TYPE
Original Research
15 March 2023
10.3389/fmicb.2023.1112941
PUBLISHED
DOI
SPECIALTY SECTION
The mobile gene cassette carrying
tetracycline resistance genes in
Aeromonas veronii strain Ah5S-24
isolated from catfish pond
sediments shows similarity with a
cassette found in other
environmental and foodborne
bacteria
This article was submitted to
Antimicrobials, Resistance and Chemotherapy,
a section of the journal
Frontiers in Microbiology
Saurabh Dubey 1, Eirill Ager-Wiick 1, Bo Peng 2, Angelo DePaola 3,
Henning Sørum 4 and Hetron Mweemba Munang’andu 1,5*
OPEN ACCESS
EDITED BY
Zhi Ruan,
Zhejiang University,
China
REVIEWED BY
Tingting Xu,
Jinan University,
China
Christopher John Grim,
United States Food and Drug Administration,
United States
*CORRESPONDENCE
Hetron Mweemba Munang’andu
hetron.m.munangandu@nord.no
RECEIVED 30
November 2022
February 2023
PUBLISHED 15 March 2023
ACCEPTED 13
CITATION
Dubey S, Ager-Wiick E, Peng B, DePaola A,
Sørum H and Munang’andu HM (2023) The
mobile gene cassette carrying tetracycline
resistance genes in Aeromonas veronii strain
Ah5S-24 isolated from catfish pond sediments
shows similarity with a cassette found in other
environmental and foodborne bacteria.
Front. Microbiol. 14:1112941.
doi: 10.3389/fmicb.2023.1112941
COPYRIGHT
© 2023 Dubey, Ager-Wiick, Peng, DePaola,
Sørum and Munang’andu. This is an openaccess article distributed under the terms of
the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted which
does not comply with these terms.
Frontiers in Microbiology
Section for Experimental Biomedicine, Department of Production Animal Clinical Sciences, Faculty of
Veterinary Medicine, Norwegian University of Life Sciences, Ås, Norway, 2 State Key Laboratory of
Biocontrol, Guangdong Key Laboratory of Pharmaceutical Functional Genes, School of Life Sciences,
Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Higher Education Mega
Center, Sun Yat-sen University, Guangzhou, China, 3 Angelo DePaola Consulting LLC, Coden, AL,
United States, 4 Department of Paraclinical Sciences, Faculty of Veterinary Medicine, Norwegian
University of Life Sciences, Ås, Norway, 5 Faculty of Biosciences and Aquaculture, Nord University, Bodø,
Norway
1
Aeromonas veronii is a Gram-negative bacterium ubiquitously found in aquatic
environments. It is a foodborne pathogen that causes diarrhea in humans and
hemorrhagic septicemia in fish. In the present study, we used whole-genome
sequencing (WGS) to evaluate the presence of antimicrobial resistance (AMR)
and virulence genes found in A. veronii Ah5S-24 isolated from catfish pond
sediments in South-East, United States. We found cphA4, dfrA3, mcr-7.1, valF,
blaFOX-7, and blaOXA-12 resistance genes encoded in the chromosome of A. veronii
Ah5S-24. We also found the tetracycline tet(E) and tetR genes placed next to
the IS5/IS1182 transposase, integrase, and hypothetical proteins that formed
as a genetic structure or transposon designated as IS5/IS1182/hp/tet(E)/tetR/
hp. BLAST analysis showed that a similar mobile gene cassette (MGC) existed in
chromosomes of other bacteria species such as Vibrio parahaemolyticus isolated
from retail fish at markets, Aeromonas caviae from human stool and Aeromonas
media from a sewage bioreactor. In addition, the IS5/IS1182/hp/tet(E)/tetR/hp
cassette was also found in the plasmid of Vibrio alginolyticus isolated from shrimp.
As for virulence genes, we found the tap type IV pili (tapA and tapY), polar flagellae
(flgA and flgN), lateral flagellae (ifgA and IfgL), and fimbriae (pefC and pefD) genes
responsible for motility and adherence. We also found the hemolysin genes (hylII,
hylA, and TSH), aerA toxin, biofilm formation, and quorum sensing (LuxS, mshA,
and mshQ) genes. However, there were no MGCs encoding virulence genes
found in A. veronii AhS5-24. Thus, our findings show that MGCs could play a vital
role in the spread of AMR genes between chromosomes and plasmids among
bacteria in aquatic environments. Overall, our findings are suggesting that MGCs
encoding AMR genes could play a vital role in the spread of resistance acquired
from high usage of antimicrobials in aquaculture to animals and humans.
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10.3389/fmicb.2023.1112941
KEYWORDS
Aeromonas veronii, antimicrobial resistance, mobile gene cassette, virulence,
tetracycline, environment, foodborne
1. Introduction
tetracycline, sulphonamide, and trimethoprim widely used in
aquaculture are packaged in MGCs. Thus, although previous studies
have focused on identifying individual genes associated with
resistance, the cassettes responsible for the spread of AMR genes has
not been widely investigated for bacteria found in aquaculture.
