Novel continuous experimental evolution methodology uncovers rapid resistance development and cross-resistance

Novel continuous experimental evolution methodology uncovers rapid resistance development and cross-resistance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Novel continuous experimental evolution methodology uncovers rapid resistance development and cross-resistance Thaddäus Echelmeyer, Markus Ellmann, Stefan E. Heiden, Kaan Kocer, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7980433/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Antimicrobial resistance poses a significant global health threat. Experimental evolution studies are crucial in understanding resistance mechanisms and thereby informing strategies to preserve antibiotic efficacy. We developed a novel continuous experimental evolution system enabling uninterrupted medium exchange with a rising antibiotic gradient, using standard laboratory equipment. We applied this system to three Enterobacter cloacae complex strains isolated from urinary tract infections in Germany between 1990 and 1992, which therefore had no prior exposure to cefepime, a fourth-generation cephalosporin approved in Germany in 2004. After four days of exposure to a cefepime gradient, resistant mutants emerged in all three strains. Notably, one mutant exhibited cross-resistance to the novel antibiotics cefiderocol and ceftazidime-avibactam, due to a single missense mutation in the β-lactamase gene bla MIR−11 . Our study demonstrates the effectiveness of this novel approach for investigating AMR development and cross-resistance mechanisms, as well as the need to understand the genetic underpinnings of AMR. Health sciences/Diseases Biological sciences/Microbiology Figures Figure 1 Figure 2 Introduction Antimicrobial resistance (AMR) is one of the most critical global health crises of the 21st century. In 2019, bacterial AMR was estimated to be directly responsible for more than 1 million deaths, threatening to undermine the foundations of modern medicine if new drugs and effective mitigation strategies are not rapidly developed and implemented [ 1 ]. This challenge is particularly acute with multidrug-resistant (MDR) Gram-negative bacteria, which can possess sophisticated mechanisms to evade nearly all available antimicrobial agents (e.g., [ 2 ]). A pathogen of increasing concern in the context of AMR is the Enterobacter cloacae complex (ECC), a leading cause of nosocomial infections, including life-threatening bloodstream infections and pneumonia [ 3 , 4 ]. ECC strains are notorious for rapidly acquiring resistance, often through overexpression or mutation of their chromosomal AmpC-type β-lactamases and the acquisition of resistance determinants on mobile genetic elements [ 5 ], resulting in MDR phenotypes. To treat these recalcitrant infections, clinicians increasingly rely on last-resort antibiotics such as cefiderocol (a siderophore cephalosporin) or the combination drug ceftazidime-avibactam (a β-lactam-β-lactamase inhibitor combination). However, the emergence of resistance and, critically, cross-resistance to these key agents is now a major clinical threat, demanding urgent investigation into the evolutionary pathways involved. Despite the urgent need to predict resistance development, current experimental evolution (EE) studies often use discontinuous, batch-culture methods [ 6 ]. These methods impose abrupt and episodic selection pressures that do not replicate the gradual, continuous antibiotic concentration gradients encountered by pathogens in vivo during patient therapy or sub-therapeutic dosing [ 7 , 8 ]. This methodological limitation prevents accurate assessment of the true speed of resistance evolution and obscures the fine-grained genetic trajectories that promote cross-resistance to multiple critical antibiotics simultaneously [ 9 ]. To address this knowledge gap, we developed a novel continuous EE system using standard laboratory equipment to maintain uninterrupted medium exchange with a precise, incrementally rising antibiotic gradient. This platform mimics the prolonged, low-concentration selection pressure characteristic of clinical treatment failure, providing a more biologically relevant model for AMR research compared to classical transfer approaches. In this study, we applied this continuous flow-through system with an increasing cefepime (CEF) gradient to three clinical ECC strains, which were isolated prior to the widespread use of CEF (between 1990 and 1992). This unique opportunity allowed us to study how resistance evolves during a bacterium's first exposure to this antibiotic. We comprehensively mapped the genomic and phenotypic mechanisms driving the observed resistance and investigated cross-resistance to the last-resort drugs cefiderocol (FDC) and ceftazidime-avibactam (CAZ-AVI). This proof-of-principle study demonstrates an affordable, reproducible and simple methodology for future high-throughput AMR studies. Results Development of a continuous experimental evolution setup The development of this novel continuous EE methodology focused on three main points compared to previously established EE methods using antibiotics as selection pressure: (i) circumventing population bottlenecks, (ii) improving accessibility and resource efficiency, and (iii) minimizing manual intervention during the experiment. First, a significant disadvantage of discontinuous EE approaches, such as serial transfer, is the introduction of population bottlenecks and the resulting undesirable effects, such as genetic drift [ 10 – 12 ]. To eliminate these issues, we designed a continuous EE system that sustains uninterrupted bacterial growth. Our system maintains the bacterial population in a constant state of exponential growth by employing the simple principle of a constant influx of fresh medium and efflux of old medium. This maintains a steady culture volume and eliminates the need for serial passaging. The continuous flow was precisely achieved using standard laboratory peristaltic pumps. Second, a limiting factor in the widespread adoption of continuous EE systems is the need for specialized equipment. Methods such as the morbidostat or even bioreactor-based systems, for example, require on non-standard and expensive components that are not typically available in most laboratories [ 9 ]. To ensure accessibility, we based our system solely on standard laboratory equipment. Additionally, to improve resource efficiency, we adapted the system to allow parallel cultivation of multiple replicates. The system consists of two peristaltic pumps, a medium reservoir, a waste reservoir, several culture flasks, and a multi-neck bottle serving as an interim reservoir. The culture flasks and interim reservoir were kept in an incubator at 37°C with constant agitation using magnetic stirrer plates. Fresh medium containing a fixed antibiotic concentration, kept at room temperature, is continuously added to the interim reservoir using the first pump at a steady flow rate (V̇ 1 ), resulting in a steadily increasing antibiotic gradient over time. The second pump distributes this medium to all culture flasks and removes old medium to the waste reservoir with an identical flow rate (V̇ 2 ), thus keeping the culture volume constant. With a V̇ 1 of 0.6 mL/min, the antibiotic concentration in the culture flasks matches that of the medium reservoir after two days (Fig. 1 ). The medium reservoir can be replaced every two days, depending on the desired final concentration and the chosen rate of antibiotic increase. Finally, the design significantly reduces the experimental burden. The only manual inputs required during the entire EE run are the initial inoculation, periodic sampling of the cultures, and replacement of the medium reservoir (antibiotic refill). This minimal intervention, combined with the ability to evolve multiple replicates in parallel, establishes this method as a versatile, high-throughput, continuous EE platform. As a proof-of-principle application, we used our novel continuous EE method to investigate the development of CEF resistance in three clinical strains belonging to the ECC. The three extended-spectrum β-lactamase producing strains were originally isolated from urine samples at the University Hospital Schleswig-Holstein in Kiel (Germany) between 1990 and 1992 during routine diagnostics for urinary tract infections and have been cryopreserved since then. The isolation dates predate the medical approval of CEF (USA: 1996; EU: 2004), suggesting that the strains had no prior clinical exposure to this specific antibiotic. Genomic analysis identified the three strains as E. roggenkampii sequence type (ST)165 (PBIO4880), E. hormaechei ST1131 (PBIO4886), and E. hormaechei ST108 (PBIO4914). Subsequent analysis using AMRFinderPlus revealed that all strains carried one class C serine β-lactamase, and PBIO4880 additionally carried the glutathione-transferase fosA . We further determined the plasmid content: PBIO4886 was plasmid-free, while the other two strains (PBIO4880 and PBIO4914) each carried three plasmids (Table 1 ). Phenotypic antimicrobial susceptibility testing using the VITEK-2 COMPACT platform confirmed an almost identical resistance pattern against penicillins and third-generation cephalosporins. Crucially for the EE study, all three strains were confirmed as susceptible to CEF and according to EUCAST guidelines [ 13 ], with minimum inhibitory concentrations (MICs) of up to 2 µg/mL (Supplementary Table S1 ). Table 1 Overview of main characteristics of tested strains. All three strains belong to the Enterobacter cloacae complex and were isolated from urinary tract infections in a German tertiary care hospital between 1990 and 1992. Resistance genes were predicted using AMRFinderPlus. PBIO4880 PBIO4886 PBIO4914 Species E. roggenkampii E. hormaechei E. hormaechei ST ST165 ST1131 ST108 Origin urine urine urine Year of sampling 1990 1991 1992 Resistance genes bla MIR−11 , fosA bla ACT−16 bla ACT−55 No. of plasmids 3 0 3 Continuous experimental evolution revealed resistance mechanisms to cefepime and cross-resistance to cefiderocol For this proof-of-principle study, three replicates of each of the three ECC strains (n = 9) were cultured in parallel. Inoculation cultures served as the parent strains for subsequent comparative analyses. To achieve a final CEF concentration equivalent to four times the EUCAST breakpoint (16 µg/mL), the system used two separate medium reservoirs containing 8 µg/mL and 16 µg/mL CEF in broth. Using the established flow rate (V̇ 1 ) of 0.6 mL/min and replacing the medium reservoir after two days, the final target concentration was reached after four days. All replicates remained culturable after sampling the evolved strains (evolvants) for phenotypic and genotypic investigation. All evolvant strains (n = 9), except for two replicates of PBIO4880, exhibited a CEF-resistant phenotype. The MIC of these resistant strains increased 8- to ≥ 64-fold compared to their respective parent strains. Although the two PBIO4880 evolvant strains (DF4880_2R and DF4880_3R) remained CEF-susceptible after the EE, their MIC increased 4- to 8-fold, reaching 2 µg/mL. Cross-resistance, which describes the emergence of resistance to antibiotics due to exposure to different antibiotics with a similar mode of action, is a major contributor to the complexity of AMR [ 10 , 14 ]. This effect has been previously described for the novel cephalosporin antibiotic FDC, which bypasses traditional resistance mechanisms using a Trojan horse strategy combined with enhanced structural stability. A single base pair duplication, occurring without exposure of the strains to FDC, in a gene encoding a siderophore receptor ( cirA ), which leads to a frameshift and early stop codon conferred resistance to this sophisticated antibiotic [ 2 ]. Therefore, the FDC MIC of all CEF-resistant strains was determined to investigate potential cross-resistance effects. Most evolvant strains were FDC-susceptible and had a 2- to 4-fold increase in their MIC. The CEF-susceptible evolvant strains DF4880_2R and DF4880_3R were also FDC-susceptible and showed no increase in their MIC compared to the respective parent strains. The CEF-resistant replicate of PBIO4880 (DF4880_1R) was FDC-resistant, with a ≥ 64-fold increase in MIC compared to the parent strain (Table 2 ). Table 2 Overview of phenotypic resistances of parent and evolvant strains as well as mutations identified in evolvant strains compared to their respective parent strains for all replicates included in the continuous experimental evolution. Minimum inhibitory concentrations for cefepime (CEF) were determined by broth micodilution and for cefiderocol (FDC) using the ComASP® Cefiderocol 0.008-128 µg/mL Kit (Liofilchem, Roseto degli Abruzzi, Italy) and interpreted according to EUCAST v15 guidelines 20 . Evolvant strains were compared to their respective parent strains using breseq 23 . *, The mutation of the F0F1 ATP synthase subunit alpha gene identified in DF4914_3R compared to the respective parent strain DF4914_3S is the identicical genotype as found in the wildtype PBIO4914; n.a., not applicable (not sequenced due to CEF susceptibility); R, resistant; S, susceptible. Strain Replicate Parent Evolvant Mutation CEF [µg/mL] FDC [µg/mL] CEF [µg/mL] FDC [µg/mL] PBIO4880 1 0.25 (S) 2 (S) ≥ 16 (R) ≥ 128 (R) bla MIR−11 (g.934G > C; p.A312P) 2 0.25 (S) 2 (S) 2 (S) 2 (S) n.a. 3 0.5 (S) 2 (S) 2 (S) 2 (S) n.a. PBIO4886 1 1 (S) 0.25 (S) 8 (R) 0.5 (S) rpoA (g.79A > C; p.T27P) 2 1 (S) 0.25 (S) ≥ 16 (R) 1 (S) ampD (g.244G > A; p.G82D) 3 1 (S) 0.25 (S) ≥ 16 (R) 0.5 (S) dnaK (g.25C > T; p.L9L), ampD (g.283G > A; p.W95*) PBIO4914 1 2 (S) 1 (S) ≥ 16 (R) 2 (S) bla ACT−55 (g.952G > A; p.V318M), malT (g.631A > T; p.Q211L) 2 2 (S) 1 (S) ≥ 16 (R) 2 (S) - 3 2 (S) 1 (S) ≥ 16 (R) 2 (S) bla ACT−55 (g.919_927dupAGCGACAGT; p.S307_S309dup), F0F1 ATP synthase subunit alpha (g.376G > T; p.V126F)* To elucidate the underlying resistance mechanism, all CEF-resistant evolvant strains, their parent strains and the wild-type strains were whole-genome sequenced. Hybrid assemblies of the wild-type strains served as references for the parent strains, and the evolvant strains were compared to their respective parent strain. Two PBIO4914 evolvant strains (DF4914_1R and DF4914_3R) had mutations in the class C serine β-lactamase bla ACT−55 . DF4914_1R had a missense mutation leading to an amino acid exchange (p.V318M), which corresponds to V298M according to the “structural alignment-based numbering of class C β-lactamases” (SANC) scheme [ 15 ]. For the third replicate of PBIO4914 (DF4914_3R), a duplication of nine nucleotides was identified, leading to three additional amino acids (p.S307_S309dup) at position 289 based on the SANC scheme. Both mutations are located in the R2 loop structure of the β-lactamase domain interface. Additionally, DF4914_1R also had a missense mutation in malT , which encodes a transcriptional activator of the maltose regulon. For the parent strain DF4914_3S a mutation in the F0F1 ATP synthase subunit alpha gene compared to the wild-type PBIO4914 was identified, which was not present in the evolvant strain DF4914_3R. No mutation was identified in the second replicate of PBIO4914 (DF4914_2R) compared to the parent strain. The second and third replicates of PBIO4886, DF4886_2R and DF4886_3R respectively, had mutations in ampD , which encodes the negative regulator of AmpC β-lactamases. In DF4886_2R, a missense mutation was identified, and in DF4886_3R, a nonsense mutation and an additional missense mutation in the dnaK gene, encoding a heat shock protein 70 family chaperone, were identified. Replicate one of PBIO4886 (DF4886_1R) carried a missense mutation in the gene encoding the RNA polymerase alpha subunit ( rpoA ). Finally, the FDC-resistant replicate of PBIO4880 (DF4880_1R) exhibited a single missense mutation in the class C serine β-lactamase gene bla MIR−11 (Table 2 ). Missense mutations in bla MIR−11 confer cross-resistance to cefiderocol and ceftazidime-avibactam The identified single missense mutation of the FDC- and CEF-resistant evolvant strain DF4880_1R resulted in an amino acid substitution in the β-lactamase MIR-11 (p.A312P). This substitution is located at position 292 according to the SANC scheme (Fig. 2 A) and, based on annotations, is part of the R2 loop structure at the domain interface [ 15 ]. Additionally, the AlphaFold3-predicted structure of the mutated MIR-11 was compared with the crystal structure of class C serine β-lactamase AmpC BER from E. coli [ 16 , 17 ] (Fig. 2 B). This β-lactamase shares 73.63% amino acid identity at 93% coverage with MIR-11. The comparison also shows the location of the substituted amino acid (shown in yellow) within the R2 loop structure (Fig. 2 C). The R2 loop is part of the active site of serine β-lactamases, along with the Ω loop at the opposite boundary and the nucleophilic Ser64 in-between. The structure of these components is crucial for accommodating the R1 and R2 side chains of β-lactams [ 17 – 21 ]. Ser64 is responsible for the nucleophilic attack on the lactam ring, which is subsequently broken in the first step of the hydrolysis of β-lactam antibiotics [ 17 , 22 ]. Mutations located in the R2 and Ω loops of various β-lactamases have previously been implicated in conferring resistance to cephalosporins, such as CAZ and CEF [ 23 – 25 ]. It has also been shown, that the insertion of two alanines in the R2 loop region of AmpC BER compared to the AmpC EC2 resulted in a wider active site and improved catalytic efficiency [ 17 ]. To validate the correlation between the missense mutation in the bla MIR−11 gene and the observed phenotypic resistance to CEF and FDC, the wild-type and mutated gene were heterologously