Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam

22 This article reports an unusual Klebsiella pneumoniae clinical isolate, KpMVR1, resistant to 23 meropenem-vaborbactam, imipenem -relebactam, and ceftazidime-avibactam, and investigates the 24 underlying genetic alterations using comparative genomics and molecular experiments. 25 Resistance to carbapenems and third -generation cephalosporins is increasing in K. pneumoniae 26 globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely 27 used to circumvent β-lactamase-mediated resistance. In 2021, isolate KpMVR1 was recovered from a 28 hospitalised patient in England. Two additional isolates with the same variable-number tandem-repeat 29 profile—KpMVS1, collected from the same patient 42 days before KpMVR1, and KpMVS2, from 30 another patient in the same hospital —were susceptible to meropenem-vaborbactam, imipenem -31 relebactam, and ceftazidime-avibactam. Illumina and nanopore whole-genome sequencing and hybrid 32 genome assembly were conducted for these three isolates . Annotated genome assemblies were 33 compared to identify genetic variation, and mutagenesis experiments were performed to verify 34 predicted functional alterations. 35 All isolates belonged to a novel clone ST8134 and carried blaKPC-2-like alleles (KpMVR1: blaKPC-36 157; KpMVS1 and KpMVS2: blaKPC-2) in presumptively conjugative plasmids. ISEc68 caused a 37 frameshift mutation in KpMVR1’s ompK36 gene, reducing the meropenem-vaborbactam and 38 imipenem-relebactam susceptibility. KPC-157 demonstrated decreased hydrolysis of imipenem and 39 ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor 40 MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. 41 In conclusion, KpMVR1 harboured complex resistance-associated genetic alterations, with evidence 42 for in vivo emergence of antimicrobial resistance. Our study underlines routine screening for resistant 43 pathogens in vulnerable patients to guide antimicrobial chemotherapy as well as the need to 44 characterise underlying resistance mechanisms to help assess the potential for onward transmission. 45 .CC-BY 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint

46 Klebsiella pneumoniae, antimicrobial resistance, combination antimicrobials, β-lactam antimicrobials, 47 β-lactamase inhibitors, porins, medical microbiology, bioinformatics , comparative genomics , 48 mutagenesis experiments 49 Data summary 50 Illumina and nanopore sequencing reads, hybrid genome assemblies, and anonymised metadata of 51 isolates KpMVS1, KpMVR1, and KpMVS2 have been deposited in databases of the National Center 52 for Biotechnology Information (www.ncbi.nlm.nih.gov) under BioProject accession PRJNA1084250, 53 with BioSample accessions SAMN46778009 (KpMVS1), SAMN46778010 (KpMVR1), and 54 SAMN46778011 (KpMVS2). The genome assemblies of these isolates have also been deposited in 55 Pasteur Institute’s database for K. pneumoniae species complex ( bigsdb.pasteur.fr/klebsiella/) under 56 ids 75608 (KpMVS1), 75609 (KpMVR1), and 75610 (KpMVS2). 57 Impact statement 58 This is the first blaKPC-positive K. pneumoniae isolate referred to the UK’s national reference 59 laboratory with resistance to three last-resort β-lactam/β-lactamase inhibitor combinations 60 meropenem-vaborbactam, imipenem -relebactam, and ceftazidime-avibactam, implicating in vivo 61 emergence of this unusual resistance profile during prolonged antimicrobial chemotherapy. This 62 isolate belonged to a novel clone ST8134 and harboured a plasmid -borne blaKPC-2-like allele blaKPC-63 157. We identified complex genetic alterations in this isolate: chromosomal large deletions, point 64 mutations, and an ISEc68-induced loss -of-function truncation of the ompK36 porin gene . We 65 determined the impact of KPC-2, KPC-157, and the ompK36 truncation on the susceptibility of K. 66 pneumoniae to meropenem, meropenem -vaborbactam, imipenem, imipenem -relebactam, imipenem-67 avibactam, aztreonam, aztreonam -avibactam, ceftazidime, ceftazidime -avibactam, and cefiderocol. 68 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Our work underscores the need to monitor emerging resistance to beta-lactam/beta-lactamase inhibitor 69 combinations in healthcare and to understand underlying resistance mechanisms for assessing the 70 potential of pathogen transmission. 71 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint

72 Meropenem and imipenem are broad-spectrum carbapenem antimicrobials parenterally administrated 73 to treat serious bacterial infections, such as those caused by Enterobacterales producing extended-74 spectrum β-lactamases (ESBLs) or AmpC-type cephalosporinases (AmpCs) [1–3]. Ceftazidime, a 75 third-generation parenteral cephalosporin, is also widely used for treating severe bacterial infections , 76 although it can be hydrolysed by ESBLs and AmpCs [4, 5]. In Gram-negative bacteria, carbapenems 77 and cephalosporins diffuse through outer-membrane porins and enter the periplasm, where they 78 inactivate penicillin binding proteins, disrupting cell-wall synthesis with a bactericidal effect [6–8]. 79 Combining β-lactams with β-lactamase inhibitors in antimicrobial chemotherapy is a widely 80 employed strategy to circumvent β-lactamase-mediated resistance in bacteria. Vaborbactam, 81 relebactam, and avibactam are non-β-lactam inhibitors of Ambler class A β-lactamases, such as ESBLs 82 and Klebsiella pneumoniae carbapenemases (KPCs), as well as class C β-lactamases (AmpCs) [9]. 83 Moreover, avibactam inhibits several class D β-lactamases such as OXA-48 and OXA-10 [10]. These 84 inhibitors penetrate the outer membrane (OM) of Gram -negative bacteria via porins , blocking the 85 active sites of β-lactamases in the periplasm [9, 11]. 86 In the UK, meropenem -vaborbactam, imipenem -relebactam, and ceftazidime -avibactam are 87 reserved for highly selected patients [12]. Resistance to one or more of these combination 88 antimicrobials in clinical isolates of KPC-producing Klebsiella pneumoniae has been previously 89 reported [13–15]. Underlying resistance mechanisms include overproduction of KPCs or the AcrAB-90 TolC multidrug efflux pump , as well as gain-of-function point mutations in the blaKPC gene [9, 16–91 18]. In addition, t he disruption or transcriptional downregulation of ompK35 (ompF) and ompK36 92 (ompC), which encode non-selective porins that facilitate the diffusion of β-lactams and β-lactamase 93 inhibitors through the OM, has also been implicated [17–20]. 94 Here, we report and characterise a clinically significant K. pneumoniae isolate exhibiting resistance 95 to ceftazidime-avibactam, meropenem-vaborbactam, and imipenem -relebactam, analysed in the 96 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint context of closely related isolates recovered in the same hospital. This was the first such isolate referred 97 to the UK’s national reference laboratory for characterisation. 98