In the present study we used WGS to profile all AMR and
virulence genes found in A. veronii Ah5S-24 isolated from pond
sediment obtained from the South East, USA by DePaola et al. (1988).
Although in the previous study, they detected presence of
Oxytetracycline-resistance (OTcr) and tetracycline-resistance (Tcr) by
selecting for isolates that replicated on MacConkey agar containing
oxytetracycline or tetracycline antibiotics, they did not determine
whether the resistance gene was located in the chromosome or
plasmids. Even though they showed the transfer of OTcr and Tcr
resistance from the Aeromonas isolate to Escherichia coli, they did not
determine whether the transfer was plasmid mediated or MGC. Thus,
we wanted to determine whether the OTcr and Tcr resistance in the
isolate was encoded in the chromosome or plasmid. We also wanted
to determine whether the resistance detected was associated with a
tetracycline genetic structure similar to that found in other bacteria
species. We anticipate that data presented herein will underscore the
importance of screening for MGCs carrying AMR genes from aquatic
organisms with potential transmission to animals and humans.
Aeromonas veronii is a Gram-negative bacterium ubiquitously
found in different aquatic environments. It was first reported by
Hickman-Brenner et al. (1987) as a new species in 1983. It is
pathogenic to several fish species that include the top farmed species
such as common carp (Cyprinus carpio), channel catfish (Ictalurus
punctatus), tilapia (Oreochromis niloticus), and pangasius (Pangasius
hypophthalmus) (González-Serrano et al., 2002; Smyrli et al., 2017,
2019; Wang et al., 2022). It causes hemorrhagic septicemia and skin
ulcers in fish (Hoai et al., 2019; Tekedar et al., 2020) and diarrhea in
humans (Roberts et al., 2006). Strain variations have been linked to
virulence leading to studies aimed at identifying the virulence factors
associated with mortalities (González-Serrano et al., 2002; Smyrli
et al., 2017, 2019; Wang et al., 2022). The high mortalities experienced
in aquaculture have led to use of antibiotics, thereby contributing to
increase of antimicrobial resistance (AMR) (Roberts et al., 2006). As
mentioned in our previous studies (Dubey et al., 2022a,b), the major
limitation with most studies aimed at identifying AMR genes in
bacteria is that they are mostly done by PCR that only detects AMR
genes based on the primers used in the assay. This poses the risk of
omitting important AMR genes whose primers are not included in
PCR assays. Besides, PCR-based assays do not determine whether the
AMR genes are intrinsically encoded in the chromosomes or
extrinsically in plasmids. So, the use of whole-genome sequencing
(WGS) able to detect all genes and their location in bacteria genomes
is a better approach for elucidating the role of different bacteria species
in the spread of AMR and virulence genes than PCR-based assays.
The spreading of AMR genes by horizontal transfer is contributing
to involvement of bacteria species outside the 12 bacteria families
enlisted to pose the greatest AMR threat to human health by the
World Health Organization (WHO) (Willyard, 2017). As pointed out
by White et al. (2001), the spread of AMR genes is enhanced when
they form part of mobile gene cassettes (MGCs) or transposons. The
MGCs were first identified as integrated AMR genes found in
integrons in the early 1980s (Ward and Grinsted, 1982; Meyer et al.,
1983; White et al., 2001). Although studies done this far have focused
on cassettes carrying AMR genes, it is likely that the packaging in
cassettes includes other genes such as virulence factors. As stated by
White et al. (2001), MGCs facilitate horizontal gene transfer using
various mechanisms that include mobilization of individual cassettes
by integrons (Collis and Hall, 1992), movement of integrons having
cassettes by transposases (Brown et al., 1996; Craig, 1996; Minakhina
et al., 1999), dissemination of larger transposons carrying integrases
(Liebert et al., 1999), and translocation of conjugative plasmids having
integrases among bacteria (White et al., 2001). It is likely that most of
the AMR genes associated with infections in aquaculture, livestock
and humans are part of MGCs (Recchia and Hall, 1995). Yet, gene
cassettes conferring resistance to antibiotics used in aquaculture have
not been widely investigated as done in mammalian studies. Hence, it
is unknown whether the AMR genes selected against drugs like
Frontiers in Microbiology
2. Methodology
2.1. Bacteria culture, characterization, and
antibiotic diffusion test
A suspected Aeromonas hydrophila isolated from pond sediments
in the South-Eastern USA by DePaola et al. (1988) in 1988 was
retrieved from the –80°C freezer at the Norwegian University of Life
Sciences (NMBU), Ås, Norway. The isolate was kindly provided by Dr.
