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

Genomic and molecular characterisation of a KPC-producing Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomic and molecular characterisation of a KPC-producing 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Yu Wan, Joshua L. C. Wong, Julia Sanchez-Garrido, Wen Wen Low, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8289965/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Background Resistance to carbapenems and third-generation cephalosporins is increasing in Klebsiella pneumoniae globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, an unusual K. pneumoniae clinical isolate, KpMVR1, was recovered from a hospitalised patient in England, exhibiting resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. To investigate this phenomenon, we characterised the genome and antimicrobial susceptibility of KpMVR1 alongside two clonally related isolates susceptible to all three β-lactam/β-lactamase inhibitor combinations: KpMVS1, collected from the same patient 42 days earlier, and KpMVS2, from another patient in the same hospital. Methods Illumina and MinION whole-genome sequencing were conducted for these three isolates, followed by hybrid genome assembly. Annotated genome assemblies were compared to identify genetic variation. Mutagenesis experiments were performed to verify predicted functional alterations. Results All isolates belonged to clone ST8134 and carried bla KPC−2 alleles (KpMVR1: bla KPC−157 ; KpMVS1 and KpMVS2: bla KPC−2 ) in presumptively conjugative plasmids. Insertion sequence IS Ec68 caused a frameshift mutation in KpMVR1’s ompK36 gene, reducing susceptibility to meropenem-vaborbactam and imipenem-relebactam. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump. Conclusions KpMVR1 carried multiple resistance-associated genetic alterations and likely developed its resistance profile through within-patient evolution. This study highlights the importance of routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy and the need to characterise underlying resistance mechanisms to assess the risk of onward dissemination. Klebsiella pneumoniae antimicrobial resistance carbapenem resistance β-lactamase inhibitors KPC-157 OmpK36 insertion sequences hybrid genome assembly comparative genomics mutagenesis experiments Figures Figure 1 Figure 2 Background Meropenem and imipenem are broad-spectrum carbapenem antimicrobials parenterally administered to treat serious bacterial infections, such as those caused by Enterobacterales producing extended-spectrum β-lactamases (ESBLs) or AmpC-type cephalosporinases (AmpCs) [1–3]. Ceftazidime, a third-generation parenteral cephalosporin, is also widely used for treating severe bacterial infections, although it can be hydrolysed by ESBLs and AmpCs [4, 5]. In Gram-negative bacteria, carbapenems and cephalosporins diffuse through outer-membrane porins and enter the periplasm, where they inactivate penicillin binding proteins, disrupting cell-wall synthesis with a bactericidal effect [6–8]. Combining β-lactams with β-lactamase inhibitors in antimicrobial chemotherapy is a widely employed strategy to circumvent β-lactamase-mediated resistance in bacteria. Vaborbactam, relebactam, and avibactam are non-β-lactam inhibitors of Ambler class A β-lactamases, such as ESBLs and Klebsiella pneumoniae carbapenemases (KPCs), as well as class C β-lactamases (AmpCs) [9]. Moreover, avibactam inhibits several class D β-lactamases such as OXA-48 and OXA-10 [10]. These inhibitors penetrate the outer membrane (OM) of Gram-negative bacteria via porins, blocking the active sites of β-lactamases in the periplasm [9, 11]. In the UK, meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam are reserved for highly selected patients [12]. Resistance to meropenem-vaborbactam and imipenem-relebactam in clinical isolates of KPC-producing K. pneumoniae (KPC- Kp ) has been reported in Italy, while ceftazidime-avibactam resistance has been documented across continental Europe and Americas [13–15]. Identified resistance mechanisms include KPC overproduction, gain-of-function point mutations in bla KPC , KPC-8 (formerly, KPC-3 V239G), the AcrAB-TolC multidrug efflux pump, and mutation of ramR [9, 15–17]. Additionally, disruption or transcriptional downregulation of ompK35 ( ompF ) and ompK36 ( ompC ), which encode non-selective porins that mediate the diffusion of β-lactams and β-lactamase inhibitors through the OM, has also been implicated [15, 16, 18, 19]. In 2021, a KPC- Kp clinical isolate (KpMVR1) resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam was identified in England with evidence suggesting in vivo development of this unusual phenotype during prolonged intensive care of a patient. Here, we describe the genomic and molecular characteristics of this isolate, analysed alongside two clonally related isolates recovered from the same hospital using comparative genomics and microbiological experiments. Methods Isolate collection and phenotyping The K. pneumoniae isolate KpMVS1 was recovered from a lung biopsy specimen of an inpatient (hereafter, Patient 1) admitted to an intensive care unit (ICU) in England in 2021. The second K. pneumoniae isolate, KpMVR1, was recovered from a groin wound of the same patient 42 days later. During this ICU stay, the patient received a broad range of antimicrobials, including meropenem-vaborbactam, ciprofloxacin, and gentamicin; however, ceftazidime-avibactam and imipenem-relebactam were not used. Species identification and carbapenemase gene screening were performed using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) mass spectrometry and the GeneXpert system (Cepheid, USA), respectively. Initial antimicrobial susceptibility testing (AST) was conducted by the hospital and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Both isolates were referred to the Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit of the UK Health Security Agency (UKHSA) for variable number tandem repeat (VNTR) typing [20] and investigation of unusual antimicrobial resistance (AMR). Additionally, a KPC- Kp isolate KpMVS2—recovered from a rectal swab of another patient (Patient 2) in the same hospital in 2020 during an outbreak investigation and sharing the same VNTR profile as KpMVS1 and KpMVR1—was retrieved from AMRHAI’s culture collection for comparison. All three isolates underwent whole-genome sequencing (WGS) and AST, including determination of minimum inhibitory concentrations (MICs) for 19 antimicrobials (Table 1 ) and inhibition zone diameters for cefiderocol, interpreted according to EUCAST clinical breakpoints v15.0 [21]. Table 1 Antimicrobial minimum inhibitory concentrations (MICs; mg/L) determined by UKHSA’s AMRHAI Reference Unit and susceptibility interpretations (according to EUCAST clinical breakpoints v15.0 where applicable) of three K. pneumoniae clinical isolates. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; ETP, ertapenem; CET, ceftolozane; TZB, tazobactam; CFD, cefiderocol; FEP, cefepime; CAZ, ceftazidime; AVI, avibactam; CTX, cefotaxime; FOX, cefoxitin; TMC, temocillin; AMP, ampicillin; AMX, amoxicillin; CAV, clavulanate; PIP, piperacillin; ATM, aztreonam; AMK, amikacin; GEN, gentamicin; CST, colistin; CIP, ciprofloxacin; MB, monobactam. Susceptibility interpretations: R, resistant; I, susceptible, increased exposure; S, susceptible. 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. Whole-genome sequencing Genomic DNA was extracted from overnight cultures using the GeneJET Kit (ThermoFisher Scientific, UK) according to the manufacturer’s protocol. Short-read sequencing was performed on a HiSeq 2500 system (Illumina, USA) at UKHSA’s Colindale Sequencing Laboratory using a paired-end 101-bp protocol. Long-read sequencing was conducted on MinION R9.4.1 flow cells (Oxford Nanopore Technologies [ONT], UK), with libraries prepared using the ONT Rapid Barcoding Kit SQK-RBK004. Bioinformatics analysis Illumina reads were trimmed and filtered with Trimmomatic v0.39 for a minimum per-read quality of Phred Q30 and minimum length of 50 bp [22]. Fast-mode basecalling and de-multiplexing of MinION reads was conducted by guppy v4 (ONT), followed by read trimming and filtering for a minimum per-read quality of Q10 and minimum length of 1 kbp with fastp v0.23.4 [23]. For species confirmation and contamination assessment, taxonomical profiling of processed Illumina and MinION reads were performed using Kraken v2.1.3, bracken v2.8, and a standard Kraken database built in September 2023 [24, 25]. Genomes of KpMVR1 and KpMVS2, with estimated MinION read depths of 185× and 243×, respectively, were assembled using hybracter v0.5.0 (Additional File 1) [26, 27]. For KpMVS1, which had an estimated MinION read depth of 64×, chromosomal and plasmid sequences were assembled using Raven v1.8.3 and plassembler, respectively, and subsequently polished with MinION reads followed by Illumina reads, as for KpMVR1 and KpMVS2. Genome assemblies were assessed for quality using CheckM2 v1.0.2 and its database Uniref100/KO [28]. The average fold-coverage of each contig was estimated from Illumina and MinION reads, respectively, using mosdepth v0.3.9 [29]. The genome assemblies were annotated using bakta v1.9.2 and its standard database v5.1 [30]. Multi-locus sequence typing (MLST), serotype prediction, and virulence-factor detection were performed using Kleborate v3.1.3, which incorporated Kaptive v3.1.0 [31, 32]. Genome assemblies of all three isolates were submitted to the Klebsiella PasteurMLST database (bigsdb.pasteur.fr) for assignment of a new sequence type (ST) number. AMR genes were detected using AMRFinderPlus v3.12.8 with a minimum query coverage of 80% [33]. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes were predicted for chromosomes using CRISPRCasFinder [34]. For plasmids, replicon types were determined at a minimum of 80% nucleotide identity and coverage using PlasmidFinder v2.1 [35], and the mobility was predicted using mob_typer of MOB-suite v3.1.8 [36]. The copy number of each KPC-encoding plasmid was estimated by dividing the plasmid’s fold-coverage by that of its host’s chromosome. Transposons and insertion sequences (ISs) were identified using TnCentral Blast (blastn) and ISFinder, respectively [37, 38]. Chromosome and plasmids of KpMVR1 and KpMVS2 were compared against those of KpMVS1 using minimap v2.26 [39]. Identified genetic variants were annotated using snpEff v5.2 [40]. Gene Ontology terms were predicted from amino acid sequences using InterProScan v5.69-101.0 with sequence alignments filtered for ≥ 60% query coverages [41]. Impacts of point mutations on protein stability were predicted from wild-type protein structures in the UniProt database using Missense3D and DDMut [42–44]. The three-dimensional structure of the plasmid-encoded donor OM protein TraN was compared between KPC-encoding, IncFII-carrying plasmids pKpMVS1_1, pKpMVR1_1, and pKpMVS2_1 to estimate the impact of TraN alterations on the conjugation specificity and efficiency (Additional File 1) [27, 45]. Comparison and annotations of these three plasmids were visualised using BRIG v0.95 and Proksee [46, 47]. Gene synteny was illustrated using R package gggenes [48]. Genetic alterations in both KpMVR1 and KpMVS2 were considered unlikely to confer the unique AMR profiles of KpMVR1 and were therefore excluded from further investigation. Functional assessment To determine and compare the impacts of bla KPC−2 and bla KPC−157 on β-lactam susceptibility in K. pneumoniae , KpMVS1’s KPC-2-encoding plasmid pKpMVS1_1 was introduced into the plasmid-free K. pneumoniae laboratory strain ICC8001 (MICs: meropenem, ≤ 0.06 mg/L; imipenem, 0.25 mg/L; aztreonam, ≤ 0.125 mg/L; ceftazidime and ceftazidime-avibactam, 0.25 mg/L) through conjugation, resulting in a transconjugant ICC8001 KPC − 2 [49]. Transgenic isolates ICC8001 KPC − 157 and KpMVS1 KPC − 157 were derived from ICC8001 KPC − 2 and KpMVS1, respectively, by substituting bla KPC−2 with bla KPC−157 . Moreover, isolates ICC8001 KPC − 2/Δ ompK36 and ICC8001 KPC − 157/Δ ompK36 were derived from ICC8001 KPC − 2 and ICC8001 KPC − 157 , respectively, through seamless, markerless homologous recombination using mutagenesis vectors and a lambda-red based recombination system generated in previous work [49]. To predict the presence or absence of OmpK36 in KpMVR1’s OM, Sec-dependent signal peptides and their cleavage sites in translated ompK36 alleles were compared between KpMVS1 and KpMVR1 using SignalP v6.0 [50]. To validate this prediction, OM proteins were purified from overnight cultures of KpMVS1, KpMVR1, KpMVS2, ICC8001, and an ompK36 -knockout derivative, ICC8001 Δ ompK36 , separated by SDS-PAGE, and visualised by Coomassie staining (Additional File 1) [27]. Both progenitor isolates (KpMVS1 and KpMVR1) and five transgenic derivatives (KpMVS1 KPC − 157 , ICC8001 KPC − 2 , ICC8001 KPC − 157 , ICC8001 KPC − 2/Δ ompK36 , and ICC8001 KPC − 157/Δ ompK36 ) were tested for susceptibility to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, ceftazidime, ceftazidime-avibactam, aztreonam, aztreonam-avibactam, and cefiderocol (in iron-depleted medium) using broth microdilution according to EUCAST guidelines [51, 52]. Any MIC change exceeding a two-fold difference between two isolates was considered notable. Results Phenotypes of isolates Isolates KpMVS1 and KpMVR1, obtained 42 days apart from Patient 1 with recurrent K. pneumoniae infections, and KpMVS2, obtained from Patient 2 in the same hospital, were identified as K. pneumoniae by both MALDI-ToF and the WGS. In the ICU where KpMVS1 and KpMVR1 were recovered, all patients were screened for carriage of carbapenemase-producing Enterobacterales (CPE) on admission using PCR. The resistance profile of KpMVR1 was unique among all CPE isolates identified in the ICU during Patient 1’s stay. Based on AST results from the AMRHAI Reference Unit (Table 1 ), KpMVR1 was resistant to meropenem-vaborbactam (MIC > 256 mg/L), ceftazidime-avibactam (MIC = 16 mg/L), and ciprofloxacin (MIC > 4 mg/L), whereas KpMVS1 and KpMVS2 were susceptible to these antimicrobials (MICs: meropenem-vaborbactam ≤ 0.064 mg/L; ceftazidime-avibactam 1 mg/L; ciprofloxacin ≤ 0.125 mg/L). KpMVS2 was resistant to cefiderocol (inhibition zone diameter: 19 mm) as determined using disc diffusion. Further AST revealed that the imipenem-relebactam MIC of KpMVR1 (512 mg/L, resistant) was 2048-fold higher than that of KpMVS1 (0.25 mg/L, susceptible) (Table 2 ). Moreover, KpMVR1 exhibited a > 4-fold increase in the temocillin MIC (> 128 mg/L) and a > 8-fold reduction in the cefepime MIC (4 mg/L, susceptible, increased exposure) compared with KpMVS1 (temocillin: 32 mg/L; cefepime: >32 mg/L, resistant). Table 2 Minimum inhibitory concentrations of β-lactam antimicrobials and susceptibility interpretations (according to EUCAST clinical breakpoints v15.0 where applicable) of progenitor and transgenic K. pneumoniae isolates determined in the experiments for functional assessment. Subscripts in isolate names indicate transgenic isolates and corresponding genotypes. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; REL, relebactam; ATM, aztreonam; AVI, avibactam; CAZ, ceftazidime; CFD, cefiderocol, tested in iron-depleted Mueller Hinton broth; NT, not tested. Interpretations of antimicrobial susceptibility: R, resistant; I, susceptible, increased exposure; S, susceptible. Notations: ompK36 fs, frameshifted ompK36 ; Δ ompK36 , deletion of ompK36 . 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 bla KPC−157 ompK36 fs 512 (R) 256 (R) 512 (R) 512 (R) NT 16 (R) 4 (S) 8 (R) 8 (S) 0.5 (S) KpMVS1 bla KPC−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) KpMVS1 KPC − 157 bla KPC−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) ICC8001 KPC − 2 bla KPC−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) ICC8001 KPC − 157 bla KPC−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) ICC8001 KPC − 2/Δ ompK36 bla KPC−2 Δ ompK36 256 (R) 2 (S) 256 (R) 2 (S) NT > 1024 (R) 0.25 (S) 16 (R) 0.5 (S) 0.25 (S) ICC8001 KPC − 157/Δ ompK36 bla KPC−157 Δ ompK36 256 (R) 4 (S) 256 (R) 128 (R) NT 4 (I) 0.25 (S) 1 (S) 0.5 (S) 0.25 (S) Genetic characteristics of isolates All three isolates belonged to K. pneumoniae clone ST8134, a single-locus variant of ST240, and were predicted to share the O1αβ,2β O-antigen type and K62 capsular polysaccharide type. Sequence lengths, plasmid replicons, and AMR genes determined in hybrid genome assemblies are summarised in Table 3 . KpMVR1 and KpMVS1 shared the same plasmid types IncFII/repB(R1701), IncFII(pMET), Col(pHAD28), and Col(pHAD28)/Col440II, whereas KpMVS2 possessed unique plasmid types IncFII/IncR and IncFII(pKP91)/FIB(K). Table 3 Genetic characteristics of the three K. pneumoniae clinical isolates. The plasmid type refers to the haplotype of plasmid replicons. Each hit of plasmid replicons in this table covered the full length of its reference sequence in the PlasmidFinder database. Abbreviation: AMR, antimicrobial resistance. Isolate Sequence Category Length (bp) Plasmid type Nucleotide identity to template Plasmid mobility AMR gene KpMVS1 KpMVS1 Chromosome 5,404,514 bla SHV−36 , fosA10 pKpMVS1_1 Plasmid 111,397 IncFII/repB(R1701) IncFII(pKP91): 100%; repB(R1701): 99.52% Conjugative bla KPC−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 bla SHV−36 , fosA10 pKpMVR1_1 Plasmid 111,174 IncFII/repB(R1701) IncFII(pKP91): 100%, repB(R1701): 99.52% Conjugative bla KPC−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 bla SHV−36 , fosA10 pKpMVS2_1 Plasmid 116,795 IncFII/IncR IncFII(pKP91): 100%; IncR: 99.6% Conjugative bla KPC−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 substitutions (95% nucleotide identity) despite their same percent identity to the template. KpMVS1 carried bla KPC−2 on the 111.4 kbp IncFII(pKP91)/repB(R1701) plasmid pKpMVS1_1. A plasmid of the same type was identified in KpMVR1 (pKpMVR1_1, 111.2 kbp) and carried bla KPC−157 , which differed from bla KPC−2 by a single missense mutation (392A > G) resulting in an N131S amino acid substitution within the enzyme’s active site (NCBI Protein accessions: KPC-2, WP_004199234.1; KPC-157, WP_259115967.1) [53], where the N131 residue binds to relebactam, avibactam, and vaborbactam through a hydrogen bond [54–56]. Notably, pKpMVR1_1 differed from pKpMVS1_1 by 285 nucleotide substitutions, 14 deletions, and three insertions. These variants were concentrated in two genomic regions involved in plasmid transfer and maintenance (Supplementary Fig. 1, Additional File 2) [57], suggesting recombination between plasmids. Another plasmid type, IncFII(pKP91)/IncR, in KpMVS2 carried bla KPC−2 . All these KPC-encoding plasmids were predicted to be conjugative (relaxase type: MOBF; mating pair formation type: MPF_F), and each carried an intact variant of the Tn 4401a transposon harbouring bla KPC−2 or bla KPC−157 , with an ATTGA target site duplication and 1–2 single-nucleotide polymorphisms (SNPs) between each pair of Tn 4401a variants (Supplementary Fig. 1, Additional File 2; Supplementary Tables 1 and 2, Additional File 3) [27, 58–60]. The comparison between fold-coverages of contigs suggested that each of these three isolates carried a single copy of the KPC-encoding plasmid. Other AMR genes detected were chromosomal β-lactamase gene bla SHV (variant bla SHV−36 ) and fosfomycin resistance gene fosA10 , which are both intrinsic to K. pneumoniae [61–63]. The chromosome of KpMVR1 differed from that of KpMVS1 by six SNPs, seven insertions, four small deletions (1–15 bp), and four large deletions (Fig. 1 ; Supplementary Figs. 2–5, Additional File 2; Supplementary Table 3–6, Additional File 3) [57, 60]. Seven variants were also identified in KpMVS2 (Table 4 ), which differed from KpMVS1 by 129 SNPs, 13 insertions, and seven deletions. The 19.7 kbp and 4.9 kbp deletions in KpMVR1 may have been mediated by IS elements, as previously reported in Escherichia coli (Supplementary Figs. 3 and 5, Additional File 2) [57, 64]. Notably, KpMVR1 exhibited a 54.7 kbp deletion encompassing operons encoding an AcrAB-like multidrug efflux pump and an additional ABC-type Fe 3+ -siderophore transport system present in both KpMVS1 and KpMVS2 (Fig. 1 ; Supplementary Table 3, Additional File 3) [60]. All three isolates carried a single copy of the acrRAB operon and tolC gene, which together produce the AcrAB-TolC multidrug efflux pump. However, the permease AcrB in KpMVR1 harboured a destabilising L667R mutation located outside the protein’s transmembrane domains. Table 4 Chromosomal genetic variation in isolate KpMVR1 identified via comparison against its progenitor KpMVS1. Coordinates refer to locations in the reference sequence of the KpMVS1 chromosome. Variants shared by both KpMVR1 and KpMVS2 against their common reference sequence of KpMVS1 are indicated by asterisks following the coordinates. The “^” sign indicates an insertion between two consecutive bases in the reference sequence. Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated genes; Del, deletion; Ins, insertion; fs, frameshift. 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 IS Ec68 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 (Fig. 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 IS 3H 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 As for the biosynthesis of siderophores and transport of the iron-siderophore complex—which together facilitate cefiderocol to penetrate the OM [65]—KpMVS1, KpMVR1, and KpMVS2 were predicted to possess complete enterobactin production and iron-enterobactin transport systems. None of the yersiniabactin, colibactin, aerobactin, or salmochelin loci were detected, producing a Kleborate virulence score of zero [31]. All three isolates shared the same 19-kbp chromosomal region harbouring a cluster of enterobactin-synthesising genes entA–F and entH , enterobactin-exporter gene entS , and iron-enterobactin transporter genes fepA–D and fepG . Compared with the ciprofloxacin-susceptible isolates KpMVS1 and KpMVS2, KpMVR1 harboured a nucleotide substitution 248C > A in the DNA gyrase gene gyrA , generating the GyrA mutation S83Y, which is known to reduce ciprofloxacin susceptibility [66]. The three isolates also carried a single copy of the marRAB operon. However, KpMVR1 exhibited a unique 15-bp in-frame deletion in the non-essential transcriptional repressor gene marR within the marRAB operon, causing a loss of five amino acids and an amino acid substitution within the DNA-binding region of MarR (Table 4 ) [67]. Both the KpMVS1 and KpMVS2 genomes harboured three identical copies of the IS 5 -family insertion sequence IS Ec68 , whereas the KpMVR1 genome harboured four. Notably, seven bases at the 5’ end of ompK36 in KpMVR1 were truncated by this additional copy of IS Ec68 , producing a frameshift mutation that replaced the first three amino acids at the N-terminal of OmpK36 with 12 amino acids (Fig. 2 ). The native N-terminal 21 amino acids of OmpK36 encode a Sec-dependent signal sequence (UniProtKB accession: A0A0H3H0Y2), which is essential for translocation of OmpK36 to the inner membrane of K. pneumoniae (Fig. 2 b) [68]. The signal sequence is subsequently cleaved and OmpK36 is then folded and inserted into the OM (where the protein is functionally active as a porin) in a Bam-complex dependent fashion, a process facilitated by a C-terminal recognition sequence [69]. Whilst ompK36 from KpMVS1 and KpMVS2 is predicted to encode a complete sec-dependent signal sequence, the 12 amino acids insertion combined with the deletion of three amino acids in OmpK36 from KpMVR1 is predicted to hinder this protein’s translocation to the OM according to the disrupted signal sequence (Supplementary Fig. 6, Additional File 2) [57]. These predictions were confirmed by the SDS-PAGE, which showed a Coomassie-stained band corresponding to OmpK36 in KpMVS1 but not in KpMVR1 (Fig. 2 c and Additional File 4) [70], indicating that disruption of the Sec-dependent signal sequence of OmpK36 is functionally equivalent to deletion of ompK36 . Regarding plasmid-encoded TraN proteins, TraN pKpMVR1_1 and TraN pKpMVS2_1 were identical (NCBI protein accession: WP_049192820.1) and differed from TraN pKpMVS1_1 (WP_436914186.1) by six amino acid substitutions (Supplementary Table 7, Additional File 3) [60]. Phylogenetic analysis revealed that these proteins belonged to the specialist TraNβ group (Supplementary Fig. 7, Additional File 2) [57], which has a narrow host range [45, 71]. Pairwise structural comparison between TraN pKpMVR1_1 (TraN pKpMVS2_1 ), TraN pKpMVS1_1 , and the prototype TraNβ protein TraN pKpQIL showed high consistency (Supplementary Figs. 8, Additional File 2) [57], and no amino acid substitution occurred in the characteristic distal β-hairpin (Supplementary Fig. 9, Additional File 2) [57], suggesting that the variation in TraN sequences across plasmids pKpMVR1_1, pKpMVS1_1, pKpMVS2_1, and pKpQIL is unlikely to affect the conjugation specificity [72]. Impact of genetic alterations on antimicrobial resistance The substitution of bla KPC−2 with bla KPC−157 in KpMVS1 (producing KpMVS1 KPC − 157 ) and the transconjugant ICC8001 KPC − 2 (producing ICC8001 KPC − 157 ) did not affect the susceptibility to meropenem, meropenem-vaborbactam, or imipenem-avibactam but led to a fourfold reduction in the imipenem MIC and a 16- to 32-fold increase in imipenem-relebactam MIC (Table 2 ). Moreover, this allelic substitution resulted in a 256- to 512-fold reduction in the aztreonam MIC, a 32- to 64-fold reduction in the ceftazidime MIC, and a ≥ 4-fold reduction in the cefiderocol MIC but had no effect on the MICs of aztreonam-avibactam or ceftazidime-avibactam. Notably, imipenem and imipenem-relebactam MICs of each KPC-157-producing isolate were identical (Table 2 ). These findings suggest that KPC-157 has a weaker capacity to hydrolyse imipenem, aztreonam, ceftazidime, and cefiderocol than KPC-2, and that—unlike vaborbactam and avibactam, which inhibit both KPC variants—relebactam inhibits KPC-2 but not KPC-157, which is consistent with a previous report [54]. Knocking out ompK36 from the ICC8001 chromosome (ICC8001 KPC − 2/Δ ompK36 and ICC8001 KPC − 157/Δ ompK36 ) resulted in a 16-fold increase in meropenem and imipenem MICs, a > 33-fold increase in meropenem-vaborbactam MIC, an 8- to 32-fold increase in imipenem-relebactam MIC, and a more than twofold increase in aztreonam MIC (Table 2 ). These findings support the role of OmpK36 as an entry route for β-lactams and β-lactamase inhibitors across the OM [73]. However, when comparing MICs of ceftazidime, ceftazidime-avibactam, and cefiderocol before and after ompK36 knockout in ICC8001 KPC − 2 and ICC8001 KPC − 157 , only two pairs exhibited notable increases (from 0.125 mg/L to 0.5 mg/L for ceftazidime-avibactam, and from ≤ 0.06 mg/L to 0.25 mg/L for cefiderocol), while others remained unchanged, suggesting alternative routes for avibactam’s entry. More broadly, comparisons of β-lactam MICs with and without β-lactamase inhibitors for KpMVR1, ICC8001 KPC − 2/Δ ompK36 , and ICC8001 KPC − 157/Δ ompK36 (Table 2 ) indicate that these inhibitors penetrated the OM via routes other than OmpK36, effectively inhibiting β-lactamases. The chromosomes of KpMVR1, KpMVS1, and ICC8001 derivatives harboured the same cluster of ent and fep genes within a 19-kbp region encoding an ABC-type Fe 3+ -siderophore transporter associated with cefiderocol susceptibility [65]. These isolates did not exhibit any notable difference in cefiderocol MICs despite KpMVR1’s loss of the 54.7-kbp chromosomal region harbouring fep -like genes (Supplementary Table 3, Additional File 3) [60], suggesting alternative entry routes of cefiderocol. Discussion Meropenem-vaborbactam and imipenem-relebactam are recommended in the UK for treating adult patients (≥ 18 years of age) with severe multidrug-resistant bacterial infections where therapeutic options are limited, and ceftazidime-avibactam is recommended as an alternative when the disease-causing bacterium produces class D carbapenemase ( e.g. , OXA-48) [74–76]. Prevalence of resistance in Enterobacterales to any of these three antimicrobial combinations was 1–5% across the globe as of 2022 despite regional variation [14, 16, 77–80]. Therefore, the discovery of KpMVR1, which exhibited unusual resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, in a seriously ill patient is particularly worrisome. No KPC- Kp clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam had been identified in the UK prior to this study, to the best of our knowledge, despite recommendation to submit metallo-carbapenemase-negative isolates exhibiting resistance to any of these agents for further characterisation by UKHSA. Nevertheless, an outbreak of KPC- Kp (ST512) resistant to all these three antimicrobial combinations occurred in Italy in 2021, with isolates co-producing KPC (not including KPC-157 and KPC-2) and OXA-181 carbapenemases, truncated OmpK35, and an OmpK36 mutant [81]. The small number of chromosomal SNPs (n = 6) and indels (n = 10; ≤15 bp each) identified in KpMVR1 when compared with KpMVS1, together with Patient 1’s exposure to meropenem, meropenem-vaborbactam, and fluoroquinolones, and the unique antibiogram of KpMVR1 in the ICU suggest in vivo development of resistance to meropenem-vaborbactam, ceftazidime-avibactam, imipenem-relebactam, and ciprofloxacin in the same bacterial strain during the patient’s prolonged hospital stay. Similar within-host evolution of resistance to imipenem-relebactam, meropenem-vaborbactam, or ceftazidime-avibactam in KPC- Kp have been reported during extended hospitalisation involving complex antimicrobial regimens, including combinations such as ceftazidime-avibactam plus meropenem or meropenem-vaborbactam [15, 82]. This study suggests within-patient emergence of bla KPC−157 through a spontaneous point mutation in bla KPC−2 during prolonged intensive care with exposure to a broad range of antimicrobials. To date, bla KPC−157 has been sporadically reported in Enterobacterales and is associated with distinct plasmids. Two bla KPC−157 -positive K. pneumoniae ST11 clinical isolates, resistant to imipenem-relebactam and ceftazidime-avibactam, were recovered from two patients in China between 2023 and 2024 [83]. In both isolates, bla KPC−157 was carried by the same IncFII(pSDP9R) plasmid (GenBank accessions: CP148070.1 and CP152056.1), as we determined using PlasmidFinder v2.1. Furthermore, bla KPC−157 has also been identified in an IncFII(Yp)-like plasmid of a K. huaxiensis isolate from hospital wastewater in China in 2022, and in an IncN2 plasmid of a Citrobacter freundii isolate from a sewage treatment plant in Brazil [83, 84]. Given the consistent association between bla KPC−157 and Tn 4401 isoforms in all aforementioned cases, continuous surveillance of KPC variants across clinical settings and environmental reservoirs is warranted to monitor emerging resistance mechanisms. KPC-2 confers carbapenem resistance in Gram-negative bacteria but can be effectively inhibited by vaborbactam, avibactam, and relebactam [85, 86]. We have experimentally determined the effect of carbapenemase KPC-157 on the susceptibility to carbapenems and cephalosporins, with or without β-lactamase inhibitors. Our results indicate that KPC-157 behaves similarly to KPC-2 in interactions with meropenem and meropenem-vaborbactam. Therefore, the presence of bla KPC−157 in the single-copy plasmid pKpMVR1_1 alone cannot explain the high-level meropenem-vaborbactam resistance observed in KpMVR1. Notably, KPC-157 appears less capable of hydrolysing imipenem, aztreonam, ceftazidime, and cefiderocol than KPC-2, and is inhibited by vaborbactam and avibactam but not by relebactam. Isolate KpMVR1 exhibited genetic changes potentially affecting the antimicrobial permeability of its OM compared with KpMVS1. The IS Ec68 -induced frameshift mutation impedes OmpK36’s translocation to the OM, decreasing the influx of β-lactams and β-lactamase inhibitors into the periplasm and resulting in elevated MICs of carbapenems and cephalosporins, with and without β-lactamase inhibitors (Table 2 ). IS Ec68 has been sporadically reported to interrupt ompK36 and mgrB in K. pneumoniae clinical and wastewater isolates, reducing the susceptibility of host bacteria to β-lactams and colistin, respectively [87–89]. Our finding further supports IS Ec68 ’s association with AMR. The gyrA S83Y mutation and loss of OmpK36 function in KpMVR1 are associated with ciprofloxacin resistance [66, 90]. Further studies are needed to assess the impact of the identified marR and acrB mutations on antimicrobial susceptibility of K. pneumoniae and to explore potential interactions between the MarR and AcrB variants. For example, marR inactivation is known to upregulate the AcrAB-TolC efflux pump, conferring low-level cross-resistance to antimicrobials including β-lactams and ciprofloxacin [91]. This study was limited to three clonally related K. pneumoniae isolates, of which only KpMVR1 exhibited elevated MICs of meropenem-vaborbactam, imipenem-relebactam, aztreonam-avibactam, and ceftazidime-avibactam. KpMVR1 carried multiple AMR-associated genetic alterations. Although the individual effects of bla KPC−157 and Δ ompK36 on antimicrobial susceptibility were determined, the transgenic derivatives did not fully reproduce MIC increases observed in KpMVR1, suggesting involvement of additional genetic or regulatory mechanisms. Broader surveillance of K. pneumoniae with resistance profiles like KpMVR1 is warranted to elucidate underlying resistance mechanisms. A review of national surveillance data showed low frequencies of ceftazidime-avibactam resistance in the UK between 2016 and 2020 [80]. However, timely identification and reporting of resistance to β-lactam/β-lactamase inhibitor combinations remain essential through UKHSA’s Second Generation Surveillance System and referral of resistant isolates to the AMRHAI Reference Unit. Our findings also underscore the importance of monitoring within-patient changes in antimicrobial susceptibility profiles of bacterial pathogens during antimicrobial therapy. Abbreviations AMR Antimicrobial resistance AMRHAI Antimicrobial Resistance and Healthcare Associated Infections AST Antimicrobial susceptibility testing CPE Carbapenemase-producing Enterobacterales CRISPR Clustered regularly interspaced short palindromic repeats ESBL Extended-spectrum β-lactamase EUCAST European Committee on Antimicrobial Susceptibility Testing ICU Intensive care unit IS Insertion sequence KPC Klebsiella pneumoniae carbapenemases KPC- Kp KPC-producing K. pneumoniae MIC Minimum inhibitory concentration MLST Multi-locus sequence typing NCBI National Center for Biotechnology Information ONT Oxford Nanopore Technologies OM Outer membrane MALDI-ToF Matrix-assisted laser desorption/ionisation time-of-flight SNP Single-nucleotide polymorphism ST Sequence type UKHSA United Kingdom Health Security Agency VNTR Variable number tandem repeat WGS Whole-genome sequencing Declarations Ethics approval and consent to participate Following an internal review by the UKHSA Research Support and Governance Office (RSGO) it was agreed that this study fell outside the remit for ethics review. This decision was made based on the information provided to the RSGO and on the understanding that the study would be conducted in accordance with the conditions stated in the applicable UKHSA policies and procedures. Consent for publication Not applicable. Competing interests None to declare. Funding This work was supported by the UKHSA, the University of Liverpool David Price Evans Endowment (grant number UGG10057 to Y. W.), and Wellcome Trust–Imperial Institutional Strategic Support Fund Springboard Research Fellowship (grant number PSN109 to Y. W.). Y. W., E. J., D. M., and K. L. H. are affiliated with the National Institute for Health and Care Research Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Imperial College London in partnership with the UKHSA, in collaboration with, Imperial Healthcare Partners, University of Cambridge and University of Warwick (grant number NIHR200876). The views expressed in this article are those of the authors and not necessarily those of the NHS, the National Institute for Health Research, or the Department of Health and Social Care. Author Contribution Conceptualisation: K. L. H. and Y. W.; Resources: K. L. H., J. T., G. F., F. M., N. W., and G. M. R.; Methodology: K. L. H., Y. W., J. L. C. W., and E. J.; Data curation: Y. W.; Investigation and formal analysis: Y. W., J. L. C. W., J. S. G., W. W. L., J. T., F. M., G. M. R., K. D., I. B., G. F., E. J., D. M., and K. L. H.; Visualisation: Y. W.; Writing – original draft: Y. W., J. L. C. W., J. S. G., W. W. L., G. M. R., and K. L. H.; Writing – review and editing: all authors. Acknowledgments We acknowledge the Colebrook Laboratory, a facility supported by the NIHR Imperial Biomedical Research Centre (BRC), for providing bioinformatics resources. Part of the bioinformatics analysis was performed on equipment purchased as part of MRC CARP fellowship award MR/T005254/1. We also thank the Institut Pasteur teams for the curation and maintenance of BIGSdb-Pasteur databases at bigsdb.pasteur.fr. Data Availability Illumina and MinION sequencing reads, genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 are available in the National Center for Biotechnology Information [92] under BioProject PRJNA1084250 [93]. The assemblies are also accessible (website login required) in the Klebsiella PasteurMLST database [94] under ids 75608 (KpMVS1) [95], 75609 (KpMVR1) [96], and 75610 (KpMVS2) [97]. References Baldwin CM, Lyseng-Williamson KA, Keam SJ. Meropenem. Drugs. 2008;68:803–38. https://doi.org/10.2165/00003495-200868060-00006. Meini S, Tascini C, Cei M, Sozio E, Rossolini GM. AmpC β-lactamase-producing Enterobacterales: what a clinician should know. Infection. 2019;47:363–75. https://doi.org/10.1007/s15010-019-01291-9. Livermore DM. Defining an extended-spectrum β-lactamase. Clinical Microbiology and Infection. 2008;14:3–10. https://doi.org/10.1111/j.1469-0691.2007.01857.x. Tooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VHA, Takebayashi Y, et al. β-Lactamases and β-Lactamase Inhibitors in the 21st Century. Journal of Molecular Biology. 2019;431:3472–500. https://doi.org/10.1016/j.jmb.2019.04.002. Jacoby GA. AmpC β-Lactamases. Clinical Microbiology Reviews. 2009;22:161–82. https://doi.org/10.1128/cmr.00036-08. Jorgensen SCJ, Rybak MJ. Meropenem and Vaborbactam: Stepping up the Battle against Carbapenem-resistant Enterobacteriaceae. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2018;38:444–61. https://doi.org/10.1002/phar.2092. Rodloff AC, Goldstein EJC, Torres A. Two decades of imipenem therapy. Journal of Antimicrobial Chemotherapy. 2006;58:916–29. https://doi.org/10.1093/jac/dkl354. Prajapati JD, Kleinekathöfer U, Winterhalter M. How to Enter a Bacterium: Bacterial Porins and the Permeation of Antibiotics. Chem Rev. 2021;121:5158–92. https://doi.org/10.1021/acs.chemrev.0c01213. Lomovskaya O, Sun D, Rubio-Aparicio D, Nelson K, Tsivkovski R, Griffith DC, et al. Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae. Antimicrobial Agents and Chemotherapy. 2017;61:10.1128/aac.01443-17. https://doi.org/10.1128/aac.01443-17. Ehmann DE, Jahić H, Ross PL, Gu R-F, Hu J, Durand-Réville TF, et al. Kinetics of Avibactam Inhibition against Class A, C, and D β-Lactamases. Journal of Biological Chemistry. 