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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell. Infect. Microbiol.</journal-id>
<journal-title>Frontiers in Cellular and Infection Microbiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell. Infect. Microbiol.</abbrev-journal-title>
<issn pub-type="epub">2235-2988</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fcimb.2022.1042350</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cellular and Infection Microbiology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Comparative genomics analysis of the multidrug-resistant <italic>Aeromonas hydrophila</italic> MX16A providing insights into antibiotic resistance genes</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Guo</surname>
<given-names>Yuxin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/2002849"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zeng</surname>
<given-names>Chenxi</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn003">
<sup>&#x2020;</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1897206"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Ma</surname>
<given-names>Chenjie</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cai</surname>
<given-names>Hongjiao</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Jiang</surname>
<given-names>Xinglong</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhai</surname>
<given-names>Shaowei</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/845236"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Xu</surname>
<given-names>Xiaojin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Lin</surname>
<given-names>Mao</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/252662"/>
</contrib>
</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Fisheries College, Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Jimei University</institution>, <addr-line>Xiamen, Fujian</addr-line>, <country>China</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture and Rural Affairs, Xiamen</institution>, <addr-line>Fujian</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Pengfei Li, Guangxi Academy of Sciences, China</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Yuting Deng, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, China; Mingyou Li, Shanghai Ocean University, China</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Mao Lin, <email xlink:href="mailto:linmao@jmu.edu.cn">linmao@jmu.edu.cn</email>
</p>
</fn>
<fn fn-type="equal" id="fn003">
<p>&#x2020;These authors have contributed equally to this work and share first authorship</p>
</fn>
<fn fn-type="other" id="fn002">
<p>This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>03</day>
<month>11</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2022</year>
</pub-date>
<volume>12</volume>
<elocation-id>1042350</elocation-id>
<history>
<date date-type="received">
<day>12</day>
<month>09</month>
<year>2022</year>
</date>
<date date-type="accepted">
<day>18</day>
<month>10</month>
<year>2022</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2022 Guo, Zeng, Ma, Cai, Jiang, Zhai, Xu and Lin</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Guo, Zeng, Ma, Cai, Jiang, Zhai, Xu and Lin</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access 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.</p>
</license>
</permissions>
<abstract>
<p>In this paper, the whole genome of the multidrug-resistant <italic>Aeromonas hydrophila</italic> MX16A was comprehensively analyzed and compared after sequencing by PacBio RS II.&#xa0;To shed light on the drug resistance mechanism of <italic>A. hydrophila</italic> MX16A, a&#xa0;Kirby-Bauer disk diffusion method was used to assess the phenotypic drug susceptibility. Importantly, resistance against &#x3b2;-lactam, sulfonamides, rifamycins, macrolides, tetracyclines and chloramphenicols was largely consistent with the prediction analysis results of drug resistance genes in the CARD database.&#xa0;The varied types of resistance genes identified from <italic>A. hydrophila</italic> MX16A revealed multiple resistance mechanisms, including enzyme inactivation, gene mutation and active effusion.&#xa0;The publicly available complete genomes of 35 <italic>Aeromonas hydrophila</italic> strains on NCBI, including MX16A, were downloaded for genomic comparison and analysis. The analysis of 33 genomes with ANI greater than 95% showed that the pan-genome consisted of 9556 genes, and the core genes converged to 3485 genes. In summary, the obtained results showed that <italic>A. hydrophila</italic> exhibited a great genomic diversity as well as diverse metabolic function and it is believed that frequent exchanges between strains lead to the horizontal transfer of drug resistance genes.</p>
</abstract>
<kwd-group>
<kwd>
<italic>Aeromonas hydrophila</italic>
</kwd>
<kwd>complete genome sequencing</kwd>
<kwd>comparative genomics</kwd>
<kwd>multidrug resistance</kwd>
<kwd>drug resistance mechanism</kwd>
</kwd-group>
<contract-num rid="cn001">2020YFD0900102</contract-num>
<contract-sponsor id="cn001">National Key Research and Development Program of China<named-content content-type="fundref-id">10.13039/501100012166</named-content>
</contract-sponsor>
<counts>
<fig-count count="8"/>
<table-count count="4"/>
<equation-count count="0"/>
<ref-count count="66"/>
<page-count count="15"/>
<word-count count="5685"/>
</counts>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<title>Introduction</title>
<p>Occurrences of multi-drug resistance (MDR) have become increasingly frequent in recent decades, complicating the treatment of certain diseases caused by microorganisms in humans (<xref ref-type="bibr" rid="B63">Williams and Bardsley, 1999</xref>; <xref ref-type="bibr" rid="B60">Thomas et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B12">Boucher et&#xa0;al., 2017</xref>). Multidrug-resistant bacteria (MDRB) could render a large number of antibiotic drugs ineffective, leading to severe public health concerns and affecting virtually all population groups around the world (<xref ref-type="bibr" rid="B36">Lautenbach and Fishman, 1999</xref>).The antibiotic-rich environment promotes horizontal gene transfer between bacteria, increasing the drug resistance of bacteria and causing a great risk to the public (<xref ref-type="bibr" rid="B57">Skwor et&#xa0;al., 2020</xref>).Furthermore, with constant evolution of MDRB, food production and safety may also be affected.</p>
<p>
<italic>Aeromonas</italic> spp., a rod-shape and facultative anaerobic bacteria species, is widely distributed in natural environments, particularly in aquatic media (<xref ref-type="bibr" rid="B13">Chauret et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B53">Scoaris et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B5">Araujo et&#xa0;al., 1991</xref>; <xref ref-type="bibr" rid="B48">Rafei et&#xa0;al., 2018</xref>). The mesophilic species exhibits optimal growth at 35&#x2013;37 &#xb0;C and is involved in a variety of human infections whereas sychrophilic species can be grown at lower temperatures of 22&#x2013;25 &#xb0;C and frequently result in nonmotile diseases in fish (<xref ref-type="bibr" rid="B32">Janda and Abbott, 2010</xref>). This species of gram-negative bacteria, <italic>A. hydrophila</italic> being one representative, is commonly associated with a variety of diseases including septicaemia, gastroenteritis, and wound infections frequently affecting aquatic animals, terrestrial animals, and humans (<xref ref-type="bibr" rid="B2">Adamski et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B44">Parker and Shaw, 2011</xref>; <xref ref-type="bibr" rid="B29">Igbinosa et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B51">Rutteman, 2017</xref>).</p>
