{"paper_id":"876c2d87-b1fb-4edd-a5d1-923334141eb0","body_text":"Genomic and molecular characterisation of a Klebsiella pneumoniae 1 \nclinical isolate resistant to meropenem-vaborbactam, imipenem-2 \nrelebactam, and ceftazidime-avibactam 3 \nYu Wan1,2,3, Joshua L. C. Wong4, Julia Sanchez-Garrido4, Wen Wen Low4, Jane F. Turton1,5, 4 \nFabio Morecchiato6, Ilaria Baccani6, Kirsty Dodgson8, Gian Maria Rossolini6,7, Neil 5 \nWoodford5, Gad Frankel4, Elita Jauneikaite2, Danièle Meunier1,5, and Katie L. Hopkins1,2,5* 6 \n 7 \n1 AMR & HCAI Division, UK Health Security Agency, London, United Kingdom 8 \n2 NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial 9 \nResistance, Department of Infectious Disease, Imperial College London, London, United Kingdom 10 \n3 David Price Evans Global Health and Infectious Diseases Research Group, University of Liverpool, 11 \nLiverpool, United Kingdom 12 \n4 Department of Life Sciences, Imperial College London, London, United Kingdom 13 \n5 Public Health Microbiology Reference Microbiology Division, UK Health Security Agency, 14 \nLondon, United Kingdom 15 \n6 Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 16 \n7 Careggi University Hospital, Florence, Italy 17 \n8 Department of Medical Microbiology, Manchester University NHS Foundation Trust, Manchester, 18 \nUnited Kingdom 19 \n 20 \nAddress correspondence to Katie L. Hopkins (katie.hopkins@ukhsa.gov.uk) 21 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nAbstract 22 \nThis article reports an unusual Klebsiella pneumoniae  clinical isolate, KpMVR1,  resistant to 23 \nmeropenem-vaborbactam, imipenem -relebactam, and  ceftazidime-avibactam, and investigates  the 24 \nunderlying genetic alterations using comparative genomics and molecular experiments. 25 \nResistance to carbapenems and third -generation cephalosporins is increasing in K. pneumoniae 26 \nglobally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely 27 \nused to circumvent β-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a 28 \nhospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat 29 \nprofile—KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from 30 \nanother patient in the same hospital —were susceptible to meropenem-vaborbactam, imipenem -31 \nrelebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid 32 \ngenome assembly  were conducted for  these three isolates . Annotated genome  assemblies were 33 \ncompared to identify genetic  variation, and mutagenesis experiments were performed  to verify 34 \npredicted functional alterations. 35 \nAll isolates belonged to a novel clone ST8134 and carried blaKPC-2-like alleles (KpMVR1: blaKPC-36 \n157; KpMVS1 and KpMVS2: blaKPC-2) in presumptively conjugative plasmids. ISEc68 caused a 37 \nframeshift mutation  in KpMVR1’s ompK36 gene, reducing the meropenem-vaborbactam and 38 \nimipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and 39 \nceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor 40 \nMarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. 41 \nIn conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence 42 \nfor in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant 43 \npathogens in vulnerable patients to guide antimicrobial chemotherapy  as well as the need to 44 \ncharacterise underlying resistance mechanisms to help assess the potential for onward transmission. 45 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nKeywords 46 \nKlebsiella pneumoniae, antimicrobial resistance, combination antimicrobials, β-lactam antimicrobials, 47 \nβ-lactamase inhibitors,  porins, medical microbiology, bioinformatics , comparative genomics , 48 \nmutagenesis experiments 49 \nData summary 50 \nIllumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of 51 \nisolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center 52 \nfor Biotechnology Information (www.ncbi.nlm.nih.gov) under BioProject accession PRJNA1084250, 53 \nwith BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and 54 \nSAMN46778011 (KpMVS2). The genome assemblies  of these isolates have also been deposited in  55 \nPasteur Institute’s database for K. pneumoniae species complex ( bigsdb.pasteur.fr/klebsiella/) under 56 \nids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). 57 \nImpact statement 58 \nThis is the first  blaKPC-positive K. pneumoniae isolate referred to the UK’s national reference 59 \nlaboratory with  resistance to three last-resort β-lactam/β-lactamase inhibitor combinations 60 \nmeropenem-vaborbactam, imipenem -relebactam, and  ceftazidime-avibactam, implicating in vivo  61 \nemergence of this  unusual resistance profile during prolonged antimicrobial chemotherapy. This 62 \nisolate belonged to a novel clone ST8134  and harboured a plasmid -borne blaKPC-2-like allele blaKPC-63 \n157. We identified complex genetic alterations in this isolate: chromosomal  large deletions, point 64 \nmutations, and  an ISEc68-induced loss -of-function truncation of the ompK36 porin gene . We 65 \ndetermined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. 66 \npneumoniae to meropenem, meropenem -vaborbactam, imipenem, imipenem -relebactam, imipenem-67 \navibactam, aztreonam, aztreonam -avibactam, ceftazidime, ceftazidime -avibactam, and cefiderocol.  68 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nOur work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor 69 \ncombinations in healthcare and to understand underlying resistance mechanisms for assessing the 70 \npotential of pathogen transmission.  71 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nIntroduction 72 \nMeropenem and imipenem are broad-spectrum carbapenem antimicrobials parenterally administrated 73 \nto treat serious bacterial infections, such as those caused by Enterobacterales producing extended-74 \nspectrum β-lactamases (ESBLs) or AmpC-type cephalosporinases (AmpCs) [1–3]. Ceftazidime, a 75 \nthird-generation parenteral cephalosporin, is also widely used for treating severe bacterial infections , 76 \nalthough it can be hydrolysed by ESBLs and AmpCs [4, 5]. In Gram-negative bacteria, carbapenems 77 \nand cephalosporins diffuse through outer-membrane porins  and enter the periplasm, where they 78 \ninactivate penicillin binding proteins, disrupting cell-wall synthesis with a bactericidal effect [6–8]. 79 \nCombining β-lactams with β-lactamase inhibitors in antimicrobial chemotherapy is a  widely 80 \nemployed strategy to  circumvent β-lactamase-mediated resistance in bacteria. Vaborbactam, 81 \nrelebactam, and avibactam are non-β-lactam inhibitors of Ambler class A β-lactamases, such as ESBLs 82 \nand Klebsiella pneumoniae carbapenemases (KPCs), as well as  class C β-lactamases (AmpCs) [9]. 83 \nMoreover, avibactam inhibits several class D β-lactamases such as OXA-48 and OXA-10 [10]. These 84 \ninhibitors penetrate the outer membrane  (OM) of Gram -negative bacteria via porins , blocking the 85 \nactive sites of β-lactamases in the periplasm [9, 11]. 