Heterogeneous multicopy of bla_CTX-M variants on the same plasmid enhances evolutionary adaptability in clinical Klebsiella pneumoniae

Domain: neuroscience
Published: 2026-02-07
Editorial outcome: major_revision

Abstract
Pathogenic bacteria continually evolve under antimicrobial pressure through acquired resistance genes, making it crucial to understand their evolutionary strategies. We identify a clinical Klebsiella pneumoniae isolate resistant to ceftazidime/avibactam (CZA), harboring heterogeneous multicopy blaCTX-M, among which a blaCTX-M-249 variant mediates CZA resistance. Both blaCTX-M-249 and its closely related allele blaCTX-M-65 are dominant within the clonal population and are located at two loci on the same plasmid, with their proportions shifting under antibiotic pressure. Using experimental and mathematical models, we demonstrate that the heterogeneous arrangement of blaCTX-M variants on the same plasmid confers greater stability and competitive advantage than that across separate plasmids, particularly during drug switching. Re-analysis of large genomic datasets supports the universality of this phenomenon. Our findings reveal an evolutionary strategy in which β-lactamase genes, through multicopy heterogeneity on a single plasmid, ensure stable inheritance of resistance and enhance bacterial adaptability under fluctuating clinical antibiotic pressures.

Introduction
Humans have combated pathogenic bacteria by discovering naturally occurring antibiotics or developing synthetic antimicrobial agents, yet bacteria have continuously evolved new resistance mechanisms1,2. Meanwhile, the pace of developing new antibiotics has lagged far behind the rate at which resistance arises, becoming the biggest challenge in the treatment of clinical infections3. β-lactam antibiotics were once the most important class of antibiotics in clinical use, with carbapenems in particular regarded as the last resort against Gram-negative bacterial infections4. However, the widespread prevalence of β-lactamase- or carbapenemase-producing strains in recent decades has driven development of alternative therapeutic approaches5, notably β-lactam/β-lactamase inhibitor combinations such as ceftazidime/avibactam (CZA), approved by the FDA in 20156,7. Nevertheless, the use of these combination drugs means that new resistance genes or mechanisms may continually be selected.

The introduction of CZA has provided an effective treatment option for infections caused by multidrug-resistant Klebsiella pneumoniae, particularly those caused by the widely prevalent KPC-producing strains7. However, the widespread use of CZA in the therapy of clinical infections has been accompanied by the emergence of various resistance mechanisms8. For instance, K. pneumoniae strains have acquired metallo-β-lactamases (MBLs), or their blaKPC genes have undergone point mutations mostly within the omega loop, or increased expression via multicopy blaKPC, all of which contribute to resistance6,9–11. Beyond KPC carbapenemase, mutations within the omega loop of extended-spectrum β-lactamases (ESBLs) can also confer CZA resistance, as seen in the rare CZA-non-susceptible CMY-172 and CTX-M-219, the latter closely related to the widespread CTX-M-1412,13.

In this study, we identified a CZA-resistant K. pneumoniae strain from a clinical patient and found that its resistance was mediated by a novel blaCTX-M variant closely related to blaCTX-M-65, a widely prevalent ESBL gene in recent years24,25. CTX-M-type β-lactamase genes comprise numerous variants that are classified into several groups based on sequence similarities, with the two major groups being the blaCTX-M-1 group and the blaCTX-M-9 group. blaCTX-M-65 belongs to the blaCTX-M-9 group and represents a variant derived from the ancestral member blaCTX-M-14. Similar to CTX-M-14, CTX-M-65 exhibits strong hydrolytic activity against cefotaxime (CTX)26. Other members of the CTX-M-9 group, such as CTX-M-27, possess an Asp240Gly substitution, which confers enhanced resistance to ceftazidime (CAZ)27. Furthermore, we found that the plasmid-borne blaCTX-M-65 and three closely related alleles in this strain appeared in a multicopy heterogeneous state. The aim of this study was to explore how variants coexist, plasmid location and multicopy heterogeneity support bacterial resistance and promote an evolutionary bet-hedging strategy under multidrug pressure.

Results
Clinically isolated CZA-resistant K. pneumoniae mediated by CTX-M variant
In a genomic sequencing-based surveillance project of resistant strains in the ICU of a tertiary hospital, we discovered two closely related bloodstream infection isolates collected from separate patients (Fig. 1, part I). One isolate from a patient with a history of CZA treatment, named ICU-3, exhibited resistance to CZA with a minimum inhibitory concentration (MIC) of 128 mg/L, while the other isolate from a patient who had not undergone CZA treatment, named ICU-18, was susceptible (Table 1). Both isolates belong to ST11 and differ by <10 single-nucleotide polymorphisms (SNPs). The most noteworthy CZA resistance-related differences included the nucleotide substitution A392G in blaKPC-2 (changed to blaKPC-157 in ICU-3) and two substitutions (A397G, C508T) in blaCTX-M-65. By testing the resistance conferred by blaKPC-157 cloned into pCR2.1, we found that this change did not cause an increase in CZA resistance, as recently demonstrated28 (Table 1). We then cloned the blaCTX-M mutant from ICU-3 into pCR2.1, but this resulted in colonies exhibiting heterogeneous genotypes. Among these, two predominant variants encoded the widely disseminated CTX-M-65 β-lactamase and a novel enzyme carrying Ser130Gly and Pro167Ser (based on the ABL scheme)29, designated CTX-M-249. Two additional rare variants, carried only one of the two substitutions and were designated CTX-M-247 and CTX-M-248, respectively (Fig. 1, part I and Fig. S1a). Among clones carrying blaCTX-M-65 or three novel alleles, only the CTX-M-249-producing clone exhibited a CZA MIC of up to 64 mg/L, indicating that this novel variant could mediate CZA resistance (Table 1). Sequencing chromatograms of directly amplified blaCTX-M from strain ICU-3 showed A/G and C/T double peaks at nucleic acid positions 397 and 508, confirming gene heterogeneity in this clinical strain (Fig. S1b).

Resistance profiles and hydrolytic activity of blaCTX-M-65 and three closely related alleles
The clones carrying blaCTX-M-65 and three closely related alleles exhibited different MICs of CAZ, CTX and CZA, indicating that distinct combinations of mutations at amino acid positions 130 and 167 result in varied resistance profiles (Fig. 1, part I). The clone expressing CTX-M-65 β-lactamase exhibited high-level resistance to CTX (>256 mg/L) and low-level resistance to CAZ (16 mg/L), but was susceptible to CZA (0.25 mg/L). In contrast, the CTX-M-249-producing clone was resistant to both CAZ (64 mg/L) and CZA (64 mg/L), but highly susceptible to CTX (0.5 mg/L). The CTX-M-247-producing clone was susceptible to both cephalosporins (CAZ: 1 mg/L, CTX: 0.125 mg/L) and to CZA (0.25 mg/L), whereas the CTX-M-248-producing clone exhibited resistance to CAZ (256 mg/L) and CTX (8 mg/L) but remained susceptible to CZA (8 mg/L) (Table 1 and Fig. S1c).

The distinct resistance profiles of CTX-M-65 and three closely related alleles displayed a trade-off pattern, indicating that point mutations at different sites of the β-lactamase altered its hydrolytic activity, as confirmed by enzymatic kinetic assays (Table S1). Both CTX-M-248 and CTX-M-249 exhibit enhanced CAZ hydrolysis compared to CTX-M-65. CTX-M-247 and CTX-M-249 showed markedly higher Ki values for avibactam and other common inhibitors, indicating reduced affinity. Thus, two point mutations in CTX-M-65 can shape interactions with substrates or inhibitors. Specifically, the Ser130Gly in CTX-M-247 diminishes enzyme affinity for inhibitors, while the Pro167Ser in CTX-M-248 enhances enzyme affinity for CAZ, and their combination in CTX-M-249 confers CZA resistance.

Proportion of blaCTX-M and three closely related alleles within a clonal population
In the clinical K. pneumoniae isolate ICU-3, long-read sequencing revealed that blaKPC-157 and two copies of blaCTX-M are co-localized on the same plasmid, pICU-3. Notably, the two copies of blaCTX-M are positioned at two loci approximately 54 kb apart. One locus consistently carries blaCTX-M-65, whereas the other locus, located near blaKPC-157, can harbor any of four blaCTX-M variants, with blaCTX-M-249 being the most frequently recovered (Fig. 2a). Plasmid pICU-18 carried by ICU-18 shares an identical backbone with pICU-3, differing only by five SNPs, including two within blaCTX-M, one within blaKPC, and two within a truncated IS26 element. Interestingly, under antibiotic pressure, both the copy number and the relative proportions of these variants shifted in ICU-3 bacterial cultures, as measured by digital qPCR and amplicon sequencing, respectively. Digital qPCR results showed that, compared with drug-free culture, exposure to 32 mg/L CZA increased blaCTX-M copy number to >10 per chromosome equivalent (p<0.0001), primarily driven by a rise in plasmid copy number (from ~2 to > 7, p=0.0013). At a higher 64 mg/L CZA, blaCTX-M copy number also approached 10 (p=0.0001), but without a marked plasmid copy number increase (p=0.9944); instead, the number of blaCTX-M copies per plasmid rose from 2 to ~3 (p=0.0006, Fig. 2b). These findings suggest that amplification of resistance genes enables adaptation to drug pressure, with distinct strategy depending on pressure intensity. Next, we applied amplicon sequencing to quantify variant proportions within the clonal population. Without the CZA drug, blaCTX-M-249 accounted for 44%, blaCTX-M-65 for 51%, and blaCTX-M-247/248 for ~2% each. Under CZA pressure, the proportion of blaCTX-M-249 increased while blaCTX-M-65 declined: at 32 mg/L, 54 vs. 41%, and at 64 mg/L, 77 vs. 19%. The frequencies of blaCTX-M-247/248 remained ~2% regardless of drug exposure (Fig. 2c). At 64 mg/L CZA, analysis of raw long reads revealed tandem multicopy formation (1–7 copies) of the blaCTX-M gene near blaKPC-157, likely explaining the increased proportions of blaCTX-M-249 at this drug concentration. This tandem amplification is probably mediated by IS26 elements bracketing the resistance gene to form a translocatable unit (TU). In contrast, no tandem copies were detected at the other blaCTX-M locus, likely because its flanking IS26 are in opposite orientations (Fig. 2a). Based on above evidence, we speculate that in the vast majority of pICU-3, blaCTX-M-65 and blaCTX-M-249 coexist in a multicopy heterogeneous state, with their copy numbers varying in response to drug selective pressure (Fig. 1, part II).

Advantages of plasmid-mediated multicopy heterogeneity
We constructed five models of plasmids carrying blaCTX-M variants (Fig. S2 and Table S2) to simulate and compare the advantages of multicopy heterogeneity at the population-level and single-cell level (Fig. 1, part III). Model 1 is DH5α with p65G (strain 65G, carrying blaCTX-M-65), and Model 2 is DH5α with p249R (strain 249R, carrying blaCTX-M-249), these two represent non-heterogeneity state. Model 3 is a 1:1 mixture of 65G and 249R (Heterogeneity in two cells, H2C), representing population-level heterogeneity. Model 4 is DH5α with both p65G and p249R, termed “Heterogeneity in two plasmids” (H2P), where heterogeneous blaCTX-M copies are duplicated on separate plasmids. Model 5 is DH5α with the single plasmid p65G-249R (carrying both blaCTX-M-65 and blaCTX-M-249), termed “Heterogeneity in one plasmid” (H1P), carrying heterogeneous blaCTX-M copies on the same plasmid (Fig. 3a). Then we evaluated the bacterial survival capability for these five models when facing two antibiotics (CAZ and CTX) simultaneously using a checkerboard susceptibility assay (Fig. 3b). Strain 65G grew up to high concentrations of CTX, but only at low concentrations of CAZ, while 249R grew up to high concentrations of CAZ, but not in CTX, both as expected. Models 3, 4, and 5, which express both CTX-M-65 and CTX-M-249 in different configurations, all showed growth up to relatively high levels of both CTX and CAZ. Among these, the single-cell level heterogeneity models (H1P and H2P) demonstrated stronger resistance to both drugs than the population-level heterogeneity model (H2C).

Stability of heterogeneity in H1P and H2P models
Next, we focused on the two single-cell level heterogeneity models with dual resistance genes, H1P and H2P (Fig. 1, part III). Both models enable bacteria to acquire a bet-hedging ability, allowing them to tackle environmental stress perturbations through genetic diversification, even with the short-term fitness cost. The existence of multiple variants of the same gene that confer distinct resistance profiles enables bacteria to withstand the combined effects of multiple drugs. However, the adaptability of bacteria in these two models still shows differences. Using fluorescence microscopy (Fig. S3a), bacteria cultured for one day under antibiotic pressure already showed distinct changes: in the H2P model, the initial orange fluorescence reverted to green in the presence of CTX (resistance conferred by CTX-M-65) and to red in the presence of CZA (resistance conferred by CTX-M-249). In the H1P model, however, orange fluorescence was consistently maintained under both CTX and CZA pressure, because CTX-M-65 and CTX-M-249 are encoded on the same plasmid. We further monitored the dynamics of the H1P and H2P models during six days of serial passage under CTX or CZA pressure using flow cytometry and colony counting. Flow cytometry revealed that, under continuous exposure to CTX or CZA, the proportion of H2P clones (dual-fluorescent cells) dropped from 80% to ~19 and ~40%, respectively. In the H2P strains, p65G was gradually lost under CZA selection (when p249R conferred resistance), whereas p249R was lost under CTX selection (when p65G was effective). By contrast, H1P strains retained >96% dual-fluorescent cells under both antibiotics, indicating stable maintenance of blaCTX-M-65, blaCTX-M-249, and p65G-249R (Fig. 4a and Fig. S3b). Bacterial counts further corroborated these results. After 6 days of serial passage in Lysogeny Broth (LB) with or without antibiotics, both models showed fitness costs with reduced dual-resistant populations. However, the H2P model exhibited a decline in dual-resistant bacteria relative to total cells under CTX or CZA selection, while nearly all surviving H1P cells maintained dual resistance (Fig. 4b and Fig. S3c). Collectively, these findings suggest that heterogeneity is more stable when both copies of blaCTX-M are on the same plasmid than distributed across separate plasmids.

H1P gains a competitive advantage when drug pressure switches
We conducted a competition assay by co-culturing H1P and H2P strains to better understand how these two models of heterogeneity balance their fitness under alternating antibiotics pressure (Fig. 1, part III). For differentiation in mixed population, the H1P strain was supplemented to express blue fluorescence (H1P-B), enabling identification by flow cytometry (Fig. S4a–c). Under antibiotic selection, H2P cells frequently lost one plasmid, giving rise to single-plasmid derivatives (65G or 249R), which were also distinguishable by flow cytometry. In a 5-day competition assay, H1P-B and H2P strains were co-cultured at an initial 1:1 ratio under increasing CTX concentrations for four days, followed by CZA on the fifth day. Flow cytometry revealed that under CTX pressure, H1P-B remained relatively stable for the first 3 days but dropped to <1% on day 4 at high CTX concentrations due to its fitness cost. In contrast, H2P rapidly diversified into three subpopulations: itself and the plasmid-loss derivatives 65G and 249R (Fig. 5a). Both H2P and 249R quickly decreased to <1% with rising CTX pressure, while 65G expanded over 98% owing to its CTX resistance and higher fitness. However, once the antibiotic pressure switched to CZA, H1P-B increased sharply to 74%, while 65G rapidly fell to 1%, and both H2P and 249R remained low (Fig. 5b and Fig. S4d). A parallel trend was observed when CZA was applied first: H2P and its derivative 65G declined rapidly over four days, while the CZA-resistant 249R expanded to 79%. H1P-B remained initially stable but then declined. On the final day, when CTX was introduced, H1P-B rapidly dominated the population at 90%, while H2P and both derivatives dropped to 1, 7, and 1%, respectively (Fig. 5c and Fig. S4e). Overall, despite its fitness cost (Fig. S5), the H1P model stably maintains both resistance genes and rapidly gains advantage upon drug switching, whereas the H2P strain suffers from plasmid loss and remains competitively disadvantaged.

Mathematical modeling of stability and competitive advantage
We developed an ordinary differential equation (ODE) model to simulate plasmid population dynamics, plasmid stability, and competitive advantage (Fig. 1, part III). The stability of plasmid heterogeneity in H1P and H2P subpopulations under exposure to CTX and CZA was simulated, with detailed predictions presented in Supplementary Fig. 6a, b. The simulations indicate that, in the presence of antibiotics, H1P exhibits significantly higher stability of plasmid heterogeneity compared to H2P. This finding is consistent with stability assay, as demonstrated in Fig. 4, where experimental observations align with the model’s predictions, confirming its reliability in assessing differential stability under antibiotic stress. Simulations of competitive interactions between H1P and H2P under alternating antibiotic exposures are shown in Fig. 6a, b and are consistent with experimental results. Under escalating CTX concentrations followed by a switch to CZA (16 mg/L) on Day 5, H2P remained stable at low CTX levels, but with increasing concentrations and continued passaging, the 65G subpopulation (one of the H2P plasmid-loss derivatives) expanded sharply and became dominant at 256 mg/L, while both H1P and H2P declined. After the switch to CZA, 65G dropped precipitously under drug pressure, and H1P emerged as the dominant subpopulation due to its broader resistance. A parallel trend was observed with escalating CZA concentrations followed by a switch to CTX (16 mg/L). H1P dominated early, but at higher concentrations the 249R subpopulation (another H2P plasmid-loss derivative) expanded substantially. Once switched to CTX, 249R rapidly declined, and H1P regained dominance owing to its enhanced resistance.

The constructed mathematical model allowed us to estimate the antibiotic pressure thresholds at which different resistant subpopulations gain dominance. Growth density comparisons across a range of CTX (0.125–4096 mg/L) and CZA (0.0625–256 mg/L) concentrations over 24 h revealed the boundary concentrations at which H1P and H2P-derived subpopulations became dominant (Fig. 6c, d). At low CTX levels, the combined density of H2P and its derivatives (65G, 249R) exceeded that of H1P, reflecting greater adaptability. However, H1P surpassed H2P and its derivatives at 37.09 mg/L, marking its competitive advantage (Fig. 6c). A similar trend was observed under CZA exposure. H2P and its derivatives predominated at low concentrations, but H1P overtook H2P at 1.25 mg/L and maintained dominance at higher levels (Fig. 6d).

Universality of resistance gene heterogeneity
To examine whether enhanced multidrug resistance adaptability from such heterogeneity represents a shared bacterial mechanism, we analyzed raw genomic data from additional K. pneumoniae isolates (derived from both humans and nonhuman sources) for evidence of multicopy heterogeneity in β-lactamase genes, such as some prevalent alleles belonging to blaSHV, blaTEM, blaCTX-M, blaNDM, blaKPC, and blaOXA (Fig. 1, part III). We used the raw genomic data of K. pneumoniae from our own surveillance projects (1580 isolates) and downloaded the raw genomic data of this bacterium from the NCBI sequence read Archive (SRA) database (6339 isolates), and evaluated the copy number and the presence of mutation heterogeneity in these β-lactamase genes by mapping raw reads. The estimated copy number distributions of the examined genes were highly variable, with a substantial fraction of isolates carrying more than one copy. Multicopy heterogeneity was observed across all β-lactamase genes analyzed (Fig. 7). Notably, as blaSHV is chromosomally carried in almost all K. pneumoniae and may additionally occur on plasmids, the proportion of isolates showing multicopy heterogeneity of blaSHV-12-like reached 20.15%. The multicopy heterogeneity rates for other β-lactamase genes, including blaTEM-1-like, blaNDM-1-like, blaCTX-M-15-like/65-like, blaKPC-2-like, and blaOXA-48-like, were 6.07, 1.20, 3.09, 0.48, and 0.30%, respectively (Table 2). Furthermore, the proportion of multicopy heterogeneity in K. pneumoniae derived from humans was higher than that from nonhuman sources. For example, the multicopy heterogeneity of blaCTX-M-65 in strains from humans was 20.96%, compared to only 4.01% in strains from nonhuman sources (p<0.0001). Even no multicopy heterogeneity of blaNDM-1-like, blaCTX-M-15-like/65-like, blaKPC-2-like and blaOXA-48-like was detected in strains from nonhuman sources, unlike human-derived isolates, which are mostly patient-associated and may have experienced stronger antibiotic selective pressure (Fig. 7 and Table 2).

Inferred evolutionary trajectory
We use an inferred evolutionary trajectory diagram to illustrate the relationships between several heterogeneity states during the evolutionary process, and a model diagram to describe the evolutionary advantage of resistance gene heterogeneity in the same plasmid (Fig. 8). Based on the evidence mentioned above, we hypothesize that the acquisition of a single blaCTX-M-65 by the plasmid represents an early stage in the evolutionary trajectory in this clinical strain. Under drug selective pressure, blaCTX-M-65 underwent gene amplification, mutations such as A397G and/or C508T, resulting in various allele forms. Interestingly, these heterogeneous alleles can coexist within a single clonal population, with two resistance genes responding to different drug pressures being carried on the same plasmid, while also acquiring higher resistance level through transient tandem multicopy (Fig. 8a). We speculate that non-heterogeneity and heterogeneity in bacterial individuals exhibit different adaptive behaviors when facing multiple drug (particularly the β-lactams) pressures. Through comparison of the two non-heterogeneity and three heterogeneity models, we found that only single plasmid-level multicopy heterogeneity enables bacteria to maximize adaptability under both constant drug pressures and during drug switching (Fig. 8b).

Methods
Strains and antimicrobial susceptibility testing
Clinical isolates were collected from intensive care unit patients of a tertiary hospital in China (Fig. 1, part I). The study was approved by the Institutional Review Board of the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine (No. 20201217-33), and written informed consent was obtained from all enrolled patients. The species identification of the bacterial strains was performed by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, bioMérieux, Craponne, France). MICs were determined using the broth microdilution method, the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) Guidelines (CLSI, 2022). Escherichia coli DH5α (Takara, Tokyo, Japan) and BL21 (DE3) (Weidibio, Shanghai, China) competent cells were used in this study. E. coli ATCC 25922 was used as reference strain for quality control.

Whole-genome sequencing and analysis
Genomic DNA was extracted using a Qiagen mini kit (Qiagen, Hilden, Germany). Whole-genome sequencing was performed using both the NovaSeq (Reagent Kit, v1.5, Illumina, San Diego, USA) and the MinION (Flow Cell, R9.4.1, Nanopore, Oxford, UK) platforms (Fig. 1, part I and II). The complete genome was finished by first assembling Illumina short reads, followed by polishing and scaffolding with Nanopore long reads using Unicycler v0.4.8 (https://github.com/rrwick/Unicycler)47. Multilocus sequence typing (MLST) was performed by MLST v2.23.0 (https://github.com/tseemann/mlst)48. Antimicrobial resistance genes were identified using ABRicate v1.0.1 (https://github.com/tseemann/abricate) with the ResFinder database49 and insertion sequences were identified using ISFinder50. The number of SNPs between K. pneumoniae isolates ICU-3 and ICU-18 was identified and assessed with Snippy v4.4.5 (https://github.com/tseemann/snippy) and snp-dists v0.6.3 (https://github.com/tseemann/snp-dists). The multiple alignment of the CTX-M amino acid sequence was performed using ClustalW (https://www.genome.jp/tools-bin/clustalw) and ESPript 3.0151. The protein secondary structure was predicted using SWISS-MODEL252. The genetic structures of plasmids and their comparative circular diagram were generated using Proksee (https://proksee.ca/)53.