<p>From 1977 to 2016, three generations of various types of sequencing technologies have been developed. The second and third generation sequencing technologies referred commonly to as next generation sequencing technology, has evolved significantly with increase in sequencing speed, decrease in sequencing cost, since its inception in 2004. Currently,  the third-generation sequencing platforms include Helicos&#x2122; Genetic Analysis System by SeqLL, SMRT Sequencing by Pacific Biosciences, Nanopore sequencing by Oxford Nanopore, to name a few (<xref ref-type="bibr" rid="B4">Ambardar et&#xa0;al., 2016</xref>). PacBio&#x2019;s DNA sequencing has been used for detailed genomic investigations of bacterial isolates. Whole genome sequencing of bacteria has become an important tool for understanding and analyzing bacterial evolution. The complete understanding of the antibiotic resistance mechanisms of MDR <italic>A. hydrophila</italic> is crucial for the&#xa0;further development of therapeutic agents. We therefore conducted PacBio&#x2019;s single molecule real time (SMRT&#x2122;) DNA sequencing and the preliminary analysis of the MDRB strain <italic>A. hydrophila</italic> MX16A. Our results highlight resistance-related genes to aid in the understanding of genetic mechanisms of drug resistance of <italic>A. hydrophila</italic> and the preliminary prediction of virulence factors present in the genome. We further conducted a comparative genomic analysis of MX16A and reference <italic>A. hydrophila</italic> strains to generate the pan-core genome in order to understand the exclusive and shared genes and characteristics of the species of <italic>A. hydrophila</italic>. The results obtained herein will mine and develop the value of the existing reference genomes, and gain complete variation information of the entire species.</p>
</sec>
<sec id="s2" sec-type="materials|methods">
<title>Materials and methods</title>
<sec id="s2_1">
<title>Bacterial strain and antimicrobial susceptibility test</title>
<p>
<italic>A. hydrophila</italic> strain MX16A was isolated from Jiulong River, Fujian, China (<xref ref-type="bibr" rid="B37">Lin et&#xa0;al., 2015</xref>), and cultured with Luria-Bertani broth (Hopebio, Qingdao, China) at 30 &#xb0;C in a shaking incubator for 24&#xa0;h. In previous experiments, MX16A was found to be insensitive to a variety of antibiotics, and contain various resistance genes and virulence genes detected by PCR.The toxicity tests showed that the median lethal concentration (LD50) of <italic>A. hydrophila</italic> MX16A to zebrafish was 1.6&#xd7;10<sup>6</sup> CFU/fish, indicating that MX16A had pathogenicity to aquatic animals. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disk diffusion method (OXOID, UK). All results were determined by the Clinical and Laboratory Standards Institude (CLSI) guidelines (M100-S23).</p>
</sec>
<sec id="s2_2">
<title>DNA extraction and 16S rRNA gene sequencing</title>
<p>Bacterial genomic DNA was extracted using the TIANamp Bacteria DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer&#x2019;s instructions. The strain identity was confirmed by 16S rRNA gene sequencing using specific primers (27F: 5&#x2019;-AGAGTTTGATCCTGGCTCA G-3&#x2019;; 1492R: 5&#x2019;-GGTTACCTTGTTACGACTT-3&#x2019;). The 16S rRNA sequence results from <italic>A. hydrophila</italic> MX16A were deposited into the GenBank Database with accession number KJ806394.1 (<uri xlink:href="https://www.ncbi.nlm.nih.gov/search/all/?term=KJ806394.1">https://www.ncbi.nlm.nih.gov/search/all/?term=KJ806394.1</uri>).</p>
</sec>
<sec id="s2_3">
<title>Library preparation and whole genome sequencing</title>
<p>High-quality DNA was sent to Majorbio Company (Shanghai, China) for sequencing by PacBio RS II, a single molecule real time (SMRT&#x2122;) DNA sequencing technology (Pacific Biosciences, Menlo Park, USA). Briefly, <italic>A. hydrophila</italic> MX16A genome DNA was first randomly sheared into 8-10 kb fragments. After repairing the damage and ends of the fragmented DNA, some appropriately sized double-standard DNA fragments called SMRTbell&#x2122; (structurally linear and topologically circular), capped by hairpin loops at both ends of the fragmented pieces. Next, a primer and a polymerase are annealed to the adapter, and the annealed templates were bound to DNA polymerase located at the bottom of zero-mode waveguides (ZMWs) in SMRT&#x2122; Cell (single molecule real time reaction pore, one SMRT&#x2122; Cell included 150 thousand ZMWs) (<xref ref-type="bibr" rid="B6">Ardui et&#xa0;al., 2018</xref>). Finally, the MX16A plate was set up for sequencing. This PacBio TGS sequencing procedure has been previously described and is shown in <xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>.</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Schematic representation of PacBio&#x2019;s DNA sequencing. <italic>A. hydrophila</italic> MX16A DNA sample was extracted by using a TIANamp Bacteria DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer&#x2019;s instructions.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g001.tif"/>
</fig>
</sec>
<sec id="s2_4">
<title>Gene prediction, rapid annotation, and data analysis</title>
<p>Gene prediction was carried out using Glimmer v3.02 while rRNA- and tRNA-encoding of genes was predicted using Barrnap v0.4.2 and tRNAscan-SE v1.3.1. Gene annotation and statistics were performed using blastp (BLAST 2.2.28+) and compared to the Non-Redundant (Nr) Protein Sequence Database (<uri xlink:href="https://blast.ncbi.nlm.nih.gov/">https://blast.ncbi.nlm.nih.gov/</uri>), Genes Database (<uri xlink:href="https://www.genome.jp/kegg/genes.html/">https://www.genome.jp/kegg/genes.html/</uri>), String Database (<uri xlink:href="http://string-db.org/">http://string-db.org/</uri>), and Gene Ontology (GO) Database (<uri xlink:href="http://www.gene-ontology.org/">http://www.gene-ontology.org/</uri>). Analysis of antibiotic-related genes of the <italic>A. hydrophila</italic> MX16A genome was performed using the Comprehensive Antibiotic Resistance Database (CARD, <uri xlink:href="https://card.mcmaster.ca/">https://card.mcmaster.ca/</uri>). A circular map based on the bacterial chromosomal genome was generated using Circos v0.64 in Perl environment.</p>
</sec>
<sec id="s2_5">
<title>Genbank accession number of MX16A&#x2019;s genome</title>
<p>The genome sequence of <italic>A. hydrophila</italic> strain MX16A has been deposited in GenBank and may be found using accession number CP018201.1.</p>
</sec>
<sec id="s2_6">
<title>Genome data</title>
<p>The genome of <italic>A. hydrophila</italic> was obtained from the publicly available genome database in the National Center for Biotechnology Information (NCBI). A total of 35 genomic sequences were analyzed in a comparative fashion throughout this study. The sequences were statistically classified by the host and source, including human, fish, or other hosts isolated from China, Asia (except China), South America, North America, Africa or Europe, respectively.</p>
</sec>
<sec id="s2_7">
<title>Determination of average nucleotide identity</title>
<p>To avoid any possible genomic misidentification, all genomes were subjected to ANI check using the web based ANI calculator available at <uri xlink:href="http://enve-omics.ce.gatech.edu/ani/index">http://enve-omics.ce.gatech.edu/ani/index</uri>. In the ANI calculation, the strain <italic>A. hydrophila</italic> MX16A was used as a standard to analyze the genetic relationship between the <italic>A. hydrophila</italic> MX16A genome and the downloaded reference genome.</p>
</sec>
<sec id="s2_8">
<title>Analysis of pan-genome and core-genome</title>
<p>Generated faa files of 33 strains were used from the Prokaryotic Dynamic Programming Genefinding Algorithm (Prodigal). These served as input files for the Bacterial Pan Genome Analysis tool (BPGA) to perform a core-pan-genome analysis. USEARCH clustering algorithm was used as parameters to run BPGA. The identity value was 0.5 and the number of combinations was 500.</p>
</sec>
<sec id="s2_9">
<title>Prediction of drug resistance genes and virulence genes</title>
<p>Prediction of drug resistance genes and virulence genes was re-performed for 35 genomic sequences. Gff files of 35 strains were generated by Prokka. Drug resistance genes were predicted by AMRfinder (<uri xlink:href="https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/">https://www.ncbi.nlm.nih.gov/pathogens/antimicrobial-resistance/AMRFinder/</uri>), and virulence factors were identified based on the core data set of the VFDB (<uri xlink:href="http://www.mgc.ac.cn-/VFs/">http://www.mgc.ac.cn-/VFs/</uri>). The identity threshold used was 90% and the coverage threshold was 60%.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<title>Results</title>