Angelo DePaola, Gulf Coast Seafood Laboratory, United States. After
thawing, the bacteria isolate was streaked on blood agar and incubated
at 10°C for 5–7 days. Single colonies were streaked on tryptone soy
agar (TSA) for purification followed by characterization using the
Matrix-assisted laser Desorption/Ionization-Time of Flight
(MALDI-TOF) mass spectrometry while DNA was extracted based
on manufacturer’s protocol (Qiagen, Germany). Identification of the
bacteria species was done by PCR using universal 16S rRNA primers
27F and 1492R. Phenotypic characterization of antibiotic resistance
was done using the Kirby-Bauer disk diffusion test (Joseph et al.,
2011). The commercial antibiotic discs (Neo-Sensitabs™, Rosco) used
consisted of Penicillin (PEN-10 μg), Amoxicillin (AMOXY-30 μg),
Ampicillin (AMP-10 μg), Ciprofloxacin (CIPR-5 μg), Cefoxitin
(CFO-30 μg), Cephalothin (CEP-30 μg), Tetracycline (TET-30 μg),
Gentamycin (GEN-10 μg), Rifampicin (RIF-5 μg), Sulfonamide
(SULFA-240 μg), Trimethoprim (TRIM-5 μg), Erythromycin
(Ery-15 μg), Nitrofurantoin (NI-300 μg), and (Colistin-CO-150 μg)
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Table 1 Overview of antibiotic resistence genes detected in the draft
genome of Aeromonas veronii AhS5-24 together with phenotypic
antibiotic susceptibility testing results using disk diffusion assay.
Resistance
mechanism
Resistance
gene
Antibiotic class
Antibiotic
blaFOX-7
Cephamycin
inactivation
Antibiotic
Results
Cefoxitin
R
followed by adding 15 μl of MagAttract Suspension G and 280 μl
Buffer MB to each vial (Tarumoto et al., 2017). The suspension from
each tube was transferred onto the MagAttract holder followed by
mixing for 1 min on an Eppendorf thermomixer. The magnetic beads
having the gDNA were separated on the MagAttract magnetic rack for
approximately 1 min. Supernatants were removed without disturbing
the beads followed by washing the magnetic beads twice using MW1
and PE buffer (Becker et al., 2016; Tarumoto et al., 2017). The
remaining suspension was removed by washing the beads twice using
1 ml RNAase free water (Qiagen GmbH, Hilden, Germany) (Becker
et al., 2016). The gDNA was harvested by eluting in 100 μl buffer EB
while purity was evaluated using the NanoDrop (Thermo Fisher,
United States) and gel electrophoresis using 1% agarose. Quantification
of gDNA was carried out using the Qubit double-stranded DNA
high-CHS kit following the manufacturer’s guidelines (Life
Technologies Inc., Carlsbad, CA, United States) (Guan et al., 2020).
(CFO30)
blaOXA-12
Cephalosporin
Cephalothin
R
(CEP 30)
cphA4
Amoxicillin
β-lactams
R
(AMOXY)
Antibiotic
tet(E)
Tetracycline
Tetracycline
efflux
R
(TET30)
MexB
CRP
Sulfonamide,
Sulfonamide
β-lactams
(SULFA)
Macrolide
Erythromycin
I
S
(ERY15)
Antibiotic
mcr-7.1
Peptide
Colistin
target
2.3. Library preparation, sequencing, and
bioinformatics analysis
S
(CO150)
alteration
Antibiotic
vatF
–
–
–
dfrA3
Diaminopyrimidine
Trimethoprim
I
target
Library preparation was carried out using Nextera DNA Flex
(Tagmentation Illumina Inc. San Diego, CA, United States) while
Illumina MiSeq were used with a paired-end read length of 2 × 300 bp.
The bacterial raw DNA reads were analyzed using the online Galaxy
platform1 version 21.05. Quality of both forward and reverse raw reads
were analyzed using the FastQC Version 0.11.9 software
(Bioinformatics B, 2011), while the Trimmometric version 0.38.1 was
used to remove the adapters and low-quality reads from paired-end
sequences (Bolger et al., 2014). The resulting paired-end sequence
reads were de novo assembled using SPAdes v. 3.12.0 (Coil et al., 2015)
with 33 to 91 k-mers (Bankevich et al., 2012) while genome annotation
was done using the prokaryotic genome annotation pipeline (PGAP)
(Tatusova et al., 2016) from the National Center for Biotechnology
and Information (NCBI) and Prokka (Seemann, 2014). Online Galaxy
platform (see Footnote 1) version 21.05 was used for
bioinformatic analysis.
(TRIM5)
replacement
Other
Fluoroquinolone
Ciprofloxin
resistance
S
(CIPR5)
mechanism
Aminoglycoside
Gentamicin
S
(GEN10)
Nitrofuran
Nitrofurantoin
S
(NI300)
Rifamycin
Rifampicin
S
RIF.5
(Table 1). A volume of 100 μl containing freshly cultured bacteria
diluted at McFarland concentration of 108 CFU/ml was spread on
Müller Hinton agar followed by putting the antibiotic discs on the
bacteria lawn. Next, the plates were incubated at 30°C overnight
followed by measuring the susceptibility or resistance based on the
Clinical and Laboratory Standards Institute (CLSI) guidelines
(Kahlmeter et al., 2006; Cockerill et al., 2012).