2013;288:27960–71. https://doi.org/10.1074/jbc.M113.485979. Carcione D, Siracusa C, Sulejmani A, Leoni V, Intra J. Old and New Beta-Lactamase Inhibitors: Molecular Structure, Mechanism of Action, and Clinical Use. Antibiotics. 2021;10:995. https://doi.org/10.3390/antibiotics10080995. UK Health Security Agency. UK Access, Watch, Reserve, and Other classification for antibiotics (UK-AWaRe antibiotic classification). 2025. Gaibani P, Bovo F, Bussini L, Lazzarotto T, Amadesi S, Bartoletti M, et al. Dynamic evolution of imipenem/relebactam resistance in a KPC-producing Klebsiella pneumoniae from a single patient during ceftazidime/avibactam-based treatments. Journal of Antimicrobial Chemotherapy. 2022;77:1570–7. https://doi.org/10.1093/jac/dkac100. Xu T, Guo Y, Ji Y, Wang B, Zhou K. Epidemiology and Mechanisms of Ceftazidime–Avibactam Resistance in Gram-Negative Bacteria. Engineering. 2022;11:138–45. https://doi.org/10.1016/j.eng.2020.11.004. Gaibani Paolo, Bianco Gabriele, Amadesi Stefano, Boattini Matteo, Ambretti Simone, Costa Cristina. Increased bla KPC Copy Number and OmpK35 and OmpK36 Porins Disruption Mediated Resistance to Imipenem/Relebactam and Meropenem/Vaborbactam in a KPC-Producing Klebsiella pneumoniae Clinical Isolate. Antimicrobial Agents and Chemotherapy. 2022;66:e00191-22. https://doi.org/10.1128/aac.00191-22. Gaibani P, Giani T, Bovo F, Lombardo D, Amadesi S, Lazzarotto T, et al. Resistance to Ceftazidime/Avibactam, Meropenem/Vaborbactam and Imipenem/Relebactam in Gram-Negative MDR Bacilli: Molecular Mechanisms and Susceptibility Testing. Antibiotics. 2022;11:628. https://doi.org/10.3390/antibiotics11050628. Satapoomin Naphat, Dulyayangkul Punyawee, Avison Matthew B. Klebsiella pneumoniae Mutants Resistant to Ceftazidime-Avibactam Plus Aztreonam, Imipenem-Relebactam, Meropenem-Vaborbactam, and Cefepime-Taniborbactam. Antimicrobial Agents and Chemotherapy. 2022;66:e02179-21. https://doi.org/10.1128/aac.02179-21. Wilson WR, Kline EG, Jones CE, Morder KT, Mettus RT, Doi Y, et al. Effects of KPC Variant and Porin Genotype on the In Vitro Activity of Meropenem-Vaborbactam against Carbapenem-Resistant Enterobacteriaceae. Antimicrobial Agents and Chemotherapy. 2019;63:10.1128/aac.02048-18. https://doi.org/10.1128/aac.02048-18. Humphries Romney M., Hemarajata Peera. Resistance to Ceftazidime-Avibactam in Klebsiella pneumoniae Due to Porin Mutations and the Increased Expression of KPC-3. Antimicrobial Agents and Chemotherapy. 2017;61:10.1128/aac.00537 − 17. https://doi.org/10.1128/aac.00537-17. Turton JF, Payne Z, Coward A, Hopkins KL, Turton JA, Doumith M, et al. Virulence genes in isolates of Klebsiella pneumoniae from the UK during 2016, including among carbapenemase gene-positive hypervirulent K1-ST23 and ‘non-hypervirulent’ types ST147, ST15 and ST383. Journal of Medical Microbiology. 2018;67:118–28. https://doi.org/10.1099/jmm.0.000653. The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints (v 15.0). 2025. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. https://doi.org/10.1093/bioinformatics/btu170. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–90. https://doi.org/10.1093/bioinformatics/bty560. Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biology. 2019;20:257. https://doi.org/10.1186/s13059-019-1891-0. Lu J, Breitwieser FP, Thielen P, Salzberg SL. Bracken: Estimating species abundance in metagenomics data. PeerJ Computer Science. 2017;3:e104. https://doi.org/10.7717/peerj-cs.104. Bouras G, Houtak G, Wick RR, Mallawaarachchi V, Roach MJ, Papudeshi B, et al. Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies. Microbial Genomics. 2024;10:001244. https://doi.org/10.1099/mgen.0.001244. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 1, PDF, Supplementary methods. Chklovski A, Parks DH, Woodcroft BJ, Tyson GW. CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nature Methods. 2023;20:1203–12. https://doi.org/10.1038/s41592-023-01940-w. Pedersen BS, Quinlan AR. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics. 2018;34:867–8. https://doi.org/10.1093/bioinformatics/btx699. Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microbial Genomics. 2021;7:000685. https://doi.org/10.1099/mgen.0.000685. Lam MMC, Wick RR, Watts SC, Cerdeira LT, Wyres KL, Holt KE. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nature Communications. 2021;12:4188. https://doi.org/10.1038/s41467-021-24448-3. Wyres KL, Wick RR, Gorrie C, Jenney A, Follador R, Thomson NR, et al. Identification of Klebsiella capsule synthesis loci from whole genome data. Microbial Genomics. 2016;2:e000102. https://doi.org/10.1099/mgen.0.000102. Feldgarden M, Brover V, Gonzalez-Escalona N, Frye JG, Haendiges J, Haft DH, et al. AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci Rep. 2021;11:12728. https://doi.org/10.1038/s41598-021-91456-0. Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Néron B, et al. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Research. 2018;46:W246–51. https://doi.org/10.1093/nar/gky425. Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, et al. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing. Antimicrobial Agents and Chemotherapy. 2014;58:3895–903. https://doi.org/10.1128/aac.02412-14. Robertson J, Nash JHE. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microbial Genomics. 2018;4. https://doi.org/10.1099/mgen.0.000206. Ross Karen, Varani Alessandro M., Snesrud Erik, Huang Hongzhan, Alvarenga Danillo Oliveira, Zhang Jian, et al. TnCentral: a Prokaryotic Transposable Element Database and Web Portal for Transposon Analysis. mBio. 2021;12:10.1128/mbio.02060-21. https://doi.org/10.1128/mbio.02060-21. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Research. 2006;34 suppl_1:D32–6. https://doi.org/10.1093/nar/gkj014. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–100. https://doi.org/10.1093/bioinformatics/bty191. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly. 2012;6:80–92. https://doi.org/10.4161/fly.19695. Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30:1236–40. https://doi.org/10.1093/bioinformatics/btu031. Ittisoponpisan S, Islam SA, Khanna T, Alhuzimi E, David A, Sternberg MJE. Can Predicted Protein 3D Structures Provide Reliable Insights into whether Missense Variants Are Disease Associated? Journal of Molecular Biology. 2019;431:2197–212. https://doi.org/10.1016/j.jmb.2019.04.009. The UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Research. 2019;47:D506–15. https://doi.org/10.1093/nar/gky1049. Zhou Y, Pan Q, Pires DEV, Rodrigues CHM, Ascher DB. DDMut: predicting effects of mutations on protein stability using deep learning. Nucleic Acids Research. 2023;51:W122–8. https://doi.org/10.1093/nar/gkad472. Low WW, Wong JLC, Beltran LC, Seddon C, David S, Kwong H-S, et al. Mating pair stabilization mediates bacterial conjugation species specificity. Nature Microbiology. 2022;7:1016–27. https://doi.org/10.1038/s41564-022-01146-4. Alikhan N-F, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12:402. https://doi.org/10.1186/1471-2164-12-402. Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C, et al. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Research. 2023;51:W484–92. https://doi.org/10.1093/nar/gkad326. Wilkins D, Kurtz Z. gggenes. https://wilkox.org/gggenes/. Wong JLC, Romano M, Kerry LE, Kwong H-S, Low W-W, Brett SJ, et al. OmpK36-mediated Carbapenem resistance attenuates ST258 Klebsiella pneumoniae in vivo . Nature Communications. 2019;10:3957. https://doi.org/10.1038/s41467-019-11756-y. Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD, et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol. 2022;40:1023–5. https://doi.org/10.1038/s41587-021-01156-3. The European Committee on Antimicrobial Susceptibility Testing. MIC determination of non-fastidious and fastidious organisms. https://www.eucast.org/ast_of_bacteria/mic_determination. The European Committee on Antimicrobial Susceptibility Testing. Guidance document on broth microdilution testing of cefiderocol. 2024. Ke W, Bethel CR, Thomson JM, Bonomo RA, van den Akker F. Crystal Structure of KPC-2: Insights into Carbapenemase Activity in Class A β-Lactamases. Biochemistry. 2007;46:5732–40. https://doi.org/10.1021/bi700300u. Gato E, Guijarro-Sánchez P, Alonso-García I, Pedraza-Merino R, Conde A, Lence E, et al. In vitro development of imipenem/relebactam resistance in KPC-producing Klebsiella pneumoniae involves multiple mutations including OmpK36 disruption and KPC modification. International Journal of Antimicrobial Agents. 2023;62:106935. https://doi.org/10.1016/j.ijantimicag.2023.106935. Krishnan NP, Nguyen NQ, Papp-Wallace KM, Bonomo RA, van den Akker F. Inhibition of Klebsiella β-Lactamases (SHV-1 and KPC-2) by Avibactam: A Structural Study. PLOS ONE. 2015;10:e0136813. https://doi.org/10.1371/journal.pone.0136813. Pemberton Orville A., Tsivkovski Ruslan, Totrov Maxim, Lomovskaya Olga, Chen Yu. Structural Basis and Binding Kinetics of Vaborbactam in Class A β-Lactamase Inhibition. Antimicrobial Agents and Chemotherapy. 2020;64:10.1128/aac.00398 − 20. https://doi.org/10.1128/aac.00398-20. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 2, PDF, Supplementary figures. Naas Thierry, Cuzon Gaelle, Villegas Maria-Virginia, Lartigue Marie-Frédérique, Quinn John P., Nordmann Patrice. Genetic Structures at the Origin of Acquisition of the β-Lactamase bla KPC Gene. Antimicrobial Agents and Chemotherapy. 2008;52:1257–63. https://doi.org/10.1128/aac.01451-07. Cuzon Gaelle, Naas Thierry, Nordmann Patrice. Functional Characterization of Tn 4401 , a Tn 3 -Based Transposon Involved in bla KPC Gene Mobilization. Antimicrobial Agents and Chemotherapy. 2011;55:5370–3. https://doi.org/10.1128/aac.05202-11. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 3, XSLX, Supplementary tables. Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae , an urgent threat to public health. Proceedings of the National Academy of Sciences. 2015. https://doi.org/10.1073/pnas.1501049112. Tsang KK, Lam MMC, Wick RR, Wyres KL, Bachman M, Baker S, et al. Diversity, functional classification and genotyping of SHV β-lactamases in Klebsiella pneumoniae . 2024;:2024.04.05.587953. https://doi.org/10.1101/2024.04.05.587953. Huang Y, Lin Q, Zhou Q, Lv L, Wan M, Gao X, et al. Identification of fosA10 , a Novel Plasmid-Mediated Fosfomycin Resistance Gene of Klebsiella pneumoniae Origin, in Escherichia coli . IDR. 2020;13:1273–9. https://doi.org/10.2147/IDR.S251360. Wan Y, Sabnis A, Mumin Z, Potterill I, Jauneikaite E, Brown CS, et al. IS 1 -related large-scale deletion of chromosomal regions harbouring the oxygen-insensitive nitroreductase gene nfsB causes nitrofurantoin heteroresistance in Escherichia coli . Microbial Genomics. 2023;9:001102. https://doi.org/10.1099/mgen.0.001102. Sato T, Yamawaki K. Cefiderocol: Discovery, Chemistry, and In Vivo Profiles of a Novel Siderophore Cephalosporin. Clinical Infectious Diseases. 2019;69 Supplement_7:S538–43. https://doi.org/10.1093/cid/ciz826. Chen F-J, Lauderdale T-L, Ho M, Lo H-J. The Roles of Mutations in gyrA , parC , and ompK35 in Fluoroquinolone Resistance in Klebsiella pneumoniae . Microbial Drug Resistance. 2003;9:265–71. https://doi.org/10.1089/107662903322286472. Praski Alzrigat L, Huseby DL, Brandis G, Hughes D. Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant Escherichia coli . Journal of Antimicrobial Chemotherapy. 2017;72:3016–24. https://doi.org/10.1093/jac/dkx270. Mori H, Ito K. The Sec protein-translocation pathway. Trends in Microbiology. 2001;9:494–500. https://doi.org/10.1016/S0966-842X(01)02174-6. Malinverni Juliana C., Silhavy Thomas J. Assembly of Outer Membrane β-Barrel Proteins: the Bam Complex. EcoSal Plus. 2011;4:10.1128/ecosalplus.4.3.8. https://doi.org/10.1128/ecosalplus.4.3.8. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 4, PNG, Original photo of the Coomassie-stained polyacrylamide gel electrophoresis illustrated in Fig. 2c. Frankel G, David S, Low WW, Seddon C, Wong JLC, Beis K. Plasmids pick a bacterial partner before committing to conjugation. Nucleic Acids Research. 2023;51:8925–33. https://doi.org/10.1093/nar/gkad678. 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. https://doi.org/10.1128/jb.00061-23. Rocker A, Lacey JA, Belousoff MJ, Wilksch JJ, Strugnell RA, Davies MR, et al. Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in Klebsiella pneumoniae . mBio. 2020;11:10.1128/mbio.00603 − 20. https://doi.org/10.1128/mbio.00603-20. National Institute for Health and Care Excellence. Antimicrobial prescribing: meropenem with vaborbactam (Evidence summary [ES21]). National Institute for Health and Care Excellence (NICE). 2019. https://www.nice.org.uk/advice/es21/chapter/Key-messages. National Institute for Health and Care Excellence. Ceftazidime with avibactam for treating severe drug-resistant gram-negative bacterial infections (Health technology evaluation [HTE1]). National Institute for Health and Care Excellence (NICE). 2022. https://www.nice.org.uk/guidance/hte1/chapter/1-Recommendations. National Institute for Health and Care Excellence. Antimicrobial prescribing: imipenem with cilastatin and relebactam (Evidence summary [ES30]). National Institute for Health and Care Excellence (NICE). 2020. https://www.nice.org.uk/advice/es30/evidence. Sader HS, Mendes RE, Duncan L, Kimbrough JH, Carvalhaes CG, Castanheira M. Ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam activities against multidrug-resistant Enterobacterales from United States Medical Centers (2018–2022). Diagnostic Microbiology and Infectious Disease. 2023;106:115945. https://doi.org/10.1016/j.diagmicrobio.2023.115945. Hernández-García Marta, García-Castillo María, Bou Germán, Cercenado Emilia, Delgado-Valverde Mercedes, Oliver Antonio, et al. Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies). Microbiology Spectrum. 2022;10:e02927-22. https://doi.org/10.1128/spectrum.02927-22. Shoulders BR, Casapao AM, Venugopalan V. An Update on Existing and Emerging Data for Meropenem-Vaborbactam. Clinical Therapeutics. 2020;42:692–702. https://doi.org/10.1016/j.clinthera.2020.01.023. Guy RL, Hopkins KL, Budd EL, Wilson K, Fountain H, Meunier D, et al. The importance of monitoring a new antibiotic: ceftazidime/avibactam usage and resistance experience from England, 2016 to 2020. Eurosurveillance. 2025;30:2400399. https://doi.org/10.2807/1560-7917.ES.2025.30.14.2400399. Bovo F, Amadesi S, Palombo M, Lazzarotto T, Ambretti S, Gaibani P. Clonal dissemination of Klebsiella pneumoniae resistant to cefiderocol, ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam co-producing KPC and OXA-181 carbapenemase. JAC-Antimicrobial Resistance. 2023;5:dlad099. https://doi.org/10.1093/jacamr/dlad099. Sauerborn E, Corredor NC, Reska T, Perlas A, Vargas da Fonseca Atum S, Goldman N, et al. Detection of hidden antibiotic resistance through real-time genomics. Nature Communications. 2024;15:5494. https://doi.org/10.1038/s41467-024-49851-4. Fu Yiqi, Zhang Yuchao, Zhao Jun, Xie Jiahong, Kong Haishen, Yang Tingting, et al. Emergence of co-resistance to imipenem/relebactam and ceftazidime/avibactam in clinical Klebsiella pneumoniae ST11 clone due to KPC-2 N132S and CTX-M-65 S130G/P167S substitutions. Antimicrobial Agents and Chemotherapy. 2025;0:e00891-25. https://doi.org/10.1128/aac.00891-25. Furlan JPR, Bueno GC, Sousa-Carmo RR, Silva RLO, Barbosa MRF, Sato MIZ, et al. KPC-157 and KPC-181 carbapenemases produced by Citrobacter freundii ST522 and Klebsiella pneumoniae ST258 isolated from wastewater. Molecular Biology Reports. 2025;52:705. https://doi.org/10.1007/s11033-025-10801-y. Theuretzbacher U, Carrara E, Conti M, Tacconelli E. Role of new antibiotics for KPC-producing Klebsiella pneumoniae . Journal of Antimicrobial Chemotherapy. 2021;76 Supplement_1:i47–54. https://doi.org/10.1093/jac/dkaa497. Papp-Wallace Krisztina M., Winkler Marisa L., Taracila Magdalena A., Bonomo Robert A. Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam. Antimicrobial Agents and Chemotherapy. 2015;59:3710–7. https://doi.org/10.1128/aac.04406-14. Wu L-T, Guo M-K, Ke S-C, Lin Y-P, Pang Y-C, Nguyen H-TV, et al. Characterization of the Genetic Background of KPC-2-Producing Klebsiella pneumoniae with Insertion Elements Disrupting the ompK36 Porin Gene. Microbial Drug Resistance. 2020;26:1050–7. https://doi.org/10.1089/mdr.2019.0410. Hamel M, Chatzipanagiotou S, Hadjadj L, Petinaki E, Papagianni S, Charalampaki N, et al. Inactivation of mgrB gene regulator and resistance to colistin is becoming endemic in carbapenem-resistant Klebsiella pneumoniae in Greece: A nationwide study from 2014 to 2017. International Journal of Antimicrobial Agents. 2020;55:105930. https://doi.org/10.1016/j.ijantimicag.2020.105930. Hayashi W, Iimura M, Soga E, Koide S, Izumi K, Yoshida S, et al. Presence of Colistin- and Tigecycline-Resistant Klebsiella pneumoniae ST29 in Municipal Wastewater Influents in Japan. Microbial Drug Resistance. 2021;27:1433–42. https://doi.org/10.1089/mdr.2020.0514. Martínez-Martínez L, Hernández-Allés S, Albertí S, Tomás J M, Benedi V J, Jacoby G A. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum-cephalosporins. Antimicrobial Agents and Chemotherapy. 1996;40:342–8. https://doi.org/10.1128/aac.40.2.342. Randall LP, Woodward MJ. The multiple antibiotic resistance ( mar ) locus and its significance. Research in Veterinary Science. 2002;72:87–93. https://doi.org/10.1053/rvsc.2001.0537. National Center for Biotechnology Information. www.ncbi.nlm.nih.gov. Accessed 27 Nov 2025. Wan Y, Wong J, Sanchez-Garrido J, Low W, Turton J, Morecchiato F, et al. Full information on isolate KpMVR1 (id:75609). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info&db=pubmlst_klebsiella_isolates&id=75609. Accessed 27 Nov 2025. Klebsiella pneumoniae species complex. Institut Pasteur. https://bigsdb.pasteur.fr/klebsiella/. Accessed 27 Nov 2025. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Full information on isolate KpMVS1 (id:75608). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info&db=pubmlst_klebsiella_isolates&id=75608. Accessed 27 Nov 2025. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Full information on isolate KpMVR1 (id:75609). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info&db=pubmlst_klebsiella_isolates&id=75609. Accessed 27 Nov 2025. Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Full information on isolate KpMVS2 (id:75610). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info&db=pubmlst_klebsiella_isolates&id=75610. Accessed 27 Nov 2025. Additional Declarations No competing interests reported. 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09:32:21","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42990,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/dc072470cfd95898a786f232.pdf"},{"id":98756322,"identity":"d429e657-68d1-4e76-8c4f-5dcab903d7f5","added_by":"auto","created_at":"2025-12-22 09:32:21","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":102530,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/774124eed94be2e0180305de.pdf"},{"id":98777760,"identity":"f2252d5b-ad67-494b-adf6-23fc4bad9a14","added_by":"auto","created_at":"2025-12-22 12:28:25","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172712,"visible":true,"origin":"","legend":"","description":"","filename":"ed551f81a0e0442289010905c293203a1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/ef95155cec39ca6c9e8ceb5c.xml"},{"id":98756326,"identity":"ad909a9e-a095-44e3-85e8-3c11af28d164","added_by":"auto","created_at":"2025-12-22 09:32:21","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":186279,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/4ad56deabe9fc387f77875eb.html"},{"id":98756307,"identity":"65f9f223-826c-48a5-8782-f1c9d81c9182","added_by":"auto","created_at":"2025-12-22 09:32:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":40888,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic 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 region. Genes without known names are not labelled. Each asterisk indicates an allele from a named gene family.