<sec id="s3_1">
<title>Strain identification and antibiotic susceptibility</title>
<p>A total of 16S rRNA gene sequences clearly identified the isolate as <italic>A. hydrophila</italic>. Antimicrobial susceptibility testing was carried out based on the Kirby-Bauer method (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>). <italic>A. hydrophila</italic> MX16A was proved to be resistant to most antibiotics tested, including cefotaxime, amoxicilli, trimethoprim-sulfamethoxazole, rifampicin, erythromycin, tetracycline, streptomycin and chloramphenicol which represent &#x3b2;-lactams, macrolides, rifamycins, tetracyclines, sulfonamides, aminoglycosides, and chloramphenicols respectively. Based on the drug sensitivity test, it can be concluded that <italic>A. hydrophila</italic> MX16A was indeed a multidrug-resistant bacterium.</p>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>Antimicrobial susceptibility profile of <italic>A. hydrophila</italic> MX16A.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="left">Antimicrobial class</th>
<th valign="top" align="center">Antimicrobial agent</th>
<th valign="top" align="center">Disk content (&#x3bc;g)</th>
<th valign="top" align="center">Inhibition<break/>zone diameter (mm)</th>
<th valign="top" colspan="3" align="center">Interpretive Criteria</th>
<th valign="top" align="center">Susceptibility</th>
</tr>
<tr>
<th valign="top" align="left"/>
<th valign="top" align="center"/>
<th valign="top" align="center"/>
<th valign="top" align="center"/>
<th valign="top" align="center">R</th>
<th valign="top" align="center">I</th>
<th valign="top" align="center">S</th>
<th valign="top" align="left"/>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" rowspan="2" align="left">&#x3b2;-lactams</td>
<td valign="top" align="left">Cefotaxime</td>
<td valign="top" align="center">30</td>
<td valign="top" align="center">14</td>
<td valign="top" align="center">&#x2264;22</td>
<td valign="top" align="center">15-22</td>
<td valign="top" align="center">&#x2265;26</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Amoxicillin</td>
<td valign="top" align="center">10</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x2264;13</td>
<td valign="top" align="center">14-17</td>
<td valign="top" align="center">&#x2265;17</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" rowspan="3" align="left">Quinolones</td>
<td valign="top" align="left">Nalidixic acid</td>
<td valign="top" align="center">30</td>
<td valign="top" align="center">11.5</td>
<td valign="top" align="center">&#x2264;13</td>
<td valign="top" align="center">14-18</td>
<td valign="top" align="center">&#x2265;19</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Norfloxacin</td>
<td valign="top" align="center">10</td>
<td valign="top" align="center">18</td>
<td valign="top" align="center">&#x2264;12</td>
<td valign="top" align="center">13-16</td>
<td valign="top" align="center">&#x2265;17</td>
<td valign="top" align="center">S</td>
</tr>
<tr>
<td valign="top" align="left">Enrofloxacin</td>
<td valign="top" align="center">5</td>
<td valign="top" align="center">18</td>
<td valign="top" align="center">&#x2264;15</td>
<td valign="top" align="center">16-20</td>
<td valign="top" align="center">&#x2265;21</td>
<td valign="top" align="center">I</td>
</tr>
<tr>
<td valign="top" align="left">Sulfonamides</td>
<td valign="top" align="left">Trimethoprim-<break/>Sulfamethoxazole</td>
<td valign="top" align="center">1.25/23.75</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x2264;10</td>
<td valign="top" align="center">11-15</td>
<td valign="top" align="center">&#x2265;16</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Rifamycins</td>
<td valign="top" align="left">Rifampicin</td>
<td valign="top" align="center">5</td>
<td valign="top" align="center">13</td>
<td valign="top" align="center">&#x2264;16</td>
<td valign="top" align="center">17-19</td>
<td valign="top" align="center">&#x2265;20</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Macrolides</td>
<td valign="top" align="left">Erythromycin</td>
<td valign="top" align="center">15</td>
<td valign="top" align="center">0</td>
<td valign="top" align="center">&#x2264;13</td>
<td valign="top" align="center">14-22</td>
<td valign="top" align="center">&#x2265;23</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Tetracyclines</td>
<td valign="top" align="left">Tetracycline</td>
<td valign="top" align="center">30</td>
<td valign="top" align="center">10</td>
<td valign="top" align="center">&#x2264;11</td>
<td valign="top" align="center">12-14</td>
<td valign="top" align="center">&#x2265;15</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Aminoglycosides</td>
<td valign="top" align="left">Streptomycin</td>
<td valign="top" align="center">10</td>
<td valign="top" align="center">11</td>
<td valign="top" align="center">&#x2264;11</td>
<td valign="top" align="center">12-14</td>
<td valign="top" align="center">&#x2265;15</td>
<td valign="top" align="center">R</td>
</tr>
<tr>
<td valign="top" align="left">Chloramphenicols</td>
<td valign="top" align="left">Chloramphenicol</td>
<td valign="top" align="center">30</td>
<td valign="top" align="center">12</td>
<td valign="top" align="center">&#x2264;12</td>
<td valign="top" align="center">13-17</td>
<td valign="top" align="center">&#x2265;18</td>
<td valign="top" align="center">R</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>S, susceptible; I, intermediate; R, resistant. The results were interpreted based on the guidelines by the Clinical and Laboratory Standards Institute (CLSI).</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s3_2">
<title>Whole genome analysis</title>
<p>The complete genome of <italic>A. hydrophila</italic> MX16A consisted of a single circular 4,168,374 bp chromosome with 4418 genes and no plasmid. The general genome features included the genomic GC content, intergeneric region length, and the number of RNA genes (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>). The circular genome map was consistent with the characteristics of bacterial genome (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>). Furthermore, we performed a GO analysis to determine the functional classification of differentially expressed genes (DEGs) in the regulation process of MX16A. All DGEs could be combined into three main GO categories, namely biological process, cellular component, and molecular function, along with 43 subcategories as shown in <xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>.</p>
<table-wrap id="T2" position="float">
<label>Table&#xa0;2</label>
<caption>
<p>General genome features of <italic>A. hydrophila</italic> MX16A.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="left">Feature</th>
<th valign="top" align="center">Value</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">Gene total length (bp)</td>
<td valign="top" align="center">4,168,374</td>
</tr>
<tr>
<td valign="top" align="left">Intergenetic region length (bp)</td>
<td valign="top" align="center">615,130</td>
</tr>
<tr>
<td valign="top" align="left">GC content (%)</td>
<td valign="top" align="center">61.57</td>
</tr>
<tr>
<td valign="top" align="left">Gene/Genome (%)</td>
<td valign="top" align="center">87.10</td>
</tr>
<tr>
<td valign="top" align="left">No. of Genes</td>
<td valign="top" align="center">4418</td>
</tr>
<tr>
<td valign="top" align="left">No. of rRNA</td>
<td valign="top" align="center">31</td>
</tr>
<tr>
<td valign="top" align="left">No. of tRNA</td>
<td valign="top" align="center">126</td>
</tr>
</tbody>
</table>
