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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.2024.1368622</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>Sporadic clone <italic>Escherichia coli</italic> ST615 as a vector and reservoir for dissemination of crucial antimicrobial resistance genes</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Carrera P&#xe1;ez</surname>
<given-names>Laura Camila</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Olivier</surname>
<given-names>Martin</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Gambino</surname>
<given-names>Anah&#xed; Samanta</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>Poklepovich</surname>
<given-names>Tom&#xe1;s</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
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<contrib contrib-type="author">
<name>
<surname>Aguilar</surname>
<given-names>Andrea Pamela</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Quiroga</surname>
<given-names>Mar&#xed;a Paula</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Centr&#xf3;n</surname>
<given-names>Daniela</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
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<aff id="aff1">
<sup>1</sup>
<institution>Laboratorio de Investigaciones en Mecanismos de Resistencia a Antibi&#xf3;ticos, Instituto de Investigaciones en Microbiolog&#xed;a y Parasitolog&#xed;a M&#xe9;dica, Facultad de Medicina, Universidad de Buenos Aires - Consejo Nacional de Investigaciones Cient&#xed;ficas y Tecnol&#xf3;gicas (IMPaM, UBA-CONICET)</institution>, <addr-line>Buenos Aires</addr-line>, <country>Argentina</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>The Research Institute of the McGill University Health Centre, McGill University</institution>, <addr-line>Montr&#xe9;al, QC</addr-line>, <country>Canada</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Plataforma de Gen&#xf3;mica y Bioinform&#xe1;tica, Instituto Nacional de Enfermedades Infecciosas - La Administraci&#xf3;n Nacional de Laboratorios e Institutos de Salud (INEI-ANLIS) &#x201c;Dr. Carlos G. Malbr&#xe1;n&#x201d;</institution>, <addr-line>Buenos Aires</addr-line>, <country>Argentina</country>
</aff>
<aff id="aff4">
<sup>4</sup>
<institution>Cl&#xfa;ster de Bioinform&#xe1;tica, Instituto de Investigaciones en Microbiolog&#xed;a y Parasitolog&#xed;a M&#xe9;dica, Facultad de Medicina, Universidad de Buenos Aires - Consejo Nacional de Investigaciones Cient&#xed;ficas y Tecnol&#xf3;gicas (IMPaM, UBACONICET)</institution>, <addr-line>Buenos Aires</addr-line>, <country>Argentina</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Angel Adri&#xe1;n Cataldi, Instituto Nacional de Tecnolog&#xed;a Agropecuaria, Argentina</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Marina Rosa Pulido, University of Seville, Spain</p>
<p>Pablo Power, Universidad de Buenos Aires, Argentina</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Daniela Centr&#xf3;n, <email xlink:href="mailto:dcentron@gmail.com">dcentron@gmail.com</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>29</day>
<month>04</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>14</volume>
<elocation-id>1368622</elocation-id>
<history>
<date date-type="received">
<day>10</day>
<month>01</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>27</day>
<month>03</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Carrera P&#xe1;ez, Olivier, Gambino, Poklepovich, Aguilar, Quiroga and Centr&#xf3;n</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Carrera P&#xe1;ez, Olivier, Gambino, Poklepovich, Aguilar, Quiroga and Centr&#xf3;n</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>There is scarce information concerning the role of sporadic clones in the dissemination of antimicrobial resistance genes (ARGs) within the nosocomial niche. We confirmed that the clinical <italic>Escherichia coli</italic> M19736 ST615 strain, one of the first isolates of Latin America that harbors a plasmid with an <italic>mcr-1</italic> gene, could receive crucial ARG by transformation and conjugation using as donors critical plasmids that harbor <italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>KPC-2</sub>, <italic>bla</italic>
<sub>NDM-5</sub>, <italic>bla</italic>
<sub>NDM-1</sub>, or <italic>aadB</italic> genes. <italic>Escherichia coli</italic> M19736 acquired <italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>KPC-2</sub>, <italic>bla</italic>
<sub>NDM-5</sub>, <italic>bla</italic>
<sub>NDM-1</sub>, and <italic>aadB</italic> genes, being only blaNDM-1 maintained at 100% on the 10th day of subculture. In addition, when the evolved MDR-<italic>E. coli</italic> M19736 acquired sequentially <italic>bla</italic>
<sub>CTX-M-15</sub> and <italic>bla</italic>
<sub>NDM-1</sub> genes, the maintenance pattern of the plasmids changed. In addition, when the evolved XDR-<italic>E. coli</italic> M19736 acquired in an ulterior step the paadB plasmid, a different pattern of the plasmid&#x2019;s maintenance was found. Interestingly, the evolved <italic>E. coli</italic> M19736 strains disseminated simultaneously the acquired conjugative plasmids in different combinations though selection was ceftazidime in all cases. Finally, we isolated and characterized the extracellular vesicles (EVs) from the native and evolved XDR-<italic>E. coli</italic> M19736 strains. Interestingly, EVs from the evolved XDR-<italic>E. coli</italic> M19736 harbored <italic>bla</italic>
<sub>CTX-M-15</sub> though the pDCAG1-CTX-M-15 was previously lost as shown by WGS and experiments, suggesting that EV could be a relevant reservoir of ARG for susceptible bacteria. These results evidenced the genetic plasticity of a sporadic clone of <italic>E. coli</italic> such as ST615 that could play a relevant transitional link in the clinical dynamics and evolution to multidrug/extensively/pandrug-resistant phenotypes of superbugs within the nosocomial niche by acting simultaneously as a vector and reservoir of multiple ARGs which later could be disseminated.</p>
</abstract>
<kwd-group>
<kwd>antimicrobial resistance</kwd>
<kwd>conjugation</kwd>
<kwd>
<italic>mcr-1</italic> gene</kwd>
<kwd>
<italic>Escherichia coli</italic>
</kwd>
<kwd>extracellular vesicles (EVs)</kwd>
</kwd-group>
<counts>
<fig-count count="8"/>
<table-count count="5"/>
<equation-count count="0"/>
<ref-count count="97"/>
<page-count count="20"/>
<word-count count="11320"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Bacteria and Host</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p>
<italic>Escherichia coli</italic> is common among the aerobic bacteria in the gastrointestinal tract microbiota of both humans and mammals (<xref ref-type="bibr" rid="B23">Denamur et&#xa0;al., 2021</xref>). Simultaneously, some lineages have developed into a pathogen well adapted to their host causing different diseases (<xref ref-type="bibr" rid="B33">Geurtsen et&#xa0;al., 2022</xref>), including adaptation to the nosocomial niche as high-risk clones or &#x201c;superbugs&#x201d; that rapidly evolve to extreme drug resistance (XDR). The lineage represented by sequence type (ST) 131 is the predominant isolate of hospital infections worldwide among <italic>E. coli</italic> strains that behave like an epidemic clone, also known as pandemic clones (<xref ref-type="bibr" rid="B69">Pitout and DeVinney, 2017</xref>; <xref ref-type="bibr" rid="B85">Soncini et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B68">Pitout and Chen, 2023</xref>). On the other hand, little is known about the role of sporadic clones of this species in the adaptation to multidrug resistance among clinical isolates. Since a few strains unrelated to outbreaks of <italic>E. coli</italic> ST615 have been reported from Tunisia (<xref ref-type="bibr" rid="B54">Maamar, 2016</xref>), Poland (<xref ref-type="bibr" rid="B40">Jamborova et&#xa0;al., 2015</xref>), and Spain (<xref ref-type="bibr" rid="B63">Ojer-Usoz et&#xa0;al., 2017</xref>) and <italic>E. coli</italic> M19736 from Argentina (<xref ref-type="bibr" rid="B88">Tijet et&#xa0;al., 2017</xref>), this ST may be considered as a sporadic clone. According to the World Health Organization, multidrug resistance (MDR) in Gram-negative bacilli (Gnb) has become a challenge due to its high global incidence and prevalence. In 2019, it was estimated that 4.95 million deaths were due to infections associated with antimicrobial resistance (AMR) (<xref ref-type="bibr" rid="B34">Global antimicrobial resistance and use surveillance system (GLASS) report: 2022, 2022</xref>). These pathogens represent a particular threat in nosocomial infections, and among those of greatest clinical interest, <italic>Acinetobacter baumannii</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Enterobacteriaceae</italic> producers of carbapenemases have been identified as a critical priority (<xref ref-type="bibr" rid="B76">Rello et&#xa0;al., 2019</xref>). MDR Gnb usually harbor multiple mobile genetic elements such as gene cassettes, transposons, and plasmids that confer their MDR phenotypes. These mobile genetic elements can be transferred by conjugation, transformation, and transduction and by the most recently discovered mechanism known as vesiduction through the extracellular vesicles (EVs).</p>
<p>The rapid increase of carbapenem-resistant Gnb due to the expression of enzymes such as KPC-2 (<italic>Klebsiella pneumoniae</italic> carbapenemase-2) and NDM-1 (New Delhi metallo-&#x3b2;-lactamase-1) is a global public health concern. Consequently, interest in another family of antibiotics, polymyxins, has recently increased as a last resort used in medical clinics despite its high nephrotoxicity (<xref ref-type="bibr" rid="B78">Rodr&#xed;guez et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B8">Binsker et&#xa0;al., 2022</xref>). Furthermore, the intensive use of polymyxins in veterinary medicine not only for the treatment of infections but also as a growth promoter has led to an increase of the isolation of Gnb strains resistant to this antibiotic in clinical settings (<xref ref-type="bibr" rid="B8">Binsker et&#xa0;al., 2022</xref>). Resistance to polymyxins includes chromosome-encoded resistance traits, as well as the mobile plasmid-encoded polymyxin resistance determinants such as the <italic>mcr-1</italic> gene (<xref ref-type="bibr" rid="B52">Liu et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B14">Caniaux et&#xa0;al., 2017</xref>). The transferable <italic>mcr-</italic>1 gene was first detected in <italic>E. coli</italic> isolates from animals, food, and patients in China (<xref ref-type="bibr" rid="B52">Liu et&#xa0;al., 2016</xref>). Aside from this gene, 10 other <italic>mcr-</italic>like genes (from <italic>mcr-2</italic> to <italic>mcr-10</italic>) as well as several of their variants designated as <italic>mcr-1.2</italic>, <italic>mcr-1.3</italic>, <italic>mcr-1.12</italic>, etc. have been identified (<xref ref-type="bibr" rid="B38">Hussein et&#xa0;al., 2021</xref>). The <italic>bla</italic>
<sub>KPC</sub>, <italic>bla</italic>
<sub>NDM</sub>, and <italic>mcr</italic>-like genes are usually found in conjugative plasmids of diverse incompatibility groups, which enhance the challenge to combat the bacteria possessing these antibiotic-resistant determinants (<xref ref-type="bibr" rid="B12">Brandt et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B16">Cejas et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B44">Knecht et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B49">Li C. et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B61">Molina et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B81">Sanz et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B11">Boutzoukas et&#xa0;al., 2023</xref>; <xref ref-type="bibr" rid="B77">Riccobono et&#xa0;al., 2023</xref>). Despite the key role that conjugative plasmids have in nosocomial isolates, understanding how they can persist in bacterial populations in the absence of positive selection is challenging for pandemic and sporadic clones.</p>
<p>The nanosize EV entities secreted by Gnb have been recognized for their cardinal importance in intercellular communication among cells and to be responsible for the modulation of various biological functions (<xref ref-type="bibr" rid="B60">McMillan and Kuehn, 2021</xref>; <xref ref-type="bibr" rid="B89">Toyofuku et&#xa0;al., 2023</xref>). EVs are now well recognized for their role as long-distance secretion&#x2013;delivery systems that eliminate the need for cell&#x2013;cell contact (<xref ref-type="bibr" rid="B89">Toyofuku et&#xa0;al., 2023</xref>). EVs transport, harbor, and deliver in a concentrated, protected, and directed way biologically active proteins, lipids, nucleic acids, metabolites, and virulence factors between two bacterial cells (<xref ref-type="bibr" rid="B90">Tran and Boedicker, 2017</xref>; <xref ref-type="bibr" rid="B60">McMillan and Kuehn, 2021</xref>; <xref ref-type="bibr" rid="B89">Toyofuku et&#xa0;al., 2023</xref>). The exchange of DNA mediated by EV has been identified as an additional form of horizontal genetic transfer (HGT) (<xref ref-type="bibr" rid="B17">Chatterjee et&#xa0;al., 2017</xref>). It has been reported that they can carry DNA associated with the membrane and protect luminal genetic material against DNases and RNases (<xref ref-type="bibr" rid="B43">Kim et&#xa0;al., 2015</xref>). Of the DNA that is transferred, there are genes associated with AMR, which has huge clinical implications since the potential of propagation of a gene depends to a great extent on the competence to transmit (<xref ref-type="bibr" rid="B17">Chatterjee et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B92">Wagner et&#xa0;al., 2018</xref>). Earlier studies have reported the transfer by EV of a penicillin resistance gene in <italic>Neisseria gonorrhoeae</italic> (<xref ref-type="bibr" rid="B27">Dorward et&#xa0;al., 1989</xref>); the <italic>bla</italic>
<sub>OXA-24</sub> (<xref ref-type="bibr" rid="B79">Rumbo et&#xa0;al., 2011</xref>) and the <italic>bla</italic>