Cloning the β-lactamase genes
PCR products of the complete blaKPC and blaCTX-M genes, amplified from DNA extraction from the clinical strain ICU-3, was ligated into pCR2.1 and transformed into a recipient strain E. coli DH5α. Each transformant carrying the recombinant plasmid (also referred to as a colony), therefore, contained only one variant of the PCR product. Colonies were selected on kanamycin-containing plates (50 mg/L), and 100 colonies were further confirmed by Sanger sequencing (Tsingke Biotech, Beijing, China). To determine the specific blaCTX-M variant at two distinct loci, we designed locus-specific primers to differentiate between the two sites, cloned the amplified products, and validated the different variant clones by Sanger sequencing (Fig. 1, part I). Primers (Tsingke Biotech, Beijing, China) used for amplification are listed in Supplementary Table 3.

Protein expression and purification
To confirm the enzymatic activity of these β-lactamase, CTX-M-65, CTX-M-247, CTX-M-248, and CTX-M-249 with His-tag were expressed and purified. Briefly, the coding sequences of blaCTX-M-65, blaCTX-M-247, blaCTX-M-248, and blaCTX-M-249 lacking the N-terminal secretion peptide sequences were cloned into the pET28a(+) by homologous recombination. The resulting expression plasmids were transformed into the BL21 (DE3) strain to express the proteins. Each enzyme was purified using the Ni-NTA method described previously54. The purified proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to quantitate and assess purity.

Enzyme kinetics
The kinetic parameters were determined using the steady-state kinetic assay. The phosphate-buffered saline (1X, pH 7.2) was used in all enzymatic assay systems. The initial rates of substrate hydrolysis for CAZ (Δε260=−9,000 M−1 cm−1) and nitrocefin (Δε482=15,000 M−1 cm−1) were measured using a D8 UV-visible spectrophotometer (Runqee, Shanghai, China) by monitoring absorbance changes in a 1-cm quartz cuvette55. For inhibitors and the low-affinity substrate, the Ki values of enzymes were determined by a direct competitive inhibition assay using 100 μM nitrocefin as a reporter substrate56,57. The data were globally fitted to the Michaelis-Menten equation in Graphpad Prism v9.0.0 to determine the values of Km, kcat, Ki and their corresponding standard errors.

Amplicon sequencing of blaCTX-M
The clinical strain ICU-3 was cultured in LB broth supplemented with different concentrations of CZA (0, 32, 64 mg/L) overnight at 37 °C and 200 rpm. Then, the genomic DNA was subsequently extracted and standardized to a final concentration of 10 ng/µL following quantification using the Qubit 4™ (Thermo Scientific, Waltham, MA, USA). PCR amplification was performed in a volume of 50 µL, using 25 µL 2× Taq PCR MasterMix (Vazyme, Nanjing, China), 1 µL genomic DNA, 1 µL (10 µM) each primer mixture, and 23 µL double-distilled water. Primers used here are listed in Supplementary Table 3. Sequencing of the PCR amplification products was performed on the NovaSeq (Illumina, San Diego, USA) sequencing platform. Raw reads were processed in CLC Genomics Workbench (QIAGEN, Hilden, Germany) by quality trimming (score limit: 0.01), trimming ambiguous nucleotides (maximum 2), and discarding reads shorter than 100 bp. Filtered reads were aligned to blaCTX-M-65, blaCTX-M-247, blaCTX-M-248, and blaCTX-M-249 reference sequences using BLAST. The percentage of each blaCTX-M variant in each sample was calculated as the proportion of reads aligned to each reference sequence across the different CZA concentrations (Fig. 1, part II).

Digital qPCR
Strain processing and DNA extraction were performed as for amplicon sequencing. Genomic DNA (10 ng/µL) was then ultrasonically sheared on ice. Ultrasonic treatment conditions were set as follows: ultrasonic interval time 10 s, ultrasonic occurrence time 5 s, ultrasonic whole time 2 min, and ultrasonic power 300 W. Following sonication, digital qPCR was performed using the Sniper DQ24 digital qPCR (Sniper, Suzhou, China) system (Fig. 1, part II). Specific primers and probes (Supplementary Table 3) targeted 16S rDNA (approximately eight copies in K. pneumoniae)58, pgi (housekeeping gene in K. pneumoniae), psiB_1 (pICU-3 specific single-copy gene), and blaCTX-M. Copy numbers based on absolute copy numbers, were calculated as: 16s rDNA (vs chromosome) = 16s rDNA/pgi, pICU-3 (vs chromosome) = psiB_1/pgi, blaCTX-M (vs chromosome) = blaCTX-M/pgi, blaCTX-M (vs pICU-3) = blaCTX-M/psiB_1.

Construction of plasmids for experimental models
We selected the relatively low-copy-number vector pACYCDuet-1 (Tsingke Biotech, Beijing, China) with p15A replicon (~13 copies), which carries a cat gene (conferring chloramphenicol resistance), to construct reporter plasmids harboring either single or dual blaCTX-M variants (Fig. 1, part III). The fluorescent protein genes were fused downstream of the blaCTX-M genes. The following fusion plasmids were constructed: CTX-M-65-eGFP (p65G), CTX-M-249-mCherry (p249R), and CTX-M-65-eGFP/CTX-M-249-mCherry (p65G-249R) (Fig. S2a). In order to successfully screen for the co-transformation of p65G and p249R using CZA and chloramphenicol, the cat gene in p249R was disrupted. The H2P strain was generated by co-transforming p65G and p249R (1:1) into E. coli DH5α, while the H1P strain carried p65G-249R. Transformants were selected on CZA (4 mg/L) and chloramphenicol (34 mg/L). The coexistence of blaCTX-M-65 and blaCTX-M-249 in H1P and H2P was confirmed by Sanger sequencing (dual peaks at positions 397: A/G, 508: C/T) and fluorescence microscopy (Olympus, Tokyo, Japan) (Fig. S2b). Images were processed with ImageJ (https://imagej.net/ij/).

Checkerboard susceptibility assay
The antibiotic susceptibility array was prepared in 96-well flat-bottom plates using the checkerboard method, a standard assay to evaluate synergistic effects of drug combinations59 (Fig. 1, part III). Strains representing different models (65G, 249R, H2C, H1P, and H2P) were inoculated into wells containing serial dilutions of CTX, CAZ, or their combinations to assess survival. The concentrations of CTX and CAZ were serially diluted two-fold, starting from 4096 and 256 mg/L, respectively. Two 96-well plates were used to perform the checkerboard assay, with the horizontal x-axis representing 12 dilution covering CAZ from 0 to 256 mg/L, and the vertical y-axis representing 16 dilution covering CTX from 0 to 4096 mg/L. Bacteria were inoculated at a final dilution of 1:200 and incubated for 18 h at 37 °C, cultures in each well were measured for optical density (OD600) as a measure of growth using Thermo Scientific Multiskan™ GO (Thermo Scientific, Waltham, MA, USA).

Heterogeneity stability assay
The stability of different heterogeneity models in constructed H1P and H2P strains was evaluated using fluorescence microscopy, flow cytometry, and bacterial counting on agar plates. (Fig. 1, part III). Fluorescence micrographs were collected using an Olympus microscope (Tokyo, Japan) and processed using ImageJ (https://imagej.net/ij/). The H1P and H2P strains were grown in LB supplemented with either CTX (16 mg/L) or CZA (4 mg/L) in a shaking bath at 37 °C, then the overnight culture was diluted 1:100 was subcultured under the same conditions, and passaged for 6 successive days. The percentages of various populations at day 0, day 1, day 3, and day 6 were enumerated using an LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were collected in FITC channel (for eGFP) and PI channel (for mCherry) by 488 and 587 nm excitation. Three biological replicates were performed for each strain. In terms of plate colony counting experiment, H1P and H2P processing methods were the same as those described above, whereas cultures of day 3 and day 6 were serially diluted in PBS and plated on the Mueller-Hinton agar (MHA), and MHA added with either CTX (16 mg/L) and CZA (4 mg/L). Three technical replicates were performed for each sample. After overnight incubation at 37 °C, CFUs were measured by colony counting.

Fitness determination
The fitness of all constructed strains was assessed by examining growth rates under drug-free and drug-pressure conditions. Three single colonies were chosen and inoculated into 2 mL of Mueller-Hinton Broth (MHB) culture solution containing the corresponding antibiotics for plasmid maintenance, followed by overnight shake culture at 37 °C. The overnight cultures were then diluted 1:1000 (or 1:100 under drug pressure) in fresh MHB medium in 96-well microtiter plates, and three technical replicates were carried out. Optical density at 600 nm (OD600) measurements were taken every 5 min over a period of 20 or 24 h at 37 °C to construct a growth curve. Relative growth rates under drug-free conditions, as well as the degree of growth inhibition under CTX or CZA compared with drug-free conditions, were calculated using an R script, and plots were generated using GraphPad Prism v9.0.0.

Competition experiment
We performed bacterial growth competition experiments under antibiotic pressure (Fig. 1, part III). On the backbone of p65G-249R, the gene encoding blue fluorescent protein (mTagBFP) was subcloned inside the BamHI restriction site of the p65G-249R (named p65G-249R-B). The fluorescence microscope (Olympus, Tokyo, Japan) was used to confirm the successful introduction of the blue fluorescence. Images were processed with ImageJ (https://imagej.net/ij/). H1P-B and H2P strains were grown overnight in LB broth added with CZA and chloramphenicol (4 and 34 mg/L) at 37 °C, and the culture was washed and adjusted to an OD of 0.5 in PBS. The two individual strains were mixed 1:1 immediately, then the mixture was cultured in LB broth that contained daily increased concentrations of corresponding antibiotics. The mixture was incubated at 37 °C for 20 h under constant shaking, percentage of various populations were enumerated using an LSRFortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Data were collected in FITC channel (for eGFP), PI channel (for mCherry) and BV421 channel (for mTgBFP) by 488, 587, and 399 nm excitation.

Population-level plasmid dynamics model
A mathematical ODE model was constructed to simulate plasmid dynamics and population changes of bacterial communities under antibiotic exposure (Fig. 1, part III). We assumed the bacterial population consists of a plasmid-free (PF) subpopulation (with density at time t denoted by DPF(t)) and plasmid-containing subpopulations (including D65G(t), D249R(t), DH2P(t), and DH1P(t)). The growth of each subpopulation, limited by environmental resource concentration, was modeled using a standard Monod term60: G(S(t)) = ρμ(S(t)), where μ(S(t)) = μmax,i * S(t) / (Ki + S(t)), characterized by the maximum growth rate μmax,i and half-saturation constant Ki. To simulate the effects of antibiotics (CTX and CZA, denoted as AT and AZ, respectively), the sensitivity of subpopulation Di to CTX and CZA was modeled with a killing rate γTi and γZi. And the hydrolysis efficiency of enzymes produced by bacteria against CTX and CZA was represented by βTi and βZi, respectively. In our model, the plasmid loss rate, denoted by λ, is based on the binomial distribution of plasmids during bacterial division, where each plasmid has a probability P = 0.5 of entering either daughter cell. For the Di subpopulation (with initial copy number n of p65G and n of p249R, we evaluated three plasmid loss scenarios: (1) the probability of losing all plasmids (p65G and p249R) is (0.5)^(np65G+np249R), resulting in a plasmid-free subpopulation; (2) the probability of losing all p249R to become D65G is P^np249R * (1 - P^np65G), where the segregational loss rate θp249R is defined as this probability; (3) the probability of losing all p65G to become D249R is P^np65G * (1 - P^np249R), where the segregational loss rate θp65G is defined as this probability. To summarize, the system of ordinary differential equations governing the plasmid dynamics at the population level can be expressed as follows:

dS/dt = -(μPF(S)DPF + μ65G(S)D65G + μ249R(S)D249R + μH2P(S)DH2P + μH1P(S)DH1P) (1)

dAT/dt = -AT(βT * DPF + βT * D65G + βT * D249R + βT * DH2P + βT * DH1P) (2)

dAZ/dt = -AZ(βZ * DPF + βZ * D65G + βZ * D249R + βZ * DH2P + βZ * DH1P) (3)

dDPF/dt = G(S)DPF + λ * G(S)D65G + G(S)D249R + G(S)DH2P + G(S)DH1P - DPF(γT * AT + γZ * AZ) (4)

dD65G/dt = (1-λ)G(S)D65G + θp249R * G(S)DH2P - D65G(γT * AT + γZ * AZ) (5)

dD249R/dt = (1-λ)G(S)D249R + θp65G * G(S)DH2P - D249R(γT * AT + γZ * AZ) (6)

dDH2P/dt = (1-λ)G(S)DH2P - DH2P(γT * AT + γZ * AZ) (7)

dDH1P/dt = (1-λ)G(S)DH1P - DH1P(γT * AT + γZ * AZ) (8)

Parameter estimation
Following a similar approach reported in the literature61, we employed a Markov Chain Monte Carlo (MCMC) method with Metropolis-Hastings sampling to estimate growth-related parameters in the Monod model. We estimated the distribution of the maximum growth rate to half-saturation constant ratio μmax,i / Ki and the resource conversion rate ρi from experimental growth curves of each subpopulation (D PF, D 65G, D 249R, D H2P, and D H1P) grown in isolation (Fig. S8 shows the MCMC diagnostic plots). We evaluated the susceptibility of each subpopulation to CTX and CZA through single-drug dose-response experiments, estimating the killing rate parameters γTi and γZi (Fig. S9). We measured plasmid copy number using digital qPCR to derive the plasmid loss rate λ, θp249R, and θp65G, with primer details provided in Supplementary Table 3. OD600 values were converted to CFU/mL as previously described62. Numerical solutions of Eqs. (1–8) were obtained using the standard ODE solver ode45 in MATLAB R2021a (The MathWorks, Inc., Natick, MA, USA). All simulations were performed with time measured in hours. The final parameter values applied in the numerical simulations of the model are listed in Supplementary Table 4. The corresponding code used is available online (see Code availability).

Raw data analysis for multicopy heterogeneity
We conducted a comprehensive analysis of β-lactamase gene multicopy heterogeneity by genome-wide raw read alignment of a broad range of K. pneumoniae isolates (Fig. 1, part III). The prevalent β-lactamase gene alleles in K. pneumoniae, such as blaSHV-12, blaTEM-1, blaCTX-M-15/65, blaNDM-1, blaKPC-2, and blaOXA-48, were used as references for raw reads mapping. Strains carrying genes similar to these alleles were all evaluated for their multicopy heterogeneity status. Considering that the heterogeneity of blaCTX-M may arise from genetically distant ancestral groups, the blaCTX-M-1-group and blaCTX-M-9-group, we selected blaCTX-M-15 and blaCTX-M-65 as representative references for the two groups, and took the union of strains carrying multicopy heterogeneity for these alleles. Gene copy number estimation was performed based on previously established methods63, while single-nucleotide variations (SNVs) and insertions/deletions (InDels) were analyzed using GATK 4.5.0.064, Samtools 1.1, and bcftools 1.20. The corresponding code used is available online (see Code availability). When mapping raw reads to the consensus sequence, sites with less than 30x coverage depth are not considered. The data sources included isolates collected by our laboratory from a large number of hospitals across China between 2017 and 201965,66. Additionally, we downloaded all publicly available K. pneumoniae Illumina sequencing reads from the SRA database (https://www.ncbi.nlm.nih.gov/sra), covering the release period from January 1 to December 31, 2023. Some of these strains are derived from humans, with most of the strains coming from patients, while others derived from nonhuman sources such as livestock farming, food, and the natural environment. The detailed BioProject lists are provided in Supplementary Tables 5 and 6.

