Multidrug-resistant bacterial infections pose a severe threat to sepsis, where uncontrolled bacterial proliferation and the accompanying inflammatory response lead to multiorgan dysfunction. Recent clinical isolation of a super-resistant Enterobacter asburiae (E. asburiae) strain co-harboring mcr-10 and blaNDM−1 underscores the urgent need for more effective antimicrobial strategies. Herein, we develop a synergistic strategy for the chemodynamic and photothermal therapy of super-resistant E. asburiae using a multifunctional mesoporous nanocomposite. This nanocomposite is composed of mesoporous silica nanoparticle loaded with polymyxin B, polydopamine, and palladium nanoparticles, possessing properties including photothermal therapy, chemodynamic therapy, and drug cargo. This synergistic approach achieves bactericidal effects through localized hyperthermia, enhanced generation of reactive oxygen species, and membrane-disrupting antibiotic action at a low dose of 42.1 µg mL−1. In a murine peritonitis-sepsis model, treatment with this multifunctional mesoporous nanocomposite reduces the bacterial burden by 100

Abstract Iodometry (iodometric method) is one of the simplest, fastest, and most accurate oxidation–reduction titrations for colorimetric detection. Starch is a key indicator for determining the reaction end point. However, the low solubility and instability of starch limit the sensitivity and application of the iodometry. Plasmonic nanomaterials have emerged as innovative tools to inject modern vitality into traditional methods. In this study, we report gold nanostars as a novel and sensitive indicator of iodometry for colorimetric sensing. Branched gold nanostars possess localized surface plasmon resonance (LSPR) properties, and their protruding tips can be specifically reshaped by iodide to tune the optical readout. As a color indicator, gold nanostars can be gradually etched with increasing iodide concentration, displaying a variety of color changes from blue to purple and finally to red. This colorimetric sensing strategy enables highly sensitive detection of iodine standards with a linear range from 0.125 μM to 20 μM and a limit of detection of 0.0375 ± 0.0013 μM. Proof-concept experiments demonstrate the colorimetric performance of gold nanostar indicators in direct iodometry, remnant iodometry, and displacement iodometry by detecting chlorine, sulfide, and copper ions in hospital wastewater. Gold nanostars have advantages such as good stability, easy handling, low cost, and high sensitivity, which provide great prospects for the widespread application of iodometry.

Sepsis remains a leading cause of mortality because current therapies fail to address its dynamically evolving pathophysiology, in which infection, oxidative stress, and immune dysfunction emerge sequentially and interdependently. Here, we present a pH-adaptive nanozyme platform (MICP@HG) that orchestrates stage-specific antibacterial and immunomodulatory activities throughout sepsis progression. The platform integrates near-infrared imaging, catalytic therapy, and immune regulation into a single construct. In acidic infectious microenvironments, the Cu-piceatannol shell exhibits peroxidase-mimicking activity and induces cuproptosis-like bacterial death through metabolic collapse and redox imbalance. As the microenvironment normalizes, the nanozyme shifts toward antioxidative and anti-inflammatory functions via superoxide dismutase (SOD)- and catalase (CAT)-like activities. Concurrently, the hyaluronic acid (HA)/β-glucan coating facilitates infection-targeted delivery and reprograms macrophages toward a reparative phenotype while restoring immune responsiveness. This dynamic functional transition enables efficient eradication of multidrug-resistant bacteria, attenuation of systemic inflammation, and preservation of organ function, ultimately achieving complete survival in polymicrobial sepsis models. Notably, the platform also elicits a vaccine-like trained immunity effect that confers protection against reinfection. This work establishes a paradigm for temporally programmed nanotherapy that aligns therapeutic function with disease progression, offering a precision strategy for the treatment of complex inflammatory disorders.