<sub>NDM-1</sub> carbapenemase genes in <italic>Acinetobacter baumannii</italic> (<xref ref-type="bibr" rid="B17">Chatterjee et&#xa0;al., 2017</xref>); the <italic>bla</italic>
<sub>CTX-M-15</sub> and <italic>bla</italic>
<sub>TEM-1</sub> genes in <italic>E. coli</italic> O104:H4 (<xref ref-type="bibr" rid="B7">Bielaszewska et&#xa0;al., 2020</xref>); and finally, the <italic>bla</italic>
<sub>NDM</sub>, <italic>bla</italic>
<sub>KPC</sub>, <italic>bla</italic>
<sub>SHV</sub>, <italic>bla</italic>
<sub>CTX-M-9</sub>, and <italic>aac(6&#x2032;)-Ib</italic> genes in <italic>K. pneumoniae</italic> strains (<xref ref-type="bibr" rid="B50">Li P. et&#xa0;al., 2022</xref>). In the present study, we wonder about the ability of the <italic>E. coli</italic> M19736 clinical strain belonging to the sporadic clone ST615, which was one of the first isolates harboring the <italic>mcr-1</italic> gene in Latin America (<xref ref-type="bibr" rid="B88">Tijet et&#xa0;al., 2017</xref>), to adapt to the XDR phenotype (<xref ref-type="bibr" rid="B55">Magiorakos et&#xa0;al., 2012</xref>) by conjugation and/or transformation assays. The conservation of ARG in the evolved MDR and XDR-<italic>E. coli</italic> M19736 strains showed different patterns of maintenance as well as variability in the capacity to transfer the acquired conjugative plasmids including the co-transfer of different plasmids in several combinations. In addition, we identified EVs in the native and in the evolved XDR-<italic>E. coli</italic> M19736 strains with crucial content for the competence of AMR in the nosocomial niche, including the <italic>bla</italic>
<sub>CTX-M-15</sub> gene though the plasmid containing this gene, pDCAG1-CTX-M-15, was previously lost. Collectively, this scenario reveals the relevant role of sporadic clones as common vectors for the dissemination of acquired AMR within the framework of the HGT processes.</p>
</sec>
<sec id="s2">
<label>2</label>
<title>Methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>Bacterial strains and growth conditions</title>
<p>
<italic>Escherichia coli</italic> M19736 was isolated in November 2015, in Argentina, from the blood of a patient with peritonitis secondary to colon cancer (<xref ref-type="bibr" rid="B88">Tijet et&#xa0;al., 2017</xref>). The strain was shown by MLST to be a single-locus variant of ST615 (<xref ref-type="bibr" rid="B74">Rapoport et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B88">Tijet et&#xa0;al., 2017</xref>). <italic>Escherichia coli</italic> M19736 has the pM19736 plasmid with the size of 63,230 bp that harbors an <italic>mcr-1</italic> gene as described previously (<xref ref-type="bibr" rid="B88">Tijet et&#xa0;al., 2017</xref>). <italic>Escherichia coli</italic> SM5 (<xref ref-type="bibr" rid="B83">Sennati et&#xa0;al., 2012</xref>), <italic>Klebsiella pneumoniae</italic> HA7Kp (<xref ref-type="bibr" rid="B44">Knecht et&#xa0;al., 2022</xref>), <italic>Klebsiella pneumoniae</italic> HA31Kp (<xref ref-type="bibr" rid="B5">&#xc1;lvarez et&#xa0;al., 2024</xref>), and <italic>Serratia marcescens</italic> SM938 (this study) (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) were used as donors for conjugation assays as described below. Also, <italic>E. coli</italic> J53 was used as a laboratory model for the control of the conjugation experiments.</p>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>Bacterial strains used for the experiments.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" align="center">Strain</th>
<th valign="middle" align="center">ST</th>
<th valign="middle" align="center">Year of isolation</th>
<th valign="middle" align="center">Origin of the sample</th>
<th valign="middle" align="center">Inc. groups</th>
<th valign="middle" align="center">Sequencing technique</th>
<th valign="middle" align="center">Antibiotic susceptibility<xref ref-type="table-fn" rid="fnT1_1">
<sup>a</sup>
</xref>
</th>
<th valign="middle" align="center">Reference</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> M19736</td>
<td valign="middle" align="center">615</td>
<td valign="middle" align="center">2015</td>
<td valign="middle" align="center">Blood culture</td>
<td valign="middle" align="center">IncI2, IncFII, IncI1-I (Alpha)</td>
<td valign="middle" align="center">Illumina MiSeq</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: AMN, AMC, CMP, CIP, FOS, TET<break/>
<bold>Intermediate</bold>: AMS</td>
<td valign="middle" align="center">
<xref ref-type="bibr" rid="B74">Rapoport et&#xa0;al. (2016)</xref>; <xref ref-type="bibr" rid="B88">Tijet et&#xa0;al. (2017)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> SM5</td>
<td valign="middle" align="center">131</td>
<td valign="middle" align="center">2010</td>
<td valign="middle" align="center">Urine</td>
<td valign="middle" align="center">IncFIB, IncFII</td>
<td valign="middle" align="center">Illumina MiSeq</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: AMN, CAZ, CRO, FEP, AZT, TAZ, AMC, AMS, CIP, TMS, FOS<break/>
<bold>Intermediate</bold>: AKN</td>
<td valign="middle" align="center">
<xref ref-type="bibr" rid="B83">Sennati et&#xa0;al. (2012)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">
<italic>Klebsiella pneumoniae</italic> HA7Kp</td>
<td valign="middle" align="center">18</td>
<td valign="middle" align="center">2018</td>
<td valign="middle" align="center">Rectal swab</td>
<td valign="middle" align="center">IncM1, IncHI1B/IncFIB</td>
<td valign="middle" align="center">Illumina MiSeq</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: AMN, CAZ, CRO, AZT, MEM, IMI, TAZ, AMC, AMS, TMS<break/>
<bold>Intermediate</bold>: FEP, TET</td>
<td valign="middle" align="center">
<xref ref-type="bibr" rid="B44">Knecht et&#xa0;al. (2022)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">
<italic>Klebsiella pneumoniae</italic> HA31Kp</td>
<td valign="middle" align="center">11</td>
<td valign="middle" align="center">2018</td>
<td valign="middle" align="center">Tracheal aspirate</td>
<td valign="middle" align="center">IncFII, Col440I, IncFIB(K)</td>
<td valign="middle" align="center">Illumina MiSeq</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: AKN, GEN, AMN, CAZ, CRO, FEP, AZT, MEM, IMI, TAZ, AMC, AMS, CMP, CIP, TMS, FOS</td>
<td valign="middle" align="center">
<xref ref-type="bibr" rid="B5">&#xc1;lvarez et&#xa0;al. (2024)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">
<italic>Serratia marcescens</italic> SM938</td>
<td valign="middle" align="center">NA</td>
<td valign="middle" align="center">2018</td>
<td valign="middle" align="center">Blood culture</td>
<td valign="middle" align="center">IncC</td>
<td valign="middle" align="center">Illumina MiSeq/MinION</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: AMN, CAZ, CRO, TAZ, AMC, AMS, MEM, IMI<break/>
<bold>Intermediate</bold>: FEP, AZT</td>
<td valign="middle" align="center">This study</td>
</tr>
<tr>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> TOP 10::paadB</td>
<td valign="middle" align="center">10</td>
<td valign="middle" align="center">NA</td>
<td valign="middle" align="center">Laboratory</td>
<td valign="middle" align="center">p15A</td>
<td valign="middle" align="center">Illumina MiSeq</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: GEN, CMP</td>
<td valign="middle" align="center">
<xref ref-type="bibr" rid="B71">Quiroga (2012)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> J53</td>
<td valign="middle" align="center">10</td>
<td valign="middle" align="center">NA</td>
<td valign="middle" align="center">Laboratory model</td>
<td valign="middle" align="center">NA</td>
<td valign="middle" align="center">Illumina NextSeq 500/MinION</td>
<td valign="middle" align="center">
<bold>Resistant</bold>: AZI</td>
<td valign="middle" align="center">
<xref ref-type="bibr" rid="B59">Matsumura et&#xa0;al. (2018)</xref>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>General information on the bacterial strains used for the experiments including sequence type (ST), source of isolation, year of isolation, sequencing technique, incompatibility groups (inc. groups), and antibiotic susceptibility.</p>
</fn>
<fn>
<p>AKN, amikacin; GEN, gentamicin; AMN, ampicillin; CAZ, ceftazidime; CRO, ceftriaxone; FEP, cefepime; AZT, aztreonam; MEM, meropenem; IMI, imipenem; TAZ, piperacillin/tazobactam; AMC, amoxicillin/clavulanic; AMS, ampicillin/sulbactam; CMP, chloramphenicol; CIP, ciprofloxacin; TMS, trimethoprim/sulfamethoxazole; FOS, fosfomycin; TET, tetracycline; AZI, sodium azide; NA, not applicable.</p>
</fn>
<fn id="fnT1_1">
<label>a</label>
<p>The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines (<xref ref-type="bibr" rid="B21">CLSI, 2023</xref>).</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>The plasmid paadB from the <italic>E. coli</italic> TOP10::paadB strain was used for the transformation assays (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>). The <italic>aadB</italic> gene cassette from <italic>S.&#xa0;marcescens</italic> SCH909 (<xref ref-type="bibr" rid="B31">Gambino et&#xa0;al., 2021</xref>) was subcloned from a pCR2.1TOPO vector (Invitrogen, Carlsbad, CA) into the commercial vector pACYC184; paadB is resistant to chloramphenicol due to the background of its vector and to gentamicin due to the <italic>Pc</italic> promoter contained upstream the <italic>aadB</italic> gene cassette (<xref ref-type="bibr" rid="B31">Gambino et&#xa0;al., 2021</xref>).</p>
<p>All the strains were grown in Luria&#x2013;Bertani (LB) broth at 37&#xb0;C with shaking (150 rpm). When needed, antibiotics were used at the following concentrations: ceftazidime (8 &#x3bc;/ml), meropenem (2 &#x3bc;g/ml), or gentamicin (25 &#x3bc;g/ml).</p>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>Antibiotic susceptibility testing, minimum inhibitory concentration, and phenotypic detection of &#x3b2;-lactamases</title>
<p>Receptor, donor, and transconjugant strains were tested by antibiotic susceptibility testing (AST) and minimum inhibitory concentration (MIC). AST was carried out by the agar disk diffusion method; MIC was determined by agar dilution. Both assays were conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (<xref ref-type="bibr" rid="B21">CLSI, 2023</xref>), and the results were interpreted under the same guidelines. On the other hand, phenotypic detection of &#x3b2;-lactamases was determined by two tests. First, the detection of carbapenemases was performed by the modified Hodge test as previously described (<xref ref-type="bibr" rid="B65">Pasteran et&#xa0;al., 2016</xref>). Secondly, the synergy produced between the extended-spectrum cephalosporins and clavulanic acid was used for the detection of extended-spectrum &#x3b2;-lactamase.</p>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>Polymerase chain reaction assays</title>
<p>Total DNA extraction was done using the boiling technique for all the experiments of conjugation and transformation. All polymerase chain reaction (PCR) reactions were done using 2 U of Taq DNA polymerase (Inbio Highway, Tandil, Argentina) in 0,5&#xd7; Taq buffer (Inbio Highway) supplemented with 2,5 mM of MgCl<sub>2</sub>, 0,2 mM dNTP mix, and 0,4 uM of each primer in a final volume of 25 &#xb5;l The PCR conditions were 5&#xa0;min at 95&#xb0;C, 30 cycles of 30 s at 95&#xb0;C, 45 s at the appropriate annealing temperature and 1&#xa0;min at 72&#xb0;C, followed by a final extension of 5&#xa0;min at 72&#xb0;C.</p>
<p>Detection of each gene of interest&#x2014;<italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>KPC-2</sub>, <italic>bla</italic>
<sub>NDM-5</sub>, <italic>bla</italic>
<sub>NDM-1</sub>, <italic>aadB</italic>, and/or <italic>mcr-1</italic> genes&#x2014;was conducted with specific primers listed in <xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>. PCR products were separated on agarose gels by electrophoresis, stained with SYBR green, and visualized by UV transillumination.</p>
<table-wrap id="T2" position="float">
<label>Table&#xa0;2</label>
<caption>
<p>Primers used for the detection of genes of interest.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" align="center">Gene/plasmid of interest</th>
<th valign="middle" align="center">Primers</th>
<th valign="middle" align="center">Sequence (5&#x2032;&#x2013;3&#x2032;)</th>
<th valign="middle" align="center">Expected size (bp)</th>
<th valign="middle" align="center">Reference</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="middle" rowspan="2" align="center">
<bold>
<italic>mcr-1</italic>
</bold>
</td>
<td valign="middle" align="center">ForMCR-1</td>
<td valign="middle" align="center">AGTCCGTTTGTTCTTGTGGC</td>
<td valign="middle" rowspan="2" align="center">320</td>
<td valign="middle" rowspan="2" align="center">
<xref ref-type="bibr" rid="B75">Rebelo et&#xa0;al. (2018)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">RevMCR-1</td>
<td valign="middle" align="center">AGATCCTTGGTCTCGGCTTG</td>
</tr>
<tr>
<td valign="middle" rowspan="2" align="center">
<bold>
<italic>bla</italic>
<sub>CTX-M-15</sub>
</bold>
</td>
<td valign="middle" align="center">CTX-M-15F</td>
<td valign="middle" align="center">CGTCACGCTGTTGTTAGGAA</td>
<td valign="middle" rowspan="2" align="center">612</td>
<td valign="middle" rowspan="2" align="center">This study</td>
</tr>
<tr>
<td valign="middle" align="center">CTX-M-15R</td>
<td valign="middle" align="center">CGGTGGTATTGCCTTTCATC</td>
</tr>
<tr>
<td valign="middle" rowspan="2" align="center">
<bold>
<italic>bla</italic>
<sub>KPC-2</sub>
</bold>
</td>
<td valign="middle" align="center">KPC-F</td>
<td valign="middle" align="center">CCGTCAGTTCTGCTGTC</td>
<td valign="middle" rowspan="2" align="center">916</td>
<td valign="middle" rowspan="2" align="center">
<xref ref-type="bibr" rid="B73">Ram&#xed;rez et&#xa0;al. (2013)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">KPC-R</td>
<td valign="middle" align="center">CGTTGTCATCCTCGTTAG</td>
</tr>
<tr>
<td valign="middle" rowspan="2" align="center">
<bold>
<italic>bla</italic>
<sub>NDM</sub>
</bold>
</td>
<td valign="middle" align="center">NDM1-F</td>
<td valign="middle" align="center">CGCGAAGCTGAGCACCGCATTAG</td>
<td valign="middle" rowspan="2" align="center">733</td>