Statistical analysis
The t-tests were performed to compare colony counting numbers, and fitness cost. One-way ANOVA was used to analyze gene copy numbers. Two-way ANOVA was performed to compare the percentages of dual-fluorescent cells under CTX or CZA pressure, as measured by flow cytometry. Chi-square tests were used for comparison of multicopy heterogeneity in strains from humans or nonhuman sources. p values <0.05 were considered significant. The statistical analyses were implemented in GraphPad Prism v9.0.0.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Weng et al presents one model case of paralogous gene evolution in one genome by focusing on a plasmid detected in a hospital. By conducting additional survey in publicly available raw reads, they found that the paralogous (beta-lactamase) gene evolution in one plasmid is common. Furthermore, the authors characterized the nature of four CTX-M variants in vitro. These are solid and important data. The statement in the abstract is valid. The manuscript is clearly written. However, I am not sure about the significance of discussion on the stability of gene heterogeneity, comprising a large part of this study. The fitness advantage of the “dual-copy genes on one plasmid” state (DCG model) over the “dual-copy plasmids” state (DCP model) under drug-switching condition (Fig 5eg) is immediately obvious as far as plasmid loss can occur, unless huge fitness cost arises from the DCG state. The points of discussion should be “when does DCP or mix model win?”. Therefore, to discuss the conditions where DCP/PCG/mix state wins, I think that the authors need to implement mathematical model for the serial passage or competition experiments considering the fitness advantage of plasmid-bearing cells and plasmid loss rate. To address the above problem, ideally, all experiments should be designed using a low copy number and stable plasmid such as mini-F to mimic the condition of the real situation. Although the authors mentioned the cost of these cell types in the absence of antibiotics (Fig S1), competition experiments and stability test were performed in the presence of antibiotics where plasmid-free cells are immediately killed. Therefore, the cost information is not really linked to experimental data. The most parts of figure are used to explain the experimental set-up. Figure information should be more condensed. For example, Figure 4 can be condensed to only two line plots: one from flow-cyto (Fig4c), one from CFU (Fig4de). Figure 5 can be condensed to only Fig5eg. It appears that to select for specific (mix model) cell types, a high concentration of CTX, 256 ug/ml, was used in the experiment. The concentration should be in clinically achievable range (< or = 100 ug/ml). MINOR POINTS: FIG1a. Gray area does not precisely connect the circular map and gene map of tandemly amplified loci. FIG3b. I think that gene map on plasmids is unnecessary as they are redundant with panel a. FIG4. Plasmid stability data measured by flow cytometer (a-c?) and by CFU count (d, e). c. Please clarify in legend whether this graph is based on CFU count or summary of flow cytometer counts. d-e: d and e show the same information. I suggest the author should show the data of proportion of the resistance gene-carrying cells by line plot like panel c. Fig5. d label. Please add ug/ml after antibiotic concentrations because it is not immediately clear whether those are symbol of CTX-M variants.", "reviewer_2": "Regarding the manuscript ”The heterogenetic multicopy of blaCTX-M variants on one plasmid optimizes the evolution adaptability in the clinically isolated Klebsiella pneumoniae” by Weng et al. This manuscript presents a novel and well-supported study on how multicopy heterogeneity of blaCTX-M variants on a single plasmid contributes to the adaptability and stability of antibiotic resistance in Klebsiella pneumoniae. Overall, the manuscript is well-written, and the study is highly relevant, novel, and scientifically rigorous, with important clinical relevance. The combination of whole-genome sequencing, digital PCR, amplicon sequencing, and in vitro modeling provides strong experimental support for the proposed mechanism. However, the comprehensiveness of the study makes it somewhat difficult to follow, and some sections appear misplaced, affecting the clarity and flow of the manuscript. Below, I have outlined major and minor comments that would benefit the manuscript. Major Comments 1. Structural Issues & Misplaced Sections: Several sections appear misplaced within the manuscript. I recommend restructuring the introduction and results sections to improve clarity and logical flow. Some specific examples: - Lines 99-111: This section describes the findings of the study, which should not be placed in the introduction. The introduction should focus on background information and knowledge gaps, leading to the study’s aim and objectives. - Lines 120-122: This section describes the aim of the study, which should be included in the introduction, not the results section. - Lines 331-337: Discussion elements appear within the results section. These should be moved to the discussion section. - Lines 359-361: Similarly, discussion/conclusion elements are found in the results section and should be relocated. I suggest a thorough review of the manuscript structure to ensure that each section follows a logical order. 2. Results Section is Long & Difficult to Follow The results section is long, which is expected with the comprehensiveness of the study, making it a little challenging to follow. Consider referring to the steps in the flow-diagram (Figure 1a) at relevant points in the results. Possibly expand the flow-diagram to further guide the reader through the workflow and experimental design. This small change could significantly improve readability and clarity. 3. Clarification of Nucleotide and Amino Acid Mutations In Lines 123-124, the authors mention the noteworthy changes, including: “A397G, C508T in the bla….” Then, in Lines 131-133, the authors mention the corresponding amino acid changes: “Ser130Gly and Pro167Ser”. However, based on the alignment in Figure 1b, these mutations appear to correspond to amino acid positions 133 and 170, not 130 and 167. Clarification needed: Are the mutation positions correctly assigned? If different numbering conventions were used, this should be explicitly stated in the Methods section. 4. Statistical Analysis Throughout the manuscript, results are stated as “significant”, but p-values are missing in multiple places. Example: Line 192 mentions significance without statistical support. Include p-values wherever statistical significance is claimed. 5. Figures are Overloaded – Consider Moving Some to Supplementary Material While only seven figures are presented, each figure contains a high number of panels. I recommend to move redundant figures to supplementary materials to streamline the main text. Recommended changes: - Move Figure 1b and 1c to supplementary material. - Move Figure 3c and 3d, but expand Figure 1b by integrating the findings for the five different models. - Move Figure 4a, 4b, and 4d to the supplementary. - Move entire Figure 5 to the supplementary. This restructuring would help reduce figure complexity while keeping essential data accessible. Minor Comments I have a few minor comments on wording and terminology - Line 55: “The antibiotics developed by humans…” What about the naturally occurring ones? - Line 58: “Carbapenems were once considered the last-resort…” Carbapenems are still a last-resort antibiotic, so this should be reworded for accuracy. - Line 457: “We postulate…” think it should be changed to “We presented” or “We constructed” - Lines 642-643: “alignment of genome raw reads in a broader range of K. pneumoniae.” Reword for clarity. Perhaps: “Genome-wide raw read alignment of a broad range of K. pneumoniae isolates.” - Figure 2 – Circular Map: What software was used to generate the circular map in Figure 2? This should be mentioned in the figure legend or methods section.", "reviewer_3": "This manuscript describes a study looking at blaCTM-65 and variants detected at different copy numbers on the same plasmid in a clinical isolate of Klebsiella pneumoniae, with different gene combinations and copy numbers influencing resistance to ceftazidime, cefotaxime and ceftazidime+avibactam. Analysis of available sequences suggested that multiple copies of AMR gene variants in a single isolate may be more common than is usually recognized. While the results are potentially interesting and useful, there are some problems. Better sequence analysis, with reference to the available literature, is needed to improve accuracy in some of the descriptions. The detail about the results of experiments is wordy and uses unnecessarily complex terms, making it rather inaccessible and overwhelming. The text would benefit from language editing to make it clearer and easier to follow (and there is some odd/unscientific phrasing in places) and its could be condensed – some examples and suggestions are given below. There are many large complex figures and some thought about how these could be reduced would also be beneficial (e.g., parts showing detailed results of experiments could be made Supplementary, with summaries only in main text) – again I have given some suggestions below. Major points 1) blaCTX-M-65 and variants, Lines 100-2,129-136, Lines 100-2 - the information about blaCTX-M-65 could be expanded, explaining that blaCTX-M-65 belongs to the blaCTX-M-9 group but does not have a mutation resulting in Asp240Gly usually associated with CAZ resistance (as found in e.g., in blaCTX-M-27). Lines 131-46 could be condensed and simplified e.g., it is not really relevant when blaCTX-M-249 or the other variants were named. Line 131-3 – this is unclear. What was cloned that revealed the variants? Line 132 – are these amino acid positions numbered according to the Ambler scheme for class A beta-lactamases (PMID: 2039479). If so, this should be clearly stated. Line 134 – it is antibiotics that have MICs, not isolates. Line 136 – should be “mutations in blaCTX-M-65” Line 138 – how were these “clonal isolates” obtained? And it should be “alleles” here. Lines 145-6 – do not make sense and needs rewording. 2) Plasmids, contexts/amplification of blaCTX-M-65 and variants, Lines 123-5, 179-216, 312-397 The assembled sequence of pICU-3 should be submitted to GenBank, along with the sequence of pICU-18, in addition to raw reads. It is possible to add comments to GenBank entries to explain that e.g. the sequence submitted is a representative of several variants of the same plasmid that can have different numbers of copies of a region that includes blaCTX-M-65 but also the other variants. The structure of pIUC-3 needs to be explained in the text and compared with pICU-3. Lines 186-208 - from Fig. 2, but not really explained in the text, it seems that the copy of blaCTX-65 that is not amplified also does not undergo the mutations? If so, this might need to be taken into account when calculating/drawing conclusions from the number of copies e.g. plasmid copy number will increase the copy numbers of both this unchanging copy of blaCTX-M-65 and whichever variant is in the other position, which could be blaCTX-M-65, 247, 248 or 249, while gene amplification will not affect the copy number of the non-amplified blaCTX-M-65 copy. It is not clear if/how this has been taken into account in the calculations. Lines 182-3 – how was this calculated? What are these numbers? Line 188-9 – are there any clues in the sequence about what is responsible for this increase in plasmid copy number? This should be looked at. Line 193 – one of the ONT raw reads provided has 7 copies of the TU, the other 3 plus probably a truncated fourth one. How does this fit with \"about three\" stated here? Lines 197–208 – it’s not entirely clear from Methods how these proportions were determined/calculated and a bit more detail might be helpful. Lines 209-10 – presumably “long reads from isolates grown in 64 mg/L CAZ” is meant? This should be rewritten. Line 212 – this needs to be looked at further and explained better and more accurately. IS903, or a truncated copy, is commonly found downstream of blaCTX-M-9 group genes in ISEcp1 “transposition units” (TPUs) responsible for moving these genes. Examination of the pICU-3 sequence and raw long reads provided shows that, as expected, IS903 has nothing to do with the amplification. The amplified region instead seems to be a 4844 bp IS26 “translocatable unit” (TU; positions 58367-63210 in pICU-3 sequence provided), something that has been reported previously for different AMR genes (e.g. Refs 33, 34 cited here) forming a chain of “pseudocompound” transposons. The authors need to look at recent reviews and/or papers on IS26 mechanisms of transposition (mainly by RM Hall and colleagues, but also M Chandler and colleagues). I also would suggest looking at whether this TU has been seen before and at papers on the plasmid contexts of blaCTX-M-65 (e.g. PMID: 22984205). Lines 213-4 – this region cannot not undergo the same type of IS26-mediated tandem amplification as the flanking IS26 copies are in opposite orientations. 3) Lines 218-329 Models of heterogeneity This part of the text gets hard to follow and could be simplified and condensed. Line 219 – “plasmid-harboring beta-lactamase genes” does not make sense. Presumably “plasmids harboring” and “blaCTX-M genes” could be specified here, rather than “beta-lactamase genes”. Lines 221-2 – genes are cloned in fusion with genes, not proteins, and the gene names are blaCTX-M-65 and blaCTX-M-249. This should be written more accurately (and maybe moved to Methods). Line 222-5 – “for example” should be deleted if you then list everything that was constructed. Maybe just saying here that plasmids encoding the following fusions were constructed: CTX-M-65-eGFP (p65G), CTX-M-249-mCherry (p249R) and both CTX-M-65-eGFP and CTX-M-249-mCherry (p65G-249R), using R to indicate the red fluorescence rather than “m” for mCherry, and explaining this and also that both colours together give orange. Lines 226 – the plasmid names could then be used to simplify the text i.e. Model 1 is DH5alpha with p65G (strain 65G), Model 2 is DH5alpha with p249R (strain 249R), Model 3 is a 1:1 mixture 65G and 249R, Model 4 is DH5alpha with both p65G and p249R and Model 5 is DH5alpha with p65G-249R (or maybe consider putting this information a table). Having two names for Model 4/DCPs and Model 5/DCGs is also confusing and seems unnecessary. The names DCP and DCG are confusing as they are too similar (and the “s” at the end is not needed). Any further necessary details (e.g. of transformation) and Lines 234-6 could be left to Methods (or Fig. 2d legend, where they are already) and also consider whether the information in Lines 236-40 would be better mainly moved to Methods. Lines 240-3 – it might be clearer to say that strain 65G grew up to high concentrations of CTX, but only at low concentrations of CAZ, while 249R grew up to high concentrations of CAZ, but not in CTX, both as expected, if this is what is meant (“tolerable drug” does not make sense here). Lines 243-7 could then say that Models 3, 4 and 5, which express both CTX-M-65 and CTX-M-249 in different configurations, all showed growth up to relatively high levels of both CTX and CAZ, then explain the differences between Model 3 and Models 4 and 5. Lines 253 – unclear, needs rewording. Line 257 – suggest “selection with”. Line 259-65 – wording does not make sense and is unclear – say that Model 4 has CTX-M-65 (green) and CTX-M-249 (red) encoded by two different plasmids in the same cell. Then that in Model 4 orange fluorescence was replaced by green in the presence of CTX (resistance conferred by CTX-M-65) and by red in the presence of CAZ (resistance conferred by CTX-M-249), then information from Lines 266-9. Lines 265-71 – could just state that Model 5 has CTX-M-65 and CTX-M-249 encoded on the same plasmid and that orange fluorescence was maintained and reword Lines 269-71 – e.g., I don’t think that using “paralogous” here is helpful. Lines 272-288, L290-329 – much of the current wording does not make sense or is unclear - the same principles as above can be applied to explain this more simply and clearly. 4) Universality of resistance gene heterogeneity, Lines 331-61 Again, the language could be simplified. Lines 332 – suggest something like “Having multiple copies of different variants of a resistance gene that confer different phenotypes”. Lines 335, 350-1, 355-7 – blaSHV is an intrinsic chromosomal gene in K. pneumoniae, which is the source of plasmid-borne variants. This needs to be properly explained and I would suggest looking at PMID: 17999968, which discusses blaSHV variants on plasmids and the chromosome. I would also suggest looking at PMID: 28812563 about blaTEM. This could all have implications for the analysis. Lines 335-57 – the name blaOXA encompasses many families that are not closely related, so it does not make sense to give a single % for multicopy heterogeneity for blaOXA. Was a particular blaOXA looked at? If so, this must be stated. If not, then the analysis may need to be redone. Line 342 – is this sequence data available and/or associated with a publication? If so, please give the BioProject and/or citation. Suggest “raw sequence data from this species” Line 351-57 – which blaCTX-M group or did you look at multiple groups? Major sequence differences between members of different blaCTX-M groups (Group 1, Group 9 etc) are not due to mutations, but because the source genes are from different Kluyvera species (there are also hybrids between Group 1 and Group 9 genes). Minor changes between blaCTX-M genes within each group are probably explained by mutations that have occurred since capture. 5) Other scientific points Lines 40, 48, 334-5 – genes encode proteins, they are not encoded. Suggest “carried”/“carry” or “plasmid-borne”, as appropriate. Lines 75, 382 – it is not clear what is meant by “the CTX-M-14 isoform” here. bla gene variants are assigned numbers by NCBI. A CTX-M-14 sequence with these amino acid changes appear to have been numbered CTX-M-219, described as “inhibitor resistant”, with the accession CP019080 that cites Ref 14. See https://www.ncbi.nlm.nih.gov/pathogens/refgene/#blaCTX-M-219. Line 90-1 – many large conjugative plasmids that typically carry AMR genes are present at around only one copy/cell and “duplication state” needs rewording. Line 116 – homology is not quantifiable, so what is really meany by “highly homologous” here? “closely-related”? This is from the SNP distance of <10? Also suggest “isolates” here, Line 122 etc. Lines 123-4 – which blaKPC allele does ICU-18 have? This must be stated. Line 136 – this should be “mutations” not “mutants” Lines 139, 403, 405 etc. – I don’t think that it makes sense to use “wildtype” – it can be deleted. Lines 171-7 – are there other studies reporting on the effects of these mutations? Line 200 – I would suggest making these % less precise. Line 210 – specify blaKPC-157 here. Line 282 – LB originally stood for “lysogeny broth”. 5) Discussion This is long and wordy and could be condensed. The discussion section should not repeat information in Results (or Introduction) and comments on the rest of the manuscript need to be considered. Line 365, 482 – is this really completely novel? Lines 365-76 – this seems more like introductory information and parts seem to repeat the Introduction. Lines 382-3 – wording does not make sense. Line 392 – “reflected on several level of meanings” does not make sense. Line 394-5 – the plasmid copy number and how it was determined and the distance between the blaCTX-M-65-like genes should be stated in Results. Line 397 - “elements” is not needed – these are IS. Line 416 - “AS” is not needed here and suggest referring to the paper without naming the authors Line 417 – the genes are blaTEM-1 and blaTEM-12 Line 428 – it is the constructed plasmids that are lost? Lines 434-6 – why wasn’t this data included? Line 436 – “inhibitor” is not needed here. Line 438 – wording does not make sense. Line 446 – I think that heteroresistance can also be due to copy number? See papers by D. Anderson. Line 452 – use “and/or” and delete “dual site mutations” Lines 457-65 – this information might be in the legend of Fig. 7, which currently very short. Lines 468-9 – unclear what is meant here. Line 470 – “studies” Lines 471-5 – is this really true for long-read if multiple copies of the gene are in different genetic locations, as for copies of blaCTX-M-65 (or variants) both in pICU-3 here? Line 484 – what is meant by “horizontal transfer elements” here? Line 492 – “introduction” seems like the wrong word here – maybe “recognition” 6) Methods These lack detail/clarity in places, but text in other places could be condensed. Primers - although the text refers to Table 3 in several places, it is not always immediately clear which primer sets are being referred to and it might be helpful to state the primer names in the text or better indicate in Table 3 which section they were used for. Line 499 – presumably “species identification” is meant here? Lines 523-7 – the wording does not make sense here and more details are needed. What was cultured for the PCR? Lines 573-84 – this section can be condensed. The first sentence is not really needed, is something like “Three reporter plasmids based on pACYCDuet1 were constructed” and state which plasmid has which gene. Genes are cloned in fusion with genes, not proteins, and first explain that pACYCDuet1 has a cat gene. 7) Other minor/formatting/English Title needs some rewording – suggest “Heterogeneity in multiple copies of blaCTX-M-65-like genes in a clinical isolate of Klebsiella pneumoniae”. Throughout – odd/unscientific phrasing needs to be changed, e.g., Line 33 “combat”, Line 42 “lock in, Line 55 – “constant battle”, Line 77 - bacteria do not really “choose their evolutionary paths”, Line 386 - “ingeniously explored”, Line 423 “solidified”, Lines 448, 454 “locked on/onto”. Throughout – “the blaCTX-M-65” is incorrect. This should either be “the blaCTX-M-65 gene” or preferably just “blaCTX-M-65” e.g. Line 102. (except Line 36, where “the” is needed before “CTX-M-65-type”). Also, for other gene/allele names. “bla” in the name indicates the gene Throughout – the abbreviation CZA is used for ceftazidime/avibactam. The common abbreviations CAZ and CTX, as used in figures, could also be defined and used in text. Line 37-8 – I don’t think that “monoclonal” is commonly used to describe bacteria? Maybe “a single strain was found to have multiple copies of blaCTX-M-65 or minor variants that conferred different spectrums of resistance”. Line 42 ,412 etc – I am don’t think that that “paralogous” is the clearest term to use. Line 47 – suggest “clinical isolates” rather than “patient-isolated strains”. Lines 47-50, 61-62, 73-74 need rewording. Line 80-1, 108, 384, 389 suggest just “trade-offs”, rather than the “the trade-offs effect” – or at least “the trade-off effect” and reword “may introduce new roles”. Line 84-5 – “Compared with… mutations, genetic amplifications” – i.e. delete “the” Line 93 – either “multicopy resistance plasmids” or “multiple copies of a resistance plasmid” Lines 95-6, 110 – need rewording. Lines 99-102 – this long sentence might be better split but certainly needs rewording. Line 111 – “considerable numbers” Line 122 – “The results showed” not needed. Suggest “Both isolates belong to ST11 and differ by <10 SNPs” Line 126 – “clones with blaKPC-157” Lines 157-60 – suggest “each has one of the two mutations seen in CTX-M-249” and “the two common third generation cephalosporins” is not needed (they have already been discussed). Line 180 – “long-read sequence assembly” Line 227 – “respectively” needs to be at the end of the sentence. Line 228 – “mixing of” Line 261 - “observation of fluorescent microscope image” is not needed Line 337 – “mutation” i.e. no “s”. Lines 339-40, 354-5, 654-5 etc. – I would suggest using “isolates from humans or other sources”. Line 513 – “was performed” Line 517 – “between ICU-3 and ICU-18 was calculated”. Line 530 – don’t need both “enzymes” and “enzymatic” here. Line 534 - “successful different” needs rewording. Lines 540-1 do not make sense and need rewording. I don’t think that “velocities” is the right word here. Line 557, 569 etc. - “are listed”, not “were listed” Line 562 – “as for amplicon sequencing”. Lines 562-3 – the wording here does not make sense. Lines 568-9 – this is unclear and needs to be explained better. Lines 578 – “removed” Line 579, 80 – “DCP strains”, “DCG strains” Line 581 – “dual antibiotic” Line 588 – not clear what is meant here and “including” is not needed. References are normally placed before figure legends. Ref 8 needs page numbers or equivalent. Something seems to be wrong with page numbers in Ref 49. Journal titles need to be correctly abbreviated. Ref 4, 12 etc., remove Title Case. Species and gene names etc. need to be correctly formatted. 8) Figures General – some parts could be moved to supplementary, shorter titles would be better and legends could be condensed. Fig. 1 I think that most parts of this figure are not needed or could be moved to supplementary. I would suggest retaining the grid showing resistance profiles from part a (with amino acid changes for each CTX-M variant listed) plus part d in the main manuscript. Maybe consider adding CTX-M-219 (“CTX-M-14 isoform”) discussed in the text to part b, if retained as Supplementary. Fig. 2 Part a - on the circular diagram of the plasmid at least the two IS26 flanking blaCTX-M-65/247/248/249 should be shown, plus preferably the other AMR genes, IS and transposons in this region and surrounding the other copy of blaCTX-M-65 (see ISfinder). The diagram to side should properly show the boundaries of IS26 and of all copies of the amplified unit. The blaSHV gene number should be added. Part b CZA 32 mg/L – colour is too pale, hard to see Fig. 3 Suggest moving parts a-c to supplementary, simplifying the plasmid diagrams in part b and showing each model above the corresponding plot in part d. The wording of this part of the legend also needs improving – e.g. it is not expression of plasmids, but of fused genes. Fig 4 Part a – suggest stating clearly what is in DCPs and DCGs on the figure, rather than having to refer to text in legend. Using the names suggested above would help with this. Part b – this could be supplementary and just show the results in part d in the main manuscript. Part c – this could be supplementary and just show the results in part e in the main manuscript Line 749 in legend – respectively appears incorrect here – it is not clear what is being referred to. Fig. 5 Parts a and b do not both need to be shown. Parts d and f could be moved to supplementary, just showing the summary of results in parts e and g. Fig. 6 See comments above on analysis of blaSHV, blaCTX-M and blaOXA genes. Line 781 – “including” is incorrect here, as all genes analysed are then listed Fig. 7 What is the evidence for multidrug resistance pressure resulting in two copies of blaCTX-65 on the plasmid? What to the following unlabeled arrows represent? 9) Supplementary Table S1 How do values compare with previous determinations for CTX-M-65, if available? Table S3 It should be possible to fit this table on a single page, for ease of reading, e.g. reduce margins, “amplification” is not needed in every line of the last column, remove “the” before “blaCTX-M-65” etc. Final column heading could be “Purpose” instead of “Usage”. PsiB should be pisB in final column, if the gene is meant. CTX-M-F/R/P – blaCTX-M should be used for the gene and which blaCTX-M genes/alleles would be amplified? Table S4 This could be split into two tables, one with all isolates from humans, with this stated in the title and the final column removed, and one for other host species giving the details. I don’t think that the “Study” column is really needed – readers can get this information from the BioProject, if required. BioProject numbers and host species would be easier to read if aligned to the left. ATTACHED REVIEWER REPORT — IMPORTANT The text below is a complete reviewer report submitted as a PDF attachment. It appears after the Creative Commons license block. Assign it to whichever reviewer wrote 'see pdf' or had no inline text. Response to Reviewer #1 (Remarks to the Author): Weng et al presents one model case of paralogous gene evolution in one genome by focusing on a plasmid detected in a hospital. By conducting additional survey in publicly available raw reads, they found that the paralogous (beta-lactamase) gene evolution in one plasmid is common. Furthermore, the authors characterized the nature of four CTX-M variants in vitro. These are solid and important data. The statement in the abstract is valid. The manuscript is clearly written. However, I am not sure about the significance of discussion on the stability of gene heterogeneity, comprising a large part of this study. The fitness advantage of the “dual-copy genes on one plasmid” state (DCG model) over the “dual-copy plasmids” state (DCP model) under drug-switching condition (Fig 5eg) is immediately obvious as far as plasmid loss can occur, unless huge fitness cost arises from the DCG state. The points of discussion should be “when does DCP or mix model win?”