Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales

Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales View ORCID Profile Mia Rondinelli , View ORCID Profile Sabhjeet Kaur , View ORCID Profile Owen A. Ledwell , Henry Wong , Prameet M. Sheth , View ORCID Profile George C. diCenzo doi: https://doi.org/10.1101/2025.09.07.674714 Mia Rondinelli 1 Department of Biology, Queen’s University , Kingston, Ontario, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mia Rondinelli Sabhjeet Kaur 1 Department of Biology, Queen’s University , Kingston, Ontario, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sabhjeet Kaur Owen A. Ledwell 1 Department of Biology, Queen’s University , Kingston, Ontario, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Owen A. Ledwell Henry Wong 2 Division of Microbiology, Kingston Health Sciences Centre , Kingston, ON, Canada 3 Department of Pathology and Molecular Medicine, Queen’s University , Kingston, ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site Prameet M. Sheth 2 Division of Microbiology, Kingston Health Sciences Centre , Kingston, ON, Canada 3 Department of Pathology and Molecular Medicine, Queen’s University , Kingston, ON, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site George C. diCenzo 1 Department of Biology, Queen’s University , Kingston, Ontario, Canada Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for George C. diCenzo For correspondence: george.dicenzo{at}queensu.ca Abstract Full Text Info/History Metrics Supplementary material Data/Code Preview PDF ABSTRACT The VIM-1 metallo-β-lactamase enzyme, encoded as a cassette within class 1 integrons, is found in Gram-negative clinical isolates worldwide and has been linked to outbreaks of bacterial pathogens in nosocomial settings. Six vim-1 + clinical isolates, from the genera Escherichia , Klebsiella , and Enterobacter , were obtained from Kingston, Ontario, Canada. Whole genome sequencing revealed that vim-1 was plasmid-borne in all strains and situated as the first gene in In916 or In110 integrons. Analysis of related plasmids suggested that these vim-1 -containing plasmids are globally disseminated and have spread via horizontal gene transfer and autochthonous vertical spread within Ontario. Interestingly, the minimum inhibitory concentrations of ertapenem and meropenem, two clinically relevant carbapenem antibiotics, against these six isolates varied more than tenfold, suggesting the effects of VIM-1 are dependent on the genomic content of the host microbe. To further study the genomic content dependency of VIM-1, we introduced vim-1 into three common Enterobacterales laboratory strains. Although introduction of vim-1 into Escherichia coli DH5α resulted in little resistance to ertapenem or meropenem, multiple rounds of adaptive laboratory evolution allowed us to identify variants with extremely high levels of resistance to both carbapenems. DNA sequencing revealed that the increase in carbapenem resistance was due to a combination of increased vim-1 gene dosage and epistatic interactions with mutations of ompC that likely would have decreased outer membrane permeability to these antibiotics. Together, these results provide additional support for the role of gene epistasis is modulating the antimicrobial resistance phenotypes of acquired resistance genes, as well as previous results suggesting that the presence of a β-lactamase gene is insufficient to confer strong resistance to carbapenems without being paired with reduced outer membrane permeability. INTRODUCTION Antimicrobial resistant pathogens are three times more likely to result in mortality than their susceptible counterparts ( Dadgostar, 2019 ), resulting in an estimated global death toll of 1.27 million in 2019 alone ( Murray et al., 2022 ). In bacteria, horizontal gene transfer (HGT) facilitates the exchange of antimicrobial resistance genes (ARGs) across diverse bacterial populations and the collection of these genes into conjugative transposons ( van der Zee et al., 2018 ) and plasmids ( De la Cruz Barrón et al., 2018 ). These, in turn, disseminate quickly across microbial ecosystems via HGT ( von Wintersdorff et al., 2016 ), occasionally recombining with each other along the way ( Y. Li et al., 2018 ). Transposons can additionally exacerbate antimicrobial resistance (AMR) by inserting into promoter regions, activating genes associated with conjugation, and improving overall conjugation frequency ( Poidevin et al., 2018 ). While HGT plays a role in ARG dissemination and evolution, there appear to be limits to how far ARGs can spread, suggesting interplay between the chromosomal genome and acquired genes ( Wong, 2017 ). Studies investigating the dissemination of resistance to antimicrobial peptides (AMPs) and antibiotics have noted that the transfer of ARGs occurs at a higher rate between more closely related bacteria ( Hu et al., 2016 ; Kintses et al., 2019 ). In one case, genes encoding AMPs from gut microbiota could not confer resistance when transferred to Escherichia coli ( Kintses et al., 2019 ). In others, the transfer of ARGs from distantly related species (such as Shewanella spp.) to E. coli resulted in toxicity and cell death ( Sorek et al., 2007 ). This is likely due to the dependence of resistance mechanisms on host physiology, suggesting that there are phylogenetic barriers that may limit the spread of certain ARGs ( Porse et al., 2018 ; Sorek et al., 2007 ). Additionally, there are examples of compensatory mutations increasing the fitness of bacterial strains that have evolved AMR mechanisms. Knopp and Andersson (2015) found that E. coli could overcome the fitness cost of losing outer membrane proteins C and F through compensatory mutations to pathways producing other porins. There is also evidence that epistatic relationships exist between acquired and chromosomal ARGs. For instance, Silva et al (2011) found that, occasionally, plasmids carrying resistance genes confer a selective advantage to strains carrying ARGs on their chromosome in the absence of antibiotics. Surveillance data from across Southern Ontario identified the emergence of clinical isolates in Gram-negative Enterobacterales carrying the Verona Integron-encoded Metallo-β-lactamase (VIM) ( P. P. Kohler et al., 2018 ) that was first identified in Europe ( Arcari et al., 2020 ; P. Kohler et al., 2020 ; Mano et al., 2015 ; Papadimitriou-Olivgeris et al., 2019 ). Unlike other metallo-β-lactamases (MBLs) identified in this Canadian surveillance program ( Kohler et al., 2020 ), VIM-positive patients had none of the ‘classical’ risk factors associated seen in patients with other MBL’s including documented travel history outside of Canada or hospital admission, suggesting local acquisition. The dissemination of these pathogens is likely through undetected colonization and transmission amongst patients in acute care ( Kohler et al., 2020 ) or acquisition via unknown community exposure. Like other MBLs, but unlike extended-spectrum β-lactamases (ESBLs), VIM can hydrolyze carbapenem antibiotics, which are often considered as a last-resort antibiotic. Of all VIM genes, vim-1 is the most common gene variant globally ( Matsumura et al., 2017 ). Like other B1 MBLs, the structure of VIM-1 displays an overall αβ/βα fold with the Zn 2+ centre forming the active site situated in a shallow groove formed by the interface of the two αβ domains ( Salimraj et al., 2019 ). The vim-1 gene is generally carried as part of class 1 integrons, which are major contributors to AMR through their ability to capture and express a diverse range of ARGs ( Gillings et al., 2008 ). Class 1 integrons can be found embedded within an extremely broad range of plasmids, including plasmids of incompatibility (Inc) groups IncA ( Botelho et al., 2018 ), IncC ( Gaibani et al., 2018 ), IncH12 ( Kohler et al., 2020 ), IncFII ( Sánchez-Romero et al., 2012 ), IncN ( Miriagou et al., 2010 ), IncP ( Zeng et al., 2019 ), IncL/M ( Peirano et al., 2014 ), and IncR ( P. Kohler et al., 2020 ). There is also evidence of these integrons integrating into the host chromosome ( Irrgang et al., 2019 ). This promiscuity results in highly mobile genetic apparatuses that can spread horizontally across a broad range of bacterial species. Here, we report the sequencing and phenotypic characterization of six Enterobacterales clinical isolates collected at Kingston Health Sciences Center, all of which were positive for vim-1. We demonstrate that the minimum inhibitory concentrations (MIC) of the carbapenem antibiotics ertapenem and meropenem varied more than tenfold across the isolates, suggesting that the effects of vim-1 are dependent on the genomic content of the host microbe. Introduction of vim-1 into common laboratory strains resulted in little ertapenem resistance. However, adaptive laboratory evolution led to massive increases in the level of vim-1 -mediated ertapenem resistance, which we show was due to a combination of increased vim-1 gene dosage and epistatic interactions with mutations of the ompC gene encoding an outer membrane protein. MATERIALS AND METHODS Bacterial strains and growth conditions Bacterial strains used in this work are listed in Table 1 . Strains were routinely grown using Lysogeny Broth (LB) medium (10 g L −1 tryptone, 5 g L −1 yeast extract, 5 g L −1 sodium chloride) and Mueller Hinton (MH) medium (Sigma Aldrich; Product No. 90922). Gentamicin (10 µg/mL in liquid media, 20 µg/mL in solid media) was used to maintain pBBR1mcs-5 plasmids in transformed strains. Super