2.4. Pangenome analysis
Pangenome analysis of A. veronii AhS5-24 together with 30
complete genomes of other A. veronii isolates retrieved from the NCBI
was carried out using Roary v. 3.13.0 using general feature files 3 (.gff)
file generated from Prokka v. 1.14.5. The phylogenetic tree was made
using the Phandango software using Gene_presence_absence and
Newick files obtained from Roary v. 3.13.0. The average nucleotide
identity (ANI) of all 31 A. veronii genomes was computed using
FastANI v1.3 using A. veronii FC951 (CP032839) as a reference strain.
Antimicrobial resistance (AMR) genes were identified using staramr
version 0.7.2 (Tran et al., 2021) and ABRicate v1.0.1 (Seemann, 2016)
in the Comprehensive antimicrobial resistance database (CARD)
(Alcock et al., 2020) and staramr v. 0.7.2 with the identification
threshold set at 80%. Plasmidfinder v 2.0 (Ullah et al., 2020) was used
2.2. DNA extraction
Genomic DNA (gDNA) was extracted as previously described
(Becker et al., 2016) using the MagAttract HMW DNA kit based on
the manufacturer protocols (Qiagen GmbH, Hilden, Germany).
Briefly, a 1 ml volume of approximately 2 × 109 CFU/ml of freshly
overnight cultured bacteria was spanned in 2 ml Eppendorf tubes
followed by suspending the pellets in 180 μl buffer ATL (tissue lysis
buffer, Qiagen GmbH, Hilden, Germany). Next, Proteinase K was
added to each vial at a concentration of 20 mg/ml followed by
incubation at 56°C in an Eppendorf thermomixer for 30 min.
Afterward, 4 μl RNase was added and the vials were pulse vortexed
®
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1 https://usegalaxy.no/
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FIGURE 1
Phylogenetic tree based on pangenome analysis of Aeromonas veronii AhS5-24 together with 30 complete genomes of other A. veronii strains
obtained from the National Center for Biotechnology and Information (NCBI). Note that A. veronii AhS5-24 is clustered together with A. veronii strains
FC951 (CP032839) isolated from hospital sewage. A total of 20352 genes were detected in all 31 A. veronii genomes by pangenome analysis using the
Roary software of which 1,429 genes were core genes, 875 soft core genes while the number of shell and cloud genes was 2,241 and 15,807 genes,
respectively.
to identify plasmids in the bacterial genomes while virulence genes
were identified using virulence factors database (VFDB). Genome
circular maps were created using Proksee.2
of 4,493 genes were predicted with 4,334 genes coding for proteins.
The genome contained a total of 108 genes of RNA consisting of 99
tRNA and 5 rRNAs. The total number of genes detected from the 31
A. veronii genomes based on pangenome analysis was 20,352 of which
1,429 genes were core-, 875 softcore-, 2,241 shell-, and 15,807 cloud
genes. The phylogenetic tree divided the genomes into three groups of
which strain AhS5-24 was closely related to the human CP032839
(FC951) and hospital sewage CP079823 (HD6454) isolates (Figure 1).
The average nucleotide identity (ANI) analysis using FastANI showed
high similarity (>93%) of all 31 A. veronii isolates despite coming from
different host species and geographic locations. The ANI of strain
AhS5-24 was 96.31% similar with the A. veronii CF951 (CP032839)
human clinical isolate and 96.20% similar with A. veronii HD6454
(CP079823) from hospital sewage.
The virulence genes found in A. veronii AhS5-24 comprised the
motility and adherence genes that included the (i) lateral flagella
proteins consisting of lfgA and lfgL, (ii) polar flagella that were
represented by flgA and flgN, (iii) members of the tap type IV pili that
included tapA, tapW and tapY, and (iv) fimbrial adherence
determinants that included pefC and pefD genes (Figure 2). The
mannose-sensitive hemagglutinin (MHSA) is encoded by the genes
mshA and mshQ (Figure 2). Genes associated with capsule formation
and immune evasion included ddhA, ddhC, and wcaG1. The
hemolysin genes detected were hlyA, hylIII, and thermostable
hemolysin (TSH) while toxin genes consisted of aerolysin aerA. Genes
associated with iron acquisition consisted of the Iron ABC transporter
while biofilm formation and quorum sensing genes were represented
by luxS and MshA-Q pilus. We detected genes belonging to the type II
secretion systems (T2SS) represented by exeA to exeN
(Supplementary Table S1) and vgrG, which is part of T6SS (Figure 2).
Overall, the virulence genes detected belonged to motility, adherence,
secretion systems, iron acquisition, biofilm formation, quorum
sensing, and immune evasion groups.
2.5. Phylogenetic analysis of antimicrobial
resistance genes
Phylogenetic comparison of the tet(E) and tetR genes from strain
AhS5-24 with other A. veronii isolates was done using the Molecular
Evolutionary Genetic Analysis version 7 (MEGA-7) software (Kumar
et al., 2016). The tet(E) and tetR sequences from strain AhS5-24 were
retrieved after screening using ABRicate version 1.0.1 followed by
comparison with tet(E) and tetR sequences from other A. veronii
isolates retrieved from NCBI. Phylogenetic trees were produced using
the Neighbor-joining and BioNJ algorithm to a pairwise matrix
estimated using JTT model and expressed as number of base
substitution per site (Jones et al., 1992).