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/3ce505f26a8e0582945a1224.png"},{"id":98778583,"identity":"a6e9570d-74a0-42ed-aafc-31f6df4c9931","added_by":"auto","created_at":"2025-12-22 12:29:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":177075,"visible":true,"origin":"","legend":"\u003cp\u003eIS\u003cem\u003eEc68\u003c/em\u003e-mediated disruption of \u003cem\u003eompK36\u003c/em\u003e in isolate KpMVR1. (\u003cstrong\u003ea\u003c/strong\u003e) Genetic environment of the disrupted \u003cem\u003eompK36\u003c/em\u003e in KpMVR1. The arrow labelled “\u003cem\u003eompK36*\u003c/em\u003e” denotes the upstream-shifted open reading frame caused by the insertion of IS\u003cem\u003eEc68\u003c/em\u003e. Abbreviations: CDS: coding sequence; IS, insertion sequence; ncRNA: non-coding RNA. (\u003cstrong\u003eb\u003c/strong\u003e) Comparison of predicted OmpK36 sequences using Clustal Omega (www.ebi.ac.uk/jdispatcher/msa/clustalo). Mismatches are highlighted in red, and the 22 N-terminal amino acids signal sequence of OmpK36 are indicated by the yellow shade. (\u003cstrong\u003ec\u003c/strong\u003e) Coomassie-stained polyacrylamide gel electrophoresis of outer membrane proteins to confirm the absence of OmpK36 in KpMVR1 and the \u003cem\u003eompK36\u003c/em\u003e-knockout isolate ICC8001\u003csub\u003eΔ\u003c/sub\u003e\u003csub\u003e\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e. See Additional File 4 for the original gel image.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/e9b227bc4dbe35d2c2181626.png"},{"id":98783533,"identity":"71b73b6a-52b3-45d0-8cb0-17c99af6b179","added_by":"auto","created_at":"2025-12-22 12:42:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1492026,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/0bc9d215-86a1-4194-9967-8b1ec3a3a457.pdf"},{"id":98756308,"identity":"1ae10a8f-6e67-470e-b63b-9a3e7aa0a315","added_by":"auto","created_at":"2025-12-22 09:32:21","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":31597,"visible":true,"origin":"","legend":"","description":"","filename":"2AdditionalFile120251205.docx","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/e68b60685fc458d8b3f79174.docx"},{"id":98756327,"identity":"3f958577-2c1b-47f8-8afe-e200ecb4d80b","added_by":"auto","created_at":"2025-12-22 09:32:21","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":6102557,"visible":true,"origin":"","legend":"","description":"","filename":"3AdditionalFile220251205.docx","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/a065e406ac959cf56e5b381f.docx"},{"id":98777874,"identity":"71d094b6-329e-4a68-966d-03e11d318e4e","added_by":"auto","created_at":"2025-12-22 12:28:36","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":57852,"visible":true,"origin":"","legend":"","description":"","filename":"4AdditionalFile320251205.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/08ec7cde8cbfbfa086c90ee1.xlsx"},{"id":98780641,"identity":"a1e107ea-4799-41fa-bb80-20b41fbc6ab5","added_by":"auto","created_at":"2025-12-22 12:31:31","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":660562,"visible":true,"origin":"","legend":"","description":"","filename":"5AdditionalFile420251210.png","url":"https://assets-eu.researchsquare.com/files/rs-8289965/v1/e45de5acb68f1c70e17cc0f7.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic and molecular characterisation of a KPC-producing Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam","fulltext":[{"header":"Background","content":"\u003cp\u003eMeropenem and imipenem are broad-spectrum carbapenem antimicrobials parenterally administered to treat serious bacterial infections, such as those caused by Enterobacterales producing extended-spectrum β-lactamases (ESBLs) or AmpC-type cephalosporinases (AmpCs) [1\u0026ndash;3]. Ceftazidime, a third-generation parenteral cephalosporin, is also widely used for treating severe bacterial infections, although it can be hydrolysed by ESBLs and AmpCs [4, 5]. In Gram-negative bacteria, carbapenems and cephalosporins diffuse through outer-membrane porins and enter the periplasm, where they inactivate penicillin binding proteins, disrupting cell-wall synthesis with a bactericidal effect [6\u0026ndash;8].\u003c/p\u003e \u003cp\u003eCombining β-lactams with β-lactamase inhibitors in antimicrobial chemotherapy is a widely employed strategy to circumvent β-lactamase-mediated resistance in bacteria. Vaborbactam, relebactam, and avibactam are non-β-lactam inhibitors of Ambler class A β-lactamases, such as ESBLs and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemases (KPCs), as well as class C β-lactamases (AmpCs) [9]. Moreover, avibactam inhibits several class D β-lactamases such as OXA-48 and OXA-10 [10]. These inhibitors penetrate the outer membrane (OM) of Gram-negative bacteria via porins, blocking the active sites of β-lactamases in the periplasm [9, 11].\u003c/p\u003e \u003cp\u003eIn the UK, meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam are reserved for highly selected patients [12]. Resistance to meropenem-vaborbactam and imipenem-relebactam in clinical isolates of KPC-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e (KPC-\u003cem\u003eKp\u003c/em\u003e) has been reported in Italy, while ceftazidime-avibactam resistance has been documented across continental Europe and Americas [13\u0026ndash;15]. Identified resistance mechanisms include KPC overproduction, gain-of-function point mutations in \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e, KPC-8 (formerly, KPC-3 V239G), the AcrAB-TolC multidrug efflux pump, and mutation of \u003cem\u003eramR\u003c/em\u003e [9, 15\u0026ndash;17]. Additionally, disruption or transcriptional downregulation of \u003cem\u003eompK35\u003c/em\u003e (\u003cem\u003eompF\u003c/em\u003e) and \u003cem\u003eompK36\u003c/em\u003e (\u003cem\u003eompC\u003c/em\u003e), which encode non-selective porins that mediate the diffusion of β-lactams and β-lactamase inhibitors through the OM, has also been implicated [15, 16, 18, 19].\u003c/p\u003e \u003cp\u003eIn 2021, a KPC-\u003cem\u003eKp\u003c/em\u003e clinical isolate (KpMVR1) resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam was identified in England with evidence suggesting \u003cem\u003ein vivo\u003c/em\u003e development of this unusual phenotype during prolonged intensive care of a patient. Here, we describe the genomic and molecular characteristics of this isolate, analysed alongside two clonally related isolates recovered from the same hospital using comparative genomics and microbiological experiments.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolate collection and phenotyping\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eK. pneumoniae\u003c/em\u003e isolate KpMVS1 was recovered from a lung biopsy specimen of an inpatient (hereafter, Patient 1) admitted to an intensive care unit (ICU) in England in 2021. The second \u003cem\u003eK. pneumoniae\u003c/em\u003e isolate, KpMVR1, was recovered from a groin wound of the same patient 42 days later. During this ICU stay, the patient received a broad range of antimicrobials, including meropenem-vaborbactam, ciprofloxacin, and gentamicin; however, ceftazidime-avibactam and imipenem-relebactam were not used.\u003c/p\u003e \u003cp\u003eSpecies identification and carbapenemase gene screening were performed using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-ToF) mass spectrometry and the GeneXpert system (Cepheid, USA), respectively. Initial antimicrobial susceptibility testing (AST) was conducted by the hospital and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Both isolates were referred to the Antimicrobial Resistance and Healthcare Associated Infections (AMRHAI) Reference Unit of the UK Health Security Agency (UKHSA) for variable number tandem repeat (VNTR) typing [20] and investigation of unusual antimicrobial resistance (AMR). Additionally, a KPC-\u003cem\u003eKp\u003c/em\u003e isolate KpMVS2\u0026mdash;recovered from a rectal swab of another patient (Patient 2) in the same hospital in 2020 during an outbreak investigation and sharing the same VNTR profile as KpMVS1 and KpMVR1\u0026mdash;was retrieved from AMRHAI\u0026rsquo;s culture collection for comparison. All three isolates underwent whole-genome sequencing (WGS) and AST, including determination of minimum inhibitory concentrations (MICs) for 19 antimicrobials (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and inhibition zone diameters for cefiderocol, interpreted according to EUCAST clinical breakpoints v15.0 [21].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial minimum inhibitory concentrations (MICs; mg/L) determined by UKHSA\u0026rsquo;s AMRHAI Reference Unit and susceptibility interpretations (according to EUCAST clinical breakpoints v15.0 where applicable) of three \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolates. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; ETP, ertapenem; CET, ceftolozane; TZB, tazobactam; CFD, cefiderocol; FEP, cefepime; CAZ, ceftazidime; AVI, avibactam; CTX, cefotaxime; FOX, cefoxitin; TMC, temocillin; AMP, ampicillin; AMX, amoxicillin; CAV, clavulanate; PIP, piperacillin; ATM, aztreonam; AMK, amikacin; GEN, gentamicin; CST, colistin; CIP, ciprofloxacin; MB, monobactam. Susceptibility interpretations: R, resistant; I, susceptible, increased exposure; S, susceptible.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"21\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c18\" colnum=\"18\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c19\" colnum=\"19\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c20\" colnum=\"20\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c21\" colnum=\"21\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eCarbapenem\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c12\" namest=\"c6\"\u003e \u003cp\u003eCephalosporin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c16\" namest=\"c13\"\u003e \u003cp\u003ePenicillin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003eMB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c19\" namest=\"c18\"\u003e \u003cp\u003eAminoglycoside\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c21\" namest=\"c20\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMEM-VAB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIPM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eETP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCET-TZB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCFD\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFEP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eCAZ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCAZ-AVI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCTX\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eFOX\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eTMC\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eAMP\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003eAMX-CAV\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c16\"\u003e \u003cp\u003ePIP-TZB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c17\"\u003e \u003cp\u003eATM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c18\"\u003e \u003cp\u003eAMK\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c19\"\u003e \u003cp\u003eGEN\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c20\"\u003e \u003cp\u003eCST\u003csup\u003e\u0026Dagger;\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c21\"\u003e \u003cp\u003eCIP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;256\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;4\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4\u003c/p\u003e \u003cp\u003e(I)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e256\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e8\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;4\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.064\u003c/p\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;4\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e128\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e64\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.125\u003c/p\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.032\u003c/p\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;4\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e64\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e1\u003c/p\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e16\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c15\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c16\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c17\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;32\u003c/p\u003e \u003cp\u003e(R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c18\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c19\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c20\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c21\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.125\u003c/p\u003e \u003cp\u003e(S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"21\"\u003e\u003csup\u003e*\u003c/sup\u003e CFD susceptibility was determined using disc diffusion. \u003csup\u003e\u0026Dagger;\u003c/sup\u003e Interpretations were not available according to EUCAST guidelines.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWhole-genome sequencing\u003c/h3\u003e\n\u003cp\u003eGenomic DNA was extracted from overnight cultures using the GeneJET Kit (ThermoFisher Scientific, UK) according to the manufacturer\u0026rsquo;s protocol. Short-read sequencing was performed on a HiSeq 2500 system (Illumina, USA) at UKHSA\u0026rsquo;s Colindale Sequencing Laboratory using a paired-end 101-bp protocol. Long-read sequencing was conducted on MinION R9.4.1 flow cells (Oxford Nanopore Technologies [ONT], UK), with libraries prepared using the ONT Rapid Barcoding Kit SQK-RBK004.\u003c/p\u003e\n\u003ch3\u003eBioinformatics analysis\u003c/h3\u003e\n\u003cp\u003eIllumina reads were trimmed and filtered with Trimmomatic v0.39 for a minimum per-read quality of Phred Q30 and minimum length of 50 bp [22]. Fast-mode basecalling and de-multiplexing of MinION reads was conducted by guppy v4 (ONT), followed by read trimming and filtering for a minimum per-read quality of Q10 and minimum length of 1 kbp with fastp v0.23.4 [23]. For species confirmation and contamination assessment, taxonomical profiling of processed Illumina and MinION reads were performed using Kraken v2.1.3, bracken v2.8, and a standard Kraken database built in September 2023 [24, 25].\u003c/p\u003e \u003cp\u003eGenomes of KpMVR1 and KpMVS2, with estimated MinION read depths of 185\u0026times; and 243\u0026times;, respectively, were assembled using hybracter v0.5.0 (Additional File 1) [26, 27]. For KpMVS1, which had an estimated MinION read depth of 64\u0026times;, chromosomal and plasmid sequences were assembled using Raven v1.8.3 and plassembler, respectively, and subsequently polished with MinION reads followed by Illumina reads, as for KpMVR1 and KpMVS2. Genome assemblies were assessed for quality using CheckM2 v1.0.2 and its database Uniref100/KO [28]. The average fold-coverage of each contig was estimated from Illumina and MinION reads, respectively, using mosdepth v0.3.9 [29].\u003c/p\u003e \u003cp\u003eThe genome assemblies were annotated using bakta v1.9.2 and its standard database v5.1 [30]. Multi-locus sequence typing (MLST), serotype prediction, and virulence-factor detection were performed using Kleborate v3.1.3, which incorporated Kaptive v3.1.0 [31, 32]. Genome assemblies of all three isolates were submitted to the \u003cem\u003eKlebsiella\u003c/em\u003e PasteurMLST database (bigsdb.pasteur.fr) for assignment of a new sequence type (ST) number. AMR genes were detected using AMRFinderPlus v3.12.8 with a minimum query coverage of 80% [33]. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes were predicted for chromosomes using CRISPRCasFinder [34]. For plasmids, replicon types were determined at a minimum of 80% nucleotide identity and coverage using PlasmidFinder v2.1 [35], and the mobility was predicted using mob_typer of MOB-suite v3.1.8 [36]. The copy number of each KPC-encoding plasmid was estimated by dividing the plasmid\u0026rsquo;s fold-coverage by that of its host\u0026rsquo;s chromosome. Transposons and insertion sequences (ISs) were identified using TnCentral Blast (blastn) and ISFinder, respectively [37, 38].\u003c/p\u003e \u003cp\u003eChromosome and plasmids of KpMVR1 and KpMVS2 were compared against those of KpMVS1 using minimap v2.26 [39]. Identified genetic variants were annotated using snpEff v5.2 [40]. Gene Ontology terms were predicted from amino acid sequences using InterProScan v5.69-101.0 with sequence alignments filtered for \u0026ge;\u0026thinsp;60% query coverages [41]. Impacts of point mutations on protein stability were predicted from wild-type protein structures in the UniProt database using Missense3D and DDMut [42\u0026ndash;44]. The three-dimensional structure of the plasmid-encoded donor OM protein TraN was compared between KPC-encoding, IncFII-carrying plasmids pKpMVS1_1, pKpMVR1_1, and pKpMVS2_1 to estimate the impact of TraN alterations on the conjugation specificity and efficiency (Additional File 1) [27, 45]. Comparison and annotations of these three plasmids were visualised using BRIG v0.95 and Proksee [46, 47]. Gene synteny was illustrated using R package gggenes [48]. Genetic alterations in both KpMVR1 and KpMVS2 were considered unlikely to confer the unique AMR profiles of KpMVR1 and were therefore excluded from further investigation.\u003c/p\u003e\n\u003ch3\u003eFunctional assessment\u003c/h3\u003e\n\u003cp\u003eTo determine and compare the impacts of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e on β-lactam susceptibility in \u003cem\u003eK. pneumoniae\u003c/em\u003e, KpMVS1\u0026rsquo;s KPC-2-encoding plasmid pKpMVS1_1 was introduced into the plasmid-free \u003cem\u003eK. pneumoniae\u003c/em\u003e laboratory strain ICC8001 (MICs: meropenem, \u0026le;\u0026thinsp;0.06 mg/L; imipenem, 0.25 mg/L; aztreonam, \u0026le;\u0026thinsp;0.125 mg/L; ceftazidime and ceftazidime-avibactam, 0.25 mg/L) through conjugation, resulting in a transconjugant ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e [49]. Transgenic isolates ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e and KpMVS1\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e were derived from ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e and KpMVS1, respectively, by substituting \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e. Moreover, isolates ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e and ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e were derived from ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e and ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e, respectively, through seamless, markerless homologous recombination using mutagenesis vectors and a lambda-red based recombination system generated in previous work [49].\u003c/p\u003e \u003cp\u003eTo predict the presence or absence of OmpK36 in KpMVR1\u0026rsquo;s OM, Sec-dependent signal peptides and their cleavage sites in translated \u003cem\u003eompK36\u003c/em\u003e alleles were compared between KpMVS1 and KpMVR1 using SignalP v6.0 [50]. To validate this prediction, OM proteins were purified from overnight cultures of KpMVS1, KpMVR1, KpMVS2, ICC8001, and an \u003cem\u003eompK36\u003c/em\u003e-knockout derivative, ICC8001\u003csub\u003eΔ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e, separated by SDS-PAGE, and visualised by Coomassie staining (Additional File 1) [27].