optimal broth with catabolite repression (SOC) (20 g L −1 tryptone, 5 g L −1 yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO4, 10 mM CaCl2, 20 mM glucose) was used as a recovery medium following transformation by electroporation. All strains were grown at 37°C. When required, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was added to the media at a final concentration of 40 µg mL −1 . View this table: View inline View popup Download powerpoint Table 1. Bacterial strains and plasmids. Whole genome sequencing, assembly, and annotation Genomic DNA (gDNA) samples were isolated from clinical isolates using phenol-chloroform extraction ( Cowie et al., 2006 ). Purified gDNA samples were then sequenced using an Oxford Nanopore Technologies (ONT) MinION Mk1B nanopore sequencer and the Rapid Barcoding Kit (RBK004) according to the manufacturer’s instructions. Basecalling and demultiplexing were performed using GPU-enabled Guppy version 5.011+2b6dbffa5 and the High-Accuracy model (Oxford Nanopore Technologies). Purified gDNA samples were also sequenced at the Microbial Genome Sequencing Center (Pittsburgh, PA, USA) using an Illumina NextSeq 550 instrument with 150 bp paired-end reads. Genome assemblies were generated using a pipeline previously described by Duan et al (2022) . Programs and versions used are as follows: Flye version 2.8.3 ( Kolmogorov et al., 2019 ), NUCmer version 4.0.0rc1 ( Kurtz et al., 2004 ), Racon version 1.4.22 ( Vaser et al., 2017 ), Minimap2 version 2.20-r1061 ( Li, 2018 ), Medaka version 1.4.1 (Oxford Nanopore Technologies), Pilon version 1.24 ( Walker et al., 2014 ), bwa version 0.7.17.r1198-dirty ( H. Li & Durbin, 2009 ), and Circlator version 1.5.5 ( Hunt et al., 2015 ). Assembly quality was checked using CheckM version 1.2.2 ( Parks et al., 2015 ). Genome assemblies were annotated using the NCBI prokaryotic genome annotation pipeline (PGAP) build 5429 ( Tatusova et al., 2016 ). Plasmid replicons were identified in the genome assemblies using PlasmidFinder version 2.1.1 ( Carattoli et al., 2014 ). Integron-associated features were identified within the genome assemblies using IntegronFinder version 2.0.2 ( Néron et al., 2022 ). Resistance genes/proteins were identified in nucleotide or amino acid sequences using the Comprehensive Antibiotic Resistance Database (CARD) 3.2.2 Resistance Gene Identifier (RGI) 5.2.1 ( Alcock et al., 2020 ). Genome assemblies were visualized in Mauve snapshot 2015-02-25 build 0 following alignment with progressiveMauve ( Darling et al., 2004 ). Taxonomic classification of strains was performed using TYGS ( Meier-Kolthoff et al., 2022 ). Identification of related vim-1 -containing plasmids To identify vim-1 -containing plasmids related to those of our clinical isolates, we first downloaded the plasmid database (PLSDB) version 2021_06_23_v2 ( Galata et al., 2019 ). Next, each of the vim-1 -containing plasmids of our clinical isolates were individually used as queries with BLASTn from BLAST version 2.10.1+ ( Camacho et al., 2009 ) to search PLSDB, and the top 20 hits for each plasmid were recorded. The exception was the vim-1 -containing plasmid of S2568, as this plasmid was not circularized. The dereplicated top hits from each plasmid were collected from PLSDB, and annotated using Prokka version 1.14.6 ( Seemann, 2014 ), as were the vim-1 -containing plasmids of our isolates. Roary version 1.7.8 ( Page et al., 2015 ) was then used to identify shared genes between all annotated plasmids. The resulting gene presence/absence matrix was used to produce a distance matrix based on Jaccard distances using the philentropy version 0.8.0 ( Drost et al., 2018 ) package in R version 4.3.0 ( R Core Team, 2021 ) which was then used to construct a dendogram using ape version 5.8 ( Paradis & Schliep, 2019 ). The distance matrix was visualized using iTOL ( Letunic & Bork, 2021 ). Sequence comparison of OmpC and OmpD porins All proteins annotated as OmpC or OmpD in the genomes of the six clinical isolates, as well as E. coli EcGQ0079 (DH5α with a pBBR1mcs-5:: vim-1 derivative), were extracted from the proteomes. All porins were aligned using the Clustal Omega ( Sievers & Higgins, 2017 ) MBL-EBI webserver (ebi.ac.uk/jdispatcher/msa/clustalo) to generate a multisequence alignment and a percent identity matrix. The untrimmed alignment was used to create a phylogeny using IQ-TREE version 2.2.2.4 ( Minh et al., 2020 ) with the Q.pfam+F+G4 model, as it was identified as the best-scoring model by ModelFinder based on Bayesian information criterion (BIC) and with model search limited to the LG, WAG, JTT, Q.pfam, JTTDCMut, DCMut, VT, PMB, BLOSUM62 and Dayhoff models. Branch supports were assessed using Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT) ( Anisimova & Gascuel, 2006 ) and an ultrafast bootstrap analysis, with both metrics calculated from 1,000 replicates. The phylogeny and percent identity matrix were then visualized with the iTOL web server ( Letunic & Bork, 2021 ). Cloning of vim-1 PCR primers MR001 and MR003 (see Table S1 for all primer sequences) were used to amplify vim-1 and the preceding attC site from E. coli F5446 using Q5 DNA polymerase (New England Biolabs [NEB]), which was then purified using a Monarch PCR & DNA Cleanup Kit (NEB). The PCR product and the pBBR1mcs-5 cloning vector ( Kovach et al., 1995 ) were individually digested with both BamHI-HF (NEB) and HindIII-HF (NEB) overnight at 37°C; pBBR1mcs-5 was subsequently dephosphorylated using Quick CIP (NEB). Lastly, the vim-1 -containing DNA fragments were ligated into pBBR1mcs-5 using T4 DNA ligase (NEB). Ligation products were transformed into chemically competent E. coli DH5α and plated on LB with gentamicin and X-gal, and correctly assembled plasmids were preliminarily identified based on blue-white screening. Plasmid DNA was purified from transformants using a Monarch Plasmid DNA Miniprep Kit (NEB) and the vim-1 sequences then were verified using Sanger sequencing with primers M13-F and M13-R (CHU de Québec-Université Laval Research Center). Plasmids containing vim-1 were also transformed into electrocompetent cultures of Klebsiella grimontii KP5022 ( Streicher et al., 1974 ) and E. coli MG1655 ( Datsenko & Wanner, 2000 ) via electroporation and plated on LB with gentamicin to select for the presence of the plasmid. Adaptive laboratory evolution (ALE) Strains of interest were grown overnight in MH broth and diluted to an optical density at 600 nm (OD 600 ) of 0.05 in 200 µL MH broth in 96-well microplates. Each microplate contained an ertapenem gradient, where the ertapenem concentration of adjacent wells differed by a factor of two. Microplates were taped closed, inserted into a Synergy H1 plate reader, and incubated for 24 hours at 37°C with shaking. OD 600 measurements were recorded every 15 minutes. Following 24 hours of incubation, 2 µL from the well with the highest concentration of ertapenem that allowed growth were sub-cultured into each well of an ertapenem concentration gradient in a fresh 96-well plate. This process was repeated until there was a sufficient increase in the concentration of ertapenem tolerated by vim-1 -containing strains. To test if the plasmids in the derived isolates were sufficient to produce an ertapenem resistance phenotype, plasmid DNA from the derived isolates was purified with a Monarch Plasmid DNA Miniprep Kit (NEB) and transformed into electrocompetent E. coli DH5α cultures via electroporation, then plated on LB with Gm to select for plasmid uptake. Plasmids from the strains recovered at the end of the ALE were purified using a Monarch Plasmid Miniprep Kit (NEB) and the full plasmid sequence was determined using ONT sequencing by Plasmidsaurus (Louisville, KY, USA). Sequencing results were visualized using the online software Benchling ( benchling.com ), with alignment performed using MAFFT ( Katoh et al., 2019 ). For one ALE experiment, genomic DNA from an ancestral strain (EcGQ0079) and two derived lineages (EcGQ0088 and EcGQ0089) was purified and sequenced at the Microbial Genome Sequencing Center using an Illumina NextSeq 550 instrument with 150 bp paired-end reads. Illumina reads were then trimmed using BBduk version 38.96 ( Bushnell, 2021 ) and trimmomatic version 0.39 ( Bolger et al., 2014 ). A reference genome was assembled using the Illumina reads of the ancestral strain and Unicycler version 0.5.0 ( Wick et al., 2017 ) with SPAdes version 3.15.4 ( Prjibelski et al., 2020 ) and then annotated using Prokka version 1.14.6 ( Seemann, 2014 ). Next, polymorphisms between the derived isolates and the reference genome were identified using Snippy version 4.6.0 ( Seemann, 2015 ) with bwa version 0.7.17-r1198-dirty ( Li & Durbin, 2009 ). Larger deletions were identified using the samtools version 1.15 coverage and depth functions ( Danecek et al., 2021 ) and the BAM files returned by Snippy. Antibiotic susceptibility Minimum inhibitory concentrations (MIC) of ceftazidime, meropenem, and ertapenem were determined with ThermoFisher Oxoid M.I.C.Evaluator (M.I.C.E.) strips according to the manufacturer’s standard protocol on MH agar plates at 37°C. Antibiotic susceptibility was determined in accordance with CLSI guidelines ( CLSI, 2023 ). Reverse transcriptase quantitative PCR (RT-qPCR) Three biological replicates of each strain of interest were inoculated into 2 mL of Mueller-Hinton broth with relevant antibiotics (10 µg/mL of gentamicin was used for strains containing the pBBR1mcs-5 construct to stabilize the plasmid, and a sub-inhibitory