. Therefore, to discuss the conditions where DCP/PCG/mix state wins, I think that the authors need to implement mathematical model for the serial passage or competition experiments considering the fitness advantage of plasmid-bearing cells and plasmid loss rate. To address the above problem, ideally, all experiments should be designed using a low copy number and stable plasmid such as mini-F to mimic the condition of the real situation. Response: We sincerely thank the reviewers for their valuable suggestions, which have provided us with important insights to enhance the scientific depth of our study. In response to the reviewers’ recommendation to redesign the experiments using low-copy-number and stable plasmids such as mini-F, we tested the low-copy plasmid pCC1FOS (Genome Res, 2002;12(9):1434-44), whose copy number and stability are comparable to mini-F. However, our experiments revealed that pCC1FOS has several limitations: (1) its extremely low copy number (approximately 1–2) likely led to insufficient fluorescent protein expression when enhanced green fluorescent protein (eGFP) or red fluorescent protein (mCherry) gene was fused downstream of the resistance genes bla or bla , resulting in CTX-M-65 CTX-M-249 fluorescence signals below the detection threshold (Chem Rev, 2002;102(3):759-81) and rendering subsequent dynamic tracking experiments infeasible. (2) it could not support the construction of the DPD model (the DCP model described in our previous manuscript, which carries both p65G and p249R plasmids), as DPD strains require the simultaneous maintenance of at least two plasmids, while pCC1FOS’s strict copy-number control prevented dual-plasmid coexistence; (3) its copy number was too low to realistically mimic clinical strains, which we have confirmed to possess plasmid copy numbers greater than two, often increasing to over a dozen under antibiotic pressure. Based on these factors, we concluded that pCC1FOS is not suitable for achieving accurate simulation and stability, whereas the plasmid pACYCDuet-1 used in our study was confirmed by digital qPCR to have a copy number of approximately 13, placing it in the medium-to-low range, which better reflects clinical conditions and facilitates fluorescent labeling for visualization and validation. Following the reviewers’ suggestion, we developed an ordinary differential equation (ODE) model using estimated parameters from the pACYCDuet-1 system to simulate the dynamics of plasmid-free (PF), 65G, 249R, DPD, and SPD (the DCG model described in our previous manuscript, which carries a single plasmid p65G-249R) strain populations under gradients of the antibiotics CTX and CZA. This model, based on Monod growth kinetics and incorporating resource limitation, antibiotic killing effects, enzymatic hydrolysis efficiency, and plasmid loss rates, produced simulation results for serial passage (Fig. S6 a-b) and competition experiments (Fig. S6 c-d) that were consistent with our earlier experimental findings, supporting its validity. In addition, we applied this model to explore the dominance boundaries between DPD and SPD, which helps address the question of “when does DPD or SPD model win?”. The results showed that when CTX concentrations were below 39.31 mg/L, the growth fitness advantage of DPD exceeded that of SPD. When CTX was between 39.31 and 367.01 mg/L, the resistance advantage of SPD outweighed the growth advantage of DPD, and SPD dominated. At CTX concentrations above 367.01 mg/L, one of the plasmid-loss derivatives of DPD, 65G, became dominant. Similarly, for CZA, when concentrations were below 1.34 mg/L, DPD had a growth advantage over SPD. When CZA was between 1.34 and 127.91 mg/L, SPD’s resistance advantage surpassed the growth advantage of DPD, allowing SPD to dominate. At concentrations above 127.91 mg/L, another plasmid-loss derivative of DPD, 249R, prevailed. Although the authors mentioned the cost of these cell types in the absence of antibiotics (Fig S1), competition experiments and stability test were performed in the presence of antibiotics where plasmid-free cells are immediately killed. Therefore, the cost information is not really linked to experimental data. Response: We thank the reviewer and have followed this recommendation. Indeed, there was an insufficient correlation between the data on fitness costs under antibiotic-free conditions and the results from stability and competition experiments. To address this, we performed additional growth curve experiments to systematically evaluate the growth fitness of PF (plasmid-free, i.e., DH5α), 65G, 249R, DPD, and SPD strains under antibiotic-free conditions (0 mg/L) as well as across a range of antibiotic concentrations. Specifically, we tested CZA at 0, 1, 4, 8, and 32 mg/L, and CTX at 0, 4, 16, 64, and 256 mg/L, which together covered the antibiotic concentration ranges used in our stability and competition assays. We measured OD over 600 time, calculated the area under the OD –time curve (AUC), and quantified growth 600 performance using the relative growth inhibition rate [100 × (1 – AUC/AUC₀), where AUC₀ represents the AUC for each strain at 0 mg/L], thereby providing an integrated measure of growth fitness across different levels of antibiotic pressure (Fig. S5). The most parts of figure are used to explain the experimental set-up. Figure information should be more condensed. For example, Figure 4 can be condensed to only two line plots: one from flow-cyto (Fig4c), one from CFU (Fig4de). Figure 5 can be condensed to only Fig5eg. Response: We sincerely appreciate your suggestion to make the figures more concise, and we agree that adjusting Figures 4 and 5 can improve the focus of data presentation. Since several other reviewers also recommended modifications or adjustments to the figures, we carefully considered all feedback and followed the principle of highlighting only the most important and essential information. The specific adjustments are as follows: We reconstructed Figure 1 as a workflow diagram and moved the original panels 1b–d to Supplementary Figure 1. From the original Figure 3, we retained panels b and d, while moving panels a and c to Supplementary Figure 2. In Figure 4, panels a, b, and d were moved to Supplementary Figure 3, panel c was retained, and panel e was changed from a bar chart to a line chart to improve clarity. From the original Figure 5, panels a, b, c, d, and f were moved to Supplementary Figure 4, leaving only panels e and g, which were revised into the current Figure 5. It appears that to select for specific (mix model) cell types, a high concentration of CTX, 256 ug/ml, was used in the experiment. The concentration should be in clinically achievable range (< or = 100 ug/ml). Response: We sincerely thank you for pointing out that the CTX concentration used in our experiments (256 μg/mL) exceeds the clinically achievable range (≤100 μg/mL), and we agree that adjusting to clinically relevant concentrations would strengthen the applicability of our study. The original in vitro experiments employed 256 μg/mL primarily to rapidly screen for dominant patterns and to investigate bacterial competition dynamics under extreme antibiotic pressure. In response to your suggestion, we applied an ordinary differential equation (ODE) model to simulate population dynamics across a broader range of CTX concentrations (0.125– 4096 μg/mL) and confirmed that the competitive dynamics observed at clinically relevant concentrations (≤100 μg/mL) were consistent, thereby providing a more detailed analysis (Fig. S6). MINOR POINTS: FIG1a. Gray area does not precisely connect the circular map and gene map of tandemly amplified loci. Response: We sincerely thank you for pointing out the issue regarding the connection of the gray-shaded regions in the figure. We think you may be referring to Fig. 2a (rather than Fig. 1a), as Fig. 2a presents the circular plasmid map and the gene diagram of its tandem amplification sites, with gray regions used to indicate the corresponding relationships. In response to your comment, we have revised Fig. 2a and optimized the rendering of the gray-shaded areas to ensure precise connections between the circular map and the gene diagram of the tandem amplification sites. FIG3b. I think that gene map on plasmids is unnecessary as they are redundant with panel a. Response: Thank you for your suggestion. Figure 3b, which illustrates bacterial strains carrying different plasmids, corresponds directly to the five models we constructed, as reflected in the checkerboard susceptibility results shown in Figure 3d. In line with your advice and other reviewers’ comments on simplifying figures, we have revised Figure 3 by retaining panels 3b and 3d into the new Figure 3. Panels 3a and 3c have been moved to Supplementary Figure S2a and S2b to remove redundancy and improve clarity. FIG4. Plasmid stability data measured by flow cytometer (a-c?) and by CFU count (d, e). c. Please clarify in legend whether this graph is based on CFU count or summary of flow cytometer counts. d-e: d and e show the same information. I suggest the author should show the data of proportion of the resistance gene-carrying cells by line plot like panel c. Response: We greatly appreciate your suggestion, which is highly valuable for improving the clarity of the figures and the effectiveness of data presentation. We have revised Figure 4 as follows: Figure 4c, which is based on flow cytometry data and shows the proportion of cells carrying resistance genes, has been updated with a legend explicitly stating that the data were obtained from flow cytometry counts, and the figure legend has been revised accordingly to ensure accuracy. Since Figures 4d and 4e conveyed overlapping information, we have moved Figure 4d to Supplementary Figure 3c, and converted Figure 4e from a bar chart to a line graph (now Figure 4b) to enhance clarity. Fig5. d label. Please add ug/ml after antibiotic concentrations because it is not immediately clear whether those are symbol of CTX-M variants. Response: Many thanks. We have added the unit “mg/L” to all antibiotic concentrations (e.g., CTX and CZA) in the labels of Figure 5d and 5f to clearly indicate that these values represent antibiotic concentrations rather than CTX-M variants. According to the reviewer’s suggestions, we have moved these two panels to the current Supplementary Figure 4.", "reviewer_author_response": "Response: We sincerely appreciate the reviewers’ valuable suggestions. We have thoroughly revised the manuscript, reconstructed the workflow diagram (Fig. 1), and reorganized the presentation of the results accordingly. Substantial language improvements were also made in the Introduction, Discussion, and Methods sections. In addition, the composition and order of the figures were adjusted to make them clearer and more concise. We hope these revisions have further strengthened and improved the study. Major Comments 1. Structural Issues & Misplaced Sections: Several sections appear misplaced within the manuscript. I recommend restructuring the introduction and results sections to improve clarity and logical flow. Some specific examples: - Lines 99-111: This section describes the findings of the study, which should not be placed in the introduction. The introduction should focus on background information and knowledge gaps, leading to the study’s aim and objectives. Response: Thank you for this valuable suggestion. We have restructured the last paragraph of the Introduction to place more emphasis on the background information and then clearly present the study’s aim and objectives. - Lines 120-122: This section describes the aim of the study, which should be included in the introduction, not the results section. Response: We moved these sentences, similar to the aim of the study, to the last paragraph of the Introduction. Here, we condensed and revised the following sentence to make the meaning complete and concise. - Lines 331-337: Discussion elements appear within the results section. These should be moved to the discussion section. Response: Thank you for your suggestion. We have moved these sentences to the appropriate section of the Discussion. In fact, we reorganized the description of the results regarding the universality of resistance gene heterogeneity, presenting only the results and keeping the sentences concise. - Lines 359-361: Similarly, discussion/conclusion elements are found in the results section and should be relocated. Response: Similar to the above point, we reorganized this part of the results, and all discussion-like statements, including the one you mentioned, have been moved to the Discussion section. I suggest a thorough review of the manuscript structure to ensure that each section follows a logical order. Response: Thank you for your suggestion. We have made substantial revisions to the manuscript, making the description of the results more concise and clear, and adjusting the logical structure of different sections to improve coherence. 2. Results Section is Long & Difficult to Follow The results section is long, which is expected with the comprehensiveness of the study, making it a little challenging to follow. Consider referring to the steps in the flow-diagram (Figure 1a) at relevant points in the results. Possibly expand the flow-diagram to further guide the reader through the workflow and experimental design. This small change could significantly improve readability and clarity. Response: We sincerely thank you for pointing out the complexity of the Results section and for your valuable suggestion to improve the workflow diagram. To enhance readability, we expanded and refined Fig. 1 (originally Fig. 1a) by dividing it into three sections, thereby optimizing the workflow to more clearly guide readers through the research process. Specifically, the revised Fig. 1 now consists of three parts corresponding to the core components of our study. Part I: discovery of clinical CZA resistant strain and identification of the novel variant which mediated the resistance; Part II: confirmation of the profile of heterogeneous multicopy bla variants on plasmids; Part III: exploring the advantages of single-plasmid CTX-M duplicates in experimental and mathematical models. Each part incorporates detailed research content and uses arrows and annotations to visually present the logical flow. In the Results section, we now refer to the relevant parts of Fig. 1 in the corresponding paragraphs. 3. Clarification of Nucleotide and Amino Acid Mutations In Lines 123-124, the authors mention the noteworthy changes, including: “A397G, C508T in the bla….” Then, in Lines 131-133, the authors mention the corresponding amino acid changes: “Ser130Gly and Pro167Ser”. However, based on the alignment in Figure 1b, these mutations appear to correspond to amino acid positions 133 and 170, not 130 and 167. Clarification needed: Are the mutation positions correctly assigned? If different numbering conventions were used, this should be explicitly stated in the Methods section. Response: Thank you for drawing attention to the description in lines 123–124 (nucleotide substitutions A397G and C508T in bla ) and lines 131–133 (amino acid substitutions CTX-M-249 Ser130Gly and Pro167Ser). To clarify the correspondence between nomenclature and numbering, we confirm that A397G and C508T are based on nucleotide numbering of the bla gene, whereas the amino acid numbering Ser130Gly and Pro167Ser follows the CTX-M-249 ABL standard numbering scheme for Class A β-lactamases (Biochem J, 1991; 276:269-70). To improve clarity, we have added an explicit note about ABL numbering and reference 30 in the main text, and indicated the amino acid numbering in Supplementary Fig. 1a. 4. Statistical Analysis Throughout the manuscript, results are stated as “significant”, but p-values are missing in multiple places. Example: Line 192 mentions significance without statistical support. Include p-values wherever statistical significance is claimed. Response: Thank you for raising this question. We have thoroughly reviewed the entire manuscript and have systematically added the precise p-values, wherever statistical significance is claimed. This includes all data concerning: gene copy numbers, derived from digital qPCR; colony counting numbers, obtained through plate counting assays; fitness cost, calculated from growth curve analysis; percentage of dual-fluorescent cells under CTX or CZA pressure analyzed by flow cytometry. For the specific example cited by the reviewer (Line 192 in the original manuscript), we have revised the sentence to include the statistical support as follows: “Digital qPCR results showed that, compared with drug-free culture, exposure to 32 mg/L CZA increased bla copy number to >10 per chromosome equivalent (p = 0.0030), CTX-M primarily driven by a rise in plasmid copy number (from ~2 to >7, p = 0.0184). At a higher 64 mg/L CZA, bla copy number also approached 10 (p = 0.0081), but without a marked CTX-M plasmid copy number increase (p = 0.8735); instead, the number of bla copies per plasmid CTX-M rose to ~3 (p = 0.0069).” These comprehensive statistical details have been added throughout the manuscript (marked in the revised file). 5. Figures are Overloaded – Consider Moving Some to Supplementary Material While only seven figures are presented, each figure contains a high number of panels. I recommend to move redundant figures to supplementary materials to streamline the main text. Recommended changes: - Move Figure 1b and 1c to supplementary material. - Move Figure 3c and 3d, but expand Figure 1b by integrating the findings for the five different models. - Move Figure 4a, 4b, and 4d to the supplementary. - Move entire Figure 5 to the supplementary. This restructuring would help reduce figure complexity while keeping essential data accessible. Response: We greatly appreciate your suggestion and agree that moving redundant subfigures to the supplementary materials can improve the focus of the main text. Taking into account both your recommendations and comments from other reviewers, we have revised the figures as follows: Figure 1: we expanded the original Figure 1a (now Figure 1), and the content of the original Figure 1d has been incorporated into Figure 1 (part I) as a grid map; the original Figures 1b, 1c, and 1d have been moved to the supplementary materials (now Figures S1a, S1b, and S1c). Figure 3: we retained Figures 3b and 3d and combined them into the new Figure 3, while Figures 3a and 3c were moved to Supplementary Figures S2a and S2b to eliminate redundancy and enhance clarity. Figure 4: we moved Figures 4a, 4b, and 4d to Supplementary Figures S3a, S3b, and S3c; the original Figure 4e was converted from a bar chart to a line graph (now Figure 4b) to improve readability; the current Figure 4 retains the original Figure 4c (now Figure 4a) and the revised Figure 4b. Figure 5: we moved Figures 5a–d and 5f to Supplementary Figures S4a–e, while retaining only the original Figure 5e and 5g(now Figure 5a and 5b), which represents the core data, in order to maintain conciseness in the main text. Minor Comments I have a few minor comments on wording and terminology - Line 55: “The antibiotics developed by humans…” What about the naturally occurring ones? Response: Thank you for pointing that out. We have revised the first sentence of the Introduction for clarity. - Line 58: “Carbapenems were once considered the last-resort…” Carbapenems are still a last-resort antibiotic, so this should be reworded for accuracy. Response: We agree with the point that carbapenems are still a last-resort antibiotic, and we have reworded this sentence accordingly. - Line 457: “We postulate…” think it should be changed to “We presented” or “We constructed” Response: We have rephrased the sentence in question and the subsequent paragraph describing Figure 7b to eliminate ambiguity. In addition, to improve the conciseness of the main text, we have moved this text to the legend of Figure 7. - Lines 642-643: “alignment of genome raw reads in a broader range of K. pneumoniae.” Reword for clarity. Perhaps: “Genome-wide raw read alignment of a broad range of K. pneumoniae isolates.” Response: We have revised this sentence as suggested. - Figure 2 – Circular Map: What software was used to generate the circular map in Figure 2? This should be mentioned in the figure legend or methods section. Response: The genetic structures of plasmids and their comparative circular maps were generated using Proksee. We have described it in the methods section with reference 52."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors responded to my comments appropriately. Simulation results based on real experimental data added strength to their message. I recommend the authors’ current results for publication in this journal. Legibility of this manuscript has been improved. However, I still found several vague descriptions/presentation in the current manuscript. Furthermore, the simulation program was not released to public yet. Currently, readers are difficult to reproduce data analysis. The following minor points should be improved. Line 117-121. This sentence is a little too long for me. I recommend the authors rephrase this sentence. Line 628: The software platform: Python, R, C++, or MATLAB etc is not described. Codes should be deposited to public repository such as Github, figshare. Line 628: Unit of time used in simulation is hour? Please mention. Line 699: I think that Code availability section is also needed. Line 1038 panel e,f: Does this assume “one-round” batch culture competition assay starting with with 1:1 ratio mixture of SPD and DPD at the density of 1/100 of final density, but not serial batch culture transfer like experiments in panel c,d ?. Please clarify. This is just suggestion: Supplementary figure S9- panel c,d,e,f are important results. I suggest the authors place these panels as a new main figure or add these to Figure 5 (competition assay results figure). Line 666 and Line1058: right panel Y-axis. The unit in these growth curves appear to be CFU/mL. How OD600nm values (source OD data) were converted to CFU/ml is not clear. Please mention in the main text or legend. Supplementary Table 5: mu.max/K should read mu.max/Ki.", "reviewer_2": "I had the opportunity to review the first version of this manuscript and have now had the pleasure of evaluating the revised version entitled “Heterogeneous multicopy resistance genes on a single plasmid enhance evolutionary adaptability in clinical Klebsiella pneumoniae”. The authors have clearly made a substantial effort to address the comments and suggestions provided by all reviewers. The revised manuscript shows significant improvement in structure, clarity, and overall readability. The reorganization of figures and sections has made the study more coherent and easier to follow. The updated workflow figure (Fig. 1) effectively guides the reader through the study and provides a clearer overview of the experimental design. Overall, the authors have responded thoroughly and constructively to the reviewers’ feedback, and the manuscript has improved notably as a result. The revisions have strengthened both the presentation and the scientific clarity. I find that all my major concerns from the first review have been addressed satisfactorily. At this point, I only have a few minor comments. Minor comments The language occasionally shifts between tenses, so a brief consistency check would also be beneficial. Line 41–46: “Within this clonal population, blaCTX-M-65 and three of its variants were present…” Please list all alleles explicitly here (e.g. blaCTX-M-65, blaCTX-M-249, …) to avoid ambiguity. Additionally, blaCTX-M-65 is a specific allele within the CTX-M-9 group, not a group itself. Therefore, the other genes are not truly “variants of blaCTX-M-65.” They are better described as other CTX-M-9 group blaCTX-M alleles closely related to blaCTX-M-65. Suggested rephrasing: “Within this clonal population, blaCTX-M-65 and additional CTX-M-9 group blaCTX-M alleles (blaCTX-M-247, blaCTX-M-248 and blaCTX-M-249*) closely related to blaCTX-M-65 were identified.” Please apply consistent terminology throughout the manuscript. Line 77–79: “…or blaKPC they carry has undergone…” Consider replacing the first “or” with “the” and moving this phrase towards the end of the sentence for smoother flow, together with the information in lines 80–81. Line 108–109: See comment about line 41-46. As correctly noted in line 112, blaCTX-M-65 belongs to the CTX-M-9 group. Therefore, the other genes mentioned should not be referred to as “variants of blaCTX-M-65,” since they represent distinct alleles within the CTX-M-9 group, not subvariants of blaCTX-M-65. Please rephrase to “other CTX-M-9 group blaCTX-M alleles (closely related to blaCTX-M-65).” blaCTX-M-65 was the first to be described in the literature; however, due to heterogeneity among these alleles, it is not possible to determine which appeared first in the isolate, and thus which may be the derivative of the other. Line 132–133: Consider clarifying this sentence to read: “…included the nucleotide substitution A392G in…” Line 415, 422, and 426: Figure 7 is first introduced in the Discussion. New material should not be presented at this stage. Consider referring to Figure 7 earlier in the Results section.. Figure 1: Consider referring explicitly to the different parts of Figure 1 within the Methods section to improve readability and guide the reader through the workflow.", "reviewer_3": "This revised manuscript describes detection of blaCTM-65 and minor variants on the same plasmid in a clinical isolate of Klebsiella pneumoniae, with different gene combinations and copy numbers influencing resistance to ceftazidime, cefotaxime and ceftazidime+avibactam. Different models with different combinations of plasmids carrying blaCTX-M-65 and/or variants were used to explore the dynamics. Available sequences were also examined to look for multiple variants of selected bla genes in a single isolate, which suggests that this may be more common than is usually recognized. While the data are substantial, interesting and useful, and the authors have addressed many points raised by all three reviewers, improving the manuscript, there are still a few problems with scientific accuracy etc, as detailed below. While I think that the manuscript structure is better, further condensing/reorganising and editing/simplifying the text could still improve accuracy, ease of reading and flow – some examples are given below, including figure legends. Scientific points/accuracy 1) ORIGINAL COMMENT: Line 90-1 – many large conjugative plasmids that typically carry AMR genes are present at around only one copy/cell RESPONSE: Yes, there are some large plasmids that exist only as one copy. However, it has been reported that plasmid copy number ranges from only slightly higher than chromosomal copy number in low copy plasmids to tens of copies per cell in high copy plasmids, which means that most plasmids have more than one copy per bacterial cell (Nat Rev Microbiol, 2021; 19(6):347–359). Our wording was not sufficiently rigorous, and we have revised “which exist” to “most of which exist”. NEW COMMENT: The paper listed here (cited as Ref 22) also states that “Low copy number plasmids (LCPs) are typically large (from tens to hundreds of kilobases), have a low copy number and are frequently conjugative”. Many plasmids carrying AMR genes fit this category – probably including pICU-3/pICU-8 studied here - so while “most of which exist” could be numerically accurate, I would still argue that the most important plasmids for spreading AMR are mainly low copy. This also relates to Reviewer 1’s first comment, about using a low-copy number plasmid for models. While I appreciate the problems with this explained in the response to this comment, I think that it is important to at least state the copy number of pACYCDuet-1 somewhere (apologies if this is already there and I missed it) and maybe also explain that there were technical problems with using a lower copy number plasmid. I think it’s also important to acknowledge (in the Discussion) that the models here may not exactly reflect what might happen “in the real world” for large conjugative plasmids with e.g., addiction systems ORIGINAL COMMENT: “duplication state” needs rewording. NEW COMMENT: Now Line 99 – I still think that “a duplication state” is an odd and unclear description for plasmids. If “as multiple copies” is meant, this should be stated, but see comment above. 