\u003c/p\u003e \u003cp\u003eBoth progenitor isolates (KpMVS1 and KpMVR1) and five transgenic derivatives (KpMVS1\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e, ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e, ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e, ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e, and ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e) were tested for susceptibility to meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam, imipenem-avibactam, ceftazidime, ceftazidime-avibactam, aztreonam, aztreonam-avibactam, and cefiderocol (in iron-depleted medium) using broth microdilution according to EUCAST guidelines [51, 52]. Any MIC change exceeding a two-fold difference between two isolates was considered notable.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypes of isolates\u003c/h2\u003e \u003cp\u003eIsolates KpMVS1 and KpMVR1, obtained 42 days apart from Patient 1 with recurrent \u003cem\u003eK. pneumoniae\u003c/em\u003e infections, and KpMVS2, obtained from Patient 2 in the same hospital, were identified as \u003cem\u003eK. pneumoniae\u003c/em\u003e by both MALDI-ToF and the WGS. In the ICU where KpMVS1 and KpMVR1 were recovered, all patients were screened for carriage of carbapenemase-producing Enterobacterales (CPE) on admission using PCR. The resistance profile of KpMVR1 was unique among all CPE isolates identified in the ICU during Patient 1\u0026rsquo;s stay.\u003c/p\u003e \u003cp\u003eBased on AST results from the AMRHAI Reference Unit (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), KpMVR1 was resistant to meropenem-vaborbactam (MIC\u0026thinsp;\u0026gt;\u0026thinsp;256 mg/L), ceftazidime-avibactam (MIC\u0026thinsp;=\u0026thinsp;16 mg/L), and ciprofloxacin (MIC\u0026thinsp;\u0026gt;\u0026thinsp;4 mg/L), whereas KpMVS1 and KpMVS2 were susceptible to these antimicrobials (MICs: meropenem-vaborbactam\u0026thinsp;\u0026le;\u0026thinsp;0.064 mg/L; ceftazidime-avibactam 1 mg/L; ciprofloxacin\u0026thinsp;\u0026le;\u0026thinsp;0.125 mg/L). KpMVS2 was resistant to cefiderocol (inhibition zone diameter: 19 mm) as determined using disc diffusion. Further AST revealed that the imipenem-relebactam MIC of KpMVR1 (512 mg/L, resistant) was 2048-fold higher than that of KpMVS1 (0.25 mg/L, susceptible) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, KpMVR1 exhibited a\u0026thinsp;\u0026gt;\u0026thinsp;4-fold increase in the temocillin MIC (\u0026gt;\u0026thinsp;128 mg/L) and a\u0026thinsp;\u0026gt;\u0026thinsp;8-fold reduction in the cefepime MIC (4 mg/L, susceptible, increased exposure) compared with KpMVS1 (temocillin: 32 mg/L; cefepime: \u0026gt;32 mg/L, resistant).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMinimum inhibitory concentrations of β-lactam antimicrobials and susceptibility interpretations (according to EUCAST clinical breakpoints v15.0 where applicable) of progenitor and transgenic \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates determined in the experiments for functional assessment. Subscripts in isolate names indicate transgenic isolates and corresponding genotypes. Abbreviations: MEM, meropenem; VAB, vaborbactam; IPM, imipenem; REL, relebactam; ATM, aztreonam; AVI, avibactam; CAZ, ceftazidime; CFD, cefiderocol, tested in iron-depleted Mueller Hinton broth; NT, not tested. Interpretations of antimicrobial susceptibility: R, resistant; I, susceptible, increased exposure; S, susceptible. Notations: \u003cem\u003eompK36\u003c/em\u003efs, frameshifted \u003cem\u003eompK36\u003c/em\u003e; Δ\u003cem\u003eompK36\u003c/em\u003e, deletion of \u003cem\u003eompK36\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c3\" namest=\"c2\" rowspan=\"2\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c13\" namest=\"c4\"\u003e \u003cp\u003eMinimum Inhibitory Concentration (mg/L) and interpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMEM-VAB\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIPM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eIPM-REL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eIPM-AVI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eATM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eATM-AVI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCAZ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eCAZ-AVI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eCFD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eompK36\u003c/em\u003efs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e512 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e256 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e512 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e512 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e4 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e8 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e512 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVS1\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2 (I)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e512 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.125 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e32 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 (I)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.125 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.125 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;0.06 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔ\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e256 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e256 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1024 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e16 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔ\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e256 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e256 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e128 (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4 (I)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.5 (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.25 (S)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGenetic characteristics of isolates\u003c/h3\u003e\n\u003cp\u003eAll three isolates belonged to \u003cem\u003eK. pneumoniae\u003c/em\u003e clone ST8134, a single-locus variant of ST240, and were predicted to share the O1αβ,2β O-antigen type and K62 capsular polysaccharide type. Sequence lengths, plasmid replicons, and AMR genes determined in hybrid genome assemblies are summarised in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. KpMVR1 and KpMVS1 shared the same plasmid types IncFII/repB(R1701), IncFII(pMET), Col(pHAD28), and Col(pHAD28)/Col440II, whereas KpMVS2 possessed unique plasmid types IncFII/IncR and IncFII(pKP91)/FIB(K).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenetic characteristics of the three \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolates. The plasmid type refers to the haplotype of plasmid replicons. Each hit of plasmid replicons in this table covered the full length of its reference sequence in the PlasmidFinder database. Abbreviation: AMR, antimicrobial resistance.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCategory\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLength (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlasmid type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNucleotide identity to template\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePlasmid mobility\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAMR gene\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKpMVS1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5,404,514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;36\u003c/sub\u003e, \u003cem\u003efosA10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS1_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e111,397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII/repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pKP91): 100%; repB(R1701): 99.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eConjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS1_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41,868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(pMET)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pMET): 98.09%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS1_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol(pHAD28): 92.37%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS1_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)/Col440II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol(pHAD28): 93.13%; Col440II: 97.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS1_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot detected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS1_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1,917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol(pHAD28): 100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKpMVR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5,292,801\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;36\u003c/sub\u003e, \u003cem\u003efosA10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVR1_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e111,174\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII/repB(R1701)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pKP91): 100%, repB(R1701): 99.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eConjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVR1_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41,868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(pMET)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pMET): 98.09%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVR1_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol(pHAD28): 92.37%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVR1_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)/Col440II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol(pHAD28): 93.13%; Col440II: 97.52%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVR1_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot detected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVR1_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1,917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol (pHAD28): 100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKpMVS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKpMVS2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5,354,507\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;36\u003c/sub\u003e, \u003cem\u003efosA10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e116,795\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII/IncR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pKP91): 100%; IncR: 99.6%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eConjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41,868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(pMET)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pMET): 98.09%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol (pHAD28): 92.37%\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4,187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol (pHAD28): 92.37%\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3,258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNot detected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNon-mobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1,917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCol(pHAD28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCol (pHAD28): 100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMobilisable\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epKpMVS2_7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlasmid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e240,297\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIncFII(pKP91)/IncFIB(K)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIncFII(pKP91): 84.98%; IncFIB(K): 98.93%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eConjugative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003e*\u003c/sup\u003e Two hits of the Col(pHAD28) template sequence in the PlasmidFinder database differed between pKpMVS2_3 and pKpMVS2_4 by seven nucleotide substitutions (95% nucleotide identity) despite their same percent identity to the template.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eKpMVS1 carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e on the 111.4 kbp IncFII(pKP91)/repB(R1701) plasmid pKpMVS1_1. A plasmid of the same type was identified in KpMVR1 (pKpMVR1_1, 111.2 kbp) and carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e, which differed from \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e by a single missense mutation (392A\u0026thinsp;\u0026gt;\u0026thinsp;G) resulting in an N131S amino acid substitution within the enzyme\u0026rsquo;s active site (NCBI Protein accessions: KPC-2, WP_004199234.1; KPC-157, WP_259115967.1) [53], where the N131 residue binds to relebactam, avibactam, and vaborbactam through a hydrogen bond [54\u0026ndash;56]. Notably, pKpMVR1_1 differed from pKpMVS1_1 by 285 nucleotide substitutions, 14 deletions, and three insertions. These variants were concentrated in two genomic regions involved in plasmid transfer and maintenance (Supplementary Fig.\u0026nbsp;1, Additional File 2) [57], suggesting recombination between plasmids. Another plasmid type, IncFII(pKP91)/IncR, in KpMVS2 carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e. All these KPC-encoding plasmids were predicted to be conjugative (relaxase type: MOBF; mating pair formation type: MPF_F), and each carried an intact variant of the Tn\u003cem\u003e4401a\u003c/em\u003e transposon harbouring \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e or \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e, with an ATTGA target site duplication and 1\u0026ndash;2 single-nucleotide polymorphisms (SNPs) between each pair of Tn\u003cem\u003e4401a\u003c/em\u003e variants (Supplementary Fig.\u0026nbsp;1, Additional File 2; Supplementary Tables\u0026nbsp;1 and 2, Additional File 3) [27, 58\u0026ndash;60]. The comparison between fold-coverages of contigs suggested that each of these three isolates carried a single copy of the KPC-encoding plasmid. Other AMR genes detected were chromosomal β-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e (variant \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;36\u003c/sub\u003e) and fosfomycin resistance gene \u003cem\u003efosA10\u003c/em\u003e, which are both intrinsic to \u003cem\u003eK. pneumoniae\u003c/em\u003e [61\u0026ndash;63].\u003c/p\u003e \u003cp\u003eThe chromosome of KpMVR1 differed from that of KpMVS1 by six SNPs, seven insertions, four small deletions (1\u0026ndash;15 bp), and four large deletions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Figs.\u0026nbsp;2\u0026ndash;5, Additional File 2; Supplementary Table\u0026nbsp;3\u0026ndash;6, Additional File 3) [57, 60]. Seven variants were also identified in KpMVS2 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), which differed from KpMVS1 by 129 SNPs, 13 insertions, and seven deletions. The 19.7 kbp and 4.9 kbp deletions in KpMVR1 may have been mediated by IS elements, as previously reported in \u003cem\u003eEscherichia coli\u003c/em\u003e (Supplementary Figs.\u0026nbsp;3 and 5, Additional File 2) [57, 64]. Notably, KpMVR1 exhibited a 54.7 kbp deletion encompassing operons encoding an AcrAB-like multidrug efflux pump and an additional ABC-type Fe\u003csup\u003e3+\u003c/sup\u003e-siderophore transport system present in both KpMVS1 and KpMVS2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Table\u0026nbsp;3, Additional File 3) [60]. All three isolates carried a single copy of the \u003cem\u003eacrRAB\u003c/em\u003e operon and \u003cem\u003etolC\u003c/em\u003e gene, which together produce the AcrAB-TolC multidrug efflux pump. However, the permease AcrB in KpMVR1 harboured a destabilising L667R mutation located outside the protein\u0026rsquo;s transmembrane domains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChromosomal genetic variation in isolate KpMVR1 identified via comparison against its progenitor KpMVS1. Coordinates refer to locations in the reference sequence of the KpMVS1 chromosome. Variants shared by both KpMVR1 and KpMVS2 against their common reference sequence of KpMVS1 are indicated by asterisks following the coordinates. The \u0026ldquo;^\u0026rdquo; sign indicates an insertion between two consecutive bases in the reference sequence. Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; Cas: CRISPR-associated genes; Del, deletion; Ins, insertion; fs, frameshift.