99 Isolate collection and phenotyping 100 The K. pneumoniae isolate KpMVS1 was recovered from a lung biopsy specimen of a n inpatient 101 (hereafter, Patient 1) admitted to an intensive care unit (ICU) in England in 2021 . The second K. 102 pneumoniae isolate, KpMVR1, was recovered from a groin wound of the same patient 42 days later. 103 During this ICU stay, the patient received a broad range of antimicrobials, including meropenem-104 vaborbactam, ciprofloxacin, and gentamicin ; however, ceftazidime-avibactam and imipenem-105 relebactam were not used. 106 Species identification of the isolates and carbapenemase gene screening were performed using 107 matrix-assisted laser desorption/ioni sation-time of flight (MALDI-ToF) method and the GeneXpert 108 system (Cepheid, USA), respectively. Initial antimicrobial susceptibility testing (AST) was conducted 109 by the hospital, with results interpreted according to the European Committee on Antimicrobial 110 Susceptibility Testing (EUCAST) guidelines. Both isolates were referred to the Antimicrobial 111 Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit of the UK Health 112 Security Agency (UKHSA) for variable number tandem repeat (VNTR) typing [21] and investigation 113 of unusual antimicrobial resistance (AMR). Furthermore, a blaKPC-positive K. pneumoniae isolate 114 KpMVS2 — recovered from a rectal swab of another patient (Patient 2) in the same hospital in 2020 115 during an outbreak investigation and sharing the same VNTR profile as KpMVS1 and KpMVR1 — 116 was retrieved from AMRHAI’s culture collection for comparison. These three isolates were subjected 117 to whole-genome sequencing (WGS) and AST for which minimum inhibitory concentrations (MICs) 118 of 19 antimicrobials (Table 1) and diameters of cefiderocol inhibition zones were interpreted as per 119 EUCAST clinical breakpoints v15.0 [22]. 120 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Whole-genome sequencing 121 Genomic DNA of each isolate was extracted from an overnight culture using the GeneJET Kit 122 (ThermoFisher Scientific, UK) as per the manufacturer’s protocol . Short-read sequencing was 123 conducted on a HiSeq 2500 system (Illumina, USA) by UKHSA’s Colindale Sequencing Laboratory 124 following its paired-end 101-bp protocol. Long-read sequencing was performed on MinION R9.4. 1 125 flow cells (Oxford Nanopore Technologies [ONT], UK), with libraries prepared using the ONT Rapid 126 Barcoding Kit SQK-RBK004. 127 Bioinformatics analysis 128 Illumina reads were trimmed and filtered with Trimmomatic v0.39 for a minimum per-read quality of 129 Phred Q30 and minimum length of 50 bp [23]. Fast-mode basecalling and de-multiplexing of nanopore 130 reads was conducted by guppy v4 ( ONT). Nanopore reads were then trimmed and filtered for a 131 minimum per-read quality of Q10 and minimum length of 1 kbp using fastp v0.23.4 [24]. For species 132 confirmation and contamination assessment, taxonomical profiling of processed Illumina and 133 nanopore reads were performed using Kraken v2.1.3, bracken v2.8, and a standard Kraken database 134 built in September 2023 [25, 26]. 135 Genomes of KpMVR1 and KpMVS2, w ith estimated nanopore read depths of 185× and 243× , 136 respectively, were assembled using hybracter v0.5.0 (assemblers: Flye v2.9.3 and plassembler v1.5.0; 137 sequence re -orientator: dnaapler v0.5.1 ; long-read polisher: medaka v 1.8.0, short -read polishers: 138 pypolca v0.2.1 and p olypolish v0.5.0 ) [27–32]. For KpMVS1, which had nanopore reads with an 139 estimated depth of 64× , the chromosome and plasmid sequences were assembled using Raven v1.8.3 140 and plassembler, respectively, and polished with nanopore and Illumina reads as for KpMVR1 and 141 KpMVS2. Evaluation of contamination and completeness of genome assemblies were conducted with 142 CheckM2 v1.0.2 and its database Uniref100/KO [33]. The average fold-coverage of each contig was 143 estimated from Illumina and nanopore reads, respectively, using mosdepth v0.3.9 [34]. 144 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint The genome assemblies were annotated using bakta v1.9.2 and its standard database v5.1 [35]. 145 Multi-locus sequence typing, serotype prediction, and virulence-factor detection were performed using 146 Kleborate v 3.1.3, which incorporated Kaptive v 3.1.0 [36, 37] . AMR genes were detected using 147 AMRFinderPlus v3.12.8 with a minimum query coverage of 80% [38]. Clustered regularly interspaced 148 short palindromic repeats (CRISPR) and CRISPR -associated (Cas) genes in chromosomes were 149 predicted using CRISPRCasFinder [39]. For plasmids, replicon types were determined at a minimum 150 of 80% nucleotide identity and coverage using PlasmidFinder v2.1 [40] and the mobility was predicted 151 using mob_typer of MOB-suite v3.1.8 [41]. The fold-coverage of each KPC-encoding plasmid was 152 divided by that of its host’s chromosome to estimate the plasmid copy number. Transposons and 153 insertion sequences (ISs) were identified using TnCentral Blast (blastn) and ISFinder, respectively [42, 154 43]. 155 Chromosome and plasmids of KpMVR1 and KpMVS2 were compared against those of KpMVS1 156 using minimap v2.26 [44]. Identified g enetic variants were annotated using snpEff v5.2 [45]. Gene 157 Ontology terms were predicted from amino acid sequences using InterProScan v5.69-101.0 [46] with 158 sequence alignments filtered for ≥60% query coverages. Impacts of point mutations on protein stability 159 were predicted from wild -type protein structures in the UniProt database using Missense3D and 160 DDMut [47–49]. The three-dimensional structure of the plasmid-encoded donor OM protein TraN was 161 compared between KPC -encoding, IncFII -carrying plasmids pKpMVS1_1, pKpMVR1_1, and 162 pKpMVS2_1 following the approach developed by Low et al (Supplementary methods) to estimate 163 the impact of TraN alterations on the conjugation specificity and efficiency [50]. Comparison and 164 annotations of these three plasmids were visualised using BRIG v0.95 and Proksee [51, 52]. Gene 165 synteny was illustrated using R package gggenes [53]. Genetic alterations found in both KpMVR1 and 166 KpMVS2 were considered unlikely to confer the unique AMR profiles of KpMVR1 and were therefore 167 excluded from further investigation. 