Iron is an essential nutrient for Klebsiella pneumoniae, a significant opportunistic pathogen. The host’s innate immune system attempts to limit iron availability through nutritional immunity; however, K. pneumoniae has evolved sophisticated strategies to circumvent these defenses. This review explores the intricate mechanisms employed by K. pneumoniae to acquire iron from the host environment. We focus on the roles of siderophores, TonB-dependent transporters (TBDTs), and other iron acquisition systems in K. pneumoniae. Additionally, we discuss the regulation of iron acquisition, emphasizing the importance of intracellular iron storage and the Ferric Uptake Regulator (Fur). Novel therapeutic approaches based on these iron acquisition systems, including siderophore-associated antimicrobials, the use of gallium as an iron analog, and vaccines targeting siderophores and TBDTs, offer new insights into curbing severe K. pneumoniae infections. By elucidating the mechanisms of iron acquisition in K. pneumoniae, this review provides valuable insights into the pathogenesis of this opportunistic pathogen and presents potential strategies to combat multidrug-resistant and hypervirulent strains of K. pneumoniae.

Carbapenem-resistant Enterobacteriales (CRE) are increasingly recognized as reemerging nosocomial pathogens due to the dissemination of dual carbapenemase production. This study investigates the coexistence of blaKPC-2 and blaNDM-1 in CRE isolates, harbored on plasmids either separately and integratively, and examines the underlying mechanisms of plasmid formation and resistance dissemination. Two Citrobacter freundii isolates co-producing Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM) carbapenemases were analyzed. S1-PFGE, Southern blot, and mating conjugation experiments were conducted to determine gene location, transferability, and transconjugation efficiency. Whole-genome sequencing (WGS) was used to elucidate plasmid evolution mechanisms. Specific PCR followed by sequencing was performed to identify hotspot integration sites. Experiments of checkerboard microdilution and time-kill were completed to test the treatment option based on aztreonam. Three plasmids were identified in C. freundii, pNDM, pKPC, and a fusion plasmid (pNDM_KPC) co-harboring blaKPC-2 and blaNDM-1. All plasmids exhibited transconjugation capability, with significant differences in transfer efficiency (P < 0.05). pKPC demonstrated the highest efficiency (ranging from 10⁻¹ to 10⁻2), followed by pNDM_KPC (10⁻4 to 10⁻6) and then pNDM (around 10⁻⁸). IS26, located downstream of the intI site, was identified as a critical hotspot mediating the disintegration and integration of pNDM_KPC. Synergy effect and bactericidal activity were observed on aztreonam plus ceftazidime/avibactam or meropenem/vaborbactam or imipenem/relebactam. The presence of CRE isolates harboring fusion plasmid pNDM_KPC would pose a significant threat in nosocomial infections and should arise wide attention. IS26 downstream of IntI would be the hotspot driving the integration state of pNDM_KPC and contributing to complex resistance mechanisms.IMPORTANCEThe global rise of carbapenem-resistant Enterobacterales (CRE) poses a critical threat to public health. Our study identifies Citrobacter freundii strains co-harboring three distinct carbapenemase-encoding plasmids: pNDM, pKPC, and a novel fusion plasmid (pNDM_KPC). We reveal significant differences in conjugation efficiency among these plasmids, with the fusion plasmid demonstrating a moderate yet concerning ability to spread dual carbapenemase genes. This finding highlights the fusion plasmid as a potential vector for the co-dissemination of blaNDM-1 and blaKPC-2, emphasizing the need for enhanced surveillance. Furthermore, we identify the IS26 element downstream of the intI site as a potential recombination hotspot driving plasmid fusion. These insights deepen our understanding of carbapenemase gene transmission and call for global attention to the fusion plasmid-mediated resistance spread.

Brain abscess (BA) represents a severe, intracranial infection associated with high morbidity and mortality. Multiple pathogens may contribute to BA development, some of which are difficult to detect using conventional diagnostic methods alone. With the wide utilization of metagenomic next-generation sequencing (mNGS), more experiences are concluded in authenticity practice except for being regarded as a comprehensive method for detecting all pathogens. Here, we report a severe case of BA in a patient diagnosed by CT imaging, who subsequently underwent surgical removal of the abscess. To identify the causative pathogen and support anti-infection treatment, Gram staining, culture, and mNGS were performed on the abscess specimen. Interesting, we found the results were discordant: the Streptococcus constellatus was identified by culture, whereas mNGS predominantly detected anaerobic bacteria. Following additional human DNA removal, sequencing detected S. constellatus in the specimen as well. In conclusion, we highlight that a combined diagnostic strategy, leveraging both conventional culture and mNGS, is critical for comprehensive pathogen identification and informed clinical decision-making in BA.

