ABSTRACT Phage therapy and phage-antibiotic combinations are promising strategies against multidrug-resistant pathogens like carbapenem-resistant Klebsiella pneumoniae (CRKP). However, the mechanisms underlying phage resistance and the molecular basis of the rational design of phage-antibiotic combinations require further investigation. Using genome-wide transposon screening, CRISPR-Cas9 knockout, and plasmid complementation in a clinical ST11-KL64 CRKP strain, we investigated susceptibility determinants for phage P545 and their impact on antibiotic susceptibility. We identified multiple phage resistance mechanisms, including disruption of phage receptor lipopolysaccharide (LPS) biosynthesis genes (involving deletions of waaQ, wabH, wabG, ugd, galU, and wcaG), inhibition of phage burst through an undefined pathway (involving deletion of sirB1), and increased mutation frequency (involving deletions of mutS and mutL). Furthermore, we found that deletions of LPS-related genes reduced resistance to multiple antibiotics, while mutS and mutL deletions increased resistance to several antibiotics. Further synergy assays showed that P545 in combination with meropenem, colistin, or ceftazidime had synergistic effects in the in vitro killing of host bacteria. By integrating genetic screening and functional validation, our approach offers a versatile platform for dissecting phage targets, understanding resistance mechanisms, and evaluating phage-antibiotic interactions. These findings provide valuable tools and insights for optimizing phage-phage and phage-antibiotic combination therapies against clinically significant multidrug-resistant pathogens.

Introduction:Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to public health. We characterized a rarely reported ST627-KL8 CRKP lineage associated with intensive care unit (ICU) transmission. Methods:Three isolates (ZJG29565, ZJG30140, and ZJG30146) were obtained from three patients in the ICU and subjected to antimicrobial susceptibility testing. Whole-genome sequencing (WGS) was performed to determine genomic characteristics, phylogenetic relationships, and plasmid content, followed by assessments of mucoviscosity, capsule quantification, serum resistance, and bacterial virulence using a Galleria mellonella (G. mellonella) infection model. Additionally, bacterial capsule morphology was observed via transmission electron microscopy (TEM). Results:SNP analysis (≤ 5 SNPs) confirmed clonal transmission within the ICU. Phylogenetic analysis placed ST627-KL8 as a distinct lineage closely related to ST14. All isolates carried an IncFIIK34 plasmid encoding bla KPC-2, consistent with their carbapenem-resistant phenotype. Phenotypic assays-including TEM, mucoviscosity testing, serum resistance, and uronic acid quantification-demonstrated a thinner capsule and reduced mucoviscosity compared with the KL2 reference strain. In the Galleria mellonella model, ST627-KL8 exhibited intermediate virulence (66.7%-76.7% survival), between the hypervirulent K. pneumoniae ATCC 43816 strain (30.0%) and the low-virulence K. pneumoniae ATCC 700603 strain (96.7%). Discussion:This study identified a novel ST627-KL8 CRKP clone with intermediate virulence, consistent with its reduced capsule phenotype and lack of classical hypervirulence genes. These features, together with the subtle clinical presentations, may contribute to reduced clinical vigilance and delayed optimization of antimicrobial therapy. Importantly, ST627-KL8 CRKP carried the IncFIIK34 bla KPC-2 plasmid, which has been reported to exhibit high conjugation frequency, posing a significant challenge in clinical settings.

ABSTRACT Klebsiella pneumoniae (K. pneumoniae) is recognized as a significant opportunistic pathogen capable of infecting both humans and animals. The emergence of multidrug-resistant strains presents a severe challenge to current antimicrobial therapies, necessitating the development of alternative treatments such as bacteriophages and their encoded enzymes. In this study, a polysaccharide depolymerase, designated DepZ57, was identified, expressed, and characterized from the K57-specific lytic phage vB_Kp_Z57. Bioinformatic analysis indicated that DepZ57 is a hydrophilic protein with a theoretical isoelectric point of 6.27 and adopts a typical β-helix structure. The purified depolymerase exhibited high physicochemical stability across a broad pH range (2.0–11.0) and temperature range (4°C–70°C). In vitro, the K57 capsule was degraded by DepZ57 at a minimum effective concentration ranging from 0.04 to 0.4 μg/mL, which subsequently sensitized the bacteria to macrophage phagocytosis and complement-mediated serum killing. In a lethal systemic mouse infection model induced by intraperitoneal injection, a 100% survival rate was achieved following the administration of 50 μg of DepZ57, compared with a 60% survival rate observed with the phage treatment. Bacterial burdens were effectively reduced by both treatments. Notably, the bacterial loads in the blood, lungs, and liver were significantly decreased in the DepZ57 treatment group. Specifically, the bacterial load in the blood was completely eliminated, and the bacterial loads in the liver and lungs were reduced by more than 99%. Histopathological analysis confirmed that DepZ57 treatment effectively prevented hepatic necrosis and pulmonary inflammatory infiltration. Collectively, these findings demonstrate the in vivo efficacy and stability of DepZ57, suggesting it may represent a viable candidate for the control of K. pneumoniae infections.IMPORTANCEThe emergence of multidrug-resistant and hypervirulent Klebsiella pneumoniae represents a severe threat to human health and the dairy industry. Capsular polysaccharide (CPS) is the primary virulence factor that shields K. pneumoniae from host immune clearance, and the hypervirulent K57 serotype is frequently linked to severe invasive infections. Phage-derived depolymerases have emerged as promising antivirulence agents capable of specifically dismantling bacterial capsules without inducing bacterial resistance. Here, we characterized a novel, highly stable phage depolymerase, DepZ57, which exhibits robust tolerance to extreme pH and temperature conditions and specifically targets K57-type CPS. Distinct from the parental phage, DepZ57 provides full protection against lethal K57 K. pneumoniae infection in vivo and effectively alleviates infection-induced tissue damage. This work highlights the potential of phage depolymerases as stable, safe, and efficient nonantibiotic therapeutics for the prevention and control of hypervirulent K. pneumoniae infections.