concentration of 0.5 µg/mL of ertapenem was used for clinical isolates to induce vim-1 expression) and grown overnight at 37°C. The following day, cells were pelleted and washed with fresh media, then sub-cultured to a starting density of OD 600 = 0.05, grown to a final density of OD 600 = 0.5 at 37°C, pelleted, flash frozen, and stored at −80°C. RNA was purified from cell pellets with ZymoBIOMICS RNA Miniprep Kit (Zymo Research) according to the manufacturer’s instructions, which was then treated with a TURBO DNA- free Kit (Thermo Fisher) following the manufacturer’s instructions, to ensure the samples were free of contaminating DNA. Next, cDNA was synthesized from 2 µg of template RNA using the SuperScript IV VILO Master Mix Kit (Invitrogen), according to the manufacturer’s instructions. To examine the expression of vim-1 , qPCR was performed using a BioRad CFX Opus 96 Real-Time PCR System. Expression of vim-1 was normalized based on expression of the 16S rRNA gene. Standard curves were created for the 16S rRNA gene (primers: SK003 and SK004) and vim-1 (primers: GD018 and GD019) in 16 µL reactions that included 8 µL of SsoAdvanced Universal SYBR Green qPCR Supermix (BioRad), 187.5 nM of each primer, and between 0.002 and 200 ng of cDNA. The standard curves for both primer sets gave efficiencies >94% and R 2 values > 0.99 ( Figure S1 ). All subsequent qPCRs used 2 ng of cDNA. Data availability The ONT and Illumina data used to generate the whole genome sequences are available through the National Center for Biotechnology Information (NCBI) database under BioProject accession PRJNA1305022. As NCBI had not yet finished processing the annotated genomes by the time this manuscript was uploaded to NCBI, copies of the annotated genome files have been uploaded to GitHub (github.com/diCenzo-Lab/015_2025_VIM-1_analyses), and will be made available via NCBI under BioProject accession PRJNA1305022 once processing is completed. All code to repeat the computational analyses reported in this study is available through GitHub (github.com/diCenzo-Lab/015_2025_VIM-1_analyses). RESULTS Whole genome sequencing of six vim-1 -positive Enterobacterales clinical isolates Six multidrug resistant clinical isolates of order Enterobacterales were isolated from patients at the Kingston Health Sciences Center, Kingston, Ontario, Canada between 2015 and 2020. Despite all six isolates testing positive for the presence of vim-1 based on a PCR screen, they displayed a wide range of resistance to the carbapenem antibiotics ertapenem and meropenem, with some isolates phenotypically testing as susceptible despite the presence of vim-1 ( Table 2 ). In contrast, all strains were equally and highly resistant to the cephalosporin antibiotic ceftazidime ( Table 2 ). View this table: View inline View popup Download powerpoint Table 2. Minimum inhibitory concentrations of ceftazidime, meropenem, and ertapenem against six clinical isolates, together with the copy numbers of vim-1 -containing plasmids and vim-1 expression levels. Whole genome sequencing, assembly, and annotation was performed for all six clinical isolates. The resulting assemblies were high quality (completeness >99% and contamination <1% as determined by CheckM) and consisted of between three and seven contigs ( Table 3 ). For all six strains, the chromosome was assembled into a single, circular replicon. In addition, three of the six genomes appeared to be complete, closed genomes, while the other three included at least one plasmid split into two contigs. Taxonomic classification of the isolates indicated that F48994, H17629, and H70375 belonged to the species Enterobacter hormaechei, F5446 and S2568 belonged to the species Escherichia coli, and T64870 belonged to the species Klebsiella pneumoniae . View this table: View inline View popup Download powerpoint Table 3. Genome assembly and annotation statistics. Each of the isolates contained at least six perfect hits (100% nucleotide sequence identity) to different ARGs ( Table S2 ). As expected, all six isolates carried the vim-1 gene, but they also contained between 0 and 6 additional β-lactamase genes of the types ACT-24, CTX-M-15, LAP-2, OXA-1, OXA-9, TEM-1, SHV-12, and SHV-28 ( Table S2 ). The β-lactamases ACT-24, CTX-M-15, LAP-2, and TEM-1 are not known to interact with carbapenems, and therefore should not contribute to ertapenem or meropenem resistance ( Alcock et al., 2020 ). Likewise, while some OXA-family β-lactamases are considered ESBLs, OXA-1 and OXA-9 are not known to interact with carbapenems ( Evans & Amyes, 2014 ), and SHV-12 (found in isolates F5446 and S2568) and SHV-28 (found in isolate T64870) are only capable of mediating carbapenem resistance in combination with major outer membrane porin modifications ( Leavitt et al., 2009 ). Although these additional β-lactamase genes likely contributed to high ceftazidime resistance in the clinical isolates, these results suggest that vim-1 is primarily responsible for the observed carbapenem resistance. Genomic context of the vim-1 genes of the six clinical isolates As expected, vim-1 was situated within an integron in each of the six clinical isolates. However, the type of integron on which vim-1 was housed differed along species lines; the E. coli isolates contain In916 integrons while the other isolates contain In110 integrons ( Matsumura et al., 2017 ). In916 and In110 are clinical class 1 integrons ( Figure 1 ) that both contain a 5’ conserved sequence that includes the intI1 integrase, a 3’ conserved sequence that includes the ARGs qacEΔ1 and sul1 , and a variable region encoding additional ARGs and that differs in content between In916 and In110 ( Alcock et al., 2020 ). Notably, in all six of our strains, vim-1 is the first gene within the variable region. Download figure Open in new tab Figure 1. The structure of the vim-1 -containing class 1 integrons identified in this study. (A) Escherichia coli F5446 and E. coli S2568 encode vim-1 within In916 integrons, whereas (B) Enterobacter hormaechei F48994, E. hormaechei H17629, E. hormaechei H70375, and Klebsiella pneumoniae T64870 encode vim-1 within In110 integrons. ( A , B ) The 5’ conserved sequences (5’ CS), variable regions, and 3’ conserved sequences (3’ CS) are indicated above each diagram. The position of attL1 is represented as a light blue triangle, while the two genes present in In916 but absent from In110 are shown in light yellow. The diagrams are not drawn to scale. All six vim- 1-containing integrons were housed on plasmids rather than the bacterial chromosome. E. coli clinical isolates F5446 and S2568 contained vim-1 in the context of IncA plasmids, while other isolates contained their vim-1 in the context of IncN ( E. hormaechei H17629), IncR ( E. hormaechei H70375), or multi-locus IncN-IncR plasmids ( E. hormaechei F48994, K. pneumoniae T64870). The copy number (as determined by the genome assembler Flye) of vim-1- containing plasmids varied across clinical isolates, but these variations did not positively correlate with increases in vim-1 expression or tolerance to carbapenem antibiotics ( Table 2 ). Likewise, relative vim-1 expression, as determined by RT-qPCR, was not positively correlated with relative carbapenem resistance ( Table 2 ). Relationships between the vim- 1-containing plasmids of the six clinical isolates To explore the introduction and dissemination of vim-1 genes within Kingston (Ontario, Canada) and the surrounding region, a mid-point rooted, bifurcating dendrogram based on shared gene content was produced for the vim-1 containing plasmids of our six clinical isolates together with a set of 75 related plasmids ( Figure 2 ). Of the 75 external plasmids that were included in the analysis, 24 carried vim-1 . Overall, the vim-1 plasmids from our clinical isolates formed three distinct clades that also included vim-1 -containing plasmids from isolates collected in Toronto (Ontario, Canada), suggesting three separate introductions of vim-1 genes into the Kingston region and the spread of these genes/pathogens between Toronto and Kingston. Download figure Open in new tab Figure 2. Relationships between the vim-1 -containing plasmids of this study and related plasmids of the plasmid database (PLSDB). Plasmids related to the vim-1 -containing plasmids identified in the six focal clinical isolates of this study were identified in PLSDB using blast, and a dendogram was constructed from a distance matrix built on gene presence/absence data. Shown in blue are the vim-1 -containing plasmids identified in the current study, while red is used to represent other vim-1 containing plasmids. Plasmids are named according to the species in which the plasmid was identified, followed by the GenBank accession of the plasmid. The vim-1 -containing plasmids of E. hormaechei F48994, E. hormaechei H17629, and K. pneumoniae T64870 (which all contain vim-1 within an In110 integron) formed a clade with two other vim-1 -containing plasmids: pKpn13-2 (CP047709) and pCfr13-2 (CP047775). These plasmids were isolated from rectal samples of K. pneumoniae and Citrobacter freundii, respectively, by the Toronto Invasive Bacterial Diseases Network (TIBDN) from a single patient in Toronto, ON ( Kohler et al., 2020 ). These results suggest that this vim-1 -containing plasmid has spread between pathogens through horizontal transfer within southern Ontario. Notably, pKpn13-2 and pCfr13-2 are nested within the F48994, H17629, and T64870 cluster, suggesting that the detection of this plasmid in Toronto