamase, ampS encoding a class 2d penicillinase
and hydrolyzing mainly penicillins (Walsh et al., 1995), and
cphA2, cphA8, and imiS encoding a class 3 metallo-β-lactamase
and active mainly against carbapenems (Walsh et al., 1998).
Among the 53 A. veronii strains, AVNIH1 strain had
resistance genes to almost all the antibiotic classes. This clinical
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Antimicrobial Resistance Diversity in Aeromonas veronii
FIGURE 6 | AMR genes distribution across the 53 A. veronii genomes. Blue color represents presence of the gene.
was present in only four strains (MS-17-88, AVNIH1, Z2-7,
and ZJ12-3) (7.5%). The prevalence of beta-lactam resistance
genes in the 53 A. veronii genomes was as follows: ampH was
detected in 41.5% of the strains, ampS was detected in 92.4%
of the strains, imiH was detected in 13.2% of the strains, imiS
was detected in 71.7% of the strains, blaCEPH-A3 was detected
in 56.6% of the strains, blaOXA-427 was detected in 45.3% of
the strains, cphA1 was detected in 17% of the strains, cphA2
was detected in 26.41% of the strains, cphA6 was detected
in 13.2% of the strains, cphA7 was detected in 20.7% of the
In regard to sulphonamide resistance, sul1 was detected in
four strains (Ae52, ANIH1, Z2-7, and ZJ12-3) representing 7.5%
of the A. veronii strains, and sul2 was present in one strain (Z27) representing 1.8%. This suggests that sul1 is the most frequent
gene encoding sulfonamides resistance in A. veronii. Nearly all
strains (100%) carried tet(34), and a significant proportion of
strains carried tet(E) (20 strains; 37%). In contrast, tet(57) and
tet(D) were detected in only one strain (ANIH1) (1.8%), and
tet(A) was detected in two strains (Ae52 and ZJ12-3) (3.8%). floR
gene conferring resistance to florfenicol and chloramphenicol
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Antimicrobial Resistance Diversity in Aeromonas veronii
DATA AVAILABILITY STATEMENT
strains, and cphA8 was detected in 44% of the strains. Of
the aminoglycoside resistance genes, aac(6′ )-Ic was the most
prevalent (32%). Interestingly, A. veronii AVNIH1 and Ae52
strains carried multiple aminoglycoside-resistance genes. Several
studies reported that the majority of Aeromonas species exhibit
only a single aminoglycoside modifying gene (Dahanayake et al.,
2019). However, Pseudomonas aeruginosa isolated from hospitals
from Iran was reported to carry up to four aminoglycoside
resistance genes (Perez-Vazquez et al., 2009).
In conclusion, we used genome sequencing to investigate
genetic variation and AMR gene distribution in 53 A. veronii
genomes. We found significant genetic differences and a high
degree of genomic plasticity in the evaluated A. veronii genomes.
Overall, the AMR gene frequency against sulfamethoxazole and
florfenicol is low, while AMR genes against tetracycline are
very high. Among tetracycline-resistant isolates, tet(34) and
tet(E) were the most frequent AMR genes. Taken together, our
results show that AMR genes are common and are distributed
among A. veronii genomes; however, the frequency of most
AMR genes in individual strains is still low. Identified phage
elements may be useful for future development of an efficient
and effective bio-treatment method to control bacterial diseases
in aquaculture. The knowledge generated from this study can
benefit our understanding of A. veronii evolution and provide
insight into how A. veronii isolates are intrinsically resistant
to multiple antimicrobials. In addition, A. veronii species are
important considerations as potential sources for resistance
determinants in the environment. Therefore, it is important to
continue surveillance of resistance and genetic mechanisms of
resistance in this species.
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and
accession number(s) can be found in the article.
AUTHOR CONTRIBUTIONS
HT, ML, and HA designed and conceived the analysis and
experiments. HT, MA, C-YH, AT, JB, and HA performed the
experiments and analyzed the data. HT, ML, and HA wrote the
manuscript. All authors read and approved the final manuscript.
FUNDING
Salary support to HA was provided by the Center
for Biomedical Research Excellence in Pathogen–Host
Interactions, National Institute of General Medical Sciences,
and National Institutes of Health awarded grant number
P20GM103646-07. This work was supported by College
of Veterinary Medicine and by USDA-ARS SCA no.
58-6066-7081 titled MS Center for Food Safety and
Post-Harvest Technology, MS Agricultural and Forestry
Experiment Station.
ACKNOWLEDGMENTS
The authors thank the Aquatic Diagnostic Laboratory at the
College of Veterinary Medicine for providing Aeromonas veronii
strain MS 17-88.
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Conflict of Interest: The authors declare that the research was conducted in the
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potential conflict of interest.
Copyright © 2020 Tekedar, Arick, Hsu, Thrash, Blom, Lawrence and Abdelhamed.
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