3. Results
3.1. Genome organization and pangenome
analysis
The draft genome of A. veronii AhS5-24 showed a high similarity
with other A. veronii genomes, as shown in Figure 1. The draft genome
of strain AhS5-24 had a size of 4,748,224 bp with G + C content of
58.48%. It contained 157 contigs with an N50 value of 115,408. A total
2 https://proksee.ca/
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FIGURE 2
Circular map showing the loci for different virulence genes in the genome of Aeromonas veronii strain AhS5-24. Motility and adherence genes
detected included the lateral flagella ifgA and IfgL genes; polar flagella flaA and flgN genes; tap type IV pili tapA and tapY; fimbriae pefC and pefD
genes. The mannose-sensitive hemagglutinin (MHSA) genes were represented by mshA and mshQ while the hemolysin genes included hylA, hylIII, and
TSH. Iron acquisition was represented by the iron ABC transporter protein and genes associated with capsule formation included the ddhA, ddhC, and
wcaG1 genes. Quorum sensing and biofilm formation genes were represented by luxS. The T2SS was represented by exeA and exeN genes while T6SS
was represented by the vgrG gene.
3.2. Phenotype characterization of
antimicrobial resistance genes
protein, and phosphoadenyl-sulfate reductase (Figure 3). The efflux
pumps detected included the resistance-nodulation-cell division
(RND) mexB and smeD that were placed next to each other together
with the IS5 transposase (Figure 3).
Our findings show that the repressor of the tetracycline resistance
element gene tetR was placed next to tet(E) together with the
IS5/IS1182 transposase, helicase, integrase, tyrosine type recombinase/
integrase, and the site-specific integrase all in one cassette (Figure 3).
The cassette found in A. veronii AhS5-24 showed a high similarity
with cassettes found in Vibrio parahaemolyticus (MN199028.1)
isolated from a fish market, Vibrio alginolyticus plasmid (MN865127.1)
from shrimp, Aeromonas caviae (CP110176) from human stool, and
Aeromonas media (CP03844.1) from a sewage bioreactor (Figure 4).
They all had a similar genetic structure or transposon consisting of the
IS5/IS1182 transposase followed by a gene encoding a hypothetical
protein (hp), Tet(E), tetR, and another hypothetical protein (hp),
thereby forming a MGC designated as 1S5/IS1182/hp/tet(E)/tetR/hp
(Figure 4). Suffice to point out that the cassette from Vibrio
alginolyticus (MN865127.1) was from a plasmid, while the cassettes
from A. veronii AhS5-24, Vibrio parahaemolyticus (MN199028.1),
A. media (CP038444.1), and Aeromonas caviae (CP110176) were from
chromosomes. This findings demonstrate that the IS5/IS1182/hp/
tet(E)/tetR/hp cassette can be found both in chromosomes and
plasmids of different bacteria species. It is noteworthy that the cassette
for Vibrio alginolyticus plasmid (MN865127.1) had the IShfr9
transposase, and not the IS5/IS1182 transposase, despite having a
Results of the disk diffusion test showed that strain Ah5S-24 was
resistant to CFO30, CEP30, AMOXY30, and TET30, whereas it
showed intermediate resistance against SULFA240 and TRIM5
(Table 1). However, it was susceptible to ERY15, CO150, CIPR5,
GEN10, NI300, and RIF5. We found an overall correlation kappa
score of 82% (Cohen’s k = 0.8235) with a specificity of 91.66% and
sensitivity of 93% between the phenotypic profile based on the disk
diffusion test and genotypic profile based on the genes identified using
the CARD (Alcock et al., 2020).
3.3. Genotype characterization of
antimicrobial resistance genes
Identification of AMR genes using the CARD (Alcock et al., 2020)
showed that strain Ah5S-24 encoded multiple AMR genes that
included the β-lactamase like blaFOX-7, blaOXA-12, and cphA4. Other
genes detected included the colistin crp and mcr-7.1 genes as well as
the streptogramin A acetyl transferase vatF gene (Figure 3). There
were no integrase and transposases located near the blaFOX-7, blaOXA-12,
cphA4, crp, mcr-7.1, and vatF genes. The trimethoprim dfrA3 gene was
placed together with the sulfurtransferase, DUF2541 family protein,
mog, DUF3135 domain-containing protein, threonine exporter
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FIGURE 3
Circular genomic map of Aeromonas veronii AhS5-24 showing the loci for antimicrobial resistance (AMR) genes. The AMR genes detected included the
β-lactam blaOXA-12 and blaFOX-7 genes together with cphA4, dfrA3, mcr-7.1, and vatF genes while the efflux pump proteins detected were CRP, smedD,
and mexB. The extended linear map (A) shows the cassette encoding the site-specific integrase, tyrosine-type recombinase/integrase, integrase,
helicase, IS5/IS1182 transposase, tet(E) efflux pump protein gene and tetR gene designated as IS5/IS1182/hp/tet(E)/tetR/hp. The extended line map
(B) shows the linear relationship between the trimethoprim dfrA3 gene and other genes that includes sulfurtransferase, DUF2541 family protein, mog,
DUF3135 domain-containing protein, threonine/serine exporter protein, dfrA3 and phosphoadenyl-sulfate reductase in the genome of A. veronii AhS524. The extended map (C) shows the linear relationship between the smeD and mexB efflux pumps in the genome of A. veronii AhS5-24.
