Resusceptibility to Ceftazidime-Avibactam in Tigecycline-Exposed NDM-Producing CRECC

Resusceptibility to Ceftazidime-Avibactam in Tigecycline-Exposed NDM-Producing CRECC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Resusceptibility to Ceftazidime-Avibactam in Tigecycline-Exposed NDM-Producing CRECC Luyao Tian, Jiming Wu, Youtao Liang, Xushan Liang, Jin Wang, Shijian Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8784943/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Purpose Carbapenem-resistant Enterobacter cloacae complex (CRECC) has become an increasingly important pathogen in healthcare-associated infections, with limited treatment options and a high mortality rate. As reports of antimicrobial resistance continue to rise, tigecycline (TGC) and ceftazidime-avibactam (CZA) have emerged as the last-line therapies for CRECC infections. The aim of this study was to investigate collateral sensitivity to ceftazidime-avibactam following the acquisition of tigecycline resistance in NDM-producing CRECC, and to elucidate the underlying molecular mechanisms, thereby providing a theoretical basis for clinical combination therapy. Methods Antimicrobial susceptibility profiles were determined using the broth microdilution method, and changes in colony morphology were analyzed. Transcriptomic sequencing was performed to characterize global gene expression alterations associated with antimicrobial resistance, and an in vivo Galleria mellonella infection model was used to assess the virulence of the mutant strains. Results Both drug-resistant mutants displayed a stable mucoid phenotype with marked collateral susceptibility to CZA, with minimum inhibitory concentrations decreasing from greater than 128 mg/L to 0.5–1 mg/L. Compared with the parental strains, these mutants showed thickened cell surface structures, impaired growth, reduced serum tolerance, and significantly attenuated virulence in the Galleria mellonella infection model. Transcriptomic analysis indicated increased extracellular polysaccharide production, impaired lipid A modification associated with reduced phoQ expression, and markedly decreased expression of metallo- β -lactamase-related genes, including NDM and CTX-M. Conclusion We hypothesize that the susceptibility of metallo- β -lactamase-producing strains to CZA represents an adaptive survival modification, which is achieved by reducing metallo- β -lactamase expression and altering bacterial metabolism at the cost of impaired bacterial growth and pathogenicity. This trade-off between antimicrobial resistance and bacterial fitness offers novel insights into the development of optimized therapeutic strategies for CRECC infections. Enterobacter cloacae complex NDM ceftazidime-avibactam Tigecycline collateral sensitivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Carbapenem-resistant Enterobacterales (CRE) represent one of the most critical threats to global public health, with infections caused by these pathogens associated with high mortality rates and an extremely limited armamentarium of effective therapeutic options[ 1 ]. the Enterobacter cloacae complex (ECC) has attracted considerable attention due to its inherent chromosomally mediated resistance background, rapid adaptive capacity to multiple classes of antimicrobial agents, and persistent transmission in intensive care units (ICUs)[ 2 ]. Such a multidrug-resistant (MDR) phenotype typically arises from the accumulation of resistance mutations or the horizontal acquisition of resistance genes, and is particularly prevalent in antibiotic combinations with distinct mechanisms of action[ 3 ]. The global dissemination of metallo- β -lactamases (MBLs), most notably New Delhi metallo- β -lactamase enzymes, has further amplified ECC resistance to carbapenems and most β -lactam antibiotics, substantially narrowing the spectrum of clinically viable treatment options for MDR-ECC infections[ 4 , 5 ]. Accordingly, the development of effective and optimized therapeutic strategies targeting clinical NDM-producing ECC strains has become an urgent priority in the field of anti-infective medicine. As a glycylcycline antibiotic, tigecycline(TGC) blocks protein synthesis by inhibiting the 30S ribosomal subunit and is recognized as one of the "last-line agents" for infections caused by MDR Gram-negative bacteria[ 6 ]. However, the widespread clinical use of TGC has driven the rapid emergence of resistance across Enterobacteriaceae species, with overexpression of efflux pump systems and aberrant activation of global transcriptional regulators ( ramA ) identified as key molecular mechanisms underlying TGC resistance[ 7 ]. CZA is a novel β -lactam/ β -lactamase inhibitor combination that demonstrates potent activity against Enterobacterales producing KPC, AmpC, and OXA-48 enzymes. Owing to its reliable in vitro and clinical efficacy, it has been established as a first-line therapeutic option for the treatment of infections caused by CRE[ 8 ]. For strains producing metallo- β -lactamases[ 9 ], especially NDM-producing ECC isolates, CZA generally shows an inherently non-susceptible or clinically ineffective phenotype[ 4 ], which significantly limits its clinical utility. Clinical studies have demonstrated that combination strategies involving CZA plus colistin (COL), aztreonam, or other agents hold application potential in the treatment of MDR infections[ 10 , 11 ]. Therefore, optimizing the use of existing antibiotics for infections caused by NDM-producing ECC remains a major challenge in current clinical practice. In the face of the ongoing global escalation of antimicrobial resistance, optimizing the use of currently available antibiotics to delay or reverse the evolutionary emergence of resistance has become a central focus of infectious disease research. Collateral sensitivity (CS), an adaptive evolutionary phenomenon in bacteria, has garnered considerable attention. It refers to a scenario where bacteria exhibit increased susceptibility to one antibiotic while acquiring resistance to another[ 12 ] This phenomenon has been observed in a variety of pathogens and is considered to provide a theoretical basis for combination or sequential antimicrobial therapy strategies[ 13 ]. CS has been documented across a broad range of bacterial pathogens, but existing research has been largely limited to laboratory-adapted bacterial strains or CRE isolates that do not produce MBLs[ 14 ]. To date, there is a lack of systematic research on the occurrence, molecular mechanisms and clinical potential of CS in CRECC strains producing NDM.[ 15 ]. The activation of efflux pump systems, alterations in outer membrane permeability, abnormal synthesis of extracellular polysaccharides or capsules, and dynamic regulation of β -lactamase expression levels could all contribute to the complex interplay between resistance and sensitivity[ 16 , 17 ]. Elucidating these molecular mechanisms is therefore essential to understanding the biological basis of CS and its translational potential for clinical anti-infective therapy. In this study, metallo- β -lactamase-producing CZA-resistant strains were found to restore susceptibility to CZA following exposure to TGC. The aim of this study was to investigate CS to CZA following the acquisition of tigecycline resistance in NDM-producing CRECC, and to elucidate the underlying molecular mechanisms, thereby providing a theoretical basis for clinical combination therapy. Materials and Methods Isolation and identification of strains and antibiotic sensitivity detection A total of 38 CRECC strains were isolated from clinical samples at a teaching hospital. Six strains sensitive to TGC but resistant to CZA were selected.(detailed information in table S1 ). Bacterial identification was performed using the Vitek 2 system (bioMérieux, Marcy I’Etoile, France) and MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA). Tigecycline powder, ceftazidime powder, and avibactam powder were purchased from MCE (USA). Minimum inhibitory concentrations (MICs) for ceftazidime-avibactam and tigecycline were determined using broth microdilution (BMD). The susceptibility results for (TGC and CZA were interpreted according to the CLSI 2020 guidelines[ 18 ] and EUCAST[ 19 ], where TGC susceptibility is defined as ≤ 0.5 mg/L, resistance as > 0.5 mg/L, CZA susceptibility (S) is ≤ 8/4 mg/L, resistance (R) is ≥ 16/4 mg/L, the denominator 4 indicates that the concentration of avibactam is fixed at 4 mg/L. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. In vitro induction and selection of resistant mutants To induce TGC resistance, CRECC strains were cultured in LB broth at 37°C with shaking at 160 rpm overnight. The cultures were then inoculated into LB broth containing different concentrations of TGC (ranging from 0.25 to 16 mg/L), with the concentration doubling eachday. The cultures were incubated at 37°C with shaking. Resistance mutants from every daywere preserved in glycerol at -80°C for subsequent antimicrobial susceptibility testing. Antimicrobial susceptibility testing of each strain was performed using the broth microdilution method, we found that the resistant progenies of CRECC112 and CRECC414 exhibited two distinct morphologies, with one morphological type of resistant mutant switching from CZA resistance to susceptibility. The latest TGC-resistant mutants of these two strains were designated as CRECC112-1, CRECC112-2, CRECC414-1, and CRECC414-2, where CRECC112-1 and CRECC414-1 were mucoid-type, and CRECC112-2 and CRECC414-2 were dry-type. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) analysis was performed by the Scientific Compass Service Center, following the procedure outlined below: Bacterial cultures with an OD₆₀₀ value of 0.5–0.8 were transferred into 1.5 mL centrifuge tubes, and the bacterial pellets were collected by centrifugation at 7000 rpm for 5 minutes. The pellets were then fixed in 2.5% glutaraldehyde/PBS solution (pH 7.4) at 4°C. After washing three times with PBS buffer, the mixture was resuspended and incubated at room temperature for 30 minutes, followed by overnight fixation at 4°C. Following fixation, the samples were dehydrated using a graded series of ethanol solutions (30%, 50%, 70%, 80%, 90%, and 100%), and then infiltrated with a mixture of anhydrous acetone and Spurr resin in a graded manner. The samples were finally embedded in Spurr resin and polymerized at 70°C for 12 hours. Ultrathin sections (70–90 nm) were prepared using the LEICA EM UC7 ultramicrotome and observed under a Tecnai G2 Spirit 120 kV transmission electron microscope, with images captured for analysis. Identification of gene mutations associated with TGC and CZA resistance. Genomic DNA was extracted from all parental strains and resistant mutants and used as the template for polymerase chain reaction (PCR). PCR primers targeting tigecycline resistance-associated genes ( acrA, acrB, oqxB, ramA ), ceftazidime-avibactam resistance-associated genes ( ompC, ompF , bla NDM , bla CTX−M ), capsular polysaccharide and extracellular polysaccharide synthesis genes ( wcaD, wcaE, wzb, wzc ), and biofilm formation-related genes were used for amplification. The PCR products were verified by gel electrophoresis in 1.2% agarose gels in 0.5× TAE buffer. Positive amplicons were sequenced using the Sanger method, and the resulting sequences were compared with reference sequences in the GenBank database using the BLAST tool ( http://www.ncbi.nlm.nih.gov/blast/ ) to analyze gene mutations. Whole Genome Sequencing (WGS) Genomic DNA from two CRECC strains was extracted using the Wizard Genomic DNA Purification Kit and sequenced using the Illumina HiSeq 2000 (2×150 bp paired-end sequencing, Illumina Inc., San Diego, CA, USA) and MinION platform (Oxford Nanopore Technologies, Oxford, UK). The genome was assembled using Canu v.1.6 and corrected with Pilon v.1.22. Annotation was performed using PGAP and Glimmer 3.02 software ( http://www.cbcb.umd.edu/software/Glimmer/ ). PlasmidFinder ( https://cge.food.dtu.dk/services/PlasmidFinder/ ), CARD, and ResFinder ( https://cge.food.dtu.dk/services/resfinder/ ) were used to identify plasmid sequences, resistance genes, and virulence factors, while ISfinder ( https://www-is.bio-toul.fr/ ) was used to locate transposons and insertion sequences. Sequence alignment was performed using BLASTn, and visualizations were created using BRIG ( http://brig.sourceforge.net ) and Easyfig ( https://github.com/mjsull/Easyfig )[ 20 ]. The genome has been deposited in GenBank. Serum resistance test and bacterial growth curve The serum resistance assay was conducted by culturing the strains in LB broth containing 10% mixed human serum for 24 hours, following the method described in reference[ 21 ]. After overnight incubation of the strains in LB broth at 37°C with shaking at 160 rpm, the cultures were diluted 1:100 in LB broth containing 10% human serum. The optical density at 600 nm (OD 600 ) was measured every hour for 24 hours to generate a growth curve. To assess the fitness cost of TGC-resistant mutants compared to their parent strains, both the mutants and parent strains were inoculated into fresh LB broth without any antibiotics and incubated at 37°C with shaking at 160 rpm until the turbidity reached 0.5 McFarland units. The cultures were then diluted 1:100 into 30 mL of LB broth and incubated at 37°C with continuous shaking. The OD 600 was measured every hour. Each strain was tested in triplicate, and the average OD was calculated. Growth curves were plotted using GraphPad Prism 9.5 software, and intergroup differences were analyzed using a t-test, with a p ≤ 0.05 indicating statistical significance. virulence assay As previously described, after some modifications[ 22 ], the optimal infection dose for the 6 CRECC strains was determined prior to the Galleria mellonella survival assay to ensure that approximately 80% of the larvae would die within 3 days of infection[ 23 ]. The Galleria mellonella larvae were purchased from Chongqing YeMai Biotechnology Co., Ltd., with an average larval weight of 400 ± 50 mg. Overnight bacterial cultures of each strain were adjusted to a concentration of 1×10⁸ CFU/mL in sterile phosphate-buffered saline (PBS, pH 7.4). A 10 µL inoculum (containing 1×10⁶ CFU) was injected into the hemocoel of the larvae via the last left leg using a 50 µL Hamilton syringe (Hamilton, Shanghai, China). Infected larvae were incubated in Petri dishes at 37°C and observed for survival every 12 hours over a 4-day period. Larvae were considered dead if there was no response when gently touched with sterile metal forceps. Galleria mellonella larvae injected with 10 µL of sterile PBS were used as negative controls. Each group consisted of 10 larvae, and the experiment was repeated three independent times. Tigecycline-resistant mutant stability assay To investigate the stability of the MICs of TGC and CZA in resistant strains, four resistant mutants were each inoculated into antibiotic-free LB broth and incubated at 37°C with shaking at 160× g . Every 12 hours, the cultures were diluted 1:100 and passaged for 10 consecutive days (approximately 200 generations). The MICs of TGC and CZA were determined every two days using broth microdilution. The final MIC values of each strain for TGC and CZA were plotted as line graphs using GraphPad Prism 9.5 software to analyze the stability of resistance. RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) Bacterial cultures in the logarithmic growth phase were collected, centrifuged at 12,000×g for 10 minutes at 4°C, and the supernatant was discarded. Total RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies) with an A₂₆₀/A₂₈₀ ratio between 1.8 and 2.0. RNA integrity was evaluated by 1% agarose gel electrophoresis. Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara, Japan) according to the protocol, with DEPC-treated water as the negative control template. The relative expression levels of target genes were quantified using SYBR Green dye (MedChemExpress) on a CFX96 Real-Time PCR System (Bio-Rad). The rpoB gene was used as the internal reference gene, and relative gene expression was calculated using the 2 ⁻ΔΔCt method. RNA sequencing was conducted on the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) with 2 µg of total RNA to construct the RNA-seq transcriptome library[ 24 ], and raw image-to-sequence conversion was performed using the Illumina GA Pipeline (version 1.6). Statistical analysis was performed using GraphPad Prism 9.5 software. Comparisons between groups were made using the unpaired t-test with Welch's correction, with P < 0.05 considered statistically significant. Primer sequences used are provided in Table S2 . Time-kill assay To determine the bactericidal effects of TGC and CZA, a time-kill assay was conducted according to the established protocol[ 25 ]. Overnight cultures of the strains were adjusted to 0.5 McFarland units and added to cation-adjusted Mueller-Hinton broth (CA-MHB) to a final volume of 20 mL, with an initial bacterial concentration of 1×10⁶ CFU/mL in the flask. The experimental groups included a blank control (no antibiotics), TGC monotherapy (2 mg/L), CZA monotherapy (32/8 mg/L, or 1/16 MIC), and the TGC + CZA combination (TGC 2 mg/L + CZA 32/8 mg/L), all incubated at 37°C with shaking at 160 rpm for 24 hours. Samples were taken at 0, 2, 4, 8, 12, and 24 hours, and serial dilutions were made in sterile PBS before plating on Mueller-Hinton (MH) agar plates. After incubating at 37°C for 18–24 hours, colony-forming units (CFU) were counted. The experiment was repeated three times, and time-kill curves were generated using GraphPad Prism 9.5 software. Group differences were analyzed using a t-test, with P < 0.05 considered statistically significant. Plasmid conjugation experiment The plasmid conjugation experiment was modified based on the study by Gong Xue et al[ 26 ]. In this experiment, CRECC112-1 and CRECC414-1 strains were used as donor bacteria, and rifampin-resistant Escherichia coli EC600 was used as the recipient. The procedure is as follows: the LB broth cultures of the donor and recipient bacteria were mixed in a 1:3 ratio, and the mixture was spotted onto a sterile filter membrane. The membrane was then placed on an LB agar plate and incubated at 35°C for 24 hours. After incubation, the membrane was transferred onto Mueller-Hinton agar plates containing imipenem (2 mg/L) and rifampin (100 mg/L) to select for conjugants. The presence of conjugants was confirmed using the VITEK-2 COMPACT system and 16S rRNA gene sequencing[ 27 ]. The presence of resistance genes in the conjugants was further verified by PCR. Bioinformatics Processing and Analysis Bioinformatics analysis was performed on RNA-seq data generated using the Illumina platform. Differentially expressed genes (DEGs) between the tigecycline-treated and control groups were identified using the DESeq2 package ( http://bioconductor.org/packages/release/bioc/html/DESeq2.html ), with a selection criterion of |log₂FC| > 1 and a false discovery rate (FDR) < 0.05. Gene ontology (GO) functional enrichment analysis was performed using the Goatools tool ( https://github.com/tanghaibao/GOatools ) combined with Fisher's exact test, selecting GO terms with statistical significance (P < 0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted using KOBAS 2.0 software ( http://kobas.cbi.pku.edu.cn ), with Fisher's exact test identifying significantly enriched pathways (P < 0.05). Multiple testing correction was performed using the Benjamini-Hochberg (BH) method to reduce type I errors. Statistical significance levels were indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Results Characteristics of CRECC clinical isolates A total of six tigecycline-sensitive, ceftazidime-avibactam-resistant CRECC strains were selected from clinical isolates in Table S1 . The results showed that after 7 days of tigecycline exposure, two strains (CRECC112 and CRECC414) developed two phenotypically distinct tigecycline-resistant mutant strains, named CRECC112-1, CRECC112-2, and CRECC414-1, CRECC414-2, respectively. Among these, CRECC112-1 and CRECC414-1 exhibited mucoid colony morphology, while CRECC112-2 and CRECC414-2 exhibited dry colony morphology, which was consistent with that of the parent strains in Fig. 1 b. Further antibiotic susceptibility testing on these two parental strains and their resistant mutants revealed that all strains were resistant to most of the tested antibiotics in Table 1 . Notably, the mucoid-resistant mutants (CRECC112-1, CRECC414-1) showed a phenotypic change from ceftazidime-avibactam resistance to susceptibility. Whole-genome sequencing revealed that CRECC112 was identified as Enterobacter hormaechei , and CRECC414 was identified as Enterobacter kobei [ 28 ]. Resistance gene detection showed that CRECC112 carried three β-lactamase genes: bla NDM−1 , bla TEM , and bla CTX−M ; CRECC414 also carried three β -lactamase genes: bla NDM−1 , bla CTX−M , and bla SHV−12 . Table 1 Antimicrobial susceptibility profiles of two CRECC strains harboring carbapenemase genes Antibiotics b Isolates TGC COL MEM IPM CAZ CZA AK TZP CZX Breakpoints(S-R)MIC(mg/L) a 112 0.5 S 64 R 16 R 16 R > 128 R > 128 R 64 R 64 R 64 R 112-1 32 R 16 R > 16 R > 128 R 0.5 S 32 R 128 R 64 R 112-2 32 R 16 R > 16 R > 128 R > 128 R > 64 R 128 R 64 R 414 0.5 S 64 R 16 R 16 R > 128 R > 128 R 64 R 64 R 64 R 414-1 32 R 16 R > 16 R > 128 R 1 S 32 R 128 R 64 R 414-2 32 R 16 R > 16 R > 128 R > 128 R 32 R 128 R 64 R a S-R represents the susceptible (S) breakpoint to the resistant (R); b TGC, tigecycline; COL, Colistin; MEM, meropenem; IPM, imipenem; CAZ, ceftazidime; CZA, ceftazidime-avibactam; AK, Amikacin; TZP, piperacillin/tazobactam; CZX, cefepime;. Susceptibility breakpoints for TGC against CRECC were interpreted according to the EUCAST guidelines, while those for the remaining antimicrobial agents were based on the CLSI 2020 criteria Changes in resistance after tigecycline induction Antimicrobial susceptibility testing of the induced resistant mutant strains was performed using the microdilution method in Fig. 1 b. The results showed that the mucoid mutants (CRECC112-1, CRECC414-1) exhibited a significant reduction in the minimum inhibitory concentration (MIC) of CZA from > 128 µg/mL in the parent strains to 0.5–1 µg/mL, whereas the dry-type mutants (CRECC112-2, CRECC414-2) showed no significant change in MIC, remaining at > 128 µg/mL. These findings indicate that tigecycline-induced mucoid-resistant mutants demonstrated a significant restoration of CZA sensitivity, and this phenotype was associated with the colony morphology. To further validate these results, Kirby-Bauer (K-B) disk diffusion tests were performed, which revealed that the inhibition zone diameters of CRECC112-1 and CRECC414-1 against CZA were > 21 mm, further confirming the presence of collateral sensitivity in Fig. 1 c. To investigate the phenotypic characteristics of the different colony morphology mutants, TEM was used to examine all six strains (two parental strains and four mutants). The results showed that CRECC112-1 and CRECC414-1 had a thick capsule structure surrounding the bacterial cell, while both the parent strains and the dry-type mutants (CRECC112-2, CRECC414-2) lacked any capsule structure in Fig. 1 d. Mutation and expression of drug-resistance-related genes Sequencing was performed on TGC resistance-associated genes, CZA resistance-associated genes and regulatory factors, efflux pump genes, capsule polysaccharide and extracellular polysaccharide synthesis genes, as well as biofilm formation-related genes to identify the molecular determinants of sensitivity and resistance. In both the parent strains and the TGC-resistant mutants, mutations in CZA resistance-related genes ( ompC , bla NDM , bla CTX−M ), efflux pump-related genes ( acrA, acrB, oqxB ), capsule polysaccharide and extracellular polysaccharide synthesis genes (wzb, wzc, wcaD , wcaE ), and biofilm formation-related genes were compared. The results showed that, except for the ramA gene in CRECC414-1, which underwent a L53R missense mutation, and multiple amino acid substitutions in the lpxA gene in Fig S3 , no mutations were detected in the aforementioned genes in the other strains in Table 2 Table 2 Gene mutations associated with TGC and CZA resistance in two clinically isolated strains and their corresponding resistant derivatives Isoles grnes acrB ramA wcaD wcaE wzB wzC oqxB ompC bla NDM bla CTX−M lpxA lpxC 112 WT WT WT WT WT WT WT WT WT WT WT WT 112-1 WT WT WT WT WT WT WT WT WT WT WT WT 414 WT WT WT WT WT WT WT WT WT WT WT WT 414-1 WT + WT WT WT WT WT WT WT WT + WT +: exist with amino acid substitution,WT: indicates that there is no mutation of the corresponding gene in all the strains in the study Fitness Cost and Stability Analysis To determine whether TGC resistance mutations incur a fitness cost and assess the stability of such resistance, four TGC-resistant mutant strains were serially passaged in antibiotic-free medium, and changes in the MICs of TGC and CZA were monitored. After 10 days of serial passage, the TGC MICs of all four mutants decreased from initial values of 32 µg/mL and 16 µg/mL to 8 µg/mL and 4 µg/mL in Fig. 2a , indicating that TGC resistance in these mutants is unstable relative to their parental strains. For CZA, the MICs of CRECC112-2 and CRECC414-2 remained > 128 µg/mL during passage, whereas the MICs of CRECC112-1 and CRECC414-1 increased from 0.25 µg/mL to 4 µg/mL in Fig. 2a , suggesting that the CZA susceptibility of mucoid mutants is gradually lost, while dry-type mutants maintain high-level resistance.Correlation analysis of colony morphology and growth kinetics revealed that the growth rates of mucoid strains (CRECC112-1 and CRECC414-1) were significantly lower than those of the parental and dry-type strains in Fig. 2b . The serum resistance assay showed that after incubation for 24 hours in LB medium supplemented with 10% human serum, the two mucoid strains exhibited almost no growth within the first 10 hours after inoculation compared with the dry-type strain in Fig. 2c . Furthermore, in the G mellonella infection model, larval mortality rates were comparable between parental and dry-type strains (survival < 50%), whereas infection with mucoid resistant strains resulted in significantly reduced mortality, indicating attenuated virulence in Fig. 2d . In vitro time-kill curve The time-kill assay results for TGC and CZA, both alone and in combination, against two parental strains (CRECC112 and CRECC414) showed the following: In the CZA monotherapy group (32/8 mg/L), bacterial growth rapidly increased within 2 hours, reaching levels comparable to the control group after 12 hours. The TGC monotherapy group (2 mg/L) inhibited bacterial growth for the first 2 hours, but bacteria rapidly regrew thereafter, maintaining a concentration of 5–6 log 10 CFU/mL by 24 hours. In contrast, the combination of TGC and CZA exhibited a synergistic bactericidal effect against the CRECC strains, with a sustained decline in bacterial concentration over the 24-hour period (Fig. 3 ). Global Transcriptomic Variations in Strains Induced by Tigecycline were analyzed via RNA sequencing Given the significant changes in the MIC and colony morphology of the CRECC112 and CRECC414 strains following tigecycline induction, as well as their ability to survive under high tigecycline pressure, it is hypothesized that transcriptional adaptation may have occurred. To investigate this, transcriptomic sequencing was employed to analyze the genome-wide expression changes induced by tigecycline. The Venn diagram illustrates the overlap and unique differentially expressed genes (DEGs) across four samples (CRECC112, CRECC112-1, CRECC414, CRECC414-1) in Fig. 4 a. Volcano plot analysis revealed significant transcriptional alterations in both strains following tigecycline treatment in Fig. 4 b. Functional enrichment analysis indicated that the DEGs in CRECC112-1, compared to CRECC112, were mainly involved in pyruvate metabolism, glycolysis/gluconeogenesis, and phosphotransferase systems. In contrast, the DEGs in CRECC414-1 relative to CRECC414 were primarily associated with the ABC transporter system and two-component regulatory systems. Both strains shared DEGs that participated in flagellar assembly, pyruvate metabolism, and nitrogen metabolism in Fig. 4 c. GO functional annotation showed that the DEGs in CRECC112 were most enriched in metabolic processes, compound binding, and small molecule binding, with a higher number of upregulated genes than downregulated genes. In CRECC414, the DEGs