Drosophila melanogaster Down Syndrome cell adhesion molecule 1 (Dscam1) gene encodes 38,016 diverse cell surface receptor proteins via alternative splicing, which have both nervous and immune functions. However, it remains elusive why organisms have evolved such an astonishing diversity of isoforms. Here, we show that fitness and immunity properties have driven the modern evolution of Dscam1 isoform diversity. We assess multiple aspects of fly fitness in deletion mutants harboring exon 4, 6, or 9 clusters, respectively, reducing ectodomain isoform diversity stepwise from 18,612 to 396. All fitness-related traits generally improved as the potential number of isoforms increased; however, the magnitude of the changes varied remarkably in a variable cluster-specific manner. Correlation analysis revealed that fitness-related traits were much more sensitive to reductions in Dscam1 diversity compared to canonical neuronal self/non-self discrimination. We conclude that the role of Dscam1 isoforms in canonical neuronal self-avoidance and self/non-self discrimination is mediated by a small fraction of all isoforms (<1/10), whereas a separate role essential for other developmental contexts and resistances, likely in fitness and immunity, requires almost full isoform diversity. Thus, fitness and immunity properties, rather than canonical neuronal functions, are the dominant drivers during the modern diversification of the Dscam1 isoform. Our findings suggest that Dscam1 diversity is closely linked to adaptation and species diversification in arthropods.

OBJECTIVE:The IMP-4 carbapenemase is an endemic cause of carbapenem resistance in the Asia-Pacific region. Our aim was to determine the dissemination mechanism of the blaIMP-4 gene. METHODS:Twelve representative Australian IMP-4 clinical isolates from The Alfred Hospital (Victoria, Australia) were characterised using antimicrobial susceptibility testing, with their genome and plasmid assemblies analysed. The conjugation efficiencies of different plasmids were investigated using filter mating with four recipient strains across two species. RESULTS:Selected IMP-4 isolates included six species and four genera (Enterobacter, Klebsiella, Serratia, and Acinetobacter), whereby isolates of the same species belonged to the same sequence type and were closely related. Four IMP-4 plasmid types were noted: IncHI2A types 1 and 2 (Klebsiella spp. and Enterobacter hormaechei, respectively), IncC (Serratia marcescens and Klebsiella pneumoniae), and a novel type in Acinetobacter pittii. Sequence homology was observed across all plasmids at the blaIMP-4 location, termed Region I, with IS26 on IncHI2A, and IS5075 and Tn3 resistance gene cassettes present on IncC plasmids. Genomic rearrangements mediated by IS26 or Tn3 and IS5075 were identified in Region I of plasmids from the same Inc type. The plasmids of each Inc type were capable of conjugative transfer with varying efficiency. IncH12A plasmids and K. pneumoniae IncC displayed higher transfer efficiencies than other plasmids examined in this study when using the recipient E. coli strain J53 (with conjugation efficiencies of 1.17×10-2 to 5.02×10-5, P < 0.001). CONCLUSIONS:Clonal spread, Inc type, homologous region, and insertion sequences are important mobility factors in the dissemination and evolution of blaIMP-4 plasmids.