86 \nIn the UK, meropenem -vaborbactam, imipenem -relebactam, and ceftazidime -avibactam are 87 \nreserved for highly selected patients [12]. Resistance to one or more  of these combination 88 \nantimicrobials in clinical isolates of KPC-producing Klebsiella pneumoniae  has been previously 89 \nreported [13–15]. Underlying resistance mechanisms include overproduction of KPCs or the AcrAB-90 \nTolC multidrug efflux pump , as well as gain-of-function point mutations in the blaKPC gene [9, 16–91 \n18]. In addition, t he disruption  or transcriptional downregulation of ompK35 (ompF) and ompK36 92 \n(ompC), which encode non-selective porins that facilitate the diffusion of β-lactams and β-lactamase 93 \ninhibitors through the OM, has also been implicated [17–20]. 94 \nHere, we report and characterise a clinically significant K. pneumoniae isolate exhibiting resistance 95 \nto ceftazidime-avibactam, meropenem-vaborbactam, and imipenem -relebactam, analysed  in the 96 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \ncontext of closely related isolates recovered in the same hospital. This was the first such isolate referred 97 \nto the UK’s national reference laboratory for characterisation. 98 \nMethods 99 \nIsolate collection and phenotyping 100 \nThe K. pneumoniae  isolate KpMVS1  was recovered from a lung biopsy specimen  of a n inpatient 101 \n(hereafter, Patient 1)  admitted to an intensive care unit (ICU) in England in 2021 . The second K. 102 \npneumoniae isolate, KpMVR1, was recovered from a groin wound of the same patient 42 days later. 103 \nDuring this ICU stay, the patient received a broad range of  antimicrobials, including meropenem-104 \nvaborbactam, ciprofloxacin, and gentamicin ; however,  ceftazidime-avibactam and imipenem-105 \nrelebactam were not used. 106 \nSpecies identification of the isolates  and carbapenemase gene screening  were performed using 107 \nmatrix-assisted laser desorption/ioni sation-time of flight  (MALDI-ToF) method and the GeneXpert 108 \nsystem (Cepheid, USA), respectively. Initial antimicrobial susceptibility testing (AST) was conducted 109 \nby the hospital, with results interpreted according to  the European Committee on Antimicrobial 110 \nSusceptibility Testing  (EUCAST) guidelines. Both isolates were referred to the Antimicrobial 111 \nResistance and Healthcare Associated Infections  (AMRHAI) Reference Unit  of the UK Health 112 \nSecurity Agency (UKHSA) for variable number tandem repeat (VNTR) typing [21] and investigation 113 \nof unusual  antimicrobial resistance (AMR). Furthermore, a blaKPC-positive K. pneumoniae  isolate 114 \nKpMVS2 — recovered from a rectal swab of another patient (Patient 2) in the same hospital in 2020 115 \nduring an outbreak investigation and sharing the same VNTR profile as KpMVS1 and KpMVR1 — 116 \nwas retrieved from AMRHAI’s culture collection for comparison. These three isolates were subjected 117 \nto whole-genome sequencing (WGS) and AST for which minimum inhibitory concentrations (MICs) 118 \nof 19 antimicrobials (Table 1) and diameters of cefiderocol inhibition zones were interpreted as per 119 \nEUCAST clinical breakpoints v15.0 [22]. 120 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nWhole-genome sequencing 121 \nGenomic DNA of each isolate was extracted  from an overnight culture  using the GeneJET Kit 122 \n(ThermoFisher Scientific, UK)  as per the manufacturer’s protocol . Short-read sequencing was 123 \nconducted on a HiSeq 2500 system (Illumina, USA) by UKHSA’s Colindale Sequencing Laboratory 124 \nfollowing its paired-end 101-bp protocol. Long-read sequencing was performed on MinION R9.4. 1 125 \nflow cells (Oxford Nanopore Technologies [ONT], UK), with libraries prepared using the ONT Rapid 126 \nBarcoding Kit SQK-RBK004. 127 \nBioinformatics analysis 128 \nIllumina reads were trimmed and filtered with Trimmomatic v0.39 for a minimum per-read quality of 129 \nPhred Q30 and minimum length of 50 bp [23]. Fast-mode basecalling and de-multiplexing of nanopore 130 \nreads was conducted by guppy v4 ( ONT). Nanopore reads were  then trimmed and filtered for a 131 \nminimum per-read quality of Q10 and minimum length of 1 kbp using fastp v0.23.4 [24]. For species 132 \nconfirmation and contamination  assessment, taxonomical profiling of processed Illumina and 133 \nnanopore reads were performed using Kraken v2.1.3, bracken v2.8, and a standard Kraken database 134 \nbuilt in September 2023 [25, 26]. 135 \nGenomes of KpMVR1 and KpMVS2, w ith estimated nanopore read depths of 185×  and 243× , 136 \nrespectively, were assembled using hybracter v0.5.0 (assemblers: Flye v2.9.3 and plassembler v1.5.0; 137 \nsequence re -orientator: dnaapler v0.5.1 ; long-read polisher: medaka v 1.8.0, short -read polishers: 138 \npypolca v0.2.1 and p olypolish v0.5.0 ) [27–32]. For KpMVS1, which had  nanopore reads with an 139 \nestimated depth of 64× , the chromosome and plasmid sequences were assembled using Raven v1.8.3 140 \nand plassembler, respectively, and polished with nanopore and Illumina reads as for KpMVR1 and 141 \nKpMVS2. Evaluation of contamination and completeness of genome assemblies were conducted with 142 \nCheckM2 v1.0.2 and its database Uniref100/KO [33]. The average fold-coverage of each contig was 143 \nestimated from Illumina and nanopore reads, respectively, using mosdepth v0.3.9 [34]. 144 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nThe genome assemblies were annotated using bakta v1.9.2 and its standard database v5.1  [35]. 145 \nMulti-locus sequence typing, serotype prediction, and virulence-factor detection were performed using 146 \nKleborate v 3.1.3, which incorporated Kaptive v 3.1.0 [36, 37] . AMR genes were detected using 147 \nAMRFinderPlus v3.12.8 with a minimum query coverage of 80% [38]. Clustered regularly interspaced 148 \nshort palindromic repeats  (CRISPR) and CRISPR -associated (Cas) genes in chromosomes were 149 \npredicted using CRISPRCasFinder [39]. For plasmids, replicon types were determined at a minimum 150 \nof 80% nucleotide identity and coverage using PlasmidFinder v2.1 [40] and the mobility was predicted 151 \nusing mob_typer of MOB-suite v3.1.8 [41]. The fold-coverage of each KPC-encoding plasmid was  152 \ndivided by that of its host’s chromosome to  estimate the plasmid  copy number. Transposons and 153 \ninsertion sequences (ISs) were identified using TnCentral Blast (blastn) and ISFinder, respectively [42, 154 \n43]. 155 \nChromosome and plasmids of KpMVR1 and KpMVS2 were compared against those of KpMVS1 156 \nusing minimap v2.26  [44]. Identified g enetic variants were annotated using snpEff v5.2 [45]. Gene 157 \nOntology terms were predicted from amino acid sequences using InterProScan v5.69-101.0 [46] with 158 \nsequence alignments filtered for ≥60% query coverages. Impacts of point mutations on protein stability 159 \nwere predicted from wild -type protein structures in the UniProt database using Missense3D and 160 \nDDMut [47–49]. The three-dimensional structure of the plasmid-encoded donor OM protein TraN was 161 \ncompared between KPC -encoding, IncFII -carrying plasmids pKpMVS1_1, pKpMVR1_1, and 162 \npKpMVS2_1 following the approach developed by Low et al (Supplementary methods) to estimate 163 \nthe impact of TraN alterations on the conjugation specificity and efficiency [50]. Comparison and 164 \nannotations of these three plasmids were visualised using BRIG v0.95 and Proksee [51, 52]. Gene 165 \nsynteny was illustrated using R package gggenes [53]. Genetic alterations found in both KpMVR1 and 166 \nKpMVS2 were considered unlikely to confer the unique AMR profiles of KpMVR1 and were therefore 167 \nexcluded from further investigation. 