</table-wrap>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Circos circular map of <italic>A. hydrophila</italic> MX16A genome. The outer circle indicates the size of the genome, every scale represents 0.5 Mb; The second and third circles represent the CDS from positive-sense and negative-sense strand of genome sequences, different colors represent various functions of COG; The fourth circle represents tRNA and rRNA; The fifth circle shows the (G+C) content, the outer red portion indicates that the GC content of the region is higher than the average GC content of the whole genome; higher peaks indicate a larger difference with the average GC content; the inner blue signals indicate that the GC content of the region was lower than the average GC content of the whole genome; higher peaks indicate a larger difference with the average GC content; the innermost circle shows the GC-Skew value. The innermost circle features the GC-Skew value, the specific algorithm was (G-C)/(G+C), useful to determine the leading strand and lagging strand. In general, the leading strand GC features skew&gt;0, the lagging strand GC features skew&lt;0. This may further help to determine the replication starting point and the cumulative offset may also assist in determining the starting point, i.e. cumulative offset minimum, and the end point, i.e. cumulative offset maximum, of replication, particularly important for cyclic genomes.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g002.tif"/>
</fig>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Gene ontology (GO) functional classification of <italic>A. hydrophila</italic> MX16A. DEGs, Differentially expressed genes.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g003.tif"/>
</fig>
</sec>
<sec id="s3_3">
<title>Types and distribution of resistance-associated genes</title>
<p>Analysis of antibiotic-related genes of <italic>A. hydrophila</italic> MX16A associated with resistance to &#x3b2;-lactams (<italic>ampC, bla2, blaZ</italic>), tetracyclines (<italic>tetA</italic>), quinolones (<italic>gyrA, parC</italic>), sulfonamides (<italic>sulA, folC</italic>), aminoglycosides (<italic>aphA, aacC, aadA</italic>), chloramphenicols (<italic>cat</italic>), macrolides (<italic>msbA</italic>), and rifamycins (<italic>rpoB</italic>) was performed using CARD databases. Some other efflux-associated genes following by <italic>acrA, acrB, emrA, emrB, emrD, emrE, mdtK, tolC, bcr</italic>, and <italic>ABCC10</italic> (gene symbol of efflux protein MRP7) were also identified (<xref ref-type="table" rid="T3">
<bold>Table&#xa0;3</bold>
</xref> and <xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>).</p>
<table-wrap id="T3" position="float">
<label>Table&#xa0;3</label>
<caption>
<p>Resistance genes of the <italic>A. hydrophila</italic> MX16A genome extracted by CARD and Nr analysis.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="left">Type</th>
<th valign="top" align="center">Antibiotic resistanceontology</th>
<th valign="top" align="center">Hit length(bp)</th>
<th valign="top" align="center">Description</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" rowspan="3" align="left">&#x3b2;-lactams</td>
<td valign="top" align="center">
<italic>ampC</italic>
</td>
<td valign="top" align="center">382</td>
<td valign="top" align="left">AmpC is a class C &#x3b2;-lactamase, commonly isolated from extended- spectrum cephalosporin-resistant gram-negative bacteria.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>bla2</italic>
</td>
<td valign="top" align="center">254</td>
<td valign="top" align="left">Bla2 is a chromosomal-encoded &#x3b2;-lactamase, found in <italic>Bacillus anthracis</italic>, exhibiting penicillin, cepha-lonsporin, and carbapenem- hydrolizing capabilities.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>blaZ</italic>
</td>
<td valign="top" align="center">299</td>
<td valign="top" align="left">BlaZ is a class A &#x3b2;-lactamase responsible for penicillin resistance in <italic>Staphylococcus aures</italic>.</td>
</tr>
<tr>
<td valign="top" align="left">Chloramphenicols</td>
<td valign="top" align="center">
<italic>cat</italic>
</td>
<td valign="top" align="center">150</td>
<td valign="top" align="left">The <italic>cat</italic> gene is used to describe many variants of the chloramphenicol acetyltransferase (CAT) gene in a range of organisms.</td>
</tr>
<tr>
<td valign="top" rowspan="3" align="left">Aminoglycosides</td>
<td valign="top" align="center">
<italic>aacC</italic>
</td>
<td valign="top" align="center">97</td>
<td valign="top" align="left">The <italic>aacC</italic> gene encodes forms of acetyltransferase (AAC).</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>aadA</italic>
</td>
<td valign="top" align="center">172</td>
<td valign="top" align="left">The <italic>aadA</italic> gene encodes forms of adenylyltransferase (ANT).</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>aphA</italic>
</td>
<td valign="top" align="center">236</td>
<td valign="top" align="left">The <italic>aphA</italic> gene encodes forms of phosphotransferase (APH).</td>
</tr>
<tr>
<td valign="top" rowspan="2" align="left">Quinolones</td>
<td valign="top" align="center">
<italic>gyrA</italic>
</td>
<td valign="top" align="center">915</td>
<td valign="top" align="left">DNA gyrase (DNAG) is responsible for DNA supercoiling and consists of two &#x3b1;- and two &#x3b2;-subunits. Mutations in the quinolone resistance-determining regions (QRDRs) of these genes (<italic>gyrA, gyrB, parC and parE</italic>) result in amino acid substitutions that structurally change the target protein and, subsequently, the drug-binding affinity of the enzyme. In <italic>E. coli</italic>, the most common mutation site in <italic>gyrA</italic> is at Ser83 followed by Asp87, and similar mutation frequencies are seen at equivalent positions for <italic>gyrA</italic> and <italic>parC</italic> in other species.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>parC</italic>
</td>
<td valign="top" align="center">764</td>
<td valign="top" align="left">ParC is a subunit of topoisomerase IV (TOPO IV) that decatenates and relaxes DNA to allow access to genes for transcription or translation. Point mutations in ParC prevent fluoroquinolone antibiotics from inhibiting DNA synthesis and confer low-level resistance.&#xa0;</td>
</tr>
<tr>
<td valign="top" align="left">Rifamycins</td>
<td valign="top" align="center">
<italic>rpoB</italic>
</td>
<td valign="top" align="center">1342</td>
<td valign="top" align="left">RNA polymerase (RNAP) is a multi-subunit enzyme necessary for transcription. The &#x3b2;-subunit of RNAP forms the active center of the enzyme and template/transcript binding sites. Mutations in <italic>rpoB</italic> gene confers antibiotic resistance.</td>
</tr>
<tr>
<td valign="top" rowspan="2" align="left">Sulfonamides</td>
<td valign="top" align="center">
<italic>sulA</italic>
</td>
<td valign="top" align="center">162</td>
<td valign="top" align="left">The <italic>sulA</italic> gene encodes forms of dihydropteroate synthase (DHPS) that confer resistance to sulfonamide.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>folC</italic>
</td>
<td valign="top" align="center">415</td>
<td valign="top" align="left">Similar to <italic>sulA</italic>.</td>
</tr>
<tr>
<td valign="top" rowspan="12" align="left">Antibiotic Efflux</td>
<td valign="top" align="center">
<italic>tetA</italic>
</td>
<td valign="top" align="center">424</td>
<td valign="top" align="left">TetA is a tetracycline efflux pump found in various species of gram-negative bacteria.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>acrA</italic>
</td>
<td valign="top" align="center">349</td>
<td valign="top" align="left">Protein subunit of AcrAB-TolC multidrug efflux complex. AcrA represents the periplasmic portion of the transport protein.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>acrB</italic>
</td>
<td valign="top" align="center">1065</td>
<td valign="top" align="left">Protein subunit of AcrAB-TolC multidrug efflux complex. AcrB functions as a heterotrimer forming the inner membrane component and is primarily responsible for substrate recognition and energy transduction by acting as a drug/proton antiporter.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>tolC</italic>
</td>
<td valign="top" align="center">441</td>