<td valign="middle" rowspan="2" align="center">This study</td>
</tr>
<tr>
<td valign="middle" align="center">NDM1-R</td>
<td valign="middle" align="center">CTATCGGGGGCGGAATGG</td>
</tr>
<tr>
<td valign="middle" rowspan="2" align="center">
<bold>
<italic>aadB</italic>
</bold>
</td>
<td valign="middle" align="center">SULPRO3</td>
<td valign="middle" align="center">GCCTGACGATGCGTGGA</td>
<td valign="middle" rowspan="2" align="center">623</td>
<td valign="middle" rowspan="2" align="center">
<xref ref-type="bibr" rid="B31">Gambino et&#xa0;al. (2021)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">aadBR5&#x2032;</td>
<td valign="middle" align="center">AAGAATCCATAGTCCAACTCC</td>
</tr>
<tr>
<td valign="middle" rowspan="2" align="center">
<bold>pACYC184</bold>
</td>
<td valign="middle" align="center">PACYC1845&#x2032;</td>
<td valign="middle" align="center">TGTAGCACCTGAAGTCAGCC</td>
<td valign="middle" rowspan="2" align="center">496</td>
<td valign="middle" rowspan="2" align="center">
<xref ref-type="bibr" rid="B36">Gravel et&#xa0;al. (1998)</xref>
</td>
</tr>
<tr>
<td valign="middle" align="center">PACYC1843&#x2032;N</td>
<td valign="middle" align="center">GTGATGTCGGCGATATAGGC</td>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>Conjugation assays</title>
<p>Conjugation experiments were performed according to a method described previously (<xref ref-type="bibr" rid="B64">Di Noto et&#xa0;al., 2016</xref>). Briefly, mating assays were carried out on LB agar plates. <italic>Escherichia coli</italic> SM5 (Caz<sup>R</sup>), HA7Kp (Mem<sup>R</sup>), <italic>K. pneumoniae</italic> HA31Kp (Mem<sup>R</sup>), and <italic>S. marcescens</italic> SM98 (Mem<sup>R</sup>) were used as donor strains, while <italic>E. coli</italic> M19736 (Cmp<sup>R</sup>) and J53 (Azi<sup>R</sup>) were used as recipient strains. Donor and recipient strains were diluted from saturated overnight cultures into 12&#xa0;ml and grown until OD<sub>600 nm</sub> ~0.6 at 37&#xb0;C. The cells were harvested by centrifugation, mixed together in a ratio of 1:1, and spotted onto LB plates. They were also spotted individually on LB plates as controls. After 18&#xa0;h of incubation at 37&#xb0;C, mating spots were washed and resuspended in saline; serial dilutions were plated onto LB agar with the specific antibiotic to select for donor, recipient, or transconjugant cells (meropenem 3 &#xb5;g/ml or ceftazidime 8 &#xb5;g/ml and chloramphenicol 50 &#xb5;g/ml or sodium azide 150 &#xb5;g/ml). Conjugation frequency was expressed as the number of transconjugant cells per donor cell in the mating mixture at the time of plating. Transconjugants obtained were checked by plating on CROMagar and LB plates with double antibiotics. Then, the different genes of interest (<italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>KPC-2</sub>, <italic>bla</italic>
<sub>NDM-5</sub>, and <italic>bla</italic>
<sub>NDM-1</sub>) were detected by PCR for each transconjugant. The original recipient strains [<italic>E. coli</italic> M19736 (Cmp<sup>R</sup>) and J53 (Azi<sup>R</sup>)] were used as negative controls, and the donor strains [<italic>E. coli</italic> SM5 (Caz<sup>R</sup>), HA7Kp (Mem<sup>R</sup>), <italic>K. pneumoniae</italic> HA31Kp (Mem<sup>R</sup>), and <italic>S. marcescens</italic> SM98 (Mem<sup>R</sup>)] were used as positive controls (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>).</p>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Transformation assays</title>
<p>
<italic>Escherichia coli</italic> M19736 was treated with 10% glycerol previously. Afterward, electroporation was performed with a Gene Pulser&#x2122; apparatus (Bio-Rad Laboratories, Denver, USA) and conducted using the following parameters: 200 &#x2126; resistance, 25 mF capacitance, and 2 kV voltage, resulting in a time constant between 4.5 and 5.0 ms. Transformed <italic>E. coli</italic> cells were recovered in a 1-ml LB broth and incubated at 37&#xb0;C with a 200-rpm shaking for 2&#xa0;h before being plated on LB agar plates supplied with gentamicin (25 &#x3bc;g/ml) using 100 &#xb5;l.</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>Plasmid maintenance assay</title>
<p>Each strain of interest was cultured in 5&#xa0;ml of LB broth without antibiotic pressure and incubated at 37&#xb0;C ON with shaking (200 rpm). Consecutive subcultures were made for 10 days. An aliquot was taken from each experiment on the 1st and 10th days and plated on LB agar without antibiotics. From each of the replicates, 30 colonies were selected and analyzed. DNA was then extracted from each of these colonies using the boiling method. Next, each of the 30 colonies taken per replicate was tested by PCR for the presence or absence of each of the acquired genes of interest (<italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>KPC-2</sub>, <italic>bla</italic>
<sub>NDM-1</sub>, <italic>bla</italic>
<sub>NDM-5</sub>, <italic>mcr-1</italic> and <italic>aadb</italic>). The maintenance percentage of each replicate was then calculated as follows: <inline-formula>
<mml:math display="inline" id="im1">
<mml:mrow>
<mml:mo>%</mml:mo>
<mml:mi>M</mml:mi>
<mml:mi>a</mml:mi>
<mml:mi>i</mml:mi>
<mml:mi>n</mml:mi>
<mml:mi>t</mml:mi>
<mml:mi>e</mml:mi>
<mml:mi>n</mml:mi>
<mml:mi>a</mml:mi>
<mml:mi>n</mml:mi>
<mml:mi>c</mml:mi>
<mml:mi>e</mml:mi>
<mml:mo>=</mml:mo>
<mml:mo>[</mml:mo>
<mml:mfrac>
<mml:mrow>
<mml:mo stretchy="false">(</mml:mo>
<mml:msub>
<mml:mi>n</mml:mi>
<mml:mrow>
<mml:mi>p</mml:mi>
<mml:mi>c</mml:mi>
</mml:mrow>
</mml:msub>
<mml:mo>+</mml:mo>
<mml:mn>100</mml:mn>
<mml:mo>%</mml:mo>
<mml:mo stretchy="false">)</mml:mo>
</mml:mrow>
<mml:mrow>
<mml:msub>
<mml:mi>n</mml:mi>
<mml:mi>t</mml:mi>
</mml:msub>
</mml:mrow>
</mml:mfrac>
<mml:mo>]</mml:mo>
</mml:mrow>
</mml:math>
</inline-formula>, where <italic>n<sub>pc</sub>
</italic> is the number of positive colonies for the gene of interest and <italic>n<sub>t</sub>
</italic> is the total number of colonies tested (<italic>n<sub>t</sub>
</italic> = 30). Then, the average <inline-formula>
<mml:math display="inline" id="im2">
<mml:mrow>
<mml:mo stretchy="false">(</mml:mo>
<mml:mover accent="true">
<mml:mi>X</mml:mi>
<mml:mo stretchy="true">&#xaf;</mml:mo>
</mml:mover>
<mml:mo stretchy="false">)</mml:mo>
</mml:mrow>
</mml:math>
</inline-formula> of three replicates performed was calculated with their respective standard deviations (SDs).</p>
</sec>
<sec id="s2_7">
<label>2.7</label>
<title>Isolation and purification of EV</title>
<p>EVs were isolated from the late log-phase (16&#xa0;h) culture of <italic>E. coli</italic> M19736 and evolved XDR-<italic>E. coli</italic> M19736 on day 1 of subculture. In brief, cells were cultivated in 600&#xa0;ml of LB broth with 10 &#xb5;g/ml of ceftazidime and subinhibitory concentrations of meropenem ~14 h at 37&#xb0;C. The next day, the cultures were adjusted to OD<sub>600</sub> ~0.7. The cells were pelleted by centrifugation (9,500 rpm, 4&#xb0;C for 20&#xa0;min), and the supernatant was filtered through a 0.22-&#x3bc;m membrane filter (Merck Millipore, Tullagreen, Carrigtwohill, Co. Cork, Ireland) to remove cells and cellular debris. The filtrate was subjected to ultracentrifugation (100,000<italic>g</italic>) for 2&#xa0;h at 4&#xb0;C using a P45AT(RP45T) fixed angle rotor (HIMAC CP80NX). For washing the EV, the pellet suspended in EV buffer (137 mM of NaCl and 20 mM of HEPES [pH 7.5]) was ultracentrifuged (100,000<italic>g</italic>) for 1&#xa0;h at 4&#xb0;C using the same rotor. The pellet was finally resuspended in 1500 &#xb5;l of buffer, filtered and frozen at &#x2212;80&#xb0;C. The EVs were grown in 2&#xa0;ml of LB broth to test for any bacterial growth.</p>
</sec>
<sec id="s2_8">
<label>2.8</label>
<title>Dynamic light scattering</title>
<p>The hydrodynamic diameter (Dh) and the size distribution (polydispersity index, PDI) of different EV sources were assayed by dynamic light scattering (DLS) (DLS, Zetasizer Nano-ZS, Malvern Instruments) at a scattering angle of 173&#xb0;. The nano-ZS contains a 4-mW He&#x2013;Ne laser operating at a wavelength of 633 nm, a digital correlator ZEN3600, and non-invasive backscatter (NIBS<sup>&#xae;</sup>) technology. For the measurement, 200 &#x3bc;l of vesicles suspended in EV buffer (137 mM NaCl and 20 mM HEPES [pH 7.5]) were used. All the samples were analyzed at 25&#xb0;C. Viscosities were between 0.8880 and 0.8872 cP. Results were expressed as mean &#xb1; standard deviation (SD) of three independent samples prepared in identical conditions. Data for each single specimen were the result of at least six runs.</p>
</sec>
<sec id="s2_9">
<label>2.9</label>
<title>Transmission electron microscopy</title>
<p>To verify the presence of intact EV, the preparations were analyzed using transmission electron microscopy (TEM). The EV suspension was fixed in nickel grids for TEM with carbon (200 mesh) (Agar Scientific Ltd., Stansted, Essex, UK), with 2% glutaraldehyde, 4% formaldehyde, and 5% sucrose in PBS; washed three times with ultrapure water; stained with 3% uranyl acetate; allowed to dry for at least 30&#xa0;min; and examined under a transmission electron microscope (Zeiss EM 109T equipped with Gatan ES1000W digital camera).</p>
</sec>
<sec id="s2_10">
<label>2.10</label>
<title>Mass spectrometry analysis</title>
<p>EV proteins were quantified by the Micro BCA&#x2122; Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Protein digestion from lysed EV of native <italic>E. coli</italic> M19736 was performed. We used 40 &#x3bc;g of protein from EV based on the Micro BCA results. The proteins were reduced and alkylated with 10 mM of DTT and 20 mM of iodoacetamide and then precipitated with 15% trichloroacetic acid/acetone and processed for liquid chromatography&#x2013;MS/MS (LC-MS/MS) analysis. Mass spectrometry analysis was performed at the Proteomics Core Facility (CEQUIBIEM), University of Buenos Aires/CONICET (National Research Council) by analyzing the digests by nanoLC-MS/MS in a Thermo Scientific Q Exactive Mass Spectrometer coupled to a nanoHPLC EASY-nLC 1000 (Thermo Scientific). For the LC-MS/MS analysis, approximately 2 &#x3bc;g of peptides were loaded onto the column and eluted for 120&#xa0;min using the reverse phase column (C18, 2 &#xb5;m, 100&#xa0;A, 50 &#xb5;m &#xd7; 150&#xa0;mm) EASY-Spray Column PepMap RSLC (P/N ES801) suitable for separating protein complexes with a high degree of resolution. The flow rate used for the nano column was 300 nl min<sup>&#x2212;1</sup>, and the solvent ranged from 7% B (5&#xa0;min) to 35% (120&#xa0;min). Solvent A was 0.1% formic acid in water, whereas solvent B was 0.1% formic acid in acetonitrile. The injection volume was 2 &#xb5;l. The MS equipment has a high collision dissociation cell (HCD) for fragmentation and an Orbitrap analyzer (Thermo Scientific, Q-Exactive). A voltage of 3.5 kV was used for electrospray ionization (Thermo Scientific, EASY-Spray).</p>
<p>XCalibur 3.0.63 (Thermo Scientific) software was used for data acquisition and equipment configuration that allows peptide identification and chromatographic separation. Full-scan mass spectra were acquired in the Orbitrap analyzer. The scanned mass range was 400&#x2013;1,800 <italic>m</italic>/<italic>z</italic>, at a resolution of 70,000 at 400 <italic>m</italic>/<italic>z</italic>, and the 12 most intense ions in each cycle were sequentially isolated, fragmented by HCD, and measured in the Orbitrap analyzer. Peptides with a charge of +1 or with an unassigned charge state were excluded from fragmentation for MS2.</p>
<sec id="s2_10_1">
<label>2.10.1</label>
<title>Analysis of mass spectrometry data</title>
<p>Q Exactive raw data were processed using Proteome Discoverer software (version 2.1.1.21 Thermo Scientific) and searched against the <italic>E. coli</italic> sequence database with trypsin specificity and a maximum of 1 missed cleavage per peptide. Carbamidomethylation of cysteine residues was set as a fixed modification, and oxidation of methionine was set as a variable modification. Proteome Discoverer searches were performed with a precursor mass tolerance of 10 ppm and product ion tolerance of 0.05 Da. Protein hits were filtered for high-confidence peptide matches with a maximum protein and peptide false discovery rate of 1% calculated by employing a reverse database strategy.</p>
</sec>
</sec>
<sec id="s2_11">
<label>2.11</label>
<title>EV protein analysis</title>
<p>The localization of proteins was mostly acquired using DAVID (<ext-link ext-link-type="uri" xlink:href="https://david.ncifcrf.gov/home.jsp">https://david.ncifcrf.gov/home.jsp</ext-link>). The biological process of EV proteins was derived from Gene Ontology, UniProt, and KEGG (<ext-link ext-link-type="uri" xlink:href="https://www.genome.jp/kegg/">https://www.genome.jp/kegg/</ext-link>). Proteins associated with antibiotic response/resistance were predicted using DAVID.</p>
<sec id="s2_11_1">
<label>2.11.1</label>
<title>Protein&#x2013;protein interaction network analysis</title>
<p>Protein&#x2013;protein interaction (PPI) data were downloaded using the STRING v10.5 database (<xref ref-type="bibr" rid="B87">Szklarczyk et&#xa0;al., 2017</xref>). A PPI network of EV proteins from STRING was incorporated in Cytoscape 3.9.0 (<xref ref-type="bibr" rid="B84">Shannon et&#xa0;al., 2003</xref>), and using the Cytoscape StringApp (<xref ref-type="bibr" rid="B26">Doncheva et&#xa0;al., 2019</xref>), we constructed, analyzed, and visualized the PPI network. Gene Ontology and KEEG enrichment analysis was performed for the EV proteins.</p>
</sec>
</sec>
<sec id="s2_12">
<label>2.12</label>
<title>Determination of DNA in EV</title>
<p>Intravesicular DNA was quantified following the method of <xref ref-type="bibr" rid="B79">Rumbo et&#xa0;al. (2011)</xref> with a few modifications. Fifty micrograms of EVs were treated with DNAse RQ1-Free RNAse 1U/&#xb5;l (Promega) at 37&#xb0;C for 30&#xa0;min to hydrolyze the free and surface-associated DNA. The reaction was stopped with a stop solution and incubation was conducted at 65&#xb0;C for 10&#xa0;min. DNase-treated EVs were then lysed with 0.125% Triton X-100 (Sigma-Aldrich, USA) solution for 30&#xa0;min at 37&#xb0;C, and DNA was purified using a QIAamp DNA Mini Kit with the protocol for crude cell lysates and other samples (Qiagen, Maryland, USA), according to the manufacturer&#x2019;s instructions. The DNA was quantified using the Nano-500 Micro-spectrophotometer. The purified DNA was used for further PCR using the primers listed in <xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>.</p>