FIGURE 4
Comparison of the IS5/IS1182/hp/tet(E)/tetR/hp gene cassettes for Aeromonas veronii strain AhS5-24 from pond sediments, Vibrio parahaemolyticus
(MN199028) isolated from retail fish from a market, Vibrio alginolyticus plasmid (MN865127.1) from shrimp, Aeromonas media (CP038444.1) from
sewage bioreactor and Aeromonas caviae (CP110176.1) from human stool. Note that all isolates had the hypothetical proteins (hp), tetR, tet(E), and
IS5/IS1182 transposase forming a gene cassette designated as IS5/IS1182/hp/tet(E)/tetR/hp. The uppermost linear map shows Aeromonas veronii strain
AhS5-24 having the IS5/IS1182/hp/tet(E)/tetR/hp cassette linked to the DNA helicase, two hypothetical proteins, integrase, tyrosine-type recombinase/
integrase and site-specific integrase.
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similar hp/tet(E)/tetR/hp component with other bacteria species used
in the comparison (Figure 4).
Phylogenetic analysis showed that the tet(E) gene from A. veronii
AhS5-24 had a 100% similarity with tet(E) genes from different
bacteria species that included Escherichia coli (AIL23572.1,
CAC20135.1, and WP_20194468.1), Aeromonas caviae (BBR12376.1,
WP_244056220.1, and WP_201964468.1), Yersinia ruckeri
(APO36645.1, APO36648.1, and APO36646.1), Klebsiella pneumoniae
(EIW8806435.1),
Aeromonas
spp.
(QEV84027.1
and
WP_017780889.1), and Enterobacter cloacae (ASF90526.1) (Figure 5).
Phylogenetic analysis also showed that the tetR gene from A. veronii
AhS5-24 had a 100% similarity with tetR genes from different bacteria
species that included E. coli (AAA98409.1), Gammaproteobacteria
(W_P011899269.1 and WP_017411289.1), Aeromonas salmonicida
(QJR83010.1), Aliivibrio salmonicida (CAC81917.1), and A. caviae
(WP_223946105.1 and WP_223945860.1) (Figure 6). Altogether, our
findings show that tet(E) and tetR genes were highly similar with those
found in different bacteria species.
designated as strain Ah5S-24 in this study. In addition, strain Ah5S-24
encoded several virulence and AMR genes of which tetracycline
resistance genes were placed in the same genetic structure with an
integrase, transposase and recombinase and can be defined as a
transposon. These findings demonstrate that Aeromonas spp. isolated
from aquatic environments have the potential to transmit AMR genes
to other bacteria using transposons carrying different AMR genes.
Pangenome analysis showed a high similarity of strain Ah5S-24
with other A. veronii strains linked to different diseases in aquatic
organism and humans. For example, strains CP032839.1 and
CP046407.1 shown to be closely related with A. veronii Ah5S-24 were
from human clinical cases (Ragupathi et al., 2020) and diseased rohu
(Labeo rohita) (Tyagi et al., 2022), respectively. Besides, A. veronii
Ah5S-24 had several virulence genes linked to adherence, biofilm
formation, quorum sensing, immune evasion, toxins and intracellular
secretion systems (TSS) found in other pathogenic A. veronii strains
(Arechaga and Cascales, 2022). Detection of the Msh pili, tap type IV
pili, lateral-and polar flagellar genes associated with intestinal
adherence, colonization, and biofilm formation (Ro, 2006; Hadi et al.,
2012) is suggestive that these genes play a vital role in the pathogenicity
of strain Ah5S-24. The presence of the LuxS and mshQ genes is
suggestive that strain Ah5S-24 has the capacity for biofilm formation
and quorum sensing as seen in other bacteria species (Enos-Berlage
et al., 2005; Trappetti et al., 2011) while presence of the iron ABC
transporter is suggestive that strain Ah5S-24 uses this protein in
acquiring iron from infected hosts (Delepelaire, 2019). Detection of
ddhA, ddhC, and wcaG1 associated with capsule formation (Mobine,
2008) is suggestive strain Ah5S-24 has the ability to form a capsule as
a defense mechanism against host immune responses while presence
of hylA, hylII,I, and TSH together with aerolysin aerA is suggestive
that these genes might be linked to pore formation and intracellular
release of enterotoxins by strain Ah5S-24 as seen in other bacteria
species (Honda et al., 1992; Baida and Kuzmin, 1996; Abrami et al.,
2000; Maté et al., 2014). Besides, several scientists (Wong et al., 1998;
Heuzenroeder et al., 1999; Wu et al., 2007; Castilho et al., 2009) have
shown that a combination of hylA(+) and aerA(+) is a major virulence
determinant in Aeromonas spp. Castilho et al. (2009) found a high
prevalence of hemolytic and cytotoxic Aeromonas spp. that had both
hylA(+) and aerA+ from human clinical, food, and environmental
samples in Brazil while Wu et al. (2007) showed that the absence of
hlyA(−) and aerA(−) in Aeromonas spp. from fish and human samples
in Taiwan was associated with low virulence. Heuzenroeder et al.