168 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Functional assessment 169 To experimentally determine and compare the impacts of blaKPC-2 and blaKPC-157 on β-lactam 170 susceptibility in K. pneumoniae, KpMVS1’s KPC-2-encoding plasmid pKpMVS1_1, was introduced 171 into the plasmid-free K. pneumoniae laboratory strain ICC8001 (MICs: meropenem, ≤0.06 mg/L; 172 imipenem, 0.25 mg/L; aztreonam, ≤0.125 mg/L; ceftazidime and ceftazidime -avibactam, 0.25 mg/L) 173 through conjugation , resulting in a transconjugant ICC8001 KPC-2 [54]. Transgenic isolates 174 ICC8001KPC-157 and KpMVS1KPC-157 were derived from ICC8001KPC-2 and KpMVS1, respectively, by 175 substituting the blaKPC-2 allele with blaKPC-157. Moreover, isolates ICC8001KPC-2/ΔompK36 and ICC8001KPC-176 157/ΔompK36 were derived from ICC8001 KPC-2 and ICC8001 KPC-157, respectively, through seamless, 177 markerless homologous recombination using mutagenesis vectors and a lambda -red based 178 recombination system generated in previous work [54]. 179 To predict the presence/absence of OmpK36 in the OM of KpMVR1, Sec-dependent signal 180 peptides and their cleavage site in translated ompK36 alleles were compared between KpMVS1 and 181 KpMVR1 using SignalP v6.0 [55]. To validate the prediction, p urification of OM proteins was 182 performed by resuspending an overnight LB-Miller culture (VWR, USA) of each isolate in 1M HEPES 183 (pH 7.4) and sonicating at 25% amplitude for 10 bursts of 10 seconds on, 15 seconds off each (Model 184 705 Sonic Dismembrator, Fisher Scientific). Isolates ICC8001 and its ompK36-knockout derivative, 185 ICC8001ΔompK36, were included as positive and negative controls, respectively . After separating 186 cellular debris by centrifugation, OM proteins were obtained by centrifugation at 14,000×g for 30 mins 187 and resuspended in 2% sarcosine/HEPES for 30 mins at room temperature. All steps were performed 188 at 4° C on ice to preserve protein integrity unless otherwise indicated. For visualisation, 10 μg protein 189 per isolate was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) 190 using 12% acrylamide gels and was stained with Coomassie solution (Sigma -Aldrich, USA ) and 191 imaged on a ChemiDoc XRS+ (Bio-Rad, USA). 192 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Both progenitor isolates (KpMVS1 and KpMVR1) and the five transgenic isolates (KpMVS1KPC-193 157, ICC8001KPC-2, ICC8001KPC-157, ICC8001KPC-2/ΔompK36, and ICC8001KPC-157/ΔompK36) were tested for 194 susceptibility to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-195 avibactam, ceftazidime, ceftazidime-avibactam, aztreonam, aztreonam-avibactam, and cefiderocol (in 196 iron-depleted medium) by the reference broth microdilution method as per the EUCAST guidance [56, 197 57]. Any MIC change above a two-fold difference between two isolates was considered notable. 198

199 Phenotypes of isolates 200 Isolates KpMVS1 and KpMVR1, obtained 42 days apart from Patient 1 with recurrent K. pneumoniae 201 infections, and KpMVS2 , obtained from Patient 2 in the same hospital , were identified as K. 202 pneumoniae by both MALDI -ToF and WGS . In t he ICU where KpMVS1 and KpMVR1 were 203 recovered, all patients were screened for carriage of carbapenemase-producing Enterobacterales (CPE) 204 on admission using PCR, and the resistance profile of KpMVR1 was unique among all identified CPE 205 isolates from the ICU during Patient 1’s stay. 206 Based on AST results from the AMRHAI Reference Unit (Table 1), KpMVR1 was resistant to 207 meropenem-vaborbactam (MIC>256 mg/L) , ceftazidime-avibactam (MIC=16 mg/L) , and 208 ciprofloxacin (MIC>4 mg/L), whereas KpMVS1 and KpMVS2 were susceptible to these antimicrobial 209 agents (MICs: meropenem-vaborbactam ≤0.064 mg/L; ceftazidime-avibactam 1 mg/L; ciprofloxacin 210 ≤0.125 mg/L). Notably, KpMVS2 was resistant to cefiderocol. Further AST discovered that the 211 imipenem-relebactam MIC of KpMVR1 (512 mg/L, resistant) was 2048 times that of KpMVS1 (0.25 212 mg/L, susceptible) (Table 2). Moreover, KpMVR1 exhibited a >4-fold increase in the temocillin MIC 213 (>128 mg/L) and a >8-fold reduction in the cefepime MIC (4 mg/L, susceptible, increased exposure) 214 compared with KpMVS1 (temocillin: 32 mg/L; cefepime: >32 mg/L, resistant). 215 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Table 1. Antimicrobial minimum inhibitory concentrations (MICs; mg/L) determined by UKHSA’s AMRHAI Reference Unit and susceptibility i nterpretations 216 (as per EUCAST clinical breakpoints v15.0) of three K. pneumoniae clinical isolates. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; 217 ETP, ertapenem; CET, ceftolozane; TZB, tazobactam; CFD, cefiderocol; FEP, cefepime; CAZ, ceftazidime; AVI, avibactam; CTX, cefotaxime; FOX, cefoxitin; 218 TMC, temocillin; AMP, ampicillin; AMX, amoxicillin; CAV, clavulanate; PIP, piperacillin; ATM, aztreonam; AMK, amikacin; GEN, gentamicin; CST, colistin; 219 CIP, ciprofloxacin; MB, monobactam. Susceptibility interpretations: R, resistant; I, susceptible, increased exposure; S, susceptible. 220 Carbapenem Cephalosporin Penicillin MB Aminoglycoside Others Isolate MEM- VAB MEM IPM ETP CET- TZB CFD* FEP CAZ CAZ- AVI CTX FOX‡ TMC‡ AMP AMX -CAV PIP- TZB ATM AMK‡ GEN‡ CST‡ CIP KpMVR1 >256 (R) >16 (R) >128 (R) >4 (R) 16 (R) (S) 4 (I) 256 (R) 16 (R) 8 (R) >64 >128 >32 (R) >32 (R) >64 (R) 16 (R) 2 0.5 ≤0.5 >4 (R) KpMVS1 0.064 (S) >16 (R) 64 (R) >4 (R) >16 (R) (S) >32 (R) 128 (R) 1 (S) 64 (R) >64 32 >32 (R) >32 (R) >64 (R) >32 (R) ≤1 ≤0.25 ≤0.5 ≤0.125 (S) KpMVS2 0.032 (S) 16 (R) 16 (R) >4 (R) >16 (R) (R) >32 (R) 64 (R) 1 (S) 16 (R) 32 8 >32 (R) >32 (R) >64 (R) >32 (R) ≤1 ≤0.25 1 ≤0.125 (S) * CFD susceptibility was determined using disc diffusion. ‡ Interpretations were not available according to EUCAST guidelines. 221 222 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Table 2. Minimum inhibitory concentrations of beta-lactam antimicrobials and susceptibility interpretations (as per EUCAST clinical breakpoints v15.0, where 223 applicable) of progenitor and transgenic K. pneumoniae isolates determined in the experiments for functional assessment. Subscripts in isolate names indicate 224 transgenic isolates and corresponding genotypes. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; REL, relebactam; ATM, aztreonam; 225 AVI, avibactam; CAZ, ceftazidime; CFD, cefiderocol, tested in iron-depleted Mueller Hinton broth; NT, not tested. Interpretations of antimicrobial susceptibility: 226 R, resistant; I, susceptible upon increased antimicrobial exposure; S, susceptible. Notations: ompK36fs, frameshifted ompK36; ΔompK36, deletion of ompK36. 227 Isolate Genotype Minimum Inhibitory Concentration (mg/L) and interpretation MEM MEM-VAB IPM IPM-REL IPM-AVI ATM ATM-AVI CAZ CAZ-AVI CFD KpMVR1 blaKPC-157 ompK36fs 512 (R) 256 (R) 512 (R) 512 (R) NT 16 (R) 4 (S) 8 (R) 8 (S) 0.5 (S) KpMVS1 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) KpMVS1KPC-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) ICC8001KPC-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) ICC8001KPC-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) ICC8001KPC-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) ICC8001KPC-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) 228 229 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Table 3. Genetic characteristics of the three K. pneumoniae clinical isolates. Abbreviation: AMR, antimicrobial resistance. Each hit of plasmid replicons covered 230 the full length of its reference sequence in the PlasmidFinder database. 231 Isolate Sequence Category Length (bp) Plasmid type Nucleotide identity to template Plasmid mobility AMR gene KpMVS1 KpMVS1 Chromosome 5,404,514 blaSHV-36, fosA10 pKpMVS1_1 Plasmid 111,397 IncFII/repB(R1701) IncFII(pKP91): 100%; repB(R1701): 99.52% Conjugative blaKPC-2 pKpMVS1_2 Plasmid 41,868 IncFII(pMET) IncFII(pMET): 98.09% Non-mobilisable pKpMVS1_3 Plasmid 4,809 Col(pHAD28) Col(pHAD28): 92.37% Non-mobilisable pKpMVS1_4 Plasmid 4,439 Col(pHAD28)/Col440II Col(pHAD28): 93.13%; Col440II: 97.52% Mobilisable pKpMVS1_5 Plasmid 3,258 Unknown Not detected Non-mobilisable pKpMVS1_6 Plasmid 1,917 Col(pHAD28) Col(pHAD28): 100% Mobilisable KpMVR1 KpMVR1 Chromosome 5,292,801 blaSHV-36, fosA10 pKpMVR1_1 Plasmid 111,174 IncFII/repB(R1701) IncFII(pKP91): 100%, repB(R1701): 99.52% Conjugative blaKPC-157 pKpMVR1_2 Plasmid 41,868 IncFII(pMET) IncFII(pMET): 98.09% Non-mobilisable pKpMVR1_3 Plasmid 4,809 Col(pHAD28) Col(pHAD28): 92.37% Non-mobilisable pKpMVR1_4 Plasmid 4,439 Col(pHAD28)/Col440II Col(pHAD28): 93.13%; Col440II: 97.52% Mobilisable pKpMVR1_5 Plasmid 3,258 Unknown Not detected Non-mobilisable pKpMVR1_6 Plasmid 1,917 Col(pHAD28) Col (pHAD28): 100% Mobilisable KpMVS2 KpMVS2 Chromosome 5,354,507 blaSHV-36, fosA10 pKpMVS2_1 Plasmid 116,795 IncFII/IncR IncFII(pKP91): 100%; IncR: 99.6% Conjugative blaKPC-2 pKpMVS2_2 Plasmid 41,868 IncFII(pMET) IncFII(pMET): 98.09% Non-mobilisable pKpMVS2_3 Plasmid 4,808 Col(pHAD28) Col (pHAD28): 92.37%* Non-mobilisable pKpMVS2_4 Plasmid 4,187 Col(pHAD28) Col (pHAD28): 92.37%* Non-mobilisable pKpMVS2_5 Plasmid 3,258 Unknown Not detected Non-mobilisable pKpMVS2_6 Plasmid 1,917 Col(pHAD28) Col (pHAD28): 100% Mobilisable pKpMVS2_7 Plasmid 240,297 IncFII(pKP91)/IncFIB(K) IncFII(pKP91): 84.98%; IncFIB(K): 98.93% Conjugative * Two hits of the Col(pHAD28) template sequence in the PlasmidFinder database differed between pKpMVS2_3 and pKpMVS2_4 by seven nucleotide 232 substitutions (95% nucleotide identity) despite their same percent identity to the template. 233 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Table 4. Chromosomal genetic variation in isolate KpMVR1 identified via comparison against its progenitor KpMVS1. Coordinates refer to locations in the 234

sequence of the KpMVS1 chromosome. Variants shared by both KpMVR1 and KpMVS2 against their common reference sequenc e of KpMVS1 are 235 indicated by asterisks following the coordinates. The “^” sign indicates an insertion between two consecutive bases in the re ference sequence. Abbreviations: 236 CRISPR, clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated genes; Del, deletion; Ins, insertion; fs, frameshift. 237 Location Locus Product Variant type DNA change Protein change 455179 rrl 23S rRNA Substitution G>T 1050491 – 1070213 Multiple Type I-E CRISPR-Cas system, etc. (Figure S3, Table S3) Deletion Deletion of 19,723 bp Loss of production 1113441 – 1113446 flhA Formate hydrogenlyase transcriptional activator Deletion Deletion of 6 bp L367Del, T368Del 1218110^1218111 * rrl 23S rRNA Insertion Insertion of base G 1578603 gyrA DNA topoisomerase (ATP-hydrolyzing) subunit A Substitution 248C>A S83Y 1588108^1588109 ompK36 Outer membrane porin OmpK36 Insertion Insertion of ISEc68 Amino acid substitutions 1724677^1724678 * xylB Xylulose kinase Insertion Insertion of base G I236fs 1792900 rfbD UDP-galactopyranose mutase Substitution 578T>A M193K 1819375^1819376 Intergenic Insertion Insertion of base C 2183835^2183836 * Intergenic Insertion Insertion of base T 2183837 * Intergenic Substitution A>T 2201799–2256547 Multiple Multiple products including transporters (Figure 1, Table S2) Deletion Deletion of 54,749 bp Loss of production 2759671–2759685 marR Multiple-AMR (Mar) transcriptional repressor MarR Deletion Deletion of bases 263–277 P88–D92Del, K93Q 3157221^3157222 * Intergenic Insertion Insertion of base C 3189754 – 3223285 * Multiple Multiple products (Figure S4, Table S4) Deletion Deletion of 33,532 bp Loss of production 3274833 phoQ Two-component system sensor histidine kinase Substitution C>T T156I 3580334 – 3585227 * Multiple IS3H composite transposon (Figure S5, Table S5) Deletion Deletion of 4,894 bp Loss of production 4104884 acrB Multidrug efflux RND transporter permease subunit AcrB Substitution T>G L667R 4262704^ 4262705 ecpR Regulator protein EcpR Insertion Insertion (2 bp) I115fs 4723928 rrl 23S ribosomal RNA Deletion Deletion of base C 5349457 rrl 23S ribosomal RNA Deletion Deletion of base C 238 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Genetic characteristics of isolates 239 All three isolates belonged to K. pneumoniae clone ST8134, a novel single-locus variant of ST240 , 240 and were predicted to share the O1αβ,2β O-antigen type and K62 capsular polysaccharide type. 241 Sequence lengths, plasmid replicons, and AMR genes determined in hybrid genome assemblies are 242 summarised in Table 3. KpMVR1 and KpMVS1 shared the same plasmid types IncFII/repB(R1701), 243 IncFII(pMET), Col(pHAD28), and Col(pHAD28)/Col440II , whereas KpMVS2 possessed unique 244 plasmid types IncFII/IncR and IncFII(pKP91)/FIB(K). 