</sec>
<sec id="s2_13">
<label>2.13</label>
<title>DNA extraction, DNA sequencing, and sequence assembly</title>
<p>DNA of <italic>E. coli</italic> M19736 and evolved XDR-<italic>E. coli</italic> M19736 on day 1 of subculture was extracted using the mini kit QIAamp DNA (Qiagen) following the manufacturer&#x2019;s protocol for Gram-negative bacteria. The concentration and purity were measured using a NanoDrop instrument (Nano-500 Micro-Spectrophotometer, Allsheng,  Hangzhou, China).</p>
<p>DNA was sequenced on a MiSeq sequencer (Illumina pair ends). The sequencing was performed in the Genomics and Bioinformatics Unit of ANLIS Malbr&#xe1;n (Argentina), and the library preparation was made according to the manufacturer&#x2019;s protocol. Read quality metrics were evaluated using FASTQC v0.11.9. To remove the low-quality reads and the remaining adapters from the sequencing, trimmomatic v0.39 was used. The parameters used were as follows: -threads 8 -phred33 ILLUMINACLIP:TruSeq3.fa:2:30:10 TRAILING:20 SLIDINGWINDOW:4:20 MINLEN:50. The trimmed Fastq files were evaluated using fastqc to determine their quality. Finally, we performed a short-read assembly using Unicycler v0.4.8 (<xref ref-type="bibr" rid="B94">Wick et&#xa0;al., 2017</xref>) with default options. Unicycler was executed by the command line in the GNU/Linux environment. Subsequently, the quality of the assembled files was evaluated using the QUAST v5.0.2 program, using default parameters.</p>
</sec>
<sec id="s2_14">
<label>2.14</label>
<title>Genomic and plasmid analysis</title>
<p>Consensus sequences of the complete assembly were imported into the RAST (<xref ref-type="bibr" rid="B4">Aziz et&#xa0;al., 2008</xref>) and PROKKA (<xref ref-type="bibr" rid="B82">Seemann, 2014</xref>) databases. The search for all ARGs and efflux pumps associated with AMR was performed using ResFinder (<xref ref-type="bibr" rid="B95">Zankari et&#xa0;al., 2012</xref>), RGI 6.0.2, and CARD 3.2.7 online databases with a minimum identity of 95%. In addition, chromosomal mutations associated with AMR were searched using PointFinder available in the ResFinder database. PlasmidFinder v 2.1.6 (<xref ref-type="bibr" rid="B20">Clausen et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B15">Carattoli and Hasman, 2020</xref>) was used to detect the plasmids in our samples using the default parameters. VRprofile2 v2.0 was used to predict mobilome (<xref ref-type="bibr" rid="B93">Wang et&#xa0;al., 2022</xref>). Conjugation systems were searched against the NCBI database, and OriT was detected using the online tool oriTfinder (<xref ref-type="bibr" rid="B46">Li et&#xa0;al., 2018</xref>). The search and detection of toxin&#x2013;antitoxin systems was performed using the online tool TADB v2.0 using the default parameters (<xref ref-type="bibr" rid="B37">Guan et&#xa0;al., 2023</xref>).</p>
</sec>
<sec id="s2_15">
<label>2.15</label>
<title>Statistical analysis</title>
<p>Statistical analysis of the data obtained was performed using the GraphPad Prism 8.0.2 program (GraphPad, La Jolla, CA, USA). Variables were expressed as median (interquartile range, IQR) and compared by Kruskal&#x2013;Wallis followed by Dunn&#x2019;s <italic>post-hoc</italic> test. We looked for statistically significant differences between conjugation frequencies of the <italic>E. coli</italic> M19736 strain and the laboratory control strain <italic>E. coli</italic> J53. <italic>p</italic>-values&lt;0.05 were regarded as statistically significant.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results</title>
<sec id="s3_1">
<label>3.1</label>
<title>Ability of <italic>Escherichia coli</italic> M19736 to acquire plasmids from different species</title>
<p>
<italic>Escherichia coli</italic> M19736 was tested as a receptor to receive crucial ARG by transformation and conjugation using different relevant plasmids as donors (<xref ref-type="table" rid="T3">
<bold>Table&#xa0;3</bold>
</xref>) including i) pDCAG1-CTX-M-15 (&gt;112.000 bp, IncFII) from the clinical strain <italic>E. coli</italic> SM5 that harbors <italic>bla</italic>
<sub>CTX-M-15</sub>, ii) pDCCK<sub>1</sub>-KPC (&gt;77.218 bp, IncM1) from the clinical strain <italic>K. pneumoniae</italic> HA7Kp that harbors <italic>bla</italic>
<sub>KPC-2</sub>, iii) pDCVA3-NDM-5 (&gt;534.520 bp, IncFII) from the clinical strain <italic>K. pneumoniae</italic> HA31Kp that harbors <italic>bla</italic>
<sub>NDM-5</sub>, iv) pDCASG-NDM-1 (137.269 bp, IncC) from the clinical strain <italic>S. marcescens</italic> SM938 that harbors <italic>bla</italic>
<sub>NDM-1</sub>, and v) recombinant plasmid paadB from <italic>Escherichia coli</italic> TOP 10::paadB (5.877 bp, p15A) that harbors the <italic>aadB</italic> gene cassette. We identified by bioinformatics analysis that the four clinical plasmids had conjugation systems (<xref ref-type="table" rid="T3">
<bold>Table&#xa0;3</bold>
</xref>), all of them being conjugative to <italic>E. coli</italic> J53 and <italic>E. coli</italic> M19736 in our experimental conditions (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>). Also, <italic>E. coli</italic> M19736 was able to acquire paadB by chemical transformation (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>). Conjugation was performed as previously described, showing that <italic>E. coli</italic> M19736 was able to acquire the four plasmids (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>). Although all conjugation efficiencies were higher when <italic>E. coli</italic> M19736 was the receptor strain, a statistical difference between <italic>E. coli</italic> M19736 and <italic>E. coli</italic> J53 was only found when <italic>S. marcescens</italic> SM938 (pDCASG6-NDM-1) was used as a donor (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>).</p>
<table-wrap id="T3" position="float">
<label>Table&#xa0;3</label>
<caption>
<p>Features of plasmids used for the experiments.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" align="center">Plasmid</th>
<th valign="middle" align="center">Bacterial strain</th>
<th valign="middle" align="center">Plasmid or pseudomolecule size</th>
<th valign="middle" align="center">Inc group<xref ref-type="table-fn" rid="fnT3_1">
<sup>a</sup>
</xref>
</th>
<th valign="middle" align="center">Antibiotic resistance genes of the plasmids<xref ref-type="table-fn" rid="fnT3_2">
<sup>b</sup>
</xref>
</th>
<th valign="middle" align="center">Conjugation genes</th>
<th valign="middle" align="center">Toxin/antitoxin system</th>
<th valign="middle" align="center">GenBank accession no.<xref ref-type="table-fn" rid="fnT3_3">
<sup>c</sup>
</xref>
</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="middle" align="center">
<bold>pM19736-MCR-1</bold>
</td>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> M19736</td>
<td valign="middle" align="center">63,230 bp</td>
<td valign="middle" align="center">IncI2</td>
<td valign="middle" align="center">
<italic>mcr-1.1</italic>
</td>
<td valign="middle" align="center">
<italic>pilVUTSRQPON/traKJIHG/traEDCB/pilML/trbJL</italic>
</td>
<td valign="middle" align="center">
<italic>relE/B</italic> and <italic>TsxA/B</italic>
</td>
<td valign="middle" align="center">FR851304 and JN983044</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>pDCAG1-CTX-M-15</bold>
</td>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> SM5</td>
<td valign="middle" align="center">&gt;112.000 bp</td>
<td valign="middle" align="center">IncFII</td>    <td valign="middle" align="center">
<italic>tet(B)</italic>, <italic>catB3</italic>, <italic>dfrA8</italic>, <italic>sul2</italic>, <italic>
<underline>bla</underline>
</italic>
<underline>
<sub>CTX-M-15</sub>
</underline>, <italic>bla</italic>
<sub>TEM-1B</sub>, <italic>bla</italic>
<sub>OXA-1</sub>, <italic>aac(6&#x2032;)-Ib-cr</italic>, <italic>aph(6)-Id</italic>, <italic>aph(3&#x2033;)-Ib</italic>
</td>
<td valign="middle" align="center">
<italic>FinO/traXIDTSGH/trbFJB/traQ/trbA/traF/trbE/traN/trbC/traUW/trbI/traCRV/trbD/traPBKELAJM</italic>
</td>
<td valign="middle" align="center">
<italic>CcdA/B</italic> and <italic>pemK/L</italic>
</td>
<td valign="middle" align="center">AY458016.1</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>pDCCK1-KPC</bold>
</td>
<td valign="middle" align="center">
<italic>Klebsiella pneumoniae</italic> HA7Kp</td>
<td valign="middle" align="center">&gt;77.218 bp</td>
<td valign="middle" align="center">IncM1</td>    <td valign="middle" align="center">
<italic>
<underline>bla</underline>
</italic>
<underline>
<sub>KPC-2</sub>
</underline>
</td>
<td valign="middle" align="left">
<italic>traHIJK/traLMNOPQRUWXY/trbCBAN</italic>
</td>
<td valign="middle" align="center">
<italic>pemK/L</italic>
</td>
<td valign="middle" align="center">AF550415.2</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>pDCVA3-NDM-5</bold>
</td>
<td valign="middle" align="center">
<italic>Klebsiella pneumoniae</italic> HA31Kp</td>
<td valign="middle" align="center">&gt;534.520 bp</td>
<td valign="middle" align="center">IncFII</td>    <td valign="middle" align="center">
<italic>fosA</italic>, <italic>catB3</italic>, <italic>sul1</italic>, <italic>aac(6&#x2032;)-Ib-cr</italic>, <italic>oqxA</italic>, <italic>qnrS1</italic>, <italic>oqxB</italic>, <italic>erm(B)</italic>, <italic>mph(A)</italic>, <italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>TEM-1B</sub>, <italic>bla</italic>
<sub>OXA-1</sub>, <italic>
<underline>bla</underline>
</italic>
<underline>
<sub>NDM-5</sub>
</underline>, <italic>qacE, aadA2</italic>, <italic>aac(6&#x2032;)-Ib-cr</italic>, <italic>aph(3&#x2032;)-Ia</italic>, <italic>dfrA12</italic>, <italic>rmtB</italic>
</td>
<td valign="middle" align="center">
<italic>FinO/traXID/traGH/trbFJB/traQ/trbA/traF/</italic>/<italic>traN/trbC/traUW/trbI/traCRV/trbD/traPBKELAJM</italic>
</td>
<td valign="middle" align="center">
<italic>AAA-ATPase/relB</italic>, <italic>pemK/L</italic>, and <italic>HipA/B</italic>
</td>
<td valign="middle" align="center">AY458016.1</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>pDCASG-NDM-1</bold>
</td>
<td valign="middle" align="center">
<italic>Serratia marcescens</italic> SM938</td>
<td valign="middle" align="center">137.269 bp</td>
<td valign="middle" align="center">IncC</td>    <td valign="middle" align="center">
<italic>
<underline>bla</underline>
</italic>
<underline>
<sub>NDM-1</sub>
</underline>, <italic>ble</italic>
<sub>MBL</sub>, <italic>bla</italic>
<sub>CMY-6</sub>, <italic>qacE&#x394;1, sul1</italic>, <italic>&#x394;bla</italic>
<sub>OXA-1</sub>/<italic>aac(6&#x2032;)-Ib3</italic>
</td>
<td valign="middle" align="center">
<italic>traFHG/traID traLEKBVA/traC/trhF</italic>/<italic>traWUN</italic>
</td>
<td valign="middle" align="center">
<italic>HigA/B</italic>
</td>
<td valign="middle" align="center">JX141473.1</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>paadB</bold>
</td>
<td valign="middle" align="center">
<italic>Escherichia coli</italic> TOP 10::paadB</td>
<td valign="middle" align="center">5.877 bp</td>
<td valign="middle" align="center">p15A</td>
<td valign="middle" align="center">
<italic>
<underline>aadB</underline>
</italic>, <italic>catA1</italic>
</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">Not applicable</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>The results found by bioinformatics analysis of the plasmids used as donors in the transformation and conjugation assays.</p>
</fn>
<fn id="fnT3_1">
<label>a</label>
<p>The replicon shown for each strain corresponded to the genetic location where the carbapenemase gene, the bla<sub>CTX-M-15</sub>, or the <italic>aadB</italic> gene cassette was found.</p>
</fn>
<fn id="fnT3_2">
<label>b</label>
<p>Underlined genes were used as conjugation/transformation biomarkers.</p>
</fn>
<fn id="fnT3_3">
<label>c</label>
<p>GenBank accession number of plasmids used as reference.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Conjugation/transformation assays of different multidrug-resistant plasmids with <italic>Escherichia coli</italic> M19736 and <italic>E. coli</italic> J53 as laboratory control. Panel <bold>(A)</bold> shows the acquisition of the different plasmids by conjugation or transformation of <italic>E. coli</italic> M19736. Each color represents the plasmids that were transferred: pDCAG1-CTX-M-15 (violet), pDCCK1-KPC (pink), pDCVA3-NDM-5 (blue), pDCASG-NDM-1 (orange), and paadB (yellow). Five transconjugants using <italic>E. coli</italic> M19736 as receptor (<italic>E. coli</italic> M19736::pDCAG1-CTX-M-15, <italic>E. coli</italic> M19736::pDCCK1-KPC, <italic>E. coli</italic> M19736:: pDCVA3-NDM-5, <italic>E. coli</italic> M19736::pDCASG-NDM-1, and evolved MDR-<italic>E. coli</italic> M19736) and transformant XDR-<italic>E. coli</italic> M19736 were generated. Panel <bold>(B)</bold> shows each donor and transconjugant (TRCJ) obtained in the different experiments using <italic>E. coli</italic> M19736 and <italic>E. coli</italic> J53 as receptors. <italic>Escherichia coli</italic> J53 as control is depicted as light gray with dotted bars, <italic>E. coli</italic> M19736 as black diagonal line bars, and evolved MDR-<italic>E. coli</italic> M19736 as dark gray with horizontal line bars. Data show the comparison of conjugation efficiencies of each plasmid between <italic>E. coli</italic> M19736 and <italic>E. coli</italic> J53. Conjugation efficiencies were calculated as the quotient between the number of transconjugants (Tc) and the number of donors (D) (Tc/D) in triplicates. Data show the median with range from three independent experiments (<italic>n</italic> = 3). Significant differences between groups are indicated: *<italic>p</italic>&lt; 0.05; ns, not significant.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g001.tif"/>
</fig>