expressed in E. coli DH5α. Both variants, including their promoter and terminator sequences, were cloned into a vector to facilitate expression at near-native levels. Comparison between both revealed a 16-fold and 8-fold increase in MIC for CEF and FDC, respectively, for the strain expressing the mutant gene compared to the strain expressing the wild-type gene. In addition, DF4880_1R also presented phenotypic resistance to CAZ-AVI, which is indicated for treatment of complicated urinary tract infections caused by MDR ECC, with a > 64-fold increase in MIC compared to the parent strain (Table 3 and Supplementary Table S1 ). The E. coli DH5α strain expressing the mutated bla MIR−11 gene showed a 16-fold increase in MIC compared to the strain expressing the wild-type gene. The novel bicyclic boronate β-lactamase inhibitor taniborbactam has been shown to be a promising candidate in combination with CEF for lowering MIC in serine and metallo-β-lactamase-producing isolates [ 26 – 28 ]. Here, we observed a 64-fold decrease in the MIC for the strain expressing the mutated bla MIR−11 , indicating that the divergence in the domain interface of the enzyme did not affect taniborbactam susceptibility. According to EUCAST guidelines, the E. coli DH5α strain expressing the mutated bla MIR−11 gene only presented phenotypic resistance to CEF, but the substantial increase in MIC for CEF, FDC, and CAZ-AVI highlights and validates the influence of the single nucleotide substitution on resistance development in the investigated E. roggenkampii strain (Table 3 ). Further investigation of fitness and virulence of the FDC-resistant mutant DF4880_1R compared to the parent strain DF4880_1S revealed no differences, as determined by growth kinetics as well as siderophore secretion quantification. Additionally, a comparative analysis of the β-lactamase activity using the chromogenic cephalosporin nitrocefin revealed no significant difference between parent and mutant strain as well as between the E. coli DH5α strains expressing the mutant and the wild-type bla MIR−11 gene (Fig. S1 ). Table 3 Overview of minimum inhibitory concentrations (MIC) of cefepime (CEF), cefiderocol (FDC), ceftazidime-avibactam (CAZ-AVI), and cefepime-taniborbactam (FTB). MICs were determined by broth microdilution, using ComASP® Cefiderocol 0.008-128 µg/mL Kit (Liofilchem, Roseto degli Abruzzi, Italy) for FDC, or by the VITEK® 2 COMPACT (bioMérieux) platform 1 . Shown are the parent and evolvant strains of PBIO4880 obtained during the experimental evolution as well as the wild-type PBIO4880 strain. Additionally, the MIC of E. coli DH5α transformed with a plasmid carrying either the wild-type or mutated bla MIR-11 gene, or the empty vector were compared together with the wild-type strain. n.a., not applicable. CEF [µg/mL] FDC [µg/mL] CAZ-AVI [mg/L] FTB [µg/mL] PBIO4880 0.25 0.5 0.5 1 n.a. DF4880_1S 0.25 2 0.25 1 n.a. DF4880_1R ≥ 16 ≥ 128 ≥ 16 1 n.a. E. coli DH5α 0.125 0.064 0.125 ≤ 0.06 E. coli DH5α pCR2.1 1 0.064 0.125 0.125 E. coli DH5α pCR2.1_blaMIR11−WT 0.5 0.064 0.25 ≤ 0.06 E. coli DH5α pCR2.1_blaMIR11−MT 8 0.5 4 0.125 Discussion Our study presents a novel, high-throughput system for continuous EE that overcomes the main limitations of conventional methods. Unlike specialized, expensive devices such as the morbidostat, our design uses standard laboratory equipment and simple peristaltic pumps to provide constant medium exchange and a continuously increasing antibiotic gradient. This simple yet straightforward setup greatly reduces hands-on time and makes continuous EE accessible to many laboratories, streamlining resistance evolution research. By eliminating the need for optical density-dependent adjustments or discontinuous drug gradients, our method maintains a steady, constant selection pressure that favors the emergence of resistant mutants while still allowing population survival [ 9 , 10 ]. Although this linear increase in antibiotic concentration differs from the fluctuating multi-dose in vivo pharmacokinetics, it shares the critical feature of being independent of bacterial population dynamics, a key advantage over other continuous methods such as the morbidostat. In addition, by avoiding the population bottlenecks inherent in most discontinuous EE approaches, our system more closely models the sustained selection environment found in vivo when considering the core interaction between bacteria and antibiotics. In just four days of exposure to an increasing CEF gradient, all replicates of three ECC strains remained culturable. Over 77% (7/9) of the total replicates cultivated simultaneously exhibited phenotypic CEF resistance, with an increase in MIC of 8- to ≥ 64-fold. For two replicates, we could not confirm phenotypic resistance according to EUCAST guidelines. This is likely due to the sampling process, which imposes an artificial bottleneck and may, by chance, select susceptible clones that survived the antibiotic challenge in the overall population. This is based on their own increase in MIC of 4- to 8-fold, reaching 2 µg/mL, just below the EUCAST breakpoint of > 4 µg/mL, and on population dynamics, such as the protection provided by other resistant clones in the population [ 10 , 29 ]. Overall, these findings highlight the effectiveness of our method and the adaptability of the ECC strains. Our system retains the major advantages of continuous EE methodologies while overcoming previous limitations of low throughput caused by the need for specialized equipment, such as the morbidostat system [ 10 ]. This is accomplished by enabling parallel cultivation of multiple replicates using only standard laboratory equipment and minimal manual intervention. Future research will determine whether the observed efficiency is consistent across diverse bacterial species and drug combinations. Four of the seven resistant evolvant strains carried mutations directly in or related to serine class C β-lactamases. Two replicates of PBIO4914 had a mutation in the bla ACT−55 gene, resulting in divergence in the R2 loop structure of the β-lactamase. Previous studies have shown that mutations in the R2 loop structure of serine class C β-lactamases can increase catalytic efficiency against cephalosporins such as CEF, leading to resistant phenotypes [ 25 ]. For the β-lactamase AmpC BER, the insertion of two amino acids in the R2 loop region could be traced to a wider active site and higher catalytic efficiency ( k cat / K m ) for cephalosporins and imipenem [ 17 ]. In contrast, two replicates of PBIO4886, carrying the β-lactamase gene bla ACT−16 , had mutations in the ampD gene. AmpD regulates AmpC β-lactamase expression, and mutations have been shown to cause overexpression of β-lactamases, resulting in higher MICs and resistance [ 30 – 33 ]. The other CEF-resistant replicate of PBIO4886 had a missense mutation in rpoA , a gene previously implicated only in ampicillin resistance in E. coli and enhanced susceptibility in Pseudomonas aeruginosa due to reduction of the MexEF-OprN efflux pump activity [ 34 , 35 ]. The mutation in rpoA may influence global transcription patterns in a way that indirectly affects either the β-lactamase expression itself or other resistance mechanisms. Notably, we were unable to identify a mutation in the other evolvant strain of PBIO4914 compared to the parent strain (Table 2 ). This is likely attributable to transcriptional effects, such as overexpression of the genomic bla ACT−55 or genes encoding efflux pumps. The identification of this CEF-resistant strain demonstrates that our method can identify resistance mechanisms based on genetic mutations as well as putative adaptations at the transcriptional level, allowing for a more holistic understanding of resistance development processes. The exact underlying mechanisms of this resistance will be elucidated in further investigations. The CEF-resistant replicate of PBIO4880 (DF4880_1R) also showed phenotypic resistance to FDC and CAZ-AVI, which we traced to a single missense mutation in the serine class C β-lactamase gene bla MIR−11 . The resulting amino acid substitution, from alanine to proline at position 292 according to the SANC scheme, is located in the R2 loop structure [ 15 ] (Fig. 2 ). As previously mentioned, these types of mutations have been implicated in resistance to various cephalosporins. Shields et al. reported a clinically evolved E. hormaechei strain that was phenotypically resistant to CAZ-AVI and CEF, among others, and also showed reduced susceptibility to FDC after the patient was treated with CEF. The resistance and cross-resistance effects were attributed to a deletion of two amino acids in the R2 loop of an AmpC β-lactamase (A292_L293del) [ 36 ]. During a clinical trial of FDC, a resistant E. cloacae strain emerged after FDC treatment in a patient with nosocomial pneumonia. The resistant isolate had a MIC of 8 µg/mL, representing an 8-fold increase, and carried a missense mutation in the β-lactamase ACT-17 (A313P), which is very similar in position and involved amino acids to the one we observed (A312P / A292P according to the SANC scheme), resulting in a ≥ 64-fold increase in MIC [ 37 , 38 ]. In both cases, expression of the mutated gene in E. coli , compared to the wild-type gene, led to an increase in MIC (> 32-fold for A292_L293del in AmpC of E. hormaechei and 2-fold for A313P in ACT-17 of E. cloacae ), comparable to the effect we observed in E. coli (8-fold increase) (Table 3 ). A comparative analysis using Clustal Omega and all bla MIR genes available in the NCBI GenBank database (accessed on 06.08.2025) revealed that bla MIR−36 also carries a missense mutation, resulting in an alanine to threonine substitution at position 292 [ 39 , 40 ]. This highlights the clinical relevance of our findings and that this type of commonly occurring mutation can even result in resistance against the novel antibiotic FDC. Similar to previous results for K. pneumoniae , where mutations conferring resistance to FDC also restored susceptibility to carbapenems, the mutation in DF4880_1R also presented an evolutionary trade-off [ 41 ]. The evolvant strain was phenotypically susceptible to the carbapenem ertapenem with a 4-fold decrease in MIC compared to the parent strain, which was phenotypically resistant (Supplementary Table S1 ). Despite the significant increase in CEF MIC attributable to the mutated bla MIR−11 gene when expressed in E. coli , the combination of CEF and the β-lactamase inhibitor taniborbactam led to a 64-fold reduction in MIC compared to CEF alone. Evidently, the amino acid exchange did not decrease taniborbactam efficacy. To our knowledge, only one case has been described in which mutations in a class C β-lactamase conferred taniborbactam non-susceptibility, as a clinical E. coli strain resistant to CAZ-AVI, showed no difference in MIC between CEF and cefepime-taniborbactam (FTB; 4 µg/mL). The strain carries an AmpC β-lactamase CMY-185 with four amino acid substitutions, one of which is located in the Ω loop and another one in helix 11 [ 42 , 43 ]. The exact influence of the amino acid exchange from alanine to proline, leading to the FDC resistant phenotype in DF4880_1R, remains to be elucidated. Previously proline residues have been implicated in enhancing secondary structure stability of β-lactamases as well as increasing protein expression levels [ 44 – 46 ]. We were not able to discern any differences in fitness or virulence between the parent strain DF4880_1S and the respective evolvant strain DF4880_1R, showing that the single amino acid exchange solely affects the phenotypical resistance (Fig. S1 ). Despite not being a significant difference, DF4880_1R as well as the E. coli strain expressing the mutant gene showed a tendency of higher nitrocefin hydrolysis activity compared to the respective wild-type variants (Fig. S1 C). It has been previously shown, that AmpC mutations leading to resistance against extended-spectrum cephalosporins can, in turn, reduce their catalytic efficiencies against narrow-spectrum cephalosporins. AmpC BER from E. coli had lower rates of hydrolysis but a higher affinity for cephalosporins compared to AmpC EC2 [ 47 ]. Overall, this highlights the significance of the identified mutant strain, exhibiting resistance to extended-spectrum cephalosporins like the novel and sophisticated FDC with evolutionary trade-offs that are limited to the overall resistance pattern and do not affect virulence or fitness. In summary, our novel continuous EE methodology enabled rapid, high-throughput detection of CEF resistance and significant cross-resistance to FDC and CAZ-AVI in historical EEC strains, traced to a single nucleotide substitution in a class C β-lactamase. The system retains the key advantages of continuous evolution methodologies while overcoming the limitations of low throughput and specialized equipment. These results highlight the low genetic barrier for cross-resistance to emerge, a major contributor to the global threat of AMR. Future research will use this methodology to investigate diverse bacterial species and drug combinations at a larger scale, and to further elucidate adaptation mechanisms at the population and transcriptional levels. Methods Bacterial strains and growth conditions The list of bacterial strains used in this study can be found in Supplementary Table S2. Generally, strains were stored at -80°C in LB containing 20% (v/v) glycerol, and 5 mL LB supplemented with 50 mg/L kanamycin (as needed for the heterologous expression) was inoculated with single colonies from LB agar plates and incubated at 37°C and 180 rpm overnight. For growth kinetics, overnight cultures were used to inoculate fresh LB medium to an optical density at 600 nm of 0.01 in a 96-well plate in triplicates. Measurements were performed every 15 min for 22 h at 37°C, with 30 sec of double orbital shaking before each measurement. The resulting growth curves were used to determine the area under the curve of the optical density at 600 nm. Continuous EE Continuous EE was performed in 100 mL flasks containing 30 mL Mueller-Hinton-Bouillon 2 (MH2) medium, employing magnetic stirring bars to facilitate agitation at 80 rpm using the MULTISTIRRER magnetic stirrer F203A0178 (Velp Scientifica™, Italy). The culture flasks and the multi-neck bottle (interim reservoir) containing fresh medium without added antibiotics, which was also continuously stirred at 300 rpm using the Hei-PLATE Mix 'n' Heat Core+ (135 mm) (Heidolph Scientific Products GmbH, Germany), were kept at 37°C for the duration of the experiment. The culture flasks were inoculated with overnight cultures to an optical density (600 nm) of 0.1. To facilitate a continuous gradient of antibiotic concentration in the culture flask, medium containing 8 µg/mL CEF from a 4 L reservoir, which was UV-protected and kept at room temperature, was transferred into the interim reservoir using an IPC ISM930 peristaltic pump (Ismatec Laboratoriumstechnik, Germany) at V̇ 1 of 0.6 mL/min over the course of two days. This interim reservoir, containing medium with a seamlessly increasing concentration of CEF, supplied all culture flasks using a second peristaltic pump, IPC ISM939 (Ismatec Laboratoriumstechnik, Germany), at V̇ 2 , which is equivalent to V̇ 1 divided by the number of culture flasks. With the same flow rate as the influx of fresh medium (V̇ 2 ), old medium was continuously removed into the waste, using the same pump. The maximum number of culture flasks that can be employed in parallel, mainly depends on the capacity of the employed pumps regarding the number of channels as well as their maximum flowrate. After two days, the reservoir was exchanged for medium with a CEF concentration of 16 µg/mL, leading to a final concentration in the culture flasks of four times the EUCAST breakpoint of CEF (16 µg/mL) after another two days 20 . Samples were collected directly after inoculation (parent) and after reaching the final antibiotic concentration (evolvant) (Fig. 1 .) Cloning The complete genes of the wild-type bla MIR− 11 from DF4880_1S and the mutated variant from DF4880_1R, including native promoter and terminator sequences (identified via bPROM and ARNold [ 48 ]), were amplified via PCR using the primers MIR11_For and MIR11_Rev (Supplementary Table S3). After purification, fragments were cloned into the pCR™2.1 vector according to the TA Cloning™ Kit (Thermo Fisher, Waltham, USA) instructions and transformed into Escherichia coli ( E. coli ) DH5α. Following selection via 50 mg/L kanamycin, and verification by PCR using the M13 primers, the insertion into the plasmids was validated by Sanger sequencing (Supplementary Table S3). Antimicrobial susceptibility testing The VITEK® 2 COMPACT (bioMérieux, Marcy-l'Étoile, France) platform was used to facilitate the phenotypic antimicrobial susceptibility testing. Additionally, to determine the minimal inhibitory concentrations (MIC) of CEF, FTB and CAZ-AVI, a broth microdilution assay was performed according to ISO 20776-1. The MIC of FDC was assessed using the ComASP® Cefiderocol 0.008-128 µg/mL Kit (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer’s instructions. Additionally, the MIC of colistin was determined using Colistin Ezy MIC™ Strips (CL) 0.016–256 mg/mL (HiMedia Laboratories, Mumbai, India) according to the manufacturer’s instructions. MIC values were evaluated according to EUCAST version 14.0 guidelines [ 13 ]. DNA isolation and whole-genome sequencing (WGS) Total genomic DNA of wild-type isolates, and parent and evolvant strains was isolated using the MasterPure DNA Purification Kit for Blood Version II (LGC Biosearch Technologies, Hoddesdon, United Kingdom). Isolated DNA was sequenced by SeqCenter (Pittsburgh, MA, USA) using Illumina sequencing by synthesis (SBS) technology. The