245 KpMVS1 carried blaKPC-2 on the 111.4 kbp IncFII(pKP91)/repB(R1701) plasmid pKpMVS1_1. A 246 plasmid of the same type was identified in KpMVR1 (pKpMVR1_1, 111.2 kbp) and carried blaKPC-157, 247 which differed from blaKPC-2 by a single missense mutation (392A>G) resulting in an N131S amino 248 acid substitution within the enzyme’s active site [58], where N131 bounds to relebactam, avibactam, 249 and vaborbactam through a hydrogen bond [59–61]. Notably, pKpMVR1_1 differed from 250 pKpMVS1_1 by 285 nucleotide substitutions, 14 deletions, and three insertions. These variants were 251 concentrated in two genomic regions involved in plasmid transfer and maintenance (Figure S 1), 252 suggesting recombination between plasmids. Another plasmid type, IncFII(pKP91)/IncR, in KpMVS2 253 harboured blaKPC-2. All these KPC-encoding plasmids were predicted to be conjugative (relaxase type: 254 MOBF; mating pair formation type: MPF_F), and each carried a variant of the Tn 4401a transposon 255 harbouring blaKPC-2 or blaKPC-157, with 1–2 SNPs between each pair of transposons (Table S 1, Figure 256 S1). The comparison between fold-coverages of contigs suggested that each of these three isolates 257 carried a single copy of the KPC-encoding plasmid. Other AMR genes detected in these isolates were 258 chromosomal β -lactamase gene blaSHV (variant blaSHV-36) and fosfomycin resistance gene fosA10, 259 which are both intrinsic to K. pneumoniae [62–64]. 260 The chromosome of KpMVR1 differed from that of KpMVS1 by six single -nucleotide 261 polymorphisms (SNPs), seven insertions, and eight deletions (including four large deletions illustrated 262 in Figures 1 and S2–5). Seven of these genetic variants were also identified in KpMVS2 (Table 4), 263 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint which differed from KpMVS1 by 129 SNPs, 13 insertions, and seven deletions. Two large deletions 264 (19.7 kbp and 4.9 kbp) in KpMVR1 could be attributed to an IS-mediated deletion that had previously 265 been observed in Escherichia coli (Figures S3 and S5) [65]. Notably, KpMVR1 exhibited deletion of 266 a 54.7-kbp region that comprised operons producing an AcrAB-like multidrug efflux pump and an 267 additional ABC-type Fe3+-siderophore transport system in both KpMVS1 and KpMVS2 (Figure 1 and 268 Table S2). Each of the three isolates carried a single copy of the acrRAB operon and tolC gene, which 269 combine to produce the AcrAB -TolC multidrug efflux pump . However, the permease AcrB in 270 KpMVR1 differed from that in KpMVS1 and KpMVS2 by a destabilising mutation L667R outside the 271 protein’s transmembrane domains. 272 As for the biosynthesis of siderophores and transport of the iron -siderophore complex, which 273 facilitate cefiderocol to penetrate the OM [66], KpMVS1, KpMVR1, and KpMVS2 were predicted to 274 possess complete enterobactin production and iron -enterobactin transport systems, while no ne of the 275 yersiniabactin, colibactin, aerobactin, or salmochelin loci were detected, corresponding to a Kleborate 276 virulence score of zero. All three isolates shared the same 19 -kbp chromosomal region harbouring a 277 cluster of enterobactin-synthesising genes entA–F and entH, enterobactin-exporter gene entS, and iron-278 enterobactin transporter genes fepA–D and fepG. 279 Compared with the ciprofloxacin-susceptible isolates KpMVS1 and KpMVS2, KpMVR1 280 harboured a nucleotide substitution 248C>A in the DNA gyrase gene gyrA, resulting in the GyrA 281 mutation S83Y, which is known to reduce ciprofloxacin susceptibility [67]. The three isolates also 282 carried a single copy of the marRAB operon. However, KpMVR1 exhibited a unique 15 -bp in-frame 283 deletion in the non-essential transcriptional repressor gene marR within the marRAB operon, causing 284 a loss of five amino acids and an amino acid substitution within the DNA-binding region of MarR 285 (Table 4) [68]. 286 Seven bases at the 5’ end of ompK36 in KpMVR1 were truncated by an additional copy of insertion 287 sequence IS Ec68 (three copies in KpMVS1 and KpMVS2, respectively) , resulting in a frameshift 288 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint mutation that replaced the first three amino acids at the N-terminal of OmpK36 with 12 amino acids 289 (Figure 2). The native 21 N-terminal amino acids of OmpK36 encode a Sec-dependent signal sequence 290 (UniProtKB accession: A0A0H3H0Y2) that is required for translocating this protein to the inner 291 membrane of K. pneumoniae (Figure 2B) [69]. The signal sequence is subsequently cleaved and 292 OmpK36 is then folded and inserted into the OM (where the protein is functionally active as a porin ) 293 in a Bam-complex dependent fashion, a process facilitated by a C-terminal recognition sequence [70]. 294 Whilst ompK36 from KpMVS1 and KpMVS2 is predicted to encode a complete sec-dependent signal 295 sequence, the 12 amino acid s insertion combined with the deletion of three amino acids in OmpK36 296 from KpMVR1 is predicted to hinder this protein’s translocation to the OM according to the disrupted 297 signal sequence (Figure S6). These predictions were confirmed by polyacrylamide gel electrophoresis 298 of OM preparations, followed by Coomassie staining, that a band corresponding to OmpK36 was 299 present in KpMVS1 but absent in KpMVR1 (Figure 2C). Therefore, the disruption of the Sec -300 dependent signal sequence of OmpK36 is functionally equivalent to deletion of ompK36. 301 Regarding the plasmid-encoded TraN proteins, TraN pKpMVR1_1 and TraNpKpMVS2_1 were identical 302 (NCBI protein accession: WP_049192820.1) and differed from TraNpKpMVS1_1 (WP_436914186.1) by 303 six amino acid substitutions (Table S6). Phylogenetic analysis revealed that these proteins belonged to 304 the specialist TraNβ group (Figure S7), which has a narrow host range [50, 71]. Pairwise structural 305 comparison between TraNpKpMVR1_1 (TraNpKpMVS2_1), TraNpKpMVS1_1, and the prototype TraNβ protein 306 TraNpKpQIL showed high consistency ( Figures S8), and no amino acid substitution occurred in the 307 characteristic distal β-hairpin (Figure S9), suggesting that the variation in TraN sequences across 308 plasmids pKpMVR1_1, pKpMVS1_1, pKpMVS2_1, and pKpQIL is unlikely to affect the conjugation 309 specificity [72]. 