2) Lines 65-71 NEW COMMENT: Lines 65-71 - I think that beta-lactams would still be considered the most important class of antibiotics, and use of beta-lactamase inhibitors is not new (clavulanic acid etc.), although there are new types such as avibactam, so I would suggest some rephrasing here. Also consider whether refs 4, 5, 6, and 7 are all really necessary here. 3) blaCTX-M-9 group variants, mutations and phenotypes, Lines 109-115. ORIGINAL COMMENT: blaCTX-M-65 and variants, Lines 100-2,129-136 Lines 100-2 - the information about blaCTX-M-65 could be expanded, explaining that blaCTX-M-65 belongs to the blaCTX-M-9 group but does not have a mutation resulting in Asp240Gly usually associated with CAZ resistance (as found in e.g., in blaCTX-M-27). RESPONSE: …“blaCTX-M-65 belongs to the blaCTX-M-9 group, but unlike typical members of this group (such as blaCTX-M-27), it does not carry the Asp240Gly substitution that enhances resistance to CAZ. Instead, blaCTX-M-65 exhibits strong hydrolytic activity against CTX, rather than CAZ” NEW COMMENT: Now Lines 100-2,129-136. The ancestral CTX-M-9 group members (CTX-M-14, CTX-M-9) do not have the Asp240Gly mutation (nor do CTX-M-1 or blaCTX-M-3, equivalent ancestors in the CTX-M-1 group). The “CTX” in CTX-M was used to indicate resistance to CTX, with mutant versions that that confer CAZ resistance arising later, so variants with the Asp240Gly mutation aren’t really “typical”. Also note that CTX-M-65 is a variant of CTX-M-14. Lines 109-10 - “originally” can be deleted. From the title Refs 26 and 27 cited here don’t seem to be specifically about CTX-M-65? ORIGINAL COMMENT: Lines 171-7 – are there other studies reporting on the effects of these mutations? RESPONSE: The dual mutations at positions 130 and 167 in blaCTX-M-65 has not yet been reported. However, the effects of single mutation at each of these sites on CTX-M enzymatic hydroactivity have been documented in several studies. For example, in Antimicrobial Agents and Chemotherapy (2003; 47(9):2958–2961), the Ser130Gly mutation was shown to reduce the hydrolytic activity of CTX-M enzymes against cefotaxime while increasing resistance to inhibitors including sulbactam, clavulanic acid, and tazobactam. In PLoS Pathogens (2010; 6(1):e1000735), the Pro167Ser mutation was reported to decrease the hydrolytic activity of group 1 CTX-M enzymes against cefotaxime. By contrast, in Antimicrobial Agents and Chemotherapy (2006; 50(2):731–738), the Pro167Ser mutation was shown to enhance the hydrolytic activity of group 9 CTX-M enzymes against ceftazidime. NEW COMMENT: Now Lines 100-2, 129-136. This information should be included in the manuscript and the relevant papers cited, to make it clear that these are not novel mutations and their effects have been described previously, even if they were first found in a new combination here. Ref 14 describing CTX-M-14 also discusses the effects of the P167S mutation. 4) Lines 125-163 - example of suggested reorganisation/simplification Sections could be combined to reduce repetition and condense. Maybe separate section on enzyme kinetics? Line 139 - this wording doesn’t make sense – the gene that you are attempting to clone is not blaCTX-M-65, but the mutant from ICU-3. Maybe something like “We attempted to clone blaCTX-M-249 from ICU-3 into pCR2.1, but this resulted in colonies with different blaCTX-M genes. Most had blaCTX-M-65 or blaCTX-M-249, but two other variants, blaCTX-M-247 and blaCTX-M-248 were also present in a few. Lines 159-163 – it is the clones encoding the CTX-M enzyme that are susceptible/resistant. 5) Lines 133-6 blaKPC-157 PMID: 40643750 (published July 2025) describes KPC-157 having the same phenotype as KPC-2. Referring to this paper would allow the text here to be simplified and would back up the result obtained here. Suggest something like “By testing the resistance conferred by blaKPC-57 cloned into pCR2.1 we found that this change did not cause an increase in CAZ resistance, as recently demonstrated (Ref)” (the carbapenem resistance is not really relevant to this manuscript). 6) Plasmid sequence differences, Lines 176-204 Line 182 - My alignment of the sequences of pICU-3 and pICU-18 provided/in GenBank shows that the two changes in addition to the ones in blaKPC and the nearby blaCTX-M gene are in adjacent bases in one copy of IS26. These changes should be carefully checked by mapping Nanopore reads, and if correct, properly described. See also PMID: 34015086. Some rewording/reorganising might also help to make things clearer. For example, maybe first say that long read sequencing showed that ICU-18 carries blaKPC-2 and two copies of blaCTX-M-65, one near blaKPC-2 and one about 54 kb away, on a single plasmid named pICU-18. Then say that ICU-3 has an almost identical plasmid, named pICU-3, with blaKPC-157, one of the four blaCTX-M-65 variants close by and blaCTX-M-65 at the other location, Note that “Backbone” in reference to a plasmid is usually taken to mean the region encoding plasmid functions, into which regions containing resistance genes are inserted. 7) Line 187 – plasmid copy number ORIGINAL COMMENT: Lines 188-9 – are there any clues in the sequence about what is responsible for this increase in plasmid copy number? This should be looked at. RESPONSE: … the molecular mechanism underlying the increase in plasmid copy number remains unclear. We performed long-read sequencing and assembly under CZA pressure and compared the results with drug-free conditions, focusing on the plasmid replicon and replication-associated regulatory genes, but detected no genetic changes that could account for the elevated plasmid copy number. NEW COMMENT: Now Lines 187 - would this analysis have identified any heterogeneity in these regions of the plasmids? If not, maybe it would be worth looking at this? I also think it would be worth mentioning in the manuscript itself that there appear to be no changes in the plasmid that would be expected to increase copy number, if this is the case. 8) ORIGINAL COMMENT: Line 212 … I also would suggest looking at whether this TU has been seen before and at papers on the plasmid contexts of blaCTX-M-65 (e.g. PMID: 22984205). RESPONSE: reports specifically involving blaCTX-M-65 TUs remain limited. In our previous work, we identified an IS26-mediated tandem multicopy structure of blaCTX-M-65 in an E. coli isolate (J Glob Antimicrob Resist, 2023; 35:202–209), though its structure differed from that observed in the present study. In addition, another study from China (J Antimicrob Chemother, 2013; 68(1):46–50) also reported a similar IS26-mediated tandem duplication unit of blaCTX-M-65 in E. coli. NEW COMMENT: Please consider referring to the JAC paper. 9) ORIGINAL COMMENT: Lines 218-329 Models of heterogeneity This part of the text gets hard to follow and could be simplified and condensed. RESPONSE: … we revised the model names and abbreviations: Model 4 is now referred to as Dual-Plasmid Duplicates (DPD), meaning heterogeneous blaCTX-M copies are duplicated on separate plasmids; Model 5 is now referred to as Single-Plasmid Duplicates (SPD), meaning heterogeneous blaCTX-M copies are duplicated on a single plasmid. The difference between the initials “D” and “S” helps readers quickly distinguish dual-plasmid from single-plasmid. NEW COMMENT: While I think that the revised model names are better, and agree that it is easier to tell the acronyms apart, to me “Dual plasmid duplicates” and “Single plasmid duplicates” are confusing descriptions. The important thing is that the two genes are different and where they are located. I would suggest considering these names further – maybe 1P for two different variants in the same plasmid and 2P for the two different variants on different plasmids, and perhaps 2C for two different variants in two cells? 10) ORIGINAL COMMENT: Line 351-57 – which blaCTX-M group or did you look at multiple groups? Major sequence differences between members of different blaCTX-M groups (Group 1, Group 9 etc) are not due to mutations, but because the source genes are from different Kluyvera species (there are also hybrids between Group 1 and Group 9 genes). Minor changes between blaCTX-M genes within each group are probably explained by mutations that have occurred since capture. RESPONSE: …this part of our analysis was performed using raw genomic data to assess the heterogeneity status of a specific β-lactamase gene in each strain across a large dataset. In practice, we first identify the target gene through homology search (BLAST), and then determine heterogeneity by analyzing sequence differences among all raw reads contributing to the assembly. Therefore, whether the reference used for BLAST belongs to a Group 1 or Group 9 blaCTX-M gene does not affect our ability to first locate a specific blaCTX-M genotype within the genome. For our analysis of blaCTX-M, we used blaCTX-M-65 as the reference sequence. As long as a blaCTX-M gene is present in the genome (even one from a divergent group such as Group 1), its presence can still be identified. We then analyze whether heterogeneity exists among all reads assembled into that gene. NEW COMMENT: I still think that giving a combined % here for all blaCTX-M genes is not ideal. It might be interesting to focus on e.g. blaCTX-1 group and 9 groups (the most common) and see whether the level of heterogeneity differs (although hybrids might interfere with this). 11) General comments to streamline text. “p” indicates a plasmid, so using “plasmid pXXX” or “the pXXX plasmid” is not necessary. Cloning something in implies a vector. e.g. Lines 134-9 - “into pCR2.1 to verify” is sufficient, “The CTX-M-65 beta lactamase” etc can just be “CTX-M-65”. Also remove “the” before CTX-M-65 on Lines 168, 169 etc - not needed. Delete things like “Results showed that” – they just add unnecessary words. Remove unnecessary descriptions of Methods in Results 12) Examples where wording would improve clarity/accuracy etc Title – while I appreciate the reason for wanting to include reference to a single plasmid, I think that the title is now too general – only a single gene type has been looked at and only in a single isolate (and all experiments models were in E. coli). Line 45 – “elevated” in relation to what? This may not be the best word here. Lines 49 – “heterogeneity duplicated within a single plasmid” is confusing. Lines 52-3 – “heterogeneity is widespread among various beta-lactamase genes in clinical isolates” is not clear – maybe something like “many clinical isolates have multiple variants of a beta-lactamase gene” if this is what is meant. Line 55- “multidrug resistance” may not be the best phrase here – this is commonly used to mean resistance to multiple classes of antibiotics, while only beta-lactams are being described here. Lines 61-3 – “Humans have” might be better. Line 74 – suggest “particularly those caused by”. Lines 77-9 – sentence gets hard to follow. More strains than what? Line 80 – I don’t think that “Beyond KPC carbapenemases” or “occurring” are needed here and “within” could be “in”. Lines 81-3 – if CMY-172 and CTX-M-219 give CZA resistance than this needs to be clearly stated. Lines 85- - wording could be improved. Line 90 – this could just say “However, gene duplications can alleviate trade-offs because”. Line 116 – suggest “plasmid-borne”. Line 118 – “location”, not “localization”. Lines 130-131 – this could just be “(Table 1). Both isolates belong…” Line 155 - as noted in previous comments, the variants do not have MICs, the antibiotics do. Line 168 - “For enzyme inhibtion” is not needed here. Lines 170 – “Thus” could be used instead of “These Results demonstrate” “point mutations” doesn’t need a dash, “enzyme’s” can be deleted Line 193 – which “drug”? Line 198 – “the blaCTX-M gene near blaKPC-157” Lines 199-200 – the IS26, not the TU, that “bracket” the blaCTX-M gene, which is part of the TU and it is a single TU that is amplified. Line 207 – “In this study” is not needed and “of plasmid-harboring blaCTX-M” does not make sense – Suggest “of plasmids carrying blaCTX-M” or “plasmid-borne blaCTX-M”. Lines 208-9 - Delete “To facilitate observation” and “corresponding” has no meaning in this context. Line 231 – maybe “The existence of multiple variants of the same gene that confer distinct resistance profiles enables bacteria to withstand the combined effects of multiple drugs” Line 239 – “are encoded” Lines 253 etc – “anchored” is not the right word here. “When both copies of blaCTX are on the same plasmid” would be simpler and more accurate, also Line 415. Line 259 – change “supplemented” to “modified” Line 270 - “surged” is not scientific. Line 281 – “of” here does not make sense. Line 308 – “gained superiority” is unscientific. Lines 314, 317, 318 – “raw genomic data” Line 316 – “heterogeneity of” and “such as” is not clear – just list all gene types examined. Line 320-1 – “by mapping raw reads” if this is what is meant? Lines 321-2 – all this needs to say is “The estimated copy number distributions of the genes examined” – it has already been made clear that they are bla genes and from K. pneumoniae. Lines 324- 5 - blaSHV is expected to be in the chromosome of all K. pneumoniae and it may or may not additionally be on plasmids – this needs to be properly explained. Line 325, 331 – sentences shouldn’t start with “And” Line 488-90 – which flow cells/chemistry was used. The assembly is not combined, the reads are combined for the assembly? Lines 494-5 – needs rewording. Line 507 – “Colonies were selected”, “Positive” is not needed. Line 510 - “…sites, cloned..” Line 528 – what is meant by “the low-affinity substrate”? Line 557 - a psiB gene is found on many plasmids, it is not specific to pICU-3 and there seem to be two genes annotated as psiB (psiB1 and psiB2) in the pICU-3 sequence in PX405700, but only ~57% identical). The psiB primers and probe in Table S3 match psiB, butthsi needs to be explained more accurately. Lines 580-581 – “12 dilutions covering”, “16 dilutions covering”? Line 589 - “grown” not “growth”. Line 602 – it’s the plasmids that have the fitness costs, not the strains? Lines 704-5 – suggest “The pICU-3 and pICU-18 sequences are available under GenBank accession numbers PX405700 and pX405 701, respectively”. 13) Discussion The Discussion is still long, and some parts need rewording to improve accuracy. Also, I think that it is important not to generalise results beyond at least bla genes, maybe not beyond blaCTX-M. See comments on Fig. 7 below. Lines 337-45 – this information still seems like it repeats and/or should be in the Introduction. Line 350 – what is meant by “ESBL mutations” here? In which gene(s)? CMY-172 and CTX-M-219 are already mentioned in the Introduction. Line 351 – needs rewording - a clonal strain is not a variant, either “trade-offs” or “a trade-off phenomenon” - see previous comments. AST is done on isolates/strains, enzyme kinetics uses proteins, neither is done with genes. Line 354 – change “explored” – see previous comments. Line 357, 360, 421 - genes are not encoded on plasmids, they are e.g. carried by plasmids, found on plasmids – see previous comments. Line 358 - “In the subsequent study” does not make sense here. Line 362 – the “locus encoding CTX-M-249” is the blaCTX-M-249 gene - this term can be used. A gene can’t undergo an “increase in plasmid copy number”. Line 367 - it is really the blaCTX-M-65 gene that is widespread. Line 370 – “the novel” not needed. Lines 371-32 – the enzymes hydrolyse, but it is the strains encoding them that shows resistance. Line 377 – it is multicopy of essentially a single gene (based on blaCTX-M-65) not multiple genes Lines 377-8 - what “functioned as a bet hedging strategy”? Lines 382-4 – “simultaneously carrying blaTEM-1 and blaTEM-12” and state differences in sequences and phenotypes, “trade-off losses”, not “trade-offs losses”. Line 385 – change “brought by” to “due to” Line 386 – “in a clinical strain” – only one strain studied here showed this effect. Line 393 – no dash needed in “single antibiotic” Line 398 – “genes split between” doesn’t really make sense here – “genes on” would be fine. Line 415 etc – “anchored in” is not the best way to say this. “on”. Lines 416-7 – unclear - do you mean that the first step was for the plasmid to acquire a single copy of blaCTX-M-65? Lines 441-2 – the wording here could still be made clearer. 14) ORIGINAL COMMENT: Lines 471-5 – is this really true for long-read if multiple copies of the gene are in different genetic locations, as for copies of blaCTX-M-65 (or variants) both in pICU-3 here? RESPONSE: …when multiple copies of a gene are located in different genetic contexts—as with blaCTX-M-65 (or its variants) present in both regions of pICU-3—the relatively lower base accuracy of long-read sequencing makes it difficult to resolve which specific variant resides in which repeat region. NEW COMMENT: I don’t really agree with this. From my own use of read mapping with ONT reads (R10), it can be pretty easy to distinguish real nt differences (occur in almost all ONT reads) from errors (each occurs in only one or two sequences). 15) References ORIGINAL COMMRENT: Ref 8 needs page numbers or equivalent. Something seems to be wrong with page numbers in Ref 49. Journal titles need to be correctly abbreviated. Ref 4, 12 etc., remove Title Case. Species and gene names etc. need to be correctly formatted. RESPONSE: and carefully checked all reference entries, correcting errors and inconsistencies in formatting. Species and gene names have also been italicized accordingly. NEW COMMENT: There are still a few problems. Ref 4 has no page numbers or equivalent Refs 4,11 – check if in vitro/vivo are in italics in these papers. Refs 8, 23, 28, 55– check if beta symbols are used in these papers. Refs 13,15, 53, 54, 55 – page number equivalents look odd. Ref 19 – probably should be “Typhimurium”, not in italics and rrn in italics. Ref 37 – “26” of IS26 should probably be in italics. Ref 61 – “a tool” 16) Figures The figures and their organisation/distribution between the main manuscript and Supplementary is improved, but more consistency in colours etc across figures/parts of figures would improve clarity For example, in the main manuscript, the bar chart at the bottom of Fig. 1 Part II uses different colours for blaCTX-M-65 and the three variants from the diagrams in Part I. What appear to be the same graphs in Fig. 1 Part I and Fig. 2 b and c use different colours again, as do Fig. 3 and Fig. 7. In Fig. 7 (a) blue is used for blaCTX-M-65 and a dark red colour is used for blaCTX-M-249, but in (b) the ancestral allele (blaCTX-M-65 in part a) is in a dark red and the evolved allele (blaCTX-M-249 in part a) is in blue. Also, in Fig. 1 Part III the colours of the blaCTX-M genes in the models are hard to see. Maybe start with green for GFP, red for mCherry, orange for both and blue for mTagBFP, as these are fixed, then find different, easily distinguishable colours to show blaCTX-M-65 and the three variants and use these consistently in diagrams, graphs etc consistently throughout, where possible. Figure legends often need some rewriting to improve accuracy/caltity Fig. 1 legend Lines 881-2 – is ICU-18 really a “clone”? Line 883 – cloning also identified the different variants Line 884 – “Plasmid location” Lines 886-7 - “different multicopy heterogeneity states”, “heterogeneity stability” can be described more clearly with less jargon. Lines 889-90 – “multiple minor variants of various bla genes in a single isolate from SRA data” Fig. 2 legend Line 892 – “diagram” not needed”. Line 893 – pICU-18 is not shown in the diagram. Line 894 – “elements” not needed – they are IS. The IS26 form the TUs, and all of the tandem TUs could be shown on the diagram – see previous comment. Line 895 – “ONT read”? Relative to what? I think that it would be helpful in part c to make it clear that these copy numbers are due to a combination of things i.e., every plasmid would have a copy of blaCTX-M-65 (non-amplified copy flanked by inverted IS26) and the % of blaCTX-M-249 is a combination of both mutation and amplification, at least under some conditions – see previous comment relating to this. Fig. 3 p15A_ori could be removed from the diagrams at the top to simplify them (it’s in all of the plasmids) and note that pACYCDuet1 has this replicon in Methods (Lines 563-71). Legend Lines 900-1 – title could be improved. Text can be simplified and condensed – see comments above and avoid explaining things that are obvious from the figure. e.g. Lines 901-2 “Model 1 (strain 65G) has p65G (blaCTX-M-65-GFP) only, Model 2 (strain 249R) has p249R (blaCTX-M-249-mCherry), Model 3 is a 1:1 mixture of 65G and 249R (population level heterogeneity, PLH). Model 4 has p65G and p249R in the same cell (2P) and Model 5 has p65G-249R, (blaCTX-M-65-GFP+blaCTX-M-249-mCherry) on a single plasmid (1P), using alternative model names suggested above. Line 970 – “results for the five models. Line 980 – “co-selection by” Lines 909-10 “represent”, not “represented” Line 911-2 – “in each cell represent the” and “values of bacterial… concentration” are not needed. Fig. 4 Part (a) - suggest stating % of what on graph, rather than in legend. Part (B) – not clear why a dotted line is used for “all” but a continuous line for CTX resistant. Might be better is all lines are dotted. Legend Title, Lines 914-5 – probably doesn’t need “during serial passages”. Lines 916-7 – probably not needed - see above (but should be “is plotted”) Lines 917-9, 922-3 – legends should describe what is in the figure, not repeat Results. Line 920 – suggest “by plating serial dilutions on CTX or CAZ and counting CFU”, Lines 920-22 are then not needed. Fig. 5 Suggest stating relative % of what on graph, rather than in legend. Maybe colours used could match the fluorescence colours? Also, 65G is used elsewhere as a strain name, but here “65G” is really the 2 plasmid strain that has lost p249R, and conversely “249R” is really the 2 plasmid strain that has lost p65G. Also “SPD” is used here, not SPD-B, as in the legend. Some simple explanatory diagrams might be useful here – showing original DPD, SPD-B and loss of one plasmid or another to give “65G” and “249R”. Legend Line 926 – suggest “(additional blue fluorescence)”. Fig. 6 See comments above on blaCTX-M genes. Line 934 – delete “such as”, “-like” should not be subscript. Line 935 – is this really mapping to a reference not “alignment”? Lines 937-8 – it’s not really the strains that are “(non)heterogeneous”, they have multiple variants of the gene of interest. Fig. 7 This scheme may be generalisable to e.g. other blaCTX-M gene families (e.g. group 1) or other bla genes and beta-lactam antibiotics (where one mutation can have a drastic effect on phenotype) but might not apply to other types of antibiotic resistance genes. See comments above on colours used for blaCTX-M genes. What do pink and blue parts signify? Suggest flipping the direction of the curved arrows on the two left hand diagrams, to make it clearer that these parts are “moving away from” the central diagram. Legend Line 942 -suggest “blaCTX-M gene heterogeneity in ICU-3” if that is what is meant? Line 945 – suggest “single mutation or two mutations” Line 946 - suggest “…pressure transient tandem copies of the mutated gene may be generated” Line 947 – suggest “of heterogeneity in blaCTX-M genes carried on the same plasmid” Line 948-53 – I would suggest trying to simplify the text, e.g. “non-heterogeneity” = single gene variant, “plasmid-level multi-copy heterogeneity” = both gene variants on the same plasmid. 17) Supplementary Table S1 - column heading says “inhabitors”, should be “inhibitors”. Table S2, 3rd column heading use “allele(s)”? Table S3 – “probes, not “Probes” in heading, suggest making it clear where CTX-M primers are only relevant for blaCTX-M group 9 genes. Table S4 – shorten and reword titles – suggest “BioProjects containing Illumina sequence reads for K. pneumoniae isolated from humans” etc. Supplementary figures – check for accuracy and condense if possible - see comments on legends for main figures. ATTACHED REVIEWER REPORT — IMPORTANT The text below is a complete reviewer report submitted as a PDF attachment. It appears after the Creative Commons license block. Assign it to whichever reviewer wrote 'see pdf' or had no inline text. Response to Reviewer #1 (Remarks to the Author): Weng et al presents one model case of paralogous gene evolution in one genome by focusing on a plasmid detected in a hospital. By conducting additional survey in publicly available raw reads, they found that the paralogous (beta-lactamase) gene evolution in one plasmid is common. Furthermore, the authors characterized the nature of four CTX-M variants in vitro. These are solid and important data. The statement in the abstract is valid. The manuscript is clearly written. However, I am not sure about the significance of discussion on the stability of gene heterogeneity, comprising a large part of this study. The fitness advantage of the “dual-copy genes on one plasmid” state (DCG model) over the “dual-copy plasmids” state (DCP model) under drug-switching condition (Fig 5eg) is immediately obvious as far as plasmid loss can occur, unless huge fitness cost arises from the DCG state. The points of discussion should be “when does DCP or mix model win?”. Therefore, to discuss the conditions where DCP/PCG/mix state wins, I think that the authors need to implement mathematical model for the serial passage or competition experiments considering the fitness advantage of plasmid-bearing cells and plasmid loss rate. To address the above problem, ideally, all experiments should be designed using a low copy number and stable plasmid such as mini-F to mimic the condition of the real situation. Response: We sincerely thank the reviewers for their valuable suggestions, which have provided us with important insights to enhance the scientific depth of our study. In response to the reviewers’ recommendation to redesign the experiments using low-copy-number and stable plasmids such as mini-F, we tested the low-copy plasmid pCC1FOS (Genome Res, 2002;12(9):1434-44), whose copy number and stability are comparable to mini-F. However, our experiments revealed that pCC1FOS has several limitations: (1) its extremely low copy number (approximately 1–2) likely led to insufficient fluorescent protein expression when enhanced green fluorescent protein (eGFP) or red fluorescent protein (mCherry) gene was fused downstream of the resistance genes bla or bla , resulting in CTX-M-65 CTX-M-249 fluorescence signals below the detection threshold (Chem Rev, 2002;102(3):759-81) and rendering subsequent dynamic tracking experiments infeasible. (2) it could not support the construction of the DPD model (the DCP model described in our previous manuscript, which carries both p65G and p249R plasmids), as DPD strains require the simultaneous maintenance of at least two plasmids, while pCC1FOS’s strict copy-number control prevented dual-plasmid coexistence; (3) its copy number was too low to realistically mimic clinical strains, which we have confirmed to possess plasmid copy numbers greater than two, often increasing to over a dozen under antibiotic pressure. Based on these factors, we concluded that pCC1FOS is not suitable for achieving accurate simulation and stability, whereas the plasmid pACYCDuet-1 used in our study was confirmed by digital qPCR to have a copy number of approximately 13, placing it in the medium-to-low range, which better reflects clinical conditions and facilitates fluorescent labeling for visualization and validation. Following the reviewers’ suggestion, we developed an ordinary differential equation (ODE) model using estimated parameters from the pACYCDuet-1 system to simulate the dynamics of plasmid-free (PF), 65G, 249R, DPD, and SPD (the DCG model described in our previous manuscript, which carries a single plasmid p65G-249R) strain populations under gradients of the antibiotics CTX and CZA. This model, based on Monod growth kinetics and incorporating resource limitation, antibiotic killing effects, enzymatic hydrolysis efficiency, and plasmid loss rates, produced simulation results for serial passage (Fig. S6 a-b) and competition experiments (Fig. S6 c-d) that were consistent with our earlier experimental findings, supporting its validity. In addition, we applied this model to explore the dominance boundaries between DPD and SPD, which helps address the question of “when does DPD or SPD model win?”