<td valign="top" align="left">TolC is a protein subunit of numerous multidrug efflux complexes in gram-negative bacteria, functions as an outer membrane efflux protein, and is constitutively open. Regulation of efflux activity often takes place at its periplasmic entrance by other components of the efflux complex.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>emrA</italic>
</td>
<td valign="top" align="center">350</td>
<td valign="top" align="left">EmrA is a membrane fusion protein, providing an efflux pathway with EmrB and TolC between the inner and the outer membranes of <italic>E.coli</italic>.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>emrB</italic>
</td>
<td valign="top" align="center">517</td>
<td valign="top" align="left">EmrB is a translocase in the EmrAB-TolC efflux protein in <italic>E.coli</italic>. It recognizes substrates including carbonyl cyanide m-chlorophenylhydrazone (CC-CP), nalidixic acid, and thioloactomycin.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>emrD</italic>
</td>
<td valign="top" align="center">399</td>
<td valign="top" align="left">EmrD is a multidrug transporter from the major facilitator (MF) superfamily primarily found in <italic>E.coli</italic>. EmrD couples efflux of amphipathic compounds with proton import across the plasma membrane.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>emrE</italic>
</td>
<td valign="top" align="center">137</td>
<td valign="top" align="left">EmrE is a small multidrug transporter that functions as a homodimer and couples the efflux of small polyaromatic cations from the cell with the import of protons down an electrochemical gradient. EmrE is found in <italic>E.coli</italic> and <italic>Pseudomonas aeruginosa</italic>.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>mdtK</italic>
</td>
<td valign="top" align="center">440</td>
<td valign="top" align="left">MdtK is a multidrug and toxic compound extrusions (MATE) family transporter conferring resistance to norfloxacin, doxorubicin, and acriflavine.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>msbA</italic>
</td>
<td valign="top" align="center">589</td>
<td valign="top" align="left">MsbA is a multidrug-resistance (MDR) transporter homolog from <italic>E.coli</italic> belonging to the adenosine triphosphate (ATP) binding cassette (ABC) superfamily. MsbA transports lipid A, a major component of the bacterial outer cell membrane, and is the only bacterial ABC transporter that is essential for cell viability.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>bcr</italic>
</td>
<td valign="top" align="center">395</td>
<td valign="top" align="left">Bcr is a transmembrane protein that expels bicyclomycin from the cell, leading to bicyclomycin resistance.</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>ABCC10</italic>
</td>
<td valign="top" align="center">1186</td>
<td valign="top" align="left">ABCC10 is a ABC superfamily human protein that can transport amphipathic anions and confer resistance to several anticancer agents such as docetaxel and vincristine.</td>
</tr>
</tbody>
</table>
</table-wrap>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Sankey diagram of distribution of resistance-associated genes in <italic>A. hydrophila</italic> MX16A. CAT, chloramphenicol acetyltransferase; AAC, acetyltransferase; ANT, adenylyltransferase; APH, phosphotransferase; DNAG, DNA gyrase; TOPO IV, topoisomerase IV; RNAP, RNA polymerase; DHPS, dihydropteroate synthase.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g004.tif"/>
</fig>
<p>However, antimicrobial susceptibility test (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) revealed that MX16A was not resistant to enrofloxacin and norfloxacin. We further compared the common mutation sites on the quinolone resistance- determining regions (QRDR) of <italic>gyrA</italic> and <italic>parC</italic> genes and found that Ser83 on <italic>gyrA</italic> and <italic>Ser87</italic> on parC were both mutated to Ile. For this reason, MX16A was performed additional drug sensitivity tests to nalidixic acid and found to be resistant to it.</p>
</sec>
<sec id="s3_4">
<title>Overview of the genomes for comparison</title>
<p>The complete genomes of 35 Aeromonas hydrophila strains isolated from different regions and hosts on NCBI, including MX16A, were downloaded for genomic comparison (<xref ref-type="table" rid="T4">
<bold>Table&#xa0;4</bold>
</xref>).</p>
<table-wrap id="T4" position="float">
<label>Table&#xa0;4</label>
<caption>
<p>Genomes information of 35 srains of <italic>A. hydrophila</italic> used in the present study.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="left">Strain name</th>
<th valign="top" align="center">GenBank assembly accession</th>
<th valign="top" align="center">Host</th>
<th valign="top" align="center">Region</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="left">MX16A</td>
<td valign="top" align="center">GCA_001895965.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">WCHAH045096</td>
<td valign="top" align="center">GCA_002850695.3</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">NUITM-VA1</td>
<td valign="top" align="center">GCA_021654355.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">M052</td>
<td valign="top" align="center">GCA_001756325.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">GSH8-2</td>
<td valign="top" align="center">GCA_004296435.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">WP7-S18-ESBL-06</td>
<td valign="top" align="center">GCA_014161955.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">CS USB2</td>
<td valign="top" align="center">GCA_020162255.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">North America</td>
</tr>
<tr>
<td valign="top" align="left">GTCBM_22</td>
<td valign="top" align="center">GCA_019348635.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">South America</td>
</tr>
<tr>
<td valign="top" align="left">BB1457</td>
<td valign="top" align="center">GCA_903684605.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">Africa</td>
</tr>
<tr>
<td valign="top" align="left">KLG1</td>
<td valign="top" align="center">GCA_901212375.1</td>
<td valign="top" align="center">water</td>
<td valign="top" align="center">Europe</td>
</tr>
<tr>
<td valign="top" align="left">ZYAH72</td>
<td valign="top" align="center">GCA_003491225.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">LHW39</td>
<td valign="top" align="center">GCA_011602425.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">JBN2301</td>
<td valign="top" align="center">GCA_001455365.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">D4</td>
<td valign="top" align="center">GCA_001518775.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">B11</td>
<td valign="top" align="center">GCA_013205705.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">GYK1</td>
<td valign="top" align="center">GCA_001683535.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">HX-3</td>
<td valign="top" align="center">GCA_009791455.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">AH10</td>
<td valign="top" align="center">GCA_000963645.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">LP0103</td>
<td valign="top" align="center">GCA_022557195.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">3019</td>
<td valign="top" align="center">GCA_018802385.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">North America</td>
</tr>
<tr>
<td valign="top" align="left">3019</td>
<td valign="top" align="center">GCA_018802385.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">North America</td>
</tr>
<tr>
<td valign="top" align="left">Aer_OnP4.2</td>
<td valign="top" align="center">GCA_017310115.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">South America</td>
</tr>
<tr>
<td valign="top" align="left">AC185</td>
<td valign="top" align="center">GCA_022631175.1</td>
<td valign="top" align="center">fish</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">ATCC 7966</td>
<td valign="top" align="center">GCA_000014805.1</td>
<td valign="top" align="center">other</td>
<td valign="top" align="center">Europe</td>
</tr>
<tr>
<td valign="top" align="left">AFG_SD03_1510_Ahy_093</td>