<p>ARG acquisition by <italic>E. coli</italic> M19736 and <italic>E. coli</italic> J53 as receptor strains was verified by PCR of conjugation markers (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>), phenotypic detection of &#x3b2;-lactamases, AST (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;1</bold>
</xref>), and MIC (<xref ref-type="table" rid="T4">
<bold>Table&#xa0;4</bold>
</xref>). In the experiments of successive conjugation assays, the evolved MDR-<italic>E. coli</italic> M19736 was able to harbor simultaneously <italic>mcr-1</italic>, <italic>bla</italic>
<sub>CTX-M-15</sub>, and <italic>bla</italic>
<sub>NDM-1</sub> genes (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>). The evolved XDR-<italic>E. coli</italic> M19736 was able to acquire also the <italic>aadB</italic> gene later by transformation assay (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>). Susceptibility tests showed that the evolved XDR-<italic>E. coli</italic> M19736 became resistant to all antibiotics tested except to trimethoprim/sulfamethoxazole (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;1</bold>
</xref>). The MIC for carbapenem antibiotics (ERT and MEM) was slightly higher in <italic>E. coli</italic> M19736::pDCVA3-NDM-5, evolved MDR-<italic>E. coli</italic> M19736, and evolved XDR-<italic>E.coli</italic> M19736 than the respective donor strains and transconjugants of <italic>E. coli</italic> J53 (<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>Minimum inhibitory concentration (MIC) of <italic>Escherichia coli</italic> strains.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" align="center"/>
<th valign="middle" align="center">CAZ</th>
<th valign="middle" align="center">ERT</th>
<th valign="middle" align="center">MEM</th>
<th valign="middle" align="center">GEN</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> ATCC 25922</bold>
</td>
<td valign="middle" align="center">0.5 (S)</td>
<td valign="middle" align="center">0.016 (S)</td>
<td valign="middle" align="center">0.03 (S)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> M19736</bold>
</td>
<td valign="middle" align="center">0.03 (S)</td>
<td valign="middle" align="center">0.5 (S)</td>
<td valign="middle" align="center">0.25 (S)</td>
<td valign="middle" align="center">0.03 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> J53</bold>
</td>
<td valign="middle" align="center">0.25 (S)</td>
<td valign="middle" align="center">0.08 (S)</td>
<td valign="middle" align="center">0.03 (S)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> SM5</bold>
</td>
<td valign="middle" align="center">64 (R)</td>
<td valign="middle" align="center">0.12 (S)</td>
<td valign="middle" align="center">0.06 (S)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> M19736::pDCAG1-CTX-M-15</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">0.5 (S)</td>
<td valign="middle" align="center">0.25 (S)</td>
<td valign="middle" align="center">0.03 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> J53::pDCAG1-CTX-M-15</bold>
</td>
<td valign="middle" align="center">64 (R)</td>
<td valign="middle" align="center">0.08 (S)</td>
<td valign="middle" align="center">0.03 (S)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>K pneumoniae</italic> HA7Kp</bold>
</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">8 (R)</td>
<td valign="middle" align="center">8 (R)</td>
<td valign="middle" align="center">0.25 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> M19736::pDCCK1-KPC</bold>
</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">4 (R)</td>
<td valign="middle" align="center">2 (I)</td>
<td valign="middle" align="center">0.03 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> J53::pDCCK1-KPC</bold>
</td>
<td valign="middle" align="center">16 (R)</td>
<td valign="middle" align="center">2 (R)</td>
<td valign="middle" align="center">4 (R)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>K. pneumoniae</italic> HA31Kp</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">&gt;64 (R)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> M19736::pDCVA3-NDM-5</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">64 (R)</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">0.03 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> J53::pDCVA3-NDM-5</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">2 (R)</td>
<td valign="middle" align="center">4 (R)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>S. marcescens</italic> SM938</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">8 (R)</td>
<td valign="middle" align="center">8 (R)</td>
<td valign="middle" align="center">2 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> M19736::pDCASG-NDM-1</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">16 (R)</td>
<td valign="middle" align="center">8 (R)</td>
<td valign="middle" align="center">0.03 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>
<italic>E. coli</italic> J53::pDCASG-NDM-1</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">16 (R)</td>
<td valign="middle" align="center">0.5 (S)</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>Evolved MDR<italic>-E. coli</italic> M19736</bold>
</td>
<td valign="middle" align="center">&gt;64 (R)</td>
<td valign="middle" align="center">32 (R)</td>
<td valign="middle" align="center">16 (R)</td>
<td valign="middle" align="center">0.03 (S)</td>
</tr>
<tr>
<td valign="top" align="center">
<bold>Evolved XDR-<italic>E. coli</italic> M19736</bold>
</td>
<td valign="top" align="center">&gt;64 (R)</td>
<td valign="top" align="center">32 (R)</td>
<td valign="top" align="center">16 (R)</td>
<td valign="top" align="center">32 (R)</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>Results were interpreted according to the Clinical and Laboratory Standards Institute guidelines (<xref ref-type="bibr" rid="B21">CLSI, 2023</xref>).</p>
</fn>
<fn>
<p>CAZ, ceftazidime; ERT, ertapenem; MEM, meropenem; GEN, gentamicin. Interpretation results: I, intermediate; R, resistance; S, susceptible.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s3_2">
<label>3.2</label>
<title>Maintenance of plasmids in native and evolved MDR and XDR-<italic>Escherichia coli</italic> M19736 strains</title>
<p>Firstly, we evaluated the maintenance of plasmids of each donor cell (<italic>Escherichia coli</italic> SM5, <italic>K. pneumoniae</italic> HA7Kp, <italic>K. pneumoniae</italic> HA31Kp, and <italic>S. marcescens</italic> SM938) on the 1st and 10th days after being subcultured without antibiotic pressure at 37&#xb0;C. All plasmids were maintained at 100% of each assay (data not shown). Then, each transconjugant of <italic>E. coli</italic> M19736, or transformant in the case of XDR-<italic>E. coli</italic> M19736 (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>), was evaluated for its ability to maintain ARG (<italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>bla</italic>
<sub>KPC-2</sub>, <italic>bla</italic>
<sub>NDM-5</sub>, <italic>bla</italic>
<sub>NDM-1</sub>, or <italic>aadB</italic>) by doing the same assay without antibiotic pressure at 37&#xb0;C (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>). At the same time, the maintenance of the <italic>mcr-1</italic> gene was evaluated in all combinations. Each one of the four clinical plasmids (pDCAG1-CTX-M-15, pDCCK1-KPC, pDCVA3-NDM-5, and pDCASG6-NDM-1) harbored a different toxin/antitoxin system (<xref ref-type="table" rid="T3">
<bold>Table&#xa0;3</bold>
</xref>). The transconjugants were able to maintain each gene of interest on the first day of subculture at 100%. Each transconjugant or transformant maintained <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15), <italic>bla</italic>
<sub>KPC-2</sub> (pDCCK1-KPC), <italic>bla</italic>
<sub>NDM-5</sub> (pDCVA3-NDM-5), <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG6-NDM-1), and <italic>aadB</italic> (paadB) at 98.9%, 73.3%, 88.9%, 100%, and 0%, respectively, on the 10th day of subculture (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>).</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Maintenance of crucial ARG harbored by plasmids. Each color/pattern shows the percentage of maintenance of each ARG that was acquired in its respective plasmid by <italic>E. coli</italic> M19736 <bold>(A)</bold> or by <italic>E. coli</italic> J53 <bold>(B)</bold>. The <italic>mcr-1</italic> gene in pM19736-MCR-1, the <italic>bla</italic>
<sub>CTX-M-15</sub> gene in pDCAG1-CTX-M-15, the <italic>bla</italic>
<sub>KPC-2</sub> gene in pDCCK<sub>1</sub>-KPC, the <italic>bla</italic>
<sub>NDM-5</sub> gene in pDCVA3-NDM-5, the <italic>bla</italic>
<sub>NDM-1</sub> gene in pDCASG-NDM-1, and the <italic>aadB</italic> gene in paadB were used as target for PCR detection with primers from <xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>, respectively. Data shows the mean &#xb1; SD from three independent experiments performed in triplicate (<italic>n</italic> = 3).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g002.tif"/>
</fig>
<p>Interestingly, when the evolved MDR and XDR-<italic>E. coli</italic> M19736 strains acquired progressively <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15) and <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG6-NDM-1) or acquired <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15), <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG6-NDM-1), and <italic>aadB</italic> (paadB) plasmids, respectively, a different pattern of maintenance was found (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>). In the case of evolved MDR-<italic>E. coli</italic> M19736, pDCAG1-CTX-M-15 and pDCASG-NDM-1 were maintained at 41.1% and 91.1%, respectively, on the 10th day of subculture (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>). When the evolved XDR-<italic>E. coli</italic> M19736 that harbored <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15), <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG6-NDM-1), and <italic>aadB</italic> (paadB) genes was tested, we found that <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15) and <italic>aadB</italic> (paadB) genes were lost while maintaining the <italic>bla</italic>
<sub>NDM-1</sub> gene (pDCASG6-NDM-1) at 98.9% on the 10th day of subculture. Remarkably, in all cases without antibiotic pressure, <italic>E. coli</italic> M19736 maintained the <italic>mcr-1</italic> gene.</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Maintenance of the ARG-plasmid located in the evolved MDR and XDR <italic>E. coli</italic> M19736 strains. The bars represent an evolved MDR-<italic>E. coli</italic> M19736 or evolved XDR-<italic>E. coli</italic> M19736 on the 1st day and 10th day of subculture. Each color/pattern and number within each bar represents the percentage of maintenance for the conjugated plasmids. Data shown are the mean &#xb1; SD from three independent experiments performed in triplicate (<italic>n</italic> = 3).</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g003.tif"/>
</fig>
<p>On the other hand, bioinformatics analysis of the WGS of the evolved XDR-<italic>E. coli</italic> M19736 from subculture on day 1 confirmed the presence of eight ARGs found in pM19736-MCR-1 and pIncFII-M19736 plasmids and chromosome from the host <italic>E. coli</italic> M19736 strain, five ARGs from pDCASG-NDM-1 as expected, and two ARGs from paadB (<xref ref-type="table" rid="T5">
<bold>Table&#xa0;5</bold>
</xref>). The replication origins of these plasmids were also found. In contrast, no antibiotic determinant or replication origins of the pDCAG1-CTX-M-15 plasmid were found in the evolved XDR-<italic>E. coli</italic> M19736 which could be due to the fact that pDCAG1-CTX-M-15 was rapidly lost on day 1 as shown in our maintenance experiments (see below) (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>).</p>
<table-wrap id="T5" position="float">
<label>Table&#xa0;5</label>
<caption>
<p>ARGs found in the genome of the evolved XDR-<italic>E. coli</italic> M19736.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="top" align="center">Evolved XDR-<italic>E. coli</italic> M19736</th>
<th valign="top" align="center">
<italic>E. coli</italic> M19736</th>
<th valign="top" align="center">pIncFII-M19736</th>
<th valign="top" align="center">pM19736-MCR-1</th>
<th valign="top" align="center">pDCASG6-NDM-1</th>
<th valign="top" align="center">paadB</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="center">
<italic>fosL1</italic>
</td>
<td valign="top" align="center">
<italic>fosL1</italic>
</td>
<td valign="top" align="center">
<italic>aph(6)-Id</italic>
</td>
<td valign="top" align="center">
<italic>mcr-1.1</italic>
</td>
<td valign="top" align="center">
<italic>aac(6&#x2032;)-Ib3</italic>
</td>
<td valign="top" align="center">
<italic>aadB</italic>
</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>tet(B)</italic>
</td>
<td valign="top" align="center">
<italic>tet(B)</italic>
</td>
<td valign="top" align="center">
<italic>aph(3&#x2033;)-Ib</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>bla</italic>
<sub>CMY-6</sub>
</td>
<td valign="top" align="center">
<italic>catA1</italic>
</td>
</tr>
<tr>
<td valign="top" align="center">
<italic>aph(6)-Id</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>bla</italic>
<sub>TEM-1B</sub>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>bla</italic>
<sub>NDM-1</sub>
</td>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>aph(3&#x2033;)-Ib</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>floR</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>sul1</italic>
</td>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>bla</italic>
<sub>TEM-1B</sub>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>sul2</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center">
<italic>qacE</italic>
</td>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>floR</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>sul2</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>mcr-1.1</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>aac(6&#x2032;)-Ib3</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>bla</italic>