310 Impact of genetic alterations on antimicrobial resistance 311 The substitution of blaKPC-2 with blaKPC-157 in KpMVS1 (KpMVS1KPC-157) and the transconjugant 312 ICC8001KPC-2 (ICC8001KPC-157) did not affect the susceptibility to meropenem, meropenem-313 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint vaborbactam, or imipenem-avibactam but led to a fourfold reduction in the imipenem MIC and a 16- 314 to 32-fold increase in imipenem-relebactam MIC (Table 2). Moreover, this allelic substitution resulted 315 in a 256- to 512-fold reduction in the aztreonam MIC , a 32 - to 64-fold reduction in the ceftazidime 316 MIC, and a ≥4-fold reduction in the cefiderocol MIC , but had no effect on the MICs of aztreonam-317 avibactam or ceftazidime -avibactam. Notably, imipenem and imipenem -relebactam MICs of each 318 KPC-157-producing isolate were identical (Table 2). These findings suggest that KPC-157 has a 319 weaker capacity to hydrolyse imipenem, aztreonam, ceftazidime, and cefiderocol than KPC -2, and 320 that—unlike vaborbactam and avibactam, which inhibit both KPC variants—relebactam inhibits KPC-321 2 but not KPC-157, which is consistent with a previous report [59]. 322 Knocking out ompK36 from the ICC8001 chromosome (ICC8001KPC-2/ΔompK36 and ICC8001 KPC-323 157/ΔompK36) led to a 16-fold increase in the MICs of both meropenem and imipenem, a >33-fold increase 324 in the meropenem-vaborbactam MIC, an 8- to 32-fold increase in the imipenem-relebactam MIC, and 325 a more than twofold increase in the aztreonam MIC (Table 2). These findings are consistent with the 326 role of OmpK36 as an entry route for β-lactams and β-lactamase inhibitors to penetrate the OM [73]. 327 Nevertheless, when comparing MICs of ceftazidime, ceftazidime-avibactam, and cefiderocol before 328 and after knocking out ompK36 from ICC8001KPC-2 and ICC8001 KPC-157, only two pairs of MICs 329 exhibited notable increases (from 0.125 mg/L to 0.5 mg/L for ceftazidime-avibactam, and from ≤0.06 330 mg/L to 0.25 mg/L for cefiderocol) , while the others showed no appreciable changes, suggesting 331 alternative routes of avibactam’s entry. More generally, the comparison between β-lactam MICs with 332 and without β-lactamase inhibitors for isolates KpMVR1, ICC8001 KPC-2/ΔompK36, and ICC8001KPC-333 157/ΔompK36 in Table 2 indicates that these inhibitors penetrated the OM via routes other than OmpK36, 334 effectively inhibiting β-lactamases. 335 The chromosomes of KpMVR1, KpMVS1, and ICC8001 derivatives harboured the same cluster 336 of ent and fep genes within a 19 -kbp region encoding an ABC-type Fe 3+-siderophore transporter 337 associated with the cefiderocol susceptibility [66]. These isolates did not exhibit any notable difference 338 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint in cefiderocol MICs despite KpMVR1’s loss of the 54.7-kbp chromosomal region harbouring fep-like 339 genes (Table S2), suggesting alternative entry routes of cefiderocol. 340

341 In the UK, m eropenem-vaborbactam and imipenem-relebactam are recommended for treating adult 342 patients (≥18 years of age) with severe multidrug-resistant infections where therapeutic options are 343 limited, and ceftazidime-avibactam is recommended as an alternative when the disease -causing 344 bacterium produces class D carbapenemase ( e.g., OXA -48) [74–76]. Prevalence of resistance in 345 Enterobacterales to any of these three combination antimicrobials was 1–5% across the globe as of 346 2022 despite regional variation [17, 77 –81]. Therefore, the discovery of K. pneumoniae isolate 347 KpMVR1, which exhibited unusual resistance to meropenem-vaborbactam, imipenem-relebactam, 348 and ceftazidime-avibactam, in a seriously ill patient is particularly worrisome . The small number of 349 chromosomal SNPs (n=6) and indels (n=10; ≤15 bp each) identified in KpMVR1 when compared with 350 KpMVS1, Patient 1’s exposure to meropenem, meropenem -vaborbactam, and fluoroquinolones, and 351 the unique antibiogram of KpMVR1 in the ICU altogether support the suspected in vivo emergence of 352 meropenem-vaborbactam, ceftazidime-avibactam, imipenem-relebactam, and ciprofloxacin resistance 353 in the same K. pneumoniae strain during this patient’s hospital stay. A similar shift in the ceftazidime-354 avibactam susceptibility profile of K. pneumoniae has been reported during treatment using 355 meropenem followed by ceftazidime-avibactam [82]. 356 KPC-2 is known to confer carbapenem resistance in Gram-negative bacteria but can be effectively 357 inhibited by vaborbactam, avibactam, and relebactam [83]. Here, we have experimentally determined 358 the effect of carbapenemase KPC-157 on the susceptibility to carbapenems and cephalosporins with 359 or without β-lactamase inhibitors. Our results indicate that KPC-157 does not differ from KPC-2 in its 360 interaction with meropenem or meropenem-vaborbactam. Therefore, the presence of blaKPC-157 in the 361 single-copy plasmid pKpMVR1_1 alone cannot explain the high -level meropenem-vaborbactam 362 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint resistance observed in KpMVR1. Notably, KPC-157 appears less capable of hydrolysing imipenem, 363 aztreonam, ceftazidime, and cefiderocol than KPC-2, and is inhibited by vaborbactam and avibactam 364 but not by relebactam. 365 Isolate KpMVR1 showed genetic changes that may alter the antimicrobial permeability of its OM 366 when compared with isolate KpMVS1. Resulting from an IS -induced sequence disruption, the 367 hindered translocation of OmpK36 to the OM is predicted to hamper the influx of β-lactams and β-368 lactamase inhibitors into the periplasm , hence elevated MIC s of carbapenems and cephalosporins 369 tested in Table 2 with and without β-lactamase inhibitors. Such hampered antimicrobial and inhibitor 370 influx might be further compromised by a decreased expression of ompK35 in KpMVR1 as a result of 371 the in-frame, presumptively inactivating deletion within the repressor gene marR and the consequent 372 upregulation of the marA gene [84]. Moreover, the inactivation of marR is known to i ncrease the 373 production of the AcrAB -TolC efflux pump , conferring low -level cross-resistance to antimicrobials 374 including β-lactams and ciprofloxacin [85]. However, this upregulation of AcrAB-TolC in KpMVR1 375 might not alter its antimicrobial susceptibility owing to the possibly destabilised AcrB. Therefore, the 376 high-level ciprofloxacin resistance in KpMVR1 could be primarily driven by the combination of the 377 gyrA mutation S83Y and the absence of OmpK36 in this isolate’s OM [67, 86]. 378 This study is limited to three K. pneumoniae isolates belonging to the same clone, with only one 379 isolate (KpMVR1) exhibiting elevated MICs of meropenem -vaborbactam, imipenem -relebactam, 380 aztreonam-avibactam, and ceftazidime -avibactam. KpMVR1 harboured multiple AMR -associated 381 genetic alterations. Broader surveillance of genetic variants similar to those identified in KpMVR1 is 382 needed to assess the prevalence and clinical relevance of these putative resistance mechanisms. 