. The results showed that when CTX concentrations were below 39.31 mg/L, the growth fitness advantage of DPD exceeded that of SPD. When CTX was between 39.31 and 367.01 mg/L, the resistance advantage of SPD outweighed the growth advantage of DPD, and SPD dominated. At CTX concentrations above 367.01 mg/L, one of the plasmid-loss derivatives of DPD, 65G, became dominant. Similarly, for CZA, when concentrations were below 1.34 mg/L, DPD had a growth advantage over SPD. When CZA was between 1.34 and 127.91 mg/L, SPD’s resistance advantage surpassed the growth advantage of DPD, allowing SPD to dominate. At concentrations above 127.91 mg/L, another plasmid-loss derivative of DPD, 249R, prevailed. Although the authors mentioned the cost of these cell types in the absence of antibiotics (Fig S1), competition experiments and stability test were performed in the presence of antibiotics where plasmid-free cells are immediately killed. Therefore, the cost information is not really linked to experimental data. Response: We thank the reviewer and have followed this recommendation. Indeed, there was an insufficient correlation between the data on fitness costs under antibiotic-free conditions and the results from stability and competition experiments. To address this, we performed additional growth curve experiments to systematically evaluate the growth fitness of PF (plasmid-free, i.e., DH5α), 65G, 249R, DPD, and SPD strains under antibiotic-free conditions (0 mg/L) as well as across a range of antibiotic concentrations. Specifically, we tested CZA at 0, 1, 4, 8, and 32 mg/L, and CTX at 0, 4, 16, 64, and 256 mg/L, which together covered the antibiotic concentration ranges used in our stability and competition assays. We measured OD over 600 time, calculated the area under the OD –time curve (AUC), and quantified growth 600 performance using the relative growth inhibition rate [100 × (1 – AUC/AUC₀), where AUC₀ represents the AUC for each strain at 0 mg/L], thereby providing an integrated measure of growth fitness across different levels of antibiotic pressure (Fig. S5). The most parts of figure are used to explain the experimental set-up. Figure information should be more condensed. For example, Figure 4 can be condensed to only two line plots: one from flow-cyto (Fig4c), one from CFU (Fig4de). Figure 5 can be condensed to only Fig5eg. Response: We sincerely appreciate your suggestion to make the figures more concise, and we agree that adjusting Figures 4 and 5 can improve the focus of data presentation. Since several other reviewers also recommended modifications or adjustments to the figures, we carefully considered all feedback and followed the principle of highlighting only the most important and essential information. The specific adjustments are as follows: We reconstructed Figure 1 as a workflow diagram and moved the original panels 1b–d to Supplementary Figure 1. From the original Figure 3, we retained panels b and d, while moving panels a and c to Supplementary Figure 2. In Figure 4, panels a, b, and d were moved to Supplementary Figure 3, panel c was retained, and panel e was changed from a bar chart to a line chart to improve clarity. From the original Figure 5, panels a, b, c, d, and f were moved to Supplementary Figure 4, leaving only panels e and g, which were revised into the current Figure 5. It appears that to select for specific (mix model) cell types, a high concentration of CTX, 256 ug/ml, was used in the experiment. The concentration should be in clinically achievable range (< or = 100 ug/ml). Response: We sincerely thank you for pointing out that the CTX concentration used in our experiments (256 μg/mL) exceeds the clinically achievable range (≤100 μg/mL), and we agree that adjusting to clinically relevant concentrations would strengthen the applicability of our study. The original in vitro experiments employed 256 μg/mL primarily to rapidly screen for dominant patterns and to investigate bacterial competition dynamics under extreme antibiotic pressure. In response to your suggestion, we applied an ordinary differential equation (ODE) model to simulate population dynamics across a broader range of CTX concentrations (0.125– 4096 μg/mL) and confirmed that the competitive dynamics observed at clinically relevant concentrations (≤100 μg/mL) were consistent, thereby providing a more detailed analysis (Fig. S6). MINOR POINTS: FIG1a. Gray area does not precisely connect the circular map and gene map of tandemly amplified loci. Response: We sincerely thank you for pointing out the issue regarding the connection of the gray-shaded regions in the figure. We think you may be referring to Fig. 2a (rather than Fig. 1a), as Fig. 2a presents the circular plasmid map and the gene diagram of its tandem amplification sites, with gray regions used to indicate the corresponding relationships. In response to your comment, we have revised Fig. 2a and optimized the rendering of the gray-shaded areas to ensure precise connections between the circular map and the gene diagram of the tandem amplification sites. FIG3b. I think that gene map on plasmids is unnecessary as they are redundant with panel a. Response: Thank you for your suggestion. Figure 3b, which illustrates bacterial strains carrying different plasmids, corresponds directly to the five models we constructed, as reflected in the checkerboard susceptibility results shown in Figure 3d. In line with your advice and other reviewers’ comments on simplifying figures, we have revised Figure 3 by retaining panels 3b and 3d into the new Figure 3. Panels 3a and 3c have been moved to Supplementary Figure S2a and S2b to remove redundancy and improve clarity. FIG4. Plasmid stability data measured by flow cytometer (a-c?) and by CFU count (d, e). c. Please clarify in legend whether this graph is based on CFU count or summary of flow cytometer counts. d-e: d and e show the same information. I suggest the author should show the data of proportion of the resistance gene-carrying cells by line plot like panel c. Response: We greatly appreciate your suggestion, which is highly valuable for improving the clarity of the figures and the effectiveness of data presentation. We have revised Figure 4 as follows: Figure 4c, which is based on flow cytometry data and shows the proportion of cells carrying resistance genes, has been updated with a legend explicitly stating that the data were obtained from flow cytometry counts, and the figure legend has been revised accordingly to ensure accuracy. Since Figures 4d and 4e conveyed overlapping information, we have moved Figure 4d to Supplementary Figure 3c, and converted Figure 4e from a bar chart to a line graph (now Figure 4b) to enhance clarity. Fig5. d label. Please add ug/ml after antibiotic concentrations because it is not immediately clear whether those are symbol of CTX-M variants. Response: Many thanks. We have added the unit “mg/L” to all antibiotic concentrations (e.g., CTX and CZA) in the labels of Figure 5d and 5f to clearly indicate that these values represent antibiotic concentrations rather than CTX-M variants. According to the reviewer’s suggestions, we have moved these two panels to the current Supplementary Figure 4. Reviewer #2 (Remarks to the Author): Regarding the manuscript ”The heterogenetic multicopy of blaCTX-M variants on one plasmid optimizes the evolution adaptability in the clinically isolated Klebsiella pneumoniae” by Weng et al. This manuscript presents a novel and well-supported study on how multicopy heterogeneity of blaCTX-M variants on a single plasmid contributes to the adaptability and stability of antibiotic resistance in Klebsiella pneumoniae. Overall, the manuscript is well-written, and the study is highly relevant, novel, and scientifically rigorous, with important clinical relevance. The combination of whole-genome sequencing, digital PCR, amplicon sequencing, and in vitro modeling provides strong experimental support for the proposed mechanism. However, the comprehensiveness of the study makes it somewhat difficult to follow, and some sections appear misplaced, affecting the clarity and flow of the manuscript. Below, I have outlined major and minor comments that would benefit the manuscript. Response: We sincerely appreciate the reviewers’ valuable suggestions. We have thoroughly revised the manuscript, reconstructed the workflow diagram (Fig. 1), and reorganized the presentation of the results accordingly. Substantial language improvements were also made in the Introduction, Discussion, and Methods sections. In addition, the composition and order of the figures were adjusted to make them clearer and more concise. We hope these revisions have further strengthened and improved the study. Major Comments 1. Structural Issues & Misplaced Sections: Several sections appear misplaced within the manuscript. I recommend restructuring the introduction and results sections to improve clarity and logical flow. Some specific examples: - Lines 99-111: This section describes the findings of the study, which should not be placed in the introduction. The introduction should focus on background information and knowledge gaps, leading to the study’s aim and objectives. Response: Thank you for this valuable suggestion. We have restructured the last paragraph of the Introduction to place more emphasis on the background information and then clearly present the study’s aim and objectives. - Lines 120-122: This section describes the aim of the study, which should be included in the introduction, not the results section. Response: We moved these sentences, similar to the aim of the study, to the last paragraph of the Introduction. Here, we condensed and revised the following sentence to make the meaning complete and concise. - Lines 331-337: Discussion elements appear within the results section. These should be moved to the discussion section. Response: Thank you for your suggestion. We have moved these sentences to the appropriate section of the Discussion. In fact, we reorganized the description of the results regarding the universality of resistance gene heterogeneity, presenting only the results and keeping the sentences concise. - Lines 359-361: Similarly, discussion/conclusion elements are found in the results section and should be relocated. Response: Similar to the above point, we reorganized this part of the results, and all discussion-like statements, including the one you mentioned, have been moved to the Discussion section. I suggest a thorough review of the manuscript structure to ensure that each section follows a logical order. Response: Thank you for your suggestion. We have made substantial revisions to the manuscript, making the description of the results more concise and clear, and adjusting the logical structure of different sections to improve coherence. 2. Results Section is Long & Difficult to Follow The results section is long, which is expected with the comprehensiveness of the study, making it a little challenging to follow. Consider referring to the steps in the flow-diagram (Figure 1a) at relevant points in the results. Possibly expand the flow-diagram to further guide the reader through the workflow and experimental design. This small change could significantly improve readability and clarity. Response: We sincerely thank you for pointing out the complexity of the Results section and for your valuable suggestion to improve the workflow diagram. To enhance readability, we expanded and refined Fig. 1 (originally Fig. 1a) by dividing it into three sections, thereby optimizing the workflow to more clearly guide readers through the research process. Specifically, the revised Fig. 1 now consists of three parts corresponding to the core components of our study. Part I: discovery of clinical CZA resistant strain and identification of the novel variant which mediated the resistance; Part II: confirmation of the profile of heterogeneous multicopy bla variants on plasmids; Part III: exploring the advantages of single-plasmid CTX-M duplicates in experimental and mathematical models. Each part incorporates detailed research content and uses arrows and annotations to visually present the logical flow. In the Results section, we now refer to the relevant parts of Fig. 1 in the corresponding paragraphs. 3. Clarification of Nucleotide and Amino Acid Mutations In Lines 123-124, the authors mention the noteworthy changes, including: “A397G, C508T in the bla….” Then, in Lines 131-133, the authors mention the corresponding amino acid changes: “Ser130Gly and Pro167Ser”. However, based on the alignment in Figure 1b, these mutations appear to correspond to amino acid positions 133 and 170, not 130 and 167. Clarification needed: Are the mutation positions correctly assigned? If different numbering conventions were used, this should be explicitly stated in the Methods section. Response: Thank you for drawing attention to the description in lines 123–124 (nucleotide substitutions A397G and C508T in bla ) and lines 131–133 (amino acid substitutions CTX-M-249 Ser130Gly and Pro167Ser). To clarify the correspondence between nomenclature and numbering, we confirm that A397G and C508T are based on nucleotide numbering of the bla gene, whereas the amino acid numbering Ser130Gly and Pro167Ser follows the CTX-M-249 ABL standard numbering scheme for Class A β-lactamases (Biochem J, 1991; 276:269-70). To improve clarity, we have added an explicit note about ABL numbering and reference 30 in the main text, and indicated the amino acid numbering in Supplementary Fig. 1a. 4. Statistical Analysis Throughout the manuscript, results are stated as “significant”, but p-values are missing in multiple places. Example: Line 192 mentions significance without statistical support. Include p-values wherever statistical significance is claimed. Response: Thank you for raising this question. We have thoroughly reviewed the entire manuscript and have systematically added the precise p-values, wherever statistical significance is claimed. This includes all data concerning: gene copy numbers, derived from digital qPCR; colony counting numbers, obtained through plate counting assays; fitness cost, calculated from growth curve analysis; percentage of dual-fluorescent cells under CTX or CZA pressure analyzed by flow cytometry. For the specific example cited by the reviewer (Line 192 in the original manuscript), we have revised the sentence to include the statistical support as follows: “Digital qPCR results showed that, compared with drug-free culture, exposure to 32 mg/L CZA increased bla copy number to >10 per chromosome equivalent (p = 0.0030), CTX-M primarily driven by a rise in plasmid copy number (from ~2 to >7, p = 0.0184). At a higher 64 mg/L CZA, bla copy number also approached 10 (p = 0.0081), but without a marked CTX-M plasmid copy number increase (p = 0.8735); instead, the number of bla copies per plasmid CTX-M rose to ~3 (p = 0.0069).” These comprehensive statistical details have been added throughout the manuscript (marked in the revised file). 5. Figures are Overloaded – Consider Moving Some to Supplementary Material While only seven figures are presented, each figure contains a high number of panels. I recommend to move redundant figures to supplementary materials to streamline the main text. Recommended changes: - Move Figure 1b and 1c to supplementary material. - Move Figure 3c and 3d, but expand Figure 1b by integrating the findings for the five different models. - Move Figure 4a, 4b, and 4d to the supplementary. - Move entire Figure 5 to the supplementary. This restructuring would help reduce figure complexity while keeping essential data accessible. Response: We greatly appreciate your suggestion and agree that moving redundant subfigures to the supplementary materials can improve the focus of the main text. Taking into account both your recommendations and comments from other reviewers, we have revised the figures as follows: Figure 1: we expanded the original Figure 1a (now Figure 1), and the content of the original Figure 1d has been incorporated into Figure 1 (part I) as a grid map; the original Figures 1b, 1c, and 1d have been moved to the supplementary materials (now Figures S1a, S1b, and S1c). Figure 3: we retained Figures 3b and 3d and combined them into the new Figure 3, while Figures 3a and 3c were moved to Supplementary Figures S2a and S2b to eliminate redundancy and enhance clarity. Figure 4: we moved Figures 4a, 4b, and 4d to Supplementary Figures S3a, S3b, and S3c; the original Figure 4e was converted from a bar chart to a line graph (now Figure 4b) to improve readability; the current Figure 4 retains the original Figure 4c (now Figure 4a) and the revised Figure 4b. Figure 5: we moved Figures 5a–d and 5f to Supplementary Figures S4a–e, while retaining only the original Figure 5e and 5g(now Figure 5a and 5b), which represents the core data, in order to maintain conciseness in the main text. Minor Comments I have a few minor comments on wording and terminology - Line 55: “The antibiotics developed by humans…” What about the naturally occurring ones? Response: Thank you for pointing that out. We have revised the first sentence of the Introduction for clarity. - Line 58: “Carbapenems were once considered the last-resort…” Carbapenems are still a last-resort antibiotic, so this should be reworded for accuracy. Response: We agree with the point that carbapenems are still a last-resort antibiotic, and we have reworded this sentence accordingly. - Line 457: “We postulate…” think it should be changed to “We presented” or “We constructed” Response: We have rephrased the sentence in question and the subsequent paragraph describing Figure 7b to eliminate ambiguity. In addition, to improve the conciseness of the main text, we have moved this text to the legend of Figure 7. - Lines 642-643: “alignment of genome raw reads in a broader range of K. pneumoniae.” Reword for clarity. Perhaps: “Genome-wide raw read alignment of a broad range of K. pneumoniae isolates.” Response: We have revised this sentence as suggested. - Figure 2 – Circular Map: What software was used to generate the circular map in Figure 2? This should be mentioned in the figure legend or methods section. Response: The genetic structures of plasmids and their comparative circular maps were generated using Proksee. We have described it in the methods section with reference 52. Reviewer #3 (Remarks to the Author): This manuscript describes a study looking at blaCTM-65 and variants detected at different copy numbers on the same plasmid in a clinical isolate of Klebsiella pneumoniae, with different gene combinations and copy numbers influencing resistance to ceftazidime, cefotaxime and ceftazidime+avibactam. Analysis of available sequences suggested that multiple copies of AMR gene variants in a single isolate may be more common than is usually recognized. While the results are potentially interesting and useful, and the authors have addressed many points raised by all three reviewers, improving the manuscript, there are still a few problems with scientific accuracy etc, as detailed below. While I think that the manuscript structure is better, further condensing/reorganising and editing/simplifying the text could still improve accuracy, ease of reading and flow – some examples are given below, including figure legends. Response: We sincerely appreciate the reviewer’s valuable suggestions. Following your advice, we have thoroughly revised the manuscript, reorganized its structure, ensured more concise and clear wording, adjusted the composition and order of the figures, and moved redundant figures to the supplementary materials. Below, we provide point-by-point responses to your comments, and we hope that our revisions have substantially improved the overall quality of the study. Major points 1) blaCTX-M-65 and variants, Lines 100-2,129-136, Lines 100-2 - the information about blaCTX-M-65 could be expanded, explaining that blaCTX-M-65 belongs to the blaCTX-M-9 group but does not have a mutation resulting in Asp240Gly usually associated with CAZ resistance (as found in e.g., in blaCTX-M-27). Response: We have added some information about bla in the Introduction section. CTX- CTX-M-65 M-type β-lactamase genes comprise numerous variants that are classified into several groups based on sequence differences, with the two major groups being the bla group and the CTX-M-1 bla group. bla belongs to the bla group, but unlike typical members of this CTX-M-9 CTX-M-65 CTX-M-9 group (such as bla ), it does not carry the Asp240Gly substitution that enhances CTX-M-27 resistance to CAZ. Instead, bla exhibits strong hydrolytic activity against CTX, rather CTX-M-65 than CAZ. Lines 131-46 could be condensed and simplified e.g., it is not really relevant when blaCTX-M- 249 or the other variants were named. Response: We substantially reorganized this paragraph to make the expression more concise and clearer. Instead of separately introducing the naming of bla and the other variants, CTX-M-65 we more concisely highlighted their heterogeneous genotypes (See the section “CTX-M-65 variant mediating CZA resistance”). Line 131-3 – this is unclear. What was cloned that revealed the variants? Response: Thank you for the suggestion. We realize that we did not explain the cloning experiment clearly. Here, cloning refers to the process in which the PCR product of the target gene from a clinical strain was ligated into the vector plasmid pCR2.1 and then transformed into a recipient strain, so that each transformant successfully obtaining the recombinant plasmid (also referred to as a colony) would carry only one variant of the PCR product. We used this method to confirm the presence of multiple variants. In cases where the target gene in the strain exhibits multicopy heterogeneity, the PCR product will consist of a mixture of different variants, and sequencing the gene fragment on the recombinant plasmid allows clear identification of which variant has been successfully cloned, since typically a single plasmid is introduced into only one clone. The proportions of different variants recovered from the clones can thus provide a rough estimate of the variant composition in the original PCR product. We have substantially reorganized this paragraph to express it more concisely and clearly. Line 132 – are these amino acid positions numbered according to the Ambler scheme for class A beta-lactamases (PMID: 2039479). If so, this should be clearly stated. Response: Yes, we confirm that the amino acid numbering Ser130Gly and Pro167Ser follows the ABL standard numbering scheme for Class A β-lactamases (Biochem J, 1991; 276:269-70). To improve clarity, we have added an explicit note about ABL numbering and reference 30 in the main text, and indicated the amino acid numbering in Supplementary Fig. 1a. Line 134 – it is antibiotics that have MICs, not isolates. Response: Thank you for the reminder. We have corrected this statement in the main text when described the MICs. Line 136 – should be “mutations in blaCTX-M-65” Response: We agree with your suggested revision; however, since this section has already been reorganized, we adopted alternative wording. Line 138 – how were these “clonal isolates” obtained? And it should be “alleles” here. Response: We have now provided a detailed description in the Methods section of how these clones were obtained. In this part, we reorganized the section and prefer to use variants to represent the different mutants of bla . CTX-M-65 Lines 145-6 – do not make sense and needs rewording. Response: Our intention here was to clarify that bla does not exhibit multicopy KPC-157 heterogeneity similar to bla . However, since the previous paragraph had already CTX-M-65 explained that bla clones cannot mediate CZA resistance, this statement was redundant KPC-157 and has been removed. 2) Plasmids, contexts/amplification of blaCTX-M-65 and variants, Lines 123-5, 179-216, 312- 397 The assembled sequence of pICU-3 should be submitted to GenBank, along with the sequence of pICU-18, in addition to raw reads. It is possible to add comments to GenBank entries to explain that e.g. the sequence submitted is a representative of several variants of the same plasmid that can have different numbers of copies of a region that includes blaCTX-M-65 but also the other variants. Response: Thank you for your suggestion. All raw genomic data for strains ICU-3 and ICU-18, under different antibiotic pressures, generated from both Illumina and Nanopore platforms, have been deposited in the SRA under BioProject accession number PRJNA1194354. The plasmid assembly files (pICU-3 and pICU-18) are also available in GenBank under accession numbers PX405700 and PX405701. In addition, we have added comments to the GenBank entries to clarify that at one locus of bla carried by plasmid pICU-3, either bla or CTX-M CTX-M-65 any of its three variants may be present. The structure of pIUC-3 needs to be explained in the text and compared with pICU-3. Response: We speculate that the reviewer intended to compare the structures of pIUC-3 and pIUC-18. We have added an explanation in the main text. Plasmid pICU-18 shared an identical backbone with pICU-3, differing only in that both bla copies corresponded to bla CTX-M CTX-M-65 without mutations at A397G or C508T, and the bla genotype was still retained. KPC-2 Lines 186-208 - from Fig. 2, but not really explained in the text, it seems that the copy of blaCTX-65 that is not amplified also does not undergo the mutations? If so, this might need to be taken into account when calculating/drawing conclusions from the number of copies e.g. plasmid copy number will increase the copy numbers of both this unchanging copy of blaCTX-M-65 and whichever variant is in the other position, which could be blaCTX-M-65, 247, 248 or 249, while gene amplification will not affect the copy number of the non-amplified blaCTX-M-65 copy. It is not clear if/how this has been taken into account in the calculations. Response: We reorganized the description of this section to provide a clearer explanation. On plasmid pICU-3, bla was present in two copies, separated by ~54 kb. The copy CTX-M corresponding to bla that is not amplified also remains unmutated, as confirmed by PCR CTX-M-65 amplification of flanking specific regions followed by cloning. The absence of tandem duplication at this locus is explained by the opposite orientations of the flanking IS26. In contrast, the other copy located near bla could represent bla or one of its variants, KPC-157 CTX-M-65 with bla being most frequently recovered. Under moderate CZA pressure (32 mg/L), CTX-M-249 the primary effect was an increase in plasmid copy number, leading to elevated bla copies CTX-M at both loci. The expansion mainly involved plasmids carrying the bla + bla CTX-M-65 CTX-M-249 combination rather than bla with the other three variants. Consequently, bla copy CTX-M-65 CTX-M number relative to the plasmid remained stable but increased relative to the chromosome, with a slight higher proportion of bla ,possibly accompanied by limited tandem amplification CTX-M-249 as later observed under higher CZA pressure. At 64 mg/L CZA, plasmid copy number reverted to the drug-free level, whereas the bla copy near bla underwent more IS26 CTX-M-249 KPC-157 “translocatable unit” (TU)–mediated tandem amplifications, resulting in increased bla CTX-M copy number per plasmid and a further rise in the proportion of bla . Digital qPCR and CTX-M-249 amplicon sequencing results support this interpretation. Lines 182-3 – how was this calculated? What are these numbers? Response: We realized that our description of the digital qPCR method and the calculation of copy numbers was not sufficiently clear. We have now reorganized this section to make the presentation of the digital qPCR results more explicit. Specifically, we used digital qPCR to quantify the absolute copy numbers of the chromosomal housekeeping gene pgi, 16S rDNA (known to have eight copies per chromosome), and the plasmid-encoded single-copy genes psiB and bla (variants not distinguished) in each sample. These values were then CTX-M normalized to pgi or psiB to calculate the relative copy numbers with respect to the chromosome or plasmid, respectively. Correspondingly, we also expanded the description of the digital qPCR methodology in the Methods section. Line 188-9 – are there any clues in the sequence about what is responsible for this increase in plasmid copy number? This should be looked at. Response: In this study, we found that the tandem amplification of one bla copy is likely CTX-M mediated by the IS26 TUs bracketing the resistance gene. However, the molecular mechanism underlying the increase in plasmid copy number remains unclear. We performed long-read sequencing and assembly under CZA pressure and compared the results with drug-free conditions, focusing on the plasmid replicon and replication-associated regulatory genes, but detected no genetic changes that could account for the elevated plasmid copy number. Line 193 – one of the ONT raw reads provided has 7 copies of the TU, the other 3 plus probably a truncated fourth one. How does this fit with \"about three\" stated here? Response: Here “about three” was estimated based on the digital qPCR results (Fig. 2b). The value of three copies can be regarded as an average representing the overall increase in copy number. In reality, the number of expanded plasmid fragments likely varies among individual bacterial cells; for example, our raw data show cases with seven copies, incomplete expansions with four copies, as well as other cases with fewer than three copies (including some cells without amplification, remaining as a single copy). Therefore, in Fig. 2a, the schematic of copy number increase is illustrated as three copies, serving only as a representation of this average situation. Lines 197–208 – it’s not entirely clear from Methods how these proportions were determined/calculated and a bit more detail