may have been due to spread from Kingston. Four vim-1- containing plasmids clustered with the E. hormaechei H70375 plasmid (which contains vim-1 within an In110 integron): pEclE2-2 (CP047717), pEcl6-3 (CP047732), pEcl5-2 (CP047738), and pEcl2-3 (CP047760). These plasmids all came from E. hormaechei isolates, one of which (pEclE2-2) was an environmental isolate collected from Toronto, ON sewage water in 2015, while the others are clinical isolates collected from hospitals by the TIBDN in Toronto, ON between 2011 and 2014 ( Kohler et al., 2020 ). These plasmids all have IncR replicons and are thought to share a common ancestral IncR plasmid ( Kohler et al., 2020 ). The plasmid from H70375 being the deepest branching lineage within this cluster, suggests that the detection of these vim-1 -containing plasmids in Toronto may have been the result of its spread from Kingston. Finally, there is a large cluster of vim-1 -containing plasmids that includes the vim-1 -containing plasmids of E. coli F5446 and S2568 (which contain vim-1 within an In916 integrons); the plasmid of S2568 is not shown in the dendrogram due to it being nearly identical to that of F5446 but not fully assembled. The most closely related plasmid to that from E. coli F5446 is pEco15-1 (CP047711), which was isolated from E. coli from a rectal swab gathered by the TIBDN in 2015 ( Kohler et al., 2020 ). Interestingly, the other plasmids within this cluster are not local to Ontario and come from isolates across the globe and from multiple species. These include pR210-2 (CP034084) from a K. pneumoniae isolate collected at the Hong Kong Polytechnic University (unpublished), pRIVM0001_VIM-1_171012_B12 (MH220284) from a Dutch Aeromonas sp. sample (unpublished), p550_IncA_VIM_1 (CP058224) from an Italian E. coli sample ( Mattioni Marchetti et al., 2020 ), pGA_VIM (MN783743) from an Italian E. coli sample ( Arcari et al., 2020 ), pKC-BO-N1-VIM (MG228427) from an Italian Kluyvera cryocrescens sample ( Gaibani et al., 2018 ), and pFDL_VIM (MN783744) from an Italian Klebsiella oxytoca sample ( Arcari et al., 2020 ). Expression of vim-1 alone facilitates limited resistance to ertapenem in laboratory strains To explore the contribution of vim-1 to the carbapenem antibiotic ertapenem, vim-1 of E. coli F5446 was PCR amplified and expressed under the control of the pLac promoter on the plasmid pBBR1mcs-5 in three non-pathogenic lab strains: E. coli DH5α, E. coli MG1655, and K. grimontii KP5022. Antibiotic susceptibility tests of E. coli DH5α, E. coli MG1655, and K. grimontii KP5022 strains carrying an empty pBBR1mcs-5 vector confirmed that the vector itself did not contribute to ertapenem, meropenem, or ceftazidime resistance ( Table 4 ). On the other hand, introduction of pBBR1mcs-5:: vim-1 resulted in an ∼ 20-fold to 64-fold increase in resistance to the cephalosporin antibiotic ceftazidime ( Table 4 ), confirming vim-1 was expressed and functional in all strains. Introduction of pBBR1mcs-5:: vim-1 into K. grimontii KP5022 also resulted in an ∼ 40-fold increase in resistance to the carbapenem meropenem but had little impact on resistance to the carbapenem ertapenem ( Table 4 ). Similarly, introduction of vim-1 into the two E. coli strains resulted in at most a 3-fold increase in resistance to meropenem and ertapenem ( Table 4 ). In all cases, introduction of vim-1 did not result in any of the three strains being classified as resistant to any of the three antibiotics according to the CLSI guidelines. View this table: View inline View popup Download powerpoint Table 4. Minimum inhibitory concentrations of ceftazidime, meropenem, and ertapenem against laboratory strains expressing vim-1 , both before and after repeated passaging in growth media containing ertapenem. To increase the level of resistance to ertapenem, adaptive laboratory evolution (ALE) experiments were performed with K. grimontii KP5022 (pBBR1mcs-5:: vim-1 ) and E. coli DH5α (pBBR1mcs-5:: vim-1 ). Replicates of each strain were grown in the presence of increasingly higher concentrations of ertapenem over a span of five days, following which individual isolates were isolated from two of the replicates of each species. Antibiotic susceptibility testing indicated that all derived lineages had extremely high resistance to ceftazidime but little to no increase in resistance to meropenem or ertapenem under the tested conditions ( Table 4 ). Transfer of the pBBR1mcs-5:: vim-1 plasmids from the K. grimontii or E. coli ALE derived isolates to otherwise wildtype K. grimontii KP5022 or E. coli DH5α, respectively, demonstrated that the mutation(s) responsible for the elevated ceftazidime resistance in the derived isolates are linked to the plasmids ( Table 4 ). To identify the responsible mutation(s), we used ONT sequencing to determine the complete sequence of the pBBR1mcs-5:: vim-1 plasmids from the derived isolates and compared them to the sequence of the original pBBR1mcs-5:: vim-1 plasmid. Unexpectedly, we observed that the plasmids isolated from the derived lineages were all approximately twice the length of the original pBBR1mcs-5:: vim-1 construct, with all genes (including vim-1 ) duplicated. This result suggested that the increased ceftazidime resistance of the derived lineages was a result an increase in vim-1 gene dosage and thus higher vim-1 expression. Indeed, RT-qPCR indicated that vim-1 expression in the derived isolates was ∼ 3.7- to 6.3-fold higher than in the ancestral strains ( Table 5 ). View this table: View inline View popup Download powerpoint Table 5. Expression of vim-1 in laboratory strains expressing vim-1 , both before and after repeated passaging in growth media containing ertapenem. E. coli DH5α resistance to ertapenem requires both vim-1 and mutations of an outer membrane porin Despite the increased vim-1 expression in the evolved lineages described above, none of the derived isolates were considered resistant to either ertapenem or meropenem under the tested conditions based on the CLSI guidelines ( Table 4 ). To evolve higher ertapenem resistance in E. coli DH5α, an E. coli DH5α strain carrying the enlarged pBBR1mcs-5:: vim-1 plasmid isolated from one of the K. grimontii KP5022 derived lineages was subjected to a six-day ALE in the presence of ertapenem, following which individual isolates were collected from two replicate cultures. Antibiotic susceptibility testing confirmed that the derived isolates displayed very high resistance to all three tested antibiotics (ceftazidime, meropenem, and ertapenem) ( Table 4 ). Curing of the vim-1 -containing plasmid from the derived lineages resulted in close to ancestral levels of resistance, while transfer of the vim-1 -containing plasmid to otherwise wildtype E. coli DH5α resulted in little resistance to meropenem or ertapenem ( Table 4 ). Together, these results suggest that the high ertapenem resistance of the derived lineages is dependent both on the presence of vim-1 and at least one additional mutation on the chromosome. To identify the chromosomal mutations responsible for elevated ertapenem resistance, whole genome sequencing of two derived isolates (strains EcGQ0088 and EcGQ0089; Table 4 ), as well as the ancestral strain (strain EcGQ0079; Table 4 ), was performed. Mapping of the EcGQ0088 sequencing reads to the EcGQ0079 reference genome identified a single polymorphism: an in-frame deletion of six nucleotides within ompC , which encodes outer membrane porin C, a transmembrane protein that facilitates the transport of beta-lactam antibiotics across the outer membrane ( Jaffe et al., 1982 ). Similarly, EcGQ0089 contained only a single mutation: a 26 kb deletion that spanned ompC , among other genes. Overall, these results indicate that high levels of vim-1 -mediated ertapenem resistance in E. coli DH5α is dependent on loss-of-function mutations within ompC . DISCUSSION The gene vim-1 is disseminated clonally and by plasmid-mediated HGT in Ontario Plasmids from all clinical isolates identified here can be linked to those associated with hospital outbreaks in Ontario and Europe and appear to form three distinct clades. The first clade, which forms around the vim-1 plasmids of E. coli F5664 and S2568, contains plasmids belonging to the IncA incompatibility group. The spread of plasmids in this clade, both geographically and across species of bacteria, suggests plasmids of this cluster are globally distributed and have been spread through HGT. The second clade contained the vim-1 -containing plasmids of E. hormaechei H17629, F48994, K. pneumoniae T64870, and two additional plasmids belonging to the multilocus IncR-IncN incompatibility group from Toronto ( Kohler et al., 2020 ). The third clade was formed around the vim-1- containing plasmid from E. hormaechei H70375 and four additional plasmids from Toronto ( Kohler et al., 2020 ), all belonging to the IncR incompatibility group. For both of these clades, the detection of similar plasmids in isolates from two cities within Ontario, Canada, but not yet elsewhere at the time this analysis was performed, suggests that these plasmids have been spread across species via HGT within Ontario. However, it is unknown whether IncR plasmids are transferable. Their broad host range suggests that they should be transferable ( Bielak et al., 2011 ; Kohler et al., 2020 ), but this plasmid complex lacks a transfer system and relaxase necessary for mobilization ( Compain et al., 2014 ; Kohler et al., 2020 ; Smillie et al., 2010 ). Therefore, it is possible that plasmids in the third