were most enriched in metabolic processes and small molecule binding, with a balanced number of upregulated and downregulated genes, although some GO categories were dominated by downregulated genes in Fig. 4 d. Transcriptomic changes associated with resistance to two antibiotics In the mucoid TGC-resistant mutants CRECC112-1 and CRECC414-1, whole-genome transcriptomic analysis revealed significant and functionally relevant changes compared with their parental strains(Fig. 5 ). Genes associated with efflux pumps ( acrA , acrB , oqxB ), the two-component regulatory system ( phoQ ), and outer membrane porins ( ompC , ompF ) were consistently downregulated in both mutants. Genes linked to CZA resistance, including ompC and bla NDM , were also broadly downregulated, with bla NDM expression reduced by 5-20-fold. Expression of bla CTX−M decreased in CRECC112-1, whereas in CRECC414-1 it showed an increase that did not reach statistical significance. The regulatory factor ramA displayed strain-specific expression: it was upregulated approximately 5-fold in CRECC112-1 but showed the opposite trend in CRECC414-1, reflecting heterogeneity in resistance regulatory pathways among strains. Notably, genes directly involved in biofilm formation ( wcaD , wcaE ) and in cell surface polysaccharide synthesis ( wzb , wzc ) were markedly upregulated in both mutants, with fold changes of 20–25 and 8–25. Plasmid analysis of bla NDM−1 harboring strains Genome sequencing revealed that the bla NDM−1 gene in CRECC112 and CRECC414 was located on an IncFII-type and an IncX3-type plasmid, designated pNDM112 (85,718 bp) and pNDM414 (74,194 bp), respectively. Comparative analysis of these two bla NDM−1 -harboring plasmids showed that the coding regions of bla NDM shared nearly 100% homology, with both carrying conserved promoter and signal peptide sequences, indicating that bla NDM is a highly conserved functional element with no significant variation in its core resistance sequence.Functional annotation demonstrated that pNDM112 harbored bla NDM (carbapenem resistance), armA (aminoglycoside resistance), conjugation-associated genes ( tra series), and replication-associated genes ( par series). In contrast, pNDM414 contained bla NDM , bla SHV ( β -lactam resistance), virulence-associated genes ( vir series), replication/transfer-related genes ( par , tra ), and the insertion sequence IS 15. Further analysis showed that the bla NDM -armA cassette in pNDM112 was flanked by transposable elements forming a composite resistance unit, whereas bla NDM and bla SHV were tandemly arranged within a resistance island in pNDM414. Both structures exhibit typical features of mobilizable resistance modules, suggesting that such elements are key drivers of bla NDM dissemination across plasmids and strains in Fig. 6a . To investigate the regulatory mechanisms governing bla NDM expression, the relative transcription levels of the plasmid replicons carrying the gene were measured. Using rpoB as an internal reference, the replicon transcription levels in both mutant strains were lower than in the parental strains in Fig. 6b , indicating that reduced bla NDM expression in the drug-resistant mutants is accompanied by decreased transcriptional activity of the associated plasmid replicons. Figure 2 Stability Assay of Tigecycline-Resistant Mutants a : Changes in CZA MICs and TGC MICs. The x-axis represents the number of subculture days. b : Monitoring of the 24-hour growth kinetics of tigecycline-susceptible strains, ceftazidime-avibactam-resistant strains, and TGC-resistant strains. c : Serum resistance of different morphotypes. Differences in serum resistance were determined by comparing the growth curves in LB broth and LB broth supplemented with 10% pooled normal human serum. d : Survival curves of the Galleria mellonella infection model. Ten larvae per group were injected with 10 µL of the bacterial suspension at a concentration of 10⁶ CFU/mL, respectively Figure 6a Circular maps of the bla NDM-1 -harboring plasmids pNDM112 (85718 bp) and pNDM414 (74194 bp). From the inner to the outer rings: scale bar (kbp); GC skew (purple, negative values; green, positive values); GC content (black histograms); sequence alignment results (red/pink/light gray indicate alignment with “112-1” or “414,” whereas dark/light blue or gray indicate alignment with the alternative reference sequence, with darker colors representing higher sequence homology); the outermost ring denotes annotated functional genes, including bla NDM (carbapenem resistance), armA(aminoglycoside resistance; unique to pNDM112), bla SHV ( β -lactam resistance; unique to pNDM414), tra and par family genes, and the insertion sequence IS15. b Relative transcription levels of plasmid replicons carrying the bla NDM gene, normalized to rpoB as the reference gene. IncFHⅡ and IncX3 represent the replicon types of plasmids harbored by strains 112 and 414, respectively. All data were calculated using the 2 method and are presented as means ± standard deviation (SD). Discussion The global dissemination of CRECC has emerged as a major challenge for clinical anti-infective therapy[ 29 ]. The limited availability of effective therapeutic options has prompted sustained efforts to explore alternative treatment strategies. We identified and conducted an in-depth analysis of the resistance profiles of CRECC to TGC and CZA. By integrating analyses of antibiotic-associated phenotypic alterations, underlying molecular mechanisms, and potential synergistic antibacterial activity, our findings provide novel insights into the therapeutic potential of TGC-CZA-based strategies and offer both theoretical and practical implications for the clinical management of CRECC infections. Overactivation of efflux pump systems was identified as one of the key mechanisms underlying TGC resistance in the mutant strains CRECC112-1 and CRECC414-1 in this study. In Enterobacterales, the AcrAB-TolC efflux pump plays a central role in mediating TGC resistance[ 30 ]. Whole-genome sequencing combined with transcriptomic analyses indicated that mutations in the global regulator ramA may drive the overexpression of efflux pump-associated genes, including acrA , acrB , and tolC , thereby contributing to tigecycline resistance. As a member of the AraC/XylS family of global transcriptional regulators, ramA mutations-such as the L53R missense substitution identified in CRECC414-1 can markedly enhance transcriptional activation of acrA , acrB , and tolC , leading to increased synthesis and activity of the AcrAB-TolC efflux system. This pump actively exports tigecycline and other antimicrobial agents out of the bacterial cell, resulting in reduced intracellular drug accumulation, a mechanism consistent with previous reports on tigecycline resistance in Enterobacterales[ 31 ]. The TGC-resistant mutants exhibited a distinct mucoid phenotype that differed markedly in functional characteristics from both the parental strains and the dry-type mutants (CRECC112-2 and CRECC414-2). Notably, the mucoid variants demonstrated restored susceptibility to CZA, with MICs decreasing from > 128 mg/L to 0.5–1 mg/L. This pronounced reduction suggests that TGC-associated resistance mechanisms may be accompanied by a restoration of CZA susceptibility, reflecting an evolutionary trade-off consistent with the phenomenon of collateral sensitivity. Transcriptomic analysis revealed a significant downregulation of bla NDM expression (by approximately 5–20-fold) in TGC-resistant mutants, which may represent a key determinant underlying the restored CZA susceptibility. In particular, in the mucoid mutants CRECC112-1 and CRECC414-1, reduced bla NDM expression is likely to attenuate ceftazidime hydrolysis[ 32 ], thereby enabling avibactam to more effectively inhibit residual β -lactamase activity and restore CZA efficacy. In parallel, genes associated with extracellular polysaccharide (EPS) biosynthesis, including wcaD , wcaE , wzb , and wzc , were markedly upregulated in the mucoid mutants. The wcaD gene cluster is responsible for the synthesis of colanic acid, a major component of the EPS matrix in Enterobacterales , while wzb encodes a phosphatase and wzc encodes a tyrosine kinase that together regulate EPS polymerization and secretion. Enhanced expression of these genes is expected to promote EPS accumulation and surface thickening[ 33 ]. Such envelope remodeling may, at least in part, alter outer membrane permeability and influence CZA influx, although this hypothesis warrants further experimental validation. Transmission electron microscopy provided direct structural evidence supporting these transcriptomic findings, revealing a characteristic thick capsular layer surrounding the mucoid mutants, a feature absent in both the parental strains and dry-type mutants. This structural divergence represents a defining hallmark distinguishing the mucoid and dry phenotypes and provides an important morphological basis for the observed TGC resistance and CZA collateral sensitivity. Additionally, transcriptomic alterations were observed in genes related to lipid biosynthesis and envelope regulation, including lpxA and phoQ . The lpxA gene is involved in lipid A biosynthesis, and its alteration may affect outer membrane integrity and permeability[ 34 ]. The phoQ gene, as part of the PhoPQ two-component regulatory system, participates in lipid A modification and has been implicated in EPS regulation. Moreover, the wzb-wzc regulatory system may interact with other signaling pathways to fine-tune phenotype-associated gene expression, thereby stabilizing the mucoid phenotype and indirectly facilitating the restoration of CZA susceptibility. MBLs represented by bla NDM−1 are the core mediators of carbapenem resistance in CRECC[ 35 ], and their expression levels directly affect bacterial susceptibility to β -lactam antibiotics. In the present study, both parental strains CRECC112 and CRECC414 harbored the bla NDM−1 gene, which was also the key reason for their high resistance to CZA (MIC > 128 mg/L). However, following TGC exposure, mucoid mutants exhibited cross-susceptibility to CZA, with MIC values decreasing from 128 mg/L to 0.5-1 mg/L. This restoration of susceptibility was not driven by a single factor, but rather resulted from the close association and synergistic effects of bla NDM expression regulation, mucoid phenotypic changes, and efflux pump system modulation.In mucoid mutants, the expression level of bla NDM was significantly downregulated (5-50-fold reduction at the mRNA level), which directly attenuated its resistance-mediating effect. On the one hand, decreased bla NDM activity reduced the hydrolysis of ceftazidime, allowing avibactam to effectively protect ceftazidime from degradation by other β -lactamases, thereby restoring the inhibitory activity of CZA against bacteria[ 36 ]. On the other hand, the downregulation of bla NDM was accompanied by alterations in bacterial metabolic status, which may affect cell wall synthesis or outer membrane permeability, indirectly regulating susceptibility to CZA[ 37 ] .The thick envelope structure of mucoid mutants physically hindered the translocation of bla NDM to the periplasmic space, reducing its hydrolysis efficiency against ceftazidime and indirectly enhancing CZA susceptibility. Meanwhile, bacteria exhibited an obvious metabolic resource trade-off effect: the synthesis of a thick capsule consumes substantial energy and substances. To maintain basic growth and environmental adaptability, bacteria may actively downregulate the expression of non-essential resistance determinants such as bla NDM , thereby indirectly promoting the restoration of susceptibility to CZA.In TGC-resistant mutants, the expression of outer membrane protein genes ompC and ompF was significantly downregulated (20-50-fold). The porins encoded by these genes are the main channels for hydrophilic antibiotics such as ceftazidime to enter bacterial cells; reduced porin levels would normally inhibit ceftazidime influx and impair the efficacy of CZA[ 38 ]. However, the significant downregulation of bla NDM in mucoid mutants effectively offset the adverse effects caused by reduced porin expression, ultimately achieving the restoration of CZA susceptibility.Results of the resistance stability assay further confirmed the above associations. After 10 passages of mucoid mutants in medium without antibiotic selection pressure, the MIC of CZA increased from 0.125 µg/mL to 4 µg/mL, accompanied by a gradual attenuation of the mucoid phenotype and upregulation of bla NDM expression. This synergistic change trend not only clarified the close correlation among the mucoid phenotype, bla NDM expression regulation, and CZA susceptibility, but also intuitively reflected the evolutionary trade-off strategy of bacteria between drug resistance and survival adaptability. The acquisition of tigecycline resistance in the mucoid mutants was accompanied by a pr The CZA resensitization observed in this study was not attributable to the loss or inactivation of resistance determinants, but rather reflected a phenotypic reversal of resistance driven by transcriptional regulation. In the mucoid mutant strains selected following tigecycline exposure, the transcription levels of bla NDM and the associated plasmid replicons were markedly reduced, leading to decreased functional expression of carbapenemase. As a consequence, CZA exhibited restored in vitro inhibitory activity against these isolates. The acquisition of TGC resistance in mucoid mutant strains was accompanied by a pronounced attenuation of virulence, highlighting an evolutionary trade-off employed by bacteria in response to antibiotic pressure[ 39 ]. Serum resistance assays demonstrated that the mucoid variants exhibited almost no detectable growth within the first 12 h of incubation in LB broth supplemented with 10% human serum, suggesting that the thick capsular layer may impair bacterial survival in the host circulatory environment by disrupting interactions with serum complement components. Consistently, results from the G mellonella infection model showed that larvae infected with mucoid mutants experienced significantly lower mortality rates compared with those infected with the parental strains or dry-type mutants. This inverse relationship