might be helpful. Response: The proportion of each variant within the clonal population was quantified by amplicon sequencing. Accordingly, we have reorganized the description of the amplicon sequencing methodology in the Methods section to provide a more details. Lines 209-10 – presumably “long reads from isolates grown in 64 mg/L CAZ” is meant? This should be rewritten. Response: We apologize for not expressing this clearly. Under different concentrations of CZA pressure, the copy number of genes on bacterial plasmids may change. Therefore, when bacteria cultured under antibiotic pressure were subjected to long-read sequencing (Nanopore platform), we analyzed the raw reads before assembly to determine whether the target gene exhibited multicopy status. In the manuscript, the intended meaning of this sentence is that the raw long reads we analyzed were derived from bacterial genomic sequences obtained under 64 mg/L CZA pressure. We have now reorganized this sentence for greater clarity. Line 212 – this needs to be looked at further and explained better and more accurately. IS903, or a truncated copy, is commonly found downstream of blaCTX-M-9 group genes in ISEcp1 “transposition units” (TPUs) responsible for moving these genes. Examination of the pICU-3 sequence and raw long reads provided shows that, as expected, IS903 has nothing to do with the amplification. The amplified region instead seems to be a 4844 bp IS26 “translocatable unit” (TU; positions 58367-63210 in pICU-3 sequence provided), something that has been reported previously for different AMR genes (e.g. Refs 33, 34 cited here) forming a chain of “pseudocompound” transposons. The authors need to look at recent reviews and/or papers on IS26 mechanisms of transposition (mainly by RM Hall and colleagues, but also M Chandler and colleagues). I also would suggest looking at whether this TU has been seen before and at papers on the plasmid contexts of blaCTX-M-65 (e.g. PMID: 22984205). Response: Thank you for your suggestion, with which we fully agree. We also consider that the tandem duplication of one bla copy on the plasmid is mediated by the IS26 TUs rather CTX-M than by ISEcp1. We have reviewed recent papers by R.M. Hall and colleagues, which elucidate the mechanism by which IS26 TUs mediate the transfer of various AMR genes through the formation of chains of “pseudocompound” transposons. In recent years, numerous reports have described the formation of IS26-mediated TUs in clinical isolates, including cases where TUs carry bla . However, reports specifically involving bla TUs remain limited. In our CTX-M CTX-M-65 previous work, we identified an IS26-mediated tandem multicopy structure of bla in an CTX-M-65 E. coli isolate (J Glob Antimicrob Resist, 2023; 35:202–209), though its structure differed from that observed in the present study. In addition, another study from China (J Antimicrob Chemother, 2013; 68(1):46–50) also reported a similar IS26-mediated tandem duplication unit of bla in E. coli. CTX-M-65 Lines 213-4 – this region cannot not undergo the same type of IS26-mediated tandem amplification as the flanking IS26 copies are in opposite orientations. Response: We sincerely thank you for pointing this out, as it helps us explain why the other copy of bla cannot undergo IS26-mediated tandem amplification. Upon doublechecking CTX-M-65 the sequence, we confirmed that the IS26 elements flanking the other bla copy are in CTX-M-65 opposite orientations, which prevents the formation of a tandem amplification structure. We have also added this explanation to the main text. 3) Lines 218-329 Models of heterogeneity This part of the text gets hard to follow and could be simplified and condensed. Line 219 – “plasmid-harboring beta-lactamase genes” does not make sense. Presumably “plasmids harboring” and “blaCTX-M genes” could be specified here, rather than “beta-lactamase genes”. Response: we have replaced beta-lactamase with bla based the suggestion. CTX-M Lines 221-2 – genes are cloned in fusion with genes, not proteins, and the gene names are blaCTX-M-65 and blaCTX-M-249. This should be written more accurately (and maybe moved to Methods). Response: We have noted that the fusion was with genes, not proteins. Therefore, the sentence has been revised to: “To facilitate observation, we fused fluorescent protein genes downstream of the corresponding resistance genes.” Here we just briefly mention the use of fluorescent proteins to aid reader understanding, while the methodological details are provided in the Methods section. Line 222-5 – “for example” should be deleted if you then list everything that was constructed. Maybe just saying here that plasmids encoding the following fusions were constructed: CTX-M-65-eGFP (p65G), CTX-M-249-mCherry (p249R) and both CTX-M-65-eGFP and CTX-M-249-mCherry (p65G-249R), using R to indicate the red fluorescence rather than “m” for mCherry, and explaining this and also that both colours together give orange. Response: Thank you very much for this excellent suggestion. First, we removed “for example” from the main text, and then we revised and simplified the description of the model construction as recommended here. We also used “R” to indicate red fluorescence instead of “m” for mCherry, which makes it clearer for readers to associate directly with the fluorescence color. Finally, in the main text we now describe the resultant color of red and green as “orange” rather than “orange-yellow.” Lines 226 – the plasmid names could then be used to simplify the text i.e. Model 1 is DH5alpha with p65G (strain 65G), Model 2 is DH5alpha with p249R (strain 249R), Model 3 is a 1:1 mixture 65G and 249R, Model 4 is DH5alpha with both p65G and p249R and Model 5 is DH5alpha with p65G-249R (or maybe consider putting this information a table). Having two names for Model 4/DCPs and Model 5/DCGs is also confusing and seems unnecessary. The names DCP and DCG are confusing as they are too similar (and the “s” at the end is not needed). Any further necessary details (e.g. of transformation) and Lines 234-6 could be left to Methods (or Fig. 2d legend, where they are already) and also consider whether the information in Lines 236-40 would be better mainly moved to Methods. Response: Thank you for the helpful suggestion. We have revised the main text according to this way of describing the five models. The five models are already labeled in Figure 3, so we no longer list them in Table separately. Models 3, 4, and 5 represent different patterns of multicopy heterogeneity that we constructed, while the original terms DCP and DCG refer to the key factors distinguishing the latter two patterns. We gave them abbreviations to make it easier to describe the results later (since our subsequent research will focus on these two models) and also to make the labeling in the figure more concise. After carefully reconsidering the key factors that distinguish these two important models, we concluded that whether the resistant gene duplicates are located on a single plasmid or on two separate plasmids is the most critical difference. Therefore, we revised the model names and abbreviations: Model 4 is now referred to as Dual-Plasmid Duplicates (DPD), meaning heterogeneous bla copies are duplicated CTX-M on separate plasmids; Model 5 is now referred to as Single-Plasmid Duplicates (SPD), meaning heterogeneous bla copies are duplicated on a single plasmid. The difference between the CTX-M initials “D” and “S” helps readers quickly distinguish dual-plasmid from single-plasmid. We have accordingly updated the main text and figures. Other further necessary details, such as transformation and confirmation of fluorescence patterns, have been moved to the Methods section. The description of the x-axis and y-axis in the checkerboard susceptibility assay has also been moved to Methods, and we have reorganized the corresponding result descriptions. Lines 240-3 – it might be clearer to say that strain 65G grew up to high concentrations of CTX, but only at low concentrations of CAZ, while 249R grew up to high concentrations of CAZ, but not in CTX, both as expected, if this is what is meant (“tolerable drug” does not make sense here). Response: Many thanks. We have revised this description as suggested. Lines 243-7 could then say that Models 3, 4 and 5, which express both CTX-M-65 and CTX-M-249 in different configurations, all showed growth up to relatively high levels of both CTX and CAZ, then explain the differences between Model 3 and Models 4 and 5. Response: We have revised this description as suggested. Lines 253 – unclear, needs rewording. Response: We reorganized this sentence as follows: “The duplication of resistance genes into two variants with distinct resistance profiles enables bacteria to withstand the combined effects of multiple drugs simultaneously.” Line 257 – suggest “selection with”. Response: This section has been rephrased. To align with the restructured text and for conciseness, the phrase “selective pressures of CTX or CZA” has been changed to “under CTX or CZA pressure”. Lines 259-65 – wording does not make sense and is unclear – say that Model 4 has CTX-M-65 (green) and CTX-M-249 (red) encoded by two different plasmids in the same cell. Then that in Model 4 orange fluorescence was replaced by green in the presence of CTX (resistance conferred by CTX-M-65) and by red in the presence of CAZ (resistance conferred by CTX-M-249), then information from Lines 266-9. Response: We have reorganized this paragraph as suggested. Lines 265-71 – could just state that Model 5 has CTX-M-65 and CTX-M-249 encoded on the same plasmid and that orange fluorescence was maintained and reword Lines 269-71 – e.g., I don’t think that using “paralogous” here is helpful. Response: We have reorganized this paragraph as suggested and removed the final sentence. However, we believe that the concept of “paralogous” remains important in this study. Paralogs are homologous genes within the same genome that originate from gene duplication, and they can evolve new or specialized functions, thereby diversifying gene functionality. This is essentially analogous to the process by which bla and its variants arise and develop CTX-M-65 differentiated resistance profiles. Therefore, we have retained the use of “paralogous” in other parts of the manuscript and further clarified this concept in the Discussion. Lines 272-288, L290-329 – much of the current wording does not make sense or is unclear - the same principles as above can be applied to explain this more simply and clearly. Response: These two sections in the Results have been rephrased to make the descriptions more concise and clear. Some method-related details were simplified or moved to the Methods section. The last paragraph of the “competition assay” section was more in line with a discussion, so it was removed here and the relevant statements were adjusted and incorporated into the Discussion section. 4) Universality of resistance gene heterogeneity, Lines 331-61 Again, the language could be simplified. Lines 332 – suggest something like “Having multiple copies of different variants of a resistance gene that confer different phenotypes”. Response: Following a suggestion from another reviewer, we have moved this sentence to the Discussion section and refined it to improve the flow and conciseness of the text. Lines 335, 350-1, 355-7 – blaSHV is an intrinsic chromosomal gene in K. pneumoniae, which is the source of plasmid-borne variants. This needs to be properly explained and I would suggest looking at PMID: 17999968, which discusses blaSHV variants on plasmids and the chromosome. I would also suggest looking at PMID: 28812563 about blaTEM. This could all have implications for the analysis. Response: In this section of the analysis, we only consider several types of heterogeneity in resistance genes, including those on a single plasmid, among multiple plasmids, and between the chromosome and plasmids. For bla , we have also taken this into account: the SHV chromosome usually carries one copy, and plasmids may also acquire another copy. This situation is actually a form of heterogeneity, which we included in our statistics. The same idea has been applied to bla . We have carefully read the two articles concerning bla TEM SHV (Antimicrob Agents Chemother, 2008;52(2):441-5) and bla (Nat Ecol Evol, 2016;1(1):10), TEM and in fact, the latter had a very significant influence on the way we developed our research approach. Lines 335-57 – the name blaOXA encompasses many families that are not closely related, so it does not make sense to give a single % for multicopy heterogeneity for blaOXA. Was a particular blaOXA looked at? If so, this must be stated. If not, then the analysis may need to be redone. Response: Thank you for this helpful suggestion. You are correct that bla encompasses many OXA families that are not closely related. Therefore, we reanalyzed the data focusing on the bla OXA- family, which is widely prevalent in K. pneumoniae. We used the bla type as the 48 OXA-48 reference for homology searches, enabling the identification of bla genes in bacterial OXA-48-like genomes and thereby allowing estimation of their copy number and heterogeneity. We also reorganized the description of these results and updated the corresponding labels and numbers in the figures and tables. Line 342 – is this sequence data available and/or associated with a publication? If so, please give the BioProject and/or citation. Suggest “raw sequence data from this species” Response: The genomic raw sequence data of K. pneumoniae from our laboratory were obtained from two surveillance projects. One is a nationwide epidemiological investigation involving 28 hospitals from China, the results of which have already been published (J Antimicrob Chemother 2024; 79: 2292–2297); the other is a regional epidemiological investigation involving 36 hospitals in southeastern China, which has not yet been published. In the main text, we cited the published article and provided the BioProject accession number (PRJNA1290706) for the raw sequence data of the unpublished study. Line 351-57 – which blaCTX-M group or did you look at multiple groups? Major sequence differences between members of different blaCTX-M groups (Group 1, Group 9 etc) are not due to mutations, but because the source genes are from different Kluyvera species (there are also hybrids between Group 1 and Group 9 genes). Minor changes between blaCTX-M genes within each group are probably explained by mutations that have occurred since capture. Response: Thank you for your suggestion, and we fully agree with your point. The major sequence differences between members of different bla groups (Group 1, Group 9, etc.) CTX-M are not due to mutations. Therefore, if a strain carries both a bla gene from Group 1 and CTX-M another from Group 9, strictly speaking, such heterogeneity does not correspond to the copy number–derived mutational heterogeneity that we describe in our study. However, this part of our analysis was performed using raw genomic data to assess the heterogeneity status of a specific β-lactamase gene in each strain across a large dataset. In practice, we first identify the target gene through homology search (BLAST), and then determine heterogeneity by analyzing sequence differences among all raw reads contributing to the assembly. Therefore, whether the reference used for BLAST belongs to a Group 1 or Group 9 bla gene does not affect our ability to first locate a specific bla genotype within the genome. For our analysis of bla CTX-M CTX-M CTX- , we used bla as the reference sequence. As long as a bla gene is present in the M CTX-M-65 CTX-M genome (even one from a divergent group such as Group 1), its presence can still be identified. We then analyze whether heterogeneity exists among all reads assembled into that gene. 5) Other scientific points Lines 40, 48, 334-5 – genes encode proteins, they are not encoded. Suggest “carried”/“carry” or “plasmid-borne”, as appropriate. Response: Thank you for the suggestions. Depending on the meaning of each sentence, we used different words to replace “encode”. For instance, in the abstract, “located” and “carried” are used instead. Please refer to the main text for details; please note that some instances may have been removed due to whole structure revisions. Lines 75, 382 – it is not clear what is meant by “the CTX-M-14 isoform” here. bla gene variants are assigned numbers by NCBI. A CTX-M-14 sequence with these amino acid changes appear to have been numbered CTX-M-219, described as “inhibitor resistant”, with the accession CP019080 that cites Ref 14. See https://www.ncbi.nlm.nih.gov/pathogens/refgene/#blaCTX-M-219. Response: Here, “bla isoform” refers to a variant of bla conferring resistance to CTX-M-14 CTX-M-14 CZA, as reported in Ref. 14, which has been numbered bla by NCBI. We have already CTX-M-219 revised the original sentence in the main text as follows: “Although such reports are currently rare, there have been reports of CMY-172 and CTX-M-219, the latter representing a variant of the widely disseminated CTX-M-14.” Line 90-1 – many large conjugative plasmids that typically carry AMR genes are present at around only one copy/cell and “duplication state” needs rewording. Response: Yes, there are some large plasmids that exist only as one copy. However, it has been reported that plasmid copy number ranges from only slightly higher than chromosomal copy number in low copy plasmids to tens of copies per cell in high copy plasmids, which means that most plasmids have more than one copy per bacterial cell (Nat Rev Microbiol, 2021; 19(6):347–359). Our wording was not sufficiently rigorous, and we have revised “which exist” to “most of which exist”. Line 116 – homology is not quantifiable, so what is really meany by “highly homologous” here? “closely-related”? This is from the SNP distance of <10? Also suggest “isolates” here, Line 122 etc. Response: Yes, here we intended to express that the two isolates were highly similar in their genomic sequences, based on the result of an SNP distance of <10. We have revised the sentence to: “we discovered two bloodstream infection isolates that were closely-related,”. At another site, we also replaced “strains” with “isolates”. Lines 123-4 – which blaKPC allele does ICU-18 have? This must be stated. Response: In ICU-18, the bla allele is bla , whereas in ICU-3, after the A392G mutation, KPC KPC-2 it is bla . We have added this description in the main text. KPC-157 Line 136 – this should be “mutations” not “mutants” Response: We agree with your suggested revision; however, since this section has already been reorganized, we adopted alternative wording. Lines 139, 403, 405 etc. – I don’t think that it makes sense to use “wildtype” – it can be deleted. Response: We reviewed the instances where “wild-type” was used in the main text and deleted most of them. Only at a sentence in Discussion(“CTX-M-247 and CTX-M-248 each have one of these substitutions, suggesting they are intermediates in the evolution from the prevalent bla to the novel bla .”), we changed “wild-type bla ” to “prevalent bla CTX-M-65 CTX-M-249 CTX-M-6 CTX- M-65 ”, to correspond with “novel bla ”. CTX-M-249 Lines 171-7 – are there other studies reporting on the effects of these mutations? Response: The dual mutations at positions 130 and 167 in bla has not yet been reported. CTX-M-65 However, the effects of single mutation at each of these sites on CTX-M enzymatic hydroactivity have been documented in several studies. For example, in Antimicrobial Agents and Chemotherapy (2003; 47(9):2958–2961), the Ser130Gly mutation was shown to reduce the hydrolytic activity of CTX-M enzymes against cefotaxime while increasing resistance to inhibitors including sulbactam, clavulanic acid, and tazobactam. In PLoS Pathogens (2010; 6(1):e1000735), the Pro167Ser mutation was reported to decrease the hydrolytic activity of group 1 CTX-M enzymes against cefotaxime. By contrast, in Antimicrobial Agents and Chemotherapy (2006; 50(2):731–738), the Pro167Ser mutation was shown to enhance the hydrolytic activity of group 9 CTX-M enzymes against ceftazidime. Line 200 – I would suggest making these % less precise. Response: We appreciate this helpful suggestion. In fact, the proportion of each variant under different drug conditions is unstable and difficult to estimate with high precision. We have made revisions and retained only the single-digit place of the percentage values. Line 210 – specify blaKPC-157 here. Response: We have revised “bla ” with “bla ” as suggested. KPC KPC-157 Line 282 – LB originally stood for “lysogeny broth”. Response: We have replaced “Luria-Bertani (LB) broth” with “Lysogeny Broth (LB)” as suggested. 5) Discussion This is long and wordy and could be condensed. The discussion section should not repeat information in Results (or Introduction) and comments on the rest of the manuscript need to be considered. Line 365, 482 – is this really completely novel? Response: What we refer to as a “novel mechanism” in our study actually describes a bet-hedging evolutionary phenomenon or strategy that we observed and proposed. Specifically, multiple copies of paralogous resistance genes allow bacteria to maintain their ability to withstand multidrug pressure, while the fact that these copies are located on the same plasmid ensures they are less likely to be lost. This is particularly significant because their placement on a single plasmid allows for more stable functionality—an aspect not previously highlighted in earlier studies. We revised corresponding sentences to describe this bacterial evolutionary phenomenon as observing a novel strategy (emphasizing the observed phenomenon) rather than identifying a novel mechanism (emphasizing the discovered principle). We have rephrased the sentence accordingly. Lines 365-76 – this seems more like introductory information and parts seem to repeat the Introduction. Response: We reorganized the first paragraph of the Discussion, removing Introduction-like statements such as “With the widespread use of antibiotics, the prevalence of resistant bacteria has become a significant clinical issue” and “has been listed as a priority pathogen due to its severe multidrug resistance”. Other sentences were streamlined to serve as a transition to the subsequent content. Lines 382-3 – wording does not make sense. Response: We have reorganized this sentence as follows: “As a variant of bla , the clonal CTX-M-65 strain carrying bla demonstrated a trade-offs phenomenon, as both antimicrobial CTX-M-249 susceptibility testing and enzyme kinetics assays showed that it gained CZA resistance while losing resistance to CTX (decreasing from 128 mg/L to 0.5 mg/L).”. Line 392 – “reflected on several level of meanings” does not make sense. Response: We have removed this sentence. Line 394-5 – the plasmid copy number and how it was determined and the distance between the blaCTX-M-65-like genes should be stated in Results. Response: The contents described in this sentence have been moved to the Results section. Line 397 - “elements” is not needed – these are IS. Response: The word “elements” has been removed as suggested. Line 416 - “AS” is not needed here and suggest referring to the paper without naming the authors Response: Following your suggestion, this sentence has been reorganized. Line 417 – the genes are blaTEM-1 and blaTEM-12 Response: They have been revised as suggested in the main text. Line 428 – it is the constructed plasmids that are lost? Response: Yes. If the heterogeneity of the resistance genes in two copies is constructed on two separate plasmids within the same bacterial cell, one of the plasmids can be easily lost in the absence of selective pressure. Correspondingly, when both resistance genes are located on the same plasmid, this heterogeneity advantage can be anchored in even under changing selective pressures. Lines 434-6 – why wasn’t this data included? Response: Because of this, we can theoretically deduce that a combination of low concentrations of cefotaxime and CZA can effectively eliminate the bacteria. We have included the results of this checkerboard susceptibility assay in the supplementary figure 7. Line 436 – “inhibitor” is not needed here. Response: The word “inhibitor” has been removed as suggested. Line 438 – wording does not make sense. Response: This sentence has been revised as: “This likely reflects avibactam’s inhibition of CTX-M-65, while CTX-M-249 exhibits only weak hydrolysis of CTX.”. Line 446 – I think that heteroresistance can also be due to copy number? See papers by D. Anderson. Response: Yes, heteroresistance can also be due to copy number (Nat Rev Microbiol. 2019; 17(8):479–496), because instability in the copy number of specific resistance genes within a population may lead to differences in gene expression levels, thereby resulting in heteroresistance. However, in our study, we would like to emphasize that gene amplifications generate multiple copies of this gene, and different point-mutations can occur among these copies, leading to heterogeneity in resistance phenotypes. This is also what we aimed to present in Fig. 7a. The coexistence of multiple variants of resistance genes together with copy number variation can further complicate heteroresistance within the population. Line 452 – use “and/or” and delete “dual site mutations” Response: We have revised this sentence as suggested. Lines 457-65 – this information might be in the legend of Fig. 7, which currently very short. Response: This paragraph has been moved to the legend of Fig. 7. Lines 468-9 – unclear what is meant here. Response: We revised the wording of this sentence to make it clearer. The revised version is: “In contrast, multicopy heterogeneity denotes diverse resistance gene alleles that generate varied resistance profiles, potentially enabling adaptation to multidrugs.” Line 470 – “studies” Response: The word “study” has been replaced by “studies”. Lines 471-5 – is this really true for long-read if multiple copies of the gene are in different genetic locations, as for copies of blaCTX-M-65 (or variants) both in pICU-3 here? Response: What we intend to convey here is that even with third-generation sequencing based on Nanopore or PacBio platforms, the copy number of tandem repeats may still be misrepresented after assembly. This is because assemblies tend to merge raw reads into consensus sequences, whereas the actual copy number of tandemly repeated genes can vary even within a single clonal population. Moreover, when multiple copies of a gene are located in different genetic contexts—as with bla (or its variants) present in both regions of CTX-M-65 pICU-3—the relatively lower base accuracy of long-read sequencing makes it difficult to resolve which specific variant resides in which repeat region. Line 484 – what is meant by “horizontal transfer elements” here? Response: Here, “horizontal transfer elements” referred to IS elements, and we have now modified it to “some ISs.” Line 492 – “introduction” seems like the wrong word here – maybe “recognition” Response: The word “introduction” has been replaced by “recognition”. 6) Methods These lack detail/clarity in places, but text in other places could be condensed. Primers - although the text refers to Table 3 in several places, it is not always immediately clear which primer sets are being referred to and it might be helpful to state the primer names in the text or better indicate in Table 3 which section they were used for. Response: Thank you for your suggestion. In Table S3, we had already indicated the purpose of each reference in the “Usage” column. Following your suggestion, we have now added another column specifying the section of the main text where each reference was cited. Line 499 – presumably “species identification” is meant here? Response: We have revised this description as suggested. Lines 523-7 – the wording does not make sense here and more details are needed. What was cultured for the PCR? Response: Regarding the “Cloning the β-lactamase genes” methodology, we have thoroughly reorganized this section and added more details. Cloning in this study refers to the process in which the PCR product of the target gene from a clinical strain was ligated into the vector plasmid pCR2.1 and then transformed into a recipient strain, so that each transformant successfully obtaining the recombinant plasmid (also referred to as a colony) would carry only one variant of the PCR product. PCR products amplified from DNA extraction from the clinical strain ICU-3. Lines 573-84 – this section can be condensed. The first sentence is not really needed, is something like “Three reporter plasmids based on pACYCDuet1 were constructed” and state which plasmid has which gene. Genes are cloned in fusion with genes, not proteins, and first explain that pACYCDuet1 has a cat gene. Response: The corresponding paragraph has been rewritten and condensed, and we revised the methodological descriptions in line with your recommendations. 