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProduct\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVariant type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDNA change\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProtein change\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e455179\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003errl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23S rRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstitution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1050491\u0026ndash;1070213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType I-E CRISPR-Cas system, \u003cem\u003eetc\u003c/em\u003e. (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of 19,723 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLoss of production\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1113441\u0026ndash;1113446\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eflhA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFormate hydrogenlyase transcriptional activator\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of 6 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL367Del, T368Del\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1218110^1218111 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003errl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23S rRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion of base G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1578603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003egyrA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDNA topoisomerase (ATP-hydrolyzing) subunit A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstitution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e248C\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eS83Y\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1588108^1588109\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eompK36\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOuter membrane porin OmpK36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion of IS\u003cem\u003eEc68\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAmino acid substitutions\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1724677^1724678 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003exylB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eXylulose kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion of base G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eI236fs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1792900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003erfbD\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUDP-galactopyranose mutase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstitution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e578T\u0026thinsp;\u0026gt;\u0026thinsp;A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eM193K\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1819375^1819376\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntergenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion of base C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2183835^2183836 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntergenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion of base T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2183837 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntergenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstitution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2201799\u0026ndash;2256547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMultiple products including transporters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of 54,749 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLoss of production\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2759671\u0026ndash;2759685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003emarR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMultiple-AMR (Mar) transcriptional repressor MarR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of bases 263\u0026ndash;277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eP88\u0026ndash;D92Del, K93Q\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3157221^3157222 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntergenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion of base C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3189754\u0026ndash;3223285 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMultiple products (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of 33,532 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLoss of production\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3274833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ephoQ\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTwo-component system sensor histidine kinase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstitution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT156I\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3580334\u0026ndash;3585227 *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMultiple\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eIS\u003cem\u003e3H\u003c/em\u003e composite transposon (Figure S5, Table S5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of 4,894 bp\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLoss of production\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4104884\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eacrB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMultidrug efflux RND transporter permease subunit AcrB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSubstitution\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eT\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eL667R\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4262704^ 4262705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eecpR\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRegulator protein EcpR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInsertion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInsertion (2 bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eI115fs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4723928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003errl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23S ribosomal RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of base C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5349457\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003errl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23S ribosomal RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDeletion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDeletion of base C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs for the biosynthesis of siderophores and transport of the iron-siderophore complex\u0026mdash;which together facilitate cefiderocol to penetrate the OM [65]\u0026mdash;KpMVS1, KpMVR1, and KpMVS2 were predicted to possess complete enterobactin production and iron-enterobactin transport systems. None of the yersiniabactin, colibactin, aerobactin, or salmochelin loci were detected, producing a Kleborate virulence score of zero [31]. All three isolates shared the same 19-kbp chromosomal region harbouring a cluster of enterobactin-synthesising genes \u003cem\u003eentA\u0026ndash;F and entH\u003c/em\u003e, enterobactin-exporter gene \u003cem\u003eentS\u003c/em\u003e, and iron-enterobactin transporter genes \u003cem\u003efepA\u0026ndash;D\u003c/em\u003e and \u003cem\u003efepG\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCompared with the ciprofloxacin-susceptible isolates KpMVS1 and KpMVS2, KpMVR1 harboured a nucleotide substitution 248C\u0026thinsp;\u0026gt;\u0026thinsp;A in the DNA gyrase gene \u003cem\u003egyrA\u003c/em\u003e, generating the GyrA mutation S83Y, which is known to reduce ciprofloxacin susceptibility [66]. The three isolates also carried a single copy of the \u003cem\u003emarRAB\u003c/em\u003e operon. However, KpMVR1 exhibited a unique 15-bp in-frame deletion in the non-essential transcriptional repressor gene \u003cem\u003emarR\u003c/em\u003e within the \u003cem\u003emarRAB\u003c/em\u003e operon, causing a loss of five amino acids and an amino acid substitution within the DNA-binding region of MarR (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) [67].\u003c/p\u003e \u003cp\u003eBoth the KpMVS1 and KpMVS2 genomes harboured three identical copies of the IS\u003cem\u003e5\u003c/em\u003e-family insertion sequence IS\u003cem\u003eEc68\u003c/em\u003e, whereas the KpMVR1 genome harboured four. Notably, seven bases at the 5\u0026rsquo; end of \u003cem\u003eompK36\u003c/em\u003e in KpMVR1 were truncated by this additional copy of IS\u003cem\u003eEc68\u003c/em\u003e, producing a frameshift mutation that replaced the first three amino acids at the N-terminal of OmpK36 with 12 amino acids (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The native N-terminal 21 amino acids of OmpK36 encode a Sec-dependent signal sequence (UniProtKB accession: A0A0H3H0Y2), which is essential for translocation of OmpK36 to the inner membrane of \u003cem\u003eK. pneumoniae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) [68]. The signal sequence is subsequently cleaved and OmpK36 is then folded and inserted into the OM (where the protein is functionally active as a porin) in a Bam-complex dependent fashion, a process facilitated by a C-terminal recognition sequence [69]. Whilst \u003cem\u003eompK36\u003c/em\u003e from KpMVS1 and KpMVS2 is predicted to encode a complete sec-dependent signal sequence, the 12 amino acids insertion combined with the deletion of three amino acids in OmpK36 from KpMVR1 is predicted to hinder this protein\u0026rsquo;s translocation to the OM according to the disrupted signal sequence (Supplementary Fig.\u0026nbsp;6, Additional File 2) [57]. These predictions were confirmed by the SDS-PAGE, which showed a Coomassie-stained band corresponding to OmpK36 in KpMVS1 but not in KpMVR1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and Additional File 4) [70], indicating that disruption of the Sec-dependent signal sequence of OmpK36 is functionally equivalent to deletion of \u003cem\u003eompK36\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding plasmid-encoded TraN proteins, TraN\u003csub\u003epKpMVR1_1\u003c/sub\u003e and TraN\u003csub\u003epKpMVS2_1\u003c/sub\u003e were identical (NCBI protein accession: WP_049192820.1) and differed from TraN\u003csub\u003epKpMVS1_1\u003c/sub\u003e (WP_436914186.1) by six amino acid substitutions (Supplementary Table\u0026nbsp;7, Additional File 3) [60]. Phylogenetic analysis revealed that these proteins belonged to the specialist TraNβ group (Supplementary Fig.\u0026nbsp;7, Additional File 2) [57], which has a narrow host range [45, 71]. Pairwise structural comparison between TraN\u003csub\u003epKpMVR1_1\u003c/sub\u003e (TraN\u003csub\u003epKpMVS2_1\u003c/sub\u003e), TraN\u003csub\u003epKpMVS1_1\u003c/sub\u003e, and the prototype TraNβ protein TraN\u003csub\u003epKpQIL\u003c/sub\u003e showed high consistency (Supplementary Figs.\u0026nbsp;8, Additional File 2) [57], and no amino acid substitution occurred in the characteristic distal β-hairpin (Supplementary Fig.\u0026nbsp;9, Additional File 2) [57], suggesting that the variation in TraN sequences across plasmids pKpMVR1_1, pKpMVS1_1, pKpMVS2_1, and pKpQIL is unlikely to affect the conjugation specificity [72].\u003c/p\u003e\n\u003ch3\u003eImpact of genetic alterations on antimicrobial resistance\u003c/h3\u003e\n\u003cp\u003eThe substitution of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e in KpMVS1 (producing KpMVS1\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e) and the transconjugant ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e (producing ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e) did not affect the susceptibility to meropenem, meropenem-vaborbactam, or imipenem-avibactam but led to a fourfold reduction in the imipenem MIC and a 16- to 32-fold increase in imipenem-relebactam MIC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, this allelic substitution resulted in a 256- to 512-fold reduction in the aztreonam MIC, a 32- to 64-fold reduction in the ceftazidime MIC, and a\u0026thinsp;\u0026ge;\u0026thinsp;4-fold reduction in the cefiderocol MIC but had no effect on the MICs of aztreonam-avibactam or ceftazidime-avibactam. Notably, imipenem and imipenem-relebactam MICs of each KPC-157-producing isolate were identical (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings suggest that KPC-157 has a weaker capacity to hydrolyse imipenem, aztreonam, ceftazidime, and cefiderocol than KPC-2, and that\u0026mdash;unlike vaborbactam and avibactam, which inhibit both KPC variants\u0026mdash;relebactam inhibits KPC-2 but not KPC-157, which is consistent with a previous report [54].\u003c/p\u003e \u003cp\u003eKnocking out \u003cem\u003eompK36\u003c/em\u003e from the ICC8001 chromosome (ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e and ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e) resulted in a 16-fold increase in meropenem and imipenem MICs, a\u0026thinsp;\u0026gt;\u0026thinsp;33-fold increase in meropenem-vaborbactam MIC, an 8- to 32-fold increase in imipenem-relebactam MIC, and a more than twofold increase in aztreonam MIC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings support the role of OmpK36 as an entry route for β-lactams and β-lactamase inhibitors across the OM [73]. However, when comparing MICs of ceftazidime, ceftazidime-avibactam, and cefiderocol before and after \u003cem\u003eompK36\u003c/em\u003e knockout in ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2\u003c/sub\u003e and ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157\u003c/sub\u003e, only two pairs exhibited notable increases (from 0.125 mg/L to 0.5 mg/L for ceftazidime-avibactam, and from \u0026le;\u0026thinsp;0.06 mg/L to 0.25 mg/L for cefiderocol), while others remained unchanged, suggesting alternative routes for avibactam\u0026rsquo;s entry. More broadly, comparisons of β-lactam MICs with and without β-lactamase inhibitors for KpMVR1, ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;2/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e, and ICC8001\u003csub\u003eKPC\u0026thinsp;\u0026minus;\u0026thinsp;157/Δ\u003cem\u003eompK36\u003c/em\u003e\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicate that these inhibitors penetrated the OM via routes other than OmpK36, effectively inhibiting β-lactamases.\u003c/p\u003e \u003cp\u003eThe chromosomes of KpMVR1, KpMVS1, and ICC8001 derivatives harboured the same cluster of \u003cem\u003eent\u003c/em\u003e and \u003cem\u003efep\u003c/em\u003e genes within a 19-kbp region encoding an ABC-type Fe\u003csup\u003e3+\u003c/sup\u003e-siderophore transporter associated with cefiderocol susceptibility [65]. These isolates did not exhibit any notable difference in cefiderocol MICs despite KpMVR1\u0026rsquo;s loss of the 54.7-kbp chromosomal region harbouring \u003cem\u003efep\u003c/em\u003e-like genes (Supplementary Table\u0026nbsp;3, Additional File 3) [60], suggesting alternative entry routes of cefiderocol.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMeropenem-vaborbactam and imipenem-relebactam are recommended in the UK for treating adult patients (\u0026ge;\u0026thinsp;18 years of age) with severe multidrug-resistant bacterial infections where therapeutic options are limited, and ceftazidime-avibactam is recommended as an alternative when the disease-causing bacterium produces class D carbapenemase (\u003cem\u003ee.g.\u003c/em\u003e, OXA-48) [74\u0026ndash;76]. Prevalence of resistance in Enterobacterales to any of these three antimicrobial combinations was 1\u0026ndash;5% across the globe as of 2022 despite regional variation [14, 16, 77\u0026ndash;80]. Therefore, the discovery of KpMVR1, which exhibited unusual resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam, in a seriously ill patient is particularly worrisome. No KPC-\u003cem\u003eKp\u003c/em\u003e clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam had been identified in the UK prior to this study, to the best of our knowledge, despite recommendation to submit metallo-carbapenemase-negative isolates exhibiting resistance to any of these agents for further characterisation by UKHSA. Nevertheless, an outbreak of KPC-\u003cem\u003eKp\u003c/em\u003e (ST512) resistant to all these three antimicrobial combinations occurred in Italy in 2021, with isolates co-producing KPC (not including KPC-157 and KPC-2) and OXA-181 carbapenemases, truncated OmpK35, and an OmpK36 mutant [81].\u003c/p\u003e \u003cp\u003eThe small number of chromosomal SNPs (n\u0026thinsp;=\u0026thinsp;6) and indels (n\u0026thinsp;=\u0026thinsp;10; \u0026le;15 bp each) identified in KpMVR1 when compared with KpMVS1, together with Patient 1\u0026rsquo;s exposure to meropenem, meropenem-vaborbactam, and fluoroquinolones, and the unique antibiogram of KpMVR1 in the ICU suggest \u003cem\u003ein vivo\u003c/em\u003e development of resistance to meropenem-vaborbactam, ceftazidime-avibactam, imipenem-relebactam, and ciprofloxacin in the same bacterial strain during the patient\u0026rsquo;s prolonged hospital stay. Similar within-host evolution of resistance to imipenem-relebactam, meropenem-vaborbactam, or ceftazidime-avibactam in KPC-\u003cem\u003eKp\u003c/em\u003e have been reported during extended hospitalisation involving complex antimicrobial regimens, including combinations such as ceftazidime-avibactam plus meropenem or meropenem-vaborbactam [15, 82].\u003c/p\u003e \u003cp\u003eThis study suggests within-patient emergence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e through a spontaneous point mutation in \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e during prolonged intensive care with exposure to a broad range of antimicrobials. To date, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e has been sporadically reported in Enterobacterales and is associated with distinct plasmids. Two \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e-positive \u003cem\u003eK. pneumoniae\u003c/em\u003e ST11 clinical isolates, resistant to imipenem-relebactam and ceftazidime-avibactam, were recovered from two patients in China between 2023 and 2024 [83]. In both isolates, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e was carried by the same IncFII(pSDP9R) plasmid (GenBank accessions: CP148070.1 and CP152056.1), as we determined using PlasmidFinder v2.1. Furthermore, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e has also been identified in an IncFII(Yp)-like plasmid of a \u003cem\u003eK. huaxiensis\u003c/em\u003e isolate from hospital wastewater in China in 2022, and in an IncN2 plasmid of a \u003cem\u003eCitrobacter freundii\u003c/em\u003e isolate from a sewage treatment plant in Brazil [83, 84]. Given the consistent association between \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e and Tn\u003cem\u003e4401\u003c/em\u003e isoforms in all aforementioned cases, continuous surveillance of KPC variants across clinical settings and environmental reservoirs is warranted to monitor emerging resistance mechanisms.\u003c/p\u003e \u003cp\u003eKPC-2 confers carbapenem resistance in Gram-negative bacteria but can be effectively inhibited by vaborbactam, avibactam, and relebactam [85, 86]. We have experimentally determined the effect of carbapenemase KPC-157 on the susceptibility to carbapenems and cephalosporins, with or without β-lactamase inhibitors. Our results indicate that KPC-157 behaves similarly to KPC-2 in interactions with meropenem and meropenem-vaborbactam. Therefore, the presence of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e in the single-copy plasmid pKpMVR1_1 alone cannot explain the high-level meropenem-vaborbactam resistance observed in KpMVR1. Notably, KPC-157 appears less capable of hydrolysing imipenem, aztreonam, ceftazidime, and cefiderocol than KPC-2, and is inhibited by vaborbactam and avibactam but not by relebactam.\u003c/p\u003e \u003cp\u003eIsolate KpMVR1 exhibited genetic changes potentially affecting the antimicrobial permeability of its OM compared with KpMVS1. The IS\u003cem\u003eEc68\u003c/em\u003e-induced frameshift mutation impedes OmpK36\u0026rsquo;s translocation to the OM, decreasing the influx of β-lactams and β-lactamase inhibitors into the periplasm and resulting in elevated MICs of carbapenems and cephalosporins, with and without β-lactamase inhibitors (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). IS\u003cem\u003eEc68\u003c/em\u003e has been sporadically reported to interrupt \u003cem\u003eompK36\u003c/em\u003e and \u003cem\u003emgrB\u003c/em\u003e in \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical and wastewater isolates, reducing the susceptibility of host bacteria to β-lactams and colistin, respectively [87\u0026ndash;89]. Our finding further supports IS\u003cem\u003eEc68\u003c/em\u003e\u0026rsquo;s association with AMR.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003egyrA\u003c/em\u003e S83Y mutation and loss of OmpK36 function in KpMVR1 are associated with ciprofloxacin resistance [66, 90]. Further studies are needed to assess the impact of the identified \u003cem\u003emarR\u003c/em\u003e and \u003cem\u003eacrB\u003c/em\u003e mutations on antimicrobial susceptibility of \u003cem\u003eK. pneumoniae\u003c/em\u003e and to explore potential interactions between the MarR and AcrB variants. For example, \u003cem\u003emarR\u003c/em\u003e inactivation is known to upregulate the AcrAB-TolC efflux pump, conferring low-level cross-resistance to antimicrobials including β-lactams and ciprofloxacin [91].\u003c/p\u003e \u003cp\u003eThis study was limited to three clonally related \u003cem\u003eK. pneumoniae\u003c/em\u003e isolates, of which only KpMVR1 exhibited elevated MICs of meropenem-vaborbactam, imipenem-relebactam, aztreonam-avibactam, and ceftazidime-avibactam. KpMVR1 carried multiple AMR-associated genetic alterations. Although the individual effects of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e and Δ\u003cem\u003eompK36\u003c/em\u003e on antimicrobial susceptibility were determined, the transgenic derivatives did not fully reproduce MIC increases observed in KpMVR1, suggesting involvement of additional genetic or regulatory mechanisms. Broader surveillance of \u003cem\u003eK. pneumoniae\u003c/em\u003e with resistance profiles like KpMVR1 is warranted to elucidate underlying resistance mechanisms.