between resistance acquisition and virulence not only provides a mechanistic explanation for the limited stability of the collateral sensitivity phenotype, but also offers theoretical insights into the optimization of anti-infective treatment strategies targeting carbapenem-resistant Enterobacter cloacae complex. Conclusion In summary, this susceptibility phenomenon was not universally observed but was restricted to a subset of NDM-producing Enterobacter cloacae complex isolates. These findings indicate that antibiotic collateral sensitivity is highly strain dependent and may require specific genetic backgrounds or metabolic regulatory states to occur. Further validation in larger collections of clinical isolates is therefore necessary to determine its prevalence and key determinants. In addition, the regulatory mechanisms linking the mucoid phenotype to bla NDM expression remain incompletely understood and warrant comprehensive investigation through integrated transcriptomic, proteomic, and metabolomic analyses, combined with gene-editing approaches to identify the critical regulatory factors and signaling pathways involved. This study delineates the coordinated regulatory pathways underlying TGC and CZA resistance in CRECC. Overexpression of the AcrAB-TolC efflux system primarily mediates TGC resistance, while EPS synthesis, driven by wcaD, wcaE, wzb , and wzc , induces the mucoid phenotype. Concurrently, downregulation of bla NDM , together with phoQ -associated defects in lipid A modification, collectively restores CZA susceptibility. The interdependence between the mucoid phenotype and bla NDM expression reflects a coordinated evolutionary strategy in bacterial resistance, with the efflux pump pathway playing a central role in TGC resistance. These findings provide a mechanistic rationale for the potential clinical application of TGC-CZA combination therapy and offer a conceptual framework for the development of novel anti-resistance strategies targeting MBL expression and efflux systems. Future our investigations will focus on optimizing combination regimens and elucidating the molecular mechanisms of the underlying regulatory networks to better address the clinical challenges posed by CRECC infections. Declarations Competing interests The authors declare no competing interests. Funding This work was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0533106/2024ZD0533100), National Innovation Center for High-Performance Medical Devices (NMED2025KF-03-003), Chongqing public health key specialty (discipline) project, Chongqing Health Commission and Science and Technology Bureau (2023MSXM018), Yongchuan Natural Science Foundation (2025yc-cxfz10114). Author Contribution L T, J W, C L and XL Z designed and coordinated the the study. LY T and J W wrote the paper and participated in the whole experiment process. Y L, C L, Y C, and J W helped with the experimental process, J W , X S L and JM W provided the samples and the clinical data.J W analyzed and interpreted the data. All authors contributed to the article and approved the submitted version. Acknowledgement The authors thank all the patients whose data were used in the study Data Availability The datasets generated during and/or analyzed during the current study are available in this manuscript. References Tompkins K, van Duin D (2021) Treatment for carbapenem-resistant Enterobacterales infections: recent advances and future directions. 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University","correspondingAuthor":false,"prefix":"","firstName":"Chunjiang","middleName":"","lastName":"Li","suffix":""},{"id":600634607,"identity":"b8338b8f-ef3f-4c79-97c7-f8aca71c6576","order_by":11,"name":"Xiaoli Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACxgYQWSAhJ8/e2PjwA/FaDCyMDXsONxtLEG+XQUViw430NgEeYhQzTzv++DOPgQRj48yHbQwSDHZyug2EHDY7x8BwhoEEM7t0YtuDAoZkY7MDhLUwJHwwkGBjnJ3YbiDBcCBxG2Et6Q8OJBhI8DDcPNgGJInSkmDYALRFguEGI9FacowZgX4xMOxJBAayARF+MZydDgyxirr6+ezHHz78UGEnR1hLAwrXgIByEJAnQs0oGAWjYBSMdAAAFhtADAyivTQAAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Yongchuan Hospital of Chongqing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2026-02-04 10:23:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8784943/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8784943/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104383638,"identity":"7797b010-c79c-4b38-b89e-66f9f1ee32ea","added_by":"auto","created_at":"2026-03-11 08:12:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":134072,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e: Schematic diagram of the broth microdilution method \u003cstrong\u003eb\u003c/strong\u003e: Parental strains and two morphologically distinct tigecycline-induced resistant strains \u003cstrong\u003ec\u003c/strong\u003e: Susceptibility of two mucoid-resistant strains to CZA determined by the K-B method \u003cstrong\u003ed\u003c/strong\u003e: TEM images of parental strains and resistant strains with different morphotypes\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/e3fc6bd10c98fc0a3da461e0.jpg"},{"id":104383641,"identity":"30916c8f-76b6-43aa-89fd-5a5d9bcb410d","added_by":"auto","created_at":"2026-03-11 08:12:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148939,"visible":true,"origin":"","legend":"\u003cp\u003eStability Assay of Tigecycline-Resistant Mutants\u003cstrong\u003e a:\u003c/strong\u003e Changes in CZA MICs and TGC MICs. The x-axis represents the number of subculture days. \u003cstrong\u003eb\u003c/strong\u003e: Monitoring of the 24-hour growth kinetics of tigecycline-susceptible strains, ceftazidime-avibactam-resistant strains, and TGC-resistant strains.\u003cstrong\u003ec\u003c/strong\u003e: Serum resistance of different morphotypes. Differences in serum resistance were determined by comparing the growth curves in LB broth and LB broth supplemented with 10% pooled normal human serum.\u003cstrong\u003ed\u003c/strong\u003e: Survival curves of the \u003cem\u003eGalleria mellonella\u003c/em\u003e infection model. Ten larvae per group were injected with 10 μL of the bacterial suspension at a concentration of 10⁶ CFU/mL, respectively\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/5ce393a856c5995a16f7dd7a.jpg"},{"id":104405676,"identity":"77547ed8-334e-450e-a3dd-1ad387268d10","added_by":"auto","created_at":"2026-03-11 12:23:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":56919,"visible":true,"origin":"","legend":"\u003cp\u003eTime-Kill Curves of Two CRECC Strains: Synergistic Bactericidal Activity of TGC and CZA.Abbreviations: TGC, tigecycline; CZA, ceftazidime-avibactam\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/345d58260a94c1cf9b824384.jpg"},{"id":104405853,"identity":"2681f832-0a58-431f-938a-478ca9575737","added_by":"auto","created_at":"2026-03-11 12:23:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e:Circles in different colors represent the differentially expressed genes (DEGs) of each sample group. The values within the circles indicate the number of unique and shared expressed genes between two or three sample groups. The sum of all values inside a circle represents the total number of expressed genes in that group, while the overlapping regions of the circles denote the number of shared genes among the respective groups.\u003cstrong\u003eb\u003c/strong\u003e: Volcano plot analysis of RNA-Seq results for the strains.\u003cstrong\u003ec\u003c/strong\u003e: Bubble plot of KEGG pathway enrichment for the strains.\u003cstrong\u003ed\u003c/strong\u003e: GO enrichment analysis of upregulated and downregulated genes in terms of cellular components, molecular functions, biological processes, and their corresponding \u003cem\u003eP\u003c/em\u003e-values\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/fb7a13930e7d30649cdfb691.jpg"},{"id":104383639,"identity":"35ba92fd-ab9f-42bf-b6aa-28df0d47432e","added_by":"auto","created_at":"2026-03-11 08:12:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of genes associated with tigecycline and ceftazidime-avibactam resistance.Relative expression levels were normalized to the \u003cem\u003erpoB\u003c/em\u003e reference gene and calculated using the 2^\u003csup\u003e−ΔΔCt\u003c/sup\u003e method. Most intergroup differences were statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 and \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/7496dec30be810ae2230bdf6.png"},{"id":104383643,"identity":"f800d4a0-5d0f-497e-8413-e76ebf697d10","added_by":"auto","created_at":"2026-03-11 08:12:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":223469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eCircular maps of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e-harboring plasmids pNDM112 (85718 bp) and pNDM414 (74194 bp). From the inner to the outer rings: scale bar (kbp); GC skew (purple, negative values; green, positive values); GC content (black histograms); sequence alignment results (red/pink/light gray indicate alignment with “112-1” or “414,” whereas dark/light blue or gray indicate alignment with the alternative reference sequence, with darker colors representing higher sequence homology); the outermost ring denotes annotated functional genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (carbapenem resistance), armA(aminoglycoside resistance; unique to pNDM112), \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e (\u003cem\u003eβ\u003c/em\u003e-lactam resistance; unique to pNDM414), tra and par family genes, and the insertion sequence IS15. \u003cstrong\u003eb\u003c/strong\u003e Relative transcription levels of plasmid replicons carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e gene, normalized to rpoB as the reference gene. IncFHⅡ and IncX3 represent the replicon types of plasmids harbored by strains 112 and 414, respectively. All data were calculated using the 2 method and are presented as means ± standard deviation (SD).\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/0df80a19d35d88e8af122291.jpg"},{"id":104409531,"identity":"b1bc9113-72e9-42e8-80ed-b4c9e8f12500","added_by":"auto","created_at":"2026-03-11 12:45:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1936568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/02627bf7-8c6f-4c1c-bc29-e2688e0c0e2f.pdf"},{"id":104383645,"identity":"11f8332a-67d7-4b83-be2d-ebf40446f8aa","added_by":"auto","created_at":"2026-03-11 08:12:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":656363,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarymaterialTableandFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8784943/v1/0945f653108b0b86e9ffc1f5.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Resusceptibility to Ceftazidime-Avibactam in Tigecycline-Exposed NDM-Producing CRECC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCarbapenem-resistant \u003cem\u003eEnterobacterales\u003c/em\u003e (CRE) represent one of the most critical threats to global public health, with infections caused by these pathogens associated with high mortality rates and an extremely limited armamentarium of effective therapeutic options[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. the Enterobacter cloacae complex (ECC) has attracted considerable attention due to its inherent chromosomally mediated resistance background, rapid adaptive capacity to multiple classes of antimicrobial agents, and persistent transmission in intensive care units (ICUs)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Such a multidrug-resistant (MDR) phenotype typically arises from the accumulation of resistance mutations or the horizontal acquisition of resistance genes, and is particularly prevalent in antibiotic combinations with distinct mechanisms of action[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The global dissemination of metallo-\u003cem\u003eβ\u003c/em\u003e-lactamases (MBLs), most notably New Delhi metallo-\u003cem\u003eβ\u003c/em\u003e-lactamase enzymes, has further amplified ECC resistance to carbapenems and most \u003cem\u003eβ\u003c/em\u003e-lactam antibiotics, substantially narrowing the spectrum of clinically viable treatment options for MDR-ECC infections[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Accordingly, the development of effective and optimized therapeutic strategies targeting clinical NDM-producing ECC strains has become an urgent priority in the field of anti-infective medicine.\u003c/p\u003e \u003cp\u003eAs a glycylcycline antibiotic, tigecycline(TGC) blocks protein synthesis by inhibiting the 30S ribosomal subunit and is recognized as one of the \"last-line agents\" for infections caused by MDR Gram-negative bacteria[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, the widespread clinical use of TGC has driven the rapid emergence of resistance across Enterobacteriaceae species, with overexpression of efflux pump systems and aberrant activation of global transcriptional regulators (\u003cem\u003eramA\u003c/em\u003e) identified as key molecular mechanisms underlying TGC resistance[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CZA is a novel \u003cem\u003eβ\u003c/em\u003e-lactam/\u003cem\u003eβ\u003c/em\u003e-lactamase inhibitor combination that demonstrates potent activity against Enterobacterales producing KPC, AmpC, and OXA-48 enzymes. Owing to its reliable in vitro and clinical efficacy, it has been established as a first-line therapeutic option for the treatment of infections caused by CRE[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. For strains producing metallo-\u003cem\u003eβ\u003c/em\u003e-lactamases[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], especially NDM-producing ECC isolates, CZA generally shows an inherently non-susceptible or clinically ineffective phenotype[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which significantly limits its clinical utility. Clinical studies have demonstrated that combination strategies involving CZA plus colistin (COL), aztreonam, or other agents hold application potential in the treatment of MDR infections[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Therefore, optimizing the use of existing antibiotics for infections caused by NDM-producing ECC remains a major challenge in current clinical practice.