<sub>CMY-6</sub>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>bla</italic>
<sub>NDM-1</sub>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>sul1</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>qacE</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>aadB</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="left"/>
</tr>
<tr>
<td valign="top" align="center">
<italic>catA1</italic>
</td>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
<td valign="top" align="center"/>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>The data show the results found by ResFinder of ARGs identified in the genome of the evolved XDR-<italic>E. coli</italic> M19736, <italic>E. coli</italic> M19736, and plasmids of interest.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s3_3">
<label>3.3</label>
<title>Ability of the evolved MDR and XDR-<italic>Escherichia coli</italic> M19736 strains to disseminate the acquired conjugative plasmids</title>
<p>The evolved MDR and XDR-<italic>E. coli</italic> M19736, which were co-infected with several plasmids, were tested as donors of clinical conjugative plasmids using again as receptor <italic>E. coli</italic> J53 (<xref ref-type="fig" rid="f4">
<bold>Figures&#xa0;4A, B</bold>
</xref>). The selection was performed with ceftazidime. The evolved MDR-<italic>E. coli</italic> M19736 and XDR-<italic>E. coli</italic> M19736 strains were able to transfer the <italic>bla</italic>
<sub>NDM-1</sub> gene located in pDCASG-NDM-1 to <italic>E. coli</italic> J53 in the three independent biological replicates. On the other hand, when the evolved MDR-<italic>E. coli</italic> M19736 was used as donor, we identified two other genotypes in the transconjugants (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4A</bold>
</xref>). The first one transferred simultaneously the <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG6-NDM-1) and <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15) genes in the transconjugants of <italic>E. coli</italic> J53. The second one harbored only the <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15) gene. Furthermore, when the&#xa0;evolved XDR-<italic>E. coli</italic> M19736 was used as donor, we were able to find another genotype in which <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG6-NDM-1) and <italic>mcr-1</italic> (pM19736-MCR-1) genes were detected simultaneously (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4B</bold>
</xref>).</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Dissemination of plasmids co-infecting evolved <italic>Escherichia coli</italic> M19736 strains to <italic>E. coli</italic> J53. Evolved MDR-<italic>E. coli</italic> M19736 and XDR-<italic>E. coli</italic> M19736 were tested as donors of co-infecting clinical conjugative plasmids using as receptor <italic>E. coli</italic> J53 <bold>(A, B)</bold>. The selection was performed with 8 &#xb5;g/ml of ceftazidime. The number of colonies detected with each gene (<italic>bla</italic>
<sub>NDM-1</sub>, <italic>bla</italic>
<sub>CTX-M-15</sub>, and/or <italic>mcr-1</italic>) in transconjugants is shown. Numbers in parentheses represent the conjugation efficiencies of each experiment for the plasmids. Conjugation efficiencies were calculated as the quotient between the number of transconjugants (Tc) and the number of donors (D) (Tc/D). Both evolved strains were able to transfer the <italic>bla</italic>
<sub>NDM-1</sub> gene located in pDCASG-NDM-1 to <italic>E. coli</italic> J53 from three independent biological replicates. When the evolved MDR-<italic>E. coli</italic> M19736 was used as donor, two other genotypes were identified in transconjugants in one replicate <bold>(A)</bold>. The first genotype harbored simultaneously plasmids of the <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG-NDM-1) and <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15) genes in <italic>E. coli</italic> J53 transconjugants. The second one harbored only the <italic>bla</italic>
<sub>CTX-M-15</sub> (pDCAG1-CTX-M-15) gene. When the evolved XDR-<italic>E. coli</italic> M19736 was used as donor, in one replicate, another genotype was identified in which the <italic>bla</italic>
<sub>NDM-1</sub> (pDCASG-NDM-1) and <italic>mcr-1</italic> (pM19736-MCR-1) genes were detected simultaneously <bold>(B)</bold>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g004.tif"/>
</fig>
</sec>
<sec id="s3_4">
<label>3.4</label>
<title>Isolation and characterization of EV from the native and evolved XDR-<italic>Escherichia coli</italic> M19736 strains</title>
<p>The native <italic>E. coli</italic> M19736 and evolved XDR-<italic>E. coli</italic> M19736 strains actively released EV at the log phase of growth and were isolated and collected from the supernatant broth. The cell-free EVs extracted from both strains were purified by filtration and ultracentrifugation. EVs were characterized in terms of morphology, size, and polydispersity index (PDI). The purified EVs appeared at TEM as electron-dense particles, with a spherical morphology, a bilayer membrane, and heterogeneous nanometer size (<xref ref-type="fig" rid="f5">
<bold>Figures&#xa0;5B, D</bold>
</xref>). The purity of the EV was confirmed as there were no bacteria visualized by TEM, and contamination controls on culture plates did not show any growth. This showed that EVs were purified successfully without contamination with other bacterial components for subsequent cell experiments. The obtained data from DLS showed that the typical diameter was approximately 193.2 &#xb1; 1.8 nm with a PDI of 0.199 &#xb1; 0.012 for the native <italic>E. coli</italic> M19736 (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5A</bold>
</xref>) and 174.7 &#xb1; 0.52 nm with a PDI of 0.2 &#xb1; 0.009 for the evolved XDR-<italic>E. coli</italic> M19736 (<xref ref-type="fig" rid="f5">
<bold>Figure&#xa0;5C</bold>
</xref>).</p>
<fig id="f5" position="float">
<label>Figure&#xa0;5</label>
<caption>
<p>Phenotypic characterization of EV from native and evolved XDR-<italic>E. coli</italic> M19736. Vesicles were purified from broth cultures by ultracentrifugation and filtered through a 0.22-&#x3bc;m filter. DLS results are presented as the mean of three independent measurements &#xb1; SD. DLS measurement shows an average size of 193.2 &#xb1; 1.8 nm for the native <italic>E. coli</italic> M19736 <bold>(A)</bold> and 174.7 &#xb1; 0.52 nm for the evolved XDR-<italic>E. coli</italic> M19736 <bold>(C)</bold>. TEM results show in both strains EV with a double membrane, spherical in shape and heterogeneous in size <bold>(B, D)</bold>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g005.tif"/>
</fig>
</sec>
<sec id="s3_5">
<label>3.5</label>
<title>Content analysis of EV from both <italic>Escherichia coli</italic> M19736 and evolved XDR-<italic>Escherichia coli</italic> M19736 strains</title>
<p>The DNA purified from EVs of the native <italic>E. coli</italic> M19736 gave a specific amplified product for the <italic>mcr-1</italic> gene. Also, EV DNA from the evolved XDR-<italic>E. coli</italic> M19736 allowed us to detect specific PCR products for the <italic>bla</italic>
<sub>CTX-M-15</sub>, <italic>mcr-1</italic>, and <italic>aadB</italic> genes. On the other hand, the total vesicular proteins were extracted via lysis buffer and then quantified by the Micro BCA kit. The amount of protein for the native and evolved strains was 1,015 &#x3bc;g/ml and 1,091 &#x3bc;g/ml, respectively. To explore the protein contents of EV from <italic>E. coli</italic> M19736, LC-MS/MS analysis was applied, and 338 different proteins were identified in EVs from the native strain (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>). Database protein was included in Vesiclepedia 2024 (<ext-link ext-link-type="uri" xlink:href="http://www.microvesicles.org">http://www.microvesicles.org</ext-link>) (<xref ref-type="bibr" rid="B18">Chitti et&#xa0;al., 2024</xref>). Proteins were categorized into different classes including the following: cellular localization site (<xref ref-type="fig" rid="f6">
<bold>Figure&#xa0;6</bold>
</xref>) and biological functions (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7</bold>
</xref>). The localization of EV proteins from <italic>E. coli</italic> M19736 was found to be distributed as follows: 10% of the proteins were located in the cell membrane, 18% in the inner membrane, 16% in the outer membrane, 50% in the cytoplasm, and 6% in the periplasm. Moreover, among the 338 identified proteins, we were able to characterize 292 of them by biological processes/functions (<xref ref-type="fig" rid="f7">
<bold>Figure&#xa0;7</bold>
</xref>). Some proteins have overlapping functions. The majority were involved in the transport, metabolism, and biosynthesis of molecules such as proteins, lipids, and carbohydrates (12.9%) and biosynthesis of secondary metabolites (10.1%). Moreover, the others were involved in microbial metabolism in diverse environments (8.7%); carbon metabolism and utilization (7.1%); tricarboxylic acid cycle/pyruvate metabolism (5.7%); amino acid biosynthesis, metabolism, and transport (5.4%); cell wall and peptidoglycan biosynthesis, metabolism, and degradation (5.1%); translation (4.4%); ion transport and storage (3.1%); transport and ABC transporters (3%); glycolysis/gluconeogenesis (2.7%); two-component system (2.7%); antibiotic response (2.6%); biosynthesis of cofactors (2.4%); nucleotide biosynthesis and metabolism (2.4%); stress response (4.1%); cell cycle and division (1.7%); oxidative phosphorylation (1.4%); rRNA, tRNA, and mRNA processing and degradation (1.4%); transcription (1.4%); vitamin biosynthesis, metabolism, and transport (1.3%); and DNA replication, recombination, repair, damage, and condensation (1.1%). The main functions and pathways enriched in our EV proteins have been related to the same as other EV protein cargoes of XDR <italic>K. pneumoniae</italic> (<xref ref-type="bibr" rid="B39">Hussein et&#xa0;al., 2023</xref>). Other functions were also found to be represented in less than one percent such as quorum sensing, lipopolysaccharide (LPS) and lipid A biosynthesis, biofilm formation, respiratory electron transport chain, glutathione metabolism, Gram-negative bacterium-type cell outer membrane assembly, nitrogen metabolism, heme and porphyrin biosynthesis and metabolism, glyoxylate and dicarboxylate metabolism, methane metabolism, organic substance metabolism, organic acid catabolism, and chemotaxis.</p>
<fig id="f6" position="float">
<label>Figure&#xa0;6</label>
<caption>
<p>Predicted localization of EV proteins from the native <italic>Escherichia coli</italic> M19736. The pie diagram represents the localization of different proteins found inside or on the surface of EVs. The results are represented as the percentage of proteins found in different localizations from the native <italic>E. coli</italic> M19736.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g006.tif"/>
</fig>
<fig id="f7" position="float">
<label>Figure&#xa0;7</label>
<caption>
<p>Biological processes associated with EV proteins from the native <italic>E. coli</italic> M19736. Bar graphs categorizing the proteins with 28 differential biological functions, with distributions in the number of proteins. The functions of EV proteins are graphed with the most abundant function at the bottom and the least abundant function at the top.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g007.tif"/>
</fig>
<p>Interestingly, proteins associated with the LPS biosynthesis pathway have been found, among others: the LPS assembly OM complex LptDE &#x3b2;-barrel component LptD and LptE (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>), proteins that form a hetero-oligomeric complex that translocates LPS to the outer membrane and allows it to anchor to the cell wall surface (<xref ref-type="bibr" rid="B53">Lucena et&#xa0;al., 2023</xref>). Proteins that form efflux bombs, such as the three proteins that make up the tripartite efflux system AcrAB-TolC (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>), have also been found. The AcrB and AcrA proteins, respectively, make up the inner membrane and periplasm-spanning regions, and the TolC protein component is located in the bacterial outer membrane and also pairs with subunits of other membrane pumps. In addition, we found other proteins that are involved in pathways of cationic antimicrobial peptide (CAMP) resistance such as D-transpeptidase linking Lpp to murein, N-acetylmuramoyl-L-alanine amidase AmiC, lipoprotein NlpE, and periplasmic serine endoprotease DegP (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>). Lastly, we detected the MCR-1 protein (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>). To the best of our knowledge, the presence of this protein in EV has not been described so far. MCR-1 mediates colistin resistance by transferring phosphoethanolamine to bacterial lipid A, thereby reducing its affinity for colistin (<xref ref-type="bibr" rid="B47">Li H. et&#xa0;al., 2021</xref>).</p>
<sec id="s3_5_1">
<label>3.5.1</label>
<title>PPI network of EV proteins from <italic>Escherichia coli</italic> M19736</title>
<p>We constructed a PPI network using the STRING database and analyzed it using the Cytoscape software. Gene Ontology and KEEG enrichment analysis permitted to generate the network diagrams. Each node and line represented a term and the correlation between terms, respectively. The color of the terms indicates the classification of nodes based on their functions. We obtained three different PPI networks based on function/biological process/pathway found in the EV protein from <italic>E. coli</italic> M19736 (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8</bold>
</xref>). The first one showed cell wall and peptidoglycan biosynthesis and metabolism, represented by 27 nodes and 228 edges, with an average local clustering coefficient of 0.666 and PPI enrichment <italic>p</italic>-value of 1.15e&#x2212;03. The second one showed an antibiotic response, represented by 23 nodes and 68 edges, and the average local clustering coefficient was 0.819 and the PPI enrichment <italic>p</italic>-value was 1.0e&#x2212;16. The third one showed a pathway related to &#x3b2;-lactam resistance, represented by 7 nodes and 12 edges, with an average local clustering coefficient of 0.762 and PPI enrichment <italic>p</italic>-value of 1.94e&#x2212;07.</p>