168 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nFunctional assessment 169 \nTo experimentally determine  and compare  the impacts of blaKPC-2 and blaKPC-157 on β-lactam 170 \nsusceptibility in K. pneumoniae, KpMVS1’s KPC-2-encoding plasmid pKpMVS1_1, was introduced 171 \ninto the plasmid-free K. pneumoniae laboratory strain  ICC8001 (MICs: meropenem, ≤0.06 mg/L; 172 \nimipenem, 0.25 mg/L; aztreonam, ≤0.125 mg/L; ceftazidime and ceftazidime -avibactam, 0.25 mg/L) 173 \nthrough conjugation , resulting in a transconjugant ICC8001 KPC-2 [54]. Transgenic isolates  174 \nICC8001KPC-157 and KpMVS1KPC-157 were derived from ICC8001KPC-2 and KpMVS1, respectively, by 175 \nsubstituting the blaKPC-2 allele with blaKPC-157. Moreover, isolates ICC8001KPC-2/ΔompK36 and ICC8001KPC-176 \n157/ΔompK36 were derived from ICC8001 KPC-2 and ICC8001 KPC-157, respectively, through seamless, 177 \nmarkerless homologous recombination using mutagenesis vectors and a lambda -red based 178 \nrecombination system generated in previous work [54]. 179 \nTo predict the  presence/absence of OmpK36 in the OM of KpMVR1, Sec-dependent signal 180 \npeptides and their cleavage site in translated ompK36 alleles were compared between KpMVS1 and 181 \nKpMVR1 using SignalP v6.0  [55]. To validate the prediction, p urification of OM proteins was 182 \nperformed by resuspending an overnight LB-Miller culture (VWR, USA) of each isolate in 1M HEPES 183 \n(pH 7.4) and sonicating at 25% amplitude for 10 bursts of 10 seconds on, 15 seconds off each (Model 184 \n705 Sonic Dismembrator, Fisher Scientific). Isolates ICC8001 and its ompK36-knockout derivative, 185 \nICC8001ΔompK36, were included as positive and negative controls, respectively . After separating 186 \ncellular debris by centrifugation, OM proteins were obtained by centrifugation at 14,000×g for 30 mins 187 \nand resuspended in 2% sarcosine/HEPES for 30 mins at room temperature. All steps were performed 188 \nat 4° C on ice to preserve protein integrity unless otherwise indicated. For visualisation, 10 μg protein  189 \nper isolate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 190 \nusing 12% acrylamide gels  and was stained with Coomassie solution (Sigma -Aldrich, USA ) and 191 \nimaged on a ChemiDoc XRS+ (Bio-Rad, USA). 192 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nBoth progenitor isolates (KpMVS1 and KpMVR1) and the five transgenic isolates (KpMVS1KPC-193 \n157, ICC8001KPC-2, ICC8001KPC-157, ICC8001KPC-2/ΔompK36, and ICC8001KPC-157/ΔompK36) were tested for 194 \nsusceptibility to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-195 \navibactam, ceftazidime, ceftazidime-avibactam, aztreonam, aztreonam-avibactam, and cefiderocol (in 196 \niron-depleted medium) by the reference broth microdilution method as per the EUCAST guidance [56, 197 \n57]. Any MIC change above a two-fold difference between two isolates was considered notable. 198 \nResults 199 \nPhenotypes of isolates 200 \nIsolates KpMVS1 and KpMVR1, obtained 42 days apart from Patient 1 with recurrent K. pneumoniae 201 \ninfections, and KpMVS2 , obtained from Patient 2 in the same hospital , were identified as K. 202 \npneumoniae by both MALDI -ToF and WGS . In t he ICU where KpMVS1 and KpMVR1 were 203 \nrecovered, all patients were screened for carriage of carbapenemase-producing Enterobacterales (CPE) 204 \non admission using PCR, and the resistance profile of KpMVR1 was unique among all identified CPE 205 \nisolates from the ICU during Patient 1’s stay. 206 \nBased on AST results from the AMRHAI Reference Unit  (Table 1), KpMVR1 was resistant to 207 \nmeropenem-vaborbactam (MIC>256 mg/L) , ceftazidime-avibactam (MIC=16 mg/L) , and 208 \nciprofloxacin (MIC>4 mg/L), whereas KpMVS1 and KpMVS2 were susceptible to these antimicrobial 209 \nagents (MICs: meropenem-vaborbactam ≤0.064 mg/L; ceftazidime-avibactam 1 mg/L; ciprofloxacin 210 \n≤0.125 mg/L). Notably, KpMVS2 was resistant to cefiderocol. Further AST discovered that  the 211 \nimipenem-relebactam MIC of KpMVR1 (512 mg/L, resistant) was 2048 times that of KpMVS1 (0.25 212 \nmg/L, susceptible) (Table 2). Moreover, KpMVR1 exhibited a >4-fold increase in the temocillin MIC 213 \n(>128 mg/L) and a >8-fold reduction in the cefepime MIC (4 mg/L, susceptible, increased exposure) 214 \ncompared with KpMVS1 (temocillin: 32 mg/L; cefepime: >32 mg/L, resistant). 215 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nTable 1. Antimicrobial minimum inhibitory concentrations (MICs; mg/L) determined by UKHSA’s AMRHAI Reference Unit and susceptibility i nterpretations 216 \n(as per EUCAST clinical breakpoints v15.0) of three K. pneumoniae clinical isolates. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; 217 \nETP, ertapenem; CET, ceftolozane; TZB, tazobactam; CFD, cefiderocol; FEP, cefepime; CAZ, ceftazidime; AVI, avibactam; CTX, cefotaxime; FOX, cefoxitin; 218 \nTMC, temocillin; AMP, ampicillin; AMX, amoxicillin; CAV, clavulanate; PIP, piperacillin; ATM, aztreonam; AMK, amikacin; GEN, gentamicin; CST, colistin; 219 \nCIP, ciprofloxacin; MB, monobactam. Susceptibility interpretations: R, resistant; I, susceptible, increased exposure; S, susceptible. 220 \n Carbapenem Cephalosporin Penicillin MB Aminoglycoside Others \nIsolate MEM-\nVAB MEM IPM ETP CET-\nTZB CFD* FEP CAZ CAZ-\nAVI CTX FOX‡ TMC‡ AMP AMX\n-CAV \nPIP-\nTZB ATM AMK‡ GEN‡ CST‡ CIP \nKpMVR1 >256 \n(R) \n>16 \n(R) \n>128 \n(R) \n>4 \n(R) \n16 \n(R) (S) 4 \n(I) \n256 \n(R) \n16 \n(R) \n8 \n(R) \n>64 \n >128 >32 \n(R) \n>32 \n(R) \n>64 \n(R) \n16 \n(R) 2 0.5 ≤0.5 >4 \n(R) \nKpMVS1 0.064 \n(S) \n>16 \n(R) \n64 \n(R) \n>4 \n(R) \n>16 \n(R) (S) >32 \n(R) \n128 \n(R) \n1 \n(S) \n64 \n(R) \n>64 \n 32 >32 \n(R) \n>32 \n(R) \n>64 \n(R) \n>32 \n(R) ≤1 ≤0.25 ≤0.5 ≤0.125 \n(S) \nKpMVS2 0.032  \n(S) \n16 \n(R) \n16 \n(R) \n>4 \n(R) \n>16 \n(R) (R) >32 \n(R) \n64 \n(R) \n1 \n(S) \n16 \n(R) \n32 \n 8 >32 \n(R) \n>32 \n(R) \n>64 \n(R) \n>32 \n(R) ≤1 ≤0.25 1 ≤0.125 \n(S) \n* CFD susceptibility was determined using disc diffusion. ‡ Interpretations were not available according to EUCAST guidelines.  221 \n  222 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nTable 2. Minimum inhibitory concentrations of beta-lactam antimicrobials and susceptibility interpretations (as per EUCAST clinical breakpoints v15.0, where 223 \napplicable) of progenitor and transgenic K. pneumoniae isolates determined in the experiments for functional assessment. Subscripts in isolate names indicate 224 \ntransgenic isolates and corresponding genotypes. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; REL, relebactam; ATM, aztreonam; 225 \nAVI, avibactam; CAZ, ceftazidime; CFD, cefiderocol, tested in iron-depleted Mueller Hinton broth; NT, not tested. Interpretations of antimicrobial susceptibility: 226 \nR, resistant; I, susceptible upon increased antimicrobial exposure; S, susceptible. Notations: ompK36fs, frameshifted ompK36; ΔompK36, deletion of ompK36. 227 \nIsolate Genotype \nMinimum Inhibitory Concentration (mg/L) and interpretation \nMEM MEM-VAB IPM IPM-REL IPM-AVI ATM ATM-AVI CAZ CAZ-AVI CFD \nKpMVR1 blaKPC-157 ompK36fs 512 (R) 256 (R) 512 (R) 512 (R) NT 16 (R) 4 (S) 8 (R) 8 (S) 0.5 (S) \nKpMVS1 blaKPC-2 ompK36 32 (R) ≤0.06 (S) 32 (R) 0.25 (S) ≤0.5 512 (R) 0.25 (S) 32 (R) 0.5 (S) 0.25 (S) \nKpMVS1KPC-157 blaKPC-157 ompK36 16 (R) ≤0.06 (S) 8 (R) 8 (R) ≤0.5 2 (I) 0.25 (S) 1 (S) 0.25 (S) ≤0.06 (S) \nICC8001KPC-2 blaKPC-2 ompK36 16 (R) ≤0.06 (S) 16 (R) 0.25 (S) ≤0.5 512 (R) 0.125 (S) 32 (R) 0.25 (S) 0.25 (S) \nICC8001KPC-157 blaKPC-157 ompK36 16 (R) ≤0.06 (S) 4 (I) 4 (R) ≤0.5 1 (S) 0.125 (S) 0.5 (S) 0.125 (S) ≤0.06 (S) \nICC8001KPC-2/ΔompK36 blaKPC-2 ΔompK36 256 (R) 2 (S) 256 (R) 2 (S) NT >1024 (R) 0.25 (S) 16 (R) 0.5 (S) 0.25 (S) \nICC8001KPC-157/ΔompK36 blaKPC-157 ΔompK36 256 (R) 4 (S) 256 (R) 128 (R) NT 4 (I) 0.25 (S) 1 (S) 0.5 (S) 0.25 (S) \n 228 \n  229 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nTable 3. Genetic characteristics of the three K. pneumoniae clinical isolates. Abbreviation: AMR, antimicrobial resistance. Each hit of plasmid replicons covered 230 \nthe full length of its reference sequence in the PlasmidFinder database. 231 \nIsolate Sequence Category Length (bp) Plasmid type Nucleotide identity to template Plasmid mobility AMR gene \nKpMVS1 KpMVS1 Chromosome 5,404,514    blaSHV-36, fosA10 \n pKpMVS1_1 Plasmid 111,397 IncFII/repB(R1701) IncFII(pKP91): 100%; repB(R1701): 99.52% Conjugative blaKPC-2 \n pKpMVS1_2 Plasmid 41,868 IncFII(pMET) IncFII(pMET): 98.09% Non-mobilisable  \n pKpMVS1_3 Plasmid 4,809 Col(pHAD28) Col(pHAD28): 92.37% Non-mobilisable  \n pKpMVS1_4 Plasmid 4,439 Col(pHAD28)/Col440II Col(pHAD28): 93.13%; Col440II: 97.52% Mobilisable  \n pKpMVS1_5 Plasmid 3,258 Unknown Not detected Non-mobilisable  \n pKpMVS1_6 Plasmid 1,917 Col(pHAD28) Col(pHAD28): 100% Mobilisable  \nKpMVR1 KpMVR1 Chromosome 5,292,801    blaSHV-36, fosA10 \n pKpMVR1_1 Plasmid 111,174 IncFII/repB(R1701) IncFII(pKP91): 100%, repB(R1701): 99.52% Conjugative blaKPC-157 \n pKpMVR1_2 Plasmid 41,868 IncFII(pMET) IncFII(pMET): 98.09% Non-mobilisable  \n pKpMVR1_3 Plasmid 4,809 Col(pHAD28) Col(pHAD28): 92.37% Non-mobilisable  \n pKpMVR1_4 Plasmid 4,439 Col(pHAD28)/Col440II Col(pHAD28): 93.13%; Col440II: 97.52% Mobilisable  \n pKpMVR1_5 Plasmid 3,258 Unknown Not detected Non-mobilisable  \n pKpMVR1_6 Plasmid 1,917 Col(pHAD28) Col (pHAD28): 100% Mobilisable  \nKpMVS2 KpMVS2 Chromosome 5,354,507    blaSHV-36, fosA10 \n pKpMVS2_1 Plasmid 116,795 IncFII/IncR IncFII(pKP91): 100%; IncR: 99.6% Conjugative blaKPC-2 \n pKpMVS2_2 Plasmid 41,868 IncFII(pMET) IncFII(pMET): 98.09% Non-mobilisable  \n pKpMVS2_3 Plasmid 4,808 Col(pHAD28) Col (pHAD28): 92.37%* Non-mobilisable  \n pKpMVS2_4 Plasmid 4,187 Col(pHAD28) Col (pHAD28): 92.37%* Non-mobilisable  \n pKpMVS2_5 Plasmid 3,258 Unknown Not detected Non-mobilisable  \n pKpMVS2_6 Plasmid 1,917 Col(pHAD28) Col (pHAD28): 100% Mobilisable  \n pKpMVS2_7 Plasmid 240,297 IncFII(pKP91)/IncFIB(K) IncFII(pKP91): 84.98%; IncFIB(K): 98.93% Conjugative  \n* Two hits of the Col(pHAD28) template sequence in the PlasmidFinder database differed between pKpMVS2_3 and pKpMVS2_4 by seven nucleotide 232 \nsubstitutions (95% nucleotide identity) despite their same percent identity to the template.  233 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nTable 4. Chromosomal genetic variation in isolate KpMVR1 identified via comparison against its progenitor KpMVS1. Coordinates refer to locations in the 234 \nreference sequence of the KpMVS1 chromosome. Variants shared by both KpMVR1 and KpMVS2 against their common reference sequenc e of KpMVS1 are 235 \nindicated by asterisks following the coordinates. The “^” sign indicates an insertion between two consecutive bases in the re ference sequence. Abbreviations: 236 \nCRISPR, clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated genes; Del, deletion; Ins, insertion; fs, frameshift. 237 \nLocation Locus Product Variant type DNA change Protein change \n455179 rrl 23S rRNA Substitution G>T  \n1050491 – 1070213 Multiple Type I-E CRISPR-Cas system, etc. (Figure S3, Table S3) Deletion Deletion of 19,723 bp Loss of production \n1113441 – 1113446 flhA Formate hydrogenlyase transcriptional activator Deletion Deletion of 6 bp L367Del, T368Del \n1218110^1218111 * rrl 23S rRNA Insertion Insertion of base G  \n1578603 gyrA DNA topoisomerase (ATP-hydrolyzing) subunit A Substitution 248C>A S83Y \n1588108^1588109 ompK36 Outer membrane porin OmpK36 Insertion Insertion of ISEc68 Amino acid substitutions \n1724677^1724678 * xylB Xylulose kinase Insertion Insertion of base G I236fs \n1792900 rfbD UDP-galactopyranose mutase Substitution 578T>A M193K \n1819375^1819376 Intergenic  Insertion Insertion of base C  \n2183835^2183836 * Intergenic  Insertion Insertion of base T  \n2183837 * Intergenic  Substitution A>T  \n2201799–2256547 Multiple Multiple products including transporters (Figure 1, Table S2) Deletion Deletion of 54,749 bp Loss of production \n2759671–2759685 marR Multiple-AMR (Mar) transcriptional repressor MarR Deletion Deletion of bases 263–277 P88–D92Del, K93Q \n3157221^3157222 * Intergenic  Insertion Insertion of base C  \n3189754 – 3223285 * Multiple Multiple products (Figure S4, Table S4) Deletion Deletion of 33,532 bp Loss of production \n3274833 phoQ Two-component system sensor histidine kinase Substitution C>T T156I \n3580334 – 3585227 * Multiple IS3H composite transposon (Figure S5, Table S5) Deletion Deletion of 4,894 bp Loss of production \n4104884 acrB Multidrug efflux RND transporter permease subunit AcrB Substitution T>G L667R \n4262704^ 4262705 ecpR Regulator protein EcpR Insertion Insertion (2 bp) I115fs \n4723928 rrl 23S ribosomal RNA Deletion Deletion of base C  \n5349457 rrl 23S ribosomal RNA Deletion Deletion of base C  \n 238 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nGenetic characteristics of isolates 239 \nAll three isolates belonged to  K. pneumoniae clone ST8134, a novel single-locus variant of ST240 , 240 \nand were predicted to share the O1αβ,2β O-antigen type and K62 capsular polysaccharide  type. 241 \nSequence lengths, plasmid replicons, and AMR genes determined in hybrid genome assemblies  are 242 \nsummarised in Table 3. KpMVR1 and KpMVS1 shared the same plasmid types IncFII/repB(R1701), 243 \nIncFII(pMET), Col(pHAD28), and Col(pHAD28)/Col440II , whereas KpMVS2 possessed unique 244 \nplasmid types IncFII/IncR and IncFII(pKP91)/FIB(K). 