\u003c/p\u003e \u003cp\u003eIn the face of the ongoing global escalation of antimicrobial resistance, optimizing the use of currently available antibiotics to delay or reverse the evolutionary emergence of resistance has become a central focus of infectious disease research. Collateral sensitivity (CS), an adaptive evolutionary phenomenon in bacteria, has garnered considerable attention. It refers to a scenario where bacteria exhibit increased susceptibility to one antibiotic while acquiring resistance to another[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] This phenomenon has been observed in a variety of pathogens and is considered to provide a theoretical basis for combination or sequential antimicrobial therapy strategies[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. CS has been documented across a broad range of bacterial pathogens, but existing research has been largely limited to laboratory-adapted bacterial strains or CRE isolates that do not produce MBLs[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To date, there is a lack of systematic research on the occurrence, molecular mechanisms and clinical potential of CS in CRECC strains producing NDM.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The activation of efflux pump systems, alterations in outer membrane permeability, abnormal synthesis of extracellular polysaccharides or capsules, and dynamic regulation of \u003cem\u003eβ\u003c/em\u003e-lactamase expression levels could all contribute to the complex interplay between resistance and sensitivity[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Elucidating these molecular mechanisms is therefore essential to understanding the biological basis of CS and its translational potential for clinical anti-infective therapy. In this study, metallo-\u003cem\u003eβ\u003c/em\u003e-lactamase-producing CZA-resistant strains were found to restore susceptibility to CZA following exposure to TGC. The aim of this study was to investigate CS to CZA following the acquisition of tigecycline resistance in NDM-producing CRECC, and to elucidate the underlying molecular mechanisms, thereby providing a theoretical basis for clinical combination therapy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and identification of strains and antibiotic sensitivity detection\u003c/h2\u003e \u003cp\u003eA total of 38 CRECC strains were isolated from clinical samples at a teaching hospital. Six strains sensitive to TGC but resistant to CZA were selected.(detailed information in \u003cb\u003etable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). Bacterial identification was performed using the Vitek 2 system (bioM\u0026eacute;rieux, Marcy I\u0026rsquo;Etoile, France) and MALDI-TOF mass spectrometer (Bruker, Billerica, MA, USA). Tigecycline powder, ceftazidime powder, and avibactam powder were purchased from MCE (USA). Minimum inhibitory concentrations (MICs) for ceftazidime-avibactam and tigecycline were determined using broth microdilution (BMD). The susceptibility results for (TGC and CZA were interpreted according to the CLSI 2020 guidelines[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and EUCAST[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], where TGC susceptibility is defined as \u0026le;\u0026thinsp;0.5 mg/L, resistance as \u0026gt;\u0026thinsp;0.5 mg/L, CZA susceptibility (S) is \u0026le;\u0026thinsp;8/4 mg/L, resistance (R) is \u0026ge;\u0026thinsp;16/4 mg/L, the denominator 4 indicates that the concentration of avibactam is fixed at 4 mg/L. \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853 were used as quality control strains.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIn vitro induction and selection of resistant mutants\u003c/h3\u003e\n\u003cp\u003eTo induce TGC resistance, CRECC strains were cultured in LB broth at 37\u0026deg;C with shaking at 160 rpm overnight. The cultures were then inoculated into LB broth containing different concentrations of TGC (ranging from 0.25 to 16 mg/L), with the concentration doubling eachday. The cultures were incubated at 37\u0026deg;C with shaking. Resistance mutants from every daywere preserved in glycerol at -80\u0026deg;C for subsequent antimicrobial susceptibility testing. Antimicrobial susceptibility testing of each strain was performed using the broth microdilution method, we found that the resistant progenies of CRECC112 and CRECC414 exhibited two distinct morphologies, with one morphological type of resistant mutant switching from CZA resistance to susceptibility. The latest TGC-resistant mutants of these two strains were designated as CRECC112-1, CRECC112-2, CRECC414-1, and CRECC414-2, where CRECC112-1 and CRECC414-1 were mucoid-type, and CRECC112-2 and CRECC414-2 were dry-type.\u003c/p\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM)\u003c/h3\u003e\n\u003cp\u003eTransmission electron microscopy (TEM) analysis was performed by the Scientific Compass Service Center, following the procedure outlined below: Bacterial cultures with an OD₆₀₀ value of 0.5\u0026ndash;0.8 were transferred into 1.5 mL centrifuge tubes, and the bacterial pellets were collected by centrifugation at 7000 rpm for 5 minutes. The pellets were then fixed in 2.5% glutaraldehyde/PBS solution (pH 7.4) at 4\u0026deg;C. After washing three times with PBS buffer, the mixture was resuspended and incubated at room temperature for 30 minutes, followed by overnight fixation at 4\u0026deg;C. Following fixation, the samples were dehydrated using a graded series of ethanol solutions (30%, 50%, 70%, 80%, 90%, and 100%), and then infiltrated with a mixture of anhydrous acetone and Spurr resin in a graded manner. The samples were finally embedded in Spurr resin and polymerized at 70\u0026deg;C for 12 hours. Ultrathin sections (70\u0026ndash;90 nm) were prepared using the LEICA EM UC7 ultramicrotome and observed under a Tecnai G2 Spirit 120 kV transmission electron microscope, with images captured for analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of gene mutations associated with TGC and CZA resistance.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGenomic DNA was extracted from all parental strains and resistant mutants and used as the template for polymerase chain reaction (PCR). PCR primers targeting tigecycline resistance-associated genes (\u003cem\u003eacrA, acrB, oqxB, ramA\u003c/em\u003e), ceftazidime-avibactam resistance-associated genes (\u003cem\u003eompC, ompF\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e), capsular polysaccharide and extracellular polysaccharide synthesis genes (\u003cem\u003ewcaD, wcaE, wzb, wzc\u003c/em\u003e), and biofilm formation-related genes were used for amplification. The PCR products were verified by gel electrophoresis in 1.2% agarose gels in 0.5\u0026times; TAE buffer. Positive amplicons were sequenced using the Sanger method, and the resulting sequences were compared with reference sequences in the GenBank database using the BLAST tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/blast/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/blast/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to analyze gene mutations.\u003c/p\u003e\n\u003ch3\u003eWhole Genome Sequencing (WGS)\u003c/h3\u003e\n\u003cp\u003eGenomic DNA from two CRECC strains was extracted using the Wizard Genomic DNA Purification Kit and sequenced using the Illumina HiSeq 2000 (2\u0026times;150 bp paired-end sequencing, Illumina Inc., San Diego, CA, USA) and MinION platform (Oxford Nanopore Technologies, Oxford, UK). The genome was assembled using Canu v.1.6 and corrected with Pilon v.1.22. Annotation was performed using PGAP and Glimmer 3.02 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbcb.umd.edu/software/Glimmer/\u003c/span\u003e\u003cspan address=\"http://www.cbcb.umd.edu/software/Glimmer/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). PlasmidFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/PlasmidFinder/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/PlasmidFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), CARD, and ResFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/resfinder/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/resfinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used to identify plasmid sequences, resistance genes, and virulence factors, while ISfinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www-is.bio-toul.fr/\u003c/span\u003e\u003cspan address=\"https://www-is.bio-toul.fr/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to locate transposons and insertion sequences. Sequence alignment was performed using BLASTn, and visualizations were created using BRIG (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://brig.sourceforge.net\u003c/span\u003e\u003cspan address=\"http://brig.sourceforge.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Easyfig (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/mjsull/Easyfig\u003c/span\u003e\u003cspan address=\"https://github.com/mjsull/Easyfig\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The genome has been deposited in GenBank.\u003c/p\u003e\n\u003ch3\u003eSerum resistance test and bacterial growth curve\u003c/h3\u003e\n\u003cp\u003eThe serum resistance assay was conducted by culturing the strains in LB broth containing 10% mixed human serum for 24 hours, following the method described in reference[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. After overnight incubation of the strains in LB broth at 37\u0026deg;C with shaking at 160 rpm, the cultures were diluted 1:100 in LB broth containing 10% human serum. The optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) was measured every hour for 24 hours to generate a growth curve. To assess the fitness cost of TGC-resistant mutants compared to their parent strains, both the mutants and parent strains were inoculated into fresh LB broth without any antibiotics and incubated at 37\u0026deg;C with shaking at 160 rpm until the turbidity reached 0.5 McFarland units. The cultures were then diluted 1:100 into 30 mL of LB broth and incubated at 37\u0026deg;C with continuous shaking. The OD\u003csub\u003e600\u003c/sub\u003e was measured every hour. Each strain was tested in triplicate, and the average OD was calculated. Growth curves were plotted using GraphPad Prism 9.5 software, and intergroup differences were analyzed using a t-test, with a p\u0026thinsp;\u0026le;\u0026thinsp;0.05 indicating statistical significance.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003evirulence assay\u003c/h2\u003e \u003cp\u003eAs previously described, after some modifications[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the optimal infection dose for the 6 CRECC strains was determined prior to the Galleria mellonella survival assay to ensure that approximately 80% of the larvae would die within 3 days of infection[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The Galleria mellonella larvae were purchased from Chongqing YeMai Biotechnology Co., Ltd., with an average larval weight of 400\u0026thinsp;\u0026plusmn;\u0026thinsp;50 mg. Overnight bacterial cultures of each strain were adjusted to a concentration of 1\u0026times;10⁸ CFU/mL in sterile phosphate-buffered saline (PBS, pH 7.4). A 10 \u0026micro;L inoculum (containing 1\u0026times;10⁶ CFU) was injected into the hemocoel of the larvae via the last left leg using a 50 \u0026micro;L Hamilton syringe (Hamilton, Shanghai, China). Infected larvae were incubated in Petri dishes at 37\u0026deg;C and observed for survival every 12 hours over a 4-day period. Larvae were considered dead if there was no response when gently touched with sterile metal forceps. Galleria mellonella larvae injected with 10 \u0026micro;L of sterile PBS were used as negative controls. Each group consisted of 10 larvae, and the experiment was repeated three independent times.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTigecycline-resistant mutant stability assay\u003c/h3\u003e\n\u003cp\u003eTo investigate the stability of the MICs of TGC and CZA in resistant strains, four resistant mutants were each inoculated into antibiotic-free LB broth and incubated at 37\u0026deg;C with shaking at 160\u0026times;\u003cem\u003eg\u003c/em\u003e. Every 12 hours, the cultures were diluted 1:100 and passaged for 10 consecutive days (approximately 200 generations). The MICs of TGC and CZA were determined every two days using broth microdilution. The final MIC values of each strain for TGC and CZA were plotted as line graphs using GraphPad Prism 9.5 software to analyze the stability of resistance.