383 Although we experimentally validated the individual contributions of blaKPC-157 and ΔompK36 to 384 antimicrobial susceptibility, the genetically reconstructed isolates could not fully replicate the same 385 level of MIC increments as KpMVR1, suggesting that other genetic or regulatory mechanisms may be 386 involved, which remain to be elucidated. Transcriptomic and proteomic profiling could be performed 387 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint in the future to determine whether regulatory mechanisms also contribute to the observed AMR in 388 KpMVR1. 389 At the nation level, a review of routine surveillance and reference laboratory samples from 2016 -390 2020 revealed only low levels of resistance to ceftazidime -avibactam in the UK [81]. However, it 391 remains essential that emerging resistance to ceftazidime -avibactam and novel β-lactam/β-lactamase 392 inhibitor combinations is promptly identified and reported through UKHSA’s Second Generation 393 Surveillance System and referral of such isolates to the AMRHAI Reference Unit . Additionally, our 394 study highlights the importance of monitoring the evolving antimicrobial susceptibility profiles of 395 bacterial pathogens within patients during antimicrobial therapy. 396 Funding information 397 This work was mainly funded by the UKHSA. YW is a research fellow funded by the David Price 398 Evans Endowment (grant number: UGG10057) at the University of Liverpool and was an Imperial 399 Institutional Strategic Support Fund Springboard Research Fellow, funded by the Wellcome Trust and 400 Imperial College London (grant number: PSN109) . YW, EJ, DM, and KLH are affiliated with the 401 National Institute for Health and Care Research Health Protection Research Unit in Healthcare 402 Associated Infections and Antimicrobial Resistance at Imperial College London in partnership with 403 the UKHSA, in collaboration with, Imperial Healthcare Partners, University of Cambridge and 404 University of Warwick (grant number: NIHR200876). The views expressed in this article are those of 405 the authors and not necessarily those of the NHS, the National Institute for Health Research, or the 406 Department of Health and Social Care. 407 Acknowledgments 408 We acknowledge the Colebrook Laboratory, a facility supported by the NIHR Imperial Biomedical 409 Research Centre (BRC) , for providing bioinformatics resources. Part of the bioinformatics analysis 410 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint was performed on equipment purchased as part of MRC CARP fellowship award MR/T005254/1. We 411 also thank the Institut Pasteur teams for the curation and maintenance of BIGSdb-Pasteur databases at 412 http://bigsdb.pasteur.fr. 413 Author contributions 414 Conceptualisation: KLH and YW; Resources: KLH, JT, GF, FM, NW, and GMR; Methodology: KLH, 415 YW, JLCW, and EJ; Data curation: YW; Investigation and formal analysis: YW, JLCW, JSG, WWL, 416 JT, FM, GMR, KD, IB, GF, EJ, DM, and KLH; Visualisation: YW; Writing – original draft: YW, 417 JLCW, JSG, WWL, GMR, and KLH; Writing – review and editing: all authors. 418 Conflicts of interest 419 Authors declare that there are no conflicts of interest. 420 Consent to publish 421 No sensitive information is disclosed in this manuscript. 422 Ethical statement 423 National surveillance of communicable diseases and outbreak investigation work at UKHSA does not 424 require individual patient consent as per Regulation 3 of The Health Service (Control of Patient 425 Information) Regulations 2002. 426

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Antimicrobial Agents and Chemotherapy 653 1996;40:342–348. 654 655 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Figures 656 Figure 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 region. Genes without known names are not labelled. Each asterisk indicates an allele from a named gene family. 658 659 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Figure 2. ISEc68-mediated disruption of ompK36 in isolate KpMVR1. (A) Genetic environment of the disrupted ompK36 in KpMVR1. The arrow labelled 660 “ompK36*” denotes the upstream-shifted open reading frame caused by the insertion of IS Ec68. Abbreviations: CDS: coding sequence; IS, insertion sequence; 661 ncRNA: non-coding RNA. ( B) Comparison of predicted OmpK36 sequences using Clustal Omega ( www.ebi.ac.uk/jdispatcher/msa/clustalo). Mismatches are 662 highlighted 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 electrophoresis of outer membrane proteins to confirm the absence of OmpK36 in KpMVR1 and the ompK36-knockout isolate ICC8001ΔompK36. 664 665 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Supplementary methods Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam Yu Wan, Joshua L. C. Wong, Julia Sanchez-Garrido, Wen Wen Low, Jane F. Turton, Fabio Morecchiato, Ilaria Baccani, Kirsty Dodgson, Gian Maria Rossolini, Neil Woodford, Gad Frankel, Elita Jauneikaite, Daniè le Meunier, and Katie L. Hopkins August 2025 Comparative structural analysis of TraN Amino acid sequences of TraNpKpMVS1_1, TraNpKpMVR1_1, and TraNpKpMVS2_1 were extracted from the sequence annotations of plasmids pKpMVS1_1 (locus tag: WAS92_RS00545), pKpMVR1_1 (ACNQKT_RS26595), and pKpMVS2_1 (ACNQKS_RS28350), respectively. To contextualise these three proteins, the previously described TraN variants [1] TraNpKpQI (NCBI protein accession: ARQ19727.1), TraN MV2 (BAS44060.1), TraNR100-1 (ABD60034.1), TraN pSLT (AAL23498.1), TraN F (WP_000821835.1), TraN MV1 (ANZ89826.1), TraNMV3 (WP_001398575.1) were downloaded from the NCBI Protein database (www.ncbi.nlm.nih.gov/protein). These 10 amino acid sequences were aligned with the ClustalW algorithm [2], and subsequently, a neighbour -joining phylogenetic tree was generated from the multi -sequence alignment, with the Poisson correction method as implemented in MEGA11 [3]. The phylogenetic tree was visualised using iTOL v7.2 [4]. Three-dimensional structures of TraNpKpMVS1_1, TraNpKpMVR1_1 (identical to TraNpKpMVS2_1), and TraNpKpQI were predicted using AlphaFold 3 [5] with its default parameters on AlphaFold Server (alphafoldserver.com). The top-ranked (model 0) structural models of TraN proteins were visualised using UCSF ChimeraX v1.9 [6]. Superimposition analysis of these models was performed with ChimeraX’s Matchmaker tool using default settings, including the use of the “best -aligning” or “bb” chain -pairing method, the Needleman -Wunsch alignment algorithm, and the BLOSUM-62 similarity matrix.