\u003c/p\u003e \u003cp\u003eA review of national surveillance data showed low frequencies of ceftazidime-avibactam resistance in the UK between 2016 and 2020 [80]. However, timely identification and reporting of resistance to β-lactam/β-lactamase inhibitor combinations remain essential through UKHSA\u0026rsquo;s Second Generation Surveillance System and referral of resistant isolates to the AMRHAI Reference Unit. Our findings also underscore the importance of monitoring within-patient changes in antimicrobial susceptibility profiles of bacterial pathogens during antimicrobial therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial resistance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAMRHAI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial Resistance and Healthcare Associated Infections\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAntimicrobial susceptibility testing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbapenemase-producing Enterobacterales\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRISPR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClustered regularly interspaced short palindromic repeats\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESBL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtended-spectrum β-lactamase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEUCAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEuropean Committee on Antimicrobial Susceptibility Testing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eICU\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIntensive care unit\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInsertion sequence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKPC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e carbapenemases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKPC-\u003cem\u003eKp\u003c/em\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKPC-producing \u003cem\u003eK. pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMLST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMulti-locus sequence typing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNCBI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNational Center for Biotechnology Information\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eONT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxford Nanopore Technologies\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOuter membrane\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMALDI-ToF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMatrix-assisted laser desorption/ionisation time-of-flight\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSNP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSingle-nucleotide polymorphism\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSequence type\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUKHSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUnited Kingdom Health Security Agency\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVNTR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVariable number tandem repeat\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole-genome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eFollowing an internal review by the UKHSA Research Support and Governance Office (RSGO) it was agreed that this study fell outside the remit for ethics review. This decision was made based on the information provided to the RSGO and on the understanding that the study would be conducted in accordance with the conditions stated in the applicable UKHSA policies and procedures.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eNone to declare.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the UKHSA, the University of Liverpool David Price Evans Endowment (grant number UGG10057 to Y. W.), and Wellcome Trust\u0026ndash;Imperial Institutional Strategic Support Fund Springboard Research Fellowship (grant number PSN109 to Y. W.). Y. W., E. J., D. M., and K. L. H. are affiliated with the National Institute for Health and Care Research Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Imperial College London in partnership with the UKHSA, in collaboration with, Imperial Healthcare Partners, University of Cambridge and University of Warwick (grant number NIHR200876). The views expressed in this article are those of the authors and not necessarily those of the NHS, the National Institute for Health Research, or the Department of Health and Social Care.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualisation: K. L. H. and Y. W.; Resources: K. L. H., J. T., G. F., F. M., N. W., and G. M. R.; Methodology: K. L. H., Y. W., J. L. C. W., and E. J.; Data curation: Y. W.; Investigation and formal analysis: Y. W., J. L. C. W., J. S. G., W. W. L., J. T., F. M., G. M. R., K. D., I. B., G. F., E. J., D. M., and K. L. H.; Visualisation: Y. W.; Writing \u0026ndash; original draft: Y. W., J. L. C. W., J. S. G., W. W. L., G. M. R., and K. L. H.; Writing \u0026ndash; review and editing: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe acknowledge the Colebrook Laboratory, a facility supported by the NIHR Imperial Biomedical Research Centre (BRC), for providing bioinformatics resources. Part of the bioinformatics analysis was performed on equipment purchased as part of MRC CARP fellowship award MR/T005254/1. We also thank the Institut Pasteur teams for the curation and maintenance of BIGSdb-Pasteur databases at bigsdb.pasteur.fr.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eIllumina and MinION sequencing reads, genome assemblies, and anonymised metadata of isolates KpMVS1, KpMVR1, and KpMVS2 are available in the National Center for Biotechnology Information [92] under BioProject PRJNA1084250 [93]. The assemblies are also accessible (website login required) in the Klebsiella PasteurMLST database [94] under ids 75608 (KpMVS1) [95], 75609 (KpMVR1) [96], and 75610 (KpMVS2) [97].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Baldwin CM, Lyseng-Williamson KA, Keam SJ. Meropenem. Drugs. 2008;68:803\u0026ndash;38. https://doi.org/10.2165/00003495-200868060-00006.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Meini S, Tascini C, Cei M, Sozio E, Rossolini GM. AmpC β-lactamase-producing Enterobacterales: what a clinician should know. Infection. 2019;47:363\u0026ndash;75. https://doi.org/10.1007/s15010-019-01291-9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Livermore DM. Defining an extended-spectrum β-lactamase. Clinical Microbiology and Infection. 2008;14:3\u0026ndash;10. https://doi.org/10.1111/j.1469-0691.2007.01857.x.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VHA, Takebayashi Y, et al. β-Lactamases and β-Lactamase Inhibitors in the 21st Century. Journal of Molecular Biology. 2019;431:3472\u0026ndash;500. https://doi.org/10.1016/j.jmb.2019.04.002.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Jacoby GA. AmpC β-Lactamases. Clinical Microbiology Reviews. 2009;22:161\u0026ndash;82. https://doi.org/10.1128/cmr.00036-08.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Jorgensen SCJ, Rybak MJ. Meropenem and Vaborbactam: Stepping up the Battle against Carbapenem-resistant Enterobacteriaceae. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2018;38:444\u0026ndash;61. https://doi.org/10.1002/phar.2092.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rodloff AC, Goldstein EJC, Torres A. Two decades of imipenem therapy. Journal of Antimicrobial Chemotherapy. 2006;58:916\u0026ndash;29. https://doi.org/10.1093/jac/dkl354.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Prajapati JD, Kleinekath\u0026ouml;fer U, Winterhalter M. How to Enter a Bacterium: Bacterial Porins and the Permeation of Antibiotics. Chem Rev. 2021;121:5158\u0026ndash;92. https://doi.org/10.1021/acs.chemrev.0c01213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lomovskaya O, Sun D, Rubio-Aparicio D, Nelson K, Tsivkovski R, Griffith DC, et al. Vaborbactam: Spectrum of Beta-Lactamase Inhibition and Impact of Resistance Mechanisms on Activity in Enterobacteriaceae. Antimicrobial Agents and Chemotherapy. 2017;61:10.1128/aac.01443-17. https://doi.org/10.1128/aac.01443-17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ehmann DE, Jahić H, Ross PL, Gu R-F, Hu J, Durand-R\u0026eacute;ville TF, et al. Kinetics of Avibactam Inhibition against Class A, C, and D β-Lactamases. Journal of Biological Chemistry. 2013;288:27960\u0026ndash;71. https://doi.org/10.1074/jbc.M113.485979.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Carcione D, Siracusa C, Sulejmani A, Leoni V, Intra J. Old and New Beta-Lactamase Inhibitors: Molecular Structure, Mechanism of Action, and Clinical Use. Antibiotics. 2021;10:995. https://doi.org/10.3390/antibiotics10080995.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e UK Health Security Agency. UK Access, Watch, Reserve, and Other classification for antibiotics (UK-AWaRe antibiotic classification). 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gaibani P, Bovo F, Bussini L, Lazzarotto T, Amadesi S, Bartoletti M, et al. Dynamic evolution of imipenem/relebactam resistance in a KPC-producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e from a single patient during ceftazidime/avibactam-based treatments. Journal of Antimicrobial Chemotherapy. 2022;77:1570\u0026ndash;7. https://doi.org/10.1093/jac/dkac100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Xu T, Guo Y, Ji Y, Wang B, Zhou K. Epidemiology and Mechanisms of Ceftazidime\u0026ndash;Avibactam Resistance in Gram-Negative Bacteria. Engineering. 2022;11:138\u0026ndash;45. https://doi.org/10.1016/j.eng.2020.11.004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gaibani Paolo, Bianco Gabriele, Amadesi Stefano, Boattini Matteo, Ambretti Simone, Costa Cristina. Increased \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e Copy Number and OmpK35 and OmpK36 Porins Disruption Mediated Resistance to Imipenem/Relebactam and Meropenem/Vaborbactam in a KPC-Producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Clinical Isolate. Antimicrobial Agents and Chemotherapy. 2022;66:e00191-22. https://doi.org/10.1128/aac.00191-22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gaibani P, Giani T, Bovo F, Lombardo D, Amadesi S, Lazzarotto T, et al. Resistance to Ceftazidime/Avibactam, Meropenem/Vaborbactam and Imipenem/Relebactam in Gram-Negative MDR Bacilli: Molecular Mechanisms and Susceptibility Testing. Antibiotics. 2022;11:628. https://doi.org/10.3390/antibiotics11050628.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Satapoomin Naphat, Dulyayangkul Punyawee, Avison Matthew B. \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Mutants Resistant to Ceftazidime-Avibactam Plus Aztreonam, Imipenem-Relebactam, Meropenem-Vaborbactam, and Cefepime-Taniborbactam. Antimicrobial Agents and Chemotherapy. 2022;66:e02179-21. https://doi.org/10.1128/aac.02179-21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wilson WR, Kline EG, Jones CE, Morder KT, Mettus RT, Doi Y, et al. Effects of KPC Variant and Porin Genotype on the In Vitro Activity of Meropenem-Vaborbactam against Carbapenem-Resistant Enterobacteriaceae. Antimicrobial Agents and Chemotherapy. 2019;63:10.1128/aac.02048-18. https://doi.org/10.1128/aac.02048-18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Humphries Romney M., Hemarajata Peera. Resistance to Ceftazidime-Avibactam in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Due to Porin Mutations and the Increased Expression of KPC-3. Antimicrobial Agents and Chemotherapy. 2017;61:10.1128/aac.00537\u0026thinsp;\u0026minus;\u0026thinsp;17. https://doi.org/10.1128/aac.00537-17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Turton JF, Payne Z, Coward A, Hopkins KL, Turton JA, Doumith M, et al. Virulence genes in isolates of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e from the UK during 2016, including among carbapenemase gene-positive hypervirulent K1-ST23 and \u0026lsquo;non-hypervirulent\u0026rsquo; types ST147, ST15 and ST383. Journal of Medical Microbiology. 2018;67:118\u0026ndash;28. https://doi.org/10.1099/jmm.0.000653.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e The European Committee on Antimicrobial Susceptibility Testing. Clinical breakpoints (v 15.0). 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114\u0026ndash;20. https://doi.org/10.1093/bioinformatics/btu170.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884\u0026ndash;90. https://doi.org/10.1093/bioinformatics/bty560.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wood DE, Lu J, Langmead B. Improved metagenomic analysis with Kraken 2. Genome Biology. 2019;20:257. https://doi.org/10.1186/s13059-019-1891-0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lu J, Breitwieser FP, Thielen P, Salzberg SL. Bracken: Estimating species abundance in metagenomics data. PeerJ Computer Science. 2017;3:e104. https://doi.org/10.7717/peerj-cs.104.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Bouras G, Houtak G, Wick RR, Mallawaarachchi V, Roach MJ, Papudeshi B, et al. Hybracter: enabling scalable, automated, complete and accurate bacterial genome assemblies. Microbial Genomics. 2024;10:001244. https://doi.org/10.1099/mgen.0.001244.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 1, PDF, Supplementary methods.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chklovski A, Parks DH, Woodcroft BJ, Tyson GW. CheckM2: a rapid, scalable and accurate tool for assessing microbial genome quality using machine learning. Nature Methods. 2023;20:1203\u0026ndash;12. https://doi.org/10.1038/s41592-023-01940-w.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Pedersen BS, Quinlan AR. Mosdepth: quick coverage calculation for genomes and exomes. Bioinformatics. 2018;34:867\u0026ndash;8. https://doi.org/10.1093/bioinformatics/btx699.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Schwengers O, Jelonek L, Dieckmann MA, Beyvers S, Blom J, Goesmann A. Bakta: rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microbial Genomics. 2021;7:000685. https://doi.org/10.1099/mgen.0.000685.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Lam MMC, Wick RR, Watts SC, Cerdeira LT, Wyres KL, Holt KE. A genomic surveillance framework and genotyping tool for \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e and its related species complex. Nature Communications. 2021;12:4188. https://doi.org/10.1038/s41467-021-24448-3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wyres KL, Wick RR, Gorrie C, Jenney A, Follador R, Thomson NR, et al. Identification of \u003cem\u003eKlebsiella\u003c/em\u003e capsule synthesis loci from whole genome data. Microbial Genomics. 2016;2:e000102. https://doi.org/10.1099/mgen.0.000102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Feldgarden M, Brover V, Gonzalez-Escalona N, Frye JG, Haendiges J, Haft DH, et al. AMRFinderPlus and the Reference Gene Catalog facilitate examination of the genomic links among antimicrobial resistance, stress response, and virulence. Sci Rep. 2021;11:12728. https://doi.org/10.1038/s41598-021-91456-0.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, N\u0026eacute;ron B, et al. CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Research. 2018;46:W246\u0026ndash;51. https://doi.org/10.1093/nar/gky425.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Carattoli A, Zankari E, Garc\u0026iacute;a-Fern\u0026aacute;ndez A, Voldby Larsen M, Lund O, Villa L, et al. \u003cem\u003eIn Silico\u003c/em\u003e Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing. Antimicrobial Agents and Chemotherapy. 2014;58:3895\u0026ndash;903. https://doi.org/10.1128/aac.02412-14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Robertson J, Nash JHE. MOB-suite: software tools for clustering, reconstruction and typing of plasmids from draft assemblies. Microbial Genomics. 2018;4. https://doi.org/10.1099/mgen.0.000206.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ross Karen, Varani Alessandro M., Snesrud Erik, Huang Hongzhan, Alvarenga Danillo Oliveira, Zhang Jian, et al. TnCentral: a Prokaryotic Transposable Element Database and Web Portal for Transposon Analysis. mBio. 2021;12:10.1128/mbio.02060-21. https://doi.org/10.1128/mbio.02060-21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Research. 2006;34 suppl_1:D32\u0026ndash;6. https://doi.org/10.1093/nar/gkj014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094\u0026ndash;100. https://doi.org/10.1093/bioinformatics/bty191.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff. Fly. 2012;6:80\u0026ndash;92. https://doi.org/10.4161/fly.19695.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Jones P, Binns D, Chang H-Y, Fraser M, Li W, McAnulla C, et al. InterProScan 5: genome-scale protein function classification. Bioinformatics. 2014;30:1236\u0026ndash;40. https://doi.org/10.1093/bioinformatics/btu031.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ittisoponpisan S, Islam SA, Khanna T, Alhuzimi E, David A, Sternberg MJE. Can Predicted Protein 3D Structures Provide Reliable Insights into whether Missense Variants Are Disease Associated? Journal of Molecular Biology. 2019;431:2197\u0026ndash;212. https://doi.org/10.1016/j.jmb.2019.04.009.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e The UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Research. 2019;47:D506\u0026ndash;15. https://doi.org/10.1093/nar/gky1049.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Zhou Y, Pan Q, Pires DEV, Rodrigues CHM, Ascher DB. DDMut: predicting effects of mutations on protein stability using deep learning. Nucleic Acids Research. 2023;51:W122\u0026ndash;8. https://doi.org/10.1093/nar/gkad472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Low WW, Wong JLC, Beltran LC, Seddon C, David S, Kwong H-S, et al. Mating pair stabilization mediates bacterial conjugation species specificity. Nature Microbiology. 2022;7:1016\u0026ndash;27. https://doi.org/10.1038/s41564-022-01146-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Alikhan N-F, Petty NK, Ben Zakour NL, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics. 2011;12:402. https://doi.org/10.1186/1471-2164-12-402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen C, et al. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Research. 2023;51:W484\u0026ndash;92. https://doi.org/10.1093/nar/gkad326.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wilkins D, Kurtz Z. gggenes. https://wilkox.org/gggenes/.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wong JLC, Romano M, Kerry LE, Kwong H-S, Low W-W, Brett SJ, et al. OmpK36-mediated Carbapenem resistance attenuates ST258 \u003cem\u003eKlebsiella pneumoniae in vivo\u003c/em\u003e. Nature Communications. 2019;10:3957. https://doi.org/10.1038/s41467-019-11756-y.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Teufel F, Almagro Armenteros JJ, Johansen AR, G\u0026iacute;slason MH, Pihl SI, Tsirigos KD, et al. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nat Biotechnol. 2022;40:1023\u0026ndash;5. https://doi.org/10.1038/s41587-021-01156-3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e The European Committee on Antimicrobial Susceptibility Testing. MIC determination of non-fastidious and fastidious organisms. https://www.eucast.org/ast_of_bacteria/mic_determination.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e The European Committee on Antimicrobial Susceptibility Testing. Guidance document on broth microdilution testing of cefiderocol. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Ke W, Bethel CR, Thomson JM, Bonomo RA, van den Akker F. Crystal Structure of KPC-2: Insights into Carbapenemase Activity in Class A β-Lactamases. Biochemistry. 2007;46:5732\u0026ndash;40. https://doi.org/10.1021/bi700300u.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Gato E, Guijarro-S\u0026aacute;nchez P, Alonso-Garc\u0026iacute;a I, Pedraza-Merino R, Conde A, Lence E, et al. \u003cem\u003eIn vitro\u003c/em\u003e development of imipenem/relebactam resistance in KPC-producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e involves multiple mutations including OmpK36 disruption and KPC modification. International Journal of Antimicrobial Agents. 2023;62:106935. https://doi.org/10.1016/j.ijantimicag.2023.106935.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Krishnan NP, Nguyen NQ, Papp-Wallace KM, Bonomo RA, van den Akker F. Inhibition of \u003cem\u003eKlebsiella\u003c/em\u003e β-Lactamases (SHV-1 and KPC-2) by Avibactam: A Structural Study. PLOS ONE. 2015;10:e0136813. https://doi.org/10.1371/journal.pone.0136813.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Pemberton Orville A., Tsivkovski Ruslan, Totrov Maxim, Lomovskaya Olga, Chen Yu. Structural Basis and Binding Kinetics of Vaborbactam in Class A β-Lactamase Inhibition. Antimicrobial Agents and Chemotherapy. 2020;64:10.1128/aac.00398\u0026thinsp;\u0026minus;\u0026thinsp;20. https://doi.org/10.1128/aac.00398-20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 2, PDF, Supplementary figures.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Naas Thierry, Cuzon Gaelle, Villegas Maria-Virginia, Lartigue Marie-Fr\u0026eacute;d\u0026eacute;rique, Quinn John P., Nordmann Patrice. Genetic Structures at the Origin of Acquisition of the β-Lactamase \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e Gene. Antimicrobial Agents and Chemotherapy. 2008;52:1257\u0026ndash;63. https://doi.org/10.1128/aac.01451-07.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Cuzon Gaelle, Naas Thierry, Nordmann Patrice. Functional Characterization of Tn\u003cem\u003e4401\u003c/em\u003e, a Tn\u003cem\u003e3\u003c/em\u003e-Based Transposon Involved in \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u003c/sub\u003e Gene Mobilization. Antimicrobial Agents and Chemotherapy. 2011;55:5370\u0026ndash;3. https://doi.org/10.1128/aac.05202-11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 3, XSLX, Supplementary tables.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Holt KE, Wertheim H, Zadoks RN, Baker S, Whitehouse CA, Dance D, et al. Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e, an urgent threat to public health. Proceedings of the National Academy of Sciences. 2015. https://doi.org/10.1073/pnas.1501049112.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tsang KK, Lam MMC, Wick RR, Wyres KL, Bachman M, Baker S, et al. Diversity, functional classification and genotyping of SHV β-lactamases in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. 2024;:2024.04.05.587953. https://doi.org/10.1101/2024.04.05.587953.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Huang Y, Lin Q, Zhou Q, Lv L, Wan M, Gao X, et al. Identification of \u003cem\u003efosA10\u003c/em\u003e, a Novel Plasmid-Mediated Fosfomycin Resistance Gene of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e Origin, in \u003cem\u003eEscherichia coli\u003c/em\u003e. IDR. 2020;13:1273\u0026ndash;9. https://doi.org/10.2147/IDR.S251360.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Sabnis A, Mumin Z, Potterill I, Jauneikaite E, Brown CS, et al. IS\u003cem\u003e1\u003c/em\u003e-related large-scale deletion of chromosomal regions harbouring the oxygen-insensitive nitroreductase gene \u003cem\u003enfsB\u003c/em\u003e causes nitrofurantoin heteroresistance in \u003cem\u003eEscherichia coli\u003c/em\u003e. Microbial Genomics. 2023;9:001102. https://doi.org/10.1099/mgen.0.001102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sato T, Yamawaki K. Cefiderocol: Discovery, Chemistry, and \u003cem\u003eIn Vivo\u003c/em\u003e Profiles of a Novel Siderophore Cephalosporin. Clinical Infectious Diseases. 2019;69 Supplement_7:S538\u0026ndash;43. https://doi.org/10.1093/cid/ciz826.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Chen F-J, Lauderdale T-L, Ho M, Lo H-J. The Roles of Mutations in \u003cem\u003egyrA\u003c/em\u003e, \u003cem\u003eparC\u003c/em\u003e, and \u003cem\u003eompK35\u003c/em\u003e in Fluoroquinolone Resistance in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Microbial Drug Resistance. 2003;9:265\u0026ndash;71. https://doi.org/10.1089/107662903322286472.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Praski Alzrigat L, Huseby DL, Brandis G, Hughes D. Fitness cost constrains the spectrum of marR mutations in ciprofloxacin-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e. Journal of Antimicrobial Chemotherapy. 2017;72:3016\u0026ndash;24. https://doi.org/10.1093/jac/dkx270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Mori H, Ito K. The Sec protein-translocation pathway. Trends in Microbiology. 2001;9:494\u0026ndash;500. https://doi.org/10.1016/S0966-842X(01)02174-6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Malinverni Juliana C., Silhavy Thomas J. Assembly of Outer Membrane β-Barrel Proteins: the Bam Complex. EcoSal Plus. 2011;4:10.1128/ecosalplus.4.3.8. https://doi.org/10.1128/ecosalplus.4.3.8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Additional File 4, PNG, Original photo of the Coomassie-stained polyacrylamide gel electrophoresis illustrated in Fig.\u0026nbsp;2c.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Frankel G, David S, Low WW, Seddon C, Wong JLC, Beis K. Plasmids pick a bacterial partner before committing to conjugation. Nucleic Acids Research. 2023;51:8925\u0026ndash;33. https://doi.org/10.1093/nar/gkad678.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e 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. https://doi.org/10.1128/jb.00061-23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Rocker A, Lacey JA, Belousoff MJ, Wilksch JJ, Strugnell RA, Davies MR, et al. Global Trends in Proteome Remodeling of the Outer Membrane Modulate Antimicrobial Permeability in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. mBio. 2020;11:10.1128/mbio.00603\u0026thinsp;\u0026minus;\u0026thinsp;20. https://doi.org/10.1128/mbio.00603-20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e National Institute for Health and Care Excellence. Antimicrobial prescribing: meropenem with vaborbactam (Evidence summary [ES21]). National Institute for Health and Care Excellence (NICE). 2019. https://www.nice.org.uk/advice/es21/chapter/Key-messages.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e National Institute for Health and Care Excellence. Ceftazidime with avibactam for treating severe drug-resistant gram-negative bacterial infections (Health technology evaluation [HTE1]). National Institute for Health and Care Excellence (NICE). 2022. https://www.nice.org.uk/guidance/hte1/chapter/1-Recommendations.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e National Institute for Health and Care Excellence. Antimicrobial prescribing: imipenem with cilastatin and relebactam (Evidence summary [ES30]). National Institute for Health and Care Excellence (NICE). 2020. https://www.nice.org.uk/advice/es30/evidence.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sader HS, Mendes RE, Duncan L, Kimbrough JH, Carvalhaes CG, Castanheira M. Ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam activities against multidrug-resistant Enterobacterales from United States Medical Centers (2018\u0026ndash;2022). Diagnostic Microbiology and Infectious Disease. 2023;106:115945. https://doi.org/10.1016/j.diagmicrobio.2023.115945.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hern\u0026aacute;ndez-Garc\u0026iacute;a Marta, Garc\u0026iacute;a-Castillo Mar\u0026iacute;a, Bou Germ\u0026aacute;n, Cercenado Emilia, Delgado-Valverde Mercedes, Oliver Antonio, et al. Imipenem-Relebactam Susceptibility in Enterobacterales Isolates Recovered from ICU Patients from Spain and Portugal (SUPERIOR and STEP Studies). Microbiology Spectrum. 2022;10:e02927-22. https://doi.org/10.1128/spectrum.02927-22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Shoulders BR, Casapao AM, Venugopalan V. An Update on Existing and Emerging Data for Meropenem-Vaborbactam. Clinical Therapeutics. 2020;42:692\u0026ndash;702. https://doi.org/10.1016/j.clinthera.2020.01.023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Guy RL, Hopkins KL, Budd EL, Wilson K, Fountain H, Meunier D, et al. The importance of monitoring a new antibiotic: ceftazidime/avibactam usage and resistance experience from England, 2016 to 2020. Eurosurveillance. 2025;30:2400399. https://doi.org/10.2807/1560-7917.ES.2025.30.14.2400399.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Bovo F, Amadesi S, Palombo M, Lazzarotto T, Ambretti S, Gaibani P. Clonal dissemination of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e resistant to cefiderocol, ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam co-producing KPC and OXA-181 carbapenemase. JAC-Antimicrobial Resistance. 2023;5:dlad099. https://doi.org/10.1093/jacamr/dlad099.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Sauerborn E, Corredor NC, Reska T, Perlas A, Vargas da Fonseca Atum S, Goldman N, et al. Detection of hidden antibiotic resistance through real-time genomics. Nature Communications. 2024;15:5494. https://doi.org/10.1038/s41467-024-49851-4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Fu Yiqi, Zhang Yuchao, Zhao Jun, Xie Jiahong, Kong Haishen, Yang Tingting, et al. Emergence of co-resistance to imipenem/relebactam and ceftazidime/avibactam in clinical \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ST11 clone due to KPC-2 N132S and CTX-M-65 S130G/P167S substitutions. Antimicrobial Agents and Chemotherapy. 2025;0:e00891-25. https://doi.org/10.1128/aac.00891-25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Furlan JPR, Bueno GC, Sousa-Carmo RR, Silva RLO, Barbosa MRF, Sato MIZ, et al. KPC-157 and KPC-181 carbapenemases produced by \u003cem\u003eCitrobacter freundii\u003c/em\u003e ST522 and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ST258 isolated from wastewater. Molecular Biology Reports. 2025;52:705. https://doi.org/10.1007/s11033-025-10801-y.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Theuretzbacher U, Carrara E, Conti M, Tacconelli E. Role of new antibiotics for KPC-producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e. Journal of Antimicrobial Chemotherapy. 2021;76 Supplement_1:i47\u0026ndash;54. https://doi.org/10.1093/jac/dkaa497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Papp-Wallace Krisztina M., Winkler Marisa L., Taracila Magdalena A., Bonomo Robert A. Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam. Antimicrobial Agents and Chemotherapy. 2015;59:3710\u0026ndash;7. https://doi.org/10.1128/aac.04406-14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wu L-T, Guo M-K, Ke S-C, Lin Y-P, Pang Y-C, Nguyen H-TV, et al. Characterization of the Genetic Background of KPC-2-Producing \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e with Insertion Elements Disrupting the \u003cem\u003eompK36\u003c/em\u003e Porin Gene. Microbial Drug Resistance. 2020;26:1050\u0026ndash;7. https://doi.org/10.1089/mdr.2019.0410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hamel M, Chatzipanagiotou S, Hadjadj L, Petinaki E, Papagianni S, Charalampaki N, et al. Inactivation of \u003cem\u003emgrB\u003c/em\u003e gene regulator and resistance to colistin is becoming endemic in carbapenem-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e in Greece: A nationwide study from 2014 to 2017. International Journal of Antimicrobial Agents. 2020;55:105930. https://doi.org/10.1016/j.ijantimicag.2020.105930.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Hayashi W, Iimura M, Soga E, Koide S, Izumi K, Yoshida S, et al. Presence of Colistin- and Tigecycline-Resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e ST29 in Municipal Wastewater Influents in Japan. Microbial Drug Resistance. 2021;27:1433\u0026ndash;42. https://doi.org/10.1089/mdr.2020.0514.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Mart\u0026iacute;nez-Mart\u0026iacute;nez L, Hern\u0026aacute;ndez-All\u0026eacute;s S, Albert\u0026iacute; S, Tom\u0026aacute;s J M, Benedi V J, Jacoby G A. \u003cem\u003eIn vivo\u003c/em\u003e selection of porin-deficient mutants of \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e with increased resistance to cefoxitin and expanded-spectrum-cephalosporins. Antimicrobial Agents and Chemotherapy. 1996;40:342\u0026ndash;8. https://doi.org/10.1128/aac.40.2.342.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Randall LP, Woodward MJ. The multiple antibiotic resistance (\u003cem\u003emar\u003c/em\u003e) locus and its significance. Research in Veterinary Science. 2002;72:87\u0026ndash;93. https://doi.org/10.1053/rvsc.2001.0537.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e National Center for Biotechnology Information. www.ncbi.nlm.nih.gov. Accessed 27 Nov 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong J, Sanchez-Garrido J, Low W, Turton J, Morecchiato F, et al. Full information on isolate KpMVR1 (id:75609). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info\u0026amp;db=pubmlst_klebsiella_isolates\u0026amp;id=75609. Accessed 27 Nov 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e species complex. Institut Pasteur. https://bigsdb.pasteur.fr/klebsiella/. Accessed 27 Nov 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Full information on isolate KpMVS1 (id:75608). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info\u0026amp;db=pubmlst_klebsiella_isolates\u0026amp;id=75608. Accessed 27 Nov 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Full information on isolate KpMVR1 (id:75609). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info\u0026amp;db=pubmlst_klebsiella_isolates\u0026amp;id=75609. Accessed 27 Nov 2025.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Wan Y, Wong JLC, Sanchez-Garrido J, Low WW, Turton J, Morecchiato F, et al. Full information on isolate KpMVS2 (id:75610). Institut Pasteur. https://bigsdb.pasteur.fr/cgi-bin/bigsdb/bigsdb.pl?page=info\u0026amp;db=pubmlst_klebsiella_isolates\u0026amp;id=75610. Accessed 27 Nov 2025.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomic-data","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gtic","sideBox":"Learn more about [BMC Genomic Data](http://bmcgenet.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gtic/default.aspx","title":"BMC Genomic Data","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Klebsiella pneumoniae, antimicrobial resistance, carbapenem resistance, β-lactamase inhibitors, KPC-157, OmpK36, insertion sequences, hybrid genome assembly, comparative genomics, mutagenesis experiments","lastPublishedDoi":"10.21203/rs.3.rs-8289965/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8289965/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eResistance to carbapenems and third-generation cephalosporins is increasing in \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e globally, restricting therapeutic options. The β-lactam/β-lactamase inhibitor combinations are widely used to circumvent β-lactamase-mediated resistance. In 2021, an unusual \u003cem\u003eK. pneumoniae\u003c/em\u003e clinical isolate, KpMVR1, was recovered from a hospitalised patient in England, exhibiting resistance to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam. To investigate this phenomenon, we characterised the genome and antimicrobial susceptibility of KpMVR1 alongside two clonally related isolates susceptible to all three β-lactam/β-lactamase inhibitor combinations: KpMVS1, collected from the same patient 42 days earlier, and KpMVS2, from another patient in the same hospital.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIllumina and MinION whole-genome sequencing were conducted for these three isolates, followed by hybrid genome assembly. Annotated genome assemblies were compared to identify genetic variation. Mutagenesis experiments were performed to verify predicted functional alterations.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAll isolates belonged to clone ST8134 and carried \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e alleles (KpMVR1: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;157\u003c/sub\u003e; KpMVS1 and KpMVS2: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eKPC\u0026minus;2\u003c/sub\u003e) in presumptively conjugative plasmids. Insertion sequence IS\u003cem\u003eEc68\u003c/em\u003e caused a frameshift mutation in KpMVR1\u0026rsquo;s \u003cem\u003eompK36\u003c/em\u003e gene, reducing susceptibility to meropenem-vaborbactam and imipenem-relebactam. KPC-157 demonstrated decreased hydrolysis of imipenem and ceftazidime when compared with KPC-2. KpMVR1 also encoded a disrupted transcriptional repressor MarR and a destabilising mutation in AcrB, a component of the AcrAB-TolC multidrug efflux pump.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eKpMVR1 carried multiple resistance-associated genetic alterations and likely developed its resistance profile through within-patient evolution. This study highlights the importance of routine screening for resistant pathogens in vulnerable patients to guide antimicrobial chemotherapy and the need to characterise underlying resistance mechanisms to assess the risk of onward dissemination.\u003c/p\u003e","manuscriptTitle":"Genomic and molecular characterisation of a KPC-producing Klebsiella pneumoniae clinical isolate resistant to meropenem-vaborbactam, imipenem-relebactam, and ceftazidime-avibactam","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-22 09:32:16","doi":"10.21203/rs.3.rs-8289965/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-23T08:04:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-22T13:38:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-21T22:10:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50835376340561168572252447150077072030","date":"2026-01-15T07:27:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"219330925290687436353834934261118416319","date":"2026-01-14T22:26:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338737023625436080231641656889506339103","date":"2026-01-13T05:58:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46512173263127342271572782368759646700","date":"2026-01-12T14:41:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T07:19:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299267428479323899657542158595785197838","date":"2025-12-15T14:39:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-15T14:09:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-15T06:27:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-12T12:45:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomic Data","date":"2025-12-12T12:36:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomic-data","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gtic","sideBox":"Learn more about [BMC Genomic Data](http://bmcgenet.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gtic/default.aspx","title":"BMC Genomic Data","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"434431c6-a005-4d7a-9142-288cd2d5af37","owner":[],"postedDate":"December 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T14:41:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-22 09:32:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8289965","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8289965","identity":"rs-8289965","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}