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and reverse transcription-quantitative PCR (RT-qPCR)\u003c/h3\u003e\n\u003cp\u003eBacterial cultures in the logarithmic growth phase were collected, centrifuged at 12,000\u0026times;g for 10 minutes at 4\u0026deg;C, and the supernatant was discarded. Total RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies) with an A₂₆₀/A₂₈₀ ratio between 1.8 and 2.0. RNA integrity was evaluated by 1% agarose gel electrophoresis. Reverse transcription was performed using the PrimeScript RT Reagent Kit (Takara, Japan) according to the protocol, with DEPC-treated water as the negative control template. The relative expression levels of target genes were quantified using SYBR Green dye (MedChemExpress) on a CFX96 Real-Time PCR System (Bio-Rad). The rpoB gene was used as the internal reference gene, and relative gene expression was calculated using the 2\u003csup\u003e⁻ΔΔCt\u003c/sup\u003e method. RNA sequencing was conducted on the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) with 2 \u0026micro;g of total RNA to construct the RNA-seq transcriptome library[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and raw image-to-sequence conversion was performed using the Illumina GA Pipeline (version 1.6). Statistical analysis was performed using GraphPad Prism 9.5 software. Comparisons between groups were made using the unpaired t-test with Welch's correction, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant. Primer sequences used are provided in \u003cb\u003eTable S2\u003c/b\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTime-kill assay\u003c/h2\u003e \u003cp\u003eTo determine the bactericidal effects of TGC and CZA, a time-kill assay was conducted according to the established protocol[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Overnight cultures of the strains were adjusted to 0.5 McFarland units and added to cation-adjusted Mueller-Hinton broth (CA-MHB) to a final volume of 20 mL, with an initial bacterial concentration of 1\u0026times;10⁶ CFU/mL in the flask. The experimental groups included a blank control (no antibiotics), TGC monotherapy (2 mg/L), CZA monotherapy (32/8 mg/L, or 1/16 MIC), and the TGC\u0026thinsp;+\u0026thinsp;CZA combination (TGC 2 mg/L\u0026thinsp;+\u0026thinsp;CZA 32/8 mg/L), all incubated at 37\u0026deg;C with shaking at 160 rpm for 24 hours. Samples were taken at 0, 2, 4, 8, 12, and 24 hours, and serial dilutions were made in sterile PBS before plating on Mueller-Hinton (MH) agar plates. After incubating at 37\u0026deg;C for 18\u0026ndash;24 hours, colony-forming units (CFU) were counted. The experiment was repeated three times, and time-kill curves were generated using GraphPad Prism 9.5 software. Group differences were analyzed using a t-test, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid conjugation experiment\u003c/h2\u003e \u003cp\u003eThe plasmid conjugation experiment was modified based on the study by Gong Xue et al[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this experiment, CRECC112-1 and CRECC414-1 strains were used as donor bacteria, and rifampin-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e EC600 was used as the recipient. The procedure is as follows: the LB broth cultures of the donor and recipient bacteria were mixed in a 1:3 ratio, and the mixture was spotted onto a sterile filter membrane. The membrane was then placed on an LB agar plate and incubated at 35\u0026deg;C for 24 hours. After incubation, the membrane was transferred onto Mueller-Hinton agar plates containing imipenem (2 mg/L) and rifampin (100 mg/L) to select for conjugants. The presence of conjugants was confirmed using the VITEK-2 COMPACT system and 16S rRNA gene sequencing[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The presence of resistance genes in the conjugants was further verified by PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics Processing and Analysis\u003c/h2\u003e \u003cp\u003eBioinformatics analysis was performed on RNA-seq data generated using the Illumina platform. Differentially expressed genes (DEGs) between the tigecycline-treated and control groups were identified using the DESeq2 package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioconductor.org/packages/release/bioc/html/DESeq2.html\u003c/span\u003e\u003cspan address=\"http://bioconductor.org/packages/release/bioc/html/DESeq2.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with a selection criterion of |log₂FC| \u0026gt; 1 and a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Gene ontology (GO) functional enrichment analysis was performed using the Goatools tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/tanghaibao/GOatools\u003c/span\u003e\u003cspan address=\"https://github.com/tanghaibao/GOatools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) combined with Fisher's exact test, selecting GO terms with statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted using KOBAS 2.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://kobas.cbi.pku.edu.cn\u003c/span\u003e\u003cspan address=\"http://kobas.cbi.pku.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with Fisher's exact test identifying significantly enriched pathways (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Multiple testing correction was performed using the Benjamini-Hochberg (BH) method to reduce type I errors. Statistical significance levels were indicated as *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of CRECC clinical isolates\u003c/h2\u003e \u003cp\u003eA total of six tigecycline-sensitive, ceftazidime-avibactam-resistant CRECC strains were selected from clinical isolates in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. The results showed that after 7 days of tigecycline exposure, two strains (CRECC112 and CRECC414) developed two phenotypically distinct tigecycline-resistant mutant strains, named CRECC112-1, CRECC112-2, and CRECC414-1, CRECC414-2, respectively. Among these, CRECC112-1 and CRECC414-1 exhibited mucoid colony morphology, while CRECC112-2 and CRECC414-2 exhibited dry colony morphology, which was consistent with that of the parent strains in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. Further antibiotic susceptibility testing on these two parental strains and their resistant mutants revealed that all strains were resistant to most of the tested antibiotics in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Notably, the mucoid-resistant mutants (CRECC112-1, CRECC414-1) showed a phenotypic change from ceftazidime-avibactam resistance to susceptibility. Whole-genome sequencing revealed that CRECC112 was identified as \u003cem\u003eEnterobacter hormaechei\u003c/em\u003e, and CRECC414 was identified as \u003cem\u003eEnterobacter kobei\u003c/em\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Resistance gene detection showed that CRECC112 carried three β-lactamase genes: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eTEM\u003c/sub\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e; CRECC414 also carried three \u003cem\u003eβ\u003c/em\u003e-lactamase genes: \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e, and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u0026minus;12\u003c/sub\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\u003eAntimicrobial susceptibility profiles of two CRECC strains harboring carbapenemase genes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e \u003cp\u003eAntibiotics\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCOL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eIPM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCAZ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCZA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAK\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTZP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCZX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"10\" nameend=\"c10\" namest=\"c1\"\u003e \u003cp\u003eBreakpoints(S-R)MIC(mg/L)\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e112-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e112-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e414-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e414-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.5\u003csup\u003eS\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;16\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e32\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e128\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e64\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"10\"\u003e\u003csup\u003ea\u003c/sup\u003eS-R represents the susceptible (S) breakpoint to the resistant (R); \u003csup\u003eb\u003c/sup\u003eTGC, tigecycline; COL, Colistin; MEM, meropenem; IPM, imipenem; CAZ, ceftazidime; CZA, ceftazidime-avibactam; AK, Amikacin; TZP, piperacillin/tazobactam; CZX, cefepime;. Susceptibility breakpoints for TGC against CRECC were interpreted according to the EUCAST guidelines, while those for the remaining antimicrobial agents were based on the CLSI 2020 criteria\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eChanges in resistance after tigecycline induction\u003c/h2\u003e \u003cp\u003eAntimicrobial susceptibility testing of the induced resistant mutant strains was performed using the microdilution method in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. The results showed that the mucoid mutants (CRECC112-1, CRECC414-1) exhibited a significant reduction in the minimum inhibitory concentration (MIC) of CZA from \u0026gt;\u0026thinsp;128 \u0026micro;g/mL in the parent strains to 0.5\u0026ndash;1 \u0026micro;g/mL, whereas the dry-type mutants (CRECC112-2, CRECC414-2) showed no significant change in MIC, remaining at \u0026gt;\u0026thinsp;128 \u0026micro;g/mL. These findings indicate that tigecycline-induced mucoid-resistant mutants demonstrated a significant restoration of CZA sensitivity, and this phenotype was associated with the colony morphology. To further validate these results, Kirby-Bauer (K-B) disk diffusion tests were performed, which revealed that the inhibition zone diameters of CRECC112-1 and CRECC414-1 against CZA were \u0026gt;\u0026thinsp;21 mm, further confirming the presence of collateral sensitivity in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec. To investigate the phenotypic characteristics of the different colony morphology mutants, TEM was used to examine all six strains (two parental strains and four mutants). The results showed that CRECC112-1 and CRECC414-1 had a thick capsule structure surrounding the bacterial cell, while both the parent strains and the dry-type mutants (CRECC112-2, CRECC414-2) lacked any capsule structure in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMutation and expression of drug-resistance-related genes\u003c/h2\u003e \u003cp\u003eSequencing was performed on TGC resistance-associated genes, CZA resistance-associated genes and regulatory factors, efflux pump genes, capsule polysaccharide and extracellular polysaccharide synthesis genes, as well as biofilm formation-related genes to identify the molecular determinants of sensitivity and resistance. In both the parent strains and the TGC-resistant mutants, mutations in CZA resistance-related genes (\u003cem\u003eompC\u003c/em\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e), efflux pump-related genes (\u003cem\u003eacrA, acrB, oqxB\u003c/em\u003e), capsule polysaccharide and extracellular polysaccharide synthesis genes \u003cem\u003e(wzb, wzc, wcaD\u003c/em\u003e, \u003cem\u003ewcaE\u003c/em\u003e), and biofilm formation-related genes were compared. The results showed that, except for the ramA gene in CRECC414-1, which underwent a L53R missense mutation, and multiple amino acid substitutions in the \u003cem\u003elpxA\u003c/em\u003e gene in \u003cb\u003eFig S3\u003c/b\u003e, no mutations were detected in the aforementioned genes in the other strains in 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\u003eGene mutations associated with TGC and CZA resistance in two clinically isolated strains and their corresponding resistant derivatives\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsoles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"12\" nameend=\"c13\" namest=\"c2\"\u003e \u003cp\u003egrnes\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eacrB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eramA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003ewcaD\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003ewcaE\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ewzB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003ewzC\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eoqxB\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eompC\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003e\u003cem\u003elpxA\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003e\u003cem\u003elpxC\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e112-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e414-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e+: exist with amino acid substitution,WT: indicates that there is no mutation of the corresponding gene in all the strains in the study\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eFitness Cost and Stability Analysis\u003c/h2\u003e \u003cp\u003eTo determine whether TGC resistance mutations incur a fitness cost and assess the stability of such resistance, four TGC-resistant mutant strains were serially passaged in antibiotic-free medium, and changes in the MICs of TGC and CZA were monitored. After 10 days of serial passage, the TGC MICs of all four mutants decreased from initial values of 32 \u0026micro;g/mL and 16 \u0026micro;g/mL to 8 \u0026micro;g/mL and 4 \u0026micro;g/mL in \u003cb\u003eFig.\u0026nbsp;2a\u003c/b\u003e, indicating that TGC resistance in these mutants is unstable relative to their parental strains. For CZA, the MICs of CRECC112-2 and CRECC414-2 remained\u0026thinsp;\u0026gt;\u0026thinsp;128 \u0026micro;g/mL during passage, whereas the MICs of CRECC112-1 and CRECC414-1 increased from 0.25 \u0026micro;g/mL to 4 \u0026micro;g/mL in \u003cb\u003eFig.\u0026nbsp;2a\u003c/b\u003e, suggesting that the CZA susceptibility of mucoid mutants is gradually lost, while dry-type mutants maintain high-level resistance.Correlation analysis of colony morphology and growth kinetics revealed that the growth rates of mucoid strains (CRECC112-1 and CRECC414-1) were significantly lower than those of the parental and dry-type strains in \u003cb\u003eFig.\u0026nbsp;2b\u003c/b\u003e. The serum resistance assay showed that after incubation for 24 hours in LB medium supplemented with 10% human serum, the two mucoid strains exhibited almost no growth within the first 10 hours after inoculation compared with the dry-type strain in \u003cb\u003eFig.\u0026nbsp;2c\u003c/b\u003e. Furthermore, in the \u003cem\u003eG mellonella\u003c/em\u003e infection model, larval mortality rates were comparable between parental and dry-type strains (survival\u0026thinsp;\u0026lt;\u0026thinsp;50%), whereas infection with mucoid resistant strains resulted in significantly reduced mortality, indicating attenuated virulence in \u003cb\u003eFig.