(1999) and Wong et al. (1998) showed that deletion or attenuation of
the hlyA(+) and aerA(+) double mutant significantly reduced the
pathogenicity of A. hydrophila in mice. They also showed that
cytotoxicity to buffalo green monkey kidney cells and hemolysis on
horse blood agar was only eliminated in the double and not in the
single mutants of A. veronii, A. hydrophila, and A. caviae. Our findings
show that A. veronii Ah5S-24 had both hlyA(+) and aerA(+),
indicating that it shares the two key virulence determinants with other
pathogenic Aeromonas spp.
Several studies have shown that Aeromonas spp. intrinsically
carry various blaOXA genes in their genomes that include the blaOXA12 gene (Dubey et al., 2022a,b) previously detected in A. media,
A. jandaei, A. sobria, A. dhakensis, and A. hydrophila (Rasmussen
et al., 1994; Alksne and Rasmussen, 1997; Hilt et al., 2020; Huang
et al., 2020; Dubey et al., 2022a) being in line with its presence in
strain Ah5S-24 while blaFOX-7 previously reported in A. media and
4. Discussion
In this study, we have shown that the bacteria isolated from pond
sediments in the South East USA previously classified as A. hydrophila
using the API 20E system in DePaola et al. (1988) was characterized
as A. veronii Ah5S-24 using WGS and pangenome analysis. We have
also shown that the Tr and TOr detected by DePaola et al. (1988) could
be linked to the tetR and tet(E) genes found in the same isolate
FIGURE 5
Phylogenetic tree comparing the tet(E) gene from different bacteria
species. Note that the tet(E) Aeromonas veronii strain AhS5-24 had
100% similarity with tet(E) genes from other bacteria species that
include Aeromonas caviae (BBR12376.1, GJA56797.1, and
WP/244056220.1 and WP_20964468.1), Yersinia ruckeri
(Apo036645.1, APO36646.1, and APO36648.1), Enterobacter cloacae
(ASF90526.1), Escherichia coli (AIL23572.1, CAC20135.1,
WP_063856076.1 and HBL6787278.1), and Klebsiella pneumoniae
(EIW8806435.1).
Frontiers in Microbiology
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Dubey et al.
10.3389/fmicb.2023.1112941
FIGURE 6
Phylogenetic tree comparing the tetR gene from different bacteria species. Note the high (100%) similarity between the tetR gene of Aeromonas
veronii strain AhS5-24 with tetR genes of Escherichia coli (AAA98409.1), Gammaproteobacteria (WP_011899269.1 and WP_017411289), Aeromonas
salmonicida (QJR83010.1), Aeromonas spp. (WP_04288770.1), and Aeromonas caviae (WP_223946105.1 and WP_223945860.1).
A. allosaccharophila was also found in strain Ah5S-24 (Ebmeyer
et al., 2019). Other AMR genes detected included the cphA4 gene
known to be intrinsically encoded in various Aeromonas spp.
(Dubey et al., 2022a,b) as well as the colistin-resistance mcr-7.1
gene also reported from different Aeromonas spp. (Dubey et al.,
2022a,b). Despite so, the blaOXA-12, blaFOX-7, and mcr-7.1 genes
detected in strain Ah5S-24 were not associated with integrases,
recombinases or transposases suggesting that these genes could not
be easily transferred or acquired from other bacteria species.
Similarly, although trimethoprim and sulfonamide are among the
most widely used antibiotics linked to AMR in aquaculture (Gao
et al., 2012; Muziasari et al., 2014; Phu et al., 2015), the trimethoprim
resistance gene dfrA3 detected in the present study was not linked
to integrases and transposases. Thus, the sulfonamide and
trimethoprim resistance observed in the disc diffusion test could
have been mediated by the MexB and smeD pumps that have been
associated with resistance of several drugs that include sulfonamide,
fluoroquinolone, cephalosporins, carbapenem, and trimethoprim.
The trimethoprim and sulfonamide resistance observed on the disc
diffusion test was intermediate (I) unlike the tetracycline resistance
(R), which was highly expressed suggesting that the impact of
trimethoprim and sulfonamide in conferring resistance was not as
high as tetracycline in strain AhS5-24. Despite so, we found a high
correlation of kappa score of 82% (Cohen’s k = 0.8235) with a
specificity of 91.66% and sensitivity of 93% between the phenotype
characterization based on the disc diffusion test and genotypic
Frontiers in Microbiology
characterization based on the CARD (Alcock et al., 2020),
indicating that the two diagnostic tests were highly in agreement.