<fig id="f8" position="float">
<label>Figure&#xa0;8</label>
<caption>
<p>PPI network of proteins of interest found in EV from the native <italic>E. coli</italic> M19736. Gene Ontology and KEEG enrichment analysis of genes/proteins from the proteomes of EVs derived from the native <italic>E. coli</italic> M19736 using the STRING software and visualization using the Cytoscape software. The node colors represent the biological process or cellular functions of the genes/proteins of interest according to significant associations of related Gene Ontology and KEEG terms. The arrow points to the MCR-1 protein.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-14-1368622-g008.tif"/>
</fig>
<p>Furthermore, the finding of the interaction of some specific proteins described above is of particular interest to our work. Some of these proteins could interact to generate antibiotic response/resistance, such as LptD and LptE proteins, which have been suggested to be involved in an antibiotic stress response leading to increased production and accumulation in the outer membrane (<xref ref-type="fig" rid="f8">
<bold>Figure&#xa0;8</bold>
</xref>) (<xref ref-type="bibr" rid="B53">Lucena et&#xa0;al., 2023</xref>). The efflux pump AcrAB-TolC is involved in conferring CAMP resistance in different bacteria, although this is controversial in <italic>E. coli</italic> (<xref ref-type="bibr" rid="B9">Blair et&#xa0;al., 2022</xref>). Overexpression of these efflux pumps also confers resistance to a variety of antibiotics (<xref ref-type="bibr" rid="B13">Brindangnanam et&#xa0;al., 2022</xref>). We also found several proteins involved in ribosomal and RNA degradation, including the chaperone Hsp70 (DnaK), and 30S ribosomal proteins that have been shown to interact with the MCR-1 protein (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>) (<xref ref-type="bibr" rid="B47">Li H. et&#xa0;al., 2021</xref>). It should be noted that in addition to these proteins, the above mentioned proteins associated with CAMP resistance and the AcrA-TolC system are also important in the MCR-1 protein interactome (<xref ref-type="bibr" rid="B47">Li&#xa0;H. et&#xa0;al., 2021</xref>).</p>
</sec>
</sec>
</sec>
<sec id="s4" sec-type="discussion">
<label>4</label>
<title>Discussion</title>
<p>HGT is a powerful force that shapes the evolution, diversification, and adaptation of bacterial communities and provides, for instance, a platform for the spread and persistence of ARGs (<xref ref-type="bibr" rid="B67">Piscon et&#xa0;al., 2023</xref>). Today, three canonical mechanisms of HGT are recognized, including transformation, transduction, and conjugation (<xref ref-type="bibr" rid="B28">Dubnau and Blokesch, 2019</xref>). Also, there are other non-canonical mechanisms including vesiduction, which involves secretion and uptake of EV (<xref ref-type="bibr" rid="B50">Li P. et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B57">Marinacci et&#xa0;al., 2023</xref>). In stressing habitats such as the nosocomial habitat, genome evolution is driven by antibiotic selection. However, there are several gaps of knowledge in this field. An area not well understood yet, it is what occurs with multidrug-resistant bacterial communities and plasmids that carry ARG in periods of time in which there is no antibiotic selection pressure. Also, the role of sporadic clones related to the spread of ARG is little studied to date. Here, we exposed the ability of a sporadic clone of <italic>E. coli</italic>, the <italic>E. coli</italic> M19736 strain belonging to ST615, harboring a plasmid with the <italic>mcr-1</italic> gene, to acquire a wide variety of clinical conjugative plasmids, including sequential co-infection of three conjugative plasmids harboring ARGs of current clinical interest (<italic>mcr-1</italic>, <italic>bla</italic>
<sub>NDM-1</sub>, and <italic>bla</italic>
<sub>CTX-M-15</sub>) from different species of bacteria (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>). In turn, its competency to disseminate the conjugative plasmids again to another bacterial host was shown (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>). At the same time, other mechanisms that were tested in this strain such as transformation for the paadB plasmid and vesiduction of native and evolved <italic>E. coli</italic> M19736 strains were identified that could be relevant reservoirs. <italic>bla</italic>
<sub>CTX-M-15</sub> was found in EV even if the evolved XDR-<italic>E. coli</italic> M19736 had rapidly lost the plasmid pDCAG1-CTX-M-15 after its acquisition, showing the essential role of EV for the dissemination of ARG in bacterial communities. Interestingly, the MICs for carbapenem antibiotics (ERT and MEM) were slightly higher in <italic>E. coli</italic> M19736::pDCVA3-NDM-5, evolved MDR-<italic>E. coli</italic> M19736, and evolved XDR-<italic>E. coli</italic> M19736 than the respective donor strains and transconjugants of <italic>E. coli</italic> J53, showing the ability of one sporadic clone to express the crucial ARG after HGT acquisition.</p>
<p>It has been a while since the population structure of <italic>E. coli</italic> has been identified; long-term stability and wide geographic distribution of individual lineages have been identified (<xref ref-type="bibr" rid="B10">Bobay et&#xa0;al., 2015</xref>). Some of these clones are pandemic, such as <italic>E. coli</italic> ST131, which is the predominant extraintestinal pathogenic <italic>E. coli</italic> that causes multidrug hospital infections, usually harboring <italic>bla</italic>
<sub>CTX-M-15</sub> and/or carbapenemases (<xref ref-type="bibr" rid="B58">Mathers et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B81">Sanz et&#xa0;al., 2022</xref>). In a recent retrospective epidemiological study performed with 71 relevant carbapenem-resistant <italic>E. coli</italic> strains isolated from 2008 to 2017 from Argentina, several pandemic clones including ST10, ST38, ST131, ST155, ST648, and ST1193 were found prevalent (<xref ref-type="bibr" rid="B81">Sanz et&#xa0;al., 2022</xref>). From this bacterial population under scrutiny, three carbapenem-resistant <italic>E. coli</italic> strains harboring the <italic>mcr-1</italic> gene were also found belonging to the pandemic clone ST10 and to sporadic clones ST12657 and ST12667. Interestingly, two of these strains (<italic>E. coli</italic> ECO 37 isolated in 2014 and <italic>E. coli</italic> ECO 81 isolated in 2015) were isolated before the first description of the <italic>mcr-1</italic> gene in isolates from animals, food, and patients in China (<xref ref-type="bibr" rid="B52">Liu et&#xa0;al., 2016</xref>). <italic>Escherichia coli</italic> ST615 was not identified in those epidemiological studies and in other studies performed with <italic>E. coli</italic> strains from Argentina isolated from the clinic or other environments before or after the COVID-19 pandemic (<xref ref-type="bibr" rid="B83">Sennati et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B25">Dominguez et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B29">Faccone et&#xa0;al., 2023</xref>; <xref ref-type="bibr" rid="B35">Gramundi et&#xa0;al., 2023</xref>; <xref ref-type="bibr" rid="B66">Piekar et&#xa0;al., 2023</xref>), confirming the sporadic condition of this clone. <italic>Escherichia coli</italic> M19736 ST615 strain isolated in 2015 was identified at that time as one of the first isolates harboring the <italic>mcr-1 </italic>gene in human infections caused by <italic>E. coli</italic> in Latin America (<xref ref-type="bibr" rid="B74">Rapoport et&#xa0;al., 2016</xref>). The importance and scope of a wide variety of sporadic clones has not yet been studied in-depth. It is likely that <italic>E. coli</italic> ST615 lineage could capture crucial ARG such as the <italic>mcr-1</italic> gene, with the ability to transfer consequently to other strains as shown in the present study and previously (<xref ref-type="bibr" rid="B74">Rapoport et&#xa0;al., 2016</xref>). We also identified that the <italic>E. coli</italic> M19736 ST615 strain was able to acquire a diversity of plasmids of different incompatibility groups harboring multiple ARGs from three different species (<italic>E. coli</italic>, <italic>K. pneumoniae</italic>, and <italic>S. marcescens</italic>) (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>). In addition, co-infection of plasmids sharing the same incompatibility group such as pDCVA3-NDM-5 and pIncFII-M19736 in <italic>E. coli</italic> M19736::pDCVA3-NDM-5 or pDCAG1-CTX-M-15 and pIncFII-M19736 in evolved MDR-<italic>E. coli</italic> M19736 strain was found (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>). Several differences were identified among the three IncFII replicons (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;3</bold>
</xref>) that could be related, in part, to their ability to co-infect and to be maintained together during 10 days of daily subcultures by <italic>E. coli</italic> M19736.</p>
<p>Our studies showed that transconjugants of <italic>E. coli</italic> M19736 ST615 maintained pDCAG1-CTX-M-15 (IncFII) and pDCASG-NDM-1 (IncC) plasmids for 10 days at 100% while maintaining its native one, pMCR-M19736 (IncI2) with the <italic>mcr-1</italic> gene, without antibiotic pressure (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2A</bold>
</xref>). The co-infection with pDCAG1-CTX-M-15 (IncFII) and pDCASG-NDM-1 (IncC) is interesting since both have shown to possess a pandemic behavior. On one hand, the pDCAG1-CTX-M-15 plasmid has an F2:B10 replicon; IncF-type replicons have shaped the evolution of the main fluoroquinolone-resistant ST131-<italic>H</italic>30 clades adding an advantage resistance to several families of antibiotics including the presence of the <italic>bla</italic>
<sub>CTX-M-15</sub> gene (<xref ref-type="bibr" rid="B41">Johnson et&#xa0;al., 2016</xref>). On the other hand, pDCASG-NDM-1 belongs to the IncC group plasmids that are widely distributed among Gnb, with a large range of hosts in which these plasmids can replicate (<xref ref-type="bibr" rid="B3">Ambrose et&#xa0;al., 2018</xref>). IncC plasmids have islands of resistance incorporated in different plasmid locations where different ARGs can be accumulated including <italic>bla</italic>
<sub>NDM</sub> and <italic>bla</italic>
<sub>KPC</sub> genes (<xref ref-type="bibr" rid="B3">Ambrose et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B2">Ambrose, 2020</xref>). At first glance, since both pDCAG1-CTX-M-15 and pDCASG-NDM-1 plasmids had toxin&#x2013;antitoxin systems (<italic>CcdA/B</italic> and <italic>pemK/L</italic> and <italic>HigA/B</italic>, respectively) and different incompatibility groups (IncFII and IncC, respectively), a low percentage of loss was expected during co-infected subcultures. Recent advances in the field profoundly questioned the role of toxin&#x2013;antitoxin systems in bacterial physiology, stress response, and antimicrobial persistence (<xref ref-type="bibr" rid="B42">Jur&#x117;nas et&#xa0;al., 2022</xref>). More investigations are needed to evaluate their role in clinical isolates harboring several plasmids.</p>
<p>Recently, it has been shown that plasmids carrying a carbapenemase such as KPC or NDM could be efficiently conjugated to strains carrying the <italic>mcr-1</italic> gene and vice versa and that these plasmids could stably co-exist in clinical <italic>Enterobacteriaceae</italic> strains (<xref ref-type="bibr" rid="B51">Liu et&#xa0;al., 2021</xref>). Although the clonality of these strains was not determined, these results are congruent with our experiments of the evolved <italic>E. coli</italic> M19736 strains (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>), which were able to transfer in turn the conjugative plasmids acquired previously. In addition, our experiments showed that when the transconjugant <italic>E. coli</italic> M19736::pDCAG1-CTX-M-15 was co-infected with additional plasmids, our evolved MDR and XDR-<italic>E. coli</italic> M19736 ST615 strains showed different patterns of ARG maintenance with the ability to keep almost at 100% the <italic>bla</italic>
<sub>NDM-1</sub> gene located in the pandemic IncC plasmid pDCASG-NDM-1.</p>
<p>Concerning co-infection of plasmids, it is generally overlooked that bacterial strains frequently harbor multiple plasmids, and understanding them is of utmost importance, especially for those relevant in the clinical context (<xref ref-type="bibr" rid="B24">Dionisio et&#xa0;al., 2019</xref>). Other studies have shown that bacteria can carry more than one type of plasmid; for example, it has been shown that 27 strains of <italic>E. coli</italic> producing extended-spectrum &#x3b2;-lactamase harbored multiple different plasmids (<xref ref-type="bibr" rid="B32">Garc&#xed;a et&#xa0;al., 2007</xref>). Positive epistasis between co-infecting plasmids has been shown which minimizes the cost of plasmid carriage and increases the ability of plasmids to persist in the absence of selection for plasmid-encoded traits, suggesting that epistasis may have an important role in resolving the &#x201c;plasmid paradox&#x201d; (<xref ref-type="bibr" rid="B80">San Millan et&#xa0;al., 2013</xref>), which is in agreement with our results. We also found that maintenance of plasmids without antibiotic selection varied depending on the plasmids that co-infected the host (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>), such as the case of the evolved MDR-<italic>E. coli</italic> M19736 and XDR-<italic>E. coli</italic> M19736 strains related to the maintenance of pDCAG1-CTX-M-15 (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>). The addition of plasmid paadB (Inc15A) triggered the loss of pDCAG1-CTX-M-15 from the evolved XDR-<italic>E. coli</italic> M19736 strain. Concerning this, a strong co-evolution has been shown between some <italic>E. coli</italic> ST131 lineages and specific plasmids including F2:B10 (<xref ref-type="bibr" rid="B62">Ny et&#xa0;al., 2019</xref>), which could explain in part its ease of getting lost in a sporadic clone such as <italic>E. coli</italic> ST615.</p>