\u0026nbsp;2d\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro time-kill curve\u003c/h2\u003e \u003cp\u003eThe time-kill assay results for TGC and CZA, both alone and in combination, against two parental strains (CRECC112 and CRECC414) showed the following: In the CZA monotherapy group (32/8 mg/L), bacterial growth rapidly increased within 2 hours, reaching levels comparable to the control group after 12 hours. The TGC monotherapy group (2 mg/L) inhibited bacterial growth for the first 2 hours, but bacteria rapidly regrew thereafter, maintaining a concentration of 5\u0026ndash;6 log\u003csub\u003e10\u003c/sub\u003eCFU/mL by 24 hours. In contrast, the combination of TGC and CZA exhibited a synergistic bactericidal effect against the CRECC strains, with a sustained decline in bacterial concentration over the 24-hour period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eGlobal Transcriptomic Variations in Strains Induced by Tigecycline were analyzed via RNA sequencing\u003c/h2\u003e \u003cp\u003eGiven the significant changes in the MIC and colony morphology of the CRECC112 and CRECC414 strains following tigecycline induction, as well as their ability to survive under high tigecycline pressure, it is hypothesized that transcriptional adaptation may have occurred. To investigate this, transcriptomic sequencing was employed to analyze the genome-wide expression changes induced by tigecycline. The Venn diagram illustrates the overlap and unique differentially expressed genes (DEGs) across four samples (CRECC112, CRECC112-1, CRECC414, CRECC414-1) in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea. Volcano plot analysis revealed significant transcriptional alterations in both strains following tigecycline treatment in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. Functional enrichment analysis indicated that the DEGs in CRECC112-1, compared to CRECC112, were mainly involved in pyruvate metabolism, glycolysis/gluconeogenesis, and phosphotransferase systems. In contrast, the DEGs in CRECC414-1 relative to CRECC414 were primarily associated with the ABC transporter system and two-component regulatory systems. Both strains shared DEGs that participated in flagellar assembly, pyruvate metabolism, and nitrogen metabolism in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. GO functional annotation showed that the DEGs in CRECC112 were most enriched in metabolic processes, compound binding, and small molecule binding, with a higher number of upregulated genes than downregulated genes. In CRECC414, the DEGs were most enriched in metabolic processes and small molecule binding, with a balanced number of upregulated and downregulated genes, although some GO categories were dominated by downregulated genes in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic changes associated with resistance to two antibiotics\u003c/h2\u003e \u003cp\u003eIn the mucoid TGC-resistant mutants CRECC112-1 and CRECC414-1, whole-genome transcriptomic analysis revealed significant and functionally relevant changes compared with their parental strains(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Genes associated with efflux pumps (\u003cem\u003eacrA\u003c/em\u003e, \u003cem\u003eacrB\u003c/em\u003e, \u003cem\u003eoqxB\u003c/em\u003e), the two-component regulatory system (\u003cem\u003ephoQ\u003c/em\u003e), and outer membrane porins (\u003cem\u003eompC\u003c/em\u003e, \u003cem\u003eompF\u003c/em\u003e) were consistently downregulated in both mutants. Genes linked to CZA resistance, including \u003cem\u003eompC\u003c/em\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, were also broadly downregulated, with \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression reduced by 5-20-fold. Expression of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eCTX\u0026minus;M\u003c/sub\u003e decreased in CRECC112-1, whereas in CRECC414-1 it showed an increase that did not reach statistical significance. The regulatory factor \u003cem\u003eramA\u003c/em\u003e displayed strain-specific expression: it was upregulated approximately 5-fold in CRECC112-1 but showed the opposite trend in CRECC414-1, reflecting heterogeneity in resistance regulatory pathways among strains. Notably, genes directly involved in biofilm formation (\u003cem\u003ewcaD\u003c/em\u003e, \u003cem\u003ewcaE\u003c/em\u003e) and in cell surface polysaccharide synthesis (\u003cem\u003ewzb\u003c/em\u003e, \u003cem\u003ewzc\u003c/em\u003e) were markedly upregulated in both mutants, with fold changes of 20\u0026ndash;25 and 8\u0026ndash;25.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePlasmid analysis of\u003c/b\u003e \u003cb\u003ebla\u003c/b\u003e\u003csub\u003e\u003cb\u003eNDM\u0026minus;1\u003c/b\u003e\u003c/sub\u003e \u003cb\u003eharboring strains\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenome sequencing revealed that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e gene in CRECC112 and CRECC414 was located on an IncFII-type and an IncX3-type plasmid, designated pNDM112 (85,718 bp) and pNDM414 (74,194 bp), respectively. Comparative analysis of these two \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e-harboring plasmids showed that the coding regions of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e shared nearly 100% homology, with both carrying conserved promoter and signal peptide sequences, indicating that \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e is a highly conserved functional element with no significant variation in its core resistance sequence.Functional annotation demonstrated that pNDM112 harbored \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (carbapenem resistance), \u003cem\u003earmA\u003c/em\u003e (aminoglycoside resistance), conjugation-associated genes (\u003cem\u003etra\u003c/em\u003e series), and replication-associated genes (\u003cem\u003epar\u003c/em\u003e series). In contrast, pNDM414 contained \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e (\u003cem\u003eβ\u003c/em\u003e-lactam resistance), virulence-associated genes (\u003cem\u003evir\u003c/em\u003e series), replication/transfer-related genes (\u003cem\u003epar\u003c/em\u003e, \u003cem\u003etra\u003c/em\u003e), and the insertion sequence \u003cem\u003eIS\u003c/em\u003e15. Further analysis showed that the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e\u003cem\u003e-armA\u003c/em\u003e cassette in pNDM112 was flanked by transposable elements forming a composite resistance unit, whereas \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e were tandemly arranged within a resistance island in pNDM414. Both structures exhibit typical features of mobilizable resistance modules, suggesting that such elements are key drivers of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e dissemination across plasmids and strains in \u003cb\u003eFig.\u0026nbsp;6a\u003c/b\u003e. To investigate the regulatory mechanisms governing \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression, the relative transcription levels of the plasmid replicons carrying the gene were measured. Using \u003cem\u003erpoB\u003c/em\u003e as an internal reference, the replicon transcription levels in both mutant strains were lower than in the parental strains in \u003cb\u003eFig.\u0026nbsp;6b\u003c/b\u003e, indicating that reduced \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression in the drug-resistant mutants is accompanied by decreased transcriptional activity of the associated plasmid replicons.\u003c/p\u003e \u003c/div\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2\u003c/b\u003e Stability Assay of Tigecycline-Resistant Mutants \u003cb\u003ea\u003c/b\u003e: Changes in CZA MICs and TGC MICs. The x-axis represents the number of subculture days. \u003cb\u003eb\u003c/b\u003e: Monitoring of the 24-hour growth kinetics of tigecycline-susceptible strains, ceftazidime-avibactam-resistant strains, and TGC-resistant strains.\u003cb\u003ec\u003c/b\u003e: Serum resistance of different morphotypes. Differences in serum resistance were determined by comparing the growth curves in LB broth and LB broth supplemented with 10% pooled normal human serum.\u003cb\u003ed\u003c/b\u003e: Survival curves of the \u003cem\u003eGalleria mellonella\u003c/em\u003e infection model. Ten larvae per group were injected with 10 \u0026micro;L of the bacterial suspension at a concentration of 10⁶ CFU/mL, respectively\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;6a\u003c/b\u003e Circular maps of the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM-1\u003c/sub\u003e-harboring plasmids pNDM112 (85718 bp) and pNDM414 (74194 bp). From the inner to the outer rings: scale bar (kbp); GC skew (purple, negative values; green, positive values); GC content (black histograms); sequence alignment results (red/pink/light gray indicate alignment with \u0026ldquo;112-1\u0026rdquo; or \u0026ldquo;414,\u0026rdquo; whereas dark/light blue or gray indicate alignment with the alternative reference sequence, with darker colors representing higher sequence homology); the outermost ring denotes annotated functional genes, including \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e (carbapenem resistance), armA(aminoglycoside resistance; unique to pNDM112), \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eSHV\u003c/sub\u003e (\u003cem\u003eβ\u003c/em\u003e-lactam resistance; unique to pNDM414), tra and par family genes, and the insertion sequence IS15. \u003cb\u003eb\u003c/b\u003e Relative transcription levels of plasmid replicons carrying the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e gene, normalized to rpoB as the reference gene. IncFHⅡ and IncX3 represent the replicon types of plasmids harbored by strains 112 and 414, respectively. All data were calculated using the 2 method and are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global dissemination of CRECC has emerged as a major challenge for clinical anti-infective therapy[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The limited availability of effective therapeutic options has prompted sustained efforts to explore alternative treatment strategies. We identified and conducted an in-depth analysis of the resistance profiles of CRECC to TGC and CZA. By integrating analyses of antibiotic-associated phenotypic alterations, underlying molecular mechanisms, and potential synergistic antibacterial activity, our findings provide novel insights into the therapeutic potential of TGC-CZA-based strategies and offer both theoretical and practical implications for the clinical management of CRECC infections.\u003c/p\u003e \u003cp\u003eOveractivation of efflux pump systems was identified as one of the key mechanisms underlying TGC resistance in the mutant strains CRECC112-1 and CRECC414-1 in this study. In Enterobacterales, the AcrAB-TolC efflux pump plays a central role in mediating TGC resistance[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Whole-genome sequencing combined with transcriptomic analyses indicated that mutations in the global regulator \u003cem\u003eramA\u003c/em\u003e may drive the overexpression of efflux pump-associated genes, including \u003cem\u003eacrA\u003c/em\u003e, \u003cem\u003eacrB\u003c/em\u003e, and \u003cem\u003etolC\u003c/em\u003e, thereby contributing to tigecycline resistance. As a member of the AraC/XylS family of global transcriptional regulators, \u003cem\u003eramA\u003c/em\u003e mutations-such as the L53R missense substitution identified in CRECC414-1 can markedly enhance transcriptional activation of \u003cem\u003eacrA\u003c/em\u003e, \u003cem\u003eacrB\u003c/em\u003e, and \u003cem\u003etolC\u003c/em\u003e, leading to increased synthesis and activity of the AcrAB-TolC efflux system. This pump actively exports tigecycline and other antimicrobial agents out of the bacterial cell, resulting in reduced intracellular drug accumulation, a mechanism consistent with previous reports on tigecycline resistance in Enterobacterales[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe TGC-resistant mutants exhibited a distinct mucoid phenotype that differed markedly in functional characteristics from both the parental strains and the dry-type mutants (CRECC112-2 and CRECC414-2). Notably, the mucoid variants demonstrated restored susceptibility to CZA, with MICs decreasing from \u0026gt;\u0026thinsp;128 mg/L to 0.5\u0026ndash;1 mg/L. This pronounced reduction suggests that TGC-associated resistance mechanisms may be accompanied by a restoration of CZA susceptibility, reflecting an evolutionary trade-off consistent with the phenomenon of collateral sensitivity. Transcriptomic analysis revealed a significant downregulation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression (by approximately 5\u0026ndash;20-fold) in TGC-resistant mutants, which may represent a key determinant underlying the restored CZA susceptibility. In particular, in the mucoid mutants CRECC112-1 and CRECC414-1, reduced \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression is likely to attenuate ceftazidime hydrolysis[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], thereby enabling avibactam to more effectively inhibit residual \u003cem\u003eβ\u003c/em\u003e-lactamase activity and restore CZA efficacy. In parallel, genes associated with extracellular polysaccharide (EPS) biosynthesis, including \u003cem\u003ewcaD\u003c/em\u003e, \u003cem\u003ewcaE\u003c/em\u003e, \u003cem\u003ewzb\u003c/em\u003e, and \u003cem\u003ewzc\u003c/em\u003e, were markedly upregulated in the mucoid mutants. The \u003cem\u003ewcaD\u003c/em\u003e gene cluster is responsible for the synthesis of colanic acid, a major component of the EPS matrix in \u003cem\u003eEnterobacterales\u003c/em\u003e, while \u003cem\u003ewzb\u003c/em\u003e encodes a phosphatase and \u003cem\u003ewzc\u003c/em\u003e encodes a tyrosine kinase that together regulate EPS polymerization and secretion. Enhanced expression of these genes is expected to promote EPS accumulation and surface thickening[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Such envelope remodeling may, at least in part, alter outer membrane permeability and influence CZA influx, although this hypothesis warrants further experimental validation.\u003c/p\u003e \u003cp\u003eTransmission electron microscopy provided direct structural evidence supporting these transcriptomic findings, revealing a characteristic thick capsular layer surrounding the mucoid mutants, a feature absent in both the parental strains and dry-type mutants. This structural divergence represents a defining hallmark distinguishing the mucoid and dry phenotypes and provides an important morphological basis for the observed TGC resistance and CZA collateral sensitivity. Additionally, transcriptomic alterations were observed in genes related to lipid biosynthesis and envelope regulation, including \u003cem\u003elpxA\u003c/em\u003e and \u003cem\u003ephoQ\u003c/em\u003e. The \u003cem\u003elpxA\u003c/em\u003e gene is involved in lipid A biosynthesis, and its alteration may affect outer membrane integrity and permeability[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The \u003cem\u003ephoQ\u003c/em\u003e gene, as part of the PhoPQ two-component regulatory system, participates in lipid A modification and has been implicated in EPS regulation. Moreover, the \u003cem\u003ewzb-wzc\u003c/em\u003e regulatory system may interact with other signaling pathways to fine-tune phenotype-associated gene expression, thereby stabilizing the mucoid phenotype and indirectly facilitating the restoration of CZA susceptibility.