sequencing libraries were prepared using the tagmentation-based and PCR-based Illumina DNA Prep kit and custom IDT 10 bp unique dual indices (UDI) with a target insert size of 280 bp. No additional DNA fragmentation or size selection steps were performed and sequencing was performed on an Illumina NovaSeq X Plus sequencer in one or more multiplexed shared-flow-cell runs, producing 2x151 bp paired-end reads. Demultiplexing, initial quality control and initial adapter trimming was performed with bcl-convert v. 4.2.4 ( https://support-docs.illumina.com/SW/BCL_Convert/Content/SW/FrontPages/BCL_Convert.htm ). The wild-type isolates (PBIO4880, PBIO4886 and PBIO4914) were additionally sequenced using Nanopore sequencing. Sample libraries were prepared using the PCR-free Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-NBD114.24) with the NEBNext® Companion Module (E7180L) to manufacturer's specifications. No additional DNA fragmentation or size selection was performed. Nanopore sequencing was performed on a MinION Mk1B (or compatible GridION) sequencer using R10.4.1 flow cells in one or more multiplexed shared-flow-cell runs. Run design utilized the 400bps sequencing mode with a minimum read length of 200 bp. Adaptive sampling was not enabled. Guppy v. 6.4.6 was used for super-accurate basecalling (SUP), demultiplexing, and adapter removal (dna_r10.4.1_e8.2_400bps_modbases_5mc_cg_sup.cfg). No quality trimming has been performed. Genome assembly, annotation and genomic analysis Raw paired-end sequencing reads were contaminant-filtered (e.g. PhiX), again adapter-trimmed and quality-trimmed using BBDuK from BBMap v. 39.03 ( https://sourceforge.net/projects/bbmap/ ). Quality control of raw and trimmed reads was done with FastQC v. 0.12.1 ( https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ). Short read-only assemblies were created using shovill v. 1.1.0 ( https://github.com/tseemann/shovill ) in combination with SPAdes v. 3.15.5 [ 49 ] at a maximum sequence coverage of 100x without using the integrated correction step. A manual polishing step was performed by mapping all trimmed paired-end reads to the draft assembly using BWA-MEM2 v. 2.2.1 [ 50 ] and polishing the assembly with Polypolish v. 0.5.0 [ 51 ]. Hybrid assemblies with a short read-first approach (using trimmed reads as obtained above) were created using Unicycler v. 0.5.0 [ 52 ] with SPAdes v. 3.15.4 [ 49 ] and Racon v. 1.5.0 [ 53 ]. Completeness/contamination of draft and completed genome assemblies was assessed with CheckM v. 1.2.2 [ 54 ]. Closed genomes of wild-type isolates were annotated using Bakta v. 1.8.2 with full database version 5.0 [ 55 ]. Species assignment was performed with logic integrated in Kleborate v. 2.3.2 [ 56 ] and Multilocus Sequence Typing (MLST) was done with mlst v. 2.23.0 ( https://github.com/tseemann/mlst ) using the scheme for the Enterobacter cloacae complex [ 57 ] hosted at the PubMLST website. Short nucleotide variant (SNV) analysis was conducted using breseq v. 0.38.2 [ 58 ]. For this, short reads of parent strains were first mapped to the closed genome of the corresponding wild-type isolate and SNVs applied to these references using the gdtools APPLY command. The resulting closed parent genomes served as reference for the mapping of short reads from evolvant strains. Siderophore quantification Siderophore quantification was performed using the SideroTec-HiSens™ Assay (Accuplex Diagnostics Ltd., Ireland) according to the manufacturer’s instructions. In short, overnight cultures were used to inoculate 5 mL of 1:50 diluted chelated TSB to an optical density at 600 nm of 0.001. After 24 h of growth, the culture supernatant was used to determine the siderophore concentration compared to a standard range, by an iron-detector complex, resulting in an increase in fluorescence in the presence of iron chelators. Chromogenic β -lactam assay The chromogenic cephalosporin nitrocefin was used to compare the activity of the wild-type and mutated beta-lactamase MIR-11 [ 59 ]. Overnight cultures were used to inoculate 5 mL LB with a 1:100 dilution and incubated at 37°C and 180 rpm for 3 h. After washing with PBS, the cultures were adjusted to an optical density at 600 nm of 1 in PBS. In a 96-well plate, 100 µL 0.5 mM nitrocefin was added to 100 µL bacterial suspension and incubated at 37°C for 20 min. Absorption at 390 nm, 486 nm, and 600 nm was measured after 30 seconds of double orbital shaking. Notes Data availability The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB97487 ( https://www.ebi.ac.uk/ena/browser/view/PRJEB97487 ). Declarations Data availability The data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB97487 (https://www.ebi.ac.uk/ena/browser/view/PRJEB97487). Acknowledgments We thank Sara-Lucia Skwara for her excellent technical support. Funding This study was funded by the German Federal Ministry of Research, Technology and Space (BMFTR) within the “DISPATch_MRGN-Disarming pathogens as a different strategy to fight antimicrobial-resistant Gram-negatives” project (grant no. 01KI2410) as well as within the “T!Raum – One Health – MDR-DEKOL – Intestinal decolonization of multi-resistant pathogens through the combined use of natural substances, probiotics and vaccines” project (grant no. 03TR12W02A). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. Author contributions TE: Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. ME: Formal analysis, Investigation. SH: Data curation, Formal analysis, Investigation, Methodology, Software, Writing – review & editing. KK: Formal analysis, Investigation. MS: Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Writing – review & editing. HF: Formal analysis. GM: Formal analysis. SG: Formal analysis. DN: Formal analysis. KS: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. EE: Conceptualization, Investigation, Methodology, Project administration, Supervision, Writing – review & editing. Competing interests Author DN has previously received compensation for speaking engagements and has been a part of an advisory board for Shionogi but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests. References Naghavi M., et al. Global burden of bacterial antimicrobial resistance 1990–2021: A systematic analysis with forecasts to 2050. The Lancet . 2024;404(10459):1199–1226. https://doi.org/10.1016/S0140-6736(24)01867-1 . Schaufler K., et al. Convergent Klebsiella pneumoniae strains belonging to a sequence type 307 outbreak clone combine cefiderocol and carbapenem resistance with hypervirulence. Emerging Microbes & Infections . 2023;12(2):2271096. https://doi.org/10.1080/22221751.2023.2271096 . Mauritz M.D., et al. The EC-COMPASS: Long-term, multi-centre surveillance of Enterobacter cloacae complex – A clinical perspective. Journal of Hospital Infection . 2024;148:11–19. https://doi.org/10.1016/j.jhin.2024.03.010 . Intra J., et al. Antimicrobial resistance patterns of Enterobacter cloacae and Klebsiella aerogenes strains isolated from clinical specimens: A twenty-year surveillance study. Antibiotics . 2023;12(4):775. Preston K.E., Radomski C.C.A., and Venezia R.A. Nucleotide sequence of the chromosomal AmpC gene of Enterobacter aerogenes . Antimicrobial Agents and Chemotherapy . 2000;44(11):3158–3162. https://doi.org/10.1128/aac.44.11.3158-3162.2000 . Bergh B.V.d., et al. Experimental design, population dynamics, and diversity in microbial experimental evolution. Microbiology and Molecular Biology Reviews . 2018;82(3): 10.1128/mmbr.00008–18 . https://doi.org/10.1128/mmbr.00008-18. Eger E., et al. Extensively drug-resistant Klebsiella pneumoniae counteracts fitness and virulence costs that accompanied ceftazidime-avibactam resistance acquisition. Microbiology Spectrum . 2022;10(3):e00148-22. https://doi.org/10.1128/spectrum.00148-22 . Lázár V., et al. Genome-wide analysis captures the determinants of the antibiotic cross-resistance interaction network. Nature Communications . 2014;5(1):4352. https://doi.org/10.1038/ncomms5352 . Toprak E., et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. Nature Genetics . 2012;44(1):101–105. https://doi.org/10.1038/ng.1034 . Maeda T. and Furusawa C. Laboratory evolution of antimicrobial resistance in bacteria to develop rational treatment strategies. Antibiotics . 2024;13(1):94. Mahrt N., et al. Bottleneck size and selection level reproducibly impact evolution of antibiotic resistance. Nature Ecology & Evolution . 2021;5(9):1233–1242. https://doi.org/10.1038/s41559-021-01511-2 . Vogwill T., et al. Divergent evolution peaks under intermediate population bottlenecks during bacterial experimental evolution. Proceedings of the Royal Society B: Biological Sciences . 2016;283(1835):20160749. https://doi.org/10.1098/rspb.2016.0749 . The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 14.0, 2024. http://www.eucast.org . Pál C., Papp B., and Lázár V. Collateral sensitivity of antibiotic-resistant microbes. Trends in Microbiology . 2015;23(7):401–407. https://doi.org/10.1016/j.tim.2015.02.009 . Mack A.R., et al. A standard numbering scheme for class C β-lactamases. Antimicrobial Agents and Chemotherapy . 2020;64(3): 10.1128/aac.01841-19 . https://doi.org/10.1128/aac.01841-19. Abramson J., et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature . 2024;630(8016):493–500. https://doi.org/10.1038/s41586-024-07487-w . Jeong B.-G., et al. Crystal structure of AmpC BER and molecular docking lead to the discovery of broad inhibition activities of halisulfates against β-lactamases. Computational and Structural Biotechnology Journal . 2021;19:145–152. https://doi.org/10.1016/j.csbj.2020.12.015 . Beadle B.M. and Shoichet B.K. Structural basis for imipenem inhibition of class C β-lactamases. Antimicrobial Agents and Chemotherapy . 2002;46(12):3978–3980. https://doi.org/10.1128/aac.46.12.3978-3980.2002 . Nukaga M., et al. Hydrolysis of third-generation cephalosporins by class C β-lactamases: Structures of a transition state analog of cefotaxime in wild-type and extended spectrum enzymes. Journal of Biological Chemistry . 2004;279(10):9344–9352. https://doi.org/10.1074/jbc.M312356200 . Usher K.C., et al. Three-dimensional structure of AmpC β-lactamase from Escherichia coli bound to a transition-state analogue: Possible implications for the oxyanion hypothesis and for inhibitor design. Biochemistry . 1998;37(46):16082–16092. https://doi.org/10.1021/bi981210f . Wouters J., et al. Crystal structure of Enterobacter cloacae 908R class C β-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue. Cellular and Molecular Life Sciences CMLS . 2003;60(8):1764–1773. https://doi.org/10.1007/s00018-003-3189-2 . Galleni M. and Frère J.M. A survey of the kinetic parameters of class C β-lactamases. Penicillins. Biochemical Journal . 1988;255(1):119–122. https://doi.org/10.1042/bj2550119 . Barnaud G., et al. Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. FEMS Microbiology Letters . 2001;195(2):185–190. https://doi.org/10.1016/S0378-1097(01)00010-6 . Nukaga M., et al. Molecular evolution of a class C β-lactamase extending its substrate specificity. Journal of Biological Chemistry . 1995;270(11):5729–5735. https://doi.org/10.1074/jbc.270.11.5729 . Philippon A., et al. Class C β-lactamases: Molecular characteristics. Clinical Microbiology Reviews . 2022;35(3):e00150-21. https://doi.org/10.1128/cmr.00150-21 . Hamrick J.C., et al. VNRX-5133 (Taniborbactam), a broad-spectrum inhibitor of serine- and metallo-β-lactamases, restores activity of cefepime in Enterobacterales and Pseudomonas aeruginosa . Antimicrobial Agents and Chemotherapy . 2020;64(3): 10.1128/aac.01963-19 . https://doi.org/10.1128/aac.01963-19. Karlowsky J.A., et al. In vitro activity of cefepime-taniborbactam and comparators against clinical isolates of Gram-negative bacilli from 2018 to 2020: results from the Global Evaluation of Antimicrobial Resistance via Surveillance (GEARS) program. Antimicrobial Agents and Chemotherapy . 2023;67(1):e01281-22. https://doi.org/10.1128/aac.01281-22 . Zhanel G.G., et al. Cefepime–taniborbactam: A novel cephalosporin/β-lactamase inhibitor combination. Drugs . 2024;84(10):1219–1250. https://doi.org/10.1007/s40265-024-02082-9 . Lee H.H., et al. Bacterial charity work leads to population-wide resistance. Nature . 2010;467(7311):82–85. https://doi.org/10.1038/nature09354 . Barceló I.M., et al. Filling knowledge gaps related to AmpC-dependent β-lactam resistance in Enterobacter cloacae . Scientific Reports . 2024;14(1):189. https://doi.org/10.1038/s41598-023-50685-1 . Babouee Flury B., et al. The differential importance of mutations within AmpD in cephalosporin resistance of Enterobacter aerogenes and Enterobacter cloacae . International Journal of Antimicrobial Agents . 2016;48(5):555–558. https://doi.org/10.1016/j.ijantimicag.2016.07.021 . Kopp U., et al. Sequences of wild-type and mutant ampD genes of C itrobacter freundii and Enterobacter cloacae . Antimicrobial Agents and Chemotherapy . 1993;37(2):224–228. https://doi.org/10.1128/aac.37.2.224 . Lindquist S., Lindberg F., and Normark S. Binding of the Citrobacter freundii AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible ampC β-lactamase gene. Journal of Bacteriology . 1989;171(7):3746–3753. https://doi.org/10.1128/jb.171.7.3746-3753.1989 . Cai W., Lu M., and Dai W. Novel antibiotic susceptibility of an RNA polymerase α-subunit mutant in Pseudomonas aeruginosa . Journal of Antimicrobial Chemotherapy . 2023;78(9):2162–2169. https://doi.org/10.1093/jac/dkad207 . Gross R., et al. β-lactamase dependent and independent evolutionary paths to high-level ampicillin resistance. Nature Communications . 2024;15(1):5383. https://doi.org/10.1038/s41467-024-49621-2 . Shields R.K., et al. Clinical evolution of AmpC-mediated ceftazidime-avibactam and cefiderocol resistance in Enterobacter cloacae complex following exposure to cefepime. Clinical Infectious Diseases . 2020;71(10):2713–2716. https://doi.org/10.1093/cid/ciaa355 . Nordmann P., et al. Mechanisms of reduced susceptibility to cefiderocol among isolates from the CREDIBLE-CR and APEKS-NP clinical trials. Microbial Drug Resistance . 2022;28(4):398–407. https://doi.org/10.1089/mdr.2021.0180 . Wunderink R.G., et al. Cefiderocol versus high-dose, extended-infusion meropenem for the treatment of Gram-negative nosocomial pneumonia (APEKS-NP): a randomised, double-blind, phase 3, non-inferiority trial. The Lancet Infectious Diseases . 2021;21(2):213–225. https://doi.org/10.1016/S1473-3099(20)30731-3 . Sievers F., et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology . 2011;7(1):539. https://doi.org/10.1038/msb.2011.75 . Clark K., et al. GenBank. Nucleic Acids Research . 2015;44(D1):D67–D72. https://doi.org/10.1093/nar/gkv1276 . Hanna S., et al. Exploring mutational possibilities of KPC variants to reach high level resistance to cefiderocol. Scientific Reports . 2025;15(1):31312. https://doi.org/10.1038/s41598-025-17044-8 . Kawai A., et al. Structural insights into the molecular mechanism of high-level ceftazidime–avibactam resistance conferred by CMY-185. mBio . 2024;15(2):e02874-23. https://doi.org/10.1128/mbio.02874-23 . Uehara T., et al. Spectrum of cefepime-taniborbactam coverage against 190 β-lactamases defined in engineered isogenic Escherichia coli strains. Antimicrobial Agents and Chemotherapy . 2025;69(5):e01699-24. https://doi.org/10.1128/aac.01699-24 . Chikunova A., et al. Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC. Protein Science . 2024;33(4):e4972. https://doi.org/10.1002/pro.4972 . Marciano D.C., et al. Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 β-lactamase. Journal of Molecular Biology . 2008;384(1):151–164. https://doi.org/10.1016/j.jmb.2008.09.009 . Meneksedag D., et al. Communication between the active site and the allosteric site in class A β-lactamases. Computational Biology and Chemistry . 2013;43:1–10. https://doi.org/10.1016/j.compbiolchem.2012.12.002 . Mammeri H., Poirel L., and Nordmann P. Extension of the hydrolysis spectrum of AmpC β-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. Journal of Antimicrobial Chemotherapy . 2008;61(4):971. https://doi.org/10.1093/jac/dkn052 . Naville M., et al. ARNold: A web tool for the prediction of Rho-independent transcription terminators. RNA Biology . 2011;8(1):11–13. https://doi.org/10.4161/rna.8.1.13346 . Prjibelski A., et al. Using SPAdes de novo assembler. Current Protocols in Bioinformatics . 2020;70(1):e102. https://doi.org/10.1002/cpbi.102 . Vasimuddin M., et al. Efficient architecture-aware acceleration of BWA-MEM for multicore systems . in 2019 IEEE International Parallel and Distributed Processing Symposium (IPDPS) . 2019. Wick R.R. and Holt K.E. Polypolish: Short-read polishing of long-read bacterial genome assemblies. PLOS Computational Biology . 2022;18(1):e1009802. https://doi.org/10.1371/journal.pcbi.1009802 . Wick R.R., et al. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology . 2017;13(6):e1005595. https://doi.org/10.1371/journal.pcbi.1005595 . Vaser R., et al. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Research . 2017;27(5):737–746. https://doi.org/10.1101/gr.214270.116 . Parks D.H., et al. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Research . 2015;25(7):1043–1055. https://doi.org/10.1101/gr.186072.114 . Schwengers O., et al. Bakta: Rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. Microbial Genomics . 2021;7(11) https://doi.org/10.1099/mgen.0.000685 . Lam M.M.C., et al. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. Nature Communications . 2021;12(1):4188. https://doi.org/10.1038/s41467-021-24448-3 . Miyoshi-Akiyama T., et al. Multilocus sequence typing (MLST) for characterization of Enterobacter cloacae . PLOS ONE . 2013;8(6):e66358. https://doi.org/10.1371/journal.pone.0066358 . Deatherage D.E. and Barrick J.E. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Engineering and analyzing multicellular systems: Methods and protocols . 2014:165–188. https://doi.org/10.1007/978-1-4939-0554-6_12 . O'Callaghan C.H., et al. Novel method for detection of β-lactamases by using a chromogenic cephalosporin substrate. Antimicrobial Agents and Chemotherapy . 1972;1(4):283–288. https://doi.org/10.1128/aac.1.4.283 . Additional Declarations Competing interest reported. Author DN has previously received compensation for speaking engagements and has been a part of an advisory board for Shionogi but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests. Supplementary Files Echelmeyeretal.SupplementalMaterialV2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Dec, 2025 Reviews received at journal 17 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviews received at journal 09 Dec, 2025 Reviewers agreed at journal 04 Dec, 2025 Reviewers invited by journal 02 Dec, 2025 Editor assigned by journal 09 Nov, 2025 Submission checks completed at journal 07 Nov, 2025 First submitted to journal 29 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7980433","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":554134780,"identity":"d435074d-19fd-49c7-877c-9a7a86eb19b5","order_by":0,"name":"Thaddäus Echelmeyer","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research","correspondingAuthor":false,"prefix":"","firstName":"Thaddäus","middleName":"","lastName":"Echelmeyer","suffix":""},{"id":554134781,"identity":"c64a04b0-0350-4480-9c76-0dca9d379249","order_by":1,"name":"Markus Ellmann","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research","correspondingAuthor":false,"prefix":"","firstName":"Markus","middleName":"","lastName":"Ellmann","suffix":""},{"id":554134782,"identity":"cdc2445e-a8e0-4316-8815-4461ceeb8a7b","order_by":2,"name":"Stefan E. Heiden","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"E.","lastName":"Heiden","suffix":""},{"id":554134783,"identity":"015bfc05-3967-4bde-ae8b-6ad6e2f5dde8","order_by":3,"name":"Kaan Kocer","email":"","orcid":"","institution":"University of Lübeck and University Medical Center Schleswig-Holstein Campus Lübeck","correspondingAuthor":false,"prefix":"","firstName":"Kaan","middleName":"","lastName":"Kocer","suffix":""},{"id":554134784,"identity":"5f9e6e8b-fe95-4b2b-8eb9-9acd611d3d7d","order_by":4,"name":"Michael Schwabe","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Schwabe","suffix":""},{"id":554134785,"identity":"ec05b14c-6605-4753-967d-476e3b3dd3fe","order_by":5,"name":"Helmut Fickenscher","email":"","orcid":"","institution":"Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein","correspondingAuthor":false,"prefix":"","firstName":"Helmut","middleName":"","lastName":"Fickenscher","suffix":""},{"id":554134786,"identity":"4aef68b9-f7ff-4050-8b1d-8043aab2d68a","order_by":6,"name":"Gregor Maschkowitz","email":"","orcid":"","institution":"Christian-Albrecht University Kiel and University Medical Center Schleswig-Holstein","correspondingAuthor":false,"prefix":"","firstName":"Gregor","middleName":"","lastName":"Maschkowitz","suffix":""},{"id":554134787,"identity":"59c937e2-ba09-4446-ae9e-143d0801d69b","order_by":7,"name":"Sebastian Guenther","email":"","orcid":"","institution":"University of Greifswald","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Guenther","suffix":""},{"id":554134788,"identity":"259bed84-cb5a-48dc-b0eb-01984f93acc1","order_by":8,"name":"Dennis Nurjadi","email":"","orcid":"","institution":"University of Lübeck and University Medical Center Schleswig-Holstein Campus Lübeck","correspondingAuthor":false,"prefix":"","firstName":"Dennis","middleName":"","lastName":"Nurjadi","suffix":""},{"id":554134789,"identity":"54d1e31f-0c5c-4f7e-b4ba-9ffce0bc9962","order_by":9,"name":"Katharina Schaufler","email":"","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research","correspondingAuthor":false,"prefix":"","firstName":"Katharina","middleName":"","lastName":"Schaufler","suffix":""},{"id":554134790,"identity":"e06a7caa-fc06-4a29-b4d7-f2bcb84075e0","order_by":10,"name":"Elias Eger","email":"data:image/png;base64,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","orcid":"","institution":"Helmholtz Institute for One Health, Helmholtz Centre for Infection Research","correspondingAuthor":true,"prefix":"","firstName":"Elias","middleName":"","lastName":"Eger","suffix":""}],"badges":[],"createdAt":"2025-10-29 13:41:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7980433/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7980433/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97417982,"identity":"3b152c31-c07a-4238-8776-3bed929d427e","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4773974,"visible":true,"origin":"","legend":"","description":"","filename":"Echelmeyeretal.ManuscriptV2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/369543887d70f1868c0eaced.docx"},{"id":97417974,"identity":"5a200fc0-7626-410a-bb94-a4c8f06fd3ad","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":11602,"visible":true,"origin":"","legend":"","description":"","filename":"c5e203c7108c4b329a37e902618ca29b.json","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/bc23b0cbe0bc1cd9dd50bcd4.json"},{"id":97666548,"identity":"15c50c67-0aee-4e50-8d3d-2cd1ea055d15","added_by":"auto","created_at":"2025-12-08 09:21:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":279737,"visible":true,"origin":"","legend":"","description":"","filename":"Echelmeyeretal.SupplementalMaterialV2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/8bd7792bf37b1457fcae09b2.docx"},{"id":97417975,"identity":"0660addf-4aaa-4db9-9ce7-3ca6139e8e76","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153222,"visible":true,"origin":"","legend":"","description":"","filename":"c5e203c7108c4b329a37e902618ca29b1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/b5beb1cc30c95bce2d5a95c3.xml"},{"id":97417983,"identity":"c7ac9120-139e-49cb-ad2e-20bee5c81e16","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":719961,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/134e9b76ebba60c9172a09a9.png"},{"id":97666662,"identity":"cb47d047-88e1-45ed-9e38-50578579ce66","added_by":"auto","created_at":"2025-12-08 09:21:47","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3925415,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/e592a9acf4afa8932ef2cf8f.png"},{"id":97417977,"identity":"9be49c5c-40d3-4c06-9942-79d73ccb01c9","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101320,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/e5d225f67a9b379ea6aa01f4.png"},{"id":97417984,"identity":"b3c7fd73-f467-4272-b126-2e30e5d5ce30","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":422502,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/2ddfc9681603df96cafd7ae6.png"},{"id":97667099,"identity":"caa58f7b-6de7-4709-8238-0bff7077fdc2","added_by":"auto","created_at":"2025-12-08 09:22:43","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154191,"visible":true,"origin":"","legend":"","description":"","filename":"c5e203c7108c4b329a37e902618ca29b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/0f6356b939149e5c0973ba75.xml"},{"id":97417980,"identity":"9369bd5e-d303-4052-a313-5faf9bf1627d","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"html","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168215,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/0022541827747403a4d7f1ff.html"},{"id":97417972,"identity":"1602fc3e-09d9-4280-be48-36a8a18792a7","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":240059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic overview of the continuous experimental evolution system.\u003c/strong\u003e A medium reservoir containing medium with appropriate antibiotic concentration (room temperature) supplies an interim reservoir (37 °C) and thereby constantly ramps the antibiotic concentration in this reservoir. The constantly stirred medium from the interim reservoir is then in turn pumped into the multiple culture flasks and old medium is removed from the culture flasks to the waste with the same flow rate. Bacteria are cultivated in the culture flasks under constant agitation and at 37 °C. Created in BioRender.com.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/ca91344b90d0877a5123050b.png"},{"id":97666511,"identity":"4b53fb30-994e-4cac-9ea0-105c887f8efc","added_by":"auto","created_at":"2025-12-08 09:21:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic overview of the mutation in bla\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eMIR-11\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e of the cefiderocol-resistant PBIO4880 evolvant.\u003c/strong\u003e A: Visualization of the single nucleotide substitution (g.934G\u0026gt;C) in bla\u003csub\u003eMIR-11 \u003c/sub\u003eof the evolvant strain (DF4880_1R) compared to the respective parent (DF4880_1S) and the resulting amino acid substitution (p.A312P). B: Cartoon representation of the AlphaFold3-predicted structure of the mutated MIR-11 protein (light blue), with the changed amino acid colored in yellow, superimposed onto the structure of AmpC BER from Escherichia coli (7CIN), with the α/β domain, helical domain, Ω\u0026nbsp;loop, and R2 loop colored gray, pink, green, and red, respectively\u003csup\u003e25,26\u003c/sup\u003e. C: Excerpt from B showing the beta-lactamase domain interface with the Ω\u0026nbsp;loop and the R2 loop in detail to highlight the position of the changed amino acid (yellow) in the protein structure.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/0c9aaa0619984ea33305ce73.png"},{"id":97677250,"identity":"b75ea542-a1a5-4004-b136-e22d12039327","added_by":"auto","created_at":"2025-12-08 09:52:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1737124,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/af5660d6-5774-4748-8585-5758ad7571af.pdf"},{"id":97417978,"identity":"543d498a-917a-472a-a8ac-e927bcc34584","added_by":"auto","created_at":"2025-12-04 07:45:29","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":279737,"visible":true,"origin":"","legend":"","description":"","filename":"Echelmeyeretal.SupplementalMaterialV2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7980433/v1/31546f624424750422332fc1.docx"}],"financialInterests":"Competing interest reported. Author DN has previously received compensation for speaking engagements and has been a part of an advisory board for Shionogi but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests.","formattedTitle":"Novel continuous experimental evolution methodology uncovers rapid resistance development and cross-resistance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAntimicrobial resistance (AMR) is one of the most critical global health crises of the 21st century. In 2019, bacterial AMR was estimated to be directly responsible for more than 1\u0026nbsp;million deaths, threatening to undermine the foundations of modern medicine if new drugs and effective mitigation strategies are not rapidly developed and implemented [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This challenge is particularly acute with multidrug-resistant (MDR) Gram-negative bacteria, which can possess sophisticated mechanisms to evade nearly all available antimicrobial agents (e.g., [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]).\u003c/p\u003e\u003cp\u003eA pathogen of increasing concern in the context of AMR is the \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex (ECC), a leading cause of nosocomial infections, including life-threatening bloodstream infections and pneumonia [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. ECC strains are notorious for rapidly acquiring resistance, often through overexpression or mutation of their chromosomal AmpC-type β-lactamases and the acquisition of resistance determinants on mobile genetic elements [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], resulting in MDR phenotypes. To treat these recalcitrant infections, clinicians increasingly rely on last-resort antibiotics such as cefiderocol (a siderophore cephalosporin) or the combination drug ceftazidime-avibactam (a β-lactam-β-lactamase inhibitor combination). However, the emergence of resistance and, critically, cross-resistance to these key agents is now a major clinical threat, demanding urgent investigation into the evolutionary pathways involved.\u003c/p\u003e\u003cp\u003eDespite the urgent need to predict resistance development, current experimental evolution (EE) studies often use discontinuous, batch-culture methods [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These methods impose abrupt and episodic selection pressures that do not replicate the gradual, continuous antibiotic concentration gradients encountered by pathogens \u003cem\u003ein vivo\u003c/em\u003e during patient therapy or sub-therapeutic dosing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This methodological limitation prevents accurate assessment of the true speed of resistance evolution and obscures the fine-grained genetic trajectories that promote cross-resistance to multiple critical antibiotics simultaneously [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo address this knowledge gap, we developed a novel continuous EE system using standard laboratory equipment to maintain uninterrupted medium exchange with a precise, incrementally rising antibiotic gradient. This platform mimics the prolonged, low-concentration selection pressure characteristic of clinical treatment failure, providing a more biologically relevant model for AMR research compared to classical transfer approaches.\u003c/p\u003e\u003cp\u003eIn this study, we applied this continuous flow-through system with an increasing cefepime (CEF) gradient to three clinical ECC strains, which were isolated prior to the widespread use of CEF (between 1990 and 1992). This unique opportunity allowed us to study how resistance evolves during a bacterium's first exposure to this antibiotic. We comprehensively mapped the genomic and phenotypic mechanisms driving the observed resistance and investigated cross-resistance to the last-resort drugs cefiderocol (FDC) and ceftazidime-avibactam (CAZ-AVI). This proof-of-principle study demonstrates an affordable, reproducible and simple methodology for future high-throughput AMR studies.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDevelopment of a continuous experimental evolution setup\u003c/h2\u003e\u003cp\u003eThe development of this novel continuous EE methodology focused on three main points compared to previously established EE methods using antibiotics as selection pressure: (i) circumventing population bottlenecks, (ii) improving accessibility and resource efficiency, and (iii) minimizing manual intervention during the experiment.\u003c/p\u003e\u003cp\u003eFirst, a significant disadvantage of discontinuous EE approaches, such as serial transfer, is the introduction of population bottlenecks and the resulting undesirable effects, such as genetic drift [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To eliminate these issues, we designed a continuous EE system that sustains uninterrupted bacterial growth. Our system maintains the bacterial population in a constant state of exponential growth by employing the simple principle of a constant influx of fresh medium and efflux of old medium. This maintains a steady culture volume and eliminates the need for serial passaging. The continuous flow was precisely achieved using standard laboratory peristaltic pumps.\u003c/p\u003e\u003cp\u003eSecond, a limiting factor in the widespread adoption of continuous EE systems is the need for specialized equipment. Methods such as the morbidostat or even bioreactor-based systems, for example, require on non-standard and expensive components that are not typically available in most laboratories [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. To ensure accessibility, we based our system solely on standard laboratory equipment. Additionally, to improve resource efficiency, we adapted the system to allow parallel cultivation of multiple replicates. The system consists of two peristaltic pumps, a medium reservoir, a waste reservoir, several culture flasks, and a multi-neck bottle serving as an interim reservoir. The culture flasks and interim reservoir were kept in an incubator at 37\u0026deg;C with constant agitation using magnetic stirrer plates. Fresh medium containing a fixed antibiotic concentration, kept at room temperature, is continuously added to the interim reservoir using the first pump at a steady flow rate (V̇\u003csub\u003e1\u003c/sub\u003e), resulting in a steadily increasing antibiotic gradient over time. The second pump distributes this medium to all culture flasks and removes old medium to the waste reservoir with an identical flow rate (V̇\u003csub\u003e2\u003c/sub\u003e), thus keeping the culture volume constant. With a V̇\u003csub\u003e1\u003c/sub\u003e of 0.6 mL/min, the antibiotic concentration in the culture flasks matches that of the medium reservoir after two days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The medium reservoir can be replaced every two days, depending on the desired final concentration and the chosen rate of antibiotic increase.\u003c/p\u003e\u003cp\u003eFinally, the design significantly reduces the experimental burden. The only manual inputs required during the entire EE run are the initial inoculation, periodic sampling of the cultures, and replacement of the medium reservoir (antibiotic refill). This minimal intervention, combined with the ability to evolve multiple replicates in parallel, establishes this method as a versatile, high-throughput, continuous EE platform.