<td valign="top" align="center">GCA_003323285.1</td>
<td valign="top" align="center">other</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">WCX23</td>
<td valign="top" align="center">GCA_004684305.1</td>
<td valign="top" align="center">other</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">RIT668</td>
<td valign="top" align="center">GCA_012641195.1</td>
<td valign="top" align="center">other</td>
<td valign="top" align="center">North America</td>
</tr>
<tr>
<td valign="top" align="left">S73-1</td>
<td valign="top" align="center">GCA_017315485.1</td>
<td valign="top" align="center">other</td>
<td valign="top" align="center">North America</td>
</tr>
<tr>
<td valign="top" align="left">A34a</td>
<td valign="top" align="center">GCA_015353175.1</td>
<td valign="top" align="center">other</td>
<td valign="top" align="center">Africa</td>
</tr>
<tr>
<td valign="top" align="left">ZYAH75</td>
<td valign="top" align="center">GCA_003491245.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">Ah2111</td>
<td valign="top" align="center">GCA_022982835.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">CN17A0135</td>
<td valign="top" align="center">GCA_016729115.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">China</td>
</tr>
<tr>
<td valign="top" align="left">AHNIH1</td>
<td valign="top" align="center">GCA_001687125.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">North America</td>
</tr>
<tr>
<td valign="top" align="left">RIMD111065</td>
<td valign="top" align="center">GCA_016592295.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">Asia (excluding China)</td>
</tr>
<tr>
<td valign="top" align="left">Aer284</td>
<td valign="top" align="center">GCA_003849735.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">South America</td>
</tr>
<tr>
<td valign="top" align="left">S-P-C-021.01</td>
<td valign="top" align="center">GCA_022488365.1</td>
<td valign="top" align="center">
<italic>Homo sapiens</italic>
</td>
<td valign="top" align="center">Africa</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="s3_5">
<title>Comparative analysis of average nucleotide identity</title>
<p>In recent studies, whole-genome average nucleotide identity (ANI) has emerged as a robust method for assessing species boundaries. Typically, organisms belonging to the same species have &#x2265;95% ANI values. ANI represents the average nucleotide identity of all orthologous genes shared between any two genomes and offers robust resolution between strains of the same or closely related species (showing 80&#x2013;100% ANI) (<xref ref-type="bibr" rid="B31">Jain et&#xa0;al., 2010</xref>). We performed ANI analysis on the whole genome of 35 Aeromonas hydrophila strains used in this study (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5</bold>
</xref>; <xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;1</bold>
</xref>). The figure shows a region with high similarity and a higher genomic diversity. The analysis revealed that strains GCA_014161955.1 and GCA_022982835.1 featured high similarity with <italic>A. hydrophila</italic> MX16A. The corresponding ANI was 99.6% and 98.6%, respectively. The similarity of strains GCA_013205705.1 and GCA_903684605.1 with MX16A was below 95%. This finding may indicate that these two strains were misclassified as <italic>A. hydrophila.</italic>
</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>Heat map of ANI pairings between any two genomes of MX16A (in red font) and other <italic>A. hydrophila</italic> strains downloaded from NCBI database.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g005.tif"/>
</fig>
</sec>
<sec id="s3_6">
<title>Comparative genome analysis</title>
<p>Compared with <italic>A. hydrophila</italic> MX16A, only GCA_014161955.1 and GCA_022982835.1 featured greater ANI than 98%. Using the genomes of the three strains as inputs, OrthoFinder was used to pally align the protein sequences of the strains and detect the shared protein between the strains. Based on the generated files (Orthogroups. GeneCount. TSV) for processing, the protein contained in each of the three genomes were sorted out. Jvenn was used to draw a comparative analysis diagram of the clustering results of the three strains protein (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6</bold>
</xref>). The results obtained were similar to the ANI prediction and strain MX16A was demonstrated to be more similar to GCA_014161955.1 as these two strains shared most of the protein. Strain GCA_022982835.1 featured a larger number of unique proteins.</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>Venn diagram showing the protein shared and exclusive among <italic>A. hydrophila</italic> GCA_001895965.1 (MX16A), GCA_014161955.1, and GCA_022982835.1.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g006.tif"/>
</fig>
</sec>
<sec id="s3_7">
<title>Pan-core genome analysis</title>
<p>Excluding the two strains with an ANI lower than 95%, the remaining 33 strains were analyzed regarding their pan-core genome. As the number of genes increased, the number of core genes gradually decreased and was then found to plateau while the pan-genome continued to expand. The final core genome size was converged to 3485 genes and the final pan-genome was converged to 9556 genes (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Tables&#xa0;2</bold>
</xref>, <xref ref-type="supplementary-material" rid="SM1">
<bold>3</bold>
</xref>), presenting an open state (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7</bold>
</xref>). It may be speculated in this context that the <italic>A. hydrophila</italic> genome featured a more stable core gene cluster with high partial genome variability. These highly variable sections of the genome potentially enable different environmental adaptations.</p>
<fig id="f7" position="float">
<label>Figure&#xa0;7</label>
<caption>
<p>Pan-core genome analysis of 33 strains of <italic>A. hydrophila</italic>, except the two possibly misclassified strains (ANI&lt;95%).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g007.tif"/>
</fig>
</sec>
<sec id="s3_8">
<title>Analysis of drug resistance and virulence factors</title>
<p>The online tool ITOL (<uri xlink:href="https://itol.embl.de/">https://itol.embl.de/</uri>) was used to generate evolutionary trees and maps of drug resistance genes and virulence factors (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8</bold>
</xref>). A total of 12 classes of resistance genes were predicted in these genomes, of which &#x3b2;-lactam and &#x3b2;-lactam:carbapenem genes were shared by 35 strains. Genes of &#x3b2;-lactam:cephalosporin were also shared by alomost all strains, except the two possibly misclassified strains GCA_013205705.1 and GCA_903684605.1 (ANI&lt;95%). Strain MX16A featured a larger number of resistance genes (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;4)</bold>
</xref>. For virulence factors, 6 classes of virulence factors could be determined, common to most strains. The strains GCA_003491225.1, GCA_001455365.1, GCA_001518775.1, GCA_001683535.1 and GCA_011602425.1 exhibited highly similar drug resistance genes and virulence factors, all isolated from China with fish being the host. However, these strains stem from different Chinese provinces such as Guangdong and Hubei, potentially resulting in spreading due to breeding and transportation.</p>
<fig id="f8" position="float">
<label>Figure&#xa0;8</label>
<caption>
<p>Phylogenetic tree, distribution of drug-resistance genes and virulence factors of 35 strains of <italic>A. hydrophila</italic>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-1042350-g008.tif"/>
</fig>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<title>Discussion</title>
<sec id="s4_1">
<title>Drug resistance and resistance-associated genes of <italic>A. hydrophila</italic> MX16A</title>
<p>From antibiotic sensitivity tests. we could determine that <italic>A. hydrophila</italic> MX16A was resistant to all clinically used drugs except for some quinolones. This finding was consistent with the results of the majority of drug resistance genes predicted by CARD.</p>