BACKGROUND:Metagenomic Third Generation Sequencing (mTGS), based on nanopore technology, has emerged as a promising tool for the rapid diagnosis of pneumonia pathogens. However, this technology currently lacks standardised technical protocols, quality control measures, and comprehensive performance evaluations for the simultaneous detection of bacteria, fungi, and viruses in clinical settings. METHODS:We optimised the mTGS workflow by refining key parameters (cell wall lysis, fragment size selection, host DNA depletion, and sequencing depth) using reference samples and bronchoalveolar lavage fluid (BALF) from eight patients with pneumonia. These optimisations formed the basis for a standardised mTGS protocol. To assess the clinical diagnostic value of the optimised mTGS, a multicentre prospective cohort study involving 313 pneumonia-suspected patients was conducted. Each BALF sample was tested using conventional microbiological testing (CMTs), metagenomic next-generation sequencing (mNGS), pre-optimised mTGS, and optimised mTGS. FINDINGS:The optimised mTGS protocol, based on the refined cell wall lysis, fragment size selection, no host DNA depletion, and 800 MB sequencing depth, achieved a tenfold increase in sensitivity compared with pre-optimised mTGS for detecting the species of Bacillus subtilis, Mycobacterium tuberculosis, Mycobacterium avium, Cryptococcus neoformans, and Human papillomavirus in reference samples. In the prospective cohort, 274 patients with a confirmed diagnosis of pneumonia were identified, yielding 376 distinct microbes. The mTGS identified more microbes than CMTs (314 vs. 115), with a 45.30% increase in sensitivity (84.70% vs. 39.40%, P < 0.01, Chi-square test/Fisher's exact test). Compared with pre-optimised mTGS, the sensitivity of optimised mTGS increased by 32.51% (84.70% vs. 52.19%, P < 0.01, Chi-square test/Fisher's exact test). mTGS showed comparable performance to mNGS (84.70% vs. 79.90%, P = 0.14,Chi-square test/Fisher's exact test), both significantly outperforming CMTs. mNGS was more sensitive for detecting Non-tuberculous mycobacteria, Pneumocystis jirovecii, and Aspergillus spp., while mTGS demonstrated higher sensitivity for M. tuberculosis, Chlamydia psittaci, and Streptococcus pneumoniae. The overall diagnostic agreement between mTGS and clinical diagnosis was 81.80%. INTERPRETATION:We optimised and validated a standardised mTGS protocol that significantly improved the ability to detect pathogens in the BALF of patients with pneumonia. Optimised mTGS demonstrated comparable performance to mNGS, making it a promising tool for the aetiological diagnosis of pneumonia. FUNDING:The Research and Development Programme of Zhejiang Province (2023C03068, 2024C03187), the National Natural Science Foundation of China (82272338), the Key R&D Plan of the Ministry of Science and Technology (China) of China (2022YFC2504502).

Background Pseudomonas aeruginosa biofilms limit the efficacy of currently available antibacterial therapies and pose significant clinical challenges. Pseudomonal biofilms are complicated further when other markers of persistence such as mucoid and hypermutable phenotypes are present. There is currently a paucity of data regarding the activity of the newer beta-lactam/beta-lactamase inhibitor combination ceftolozane/tazobactam against P. aeruginosa biofilms. Methods We evaluated the efficacy of ceftolozane/tazobactam against clinical P. aeruginosa isolates, the laboratory isolate PAO1 and its isogenic mutS-deficient hypermutator derivative (PAOMS) grown under static and dynamic biofilm conditions. The clinical isolate collection included strains with mucoid and hypermutable phenotypes. Results Ceftolozane/tazobactam exposure led to a bactericidal (>= 3 log cfu/cm(2)) biofilm reduction in 15/18 (83%) clinical isolates grown under static conditions, irrespective of carbapenem susceptibility or mucoid phenotype, with greater activity compared with colistin (P < 0.05). Dynamically grown biofilms were less susceptible to ceftolozane/tazobactam with active biofilm reduction (>= 1 log cfu/cm(2)) observed in 2/3 isolates. Hypermutability did not affect the antibiofilm efficacy of ceftolozane/tazobactam in either static or dynamic conditions when comparing PAO1 and PAOMS. Consistent with the activity of ceftolozane/tazobactam as a potent inhibitor of PBP3, dramatic impacts on P. aeruginosa morphology were observed. Conclusions Our data demonstrate that ceftolozane/tazobactam has encouraging properties in the treatment of P. aeruginosa biofilm infections, and its activity is not diminished against mucoid or hypermutable variants at the timepoints examined.