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAs a proof-of-principle application, we used our novel continuous EE method to investigate the development of CEF resistance in three clinical strains belonging to the ECC. The three extended-spectrum β-lactamase producing strains were originally isolated from urine samples at the University Hospital Schleswig-Holstein in Kiel (Germany) between 1990 and 1992 during routine diagnostics for urinary tract infections and have been cryopreserved since then. The isolation dates predate the medical approval of CEF (USA: 1996; EU: 2004), suggesting that the strains had no prior clinical exposure to this specific antibiotic.\u003c/p\u003e\u003cp\u003eGenomic analysis identified the three strains as \u003cem\u003eE. roggenkampii\u003c/em\u003e sequence type (ST)165 (PBIO4880), \u003cem\u003eE. hormaechei\u003c/em\u003e ST1131 (PBIO4886), and \u003cem\u003eE. hormaechei\u003c/em\u003e ST108 (PBIO4914). Subsequent analysis using AMRFinderPlus revealed that all strains carried one class C serine β-lactamase, and PBIO4880 additionally carried the glutathione-transferase \u003cem\u003efosA\u003c/em\u003e. We further determined the plasmid content: PBIO4886 was plasmid-free, while the other two strains (PBIO4880 and PBIO4914) each carried three plasmids (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Phenotypic antimicrobial susceptibility testing using the VITEK-2 COMPACT platform confirmed an almost identical resistance pattern against penicillins and third-generation cephalosporins. Crucially for the EE study, all three strains were confirmed as susceptible to CEF and according to EUCAST guidelines [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], with minimum inhibitory concentrations (MICs) of up to 2 \u0026micro;g/mL (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eOverview of main characteristics of tested strains.\u003c/b\u003e All three strains belong to the \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex and were isolated from urinary tract infections in a German tertiary care hospital between 1990 and 1992. Resistance genes were predicted using AMRFinderPlus.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePBIO4880\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePBIO4886\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePBIO4914\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eE. roggenkampii\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eE. hormaechei\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eE. hormaechei\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eST\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eST165\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eST1131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eST108\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOrigin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eurine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eurine\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eurine\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eYear of sampling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1990\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1991\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1992\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResistance genes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e, \u003cem\u003efosA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;16\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;55\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNo. of plasmids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eContinuous experimental evolution revealed resistance mechanisms to cefepime and cross-resistance to cefiderocol\u003c/h3\u003e\n\u003cp\u003eFor this proof-of-principle study, three replicates of each of the three ECC strains (n\u0026thinsp;=\u0026thinsp;9) were cultured in parallel. Inoculation cultures served as the parent strains for subsequent comparative analyses. To achieve a final CEF concentration equivalent to four times the EUCAST breakpoint (16 \u0026micro;g/mL), the system used two separate medium reservoirs containing 8 \u0026micro;g/mL and 16 \u0026micro;g/mL CEF in broth. Using the established flow rate (V̇\u003csub\u003e1\u003c/sub\u003e) of 0.6 mL/min and replacing the medium reservoir after two days, the final target concentration was reached after four days. All replicates remained culturable after sampling the evolved strains (evolvants) for phenotypic and genotypic investigation.\u003c/p\u003e\u003cp\u003eAll evolvant strains (n\u0026thinsp;=\u0026thinsp;9), except for two replicates of PBIO4880, exhibited a CEF-resistant phenotype. The MIC of these resistant strains increased 8- to \u0026ge;\u0026thinsp;64-fold compared to their respective parent strains. Although the two PBIO4880 evolvant strains (DF4880_2R and DF4880_3R) remained CEF-susceptible after the EE, their MIC increased 4- to 8-fold, reaching 2 \u0026micro;g/mL. Cross-resistance, which describes the emergence of resistance to antibiotics due to exposure to different antibiotics with a similar mode of action, is a major contributor to the complexity of AMR [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This effect has been previously described for the novel cephalosporin antibiotic FDC, which bypasses traditional resistance mechanisms using a Trojan horse strategy combined with enhanced structural stability. A single base pair duplication, occurring without exposure of the strains to FDC, in a gene encoding a siderophore receptor (\u003cem\u003ecirA\u003c/em\u003e), which leads to a frameshift and early stop codon conferred resistance to this sophisticated antibiotic [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, the FDC MIC of all CEF-resistant strains was determined to investigate potential cross-resistance effects. Most evolvant strains were FDC-susceptible and had a 2- to 4-fold increase in their MIC. The CEF-susceptible evolvant strains DF4880_2R and DF4880_3R were also FDC-susceptible and showed no increase in their MIC compared to the respective parent strains. The CEF-resistant replicate of PBIO4880 (DF4880_1R) was FDC-resistant, with a\u0026thinsp;\u0026ge;\u0026thinsp;64-fold increase in MIC compared to the parent strain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eOverview of phenotypic resistances of parent and evolvant strains as well as mutations identified in evolvant strains compared to their respective parent strains for all replicates included in the continuous experimental evolution.\u003c/b\u003e Minimum inhibitory concentrations for cefepime (CEF) were determined by broth micodilution and for cefiderocol (FDC) using the ComASP\u0026reg; Cefiderocol 0.008-128 \u0026micro;g/mL Kit (Liofilchem, Roseto degli Abruzzi, Italy) and interpreted according to EUCAST v15 guidelines\u003csup\u003e\u003cem\u003e20\u003c/em\u003e\u003c/sup\u003e. Evolvant strains were compared to their respective parent strains using breseq\u003csup\u003e23\u003c/sup\u003e. *, The mutation of the F0F1 ATP synthase subunit alpha gene identified in DF4914_3R compared to the respective parent strain DF4914_3S is the identicical genotype as found in the wildtype PBIO4914; n.a., not applicable (not sequenced due to CEF susceptibility); R, resistant; S, susceptible.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eStrain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eReplicate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eParent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eEvolvant\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMutation\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCEF [\u0026micro;g/mL]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFDC [\u0026micro;g/mL]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCEF [\u0026micro;g/mL]\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFDC [\u0026micro;g/mL]\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003ePBIO4880\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;128 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e (g.934G\u0026thinsp;\u0026gt;\u0026thinsp;C; p.A312P)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.25 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003en.a.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003en.a.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003ePBIO4886\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003erpoA\u003c/em\u003e (g.79A\u0026thinsp;\u0026gt;\u0026thinsp;C; p.T27P)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eampD\u003c/em\u003e (g.244G\u0026thinsp;\u0026gt;\u0026thinsp;A; p.G82D)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ednaK\u003c/em\u003e (g.25C\u0026thinsp;\u0026gt;\u0026thinsp;T; p.L9L), \u003cem\u003eampD\u003c/em\u003e (g.283G\u0026thinsp;\u0026gt;\u0026thinsp;A; p.W95*)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003ePBIO4914\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;55\u003c/sub\u003e (g.952G\u0026thinsp;\u0026gt;\u0026thinsp;A; p.V318M), \u003cem\u003emalT\u003c/em\u003e (g.631A\u0026thinsp;\u0026gt;\u0026thinsp;T; p.Q211L)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16 (R)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2 (S)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;55\u003c/sub\u003e (g.919_927dupAGCGACAGT; p.S307_S309dup), \u003c/p\u003e\u003cp\u003eF0F1 ATP synthase subunit alpha (g.376G\u0026thinsp;\u0026gt;\u0026thinsp;T; p.V126F)*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the underlying resistance mechanism, all CEF-resistant evolvant strains, their parent strains and the wild-type strains were whole-genome sequenced. Hybrid assemblies of the wild-type strains served as references for the parent strains, and the evolvant strains were compared to their respective parent strain. Two PBIO4914 evolvant strains (DF4914_1R and DF4914_3R) had mutations in the class C serine β-lactamase \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;55\u003c/sub\u003e. DF4914_1R had a missense mutation leading to an amino acid exchange (p.V318M), which corresponds to V298M according to the \u0026ldquo;structural alignment-based numbering of class C β-lactamases\u0026rdquo; (SANC) scheme [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. For the third replicate of PBIO4914 (DF4914_3R), a duplication of nine nucleotides was identified, leading to three additional amino acids (p.S307_S309dup) at position 289 based on the SANC scheme. Both mutations are located in the R2 loop structure of the β-lactamase domain interface. Additionally, DF4914_1R also had a missense mutation in \u003cem\u003emalT\u003c/em\u003e, which encodes a transcriptional activator of the maltose regulon. For the parent strain DF4914_3S a mutation in the F0F1 ATP synthase subunit alpha gene compared to the wild-type PBIO4914 was identified, which was not present in the evolvant strain DF4914_3R. No mutation was identified in the second replicate of PBIO4914 (DF4914_2R) compared to the parent strain. The second and third replicates of PBIO4886, DF4886_2R and DF4886_3R respectively, had mutations in \u003cem\u003eampD\u003c/em\u003e, which encodes the negative regulator of AmpC β-lactamases. In DF4886_2R, a missense mutation was identified, and in DF4886_3R, a nonsense mutation and an additional missense mutation in the \u003cem\u003ednaK\u003c/em\u003e gene, encoding a heat shock protein 70 family chaperone, were identified. Replicate one of PBIO4886 (DF4886_1R) carried a missense mutation in the gene encoding the RNA polymerase alpha subunit (\u003cem\u003erpoA\u003c/em\u003e). Finally, the FDC-resistant replicate of PBIO4880 (DF4880_1R) exhibited a single missense mutation in the class C serine β-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eMissense mutations in\u003c/b\u003e \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eMIR\u0026minus;11\u003c/b\u003e\u003c/sub\u003e \u003cb\u003econfer cross-resistance to cefiderocol and ceftazidime-avibactam\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe identified single missense mutation of the FDC- and CEF-resistant evolvant strain DF4880_1R resulted in an amino acid substitution in the β-lactamase MIR-11 (p.A312P). This substitution is located at position 292 according to the SANC scheme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and, based on annotations, is part of the R2 loop structure at the domain interface [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Additionally, the AlphaFold3-predicted structure of the mutated MIR-11 was compared with the crystal structure of class C serine β-lactamase AmpC BER from \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This β-lactamase shares 73.63% amino acid identity at 93% coverage with MIR-11. The comparison also shows the location of the substituted amino acid (shown in yellow) within the R2 loop structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The R2 loop is part of the active site of serine β-lactamases, along with the Ω loop at the opposite boundary and the nucleophilic Ser64 in-between. The structure of these components is crucial for accommodating the R1 and R2 side chains of β-lactams [\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Ser64 is responsible for the nucleophilic attack on the lactam ring, which is subsequently broken in the first step of the hydrolysis of β-lactam antibiotics [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Mutations located in the R2 and Ω loops of various β-lactamases have previously been implicated in conferring resistance to cephalosporins, such as CAZ and CEF [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. It has also been shown, that the insertion of two alanines in the R2 loop region of AmpC BER compared to the AmpC EC2 resulted in a wider active site and improved catalytic efficiency [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo validate the correlation between the missense mutation in the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e gene and the observed phenotypic resistance to CEF and FDC, the wild-type and mutated gene were heterologously expressed in \u003cem\u003eE. coli\u003c/em\u003e DH5α. Both variants, including their promoter and terminator sequences, were cloned into a vector to facilitate expression at near-native levels. Comparison between both revealed a 16-fold and 8-fold increase in MIC for CEF and FDC, respectively, for the strain expressing the mutant gene compared to the strain expressing the wild-type gene. In addition, DF4880_1R also presented phenotypic resistance to CAZ-AVI, which is indicated for treatment of complicated urinary tract infections caused by MDR ECC, with a\u0026thinsp;\u0026gt;\u0026thinsp;64-fold increase in MIC compared to the parent strain (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The \u003cem\u003eE. coli\u003c/em\u003e DH5α strain expressing the mutated \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e gene showed a 16-fold increase in MIC compared to the strain expressing the wild-type gene. The novel bicyclic boronate β-lactamase inhibitor taniborbactam has been shown to be a promising candidate in combination with CEF for lowering MIC in serine and metallo-β-lactamase-producing isolates [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Here, we observed a 64-fold decrease in the MIC for the strain expressing the mutated \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e, indicating that the divergence in the domain interface of the enzyme did not affect taniborbactam susceptibility. According to EUCAST guidelines, the \u003cem\u003eE. coli\u003c/em\u003e DH5α strain expressing the mutated \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e gene only presented phenotypic resistance to CEF, but the substantial increase in MIC for CEF, FDC, and CAZ-AVI highlights and validates the influence of the single nucleotide substitution on resistance development in the investigated \u003cem\u003eE. roggenkampii\u003c/em\u003e strain (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Further investigation of fitness and virulence of the FDC-resistant mutant DF4880_1R compared to the parent strain DF4880_1S revealed no differences, as determined by growth kinetics as well as siderophore secretion quantification. Additionally, a comparative analysis of the β-lactamase activity using the chromogenic cephalosporin nitrocefin revealed no significant difference between parent and mutant strain as well as between the \u003cem\u003eE. coli\u003c/em\u003e DH5α strains expressing the mutant and the wild-type \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e gene (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eOverview of minimum inhibitory concentrations (MIC) of cefepime (CEF), cefiderocol (FDC), ceftazidime-avibactam (CAZ-AVI), and cefepime-taniborbactam (FTB).\u003c/b\u003e MICs were determined by broth microdilution, using ComASP\u0026reg; Cefiderocol 0.008-128 \u0026micro;g/mL Kit (Liofilchem, Roseto degli Abruzzi, Italy) for FDC, or by the VITEK\u0026reg; 2 COMPACT (bioM\u0026eacute;rieux) platform\u003csup\u003e1\u003c/sup\u003e. Shown are the parent and evolvant strains of PBIO4880 obtained during the experimental evolution as well as the wild-type PBIO4880 strain. Additionally, the MIC of \u003cem\u003eE. coli\u003c/em\u003e DH5α transformed with a plasmid carrying either the wild-type or mutated \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR-11\u003c/sub\u003e gene, or the empty vector were compared together with the wild-type strain. n.a., not applicable.