1. Low Wen Wen, Seddon Chloe, Beis Konstantinos, Frankel Gad. The Interaction of the F-Like Plasmid- Encoded TraN Isoforms with Their Cognate Outer Membrane Receptors. Journal of Bacteriology 2023;205:e00061-23. 2. Thompson JD, Gibson TobyJ, Higgins DG. Multiple Sequence Alignment Using ClustalW and ClustalX. Current Protocols in Bioinformatics 2003;00:2.3.1-2.3.22. 3. Tamura K, Stecher G, Kumar S . MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular Biology and Evolution 2021;38:3022–3027. 4. Letunic I, Bork P . Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 2007;23:127–128. 5. Abramson J, Adler J, Dunger J, Evans R, Green T, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 2024;630:493–500. 6. Meng EC, Goddard TD, Pettersen EF, Couch GS, Pearson ZJ, et al. UCSF ChimeraX: Tools for structure building and analysis. Protein Science 2023;32:e4792. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint Supplementary figures Genomic and molecular characterisation of a Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam Yu Wan, Joshua L. C. Wong, Julia Sanchez-Garrido, Wen Wen Low, Jane F. Turton, Fabio Morecchiato, Ilaria Baccani, Kirsty Dodgson, Gian Maria Rossolini, Neil Woodford, Gad Frankel, Elita Jauneikaite, Daniè le Meunier, and Katie L. Hopkins August 2025 Table of contents Figure S1 …………………………………………………………………………………………… 1 Figure S2 …………………………………………………………………………………………… 2 Figure S3 …………………………………………………………………………………………… 3 Figure S4 …………………………………………………………………………………………… 4 Figure S5 …………………………………………………………………………………………… 4 Figure S6 …………………………………………………………………………………………… 5 Figure S7 …………………………………………………………………………………………… 6 Figure S8 …………………………………………………………………………………………… 7 Figure S9 …………………………………………………………………………………………… 8

…………………………………………………………………………………………… 9 .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 1 Figure S1. Alignment of plasmids pKpMVR1_1 and pKpMVS2_1 against plasmid pKpMVS1_1 using nucleotide BLAST as implemented in Proksee. Plasmids pKpMVR1_1 and pKpMVS1_1 belonged to replicon type IncFII(pKP91)/repB(R1701), and pKpMVS2_1 belonged to IncFII(pKP91)/IncR. Th e innermost ring represents the number of genetic variants per kbp (calculated using VCFtools v0.1.17) [1] in pKpMVR2_1 compared with pKpMVS1_1. The two middle rings display genes, insertion sequences, and transposons identified in the reference sequence pKpMVS1_1, with arrows indicating orientations of these genetic features (Table S1). Genes without known names are not labelled. The two outer rings show regions of pKpMVR_1 (pink) and pKpMVS2_1 (blue) aligned to pKpMVS1, respectively. This figure was created using Proksee (proksee.ca). "Δ" in gene labels represents a truncated or interrupted feature, and each asterisk represents a variant of an insertion sequence or transposon. Abbreviations: CDS, coding sequence; IS, insertion sequence. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 2 Figure S2. A BRIG diagram comparing chromosomes of KpMVR1 and KpMVS2 with that of KpMVS1. Parameters for BLASTn sequence alignment: “-task megablast -ungapped -qcov_hsp_perc 0.8”. Four large deletions (>4 kbp) in the chromosome of KpMVR1 when compared to that of KpMVS1 (Table 3) are denoted by digits in filled circles. Genetic structures of these deleted regions are illustrated in Figure 1 and supplementary Figures S3–5. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 3 Figure S3. Genetic structure of a 19.7-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels “start” and “ end” indicate boundaries of the deleted region. Genes without known names are not labelled. “IS1X2*” denotes a variant of the IS1-family insertion sequence IS1X2 (98% nucleotide identity and 100% query coverage). “CRISPR” indicates an array of clustered regularly interspaced short palindromic repeats . See Table S3 for detailed annotations of this region. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 4 Figure S4. Genetic structure of a 33.5-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels “start” and “end” indicate boundaries of the deleted region. Genes without known names are not labelled. See Table S4 for detailed annotations of this region. Figure S5. Genetic structure of a 4.9-kbp region in KpMVS1 that was deleted in KpMVR1 (Table 4). Labels “start” and “ end” indicate boundaries of the deleted region. Genes without known names are not labelled. “IS3H*” denotes a variant of the IS 3-family insertion sequence IS 3H (79% nucleotide identity and 100% query coverage). There were nine copies of this variant in the KpMVS1 chromosome and eight copies in the KpMVR1 chromosome, which is consistent with the content of the 4.9-kbp deletion (Table S5). .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 5 Figure S 6. Prediction of signal peptides and cleavage site in the wild type OmpK36 (KpMVS1) and its frameshifted variant (KpMVR1), respectively, using the slow model mode of SignalP v6.0. “N”, “H”, and “C” regions in each protein sequence denote the N-terminal region (Sec/SPI n) , centre hydrophobic region (Sec/SPI h), and C-terminal region (Sec/SPI c) of the signal peptide, respectively , whereas “O” denotes the non-signal peptide region (OTHER). The cleavage site (CS) is indicated by the red vertical dashed line. The probability of each amino acid to be part of each peptide region is indicated by a coloured s olid curve throughout the protein sequence. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 6 Figure S7. Midpoint-rooted neighbour-joining phylogenetic tree of 10 TraN amino acid sequences , where TraNpKpMVR1_1 was identical to Tra pKpMVS2_1. The sequences are named after source plasmids. The structural groups (TraNα, TraNβ, TraNγ, and TraNδ) [2] of TraN are indicated by shaded boxes and labelled. The scale bar represents the number of amino acid substitutions per residue. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 7 Figure S8. Comparison of predicted TraN structures for plasmids pKpQIL, pKpMVS1_1, and pKpMVR1_1. Amino acid chains are represented by ribbons. ( A) Predicted 3D structures with residues coloured by scores from the predicted local distance difference test (pLDDT) . The pLDDT was performed by AlphaFold3 to evaluate the per -residue local confidence of the predicted 3D structure. ( B) Pairwise structural comparison through the super-imposition analysis. Dashed boxes indicate the tip/sensor domains of TraN [2]. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 8 Figure S9. Locations of amino acid variation in the tip/sensor domains of TraN from plasmids pKpQIL, pKpMVS1_1, and pKpMVR1_1, with amino acid chains represented by ribbons in the superimposition view. The variable sites are highlighted in yellow, blue, and magenta. In comparison with Figure S8B, the proteins are arbitrarily rotated around the vertical axis to expose all variable sites. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint 9

1. Danecek P, Auton A, Abecasis G, Albers CA, Banks E, et al. The variant call format and VCFtools. Bioinformatics 2011;27:2156–2158. 2. Frankel G, David S, Low WW, Seddon C, Wong JLC, et al. Plasmids pick a bacterial partner before committing to conjugation. Nucleic Acids Res 2023;51:8925–8933. .CC-BY 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.11.669739doi: bioRxiv preprint