</sec>
<sec id="s4_2">
<title>Enzyme mediated drug resistance</title>
<p>&#x3b2;-lactam antibiotics have been extensively used to against gram-negative pathogens due to their broad spectrum of antibacterial activity these drugs exhibit until the appearance of the &#x3b2;-lactam inactivator &#x3b2;-lactamase (<xref ref-type="bibr" rid="B1">Abraham and Chain, 1988</xref>). The latter enzymes hydrolyze the &#x3b2;-lactam ring and therefore reduce the overall activity of &#x3b2;-lactam drugs (<xref ref-type="bibr" rid="B15">Davies, 1994</xref>). The process of enzymatic hydrolization is regarded as the primary resistance mechanism to &#x3b2;-lactam antibiotics in drug-resistant pathogens. Overexpression of various chromosomally-mediated-&#x3b2;-lactamases in <italic>A. hydrophila</italic> is generally associated with &#x3b2;-lactam as reported previously (<xref ref-type="bibr" rid="B33">Ko et&#xa0;al., 1998</xref>; <xref ref-type="bibr" rid="B52">Saavedra et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B7">Balsalobre et&#xa0;al., 2009</xref>). As such, the chromosome-borne enzyme AmpC coded as gene <italic>ampC</italic> has been found to inactivate carbapenem antibiotics in <italic>Enterobacteriaceae</italic> bacteria (<xref ref-type="bibr" rid="B58">Suh et&#xa0;al., 2010</xref>) while the &#x3b2;-lactamase Bla2, coded as chromosomal gene <italic>bla2</italic>, has been found to be expressed in <italic>Escherichia coli</italic>, resulting in &#x3b2;-lactam hydrolyzation and drug resistance against antibiotics based on carbapenem, cephalosporinase, and penicillinase (<xref ref-type="bibr" rid="B40">Materon et&#xa0;al., 2003</xref>).</p>
<p>Chloramphenicol acetyltransferase (CAT), encoded by the gene <italic>cat</italic>, represents the main factor for chloramphenicol resistance demonstrated by some bacteria strains. This type of antibiotic inactivating enzyme may catalyze the acetyl-S-CoA-dependent acetylation of chloramphenicol at the 3-hydroxyl group to transfer chloramphenicol to a metabolite without antimicrobial activity (<xref ref-type="bibr" rid="B54">Shaw, 1983</xref>). Moreover, in bacteria, resistance to aminoglycosides is usually deemed to enzymatic inactivation by acetyltransferases (encoded by gene <italic>aac</italic>), adenylyltransferases (encoded by gene <italic>aad</italic>), and phosphotransferases (encoded by gene <italic>aph</italic>) (<xref ref-type="bibr" rid="B55">Shaw et&#xa0;al., 1993</xref>).</p>
</sec>
<sec id="s4_3">
<title>Genetic mutation-mediated drug resistance</title>
<p>Naturally occurring resistance to fluoroquinolones in gram-negative bacteria is usually due to the genetic mutation of DNA gyrase (encoded by <italic>gyrA</italic>) and topoisomerase IV (encoded by <italic>parC</italic>), both of which represent the enzyme targets of fluoroquinolones in the bacterial cell and play a key role in DNA replication (<xref ref-type="bibr" rid="B17">Drlica and Zhao, 1997</xref>; <xref ref-type="bibr" rid="B26">Hooper, 2000</xref>). These enzymatic changes in the target site may block the formation of compounds by these enzymes and fluoroquinolones, usually responsible for bacterial DNA replication inhibition (<xref ref-type="bibr" rid="B26">Hooper, 2000</xref>). Furthermore, the region for mutations within <italic>gyrA</italic> and <italic>parC</italic> has been described as the quinolone-resistance determining region (QRDR) (<xref ref-type="bibr" rid="B65">Yoshida et&#xa0;al., 1990</xref>) and is associated with the mechanism of quinolone-resistance in <italic>Aeromonas</italic> spp. as reported previously (<xref ref-type="bibr" rid="B3">Alcaide et&#xa0;al., 2010</xref>). Moreover, anti-rifampicin strains have been reported with alteration of the <italic>rpoB</italic> gene encoding the beta subunit of RNA polymerase. A large majority of rifampicin-resistant isolates including Aeromonas strains are related to specific mutations within the 81-bp region (rifampicin-resistance determining region, RRDR) of the <italic>rpoB</italic> gene as reported in the literature (<xref ref-type="bibr" rid="B25">Herrera et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B66">Zhou et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B47">Pridgeon et&#xa0;al., 2013</xref>).</p>
<p>For living cells, folate derivatives, synthesized by most bacterial species, generally represent the requisite cofactors participating in the biosynthesis of purines, pyrimidines, and amino acids. Thus, enzymes in the pathway of folate biosynthesis represent the paramount targets to folate-inhibitors, including sulfonamides, to antagonize pathogens (<xref ref-type="bibr" rid="B22">Goulding et&#xa0;al., 2005</xref>). Earlier reports have revealed that sulfonamide-resistance was closely associated with genetic mutation of the dihydropteroate synthase (DHPS) encoded by the chromosomal gene <italic>sul</italic> and <italic>fol</italic> (e.g. <italic>sulA</italic> and <italic>folC</italic>) in bacteria (<xref ref-type="bibr" rid="B39">Maskell et&#xa0;al., 1997</xref>; <xref ref-type="bibr" rid="B59">Swedberg and Ringertz, 1998</xref>; <xref ref-type="bibr" rid="B23">Haasum et&#xa0;al., 2001</xref>).</p>
</sec>
<sec id="s4_4">
<title>Efflux protein-mediated drug resistance</title>
<p>Efflux pumps are one of the major causes of MDR in almost all bacterial species with gene-encoding proteins found on the chromosomes or plasmids (<xref ref-type="bibr" rid="B62">Webber and Piddock, 2003</xref>; <xref ref-type="bibr" rid="B45">Poole, 2007</xref>). According to the structural diversity, energy sources, and substrates, efflux pumps of gram-negative bacteria can be divided into five families: ATP-binding cassette (ABC) superfamily, major facilitator (MF) superfamily, resistance nodulation division (RND) family, multi-antimicrobial extrusion (MATE) family, and small multidrug-resistance (SMR) family (<xref ref-type="bibr" rid="B11">Blair et&#xa0;al., 2014</xref>).</p>
<p>As members of the ABC superfamily proteins, MRP7/ABCC10 (gene symbol <italic>ABCC10</italic>) may transport amphipathic anions and cause resistance to several anticancer agents such as docetaxel and vincristine (<xref ref-type="bibr" rid="B27">Hopperborge et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B34">Kruh et&#xa0;al., 2007</xref>) while MsbA protein (encoded by gene <italic>msbA</italic> and localized to the inner membrane) was found to cause resistance to erythromycin by means of ATP binding and hydrolytic properties in both gram-positive (<xref ref-type="bibr" rid="B64">Woebking et&#xa0;al., 2005</xref>) and gram-negative (<xref ref-type="bibr" rid="B20">Ghanei et&#xa0;al., 2007</xref>) microorganisms. The corresponding gene <italic>tetA</italic> located on the chromosome encoding TetA efflux protein (generally associated with MF superfamily) frequently observed in <italic>Aeromonas</italic> spp. isolates (<xref ref-type="bibr" rid="B30">Jacobs and Chenia, 2007</xref>) has been described to leave the host bacterium unharmed by actively transporting intracellular tetracycline out of cell cytoplasm (<xref ref-type="bibr" rid="B49">Roberts, 2005</xref>; <xref ref-type="bibr" rid="B61">Wang et&#xa0;al., 2014</xref>). Previous studies have revealed that overproduction of MdtK, a MATE family membrane transport protein, encoded by gene <italic>mdtK</italic>, could induce resistance to norfloxacin, doxorubicin, and acriflavine in <italic>Salmonella enterica</italic> (<xref ref-type="bibr" rid="B28">Horiyama et&#xa0;al., 2010</xref>).</p>