\u003c/p\u003e \u003cp\u003eMBLs represented by \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e are the core mediators of carbapenem resistance in CRECC[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and their expression levels directly affect bacterial susceptibility to \u003cem\u003eβ\u003c/em\u003e-lactam antibiotics. In the present study, both parental strains CRECC112 and CRECC414 harbored the \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u0026minus;1\u003c/sub\u003e gene, which was also the key reason for their high resistance to CZA (MIC\u0026thinsp;\u0026gt;\u0026thinsp;128 mg/L). However, following TGC exposure, mucoid mutants exhibited cross-susceptibility to CZA, with MIC values decreasing from 128 mg/L to 0.5-1 mg/L. This restoration of susceptibility was not driven by a single factor, but rather resulted from the close association and synergistic effects of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression regulation, mucoid phenotypic changes, and efflux pump system modulation.In mucoid mutants, the expression level of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e was significantly downregulated (5-50-fold reduction at the mRNA level), which directly attenuated its resistance-mediating effect. On the one hand, decreased \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e activity reduced the hydrolysis of ceftazidime, allowing avibactam to effectively protect ceftazidime from degradation by other \u003cem\u003eβ\u003c/em\u003e-lactamases, thereby restoring the inhibitory activity of CZA against bacteria[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. On the other hand, the downregulation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e was accompanied by alterations in bacterial metabolic status, which may affect cell wall synthesis or outer membrane permeability, indirectly regulating susceptibility to CZA[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] .The thick envelope structure of mucoid mutants physically hindered the translocation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e to the periplasmic space, reducing its hydrolysis efficiency against ceftazidime and indirectly enhancing CZA susceptibility. Meanwhile, bacteria exhibited an obvious metabolic resource trade-off effect: the synthesis of a thick capsule consumes substantial energy and substances. To maintain basic growth and environmental adaptability, bacteria may actively downregulate the expression of non-essential resistance determinants such as \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, thereby indirectly promoting the restoration of susceptibility to CZA.In TGC-resistant mutants, the expression of outer membrane protein genes ompC and ompF was significantly downregulated (20-50-fold). The porins encoded by these genes are the main channels for hydrophilic antibiotics such as ceftazidime to enter bacterial cells; reduced porin levels would normally inhibit ceftazidime influx and impair the efficacy of CZA[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, the significant downregulation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e in mucoid mutants effectively offset the adverse effects caused by reduced porin expression, ultimately achieving the restoration of CZA susceptibility.Results of the resistance stability assay further confirmed the above associations. After 10 passages of mucoid mutants in medium without antibiotic selection pressure, the MIC of CZA increased from 0.125 \u0026micro;g/mL to 4 \u0026micro;g/mL, accompanied by a gradual attenuation of the mucoid phenotype and upregulation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression. This synergistic change trend not only clarified the close correlation among the mucoid phenotype, \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression regulation, and CZA susceptibility, but also intuitively reflected the evolutionary trade-off strategy of bacteria between drug resistance and survival adaptability.\u003c/p\u003e \u003cp\u003eThe acquisition of tigecycline resistance in the mucoid mutants was accompanied by a pr The CZA resensitization observed in this study was not attributable to the loss or inactivation of resistance determinants, but rather reflected a phenotypic reversal of resistance driven by transcriptional regulation. In the mucoid mutant strains selected following tigecycline exposure, the transcription levels of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e and the associated plasmid replicons were markedly reduced, leading to decreased functional expression of carbapenemase. As a consequence, CZA exhibited restored in vitro inhibitory activity against these isolates. The acquisition of TGC resistance in mucoid mutant strains was accompanied by a pronounced attenuation of virulence, highlighting an evolutionary trade-off employed by bacteria in response to antibiotic pressure[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Serum resistance assays demonstrated that the mucoid variants exhibited almost no detectable growth within the first 12 h of incubation in LB broth supplemented with 10% human serum, suggesting that the thick capsular layer may impair bacterial survival in the host circulatory environment by disrupting interactions with serum complement components. Consistently, results from the \u003cem\u003eG mellonella\u003c/em\u003e infection model showed that larvae infected with mucoid mutants experienced significantly lower mortality rates compared with those infected with the parental strains or dry-type mutants. This inverse relationship between resistance acquisition and virulence not only provides a mechanistic explanation for the limited stability of the collateral sensitivity phenotype, but also offers theoretical insights into the optimization of anti-infective treatment strategies targeting carbapenem-resistant \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, this susceptibility phenomenon was not universally observed but was restricted to a subset of NDM-producing \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex isolates. These findings indicate that antibiotic collateral sensitivity is highly strain dependent and may require specific genetic backgrounds or metabolic regulatory states to occur. Further validation in larger collections of clinical isolates is therefore necessary to determine its prevalence and key determinants. In addition, the regulatory mechanisms linking the mucoid phenotype to \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression remain incompletely understood and warrant comprehensive investigation through integrated transcriptomic, proteomic, and metabolomic analyses, combined with gene-editing approaches to identify the critical regulatory factors and signaling pathways involved. This study delineates the coordinated regulatory pathways underlying TGC and CZA resistance in CRECC. Overexpression of the AcrAB-TolC efflux system primarily mediates TGC resistance, while EPS synthesis, driven by \u003cem\u003ewcaD, wcaE, wzb\u003c/em\u003e, and \u003cem\u003ewzc\u003c/em\u003e, induces the mucoid phenotype. Concurrently, downregulation of \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e, together with \u003cem\u003ephoQ\u003c/em\u003e-associated defects in lipid A modification, collectively restores CZA susceptibility. The interdependence between the mucoid phenotype and \u003cem\u003ebla\u003c/em\u003e\u003csub\u003eNDM\u003c/sub\u003e expression reflects a coordinated evolutionary strategy in bacterial resistance, with the efflux pump pathway playing a central role in TGC resistance. These findings provide a mechanistic rationale for the potential clinical application of TGC-CZA combination therapy and offer a conceptual framework for the development of novel anti-resistance strategies targeting MBL expression and efflux systems. Future our investigations will focus on optimizing combination regimens and elucidating the molecular mechanisms of the underlying regulatory networks to better address the clinical challenges posed by CRECC infections.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (2024ZD0533106/2024ZD0533100), National Innovation Center for High-Performance Medical Devices (NMED2025KF-03-003), Chongqing public health key specialty (discipline) project, Chongqing Health Commission and Science and Technology Bureau (2023MSXM018), Yongchuan Natural Science Foundation (2025yc-cxfz10114).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL T, J W, C L and XL Z designed and coordinated the the study. LY T and J W wrote the paper and participated in the whole experiment process. Y L, C L, Y C, and J W helped with the experimental process, J W , X S L and JM W provided the samples and the clinical data.J W analyzed and interpreted the data. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank all the patients whose data were used in the study\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTompkins K, van Duin D (2021) Treatment for carbapenem-resistant Enterobacterales infections: recent advances and future directions. 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J Intern Med 287(3):283\u0026ndash;300. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dx.doi.org/10.1111/joim.13007\u003c/span\u003e\u003cspan address=\"10.1111/joim.13007\" 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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Enterobacter cloacae complex, NDM, ceftazidime-avibactam, Tigecycline, collateral sensitivity","lastPublishedDoi":"10.21203/rs.3.rs-8784943/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8784943/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eCarbapenem-resistant \u003cem\u003eEnterobacter cloacae\u003c/em\u003e complex (CRECC) has become an increasingly important pathogen in healthcare-associated infections, with limited treatment options and a high mortality rate. As reports of antimicrobial resistance continue to rise, tigecycline (TGC) and ceftazidime-avibactam (CZA) have emerged as the last-line therapies for CRECC infections. The aim of this study was to investigate collateral sensitivity to ceftazidime-avibactam following the acquisition of tigecycline resistance in NDM-producing CRECC, and to elucidate the underlying molecular mechanisms, thereby providing a theoretical basis for clinical combination therapy.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eAntimicrobial susceptibility profiles were determined using the broth microdilution method, and changes in colony morphology were analyzed. Transcriptomic sequencing was performed to characterize global gene expression alterations associated with antimicrobial resistance, and an in vivo \u003cem\u003eGalleria mellonella\u003c/em\u003e infection model was used to assess the virulence of the mutant strains.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBoth drug-resistant mutants displayed a stable mucoid phenotype with marked collateral susceptibility to CZA, with minimum inhibitory concentrations decreasing from greater than 128 mg/L to 0.5\u0026ndash;1 mg/L. Compared with the parental strains, these mutants showed thickened cell surface structures, impaired growth, reduced serum tolerance, and significantly attenuated virulence in the \u003cem\u003eGalleria mellonella\u003c/em\u003e infection model. Transcriptomic analysis indicated increased extracellular polysaccharide production, impaired lipid A modification associated with reduced \u003cem\u003ephoQ\u003c/em\u003e expression, and markedly decreased expression of metallo-\u003cem\u003eβ\u003c/em\u003e-lactamase-related genes, including NDM and CTX-M.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWe hypothesize that the susceptibility of metallo-\u003cem\u003eβ\u003c/em\u003e-lactamase-producing strains to CZA represents an adaptive survival modification, which is achieved by reducing metallo-\u003cem\u003eβ\u003c/em\u003e-lactamase expression and altering bacterial metabolism at the cost of impaired bacterial growth and pathogenicity. This trade-off between antimicrobial resistance and bacterial fitness offers novel insights into the development of optimized therapeutic strategies for CRECC infections.\u003c/p\u003e","manuscriptTitle":"Resusceptibility to Ceftazidime-Avibactam in Tigecycline-Exposed NDM-Producing CRECC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 08:12:36","doi":"10.21203/rs.3.rs-8784943/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-22T12:16:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-28T15:54:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23576156142711107482909204447459391851","date":"2026-03-04T12:51:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137275327434152755527499726144361204092","date":"2026-03-04T09:58:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-04T09:33:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T10:15:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-05T03:02:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","date":"2026-02-04T09:43:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-clinical-microbiology-and-infectious-diseases","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejcm","sideBox":"Learn more about [European Journal of Clinical Microbiology \u0026 Infectious Diseases](https://www.springer.com/journal/10096)","snPcode":"10096","submissionUrl":"https://submission.nature.com/new-submission/10096/3","title":"European Journal of Clinical Microbiology \u0026 Infectious Diseases","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e968b744-7d9a-42ea-9813-1843b0a792e9","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-02T11:53:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 08:12:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8784943","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8784943","identity":"rs-8784943","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}