clade can be found in E. hormaechei isolates across Ontario due to vertical transfer and subsequent evolution, but such conclusions would require further phylogenomic analysis. Overall, these findings support the current consensus on vim-1 in Ontario; that the gene is spread both clonally and by plasmid-mediated HGT ( Jamal et al., 2021 ; Kohler et al., 2020 ; Tijet et al., 2013 ). Genomic background influences vim-1 activity Among the six clinical isolates, the MICs of ertapenem ranged from 0.25 μg/mL, which is considered sensitive by CLSI guidelines, to 3 μg/mL, which is considered resistant. Ertapenem resistance was even lower (0.006 to 0.012 µg/mL) when vim-1 was cloned and introduced into three common Enterobacteriaceae laboratory strains. This phenotypic variation is consistent with previous studies noting phenotypic variability of MBLs when introduced into diverse bacterial hosts ( Socha et al., 2019 ). The differences in resistance phenotype do not fall cleanly around species lines, since there are variations in MICs of ertapenem and meropenem between individual E. coli and E. hormaechei isolates, although in general, the E. coli clinical isolates show higher ertapenem resistance than the other four isolates. The above observations led us to consider possible mechanistic explanations for the phenotypic variability across our six clinical isolates. Since vim-1 is integron-encoded and appears in identical positions relative to the integron promoter in all six clinical isolates, differences in promoter proximity and promoter strength are unlikely to explain the differences in resistance phenotypes conferred by vim-1 ; indeed, no obvious relationship was observed between resistance and vim-1 expression as measured by RT-qPCR. However, mRNA abundance and protein periplasmic concentrations are not necessarily correlated, and it has been noted that variation in MBL periplasmic concentration contributes to the phenotypic variation of MBLs across strains ( Socha et al., 2019 ). Another possibility is that the variations in VIM-1-mediated ertapenem resistance across the strains is due to epistatic interactions between vim-1 and other genomic loci. Previous studies have observed that high resistance to ertapenem and other carbapenems depends on the presence of a β-lactamase paired with reduced outer membrane permeability ( Codjoe et al., 2025 ; Doumith et al., 2009 ; Jacoby et al., 2004 ; Leavitt et al., 2009 ; Szabó et al., 2006 ; Woodford et al., 2007 ). With one exception ( Codjoe et al., 2025 ), those studies focused on variations in membrane permeability in strains with ESBLs rather than strains with true carbapenemases (like VIM-1). Examining the genomes of our six clinical isolates for outer membrane porins revealed that four of the six strains had two copies of ompC , and that the E. hormaechei strains also carried ompD . In addition, the OmpC amino acid sequences of our six isolates were highly variable, sharing between 62.9% and 100% sequence identity ( Figure S2 ). Thus, we hypothesize that variation in membrane permeability contributed to the differences in ertapenem resistance observed in our clinical isolates, which would suggest that reduced membrane permeability is likely an important factor impacting carbapenem resistance even in isolates with true carbapenemases. VIM-1 alone was not sufficient to confer carbapenem resistance To experimentally explore factors influencing VIM-1-mediated ertapenem resistance, vim-1 was amplified from a vim-1 + clinical isolate without its native promoter and expressed on a plasmid in the laboratory strains E. coli DH5α, MG1655, and K. grimontii KP5022. For all strains, the presence of vim-1 alone was insufficient to confer resistance to the carbapenem antibiotics meropenem and ertapenem, but did confer resistance to the cephalosporin antibiotic ceftazidime. These results confirmed vim-1 was expressed and the encoded enzyme was active and suggested that vim-1 alone is a strong resistance determinant for cephalosporin antibiotics (like ceftazidime), but not necessarily for carbapenems like ertapenem and meropenem. ALE was used to identify mutations increasing ertapenem resistance in the laboratory strains carrying vim-1 . In all cases, the primary mutation that we identified was an increase in the vim-1 copy number, and thus expression. Although the increase in vim-1 expression increased the concentration of ertapenem that the strains could survive in the liquid-based ALE experiments, this mutation had little impact on ertapenem MIC as determined by plate-based assays. We hypothesize that the difference between the liquid-based and agar-based assays reflects that in the liquid-assays, the concentration of ertapenem constantly decreased as VIM-1 degraded the antibiotic, allowing growth when the concentration was sufficiently low. On the other hand, the concentration of ertapenem may not have changed significantly in the plate-based assay due to diffusion of the antibiotic throughout the plate. On the other hand, strong VIM-1-mediated ertapenem resistance in the plate-based assay was observed when ompC , encoding an outer membrane porin, was mutated or deleted, although these mutations came with a trade-off of reduced growth in the absence of antibiotics. This is consistent with previous studies showing synergistic effects of pairing an ESBL with reduced outer membrane permeability ( Codjoe et al., 2025 ; Doumith et al., 2009 ; Jacoby et al., 2004 ; Szabó et al., 2006 ; Woodford et al., 2007 ). Porins are pore-forming proteins with a β-barrel structure that allow for the passive transport of hydrophilic compounds across the bacterial outer membrane ( Schulz, 2002 ). OmpC is a non-specific porin that plays a role in both membrane integrity and antibiotic transport ( Choi & Lee, 2019 ), meaning its mutation or deletion would result in reduced transport of β-lactam antibiotics across the outer membranes, and reduce their concentration in the periplasm to a level that does not overwhelm VIM-1. Conclusions Here, we describe six Enterobacterales clinical isolates carrying VIM-1 on horizontally transmissible plasmids. These plasmids appear to be globally distributed and to have spread both clonally and via plasmid-mediated HGT. In addition, the six isolates vary in carbapenem resistance, which we hypothesize is driven primarily by variations in outer membrane permeability. In support of this, we observed that transfer of vim-1 to common laboratory strains failed to result in ertapenem resistance unless paired with mutation or deletion of ompC encoding an outer membrane porin. This result is consistent with studies of strains expressing ESBLs, although the requirement for reduced outer membrane permeability for VIM-1-mediated ertapenem resistance has been less studied. Finally, these results further highlight that PCR-based detection of vim-1 is not necessarily sufficient to demonstrate that an isolate is resistant to carbapenems without functional verification. ACKNOWLEDGEMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Discovery Grant to G.C.D. (RGPIN-2020-0700). Funder Information Declared Natural Sciences and Engineering Research Council of Canada (NSERC) , RGPIN-2020-0700 Footnotes https://github.com/diCenzo-Lab/015_2025_VIM-1_analyses REFERENCES ↵ Alcock , B. P. , Raphenya , A. R. , Lau , T. T. Y. , Tsang , K. K. , Bouchard , M. , Edalatmand , A. , Huynh , W. , Nguyen , A.-L. V. , Cheng , A. A. , Liu , S. , Min , S. Y. , Miroshnichenko , A. , Tran , H.-K. , Werfalli , R. E. , Nasir , J. A. , Oloni , M. , Speicher , D. J. , Florescu , A. , Singh , B. , Faltyn , M. , Hernandez-Koutoucheva , A. , Sharma A. N. , Bordeleau , E. , Pawlowski , A. C. , Zubyk , H. L. , Dooley , D. , Griffiths , E. , Maguire , F. , Winsor , G. L. , Beiko , R. G. , Brinkman , F. S. L. , Hsiao , W. W. L. , Domselaar , G. V. & McArthur , A. G . ( 2020 ). CARD 2020: Antibiotic resistome surveillance with the comprehensive antibiotic resistance database . Nucleic Acids Res , 48 ( D1 ), D517 – D525 . doi: 10.1093/nar/gkz935 OpenUrl CrossRef PubMed ↵ Anisimova , M. , & Gascuel , O . ( 2006 ). Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative . Syst Biol , 55 ( 4 ), 539 – 552 . doi: 10.1080/10635150600755453 OpenUrl CrossRef PubMed Web of Science ↵ Arcari , G. , Di Lella , F. M. , Bibbolino , G. , Mengoni , F. , Beccaccioli , M. , Antonelli , G. , Faino , L. , & Carattoli , A. ( 2020 ). A multispecies cluster of VIM-1 carbapenemase-producing Enterobacterales linked by a novel, highly conjugative, and broad-host-range IncA plasmid forebodes the reemergence of VIM-1 . Antimicrob Agents Chemother , 64 ( 4 ), e02435 – 19 . doi: 10.1128/AAC.02435-19 OpenUrl Abstract / FREE Full Text ↵ Bielak , E. , Bergenholtz , R. D. , Jørgensen , M. S. , Sørensen , S. J. , Hansen , L. H. , & Hasman , H . ( 2011 ). Investigation of diversity of plasmids carrying the bla TEM-52 gene . J Antimicrob Chemother , 66 ( 11 ), 2465 – 2474 . doi: 10.1093/jac/dkr331 OpenUrl CrossRef PubMed Web of Science ↵ Bolger , A. M. , Lohse , M. , & Usadel , B . ( 2014 ). Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinformatics , 30 ( 15 ), 2114 – 2120 . doi: 10.1093/bioinformatics/btu170 OpenUrl CrossRef PubMed Web of Science ↵ Botelho , J. , Grosso , F. , Quinteira , S. , Brilhante , M. , Ramos , H. , & Peixe , L . ( 2018 ). Two decades of bla VIM-2-producing Pseudomonas aeruginosa dissemination: An interplay between mobile genetic elements and successful clones . Journal Antimicrob Chemother , 73 ( 4 ), 873 – 882 . doi: 10.1093/jac/dkx517 OpenUrl CrossRef PubMed ↵ Bushnell B. ( 2021 ). BBMap: BBMap short read aligner, and other bioinformatic tools. [Computer software] https://sourceforge.net/projects/bbmap/ ↵ Camacho , C. , Coulouris , G. , Avagyan , V. , Ma , N. , Papadopoulos , J. , Bealer , K. , & Madden , T.L . ( 2009 ). BLAST+: architecture and applications . BMC Bioinform , 10 ( 421 ). doi: 10.1186/1471-2105-10-421 OpenUrl CrossRef PubMed ↵ Carattoli , A. , Zankari , E. , García-Fernández , A. , Voldby Larsen , M. , Lund , O. , Villa , L. , Møller Aarestrup , F. , & Hasman , H . ( 2014 ). In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing . Antimicrob Agents Chemother , 58 ( 7 ), 3895 – 3903 . doi: 10.1128/AAC.02412-14 OpenUrl Abstract / FREE Full Text ↵ Choi , U. , & Lee , C.-R. ( 2019 ). Distinct roles of outer membrane porins in antibiotic resistance and membrane integrity in Escherichia coli. Front Microbiol , 10 ( 2019 ). https://www.frontiersin.org/articles/10.3389/fmicb.2019.00953 ↵ CLSI . ( 2023 ). CLSI M100-ED33:2023 Performance Standards for Antimicrobial Susceptibility Testing ( 33rd Edition ). ↵ Codjoe , F. S. , Kotey , F. C. N. , & Donkor , E. S . 2025 . Profile of outer membrane proteins of carbapenem-resistant Gram-negative bacilli in Ghana . BMC Res Notes , 18 ( 49 ). doi: 10.1186/s13104-024-07070-6 OpenUrl CrossRef ↵ Compain , F. , Frangeul , L. , Drieux , L. , Verdet , C. , Brisse , S. , Arlet , G. , & Decré , D . ( 2014 ). Complete nucleotide sequence of two multidrug-resistant IncR plasmids from Klebsiella pneumoniae . Antimicrob Agents Chemother , 58 ( 7 ), 4207 – 4210 . doi: 10.1128/AAC.02773-13 OpenUrl Abstract / FREE Full Text Council of Canadian Academies . ( 2019 ). When Antibiotics Fail: The Expert Panel on the Potential Socio-Economic Impacts of Antimicrobial Resistance in Canada . http://www.deslibris.ca/ID/10102747 ↵ Cowie , A. , Cheng , J. , Sibley , C. D. , Fong , Y. , Zaheer , R. , Patten , C. L. , Morton , R. M. , Golding , G. B. , & Finan , T. M . ( 2006 ). An integrated approach to functional genomics: construction of a novel reporter gene fusion library for Sinorhizobium meliloti . Appl Environ Microbiol , 72 ( 11 ), 7156 – 7167 . doi: 10.1128/AEM.01397-06 OpenUrl Abstract / FREE Full Text Currie , C. J. , Berni , E. , Jenkins-Jones , S. , Poole , C. D. , Ouwens , M. , Driessen , S. , Voogd , H. de , Butler , C. C. , & Morgan , C. L. ( 2014 ). Antibiotic treatment failure in four common infections in UK primary care 1991-2012: Longitudinal analysis . BMJ , 349 , g5493 . doi: 10.1136/bmj.g5493 OpenUrl Abstract / FREE Full Text ↵ Dadgostar , P . ( 2019 ). Antimicrobial resistance: Implications and costs . Infect Drug Resist , 12 , 3903 – 3910 . doi: 10.2147/IDR.S234610 OpenUrl CrossRef PubMed ↵ Danecek , P. , Bonfield , J. K. , Liddle , J. , Marshall , J. , Ohan , V. , Pollard , M. O. , Whitwham , A. , Keane , T. , McCarthy , S. A. , Davies , R. M. , & Li , H . ( 2021 ). Twelve years of SAMtools and BCFtools . GigaScience , 10 ( 2 ), giab008 . doi: 10.1093/gigascience/giab008 OpenUrl CrossRef PubMed ↵ Darling , A. C. E. , Mau , B. , Blattner , F. R. , & Perna , N. T . ( 2004 ). Mauve: Multiple alignment of conserved genomic sequence with rearrangements . Genome Res , 14 ( 7 ), 1394 – 1403 . doi: 10.1101/gr.2289704 OpenUrl Abstract / FREE Full Text ↵ Datsenko , K. A. , & Wanner , B. L . ( 2000 ). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products . Proc Natl Acad Sci , 97 ( 12 ), 6640 – 6645 . doi: 10.1073/pnas.120163297 OpenUrl Abstract / FREE Full Text ↵ De la Cruz Barrón , M. , Merlin , C. , Guilloteau , H. , Montargès-Pelletier , E. , & Bellanger , X. ( 2018 ). Suspended materials in river waters differentially enrich class 1 integron- and IncP-1 plasmid-carrying bacteria in sediments . Front Microbiol , 9 ( 2018 ). https://www.frontiersin.org/article/10.3389/fmicb.2018.01443 ↵ Doumith , M. , Ellington , M. J. , Livermore , D. M. , & Woodford , N . 2009 . Molecular mechanisms disrupting porin expression in ertapenem-resistant Klebsiella and Enterobacter spp. clinical isolates from the UK . J Antimicrob Chemother , 63 ( 4 ), 659 – 667 . doi: 10.1093/jac/dkp029 OpenUrl CrossRef PubMed Web of Science ↵ Drost , H . ( 2018 ). Philentropy: Information Theory and Distance Quantification with R . J Open Source Softw , 3 ( 26 ), 765 . doi: 10.21105/joss.00765 OpenUrl CrossRef ↵ Duan , Y. F. , Grogan , P. , Walker , V. K. , & diCenzo , G. C. ( 2022 ). Whole genome sequencing of three mesorhizobia isolated from northern Canada to identify genomic adaptations promoting nodulation in cold climates . Can J Microbiol , 68 ( 11 ): 661 – 673 . doi: 10.1139/cjm-2022-0102 OpenUrl CrossRef PubMed ↵ Evans , B. A. , & Amyes , S. G. B . ( 2014 ). OXA β-Lactamases . Clin Microbiol Rev , 27 ( 2 ), 241 – 263 . doi: 10.1128/CMR.00117-13 OpenUrl Abstract / FREE Full Text ↵ Galata , V. , Fehlmann , T. , Backes , C. , & Keller , A . ( 2019 ). PLSDB: a resource of complete bacterial plasmids . Nucleic Acids Res , 47 ( D1 ): D195 – D202 . doi: 10.1093/nar/gky1050 OpenUrl CrossRef PubMed ↵ Gaibani , P. , Ambretti , S. , Scaltriti , E. , Cordovana , M. , Berlingeri , A. , Pongolini , S. , Landini , M. P. , & Re , M. C . ( 2018 ). A novel IncA plasmid carrying bla VIM-1 in a Kluyvera cryocrescens strain . J Antimicrob Chemother , 73 ( 11 ), 3206 – 3208 . doi: 10.1093/jac/dky304 OpenUrl CrossRef PubMed ↵ Gillings , M. , Boucher , Y. , Labbate , M. , Holmes , A. , Krishnan , S. , Holley , M. , & Stokes , H. W . ( 2008 ). The evolution of class 1 integrons and the rise of antibiotic resistance . J Bacteriol , 190 ( 14 ), 5095 – 5100 . doi: 10.1128/JB.00152-08 OpenUrl Abstract / FREE Full Text Grant , S. G. , Jessee , J. , Bloom , F. R. , & Hanahan , D . ( 1990 ). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants . Proc Natl Acad Sci , 87 ( 12 ), 4645 – 4649 . OpenUrl Abstract / FREE Full Text Guyer , M. S. , Reed , R. R. , Steitz , J. A. , & Low , K. B . ( 1981 ). Identification of a sex-factor- affinity site in E. coli as gamma delta . Cold Spring Harb Symp Quant Biol , 44 ( 1 ), 135 – 140 . doi: 10.1101/sqb.1981.045.01.022 pmid: . PMID: 6271456 . OpenUrl CrossRef PubMed Hawkey , P. M. , & Livermore , D. M . ( 2012 ). Carbapenem antibiotics for serious infections . BMJ , 344 , e3236 . doi: 10.1136/bmj.e3236 OpenUrl FREE Full Text ↵ Hu , Y. , Yang , X. , Li , J. , Lv , N. , Liu , F. , Wu , J. , Lin , I. Y. C. , Wu , N. , Weimer , B. C. , Gao , G. F. , Liu , Y. , & Zhu , B . ( 2016 ). The bacterial mobile resistome transfer network connecting the animal and human microbiomes . Appl Environ Microbiol , 82 ( 22 ), 6672 – 6681 . doi: 10.1128/AEM.01802-16 OpenUrl Abstract / FREE Full Text ↵ Hunt , M. , Silva , N. D. , Otto , T. D. , Parkhill , J. , Keane , J. A. , & Harris , S. R . ( 2015 ). Circlator: Automated circularization of genome assemblies using long sequencing reads . Genome Biol , 16 ( 1 ), 294 . doi: 10.1186/s13059-015-0849-0 OpenUrl CrossRef PubMed ↵ Irrgang , A. , Tenhagen , B.-A. , Pauly , N. , Schmoger , S. , Kaesbohrer , A. , & Hammerl , J. A. ( 2019 ). Characterization of VIM-1-producing E. coli isolated from a German fattening pig farm by an improved isolation procedure . Front Microbiol , 10 ( 2019 ). https://www.frontiersin.org/article/10.3389/fmicb.2019.02256 ↵ Jacoby , G. A. , Mills , D. M. , & Chow , N . 2004 . Role of β-lactamases and porins in resistance to ertapenem and other β-lactams in Klebsiella pneumoniae . Antimicrob Agents Chemother , 48 ( 8 ), 3203 – 3206 . doi: 10.1128/aac.48.8.3203-3206.2004 OpenUrl Abstract / FREE Full Text ↵ Jaffe , A. , Chabbert , Y. A. , & Semonin , O . ( 1982 ). Role of porin proteins OmpF and OmpC in the permeation of beta-lactams . Antimicrob Agents Chemother , 22 ( 6 ), 942 – 948 . OpenUrl Abstract / FREE Full Text ↵ Jamal , A. J. , Mataseje , L. F. , Williams , V. , Leis , J. A. , Tijet , N. , Zittermann , S. , Melano , R. G. , Mulvey , M. R. , Katz , K. , Allen , V. G. , & McGeer , A. J . ( 2021 ). Genomic epidemiology of carbapenemase-producing Enterobacterales at a hospital system in Toronto, Ontario, Canada, 2007 to 2018 . Antimicrob Agents Chemother , 65 ( 8 ), e00360 – 21 . doi: 10.1128/AAC.00360-21 OpenUrl CrossRef Johansson , Å. , Ekelöf , J. , Giske , C. G. , & Sundqvist , M . ( 2014 ). The detection and verification of carbapenemases using ertapenem and Matrix Assisted Laser Desorption Ionization-Time of Flight . BMC Microbiol , 14 , 89 . doi: 10.1186/1471-2180-14-89 OpenUrl CrossRef PubMed ↵ Katoh , K. , Rozewicki , J. , & Yamada , K. D . ( 2019 ). MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization . Brief Bioinform , 20 ( 4 ), 1160 – 1166 . doi: 10.1093/bib/bbx108 OpenUrl CrossRef PubMed ↵ Kintses , B. , Méhi , O. , Ari , E. , Számel , M. , Györkei , Á. , Jangir , P. K. , Nagy , I. , Pál , F. , Fekete , G. , Tengölics , R. , Nyerges , Á. , Likó , I. , Bálint , A. , Molnár , T. , Bálint , B. , Vásárhelyi , B. M. , Bustamante , M. , Papp , B. , & Pál , C . ( 2019 ). Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota . Nat Microbiol , 4 ( 3 ), 447 – 458 . doi: 10.1038/s41564-018-0313-5 OpenUrl CrossRef PubMed ↵ Knopp , M. , & Andersson , D. I . ( 2015 ). Amelioration of the fitness costs of antibiotic resistance due to reduced outer membrane permeability by upregulation of alternative porins . Mol Biol Evol , 32 ( 12 ), 3252 – 3263 . doi: 10.1093/molbev/msv195 OpenUrl CrossRef PubMed ↵ Kohler , P. P. , Melano , R. G. , Patel , S. N. , Shafinaz , S. , Faheem , A. , Coleman , B. L. , Green , K. , Armstrong , I. , Almohri , H. , Borgia , S. , Borgundvaag , E. , Johnstone , J. , Katz , K. , Lam , F. , Muller , M. P. , Powis , J. , Poutanen , S. M. , Richardson , D. , Rebbapragada , A. , Sarabia , A. , Simor , A. , McGeer , A. , & the Toronto Invasive Bacterial Diseases Network (TIBDN) . ( 2018 ). Emergence of Carbapenemase-Producing Enterobacteriaceae, South-Central Ontario, Canada . Emerg Infect Dis , 24 ( 9 ), 1674 – 1682 . doi: 10.3201/eid2409.180164 OpenUrl CrossRef PubMed ↵ Kohler , P. , Tijet , N. , Kim , H. C. , Johnstone , J. , Edge , T. , Patel , S. N. , Seah , C. , Willey , B. , Coleman , B. , Green , K. , Armstrong , I. , Katz , K. , Muller , M. P. , Powis , J. , Poutanen , S. M. , Richardson , D. , Sarabia , A. , Simor , A. , McGeer , A. , & Melano , R. G. ( 2020 ). Dissemination of Verona Integron-encoded Metallo-β-lactamase among clinical and environmental Enterobacteriaceae isolates in Ontario, Canada . Sci Rep , 10 ( 1 ), Article 1. doi: 10.1038/s41598-020-75247-7 OpenUrl CrossRef PubMed ↵ Kolmogorov , M. , Yuan , J. , Lin , Y. , & Pevzner , P. A . ( 2019 ). Assembly of long, error-prone reads using repeat graphs . Nat Biotechnol , 37 ( 5 ), 540 – 546 . doi: 10.1038/s41587-019-0072-8 OpenUrl CrossRef PubMed ↵ Kovach , M. E. , Elzer , P. H. , Steven Hill , D. , Robertson , G. T. , Farris , M. A. , Roop , R. M. , & Peterson , K. M . ( 1995 ). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes . Gene , 166 ( 1 ), 175 – 176 . doi: 10.1016/0378-1119(95)00584-1 OpenUrl CrossRef PubMed Web of Science ↵ Kurtz , S. , Phillippy , A. , Delcher , A. L. , Smoot , M. , Shumway , M. , Antonescu , C. , & Salzberg , S. L . ( 2004 ). Versatile and open software for comparing large genomes . Genome Biol , 5 ( 2 ), R12 . doi: 10.1186/gb-2004-5-2-r12 OpenUrl CrossRef PubMed ↵ Leavitt , A. , Chmelnitsky , I. , Colodner , R. , Ofek , I. , Carmeli , Y. , & Navon-Venezia , S . ( 2009 ). Ertapenem resistance among extended-spectrum-β-lactamase-producing Klebsiella pneumoniae isolates . J Clin Microbiol , 47 ( 4 ), 969 – 974 . doi: 10.1128/JCM.00651-08 OpenUrl Abstract / FREE Full Text ↵ Letunic , I. , & Bork , P . ( 2021 ). Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation . Nucleic Acids Res ., 49 ( W1 ), W293 – W296 . doi: 10.1093/nar/gkab301 OpenUrl CrossRef PubMed ↵ Li , H . ( 2018 ). Minimap2: Pairwise alignment for nucleotide sequences . Bioinformatics , 34 ( 18 ), 3094 – 3100 . doi: 10.1093/bioinformatics/bty191 OpenUrl CrossRef PubMed ↵ Li , H. , & Durbin , R . ( 2009 ). Fast and accurate short read alignment with Burrows-Wheeler transform . Bioinformatics , 25 ( 14 ), 1754 – 1760 . doi: 10.1093/bioinformatics/btp324 OpenUrl CrossRef PubMed Web of Science ↵ Li , Y. , da Silva , G. C. , Li , Y. , Rossi , C. C. , Fernandez Crespo , R. , Williamson , S. M. , Langford , P. R. , Bazzolli , D. M. S. , & Bossé , J. T. ( 2018 ). Evidence of illegitimate recombination between two Pasteurellaceae plasmids resulting in a novel multi-resistance replicon, pM3362MDR, in Actinobacillus pleuropneumoniae . Front Microbiol , 9 ( 2018 ). https://www.frontiersin.org/article/10.3389/fmicb.2018.02489 Liu , L. , Feng , Y. , Hu , Y. , Kang , M. , Xie , Y. , & Zong , Z . ( 2018 ). Klebsiella grimontii , a new species acquired carbapenem resistance . Front Microbiol , 9 ( 2018 ). doi: 10.3389/fmicb.2018.02170 OpenUrl CrossRef ↵ Mano , Y. , Saga , T. , Ishii , Y. , Yoshizumi , A. , Bonomo , R. A. , Yamaguchi , K. , & Tateda , K . ( 2015 ). Molecular analysis of the integrons of metallo-β-lactamase-producing Pseudomonas aeruginosa isolates collected by nationwide surveillance programs across Japan . BMC Microbiol , 15 ( 1 ), 41 . doi: 10.1186/s12866-015-0378-8 OpenUrl CrossRef PubMed ↵ Matsumura , Y. , Peirano , G. , Devinney , R. , Bradford , P. A. , Motyl , M. R. , Adams , M. D. , Chen , L. , Kreiswirth , B. , & Pitout , J. D. D . ( 2017 ). Genomic epidemiology of global VIM-producing Enterobacteriaceae . J Antimicrob Chemother , 72 ( 8 ), 2249 – 2258 . doi: 10.1093/jac/dkx148 OpenUrl CrossRef PubMed ↵ Mattioni Marchetti , V. , Bitar , I. , Piazza , A. , Mercato , A. , Fogato , E. , Hrabak , J. , & Migliavacca , R. ( 2020 ). Genomic insight of VIM-harboring IncA plasmid from a clinical ST69 Escherichia coli strain in Italy . Microorganisms , 8 ( 8 ), 1232 . doi: 10.3390/microorganisms8081232 OpenUrl CrossRef PubMed ↵ Meier-Kolthoff , J. P. , Carbasse , J. S. , Peinado-Olarte , R. L. , & Göker , M . ( 2022 ). TYGS and LPSN: A database tandem for fast and reliable genome-based classification and nomenclature of prokaryotes . Nucleic Acids Res , 50 ( D1 ), D801 – D807 . doi: 10.1093/nar/gkab902 OpenUrl CrossRef PubMed ↵ Minh , B. Q. , Schmidt , H. A. , Chernomor , O. , Schrempf , D. , Woodhams , M. D. , von Haeseler , A. , & Lanfear , R. ( 2020 ). IQ-TREE 2: New models and efficient methods for phylogenetic inference in the genomic era . Mol Bol Evol , 37 ( 5 ), 1530 – 1534 . doi: 10.1093/molbev/msaa015 OpenUrl CrossRef PubMed ↵ Miriagou , V. , Cornaglia , G. , Edelstein , M. , Galani , I. , Giske , C. G. , Gniadkowski , M. , Malamou-Lada , E. , Martinez-Martinez , L. , Navarro , F. , Nordmann , P. , Peixe , L. , Pournaras , S. , Rossolini , G. M. , Tsakris , A. , Vatopoulos , A. , & Cantón , R . ( 2010 ). Acquired carbapenemases in Gram-negative bacterial pathogens: Detection and surveillance issues . Clin Microbiol Infect , 16 ( 2 ), 112 – 122 . doi: 10.1111/j.1469-0691.2009.03116.x OpenUrl CrossRef PubMed Web of Science ↵ Murray , C. J. , Ikuta , K. S. , Sharara , F. , Swetschinski , L. , Aguilar , G. R. , Gray , A. , Han , C. , Bisignano , C. , Rao , P. , Wool , E. , Johnson , S. C. , Browne , A. J. , Chipeta , M. G. , Fell , F. , Hackett , S. , Haines-Woodhouse , G. , Hamadani , B. H. K. , Kumaran , E. A. P. , McManigal , B. , Agarwal , R. , Akech , S. , Albertson , S. , Amuasi , J. , Andrews , J. , Aravkin , A. , Ashley , E. , Bailey , F. , Baker , S. , Basnyat , B. , Bekker , A. , Bender , R. , Bethou , A. , Bielicki , J. , Boonkasidecha , S. , Bukosia , J. , Carvalheiro , C. , Castañeda-Orjuela , C. , Chansamouth , V. , Chaurasia , S. , Chiurchiù , S. , Chowdhury , F. , Cook , A. J. , Cooper , B. , Cressey , T. R. , Criollo-Mora , E. , Cunningham , M. , Darboe , S. , Day , N. P. J. , De Luca , M. , Dokova , K. , Dramowski , A. , Dunachie , S. J. , Eckmanns , T. , Eibach , D. , Emami , A. , Feasey , N. , Fisher-Pearson , N. , Forrest , K. , Garrett , D. , Gastmeier , P. Giref , A. Z. , Greer , R. C. , Gupta , V. , Haller , S. , Haselbeck , A. , Hay , S. I. , Holm , M. , Hopkins , S. , Iregbu , K. C. , Jacobs , J. , Jarovsky , D. , Javanmardi , F. , Khorana , M. , Kissoon , N. , Kobeissi , E. , Kostyanev , T. , Krapp , F. , Krumkamp , R. , Kumar , A. , Kyu , H. H. , Lim , C. , Limmathurotsakul , D. , Loftus , M. J. , Lunn , M. , Ma , J. , Mturi , N. , Munera-Huertas , T. , Musicha , P. , Mussi-Pinhata , M. M. , Nakamura , T. , Nanavati , R. , Nangia , S. , Newton , P. , Ngoun , C. , Novotney , A. , Nwakanma , D. , Obiero , C. W. , Olivas-Martinez , A. , Olliaro , P. , Ooko , E. , Ortiz-Brizuela , E. , Peleg , A. Y. , Perrone , C. , Plakkal , N. , Ponce-de-Leon , A. , Raad , M. , Ramdin , T. , Riddell , A. , Roberts , T. , Robotham , J. V. , Roca , A. , Rudd , K. E. , Russell , N. , Schnall , J. , Scott , J. A. G. , Shivamallappa , M. , Sifuentes-Osornio , J. , Steenkeste , N. , Stewardson , A. J. , Stoeva , T. , Tasak , N. , Thaiprakong , A. , Thwaites , G. , Turner , C. , Turner , P. , Rogier van Doorn , H. , Velaphi , S. , Vongpradith , A. , Vu , H. , Walsh , T. , Waner , S. , Wangrangsimakul , T. , Wozniak , T. , Zheng , P. , Sartorius , B. , Lopez , A. D. , Stergachis , A. , Moore , C. , Dolecek , C. & Naghavi , M. ( 2022 ). Global burden of bacterial antimicrobial resistance in 2019: A systematic analysis . Lancet , 399 ( 10325 ), 629 – 655 . doi: 10.1016/S0140-6736(21)02724-0 OpenUrl CrossRef PubMed ↵ Néron , B. , Littner , E. , Haudiquet , M. , Perrin , A. , Cury , J. , & Rocha , E. P. C . ( 2022 ). IntegronFinder 2.0: Identification and analysis of integrons across bacteria, with a focus on antibiotic resistance in Klebsiella . Microorganisms , 10 ( 4 ), Article 4. doi: 10.3390/microorganisms10040700 OpenUrl CrossRef PubMed ↵ Page , A. J. , Cummins , C. A. , Hunt , M. , Wong , V. , K., Reuter , S. , Holden , M. T. G. , Fookes , M. , Falush , D. , Keane , J. A. , & Parkhill , J. ( 2015 ). Roary: rapid large-scale prokaryote pan genome analysis . Bioinformatics , 31 ( 22 ), 3691 – 3693 . doi: 10.1093/bioinformatics/btv421 OpenUrl CrossRef PubMed ↵ Papadimitriou-Olivgeris , M. , Bartzavali , C. , Lambropoulou , A. , Solomou , A. , Tsiata , E. , Anastassiou , E. D. , Fligou , F. , Marangos , M. , Spiliopoulou , I. , & Christofidou , M . ( 2019 ). Reversal of carbapenemase-producing Klebsiella pneumoniae epidemiology from blaKPC- to blaVIM-harbouring isolates in a Greek ICU after introduction of ceftazidime/avibactam . Journal Antimicrob Chemother , 74 ( 7 ), 2051 – 2054 . doi: 10.1093/jac/dkz125 OpenUrl CrossRef PubMed ↵ Paradis , E. , & Schliep , K . ( 2019 ). ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R . Bioinformatics , 35 ( 3 ), 526 – 528 . doi: 10.1093/bioinformatics/bty633 OpenUrl CrossRef PubMed ↵ Parks , D.H. , Imelfort , M. , Skennerton , C.T. , Hugenholtz , P. , Tyson , G.W . ( 2015 ). Assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes . Genome Res , 25 : 1043 – 1055 . OpenUrl Abstract / FREE Full Text ↵ Peirano , G. , Lascols , C. , Hackel , M. , Hoban , D. J. , & Pitout , J. D. D . ( 2014 ). Molecular epidemiology of Enterobacteriaceae that produce VIMs and IMPs from the SMART surveillance program . Diagn Microbiol Infect Dis , 78 ( 3 ), 277 – 281 . doi: 10.1016/j.diagmicrobio.2013.11.024 OpenUrl CrossRef PubMed ↵ Poidevin , M. , Sato , M. , Altinoglu , I. , Delaplace , M. , Sato , C. , & Yamaichi , Y . ( 2018 ). Mutation in ESBL plasmid from Escherichia coli O104:H4 leads autoagglutination and enhanced plasmid dissemination . Front Microbiol , 9 ( 2018 ). https://www.frontiersin.org/article/10.3389/fmicb.2018.00130 Poirel , L. , Naas , T. , Nicolas , D. , Collet , L. , Bellais , S. , Cavallo , J.-D. , & Nordmann , P . ( 2000 ). Characterization of VIM-2, a carbapenem-hydrolyzing metallo-β-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France . Antimicrob Agents Chemother , 44 ( 4 ), 891 – 897 . OpenUrl Abstract / FREE Full Text ↵ Porse , A. , Schou , T. S. , Munck , C. , Ellabaan , M. M. H. , & Sommer , M. O. A . ( 2018 ). Biochemical mechanisms determine the functional compatibility of heterologous genes . Nat Commun , 9 , 522 . doi: 10.1038/s41467-018-02944-3 OpenUrl CrossRef PubMed ↵ Prjibelski , A. , Antipov , D. , Meleshko , D. , Lapidus , A. , & Korobeynikov , A . ( 2020 ). Using SPAdes de novo assembler . Curr Protoc Bioinform , 70 ( 1 ), e102 . doi: 10.1002/cpbi.102 OpenUrl CrossRef PubMed ↵ R Core Team ( 2021 ). R: A language and environment for statistical computing . R Foundation for Statistical Computing , Vienna, Austria . URL https://www.R-project.org/ . Rodriguez-Martinez , J.-M. , Nordmann , P. , Fortineau , N. , & Poirel , L . ( 2010 ). VIM-19, a metallo-β-lactamase with increased carbapenemase activity from Escherichia coli and Klebsiella pneumoniae . Antimicrob Agents Chemother , 54 ( 1 ), 471 – 476 . doi: 10.1128/AAC.00458-09 OpenUrl Abstract / FREE Full Text ↵ Salimraj , R. , Hinchliffe , P. , Kosmopoulou , M. , Tyrrell , J. M. , Brem , J. , van Berkel , S. S. , Verma , A. , Owens , R. J. , McDonough , M. A. , Walsh , T. R. , Schofield , C. J. , & Spencer , J. ( 2019 ). Crystal structures of VIM-1 complexes explain active site heterogeneity in VIM-class metallo-β-lactamases . FEBS J , 286 ( 1 ), 169 – 183 . doi: 10.1111/febs.14695 OpenUrl CrossRef PubMed ↵ Sánchez-Romero , I. , Asensio , Á. , Oteo , J. , Muñoz-Algarra , M. , Isidoro , B. , Vindel , A. , Álvarez-Avello , J. , Balandín-Moreno , B. , Cuevas , O. , Fernández-Romero , S. , Azañedo , L. , Sáez , D. , & Campos , J . ( 2012 ). Nosocomial outbreak of VIM-1-producing Klebsiella pneumoniae isolates of multilocus sequence Type 15: Molecular basis, clinical risk factors, and outcome . Antimicrob Agents Chemother , 56 ( 1 ), 420 – 427 . doi: 10.1128/AAC.05036-11 OpenUrl Abstract / FREE Full Text ↵ Schulz , G. E . ( 2002 ). The structure of bacterial outer membrane proteins . Biochim Biophys Acta , 1565 ( 2 ), 308 – 317 . doi: 10.1016/s0005-2736(02)00577-1 OpenUrl CrossRef PubMed Web of Science ↵ Seeman , T . ( 2014 ). Prokka: rapid prokaryotic genome annotation . Bioinformatics , 30 ( 14 ), 2068 – 2069 . doi: 10.1093/bioinformatics/btu153 OpenUrl CrossRef PubMed Web of Science ↵ Seemann , T. ( 2015 ). snippy: Fast bacterial variant calling from NGS reads [Computer software] . https://github.com/tseemann/snippy ↵ Sievers , F. , & Higgins , D. G . ( 2017 ). Clustal Omega for making accurate alignments of many protein sequences . Protein Sci ., 27 , 135 – 145 . doi: 10.1002/pro.3290 OpenUrl CrossRef PubMed ↵ Silva , R. F. , Mendonça , S. C. M. , Carvalho , L. M. , Reis , A. M. , Gordo , I. , Trindade , S. , & Dionisio , F . ( 2011 ). Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations . PLOS Genet , 7 ( 7 ), e1002181 . doi: 10.1371/journal.pgen.1002181 OpenUrl CrossRef PubMed ↵ Smillie , C. , Garcillán-Barcia , M. P. , Francia , M. V. , Rocha , E. P. C. , & de la Cruz , F. ( 2010 ). Mobility of plasmids . Microbiol Mol Biol Rev , 74 ( 3 ), 434 – 452 . doi: 10.1128/MMBR.00020-10 OpenUrl Abstract / FREE Full Text ↵ Socha , R. D. , Chen , J. , & Tokuriki , N . ( 2019 ). The molecular mechanisms underlying hidden phenotypic variation among metallo-β-lactamases . J Mol Biol , 431 ( 6 ), 1172 – 1185 . doi: 10.1016/j.jmb.2019.01.041 OpenUrl CrossRef PubMed ↵ Sorek , R. , Zhu , Y. , Creevey , C. J. , Francino , M. P. , Bork , P. , & Rubin , E. M . ( 2007 ). Genome-wide experimental determination of barriers to horizontal gene transfer . Science , 318 ( 5855 ), 1449 – 1452 . doi: 10.1126/science.1147112 OpenUrl Abstract / FREE Full Text ↵ Streicher , S. L. , Shanmugam , K. T. , Ausubel , F. , Morandi , C. & Goldberg , R. B . ( 1974 ). Regulation of nitrogen fixation in Klebsiella pneumoniae : evidence for a role of glutamine synthetase as a regulator of nitrogenase synthesis . J Bacteriol , 120 ( 2 ), 815 – 821 . doi: 10.1128/jb.120.2.815-821.1974 OpenUrl Abstract / FREE Full Text ↵ Szabó , D. , Silveira , F. , Hujer , A. M. , Bonomo , R. A. , Hujer , K. M. , Marsh , J. M. , Bethel , C. R. , Doi , Y. , Deeley , K. & Paterson , D. L . ( 2006 ). Outer membrane protein changes and efflux pump expression together may confer resistance to ertapenem in Enterobacter cloacae . Antimicrob Agents Chemother , 50 ( 8 ), 2833 – 2835 . doi: 10.1128/aac.01591-05 OpenUrl Abstract / FREE Full Text ↵ Tatusova , T. , DiCuccio , M. , Badretdin , A. , Chetvernin , V. , Nawrocki , E. P. , Zaslavsky , L. , Lomsadze , A. , Pruitt , K. D. , Borodovsky , M. , & Ostell , J . ( 2016 ). NCBI prokaryotic genome annotation pipeline . Nucleic Acids Res , 44 ( 14 ), 6614 – 6624 . doi: 10.1093/nar/gkw569 OpenUrl CrossRef PubMed ↵ Tijet , N. , Macmullin , G. , Lastovetska , O. , Vermeiren , C. , Wenzel , P. , Stacey-Works , T. , Low , D. E. , Patel , S. N. , & Melano , R. G . ( 2013 ). Verona Integron–encoded Metallo-β-Lactamase 1 in Enterobacteria , Ontario, Canada . Emerg Infect Dis , 19 ( 7 ), 1156 – 1158 . doi: 10.3201/eid1907.121294 OpenUrl CrossRef PubMed ↵ van der Zee , A. , Kraak , W. B. , Burggraaf , A. , Goessens , W. H. F. , Pirovano , W. , Ossewaarde , J. M. , & Tommassen , J. ( 2018 ). Spread of carbapenem resistance by transposition and conjugation among Pseudomonas aeruginosa . Front Microbiol , 9 ( 2018 ). https://www.frontiersin.org/article/10.3389/fmicb.2018.02057 ↵ Vaser , R. , Sović , I. , Nagarajan , N. , & Šikić , M . ( 2017 ). Fast and accurate de novo genome assembly from long uncorrected reads . Genome Res , 27 ( 5 ), 737 – 746 . doi: 10.1101/gr.214270.116 OpenUrl Abstract / FREE Full Text Ventola , C. L . ( 2015 ). The antibiotic resistance crisis . Pharm Ther , 40 ( 4 ), 277 – 283 . OpenUrl CrossRef ↵ von Wintersdorff , C. J. H. , Penders , J. , van Niekerk , J. M. , Mills , N. D. , Majumder , S. , van Alphen , L. B. , Savelkoul , P. H. M. , & Wolffs , P. F. G. ( 2016 ). Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer . Front Microbiol , 7 ( 2016 ). https://www.frontiersin.org/article/10.3389/fmicb.2016.00173 ↵ Walker , B. J. , Abeel , T. , Shea , T. , Priest , M. , Abouelliel , A. , Sakthikumar , S. , Cuomo , C. A. , Zeng , Q. , Wortman , J. , Young , S. K. , & Earl , A. M . ( 2014 ). Pilon: An integrated tool for comprehensive microbial variant detection and genome assembly improvement . PloS One , 9 ( 11 ), e112963 . doi: 10.1371/journal.pone.0112963 OpenUrl CrossRef PubMed ↵ Wick , R. R. , Judd , L. M. , Gorrie , C. L. , & Holt , K. E . ( 2017 ). Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads . PLOS Comput Biol , 13 ( 6 ), e1005595 . doi: 10.1371/journal.pcbi.1005595 OpenUrl CrossRef PubMed ↵ Wong , A . ( 2017 ). Epistasis and the evolution of antimicrobial resistance . Front Microbiol , 8 ( 2017 ). https://www.frontiersin.org/articles/10.3389/fmicb.2017.00246 ↵ Woodford , D. , Dallow , J. W. T. , Hill , R. L. R. , Palepou , M. I. , Pike , R. , Ward , M. E. , Warner , M. & Livermore , D. M . 2007 . Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory . Int J Antimicrob Agents , 29 ( 4 ), 456 – 459 . doi: 10.1016/j.ijantimicag.2006.11.020 OpenUrl CrossRef PubMed Web of Science ↵ Zeng , L. , Zhan , Z. , Hu , L. , Jiang , X. , Zhang , Y. , Feng , J. , Gao , B. , Zhao , Y. , Yang , W. , Yang , H. , Yin , Z. , & Zhou , D . ( 2019 ). Genetic characterization of a blaVIM-24-carrying IncP-7β plasmid p1160-VIM and a blaVIM-4-harboring integrative and conjugative element Tn6413 from clinical Pseudomonas aeruginosa . Front Microbiol , 10 , 213 . doi: 10.3389/fmicb.2019.00213 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted September 07, 2025. Download PDF Supplementary Material Data/Code Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales Mia Rondinelli , Sabhjeet Kaur , Owen A. Ledwell , Henry Wong , Prameet M. Sheth , George C. diCenzo bioRxiv 2025.09.07.674714; doi: https://doi.org/10.1101/2025.09.07.674714 Share This Article: Copy Citation Tools Variations in carbapenem resistance associated with the VIM-1 metallo-β-lactamase across the Enterobacterales Mia Rondinelli , Sabhjeet Kaur , Owen A. Ledwell , Henry Wong , Prameet M. Sheth , George C. diCenzo bioRxiv 2025.09.07.674714; doi: https://doi.org/10.1101/2025.09.07.674714 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42005) Biophysics (21472) Cancer Biology (18624) Cell Biology (25534) Clinical Trials (138) Developmental Biology (13391) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40437) Molecular Biology (17200) Neuroscience (88697) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4829) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9827) Zoology (2272)