OBJECTIVES:Neisseria gonorrhoeae strains associated with the high-level ceftriaxone-resistant FC428 clone or containing its main resistance determinant, penA allele 60.001, have shown global transmission. In Hangzhou, China, 10% of the isolates were associated with the FC428 clone in 2019. Here, we investigated ceftriaxone resistance and the prevalence of FC428-associated strains in Hangzhou in 2020-22. METHODS:A total of 209 gonococcal isolates were investigated for antimicrobial susceptibility to ceftriaxone and other antibiotics by agar dilution method. Sequence types and penA alleles were determined by PCR and sequence analysis. RESULTS:Resistance to ceftriaxone (MIC > 0.125 mg/L) was observed for 16% (33/209) of the isolates, whereas 6.7% (14/209) of the isolates displayed high-level ceftriaxone resistance (MIC = 1 mg/L). These 14 high-level ceftriaxone-resistant isolates and another isolate displaying an MIC = 0.25 mg/L contained penA allele 60.001, with eight of these isolates, all from 2020 to 2021 belonging to MLST ST1903, the sequence type commonly associated with the original FC428 clone. Importantly, the six penA allele 60.001-containing isolates from 2022 belonged to MLST ST8123, ST7365 and ST7367, which are among the most frequently encountered sequence types found in China. Therefore, these results indicate that endemic lineages in China have acquired penA allele 60.001. CONCLUSIONS:Here, we report continued transmission of gonococcal strains associated with the FC428 clone or containing penA allele 60.001 in Hangzhou. A major concern for public health is the acquisition of penA allele 60.001 by successful endemic lineages, which might enhance the transmission of this high-level ceftriaxone resistance trait.

OBJECTIVES:To assess the impact of mutations in the two-component sensor envZ on antibiotic resistance and virulence in the evolutionary dynamics of MDR Salmonella enteritidis (S. enteritidis). METHODS:Five S. enteritidis isolates obtained from a patient with multisite invasive infections were analysed. Analysis of antibiotic resistance genes, virulence genes and SNP was performed through WGS. RNA sequencing, quantitative RT-PCR, virulence testing in a Galleria mellonella (G. mellonella) infection model and in vitro cell experiments were used to examine the effects of envZ mutations. RESULTS:WGS revealed identical resistance and virulence genes on an IncFIB(S)/IncFII(S)/IncX1 fusion plasmid in all strains. The faecal strains harboured envZ mutations, reducing outer membrane protein ompD and ompF transcriptional level. Virulence testing demonstrated elevated virulence in envZ mutant strains. In vitro experiments revealed increased adhesion, invasion and phagocytosis resistance in envZ mutants, along with reduced biofilm formation and growth rates. CONCLUSIONS:These findings highlight novel genetic locations on envZ influencing antibiotic resistance and virulence in clinical S. enteritidis strains. envZ mutations impact antibiotic resistance by down-regulating ompD and ompF expression and enhance virulence, contributing to multisite infections with increased fitness costs.

Abstract Background The study aims were to evaluate the species distribution and antimicrobial resistance profile of Gram-negative pathogens isolated from specimens of intra-abdominal infections (IAI), urinary tract infections (UTI), respiratory tract infections (RTI), and blood stream infections (BSI) in emergency departments (EDs) in China. Methods From 2016 to 2019, 656 isolates were collected from 18 hospitals across China. Minimum inhibitory concentrations were determined by CLSI broth microdilution and interpreted according to CLSI M100 (2021) guidelines. In addition, organ-specific weighted incidence antibiograms (OSWIAs) were constructed. Results Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) were the most common pathogens isolated from BSI, IAI and UTI, accounting for 80% of the Gram-negative clinical isolates, while Pseudomonas aeruginosa (P. aeruginosa) was mainly isolated from RTI. E. coli showed < 10% resistance rates to amikacin, colistin, ertapenem, imipenem, meropenem and piperacillin/tazobactam. K. pneumoniae exhibited low resistance rates only to colistin (6.4%) and amikacin (17.5%) with resistance rates of 25–29% to carbapenems. P. aeruginosa exhibited low resistance rates only to amikacin (13.4%), colistin (11.6%), and tobramycin (10.8%) with over 30% resistance to all traditional antipseudomonal antimicrobials including ceftazidime, cefepime, carbapenems and levofloxacin. OSWIAs were different at different infection sites. Among them, the susceptibility of RTI to conventional antibiotics was lower than for IAI, UTI or BSI. Conclusions Gram-negative bacteria collected from Chinese EDs exhibited high resistance to commonly used antibiotics. Susceptibilities were organ specific for different infection sites, knowledge which will be useful for guiding empirical therapies in the clinic.