<p>TolC (encoded by <italic>tolC</italic>),&#xa0;an outer membrane protein, functions as the outer membrane channel to several MDR efflux systems (<xref ref-type="bibr" rid="B10">Benz et&#xa0;al., 1993</xref>), including the AcrAB-TolC (RND-type) and EmrAB-TolC (MF-type). AcrAB-TolC represents the main efflux system commonly found in various gram-negative bacteria (<xref ref-type="bibr" rid="B46">Pradel and Pag&#xe8;s, 2002</xref>; <xref ref-type="bibr" rid="B8">Baucheron et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B16">de Crist&#xf3;bal et&#xa0;al., 2006</xref>), forming by a tripartite complex comprised of a cytosolic membrane pump (AcrB, encoded by acrB), a periplasmic membrane fusion protein (AcrA, encoded by <italic>acrA</italic>), and a TolC protein (<xref ref-type="bibr" rid="B18">Du et&#xa0;al., 2014</xref>). The EmrAB-TolC efflux system is also composed of three componentes including the membrane protein EmrA (encoded by <italic>emrA</italic>), transporter protein EmrB (encoded by <italic>emrB</italic>), and outer membrane protein TolC (<xref ref-type="bibr" rid="B38">Lomovskaya and Lewis, 1992</xref>). These two above-mentioned efflux systems, particularly AcrAB-TolC, have been reported to significantly affect the antimicrobial resistance to multiple antibiotic classes such as tetracyclines, chloramphenicols, rifamicins, &#x3b2;-lactams, quinolones, and macrolides in gram-native bacteria (<xref ref-type="bibr" rid="B43">Okusu et&#xa0;al., 1996</xref>; <xref ref-type="bibr" rid="B42">Nishino et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B8">Baucheron et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B14">Chollet et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B19">Elkins and Mullis, 2007</xref>). Moreover, EmrD (MF superfamily) and EmrE (SMR family) represent two membrane transporter proteins codified by genes <italic>emrD</italic> and <italic>emrE</italic>, respectively. Both of these proteins may induce drug resistance by involving adaptation to low energy shock or exporting diverse drug substrates protecting bacteria from antibiotic damage according to various studies reported in the literature (<xref ref-type="bibr" rid="B41">Naroditskaya et&#xa0;al., 1993</xref>; <xref ref-type="bibr" rid="B50">Rotem and Schuldiner, 2004</xref>). Moreover, gene <italic>bcr</italic> encoding a drug resistance bacterial transmembrane protein (Bcr), was also identified from the <italic>A. hydrophila</italic> MX16A genome involved in bicyclomycin and sulfathiazole resistance in <italic>E. coli</italic> (<xref ref-type="bibr" rid="B9">Bentley et&#xa0;al., 1993</xref>; <xref ref-type="bibr" rid="B24">Hayashi et&#xa0;al., 2010</xref>).</p>
</sec>
<sec id="s4_5">
<title>Comparative genome analysis</title>
<p>As described above, we were able to assess the diversity level of the biological genome to some extent related to the ratio of core genome to pangenome. The pangenome analysis of <italic>A. hydrophila</italic> described in this paper showed a large genomic diversity and further demonstrated that this species could adapt to and survive in different environmental conditions. In a previous study found in the literature (<xref ref-type="bibr" rid="B21">Ghatak et&#xa0;al., 2016</xref>), the pan-genome and core genes of 16 strains of <italic>Aeromonas</italic> in the genus <italic>Aeromonas</italic> spp. were analyzed and the results showed that <italic>A. hydrophila</italic> exhibited a higher genome diversity than <italic>A. veronii</italic> and <italic>A. caviae</italic>. Combined with our analytical data, the number of genes in the core genome did not change significantly, further confirming that the core genome of <italic>A. hydrophila</italic> is indeed stable. Finally, it may also be speculated that the genome of <italic>A. hydrophila</italic> features rich metabolic functions.</p>
</sec>
<sec id="s4_6">
<title>Analysis of virulence and drug resistance factors</title>
<p>Type III secretion systems are considered an important virulence mechanism for aeromonads (<xref ref-type="bibr" rid="B56">Sierra et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B35">Krzymi&#x144;ska et&#xa0;al., 2012</xref>). In this study, we did not predict genes for the type III virulence system, however, Ghatak et&#xa0;al., reported genes related to the type III secretion systems are present in all genomes of <italic>A. hydrophila</italic>, presumably due to the use of different databases. Importantly, a different drug resistance gene database for <italic>A. hydrophila</italic> MX16A was used in this report, resulting in some differences in the forecasted results. Several strains with more and roughly similar resistance genes stemed from different host types and were distributed in different regions. Due to the wide geographical distribution of <italic>A. hydrophila</italic> and the wide distribution of its hosts, it is hypothesized that frequent gene exchange between strains may lead to the transfer of corresponding drug resistance genes.</p>
</sec>
</sec>
<sec id="s5" sec-type="conclusions">
<title>Conclusions</title>
<p>In this study, we report preliminary results regarding the environmental MDR <italic>Aeromonas hydrophila</italic> strain MX16A based on complete genome sequence analysis. The results of resistance gene prediction analysis were mostly consistent with the phenotypic antimicrobial susceptibility test. A variety of antibiotic-related genes identified from the <italic>A. hydrophila</italic> MX16A genome indicates that this bacterium may pose a potential threat to both human and aquatic animal health with phylogenetic analysis further confirming this conclusion. The diverse types and distributions of antibiotic genes of <italic>A. hydrophila</italic> MX16A suggested that the bacterial resistance mechanism was variable as follows: enzyme inactivation, genetic mutation, and active efflux. However, it still remains unclear which and how these three variables influence the overall drug resistance mechanism of <italic>A. hydrophila</italic> MX16A. Thus, future studies may now focus on the genomic characteristics potentially further leading to the development of targeted antibiotics. <italic>A. hydrophila</italic> MX16A exhibited close similarities to GCA_022982835.1. Due to the finding that humans are the primary host for the GCA_022982835.1 strain, it may be concluded that the pathway of human infection with <italic>Aeromonas</italic> may be due to the presence of this bacterial species in water and infected aquatic animals. Genome comparison studies carried out throughout this research further revealed that <italic>A. hydrophila</italic> displays a wide host and geographic distribution pattern, is not conserved, features high genome variability, and horizontal gene transfer may promote the evolution of other strains.</p>
</sec>
<sec id="s6" sec-type="data-availability">
<title>Data availability statement</title>
<p>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/<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Material.</bold>
</xref>
</p>
</sec>
<sec id="s7" sec-type="author-contributions">
<title>Author contributions</title>
<p>YG and CZ carried out the experiments and drafted the manuscript. ML directed the research and revised the manuscript. CM isolated the bacterial strain. HC, XJ, SZ, and XX analyzed the genomic data and revised the manuscript. All authors contributed to the article and approved the submitted version.</p>
</sec>
<sec id="s8" sec-type="funding-information">
<title>Funding</title>
<p>This research was supported by the National Key R&amp;D Program of China (2020YFD0900102), the Earmarked Fund for China Agriculture Research System (No.CARS-46), the Xiamen Ocean and Fishery Development Special Fund (21CZP007HJ07).</p>
</sec>
<sec id="s9" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The 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.</p>
</sec>
<sec id="s10" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>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.</p>
</sec>
</body>
<back>
<sec id="s11" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fcimb.2022.1042350/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcimb.2022.1042350/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="Table_1.xlsx" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
<supplementary-material xlink:href="Table_2.xlsx" id="SM2" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
<supplementary-material xlink:href="Table_3.xlsx" id="SM3" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
<supplementary-material xlink:href="Table_4.xlsx" id="SM4" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
</sec>
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