245 \nKpMVS1 carried blaKPC-2 on the 111.4 kbp IncFII(pKP91)/repB(R1701) plasmid pKpMVS1_1. A 246 \nplasmid of the same type was identified in KpMVR1 (pKpMVR1_1, 111.2 kbp) and carried blaKPC-157, 247 \nwhich differed from blaKPC-2 by a single missense mutation (392A>G) resulting in an N131S amino 248 \nacid substitution within the enzyme’s active site [58], where N131 bounds to relebactam, avibactam, 249 \nand vaborbactam  through a hydrogen bond [59–61]. Notably, pKpMVR1_1 differed from 250 \npKpMVS1_1 by 285 nucleotide substitutions, 14 deletions,  and three insertions. These variants were 251 \nconcentrated in two  genomic regions involved in  plasmid transfer and maintenance  (Figure S 1), 252 \nsuggesting recombination between plasmids. Another plasmid type, IncFII(pKP91)/IncR, in KpMVS2 253 \nharboured blaKPC-2. All these KPC-encoding plasmids were predicted to be conjugative (relaxase type: 254 \nMOBF; mating pair formation type: MPF_F), and each carried a variant of the Tn 4401a transposon 255 \nharbouring blaKPC-2 or blaKPC-157, with 1–2 SNPs between each pair of transposons (Table S 1, Figure 256 \nS1). The comparison between fold-coverages of contigs suggested that each of these three isolates 257 \ncarried a single copy of the KPC-encoding plasmid. Other AMR genes detected in these isolates were 258 \nchromosomal β -lactamase gene blaSHV (variant blaSHV-36) and fosfomycin resistance gene fosA10, 259 \nwhich are both intrinsic to K. pneumoniae [62–64]. 260 \nThe chromosome of KpMVR1 differed from that of KpMVS1 by six single -nucleotide 261 \npolymorphisms (SNPs), seven insertions, and eight deletions (including four large deletions illustrated 262 \nin Figures 1 and  S2–5). Seven of these genetic variants were also identified in KpMVS2  (Table 4), 263 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nwhich differed from KpMVS1 by 129  SNPs, 13 insertions, and seven deletions. Two large deletions 264 \n(19.7 kbp and 4.9 kbp) in KpMVR1 could be attributed to an IS-mediated deletion that had previously 265 \nbeen observed in Escherichia coli (Figures S3 and S5) [65]. Notably, KpMVR1 exhibited deletion of 266 \na 54.7-kbp region that comprised operons producing an AcrAB-like multidrug efflux pump  and an 267 \nadditional ABC-type Fe3+-siderophore transport system in both KpMVS1 and KpMVS2 (Figure 1 and 268 \nTable S2). Each of the three isolates carried a single copy of the acrRAB operon and tolC gene, which 269 \ncombine to produce the AcrAB -TolC multidrug efflux pump . However, the permease AcrB in 270 \nKpMVR1 differed from that in KpMVS1 and KpMVS2 by a destabilising mutation L667R outside the 271 \nprotein’s transmembrane domains. 272 \nAs for the biosynthesis of siderophores and transport of the iron -siderophore complex, which 273 \nfacilitate cefiderocol to penetrate the OM [66], KpMVS1, KpMVR1, and KpMVS2 were predicted to 274 \npossess complete enterobactin production and iron -enterobactin transport systems, while no ne of the 275 \nyersiniabactin, colibactin, aerobactin, or salmochelin loci were detected, corresponding to a Kleborate 276 \nvirulence score of zero. All three isolates shared the same 19 -kbp chromosomal region harbouring a 277 \ncluster of enterobactin-synthesising genes entA–F and entH, enterobactin-exporter gene entS, and iron-278 \nenterobactin transporter genes fepA–D and fepG. 279 \nCompared with the ciprofloxacin-susceptible isolates KpMVS1 and KpMVS2, KpMVR1 280 \nharboured a nucleotide substitution 248C>A in  the DNA gyrase gene gyrA, resulting in the GyrA 281 \nmutation S83Y, which is known to reduce ciprofloxacin susceptibility [67]. The three isolates also 282 \ncarried a single copy of the marRAB operon. However, KpMVR1 exhibited a unique 15 -bp in-frame 283 \ndeletion in the non-essential transcriptional repressor gene marR within the marRAB operon, causing 284 \na loss of five amino acids and an amino acid substitution  within the DNA-binding region of MarR 285 \n(Table 4) [68]. 286 \nSeven bases at the 5’ end of ompK36 in KpMVR1 were truncated by an additional copy of insertion 287 \nsequence IS Ec68 (three copies in KpMVS1 and KpMVS2, respectively) , resulting in a frameshift 288 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nmutation that replaced the first three amino acids at the N-terminal of OmpK36 with 12 amino acids  289 \n(Figure 2). The native 21 N-terminal amino acids of OmpK36 encode a Sec-dependent signal sequence 290 \n(UniProtKB accession: A0A0H3H0Y2) that is required for translocating this protein to the inner 291 \nmembrane of K. pneumoniae  (Figure 2B) [69]. The signal sequence is subsequently cleaved and 292 \nOmpK36 is then folded and inserted into the OM (where the protein is functionally active as a porin ) 293 \nin a Bam-complex dependent fashion, a process facilitated by a C-terminal recognition sequence [70]. 294 \nWhilst ompK36 from KpMVS1 and KpMVS2 is predicted to encode a complete sec-dependent signal 295 \nsequence, the 12 amino acid s insertion combined with the  deletion of three amino acids in OmpK36 296 \nfrom KpMVR1 is predicted to hinder this protein’s translocation to the OM according to the disrupted 297 \nsignal sequence (Figure S6). These predictions were confirmed by polyacrylamide gel electrophoresis 298 \nof OM preparations, followed by Coomassie staining, that a band corresponding to OmpK36 was 299 \npresent in KpMVS1 but absent in KpMVR1  (Figure 2C). Therefore, the disruption of the Sec -300 \ndependent signal sequence of OmpK36 is functionally equivalent to deletion of ompK36. 301 \nRegarding the plasmid-encoded TraN proteins, TraN pKpMVR1_1 and TraNpKpMVS2_1 were identical 302 \n(NCBI protein accession: WP_049192820.1) and differed from TraNpKpMVS1_1 (WP_436914186.1) by 303 \nsix amino acid substitutions (Table S6). Phylogenetic analysis revealed that these proteins belonged to 304 \nthe specialist TraNβ group (Figure S7), which has a narrow host range [50, 71]. Pairwise structural 305 \ncomparison between TraNpKpMVR1_1 (TraNpKpMVS2_1), TraNpKpMVS1_1, and the prototype TraNβ protein 306 \nTraNpKpQIL showed high consistency ( Figures S8), and no amino acid substitution occurred in the 307 \ncharacteristic distal β-hairpin (Figure S9), suggesting  that the variation in TraN sequences across 308 \nplasmids pKpMVR1_1, pKpMVS1_1, pKpMVS2_1, and pKpQIL is unlikely to affect the conjugation 309 \nspecificity [72]. 310 \nImpact of genetic alterations on antimicrobial resistance 311 \nThe substitution of blaKPC-2 with blaKPC-157 in KpMVS1 (KpMVS1KPC-157) and the transconjugant 312 \nICC8001KPC-2 (ICC8001KPC-157) did not affect the susceptibility to  meropenem, meropenem-313 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nvaborbactam, or imipenem-avibactam but led to a fourfold reduction in the imipenem MIC and a 16- 314 \nto 32-fold increase in imipenem-relebactam MIC (Table 2). Moreover, this allelic substitution resulted 315 \nin a 256- to 512-fold reduction in the aztreonam MIC , a 32 - to 64-fold reduction in the ceftazidime 316 \nMIC, and a ≥4-fold reduction in the cefiderocol MIC , but had no effect on the MICs of aztreonam-317 \navibactam or ceftazidime -avibactam. Notably, imipenem and imipenem -relebactam MICs of each 318 \nKPC-157-producing isolate were identical (Table 2). These findings suggest that KPC-157 has a 319 \nweaker capacity to hydrolyse imipenem, aztreonam, ceftazidime, and cefiderocol than KPC -2, and 320 \nthat—unlike vaborbactam and avibactam, which inhibit both KPC variants—relebactam inhibits KPC-321 \n2 but not KPC-157, which is consistent with a previous report [59]. 