Tetracycline is one of the most widely used antibiotics in
aquaculture, which has been linked to resistance in farmed aquatic
organisms (Seyfried et al., 2010; Tamminen et al., 2011). Thus, it is
likely that selection of the Tet E operon in strain AhS5-24 occurred in
pond sediments used for aquaculture where tetracycline was used for
the treatment of fish diseases. Although the absence of plasmids is
suggestive that strain Ah5S-24 had lesser chances of transferring AMR
genes to other bacteria, detection of the Tet E operon together with
the integrase and IS5/IS1182 transposase suggests that tetR and tet(E)
genes could be transferred or acquired from other bacteria using the
IS5/IS1182/hp/tet(E)/tetR/hp cassette encoded in strain Ah5S-24.
Besides, DePaola et al. (1988) used the same isolate to transfer the OTr
and Tr resistance to E. coli suggesting that the IS5/IS1182/hp/tet(E)/
tetR/hp cassette found in strain AhS5-24 could have been responsible
for transferring the tetracycline resistance to E. coli. Also, detection of
the same cassette in V. parahaemolyticus, V. alginolyticus
(MN199028.1), A. media (CP038444.1), A. caviae (CP110176.1), and
A. caviae (CP038445.1) emanating from fish market, shrimp, sewage
bioreactor and human stool is suggesting that the IS5/IS1182/hp/
tet(E)/tetR/hp transposon could be involved in interspecies
transmission of the tet(E) and tetR genes in different bacteria species.
These findings also suggest that the IS5/IS1182/hp/tet(E)/tetR/hp
transposon might be in existence in different bacteria species found
in different aquatic environments hosted by species that include
08
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10.3389/fmicb.2023.1112941
shrimps, fish, animals and humans. Its presence in V. parahaemolyticus
(MN199028.1) isolated from retail fish at markets and V. alginolyticus
(MN865127.1) from shrimps is indicative that it could play a vital role
in transmission of tet(E) and tetR genes to humans through food.
The similarity of the IS5/IS1182/hp/tet(E)/tetR/hp cassette found in
the chromosomes of strain A. veronii AhS5-24, V. parahaemolyticus
(MN199028.1) and A. media (CP038445.1), with the transposon found
in the plasmid of V. alginolyticus (MN865127.1) is suggesting that the
IS5/IS1182/hp/tet(E)/tetR/hp transposon can be transferable between
chromosomes and plasmids of different bacteria species. Also, the high
similarity of the tet(E) and tetR genes detected in strain AhS5-24 with
those found in E. coli, K. pneumoniae, and Aeromonas spp. shown in the
phylogenetic analysis consolidates our view that tet(E) and tetR genes
could be transmissible between different bacteria species using MGCs.
Thus, it is likely that the transfer of the OTr and Tr resistance to E. coli
observed by DePaola et al. (1988) was not plasmid mediated but it was
done by the IS5/IS1182/hp/tet(E)/tetR/hp transposon found in strain
Ah5S-24. Therefore, our findings indicate that the resistance acquired
by different Aeromonas spp. in aquatic environments could play a vital
role in the transfer of AMR genes to foodborne, environmental,
nosocomial and other bacteria species using MGCs. However, future
studies should seek to demonstrate the transfer of tet(E) and tetR genes
using the IS5/IS1182/hp/tet(E)/tetR/hp cassette to other bacteria spp.
including nosocomial, foodborne and environmental bacteria.
Author contributions
5. Conclusion
AD was employed by Angelo DePaola Consulting LLC.
The remaining authors declare that the research was conducted in
the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
SD, HS, and HM: conceptualization, methodology, mobilizing
resources, supervision, data curation, and bioinformatics analysis. SD,
EA-W, BP, AD, HS, and HM: manuscript preparation, editing, and
submission. All authors contributed to the article and approved the
submitted version.
Funding
This study was financed by the Research Council of Norway
(FIFOSA-21 Project) Grant Number 320692 and the National Natural
Science Foundation of China (NSFC) Grant Number 32061133007.
Acknowledgments
The authors are grateful to Erik Hjerde from the Arctic University
of Norway and ELIXIR Norway for guidance on Bioinformatics. Sofie
Persdatter Sangnæs, at the Norwegian University of Life Sciences
(NMBU) for technical support.
Conflict of interest
In this study, we have shown that A. veronii AhS5-24 is a
multidrug-resistant bacterium encoding several AMR and virulence
genes. It encoded a tetracycline resistance operon Tet E placed in a
transposon designated as IS5/IS1182/hp/tet(E)/tetR/hp found in
different bacteria species inhabiting different aquatic environments
and infecting different host species suggesting that the Tet E operon
could be transferred to other bacteria. Overall, this study shows that
MGCs encoding AMR genes found in bacteria inhabiting aquatic
environments could play a vital role in the spread of AMR genes to
other bacteria infecting animals and humans.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated organizations,
or those of the publisher, the editors and the reviewers. Any product
that may be evaluated in this article, or claim that may be made by its
manufacturer, is not guaranteed or endorsed by the publisher.
Data availability statement
Supplementary material
The Aeromonas veronii whole genome shotgun (WGS) project has
the project accession JAJVCX000000000. This version of the project
(01) has the accession number JAJVCX010000000 and consists of
sequences JAJVCX010000001-JAJVCX010000157.
The Supplementary material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1112941/
full#supplementary-material
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