IntroductionThe dissemination of carbapenem-resistant Enterobacteriales (CRE) in nosocomial settings is primarily associated with the horizontal transfer of plasmids. However, limited research has focused on the in-host transferability of carbapenem resistance. In this study, ten isolates were collected from gut specimens of five individuals, each hosting two different species, including Escherichia coli, Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae, or Citrobacter koseri.MethodsSpecies identification and antimicrobial susceptibility were determined by MALDI-TOF MS and broth microdilution method. Carbapenemase genes were detected and localized using PCR, S1-PFGE and southern blot. The transferability of carbapenemase genes between species was investigated through filter mating experiments, and the genetic contexts of the plasmids were analyzed using whole genome sequencing.Results and discussionOur results revealed that each of the ten isolates harbored a carbapenemase gene, including blaNDM-5, blaNDM-1, or blaKPC-2, on a plasmid. Five different plasmids were successfully transferred to recipient cells of E. coli, K. pneumoniae or A. baumannii by transconjugation. The genetic contexts of the carbapenemase gene were remarkably similar between the two CRE isolates from each individual. This study highlights the potential for interspecies plasmid transmission in human gut, emphasizing the colonization of CRE as a significant risk factor for the dissemination of carbapenemase genes within the host. These findings underscore the need for appropriate intestinal CRE screening and colonization prevention.

The hospital setting serves as a critical conduit for pathogen dissemination, particularly amid the escalating concern of nosocomial infections in China. Currently, most studies use metagenomics to investigate microbial communities in hospital settings, with less focus on the transmission strategies of individual bacteria. In our study, we identified two Klebsiella pneumoniae strains exhibiting different mucoid characteristics. The strain designated as KPE was obtained from a well sanitized ward, while the strain KPH was isolated from the sputum samples of patients within the identical ward. We characterized the KPE strain as not lethal to mice and showed a distinct hypomuciod phenotype, strikingly different from the virulent KPH isolate. Two strains harbored the single nucleotide polymorphism (SNP) mutations in the virulence-related gene rmpA and wcaJ promoter regions, resulting in the downregulation of mucoid regulatory gene rmpA and capsule synthesis genes. Consequently, this led to diminished production of capsular polysaccharides and weakened virulence in the KPE strain. Furthermore, the KPE strain exhibited an elevated capacity for acquiring antibiotic-resistant plasmids and greater material survival ability. These findings indicated that mucoid changes enable K. pneumoniae strains to survive better on inanimate surfaces, promoting their persistence ward environment and further transmission in patients.

Human infectious diseases caused by bacterial pathogens, such as meningitis and pyaemia, threaten the public health worldwide due to their acute onset and high mortality. Sensitive and rapid detection of bacterial pathogens is urgent for the treatment of infectious diseases clinically. However, current techniques are generally time-consuming and require large-scale equipment. Plasmonic nanosensors built on metallic nanomaterials have emerged as excellent tools for biomedical diagnostics. Herein, we report an etching suppression strategy for the sensitive and rapid detection of bacterial pathogens through gold nanostars. Sulfhydryl-containing ligands suppress the iodide-mediated surface etching of gold nanostars, depending on the type, concentration, and size of sulfhydryl-containing ligands. Using mercaptophenylboronic acid (MBA) and Pseudomonas aeruginosa (P. aeruginosa)-specific aptamer as multifunctional ligands, we specifically identify bacterial pathogens through sulfhydryl-mediated etching suppression with a linear detection range from 10 to 106 CFU/mL and a limit of detection of 2 CFU/mL. This etching suppression-based strategy enables the detection of Neisseria meningitidis in the cerebrospinal fluid and P. aeruginosa in the serum within 15 min, showing great potential as a point-of-care platform for clinical diagnosis.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.