322 \nKnocking out ompK36 from the ICC8001 chromosome (ICC8001KPC-2/ΔompK36 and ICC8001 KPC-323 \n157/ΔompK36) led to a 16-fold increase in the MICs of both meropenem and imipenem, a >33-fold increase 324 \nin the meropenem-vaborbactam MIC, an 8- to 32-fold increase in the imipenem-relebactam MIC, and 325 \na more than twofold increase in the aztreonam MIC (Table 2). These findings are consistent with the 326 \nrole of OmpK36 as an entry route for β-lactams and β-lactamase inhibitors to penetrate the OM [73]. 327 \nNevertheless, when comparing MICs of ceftazidime, ceftazidime-avibactam, and cefiderocol  before 328 \nand after knocking out ompK36 from ICC8001KPC-2 and ICC8001 KPC-157, only two  pairs of MICs 329 \nexhibited notable increases (from 0.125 mg/L to 0.5 mg/L for ceftazidime-avibactam, and from ≤0.06 330 \nmg/L to 0.25 mg/L for cefiderocol) , while the others showed no appreciable changes, suggesting 331 \nalternative routes of avibactam’s entry. More generally, the comparison between β-lactam MICs with 332 \nand without β-lactamase inhibitors for isolates KpMVR1, ICC8001 KPC-2/ΔompK36, and ICC8001KPC-333 \n157/ΔompK36 in Table 2 indicates that these inhibitors penetrated the OM via routes other than OmpK36, 334 \neffectively inhibiting β-lactamases. 335 \nThe chromosomes of KpMVR1, KpMVS1, and ICC8001 derivatives harboured the same  cluster 336 \nof ent and fep genes within a 19 -kbp region  encoding an ABC-type Fe 3+-siderophore transporter  337 \nassociated with the cefiderocol susceptibility [66]. These isolates did not exhibit any notable difference 338 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nin cefiderocol MICs despite KpMVR1’s loss of the 54.7-kbp chromosomal region harbouring fep-like 339 \ngenes (Table S2), suggesting alternative entry routes of cefiderocol. 340 \nDiscussion 341 \nIn the UK, m eropenem-vaborbactam and imipenem-relebactam are recommended for treating adult 342 \npatients (≥18 years of age) with severe multidrug-resistant infections where therapeutic options are 343 \nlimited, and ceftazidime-avibactam is recommended as an alternative when the disease -causing 344 \nbacterium produces class D carbapenemase ( e.g., OXA -48) [74–76]. Prevalence of resistance in 345 \nEnterobacterales to any of these three combination antimicrobials was 1–5% across the globe  as of 346 \n2022 despite regional variation  [17, 77 –81]. Therefore, the discovery of  K. pneumoniae  isolate 347 \nKpMVR1, which  exhibited unusual resistance to meropenem-vaborbactam, imipenem-relebactam, 348 \nand ceftazidime-avibactam, in a seriously ill patient is particularly worrisome . The small number of 349 \nchromosomal SNPs (n=6) and indels (n=10; ≤15 bp each) identified in KpMVR1 when compared with 350 \nKpMVS1, Patient 1’s exposure to meropenem, meropenem -vaborbactam, and fluoroquinolones, and 351 \nthe unique antibiogram of KpMVR1 in the ICU altogether support the suspected in vivo emergence of 352 \nmeropenem-vaborbactam, ceftazidime-avibactam, imipenem-relebactam, and ciprofloxacin resistance 353 \nin the same K. pneumoniae strain during this patient’s hospital stay. A similar shift in the ceftazidime-354 \navibactam susceptibility profile of K. pneumoniae  has been reported during treatment using 355 \nmeropenem followed by ceftazidime-avibactam [82]. 356 \nKPC-2 is known to confer carbapenem resistance in Gram-negative bacteria but can be effectively 357 \ninhibited by vaborbactam, avibactam, and relebactam [83]. Here, we have experimentally determined 358 \nthe effect of carbapenemase KPC-157 on the susceptibility  to carbapenems and cephalosporins with 359 \nor without β-lactamase inhibitors. Our results indicate that KPC-157 does not differ from KPC-2 in its 360 \ninteraction with meropenem or meropenem-vaborbactam. Therefore, the presence of blaKPC-157 in the 361 \nsingle-copy plasmid pKpMVR1_1 alone cannot explain the high -level meropenem-vaborbactam 362 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nresistance observed in KpMVR1. Notably, KPC-157 appears less capable of hydrolysing imipenem, 363 \naztreonam, ceftazidime, and cefiderocol than KPC-2, and is inhibited by vaborbactam and avibactam 364 \nbut not by relebactam. 365 \nIsolate KpMVR1 showed genetic changes that may alter the antimicrobial permeability of its OM 366 \nwhen compared with isolate KpMVS1.  Resulting from an IS -induced sequence disruption, the  367 \nhindered translocation of OmpK36 to the OM is predicted to hamper the influx of β-lactams and β-368 \nlactamase inhibitors  into the periplasm , hence elevated MIC s of carbapenems and cephalosporins 369 \ntested in Table 2 with and without β-lactamase inhibitors. Such hampered antimicrobial and inhibitor 370 \ninflux might be further compromised by a decreased expression of ompK35 in KpMVR1 as a result of 371 \nthe in-frame, presumptively inactivating deletion within the repressor gene marR and the consequent 372 \nupregulation of the marA gene [84]. Moreover, the inactivation of marR is known to i ncrease the 373 \nproduction of the AcrAB -TolC efflux pump , conferring low -level cross-resistance to antimicrobials 374 \nincluding β-lactams and ciprofloxacin [85]. However, this upregulation of AcrAB-TolC in KpMVR1 375 \nmight not alter its antimicrobial susceptibility owing to the possibly destabilised AcrB. Therefore, the 376 \nhigh-level ciprofloxacin resistance in KpMVR1 could be primarily driven by the combination of the 377 \ngyrA mutation S83Y and the absence of OmpK36 in this isolate’s OM [67, 86]. 378 \nThis study is limited to three K. pneumoniae isolates belonging to the same clone, with only one 379 \nisolate (KpMVR1) exhibiting elevated MICs of meropenem -vaborbactam, imipenem -relebactam, 380 \naztreonam-avibactam, and ceftazidime -avibactam. KpMVR1 harboured multiple AMR -associated 381 \ngenetic alterations. Broader surveillance of genetic variants similar to those identified in KpMVR1 is 382 \nneeded to assess the prevalence and clinical relevance of these  putative resistance mechanisms. 383 \nAlthough we experimentally validated the individual contributions of blaKPC-157 and ΔompK36 to 384 \nantimicrobial susceptibility, the genetically reconstructed isolates could not fully replicate the same 385 \nlevel of MIC increments as KpMVR1, suggesting that other genetic or regulatory mechanisms may be 386 \ninvolved, which remain to be elucidated. Transcriptomic and proteomic profiling could be performed 387 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nin the future to determine whether regulatory mechanisms also contribute to the observed AMR in 388 \nKpMVR1. 