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCEF\u003c/p\u003e\u003cp\u003e[\u0026micro;g/mL]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFDC\u003c/p\u003e\u003cp\u003e[\u0026micro;g/mL]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCAZ-AVI\u003c/p\u003e\u003cp\u003e[mg/L]\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFTB\u003c/p\u003e\u003cp\u003e[\u0026micro;g/mL]\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePBIO4880\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.5\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en.a.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDF4880_1S\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en.a.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDF4880_1R\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;16\u003csup\u003e1\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003en.a.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eDH5α\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eDH5α\u003c/b\u003e\u003csup\u003e\u003cb\u003epCR2.1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eDH5α\u003c/b\u003e\u003csup\u003e\u003cb\u003epCR2.1_blaMIR11\u0026minus;WT\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.064\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026le;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eE. coli\u003c/b\u003e \u003cb\u003eDH5α\u003c/b\u003e\u003csup\u003e\u003cb\u003epCR2.1_blaMIR11\u0026minus;MT\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.125\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study presents a novel, high-throughput system for continuous EE that overcomes the main limitations of conventional methods. Unlike specialized, expensive devices such as the morbidostat, our design uses standard laboratory equipment and simple peristaltic pumps to provide constant medium exchange and a continuously increasing antibiotic gradient. This simple yet straightforward setup greatly reduces hands-on time and makes continuous EE accessible to many laboratories, streamlining resistance evolution research.\u003c/p\u003e\u003cp\u003eBy eliminating the need for optical density-dependent adjustments or discontinuous drug gradients, our method maintains a steady, constant selection pressure that favors the emergence of resistant mutants while still allowing population survival [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although this linear increase in antibiotic concentration differs from the fluctuating multi-dose \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetics, it shares the critical feature of being independent of bacterial population dynamics, a key advantage over other continuous methods such as the morbidostat. In addition, by avoiding the population bottlenecks inherent in most discontinuous EE approaches, our system more closely models the sustained selection environment found \u003cem\u003ein vivo\u003c/em\u003e when considering the core interaction between bacteria and antibiotics.\u003c/p\u003e\u003cp\u003eIn just four days of exposure to an increasing CEF gradient, all replicates of three ECC strains remained culturable. Over 77% (7/9) of the total replicates cultivated simultaneously exhibited phenotypic CEF resistance, with an increase in MIC of 8- to \u0026ge;\u0026thinsp;64-fold. For two replicates, we could not confirm phenotypic resistance according to EUCAST guidelines. This is likely due to the sampling process, which imposes an artificial bottleneck and may, by chance, select susceptible clones that survived the antibiotic challenge in the overall population. This is based on their own increase in MIC of 4- to 8-fold, reaching 2 \u0026micro;g/mL, just below the EUCAST breakpoint of \u0026gt;\u0026thinsp;4 \u0026micro;g/mL, and on population dynamics, such as the protection provided by other resistant clones in the population [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Overall, these findings highlight the effectiveness of our method and the adaptability of the ECC strains. Our system retains the major advantages of continuous EE methodologies while overcoming previous limitations of low throughput caused by the need for specialized equipment, such as the morbidostat system [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This is accomplished by enabling parallel cultivation of multiple replicates using only standard laboratory equipment and minimal manual intervention. Future research will determine whether the observed efficiency is consistent across diverse bacterial species and drug combinations.\u003c/p\u003e\u003cp\u003eFour of the seven resistant evolvant strains carried mutations directly in or related to serine class C β-lactamases. Two replicates of PBIO4914 had a mutation in the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;55\u003c/sub\u003e gene, resulting in divergence in the R2 loop structure of the β-lactamase. Previous studies have shown that mutations in the R2 loop structure of serine class C β-lactamases can increase catalytic efficiency against cephalosporins such as CEF, leading to resistant phenotypes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For the β-lactamase AmpC BER, the insertion of two amino acids in the R2 loop region could be traced to a wider active site and higher catalytic efficiency (\u003cem\u003ek\u003c/em\u003e\u003csub\u003e\u003cem\u003ecat\u003c/em\u003e\u003c/sub\u003e/\u003cem\u003eK\u003c/em\u003e\u003csub\u003e\u003cem\u003em\u003c/em\u003e\u003c/sub\u003e) for cephalosporins and imipenem [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In contrast, two replicates of PBIO4886, carrying the β-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;16\u003c/sub\u003e, had mutations in the \u003cem\u003eampD\u003c/em\u003e gene. AmpD regulates AmpC β-lactamase expression, and mutations have been shown to cause overexpression of β-lactamases, resulting in higher MICs and resistance [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The other CEF-resistant replicate of PBIO4886 had a missense mutation in \u003cem\u003erpoA\u003c/em\u003e, a gene previously implicated only in ampicillin resistance in \u003cem\u003eE. coli\u003c/em\u003e and enhanced susceptibility in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e due to reduction of the MexEF-OprN efflux pump activity [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The mutation in \u003cem\u003erpoA\u003c/em\u003e may influence global transcription patterns in a way that indirectly affects either the β-lactamase expression itself or other resistance mechanisms. Notably, we were unable to identify a mutation in the other evolvant strain of PBIO4914 compared to the parent strain (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This is likely attributable to transcriptional effects, such as overexpression of the genomic \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eACT\u0026minus;55\u003c/sub\u003e or genes encoding efflux pumps. The identification of this CEF-resistant strain demonstrates that our method can identify resistance mechanisms based on genetic mutations as well as putative adaptations at the transcriptional level, allowing for a more holistic understanding of resistance development processes. The exact underlying mechanisms of this resistance will be elucidated in further investigations.\u003c/p\u003e\u003cp\u003eThe CEF-resistant replicate of PBIO4880 (DF4880_1R) also showed phenotypic resistance to FDC and CAZ-AVI, which we traced to a single missense mutation in the serine class C β-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e. The resulting amino acid substitution, from alanine to proline at position 292 according to the SANC scheme, is located in the R2 loop structure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). As previously mentioned, these types of mutations have been implicated in resistance to various cephalosporins. Shields et al. reported a clinically evolved E. hormaechei strain that was phenotypically resistant to CAZ-AVI and CEF, among others, and also showed reduced susceptibility to FDC after the patient was treated with CEF. The resistance and cross-resistance effects were attributed to a deletion of two amino acids in the R2 loop of an AmpC β-lactamase (A292_L293del) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. During a clinical trial of FDC, a resistant \u003cem\u003eE. cloacae\u003c/em\u003e strain emerged after FDC treatment in a patient with nosocomial pneumonia. The resistant isolate had a MIC of 8 \u0026micro;g/mL, representing an 8-fold increase, and carried a missense mutation in the β-lactamase ACT-17 (A313P), which is very similar in position and involved amino acids to the one we observed (A312P / A292P according to the SANC scheme), resulting in a\u0026thinsp;\u0026ge;\u0026thinsp;64-fold increase in MIC [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In both cases, expression of the mutated gene in \u003cem\u003eE. coli\u003c/em\u003e, compared to the wild-type gene, led to an increase in MIC (\u0026gt;\u0026thinsp;32-fold for A292_L293del in AmpC of \u003cem\u003eE. hormaechei\u003c/em\u003e and 2-fold for A313P in ACT-17 of \u003cem\u003eE. cloacae\u003c/em\u003e), comparable to the effect we observed in \u003cem\u003eE. coli\u003c/em\u003e (8-fold increase) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). A comparative analysis using Clustal Omega and all \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u003c/sub\u003e genes available in the NCBI GenBank database (accessed on 06.08.2025) revealed that \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;36\u003c/sub\u003e also carries a missense mutation, resulting in an alanine to threonine substitution at position 292 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This highlights the clinical relevance of our findings and that this type of commonly occurring mutation can even result in resistance against the novel antibiotic FDC. Similar to previous results for \u003cem\u003eK. pneumoniae\u003c/em\u003e, where mutations conferring resistance to FDC also restored susceptibility to carbapenems, the mutation in DF4880_1R also presented an evolutionary trade-off [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The evolvant strain was phenotypically susceptible to the carbapenem ertapenem with a 4-fold decrease in MIC compared to the parent strain, which was phenotypically resistant (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Despite the significant increase in CEF MIC attributable to the mutated \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e gene when expressed in \u003cem\u003eE. coli\u003c/em\u003e, the combination of CEF and the β-lactamase inhibitor taniborbactam led to a 64-fold reduction in MIC compared to CEF alone. Evidently, the amino acid exchange did not decrease taniborbactam efficacy. To our knowledge, only one case has been described in which mutations in a class C β-lactamase conferred taniborbactam non-susceptibility, as a clinical \u003cem\u003eE. coli\u003c/em\u003e strain resistant to CAZ-AVI, showed no difference in MIC between CEF and cefepime-taniborbactam (FTB; 4 \u0026micro;g/mL). The strain carries an AmpC β-lactamase CMY-185 with four amino acid substitutions, one of which is located in the Ω loop and another one in helix 11 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The exact influence of the amino acid exchange from alanine to proline, leading to the FDC resistant phenotype in DF4880_1R, remains to be elucidated. Previously proline residues have been implicated in enhancing secondary structure stability of β-lactamases as well as increasing protein expression levels [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We were not able to discern any differences in fitness or virulence between the parent strain DF4880_1S and the respective evolvant strain DF4880_1R, showing that the single amino acid exchange solely affects the phenotypical resistance (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Despite not being a significant difference, DF4880_1R as well as the \u003cem\u003eE. coli\u003c/em\u003e strain expressing the mutant gene showed a tendency of higher nitrocefin hydrolysis activity compared to the respective wild-type variants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). It has been previously shown, that AmpC mutations leading to resistance against extended-spectrum cephalosporins can, in turn, reduce their catalytic efficiencies against narrow-spectrum cephalosporins. AmpC BER from \u003cem\u003eE. coli\u003c/em\u003e had lower rates of hydrolysis but a higher affinity for cephalosporins compared to AmpC EC2 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Overall, this highlights the significance of the identified mutant strain, exhibiting resistance to extended-spectrum cephalosporins like the novel and sophisticated FDC with evolutionary trade-offs that are limited to the overall resistance pattern and do not affect virulence or fitness.\u003c/p\u003e\u003cp\u003eIn summary, our novel continuous EE methodology enabled rapid, high-throughput detection of CEF resistance and significant cross-resistance to FDC and CAZ-AVI in historical EEC strains, traced to a single nucleotide substitution in a class C β-lactamase. The system retains the key advantages of continuous evolution methodologies while overcoming the limitations of low throughput and specialized equipment. These results highlight the low genetic barrier for cross-resistance to emerge, a major contributor to the global threat of AMR. Future research will use this methodology to investigate diverse bacterial species and drug combinations at a larger scale, and to further elucidate adaptation mechanisms at the population and transcriptional levels.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains and growth conditions\u003c/h2\u003e\u003cp\u003eThe list of bacterial strains used in this study can be found in Supplementary Table S2. Generally, strains were stored at -80\u0026deg;C in LB containing 20% (v/v) glycerol, and 5 mL LB supplemented with 50 mg/L kanamycin (as needed for the heterologous expression) was inoculated with single colonies from LB agar plates and incubated at 37\u0026deg;C and 180 rpm overnight. For growth kinetics, overnight cultures were used to inoculate fresh LB medium to an optical density at 600 nm of 0.01 in a 96-well plate in triplicates. Measurements were performed every 15 min for 22 h at 37\u0026deg;C, with 30 sec of double orbital shaking before each measurement. The resulting growth curves were used to determine the area under the curve of the optical density at 600 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eContinuous EE\u003c/h2\u003e\u003cp\u003eContinuous EE was performed in 100 mL flasks containing 30 mL Mueller-Hinton-Bouillon 2 (MH2) medium, employing magnetic stirring bars to facilitate agitation at 80 rpm using the MULTISTIRRER magnetic stirrer F203A0178 (Velp Scientifica\u0026trade;, Italy). The culture flasks and the multi-neck bottle (interim reservoir) containing fresh medium without added antibiotics, which was also continuously stirred at 300 rpm using the Hei-PLATE Mix 'n' Heat Core+ (135 mm) (Heidolph Scientific Products GmbH, Germany), were kept at 37\u0026deg;C for the duration of the experiment. The culture flasks were inoculated with overnight cultures to an optical density (600 nm) of 0.1. To facilitate a continuous gradient of antibiotic concentration in the culture flask, medium containing 8 \u0026micro;g/mL CEF from a 4 L reservoir, which was UV-protected and kept at room temperature, was transferred into the interim reservoir using an IPC ISM930 peristaltic pump (Ismatec Laboratoriumstechnik, Germany) at V̇\u003csub\u003e1\u003c/sub\u003e of 0.6 mL/min over the course of two days. This interim reservoir, containing medium with a seamlessly increasing concentration of CEF, supplied all culture flasks using a second peristaltic pump, IPC ISM939 (Ismatec Laboratoriumstechnik, Germany), at V̇\u003csub\u003e2\u003c/sub\u003e, which is equivalent to V̇\u003csub\u003e1\u003c/sub\u003e divided by the number of culture flasks. With the same flow rate as the influx of fresh medium (V̇\u003csub\u003e2\u003c/sub\u003e), old medium was continuously removed into the waste, using the same pump. The maximum number of culture flasks that can be employed in parallel, mainly depends on the capacity of the employed pumps regarding the number of channels as well as their maximum flowrate. After two days, the reservoir was exchanged for medium with a CEF concentration of 16 \u0026micro;g/mL, leading to a final concentration in the culture flasks of four times the EUCAST breakpoint of CEF (16 \u0026micro;g/mL) after another two days\u003csup\u003e20\u003c/sup\u003e. Samples were collected directly after inoculation (parent) and after reaching the final antibiotic concentration (evolvant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.)\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCloning\u003c/h3\u003e\n\u003cp\u003eThe complete genes of the wild-type \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;\u0026thinsp;11\u003c/sub\u003e from DF4880_1S and the mutated variant from DF4880_1R, including native promoter and terminator sequences (identified via bPROM and ARNold [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]), were amplified via PCR using the primers MIR11_For and MIR11_Rev (Supplementary Table S3). After purification, fragments were cloned into the pCR\u0026trade;2.1 vector according to the TA Cloning\u0026trade; Kit (Thermo Fisher, Waltham, USA) instructions and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) DH5α. Following selection via 50 mg/L kanamycin, and verification by PCR using the M13 primers, the insertion into the plasmids was validated by Sanger sequencing (Supplementary Table S3).