<p>In order to follow the trajectory of plasmids that were co-infecting our evolved strains, conjugation assays were performed revealing that the evolved MDR and XDR-<italic>E. coli</italic>-M19736 strains were able to transfer one or two plasmids simultaneously while keeping always the pM19736 plasmid (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). Previous plasmid co-transfer in <italic>E. coli</italic> strains showed that when hosts harbor two, three, or four distinct plasmids, the co-transfer of both plasmids tends to be limited by the plasmid exhibiting the lowest conjugation rate (<xref ref-type="bibr" rid="B30">Gama et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B22">Darphorn et&#xa0;al., 2022</xref>). In disagreement with this, we did not find significant differences between the conjugation efficiencies of these plasmids (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1B</bold>
</xref>), so in the cases where the co-transfer happens, we cannot associate with the conjugation rate of each plasmid (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). Also, our results are congruent with the hypothesis that suggests that de-repression could occur simultaneously on co-resident plasmids. In fact, this could happen as a response to a common stimulus. The idea that a common stimulus triggers the transfer of one or the other plasmid could explain why one or the other plasmid is co-transferred as proposed by <xref ref-type="bibr" rid="B30">Gama et&#xa0;al. (2017)</xref>. However, it is not clear in our study what stimulus could have triggered co-transfection in our experiments, but selection with ceftazidime may be studied deeply. The tendency of bacteria to maintain several plasmids simultaneously independently of the antibiotic pressure imposed on the pathogens has been shown (<xref ref-type="bibr" rid="B80">San Millan et&#xa0;al., 2013</xref>). There are two predictable ways for a strain to harbor multiple plasmids: i) through sequential plasmid acquisition as we showed in the experiments using the sporadic clone <italic>E. coli</italic> M19736 as the recipient strain that evolved to MDR-<italic>E. coli</italic> M19736 and XDR-<italic>E. coli</italic> M19736 strains (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1A</bold>
</xref>) and ii) by simultaneous transmission of several plasmids as we showed in the experiments that we performed using the evolved MDR and XDR-<italic>E. coli</italic> M19736 strains as donors to <italic>E. coli</italic> J53 (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). Our results showed that one strain belonging to a sporadic clone is able to be co-infected with several plasmids in both ways as previously suggested (<xref ref-type="bibr" rid="B24">Dionisio et&#xa0;al., 2019</xref>).</p>
<p>Since recent research has revealed that EVs may play an important role as a reservoir of genes and proteins associated with AMR contributing to this global threat through several other mechanisms (<xref ref-type="bibr" rid="B19">Ciofu et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B79">Rumbo et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B45">Lee et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B17">Chatterjee et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B7">Bielaszewska et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B56">Marchant et&#xa0;al., 2021</xref>), we also investigated the ability of the EV of the <italic>E. coli</italic> M19736 strain to harbor AMR determinants. First, we analyzed the EV of <italic>E. coli</italic> M19736 and evolved XDR-<italic>E. coli</italic> M19736 strains by PCR searching for the ARG. We found in both strains the presence of the <italic>mcr-1</italic> gene which has been also previously described (<xref ref-type="bibr" rid="B48">Li X. et&#xa0;al., 2021</xref>). Interestingly, EVs from the evolved XDR-<italic>E. coli</italic> M19736 strains harbored <italic>bla</italic>
<sub>CTX-M-15</sub> though the pDCAG1-CTX-M-15 was previously lost as shown by WGS analysis and maintenance experiments, suggesting that EVs could be a relevant reservoir of ARG for susceptible bacteria. Unexpectedly, we did not find the <italic>bla</italic>
<sub>NDM-1</sub> gene in the EV content of the evolved strain by DNA PCR. This result was surprising because the <italic>bla</italic>
<sub>NDM</sub> gene is the most described gene reported in EV so far (<xref ref-type="bibr" rid="B17">Chatterjee et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B48">Li X. et&#xa0;al., 2021</xref>). These findings lead us to consider what might play a role in DNA cargo selection in EV, as the mechanism by which DNA cargo is enriched or excluded from EV remains unclear (<xref ref-type="bibr" rid="B97">Zavan et&#xa0;al., 2023</xref>). Plasmid type has been shown to influence gene loading (<xref ref-type="bibr" rid="B90">Tran and Boedicker, 2017</xref>). However, this is not the only reason explaining why <italic>bla</italic>
<sub>NDM-1</sub> located in pDCASG6-NDM-1 was not found in EV. Other features could be involved in the selection of DNA loading in EV, such as the location of each type of plasmid within the cell (<xref ref-type="bibr" rid="B70">Pogliano, 2002</xref>). Currently, there are more studies focusing on the effects of growth stage on EV selection load. For example, analysis of EV produced by <italic>Helicobacter pylori</italic> reveals that bacterial growth stage affects the size, composition, and selection of protein load (<xref ref-type="bibr" rid="B96">Zavan et&#xa0;al., 2019</xref>). Other research showed that plasmids belonging to different incompatibility groups bind to different sites within the cell (<xref ref-type="bibr" rid="B70">Pogliano, 2002</xref>). <xref ref-type="bibr" rid="B1">Aktar et&#xa0;al. (2021)</xref> also performed some experiments to evaluate the incorporation of plasmid DNA into EV. The results showed that peptidoglycan defects increased plasmid sorting into EV. It is well known that the use of cell wall antibiotics increases EV production (<xref ref-type="bibr" rid="B6">Bauwens et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B53">Lucena et&#xa0;al., 2023</xref>). In summary, since EV DNA packaging is not a random process, more work is needed to understand the rules of plasmid packaging into vesicles.</p>
<p>Furthermore, in EVs from the <italic>E. coli</italic> M19736 strain, the proteins from the LPS biosynthesis pathway were found (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>), and 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA) is responsible for linking lipid A and core oligosaccharides through the synthesis of other proteins (<xref ref-type="bibr" rid="B39">Hussein et&#xa0;al., 2023</xref>). The experiments of <xref ref-type="bibr" rid="B86">Strohmaier et&#xa0;al. (1995)</xref> demonstrate that KdsA undergoes growth phase-dependent regulation at the transcriptional level, which again supports the idea that the growth phase is related to the selection of cargo in EV. Also, this protein increases following polymyxin B treatment (<xref ref-type="bibr" rid="B39">Hussein et&#xa0;al., 2023</xref>) which supports the idea that treatment with each antibiotic could collaborate in the selection of load in EV. Also, in EV from the <italic>E. coli</italic> M19736 strain, the MCR-1 protein was found (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>). To our knowledge, the presence of this protein has not been previously described. It has been demonstrated that MCR-1-interacting proteins were mainly involved in ribosome and RNA degradation such as the 30S ribosomal subunit proteins S5 and S10 (<xref ref-type="bibr" rid="B47">Li H. et&#xa0;al., 2021</xref>). These proteins were also identified in our EV extractions and among other ribosomal proteins as well (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Table&#xa0;2</bold>
</xref>). Also, the two-component AcrA-TolC multidrug efflux pump interacts with the MCR-1 protein and is involved in <italic>mcr-1</italic>-mediated colistin resistance (<xref ref-type="bibr" rid="B47">Li H. et&#xa0;al., 2021</xref>). In addition, EVs have shown to have an effect of protection of colistin&#x2019;s administration over other strains (<xref ref-type="bibr" rid="B56">Marchant et&#xa0;al., 2021</xref>) that could apply to the <italic>E. coli</italic> M19736 strain under scrutiny in the present work. For relevance in the nosocomial niche, the main functions and pathways enriched in our EV proteins were related to EV protein&#x2019;s cargo of XDR <italic>K. pneumoniae</italic> strains as previously shown (<xref ref-type="bibr" rid="B39">Hussein et&#xa0;al., 2023</xref>).</p>
<p>Since a few clinical sporadic clones have shown to be prone to accept plasmids by transformation (<xref ref-type="bibr" rid="B72">Ramirez et&#xa0;al., 2011</xref>), deeply analyzing the genomic plasticity of <italic>E. coli</italic> M19736, artificial competency was also achieved. This assay evidenced the ability of this strain to accept the paadB plasmid by chemical competency of the native and evolved MDR-<italic>E. coli</italic> M19736 strains, which could be maintained by <italic>E. coli</italic> M19736 during at least 58 generations (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>). Furthermore, sporadic clones were found to capture relevant features of environmental plasmids that later could spread within the nosocomial niche such as the case of pDCPR1 which has a mosaic structure with parts of successful phytopathogen plasmids such as pIII from pathogenic <italic>Xanthomonas albilineans</italic> and pPSV48C from another pathogenic species <italic>Pseudomonas savastanoi</italic> (<xref ref-type="bibr" rid="B91">Vilacoba et&#xa0;al., 2014</xref>). This ability to capture genetic platforms from the bacterial communities of the open environment adds a relevant role to sporadic clones in the framework necessary for the success of AMR in the nosocomial niche.</p>
<p>In summary, our studies showed that <italic>E. coli</italic> M19736 strains belonging to the sporadic clone ST615 have a range of valuable tools for survival under antibiotic pressure as we identified its genomic plasticity to acquire plasmids from various species with crucial ARGs, with proficiency to keep them and disseminate in turn by conjugation to other strains. Furthermore, its competence to produce EVs containing several valuable AMR determinants with the potential to be disseminated and its ability to receive plasmids by transformation as demonstrated in the present study indicated a great adaptation to the in-hospital environment. Based on this, sporadic clones could play a crucial role in a clinical context by adapting to different selection pressures and at the same time by spreading plasmids to other bacteria which guarantees an important link in the chain of events that leads bacteria to resist in the nosocomial niche.</p>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The <italic>E. coli</italic> M19736 genome data presented in the study is deposited in GenBank under the accession numbers PRJNA1080650 and PRJNA1102182. The assembled plasmids pDCASGNDM-1 and pDCAG1-CTX-M-15 were deposited in GeneBank under the accession numbers PP622803 and PP632099. The data base protein of EV was included in Vesiclepedia 2024 (<uri xlink:href="http://www.microvesicles.org">http://www.microvesicles.org</uri>) study ID 3592.</p>
</sec>
<sec id="s6" sec-type="author-contributions">
<title>Author contributions</title>
<p>LCP: Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing &#x2013; original draft, Writing &#x2013; review &amp; editing. MO: Conceptualization, Investigation, Project administration, Writing &#x2013; review &amp; editing. AG: Data curation, Writing &#x2013; review &amp; editing. TP: Investigation, Methodology, Writing &#x2013; review &amp; editing. AA: Data curation, Investigation, Writing &#x2013; review &amp; editing. MQ: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Resources, Supervision, Writing &#x2013; review &amp; editing. DC: Conceptualization, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing &#x2013; original draft, Writing &#x2013; review &amp; editing, Validation.</p>
</sec>
</body>
<back>
<sec id="s7" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grant ISID/Pfizer 2019 given to DC, grant PUE 2522 from CONICET given to IMPaM (UBA/CONICET), and PICT 2021 (1120) given to DC.</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>We are very grateful to Dr. Alejandro Petroni from INEI-ANLIS &#x201c;Dr. Carlos G. Malbr&#xe1;n&#x201d; for providing us with the <italic>Escherichia coli</italic> M19736. Also, we would like to thank Claudia Vanessa Vargas and Nicolas Donis for their help in the experimental assays. DC and MQ are career investigators of CONICET. LCP is a recipient of a doctoral fellowship from CONICET.</p>
</ack>
<sec id="s8" 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>
<p>The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.</p>
</sec>
<sec id="s9" 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>
<sec id="s10" 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.2024.1368622/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcimb.2024.1368622/full#supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="Table_1.xlsx" id="SM1" mimetype="application/vnd.openxmlformats-officedocument.spreadsheetml.sheet"/>
</sec>
<fn-group>
<title>Abbreviations</title>
<fn fn-type="abbr">
<p>ARG, antimicrobial resistance gene; EV, extracellular vesicle; ST, sequence type; XDR, extreme drug resistance; MDR, multidrug resistance; Gnb, Gram-negative bacilli; AMR, antimicrobial resistance; LB, Luria&#x2013;Bertani; PDI, polydispersity index; LPS, lipopolysaccharide; CAMP, cationic antimicrobial peptide.</p>
</fn>
</fn-group>
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