389 \nAt the nation level, a review of routine surveillance and reference laboratory samples from 2016 -390 \n2020 revealed only low levels of resistance to ceftazidime -avibactam in the UK  [81]. However, it 391 \nremains essential that emerging resistance to ceftazidime -avibactam and novel β-lactam/β-lactamase 392 \ninhibitor combinations is promptly identified and reported through UKHSA’s Second Generation 393 \nSurveillance System and referral of such isolates to the AMRHAI Reference Unit . Additionally, our 394 \nstudy highlights the importance of monitoring the evolving antimicrobial susceptibility profiles of 395 \nbacterial pathogens within patients during antimicrobial therapy. 396 \nFunding information 397 \nThis work was mainly funded by the UKHSA. YW is a research fellow  funded by the David Price 398 \nEvans Endowment (grant number: UGG10057) at the University of Liverpool and was an Imperial 399 \nInstitutional Strategic Support Fund Springboard Research Fellow, funded by the Wellcome Trust and 400 \nImperial College London (grant number: PSN109) . YW, EJ, DM, and KLH are affiliated with the 401 \nNational Institute for Health and Care Research Health Protection Research Unit in Healthcare 402 \nAssociated Infections and Antimicrobial Resistance at Imperial College London in partnership with 403 \nthe UKHSA, in collaboration with, Imperial Healthcare Partners, University of Cambridge and 404 \nUniversity of Warwick (grant number: NIHR200876). The views expressed in this article are those of 405 \nthe authors and not necessarily those of the NHS, the National Institute for Health Research, or the 406 \nDepartment of Health and Social Care.  407 \nAcknowledgments 408 \nWe acknowledge the Colebrook Laboratory, a facility supported by the NIHR Imperial Biomedical 409 \nResearch Centre (BRC) , for providing bioinformatics resources. Part of the bioinformatics analysis 410 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nwas performed on equipment purchased as part of MRC CARP fellowship award MR/T005254/1. We 411 \nalso thank the Institut Pasteur teams for the curation and maintenance of BIGSdb-Pasteur databases at 412 \nhttp://bigsdb.pasteur.fr. 413 \nAuthor contributions 414 \nConceptualisation: KLH and YW; Resources: KLH, JT, GF, FM, NW, and GMR; Methodology: KLH, 415 \nYW, JLCW, and EJ; Data curation: YW; Investigation and formal analysis: YW, JLCW, JSG, WWL, 416 \nJT, FM, GMR, KD,  IB, GF, EJ, DM, and KLH; Visualisation: YW; Writing – original draft: YW, 417 \nJLCW, JSG, WWL, GMR, and KLH; Writing – review and editing: all authors. 418 \nConflicts of interest 419 \nAuthors declare that there are no conflicts of interest. 420 \nConsent to publish 421 \nNo sensitive information is disclosed in this manuscript. 422 \nEthical statement 423 \nNational surveillance of communicable diseases and outbreak investigation work at UKHSA does not 424 \nrequire individual patient consent as per  Regulation 3 of The Health Service (Control of Patient 425 \nInformation) Regulations 2002. 426 \nReferences 427 \n1. 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The multiple antibiotic resistance (mar) locus and its significance. 649 \nResearch in Veterinary Science 2002;72:87–93. 650 \n86. Martí nez-Martí nez L, Herná ndez-Allé s S, Albertí  S, Tomá s J M, Benedi V J, et al. In vivo 651 \nselection of porin -deficient mutants of Klebsiella pneumoniae  with increased resistance to 652 \ncefoxitin and expanded -spectrum-cephalosporins. Antimicrobial Agents and Chemotherapy  653 \n1996;40:342–348. 654 \n655 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nFigures 656 \nFigure 1. Genetic structure of a 54.7-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels “start” and “end” indicate boundaries of the deleted 657 \nregion. Genes without known names are not labelled. Each asterisk indicates an allele from a named gene family. 658 \n  659 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \n \nFigure 2. ISEc68-mediated disruption of ompK36 in isolate KpMVR1. (A) Genetic environment of the disrupted ompK36 in KpMVR1. The arrow labelled 660 \n“ompK36*” denotes the upstream-shifted open reading frame caused by the insertion of IS Ec68. Abbreviations: CDS: coding sequence; IS, insertion sequence; 661 \nncRNA: non-coding RNA. ( B) Comparison of predicted OmpK36 sequences using Clustal Omega ( www.ebi.ac.uk/jdispatcher/msa/clustalo). Mismatches are 662 \nhighlighted in red, and the 22 N-terminal amino acids signal sequence of OmpK36 are indicated by the yellow shade. (C) Coomassie-stained polyacrylamide gel 663 \nelectrophoresis of outer membrane proteins to confirm the absence of OmpK36 in KpMVR1 and the ompK36-knockout isolate ICC8001ΔompK36. 664 \n 665 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \nSupplementary methods \nGenomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to \nmeropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam \nYu Wan, Joshua L. C. Wong, Julia Sanchez-Garrido, Wen Wen Low, Jane F. Turton, Fabio Morecchiato, \nIlaria Baccani, Kirsty Dodgson, Gian Maria Rossolini, Neil Woodford, Gad Frankel, Elita Jauneikaite, \nDaniè le Meunier, and Katie L. Hopkins \nAugust 2025 \nComparative structural analysis of TraN \nAmino acid sequences of TraNpKpMVS1_1, TraNpKpMVR1_1, and TraNpKpMVS2_1 were extracted from the sequence \nannotations of plasmids pKpMVS1_1  (locus tag: WAS92_RS00545), pKpMVR1_1 (ACNQKT_RS26595), \nand pKpMVS2_1 (ACNQKS_RS28350), respectively. To contextualise these three proteins,  the previously \ndescribed TraN variants [1] TraNpKpQI (NCBI protein accession: ARQ19727.1), TraN MV2 (BAS44060.1), \nTraNR100-1 (ABD60034.1), TraN pSLT (AAL23498.1), TraN F (WP_000821835.1), TraN MV1 (ANZ89826.1), \nTraNMV3 (WP_001398575.1) were downloaded from the NCBI Protein database \n(www.ncbi.nlm.nih.gov/protein). These 10 amino acid sequences were aligned with the ClustalW algorithm  \n[2], and subsequently, a neighbour -joining phylogenetic tree was generated from the multi -sequence \nalignment, with the Poisson correction method as implemented in MEGA11 [3]. The phylogenetic tree was \nvisualised using iTOL v7.2 [4]. \nThree-dimensional structures of TraNpKpMVS1_1, TraNpKpMVR1_1 (identical to TraNpKpMVS2_1), and TraNpKpQI \nwere predicted using AlphaFold 3 [5] with its default parameters on AlphaFold Server (alphafoldserver.com). \nThe top-ranked (model 0) structural models of TraN proteins were visualised using UCSF ChimeraX v1.9 [6]. \nSuperimposition analysis of these models was performed with ChimeraX’s Matchmaker tool using default \nsettings, including the use of the “best -aligning” or “bb” chain -pairing method, the Needleman -Wunsch \nalignment algorithm, and the BLOSUM-62 similarity matrix. \nReferences \n1. Low Wen Wen, Seddon Chloe, Beis Konstantinos, Frankel Gad. The Interaction of the F-Like Plasmid-\nEncoded TraN Isoforms with Their Cognate Outer Membrane Receptors. Journal of Bacteriology  \n2023;205:e00061-23. \n2. Thompson JD, Gibson TobyJ, Higgins DG. Multiple Sequence Alignment Using ClustalW and ClustalX. \nCurrent Protocols in Bioinformatics 2003;00:2.3.1-2.3.22. \n3. Tamura K, Stecher G, Kumar S . MEGA11: Molecular Evolutionary Genetics Analysis Version 11. \nMolecular Biology and Evolution 2021;38:3022–3027. \n4. Letunic I, Bork P . Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and \nannotation. Bioinformatics 2007;23:127–128. \n5. Abramson J, Adler J, Dunger J, Evans R, Green T, et al. Accurate structure prediction of biomolecular \ninteractions with AlphaFold 3. Nature 2024;630:493–500. \n6. Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, et al.  UCSF ChimeraX: Tools for \nstructure building and analysis. Protein Science 2023;32:e4792. \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n \n \nSupplementary figures \nGenomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to \nmeropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam \nYu Wan, Joshua L. C. Wong, Julia Sanchez-Garrido, Wen Wen Low, Jane F. Turton, Fabio Morecchiato, \nIlaria Baccani, Kirsty Dodgson, Gian Maria Rossolini, Neil Woodford, Gad Frankel, Elita Jauneikaite, \nDaniè le Meunier, and Katie L. Hopkins \nAugust 2025 \n \n \nTable of contents \nFigure S1 …………………………………………………………………………………………… 1 \nFigure S2 …………………………………………………………………………………………… 2 \nFigure S3 …………………………………………………………………………………………… 3 \nFigure S4 …………………………………………………………………………………………… 4 \nFigure S5 …………………………………………………………………………………………… 4 \nFigure S6 …………………………………………………………………………………………… 5 \nFigure S7 …………………………………………………………………………………………… 6 \nFigure S8 …………………………………………………………………………………………… 7 \nFigure S9 …………………………………………………………………………………………… 8 \nReference …………………………………………………………………………………………… 9 \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n1 \n \nFigure S1. Alignment of plasmids pKpMVR1_1 and pKpMVS2_1 against plasmid pKpMVS1_1 using \nnucleotide BLAST as implemented in Proksee. Plasmids pKpMVR1_1 and pKpMVS1_1 belonged to replicon \ntype IncFII(pKP91)/repB(R1701), and pKpMVS2_1 belonged to IncFII(pKP91)/IncR. Th e innermost ring \nrepresents the number of genetic variants per kbp (calculated using VCFtools v0.1.17) [1] in pKpMVR2_1 \ncompared with pKpMVS1_1. The two middle rings display genes, insertion sequences, and transposons \nidentified in the reference sequence pKpMVS1_1, with arrows indicating orientations of these genetic features \n(Table S1). Genes without known names are not labelled. The two outer rings show regions of pKpMVR_1 \n(pink) and pKpMVS2_1 (blue) aligned to pKpMVS1, respectively. This figure was created using Proksee \n(proksee.ca). \"Δ\" in gene labels represents a truncated or interrupted feature, and each asterisk represents a \nvariant of an insertion sequence or transposon. Abbreviations: CDS, coding sequence; IS, insertion sequence. \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n2 \n \nFigure S2. A BRIG diagram comparing chromosomes of KpMVR1 and KpMVS2 with that of KpMVS1. \nParameters for BLASTn  sequence alignment: “-task megablast -ungapped -qcov_hsp_perc 0.8”. Four large \ndeletions (>4 kbp) in the chromosome of KpMVR1 when compared to that of KpMVS1 (Table 3) are denoted \nby digits in filled circles.  Genetic structures of these deleted regions  are illustrated in Figure 1 and  \nsupplementary Figures S3–5. \n \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n3 \n \nFigure S3. Genetic structure of a 19.7-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels \n“start” and “ end” indicate boundaries of the deleted region. Genes without known names are not labelled.  \n“IS1X2*” denotes a variant of the IS1-family insertion sequence IS1X2 (98% nucleotide identity and 100% \nquery coverage). “CRISPR” indicates an array of clustered regularly interspaced short palindromic repeats . \nSee Table S3 for detailed annotations of this region. \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n4 \n \nFigure S4. Genetic structure of a 33.5-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels \n“start” and “end” indicate boundaries of the deleted region. Genes without known names are not labelled. See \nTable S4 for detailed annotations of this region. \n \n \nFigure S5. Genetic structure of a 4.9-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels \n“start” and “ end” indicate boundaries of the deleted region. Genes without known names are not labelled. \n“IS3H*” denotes a variant of the IS 3-family insertion sequence IS 3H (79% nucleotide identity and 100% \nquery coverage). There were nine copies of this variant in the KpMVS1 chromosome and eight copies in the \nKpMVR1 chromosome, which is consistent with the content of the 4.9-kbp deletion (Table S5). \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n5 \n \nFigure S 6. Prediction of signal peptides and cleavage site in the wild type OmpK36 (KpMVS1) and its \nframeshifted variant (KpMVR1), respectively, using the slow model mode of SignalP v6.0. “N”, “H”, and “C” \nregions in each protein sequence denote the N-terminal region  (Sec/SPI n) , centre hydrophobic region  \n(Sec/SPI h), and C-terminal region (Sec/SPI c) of the signal peptide, respectively , whereas “O” denotes the \nnon-signal peptide region (OTHER). The cleavage site (CS) is indicated by the red vertical dashed line. The \nprobability of each amino acid to be part of each peptide region is indicated by a coloured s olid curve \nthroughout the protein sequence. \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n6 \n \nFigure S7. Midpoint-rooted neighbour-joining phylogenetic tree of 10 TraN amino acid sequences , where \nTraNpKpMVR1_1 was identical to Tra pKpMVS2_1. The sequences are named after source plasmids. The structural \ngroups (TraNα, TraNβ, TraNγ, and TraNδ) [2] of TraN are indicated by shaded boxes and labelled. The scale \nbar represents the number of amino acid substitutions per residue. \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n7 \n \nFigure S8. Comparison of predicted TraN structures for plasmids pKpQIL, pKpMVS1_1, and pKpMVR1_1. \nAmino acid chains are represented by ribbons. ( A) Predicted 3D structures with residues coloured by scores \nfrom the predicted local distance difference test (pLDDT) . The pLDDT was performed by AlphaFold3 to \nevaluate the per -residue local confidence of the predicted 3D structure. ( B) Pairwise structural comparison \nthrough the super-imposition analysis. Dashed boxes indicate the tip/sensor domains of TraN [2]. \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n8 \n \nFigure S9. Locations of amino acid variation  in the tip/sensor domains of TraN from plasmids pKpQIL, \npKpMVS1_1, and pKpMVR1_1, with amino acid chains represented by ribbons in the superimposition view. \nThe variable sites are highlighted in yellow, blue, and magenta. In comparison with Figure S8B, the proteins \nare arbitrarily rotated around the vertical axis to expose all variable sites. \n \n \n  \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint \n\n9 \n \nReference \n1. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, et al. The variant call format and VCFtools. \nBioinformatics 2011;27:2156–2158. \n2. Frankel G, David S, Low WW, Seddon C, Wong JLC, et al. Plasmids pick a bacterial partner before \ncommitting to conjugation. Nucleic Acids Res 2023;51:8925–8933. \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}