7) Other minor/formatting/English Title needs some rewording – suggest “Heterogeneity in multiple copies of blaCTX-M-65-like genes in a clinical isolate of Klebsiella pneumoniae”. Response: We have reconsidered the title, striving for conciseness, focus, and a clear reflection of the significance of the study. Your suggestions were very helpful, and we referred to them during the revision. However, the location of multiple-copy genes is very important, because one of our novel findings is that when two paralogous genes with different resistance profiles are located on same plasmid, this bet-hedging feature can be stably maintained. In addition, another important significance of our study is that the heterogeneous multicopy enhances evolutionary adaptability, so this also needs to be reflected in the title. After considering all opinions, we have revised the title to: “Heterogeneous multicopy resistance genes on a single plasmid enhance evolutionary adaptability in clinical Klebsiella pneumoniae”. Now we believe this version best captures the focus and significance of our study. Throughout – odd/unscientific phrasing needs to be changed, e.g., Line 33 “combat”, Line 42 “lock in, Line 55 – “constant battle”, Line 77 - bacteria do not really “choose their evolutionary paths”, Line 386 - “ingeniously explored”, Line 423 “solidified”, Lines 448, 454 “locked on/onto”. Response: We greatly appreciate these very detailed suggestions and have revised the text accordingly, word by word. The terms “combat”, “lock in” in the abstract, and “constant battle” have been removed in the reorganized paragraph. “how these bacteria choose their evolutionary paths” has been changed to “the trajectory of bacterial resistance evolution”; “ingeniously explored” has been simplified to “explored”; “solidified” has been revised to “stably co-localized”; and expressions such as “locked on/onto” have all been replaced with “anchored in” Throughout – “the blaCTX-M-65” is incorrect. This should either be “the blaCTX-M-65 gene” or preferably just “blaCTX-M-65” e.g. Line 102. (except Line 36, where “the” is needed before “CTX-M-65-type”). Also, for other gene/allele names. “bla” in the name indicates the gene Response: We carefully checked all occurrences of bla and other gene names throughout CTX-M-65 the manuscript and corrected any inappropriate expressions. Throughout – the abbreviation CZA is used for ceftazidime/avibactam. The common abbreviations CAZ and CTX, as used in figures, could also be defined and used in text. Response: We revised the abbreviations for antibiotics in both the main text, figures and tables. Specifically, CZA is used for ceftazidime/avibactam, while CAZ and CTX represent ceftazidime and cefotaxime, respectively. Line 37-8 – I don’t think that “monoclonal” is commonly used to describe bacteria? Maybe “a single strain was found to have multiple copies of blaCTX-M-65 or minor variants that conferred different spectrums of resistance”. Response: We agree that “monoclonal” was not the most accurate wording and have replaced it with “clonal” or “a single clonal,” as appropriate. Line 42 ,412 etc – I am don’t think that that “paralogous” is the clearest term to use. Response: Thank you for your suggestion. We have revised the use of “paralogous” in certain paragraphs. However, we still believe that the concept of “paralogous” remains important in this study. Paralogs are homologous genes within the same genome that originate from gene duplication, and they can evolve new or specialized functions, thereby diversifying gene functionality. This is essentially analogous to the process by which bla and its variants CTX-M-65 arise and develop differentiated resistance profiles. Therefore, we have retained the use of “paralogous” in some parts of the manuscript and further clarified this concept in the Discussion. Line 47 – suggest “clinical isolates” rather than “patient-isolated strains”. Response: We have revised this sentence as suggested. Lines 47-50, 61-62, 73-74 need rewording. Response: All of the sentences you mentioned have been reorganized. Line 80-1, 108, 384, 389 suggest just “trade-offs”, rather than the “the trade-offs effect” – or at least “the trade-off effect” and reword “may introduce new roles”. Response: In the sections you noted, we have replaced the wording with “trade-offs.” The sentense “may introduce new roles” has also been rewritten. Line 84-5 – “Compared with… mutations, genetic amplifications” – i.e. delete “the” Response: We have revised this sentence as suggested. Line 93 – either “multicopy resistance plasmids” or “multiple copies of a resistance plasmid” Response: We have revised this phrase with “multiple copies of a resistance plasmid”. Lines 95-6, 110 – need rewording. Response: All of the sentences you mentioned have been reworded. Lines 99-102 – this long sentence might be better split but certainly needs rewording. Response: This sentence has been reworded. Line 111 – “considerable numbers” Response: Since the paragraph was reorganized, this sentence has been deleted. Line 122 – “The results showed” not needed. Suggest “Both isolates belong to ST11 and differ by <10 SNPs” Response: We have revised this sentence as suggested. Line 126 – “clones with blaKPC-157” Response: We have replaced the “clones of bla gene” with “clones with bla gene”. KPC-157 KPC-157 Lines 157-60 – suggest “each has one of the two mutations seen in CTX-M-249” and “the two common third generation cephalosporins” is not needed (they have already been discussed). Response: This paragraph has been reorganized and the sentence you mentioned has been removed. Line 180 – “long-read sequence assembly” Response: We have revised this phrase as suggested. Line 227 – “respectively” needs to be at the end of the sentence. Response: This paragraph has been condensed and the sentence has been thoroughly revised. Line 228 – “mixing of” Response: This paragraph has been thoroughly revised. Line 261 - “observation of fluorescent microscope image” is not needed Response: We have reorganized this sentence according to your suggestion. Line 337 – “mutation” i.e. no “s”. Response: This paragraph has been reorganized and the sentence you mentioned has been removed. Lines 339-40, 354-5, 654-5 etc. – I would suggest using “isolates from humans or other sources”. Response: All of the description about “Homo sapiens-derived” or “non-H. sapiens-derived” in the main text, figures and tables have been revised by “Humans” or “non-human sources”. Line 513 – “was performed” Response: We have revised “were performed” with “was performed” as suggested. Line 517 – “between ICU-3 and ICU-18 was calculated”. Response: We have revised this sentence as suggested. Line 530 – don’t need both “enzymes” and “enzymatic” here. Response: We revised this sentence as “To confirm the enzymatic activity of these β-lactamase,”. Line 534 - “successful different” needs rewording. Response: The “successful different” has been revised as “The resulting”. Lines 540-1 do not make sense and need rewording. I don’t think that “velocities” is the right word here. Response: This sentence has been revised and the “initial velocities” has been modified with “initial rates”. Line 557, 569 etc. - “are listed”, not “were listed” Response: We have replaced the “were listed” with “are listed”. Line 562 – “as for amplicon sequencing”. Response: We have revised this sentence as suggested. Lines 562-3 – the wording here does not make sense. Response: This sentence has been revised as follows: Strain processing and DNA extraction were performed as for amplicon sequencing. Genomic DNA (10 ng/µL) was then ultrasonically sheared on ice. Lines 568-9 – this is unclear and needs to be explained better. Response: This section has been reorganized with additional methodological details. Lines 578 – “removed” Response: We have revised this word as suggested. Line 579, 80 – “DCP strains”, “DCG strains” Response: This section has been thoroughly reorganized, and the terms DCP and DCG have been replaced with DPD and SPD throughout the main text to improve clarity. Line 581 – “dual antibiotic” Response: This sentence has been revised and the “dual antibiotic” has been removed. Line 588 – not clear what is meant here and “including” is not needed. Response: This sentence has been revised as follows: Strains representing different models (65G, 249R, a 65G/249R mix, DPD, and SPD) were inoculated into wells containing serial dilutions of CTX, CAZ, or their combinations to assess survival. References are normally placed before figure legends. Ref 8 needs page numbers or equivalent. Something seems to be wrong with page numbers in Ref 49. Journal titles need to be correctly abbreviated. Ref 4, 12 etc., remove Title Case. Species and gene names etc. need to be correctly formatted. Response: We have placed the references before the figure legends and carefully checked all reference entries, correcting errors and inconsistencies in formatting. Species and gene names have also been italicized accordingly. 8) Figures General – some parts could be moved to supplementary, shorter titles would be better and legends could be condensed. Response: We sincerely appreciate your thoughtful suggestions. Taking into account comments from multiple reviewers, we have made substantial adjustments to the composition and order of the figures. Several panels have been moved to the Supplementary Materials, concise titles have been added to each figure, and the legends have been condensed. Fig. 1 I think that most parts of this figure are not needed or could be moved to supplementary. I would suggest retaining the grid showing resistance profiles from part a (with amino acid changes for each CTX-M variant listed) plus part d in the main manuscript. Maybe consider adding CTX-M-219 (“CTX-M-14 isoform”) discussed in the text to part b, if retained as Supplementary. Response: In line with other reviewers’ feedback, we expanded the workflow originally shown in Fig. 1a to help readers more easily follow the overall logic of the study. Panels b, c, and d of the original Fig. 1 have been moved to Supplementary Fig. 1, with panel b now including an amino acid sequence alignment of CTX-M-14 and CTX-M-219. The resistance profiles previously shown in panel d are now represented in the new Fig. 1 (part I) using a grid map, which also lists the amino acid changes for each CTX-M variant. Fig. 2 Part a - on the circular diagram of the plasmid at least the two IS26 flanking blaCTX-M-65/247/248/249 should be shown, plus preferably the other AMR genes, IS and transposons in this region and surrounding the other copy of blaCTX-M-65 (see ISfinder). The diagram to side should properly show the boundaries of IS26 and of all copies of the amplified unit. The blaSHV gene number should be added. Part b CZA 32 mg/L – colour is too pale, hard to see Response: We updated the content and color scheme of Fig. 2 to improve clarity and visual contrast. In the circular diagram of plasmid pICU-3, both loci of bla and its variants are CTX-M-65 highlighted in red, and their flanking IS26 elements are annotated along with other AMR genes, ISs, or transposons. In the enlarged gray-background inset, the IS26-TU structure is delineated, with an example of three tandem TU copies shown as a multicopy state. The bla gene SHV-12-like is also annotated. In panel b, colors were deepened for better visibility. Fig. 3 Suggest moving parts a-c to supplementary, simplifying the plasmid diagrams in part b and showing each model above the corresponding plot in part d. The wording of this part of the legend also needs improving – e.g. it is not expression of plasmids, but of fused genes. Response: Following your advice, we moved panels a and c of Fig. 3 to the Supplementary Materials, while panels b and d were reorganized to place the plasmid structures of each model directly above their corresponding checkerboard assay results. The legend has been rewritten accordingly. Fig 4 Part a – suggest stating clearly what is in DCPs and DCGs on the figure, rather than having to refer to text in legend. Using the names suggested above would help with this. Part b – this could be supplementary and just show the results in part d in the main manuscript. Part c – this could be supplementary and just show the results in part e in the main manuscript Line 749 in legend – respectively appears incorrect here – it is not clear what is being referred to. Response: Considering feedback from several reviewers, we moved panels a, b, and d of Fig. 4 to the Supplementary Materials. Panel c, which shows flow cytometry counts, was revised and retained. Panel e, which reflects colony counts from dilution plating, has been converted from a bar chart to a line chart for consistency with panel c, and both are now presented together in the new Fig. 4. Legends were reorganized and simplified. Fig. 5 Parts a and b do not both need to be shown. Parts d and f could be moved to supplementary, just showing the summary of results in parts e and g. Response: For Fig. 5, only panels e and g were retained in the revised version. Panels a, b, c, d, and f were moved to the Supplementary Materials, with the legend correspondingly reorganized. Fig. 6 See comments above on analysis of blaSHV, blaCTX-M and blaOXA genes. Line 781 – “including” is incorrect here, as all genes analysed are then listed Response: As described in our response to earlier comments, Fig. 6 was also revised. In the bla analysis, the reference sequence was updated to bla , and annotations for human OXA OXA-48 versus non-human sources were updated. The related results section in the main text was substantially revised to improve accuracy and clarity. Fig. 7 What is the evidence for multidrug resistance pressure resulting in two copies of blaCTX-65 on the plasmid? What to the following unlabeled arrows represent? Response: We also modified Fig. 7a to make the schematic more intuitive. Fig. 7 is a conceptual model figure, where the proposed evolutionary trajectories are hypotheses based on current evidence, intended to stimulate further thought. In Fig. 7a, the dashed box represents several possible states of multicopy heterogeneity of resistance genes within a single clonal population. The unlabeled arrows indicate inferred evolutionary trajectories: at one locus, bla may CTX-M-65 first acquire either of two point-mutations (yielding bla or bla ). If both CTX-M-247 CTX-M-248 mutations occur together, bla may emerge, conferring CZA resistance. Furthermore, CTX-M-249 multicopy tandem duplication of bla at this locus could occur, further enhancing CTX-M-249 resistance levels. 9) Supplementary Table S1 How do values compare with previous determinations for CTX-M-65, if available? Response: Thank you for your valuable suggestions. In this study, we measured the kinetic parameters (e.g., K , k ) of CTX-M-65 and its variants (CTX-M-247, CTX-M-248, and CTX- m cat M-249) to evaluate their catalytic properties toward antibiotics and inhibitors. In response to your suggestion regarding comparison with previous studies, we searched the literature and found only one relevant report on CTX-M-65 kinetics (J Med Microbiol, 2009; 58(Pt 6):811– 815). However, this publication did not provide sufficient experimental details (e.g., buffer pH, temperature, substrate concentrations, or enzyme purification methods), which limited the feasibility of a direct comparison. To ensure comparability in our own work, we purified both the CTX-M-65 and its three variants and performed kinetic assays under identical experimental conditions, thereby minimizing variability. Table S3 It should be possible to fit this table on a single page, for ease of reading, e.g. reduce margins, “amplification” is not needed in every line of the last column, remove “the” before “blaCTX-M-65” etc. Final column heading could be “Purpose” instead of “Usage”. PsiB should be pisB in final column, if the gene is meant. Response: We also appreciate your suggestions on optimizing Table S3. Following your advice, we adjusted margins and fonts so that the table now fits on a single page. “Usage” was changed to “Purpose,” “Name” to “Primer/Probe,” and the unnecessary “the” preceding genes such as bla was removed. In addition, “PsiB” was corrected to “pisB” . CTX-M-65 CTX-M-F/R/P – blaCTX-M should be used for the gene and which blaCTX-M genes/alleles would be amplified? Response: In the “Purpose” column of Table S3, we replaced “CTX-M” with bla . The CTX-M CTX-M-F/R/P primers were used for digital qPCR to amplify all bla and its variants CTX-M (bla , bla , bla ) in order to quantify total copy numbers, while the CTX-M-65 CTX-M-247 CTX-M-248 CTX-M-249 relative proportions of each variant in a single clonal population were determined by amplicon sequencing. Table S4 This could be split into two tables, one with all isolates from humans, with this stated in the title and the final column removed, and one for other host species giving the details. I don’t think that the “Study” column is really needed – readers can get this information from the BioProject, if required. BioProject numbers and host species would be easier to read if aligned to the left. Response: As you suggested, we split Table S4 into two separate tables and revised the column titles. The final column was removed, and BioProject numbers as well as host species entries are now left-aligned. Supplementary figures – check for accuracy and condense if possible - see comments on legends for main figures. Response: All supplementary figures and their legends have been carefully checked for accuracy and consistency with the main figures. REVIEWER COMMENTS Reviewer #1 (Remarks to the Author): The authors responded to my comments appropriately. Simulation results based on real experimental data added strength to their message. I recommend the authors’ current results for publication in this journal. Legibility of this manuscript has been improved. However, I still found several vague descriptions/presentation in the current manuscript. Furthermore, the simulation program was not released to public yet. Currently, readers are difficult to reproduce data analysis. The following minor points should be improved. Line 117-121. This sentence is a little too long for me. I recommend the authors rephrase this sentence. Response: Thank you for your suggestion. This sentence here states the aim of this study. We have condensed it as follows: The aim of this study was to explore how variants coexistence, plasmid location and multicopy heterogeneity support bacteria resistance and promote an evolutionary bet-hedging strategy under multidrug pressure. Line 628: The software platform: Python, R, C++, or MATLAB etc is not described. Codes should be deposited to public repository such as Github, figshare. Response: We thank the reviewer for pointing this out. All simulations presented in this study were performed using MATLAB R2021a (The MathWorks, Inc., Natick, MA, USA); this information has now been clearly stated in the Methods section. To ensure transparency and reproducibility, the complete simulation codes have now been deposited in a public GitHub repository: https://github.com/Rui-Weng/Heterogeneous-multicopy. The corresponding links have been added to the “Code availability” section of the revised manuscript. Line 628: Unit of time used in simulation is hour? Please mention. Response: Yes, the unit of time in all simulations is hour. This has now been explicitly stated in the revised manuscript in the Methods subsection “Parameter estimation” as: “All simulations were performed with time measured in hours.” Line 699: I think that Code availability section is also needed. Response: We thank the reviewer for the suggestion. Simulation codes and the codes for GATK-based SNV/InDel calling are available at https://github.com/Rui-Weng/Heterogeneous-multicopy. Line 1038 panel e,f: Does this assume “one-round” batch culture competition assay starting with with 1:1 ratio mixture of SPD and DPD at the density of 1/100 of final density, but not serial batch culture transfer like experiments in panel c,d ?. Please clarify. Response: Yes, previous Supplementary Fig. 6 e-f represents a one-round batch culture competition simulation: DPD and SPD were initially mixed at a 1:1 ratio and cultured for 24 h without any dilution or serial transfer. The results show the final population densities after 24 h under different antibiotic concentrations. The black dashed line indicates the antibiotic concentration at which the total amount of SPD equals the total population derived from DPD (DPD + 65G + 249R), defining the boundary. This setup differs from the serial (1:100 dilution) transfer simulations shown in panels a–b. To avoid confusion, we have clearly annotated this in the revised figure legend and the corresponding MATLAB code as “single-cycle 24-h batch culture simulation (no serial transfer).” The previously referenced Supplementary Fig. 6 c-f have been moved to new Fig. 6 in the revised version. The models formerly referred to as SPD and DPD have been renamed in the updated manuscript as H1P (Heterogeneity in One Plasmid) and H2P (Heterogeneity in Two Plasmids), respectively. This is just suggestion: Supplementary figure S9- panel c,d,e,f are important results. I suggest the authors place these panels as a new main figure or add these to Figure 5 (competition assay results figure). Response: We believe the reviewer is kindly referring to Supplementary Fig. S6. We have promoted panels c-f of Supplementary Fig. S6 to a new main Fig. 6. Panels a and b of the original Supplementary Fig. S6 are retained in the Supplementary Materials as the revised Supplementary Fig. S6. Consequently, the original main Figures 6 and 7 have been renumbered as Figures 7 and 8, respectively, and all figure citations in the manuscript have been updated accordingly. Line 666 and Line1058: right panel Y-axis. The unit in these growth curves appear to be CFU/mL. How OD600nm values (source OD data) were converted to CFU/ml is not clear. Please mention in the main text or legend. Response: We thank the reviewer for pointing out this ambiguity. In our experiments, we used the Monod growth model (ref. 60 and 61) to simulate strain growth, where population size is expressed in CFU/mL. To convert the experimental OD₆₀₀ measurements to CFU/mL, we followed the method and formulation described in Mol Biol Evol. 2014;31(1):232-8, establishing a standard calibration curve to determine a linear conversion function. Therefore, all simulation results are presented in CFU/mL. This has now been explicitly stated in the revised manuscript in the Methods subsection “Parameter estimation” as: “OD₆₀₀ values were converted to CFU/mL as previously described”. Supplementary Table 5: mu.max/K should read mu.max/Ki. Response：We thank the reviewer for this comment. All parameters（𝜇 /Κ ，𝜌 , 𝛾T, 𝛾𝑍, max,𝑖 𝑖 𝑖 𝑖 𝑖 𝛽T, 𝛽Z) in Supplementary Table 5 (now Supplementary Table 4) have been corrected to 𝑖 𝑖 include proper subscripts. The revised table is included in the resubmitted supplementary materials. Reviewer #2 (Remarks to the Author): I had the opportunity to review the first version of this manuscript and have now had the pleasure of evaluating the revised version entitled “Heterogeneous multicopy resistance genes on a single plasmid enhance evolutionary adaptability in clinical Klebsiella pneumoniae”. The authors have clearly made a substantial effort to address the comments and suggestions provided by all reviewers. The revised manuscript shows significant improvement in structure, clarity, and overall readability. The reorganization of figures and sections has made the study more coherent and easier to follow. The updated workflow figure (Fig. 1) effectively guides the reader through the study and provides a clearer overview of the experimental design. Overall, the authors have responded thoroughly and constructively to the reviewers’ feedback, and the manuscript has improved notably as a result. The revisions have strengthened both the presentation and the scientific clarity. I find that all my major concerns from the first review have been addressed satisfactorily. At this point, I only have a few minor comments. Minor comments The language occasionally shifts between tenses, so a brief consistency check would also be beneficial. Line 41–46: “Within this clonal population, blaCTX-M-65 and three of its variants were present…” Please list all alleles explicitly here (e.g. blaCTX-M-65, blaCTX-M-249, …) to avoid ambiguity. Additionally, blaCTX-M-65 is a specific allele within the CTX-M-9 group, not a group itself. Therefore, the other genes are not truly “variants of blaCTX-M-65.” They are better described as other CTX-M-9 group blaCTX-M alleles closely related to blaCTX-M-65. Suggested rephrasing: “Within this clonal population, blaCTX-M-65 and additional CTX-M-9 group blaCTX-M alleles (blaCTX-M-247, blaCTX-M-248 and blaCTX-M-249*) closely related to blaCTX-M-65 were identified.” Please apply consistent terminology throughout the manuscript. Response: Thank you for your valuable suggestion. We have carefully revised the manuscript, especially the language, throughout the manuscript. During the revision process, we paid particular attention to maintaining tense consistency and ensuring linguistic accuracy and fluency. This is an excellent suggestion. We agree that it is more accurate to describe bla , bla and bla as “alleles closely related to bla ” Accordingly, we have CTX-M-247 CTX-M-248 CTX-M-249 CTX-M-65 revised the sentence in the Abstract following your advice and slightly adjusted the surrounding sentences to improve accuracy and conciseness. Following the same principle, we also modified similar expressions throughout the manuscript, replacing phrases such as “bla and three CTX-M-65 of its variants” with “bla and three closely related alleles”. In some contexts, we used CTX-M-65 the more general term “bla variant” when appropriate. CTX-M Line 77–79: “…or blaKPC they carry has undergone…” Consider replacing the first “or” with “the” and moving this phrase towards the end of the sentence for smoother flow, together with the information in lines 80–81. Response: We have revised this one and the following related sentence as: “For instance, K. pneumoniae strains have acquired metallo-β-lactamases (MBLs) or exhibit increased expression mediated by multicopy of bla , both of which contribute to resistance. The bla KPC KPC they carry has also undergone point-mutations, mostly within the omega loop, similar to those observed in extended-spectrum β-lactamases (ESBLs) that mediate resistance to CZA. However, such omega loop mutations in ESBLs remain rare, with only a few CZA-non-susceptible cases reported, including CMY-172 and CTX-M-219, the latter being a closely related allele of the widely disseminated CTX-M-14.”. Line 108–109: See comment about line 41-46. As correctly noted in line 112, blaCTX-M-65 belongs to the CTX-M-9 group. Therefore, the other genes mentioned should not be referred to as “variants of blaCTX-M-65,” since they represent distinct alleles within the CTX-M-9 group, not subvariants of blaCTX-M-65. Please rephrase to “other CTX-M-9 group blaCTX-M alleles (closely related to blaCTX-M-65).” blaCTX-M-65 was the first to be described in the literature; however, due to heterogeneity among these alleles, it is not possible to determine which appeared first in the isolate, and thus which may be the derivative of the other. Response: We fully agree with your comment. We have revised this sentence according to your suggestion and made minor syntactic adjustments to improve accuracy and conciseness. We also rechecked the entire manuscript for similar phrases such as “bla and three of its CTX-M-65 variants” and revised them accordingly. Wherever applicable, we now use expressions such as “bla and three closely related alleles” or, in some cases, the general term “bla CTX-M-65 CTX-M variant”. Line 132–133: Consider clarifying this sentence to read: “…included the nucleotide substitution A392G in…” Response: We have revised this description as suggested. Line 415, 422, and 426: Figure 7 is first introduced in the Discussion. New material should not be presented at this stage. Consider referring to Figure 7 earlier in the Results section.. Response: Thank you for this valuable suggestion. Considering that Figure 7 in the original manuscript presents a diagram of an inferred evolutionary trajectory, we also agree that this content fits better in the Results section. Therefore, we have added a new subsection at the end of the Results section and moved this part from the Discussion to there. The title of this new subsection is “Inferred evolutionary trajectory”. Figure 1: Consider referring explicitly to the different parts of Figure 1 within the Methods section to improve readability and guide the reader through the workflow. Response: We have now specified in the Methods section which experimental methods correspond to each part of Figure 1, so that readers can clearly understand which techniques were used in each part of the study design."}, "author_response": ""}]

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