\u003c/p\u003e\n\u003ch3\u003eAntimicrobial susceptibility testing\u003c/h3\u003e\n\u003cp\u003eThe VITEK\u0026reg; 2 COMPACT (bioM\u0026eacute;rieux, Marcy-l'\u0026Eacute;toile, France) platform was used to facilitate the phenotypic antimicrobial susceptibility testing. Additionally, to determine the minimal inhibitory concentrations (MIC) of CEF, FTB and CAZ-AVI, a broth microdilution assay was performed according to ISO 20776-1. The MIC of FDC was assessed using the ComASP\u0026reg; Cefiderocol 0.008-128 \u0026micro;g/mL Kit (Liofilchem, Roseto degli Abruzzi, Italy) according to the manufacturer\u0026rsquo;s instructions. Additionally, the MIC of colistin was determined using Colistin Ezy MIC\u0026trade; Strips (CL) 0.016\u0026ndash;256 mg/mL (HiMedia Laboratories, Mumbai, India) according to the manufacturer\u0026rsquo;s instructions. MIC values were evaluated according to EUCAST version 14.0 guidelines [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDNA isolation and whole-genome sequencing (WGS)\u003c/h2\u003e\u003cp\u003eTotal genomic DNA of wild-type isolates, and parent and evolvant strains was isolated using the MasterPure DNA Purification Kit for Blood Version II (LGC Biosearch Technologies, Hoddesdon, United Kingdom). Isolated DNA was sequenced by SeqCenter (Pittsburgh, MA, USA) using Illumina sequencing by synthesis (SBS) technology. The sequencing libraries were prepared using the tagmentation-based and PCR-based Illumina DNA Prep kit and custom IDT 10 bp unique dual indices (UDI) with a target insert size of 280 bp. No additional DNA fragmentation or size selection steps were performed and sequencing was performed on an Illumina NovaSeq X Plus sequencer in one or more multiplexed shared-flow-cell runs, producing 2x151 bp paired-end reads. Demultiplexing, initial quality control and initial adapter trimming was performed with bcl-convert v. 4.2.4 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://support-docs.illumina.com/SW/BCL_Convert/Content/SW/FrontPages/BCL_Convert.htm\u003c/span\u003e\u003cspan address=\"https://support-docs.illumina.com/SW/BCL_Convert/Content/SW/FrontPages/BCL_Convert.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe wild-type isolates (PBIO4880, PBIO4886 and PBIO4914) were additionally sequenced using Nanopore sequencing. Sample libraries were prepared using the PCR-free Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-NBD114.24) with the NEBNext\u0026reg; Companion Module (E7180L) to manufacturer's specifications. No additional DNA fragmentation or size selection was performed. Nanopore sequencing was performed on a MinION Mk1B (or compatible GridION) sequencer using R10.4.1 flow cells in one or more multiplexed shared-flow-cell runs. Run design utilized the 400bps sequencing mode with a minimum read length of 200 bp. Adaptive sampling was not enabled. Guppy v. 6.4.6 was used for super-accurate basecalling (SUP), demultiplexing, and adapter removal (dna_r10.4.1_e8.2_400bps_modbases_5mc_cg_sup.cfg). No quality trimming has been performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eGenome assembly, annotation and genomic analysis\u003c/h2\u003e\u003cp\u003eRaw paired-end sequencing reads were contaminant-filtered (e.g. PhiX), again adapter-trimmed and quality-trimmed using BBDuK from BBMap v. 39.03 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sourceforge.net/projects/bbmap/\u003c/span\u003e\u003cspan address=\"https://sourceforge.net/projects/bbmap/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Quality control of raw and trimmed reads was done with FastQC v. 0.12.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Short read-only assemblies were created using shovill v. 1.1.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/shovill\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/shovill\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) in combination with SPAdes v. 3.15.5 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] at a maximum sequence coverage of 100x without using the integrated correction step. A manual polishing step was performed by mapping all trimmed paired-end reads to the draft assembly using BWA-MEM2 v. 2.2.1 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and polishing the assembly with Polypolish v. 0.5.0 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Hybrid assemblies with a short read-first approach (using trimmed reads as obtained above) were created using Unicycler v. 0.5.0 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] with SPAdes v. 3.15.4 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and Racon v. 1.5.0 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Completeness/contamination of draft and completed genome assemblies was assessed with CheckM v. 1.2.2 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Closed genomes of wild-type isolates were annotated using Bakta v. 1.8.2 with full database version 5.0 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSpecies assignment was performed with logic integrated in Kleborate v. 2.3.2 [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and Multilocus Sequence Typing (MLST) was done with mlst v. 2.23.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tseemann/mlst\u003c/span\u003e\u003cspan address=\"https://github.com/tseemann/mlst\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using the scheme for the \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] hosted at the PubMLST website. Short nucleotide variant (SNV) analysis was conducted using breseq v. 0.38.2 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. For this, short reads of parent strains were first mapped to the closed genome of the corresponding wild-type isolate and SNVs applied to these references using the gdtools APPLY command. The resulting closed parent genomes served as reference for the mapping of short reads from evolvant strains.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSiderophore quantification\u003c/h2\u003e\u003cp\u003eSiderophore quantification was performed using the SideroTec-HiSens\u0026trade; Assay (Accuplex Diagnostics Ltd., Ireland) according to the manufacturer\u0026rsquo;s instructions. In short, overnight cultures were used to inoculate 5 mL of 1:50 diluted chelated TSB to an optical density at 600 nm of 0.001. After 24 h of growth, the culture supernatant was used to determine the siderophore concentration compared to a standard range, by an iron-detector complex, resulting in an increase in fluorescence in the presence of iron chelators.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromogenic\u003c/b\u003e β\u003cb\u003e-lactam assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chromogenic cephalosporin nitrocefin was used to compare the activity of the wild-type and mutated beta-lactamase MIR-11 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Overnight cultures were used to inoculate 5 mL LB with a 1:100 dilution and incubated at 37\u0026deg;C and 180 rpm for 3 h. After washing with PBS, the cultures were adjusted to an optical density at 600 nm of 1 in PBS. In a 96-well plate, 100 \u0026micro;L 0.5 mM nitrocefin was added to 100 \u0026micro;L bacterial suspension and incubated at 37\u0026deg;C for 20 min. Absorption at 390 nm, 486 nm, and 600 nm was measured after 30 seconds of double orbital shaking.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eNotes\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB97487 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/ena/browser/view/PRJEB97487\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/ena/browser/view/PRJEB97487\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB97487 (https://www.ebi.ac.uk/ena/browser/view/PRJEB97487).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Sara-Lucia Skwara for her excellent technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the German Federal Ministry of Research, Technology and Space (BMFTR) within the \u0026ldquo;DISPATch_MRGN-Disarming pathogens as a different strategy to fight antimicrobial-resistant Gram-negatives\u0026rdquo; project (grant no. 01KI2410) as well as within the \u0026ldquo;T!Raum \u0026ndash; One Health \u0026ndash; MDR-DEKOL \u0026ndash; Intestinal decolonization of multi-resistant pathogens through the combined use of natural substances, probiotics and vaccines\u0026rdquo; project (grant no. 03TR12W02A). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTE: Formal analysis, Investigation, Methodology, Visualization, Writing \u0026ndash; original draft. ME: Formal analysis, Investigation. SH: Data curation, Formal analysis, Investigation, Methodology, Software, Writing \u0026ndash; review \u0026amp; editing. KK: Formal analysis, Investigation. MS: Data curation, Formal analysis, Investigation, Methodology, Software, Supervision, Writing \u0026ndash; review \u0026amp; editing. HF: Formal analysis. GM: Formal analysis. SG: Formal analysis. DN: Formal analysis. KS: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing \u0026ndash; review \u0026amp; editing. EE: Conceptualization, Investigation, Methodology, Project administration, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthor DN has previously received compensation for speaking engagements and has been a part of an advisory board for Shionogi but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNaghavi M., et al. Global burden of bacterial antimicrobial resistance 1990\u0026ndash;2021: A systematic analysis with forecasts to 2050. \u003cem\u003eThe Lancet\u003c/em\u003e. 2024;404(10459):1199\u0026ndash;1226. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0140-6736(24)01867-1\u003c/span\u003e\u003cspan address=\"10.1016/S0140-6736(24)01867-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchaufler K., et al. Convergent \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e strains belonging to a sequence type 307 outbreak clone combine cefiderocol and carbapenem resistance with hypervirulence. \u003cem\u003eEmerging Microbes \u0026amp; Infections\u003c/em\u003e. 2023;12(2):2271096. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/22221751.2023.2271096\u003c/span\u003e\u003cspan address=\"10.1080/22221751.2023.2271096\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMauritz M.D., et al. The EC-COMPASS: Long-term, multi-centre surveillance of \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex \u0026ndash; A clinical perspective. \u003cem\u003eJournal of Hospital Infection\u003c/em\u003e. 2024;148:11\u0026ndash;19. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhin.2024.03.010\u003c/span\u003e\u003cspan address=\"10.1016/j.jhin.2024.03.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIntra J., et al. Antimicrobial resistance patterns of \u003cem\u003eEnterobacter cloacae\u003c/em\u003e and \u003cem\u003eKlebsiella aerogenes\u003c/em\u003e strains isolated from clinical specimens: A twenty-year surveillance study. \u003cem\u003eAntibiotics\u003c/em\u003e. 2023;12(4):775.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePreston K.E., Radomski C.C.A., and Venezia R.A. Nucleotide sequence of the chromosomal AmpC gene of \u003cem\u003eEnterobacter aerogenes\u003c/em\u003e. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 2000;44(11):3158\u0026ndash;3162. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.44.11.3158-3162.2000\u003c/span\u003e\u003cspan address=\"10.1128/aac.44.11.3158-3162.2000\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBergh B.V.d., et al. Experimental design, population dynamics, and diversity in microbial experimental evolution. \u003cem\u003eMicrobiology and Molecular Biology Reviews\u003c/em\u003e. 2018;82(3):\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/mmbr.00008\u0026ndash;18\u003c/span\u003e\u003cspan address=\"10.1128/mmbr.00008\u0026ndash;18\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org/10.1128/mmbr.00008-18.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEger E., et al. Extensively drug-resistant \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e counteracts fitness and virulence costs that accompanied ceftazidime-avibactam resistance acquisition. \u003cem\u003eMicrobiology Spectrum\u003c/em\u003e. 2022;10(3):e00148-22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/spectrum.00148-22\u003c/span\u003e\u003cspan address=\"10.1128/spectrum.00148-22\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eL\u0026aacute;z\u0026aacute;r V., et al. Genome-wide analysis captures the determinants of the antibiotic cross-resistance interaction network. \u003cem\u003eNature Communications\u003c/em\u003e. 2014;5(1):4352. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms5352\u003c/span\u003e\u003cspan address=\"10.1038/ncomms5352\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eToprak E., et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. \u003cem\u003eNature Genetics\u003c/em\u003e. 2012;44(1):101\u0026ndash;105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ng.1034\u003c/span\u003e\u003cspan address=\"10.1038/ng.1034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaeda T. and Furusawa C. Laboratory evolution of antimicrobial resistance in bacteria to develop rational treatment strategies. \u003cem\u003eAntibiotics\u003c/em\u003e. 2024;13(1):94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMahrt N., et al. Bottleneck size and selection level reproducibly impact evolution of antibiotic resistance. \u003cem\u003eNature Ecology \u0026amp; Evolution\u003c/em\u003e. 2021;5(9):1233\u0026ndash;1242. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41559-021-01511-2\u003c/span\u003e\u003cspan address=\"10.1038/s41559-021-01511-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVogwill T., et al. Divergent evolution peaks under intermediate population bottlenecks during bacterial experimental evolution. \u003cem\u003eProceedings of the Royal Society B: Biological Sciences\u003c/em\u003e. 2016;283(1835):20160749. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1098/rspb.2016.0749\u003c/span\u003e\u003cspan address=\"10.1098/rspb.2016.0749\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eThe European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 14.0, 2024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.eucast.org\u003c/span\u003e\u003cspan address=\"http://www.eucast.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eP\u0026aacute;l C., Papp B., and L\u0026aacute;z\u0026aacute;r V. Collateral sensitivity of antibiotic-resistant microbes. \u003cem\u003eTrends in Microbiology\u003c/em\u003e. 2015;23(7):401\u0026ndash;407. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tim.2015.02.009\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2015.02.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMack A.R., et al. A standard numbering scheme for class C β-lactamases. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 2020;64(3):\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac.01841-19\u003c/span\u003e\u003cspan address=\"10.1128/aac.01841-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org/10.1128/aac.01841-19.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbramson J., et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. \u003cem\u003eNature\u003c/em\u003e. 2024;630(8016):493\u0026ndash;500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-024-07487-w\u003c/span\u003e\u003cspan address=\"10.1038/s41586-024-07487-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJeong B.-G., et al. Crystal structure of AmpC BER and molecular docking lead to the discovery of broad inhibition activities of halisulfates against β-lactamases. \u003cem\u003eComputational and Structural Biotechnology Journal\u003c/em\u003e. 2021;19:145\u0026ndash;152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.csbj.2020.12.015\u003c/span\u003e\u003cspan address=\"10.1016/j.csbj.2020.12.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBeadle B.M. and Shoichet B.K. Structural basis for imipenem inhibition of class C β-lactamases. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 2002;46(12):3978\u0026ndash;3980. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.46.12.3978-3980.2002\u003c/span\u003e\u003cspan address=\"10.1128/aac.46.12.3978-3980.2002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNukaga M., et al. Hydrolysis of third-generation cephalosporins by class C β-lactamases: Structures of a transition state analog of cefotaxime in wild-type and extended spectrum enzymes. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e. 2004;279(10):9344\u0026ndash;9352. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M312356200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M312356200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUsher K.C., et al. Three-dimensional structure of AmpC β-lactamase from \u003cem\u003eEscherichia coli\u003c/em\u003e bound to a transition-state analogue: Possible implications for the oxyanion hypothesis and for inhibitor design. \u003cem\u003eBiochemistry\u003c/em\u003e. 1998;37(46):16082\u0026ndash;16092. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bi981210f\u003c/span\u003e\u003cspan address=\"10.1021/bi981210f\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWouters J., et al. Crystal structure of \u003cem\u003eEnterobacter cloacae\u003c/em\u003e 908R class C β-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue. \u003cem\u003eCellular and Molecular Life Sciences CMLS\u003c/em\u003e. 2003;60(8):1764\u0026ndash;1773. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00018-003-3189-2\u003c/span\u003e\u003cspan address=\"10.1007/s00018-003-3189-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGalleni M. and Fr\u0026egrave;re J.M. A survey of the kinetic parameters of class C β-lactamases. Penicillins. \u003cem\u003eBiochemical Journal\u003c/em\u003e. 1988;255(1):119\u0026ndash;122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1042/bj2550119\u003c/span\u003e\u003cspan address=\"10.1042/bj2550119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarnaud G., et al. Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an \u003cem\u003eEnterobacter cloacae\u003c/em\u003e clinical isolate. \u003cem\u003eFEMS Microbiology Letters\u003c/em\u003e. 2001;195(2):185\u0026ndash;190. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0378-1097(01)00010-6\u003c/span\u003e\u003cspan address=\"10.1016/S0378-1097(01)00010-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNukaga M., et al. Molecular evolution of a class C β-lactamase extending its substrate specificity. \u003cem\u003eJournal of Biological Chemistry\u003c/em\u003e. 1995;270(11):5729\u0026ndash;5735. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.270.11.5729\u003c/span\u003e\u003cspan address=\"10.1074/jbc.270.11.5729\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePhilippon A., et al. Class C β-lactamases: Molecular characteristics. \u003cem\u003eClinical Microbiology Reviews\u003c/em\u003e. 2022;35(3):e00150-21. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/cmr.00150-21\u003c/span\u003e\u003cspan address=\"10.1128/cmr.00150-21\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHamrick J.C., et al. VNRX-5133 (Taniborbactam), a broad-spectrum inhibitor of serine- and metallo-β-lactamases, restores activity of cefepime in \u003cem\u003eEnterobacterales\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 2020;64(3):\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/aac.01963-19\u003c/span\u003e\u003cspan address=\"10.1128/aac.01963-19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. https://doi.org/10.1128/aac.01963-19.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKarlowsky J.A., et al. \u003cem\u003eIn vitro\u003c/em\u003e activity of cefepime-taniborbactam and comparators against clinical isolates of Gram-negative bacilli from 2018 to 2020: results from the Global Evaluation of Antimicrobial Resistance via Surveillance (GEARS) program. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 2023;67(1):e01281-22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.01281-22\u003c/span\u003e\u003cspan address=\"10.1128/aac.01281-22\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhanel G.G., et al. Cefepime\u0026ndash;taniborbactam: A novel cephalosporin/β-lactamase inhibitor combination. \u003cem\u003eDrugs\u003c/em\u003e. 2024;84(10):1219\u0026ndash;1250. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40265-024-02082-9\u003c/span\u003e\u003cspan address=\"10.1007/s40265-024-02082-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLee H.H., et al. Bacterial charity work leads to population-wide resistance. \u003cem\u003eNature\u003c/em\u003e. 2010;467(7311):82\u0026ndash;85. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature09354\u003c/span\u003e\u003cspan address=\"10.1038/nature09354\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBarcel\u0026oacute; I.M., et al. Filling knowledge gaps related to AmpC-dependent β-lactam resistance in \u003cem\u003eEnterobacter cloacae\u003c/em\u003e. \u003cem\u003eScientific Reports\u003c/em\u003e. 2024;14(1):189. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-023-50685-1\u003c/span\u003e\u003cspan address=\"10.1038/s41598-023-50685-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBabouee Flury B., et al. The differential importance of mutations within AmpD in cephalosporin resistance of \u003cem\u003eEnterobacter aerogenes\u003c/em\u003e and \u003cem\u003eEnterobacter cloacae\u003c/em\u003e. \u003cem\u003eInternational Journal of Antimicrobial Agents\u003c/em\u003e. 2016;48(5):555\u0026ndash;558. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijantimicag.2016.07.021\u003c/span\u003e\u003cspan address=\"10.1016/j.ijantimicag.2016.07.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKopp U., et al. Sequences of wild-type and mutant \u003cem\u003eampD\u003c/em\u003e genes of C\u003cem\u003eitrobacter freundii\u003c/em\u003e and \u003cem\u003eEnterobacter cloacae\u003c/em\u003e. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 1993;37(2):224\u0026ndash;228. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.37.2.224\u003c/span\u003e\u003cspan address=\"10.1128/aac.37.2.224\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLindquist S., Lindberg F., and Normark S. Binding of the \u003cem\u003eCitrobacter freundii\u003c/em\u003e AmpR regulator to a single DNA site provides both autoregulation and activation of the inducible \u003cem\u003eampC\u003c/em\u003e β-lactamase gene. \u003cem\u003eJournal of Bacteriology\u003c/em\u003e. 1989;171(7):3746\u0026ndash;3753. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/jb.171.7.3746-3753.1989\u003c/span\u003e\u003cspan address=\"10.1128/jb.171.7.3746-3753.1989\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCai W., Lu M., and Dai W. Novel antibiotic susceptibility of an RNA polymerase α-subunit mutant in \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. \u003cem\u003eJournal of Antimicrobial Chemotherapy\u003c/em\u003e. 2023;78(9):2162\u0026ndash;2169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jac/dkad207\u003c/span\u003e\u003cspan address=\"10.1093/jac/dkad207\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGross R., et al. β-lactamase dependent and independent evolutionary paths to high-level ampicillin resistance. \u003cem\u003eNature Communications\u003c/em\u003e. 2024;15(1):5383. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-024-49621-2\u003c/span\u003e\u003cspan address=\"10.1038/s41467-024-49621-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShields R.K., et al. Clinical evolution of AmpC-mediated ceftazidime-avibactam and cefiderocol resistance in \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex following exposure to cefepime. \u003cem\u003eClinical Infectious Diseases\u003c/em\u003e. 2020;71(10):2713\u0026ndash;2716. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/cid/ciaa355\u003c/span\u003e\u003cspan address=\"10.1093/cid/ciaa355\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNordmann P., et al. Mechanisms of reduced susceptibility to cefiderocol among isolates from the CREDIBLE-CR and APEKS-NP clinical trials. \u003cem\u003eMicrobial Drug Resistance\u003c/em\u003e. 2022;28(4):398\u0026ndash;407. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/mdr.2021.0180\u003c/span\u003e\u003cspan address=\"10.1089/mdr.2021.0180\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWunderink R.G., et al. Cefiderocol versus high-dose, extended-infusion meropenem for the treatment of Gram-negative nosocomial pneumonia (APEKS-NP): a randomised, double-blind, phase 3, non-inferiority trial. \u003cem\u003eThe Lancet Infectious Diseases\u003c/em\u003e. 2021;21(2):213\u0026ndash;225. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1473-3099(20)30731-3\u003c/span\u003e\u003cspan address=\"10.1016/S1473-3099(20)30731-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSievers F., et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. \u003cem\u003eMolecular Systems Biology\u003c/em\u003e. 2011;7(1):539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/msb.2011.75\u003c/span\u003e\u003cspan address=\"10.1038/msb.2011.75\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eClark K., et al. GenBank. \u003cem\u003eNucleic Acids Research\u003c/em\u003e. 2015;44(D1):D67\u0026ndash;D72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkv1276\u003c/span\u003e\u003cspan address=\"10.1093/nar/gkv1276\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHanna S., et al. Exploring mutational possibilities of KPC variants to reach high level resistance to cefiderocol. \u003cem\u003eScientific Reports\u003c/em\u003e. 2025;15(1):31312. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-025-17044-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-025-17044-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKawai A., et al. Structural insights into the molecular mechanism of high-level ceftazidime\u0026ndash;avibactam resistance conferred by CMY-185. \u003cem\u003emBio\u003c/em\u003e. 2024;15(2):e02874-23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/mbio.02874-23\u003c/span\u003e\u003cspan address=\"10.1128/mbio.02874-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eUehara T., et al. Spectrum of cefepime-taniborbactam coverage against 190 β-lactamases defined in engineered isogenic \u003cem\u003eEscherichia coli\u003c/em\u003e strains. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 2025;69(5):e01699-24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.01699-24\u003c/span\u003e\u003cspan address=\"10.1128/aac.01699-24\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChikunova A., et al. Conserved proline residues prevent dimerization and aggregation in the β-lactamase BlaC. \u003cem\u003eProtein Science\u003c/em\u003e. 2024;33(4):e4972. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pro.4972\u003c/span\u003e\u003cspan address=\"10.1002/pro.4972\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMarciano D.C., et al. Genetic and structural characterization of an L201P global suppressor substitution in TEM-1 β-lactamase. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e. 2008;384(1):151\u0026ndash;164. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jmb.2008.09.009\u003c/span\u003e\u003cspan address=\"10.1016/j.jmb.2008.09.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMeneksedag D., et al. Communication between the active site and the allosteric site in class A β-lactamases. \u003cem\u003eComputational Biology and Chemistry\u003c/em\u003e. 2013;43:1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.compbiolchem.2012.12.002\u003c/span\u003e\u003cspan address=\"10.1016/j.compbiolchem.2012.12.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMammeri H., Poirel L., and Nordmann P. Extension of the hydrolysis spectrum of AmpC β-lactamase of \u003cem\u003eEscherichia coli\u003c/em\u003e due to amino acid insertion in the H-10 helix. \u003cem\u003eJournal of Antimicrobial Chemotherapy\u003c/em\u003e. 2008;61(4):971. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jac/dkn052\u003c/span\u003e\u003cspan address=\"10.1093/jac/dkn052\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNaville M., et al. ARNold: A web tool for the prediction of Rho-independent transcription terminators. \u003cem\u003eRNA Biology\u003c/em\u003e. 2011;8(1):11\u0026ndash;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4161/rna.8.1.13346\u003c/span\u003e\u003cspan address=\"10.4161/rna.8.1.13346\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrjibelski A., et al. Using SPAdes \u003cem\u003ede novo\u003c/em\u003e assembler. \u003cem\u003eCurrent Protocols in Bioinformatics\u003c/em\u003e. 2020;70(1):e102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/cpbi.102\u003c/span\u003e\u003cspan address=\"10.1002/cpbi.102\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVasimuddin M., et al. \u003cem\u003eEfficient architecture-aware acceleration of BWA-MEM for multicore systems\u003c/em\u003e. in \u003cem\u003e2019 IEEE International Parallel and Distributed Processing Symposium (IPDPS)\u003c/em\u003e. 2019.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWick R.R. and Holt K.E. Polypolish: Short-read polishing of long-read bacterial genome assemblies. \u003cem\u003ePLOS Computational Biology\u003c/em\u003e. 2022;18(1):e1009802. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pcbi.1009802\u003c/span\u003e\u003cspan address=\"10.1371/journal.pcbi.1009802\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWick R.R., et al. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. \u003cem\u003ePLOS Computational Biology\u003c/em\u003e. 2017;13(6):e1005595. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pcbi.1005595\u003c/span\u003e\u003cspan address=\"10.1371/journal.pcbi.1005595\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVaser R., et al. Fast and accurate de novo genome assembly from long uncorrected reads. \u003cem\u003eGenome Research\u003c/em\u003e. 2017;27(5):737\u0026ndash;746. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gr.214270.116\u003c/span\u003e\u003cspan address=\"10.1101/gr.214270.116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eParks D.H., et al. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. \u003cem\u003eGenome Research\u003c/em\u003e. 2015;25(7):1043\u0026ndash;1055. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/gr.186072.114\u003c/span\u003e\u003cspan address=\"10.1101/gr.186072.114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSchwengers O., et al. Bakta: Rapid and standardized annotation of bacterial genomes via alignment-free sequence identification. \u003cem\u003eMicrobial Genomics\u003c/em\u003e. 2021;7(11) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1099/mgen.0.000685\u003c/span\u003e\u003cspan address=\"10.1099/mgen.0.000685\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLam M.M.C., et al. A genomic surveillance framework and genotyping tool for Klebsiella pneumoniae and its related species complex. \u003cem\u003eNature Communications\u003c/em\u003e. 2021;12(1):4188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-021-24448-3\u003c/span\u003e\u003cspan address=\"10.1038/s41467-021-24448-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMiyoshi-Akiyama T., et al. Multilocus sequence typing (MLST) for characterization of \u003cem\u003eEnterobacter cloacae\u003c/em\u003e. \u003cem\u003ePLOS ONE\u003c/em\u003e. 2013;8(6):e66358. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0066358\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0066358\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDeatherage D.E. and Barrick J.E. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. \u003cem\u003eEngineering and analyzing multicellular systems: Methods and protocols\u003c/em\u003e. 2014:165\u0026ndash;188. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4939-0554-6_12\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-0554-6_12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eO'Callaghan C.H., et al. Novel method for detection of β-lactamases by using a chromogenic cephalosporin substrate. \u003cem\u003eAntimicrobial Agents and Chemotherapy\u003c/em\u003e. 1972;1(4):283\u0026ndash;288. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/aac.1.4.283\u003c/span\u003e\u003cspan address=\"10.1128/aac.1.4.283\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7980433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7980433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntimicrobial resistance poses a significant global health threat. Experimental evolution studies are crucial in understanding resistance mechanisms and thereby informing strategies to preserve antibiotic efficacy. We developed a novel continuous experimental evolution system enabling uninterrupted medium exchange with a rising antibiotic gradient, using standard laboratory equipment. We applied this system to three \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex strains isolated from urinary tract infections in Germany between 1990 and 1992, which therefore had no prior exposure to cefepime, a fourth-generation cephalosporin approved in Germany in 2004.\u003c/p\u003e\u003cp\u003eAfter four days of exposure to a cefepime gradient, resistant mutants emerged in all three strains. Notably, one mutant exhibited cross-resistance to the novel antibiotics cefiderocol and ceftazidime-avibactam, due to a single missense mutation in the β-lactamase gene \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eMIR\u0026minus;11\u003c/sub\u003e. Our study demonstrates the effectiveness of this novel approach for investigating AMR development and cross-resistance mechanisms, as well as the need to understand the genetic underpinnings of AMR.\u003c/p\u003e","manuscriptTitle":"Novel continuous experimental evolution methodology uncovers rapid resistance development and cross-resistance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 07:45:24","doi":"10.21203/rs.3.rs-7980433/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-18T16:29:05+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T21:42:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"285488826688701525460159813461537742285","date":"2025-12-10T18:36:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T16:24:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187041438411859286752811554633155856084","date":"2025-12-04T14:11:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-02T14:05:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-09T13:33:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-07T17:14:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Antimicrobials and Resistance","date":"2025-10-29T13:28:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-antimicrobials-and-resistance","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjamar","sideBox":"Learn more about [npj Antimicrobials and Resistance](http://www.nature.com/npjamar/)","snPcode":"44259","submissionUrl":"https://submission.springernature.com/new-submission/44259/3","title":"npj Antimicrobials and Resistance","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b3fc1d39-12a8-4593-9ef2-9b82861870a5","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58968454,"name":"Health sciences/Diseases"},{"id":58968455,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-02-12T13:54:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 07:45:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7980433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7980433","identity":"rs-7980433","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}