Epidermal growth factor receptor(EGFR)mutations have been classified into four distinct subgroups based on the characteristics of the kinase domain and their sensitivity to tyrosine kinase inhibitors (TKIs).However, literature regarding the distribution and biological features of these structural subtypes among Chinese patients with non-small cell lung cancer (NSCLC) remains limited.This retrospective study aims to further investigate the distribution characteristics of EGFR mutations and the sensitivity of complex EGFR mutations to TKIs in Chinese patients. A total of 452 patients diagnosed with NSCLC were enrolled in this study. All samples were screened for EGFR, KRAS, ALK, ROS1, MET, ERBB2, RET, BRAF, and NTRK1/2/3 mutations using next-generation sequencing (NGS). EGFR mutations were systematically categorized into four subgroups based on the structural classification method. Drug sensitivities to various EGFR-TKIs were analyzed separately in 28 patients with compound EGFR mutations and 39 patients with EGFR co-mutations. Additionally, the correlation between driver gene mutations and clinicopathological characteristics was also evaluated. Approximately 81.64

Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a significant threat in neonatal intensive care units (NICU) because of its high antimicrobial resistance and association with increased mortality rates. In our study, we identified three blaNDM-21-harboring CRKP isolates from three critically ill pediatric inpatients; these isolates underwent comprehensive analysis. Our investigations included assessments of their clinical spatiotemporal distribution, antimicrobial susceptibilities, whole-genome sequencing (WGS), biological properties, and genomic and plasmid replicon typing. The three blaNDM-21-harboring CRKP strains, which belong to sequence type 35 (ST35), harbored the gene on IncX3 plasmids. Clinical spatiotemporal and WGS analyses indicated that the third case resulted from transmission from the second case within the NICU, whereas the first case remained epidemiologically unrelated. All three isolates displayed robust growth, formed strong biofilms, exhibited low mucoviscosity, possessed distinct capsule structures observable under an electron microscope, and demonstrated pathogenicity and virulence via in vitro experiments. Moreover, in mouse models, blaNDM-21-harboring CRKP showed significant multi-organ invasiveness and pathologic damage at an inoculation level of 106 CFU, leading to the death of all infected mice. This study provides evidence of the emergence of blaNDM-21-harboring CRKP with enhanced virulence-associated features in pediatric/NICU-associated infections, thereby highlighting the convergence of rare carbapenemase-mediated resistance, virulence potential, and a probable risk of nosocomial transmission in a vulnerable patient population.

The global emergence of hypervirulent Klebsiella pneumoniae (hvKP) and extensively drug-resistant (XDR) phenotypes has precipitated a severe public health crisis with limited therapeutic options. In particular, the hypervirulent ST65 and ST23 lineages, alongside the carbapenem-resistant ST11 lineage, have been reported to be emerging globally with the potential for continued large-scale dissemination. Despite the urgent need for preventive strategies, the regulatory networks governing hvKP virulence and their potential as targets for live attenuated vaccines remain inadequately explored. In this study, we elucidate a pathogenic mechanism driven by the transcription factor RpoE. Deletion of the rpoE gene in hvKP significantly attenuated virulence by directly downregulating the expression of type 3 fimbriae (T3F) and subsequently impairing biofilm formation. Based on this marked virulence attenuation, we evaluated the ΔrpoE strain as a potential live attenuated vaccine candidate. Remarkably, immunization elicited protective immune responses involving functional antibody responses and CD4+ T cell-associated immunity, conferring host protection against the tested ST11, ST23, and ST65 clinical isolates. Our findings define an RpoE-T3F regulatory axis involved in hvKP virulence and support ΔrpoE as a potential live attenuated vaccine candidate with protective efficacy against these tested clinically relevant KP strains.

Objectives. Carbapenem-resistant Klebsiella pneumoniae (CRKP) strains harbouring bla KPC and bla NDM are prevalent in China, while bla OXA-48-positive CRKP remains uncommon. This study reports one of the first outbreaks of a nosocomial infection caused by ST101 bla OXA-48-positive CRKP in China.Methods. We conducted a retrospective molecular epidemiological study of 269 CRKP isolates collected at a tertiary hospital in China, between 2012 and 2022. Antimicrobial susceptibility testing, whole-genome sequencing, phylogenetic analyses and plasmid characterization were performed to investigate the clinical and molecular features of bla OXA-48-CRKP. Conjugation assays were used to assess plasmid transferability.Results. Among the 269 CRKP isolates, we identified 61 bla OXA-48-positive strains from 38 patients. Most cases were linked to the intensive care unit (ICU), with patient transfers facilitating spread to non-ICU wards and branch hospitals. The outbreak was associated with a high mortality rate (63.2%), including 42.1% within 30 days. Genomic analysis revealed that 59 isolates belonged to ST101 and carried multiple resistance determinants. All bla OXA-48 genes were located on IncL plasmids, which evolved into three structural variants during the outbreak. Notably, the late-emerging Type III plasmid had lost conjugative transferability. Core-genome SNP analysis demonstrated clonal expansion within the hospital, while phylogenetic comparisons indicated the likely introduction of ST101 strains from Europe. Global phylogenomic analysis revealed complex intercontinental transmission pathways, underscoring the lineage's high potential for global dissemination.Conclusions. We documented the first hospital outbreak in China caused by ST101 bla OXA-48-positive CRKP. This study improves our understanding of plasmid evolution during nosocomial outbreaks and provides valuable insights to guide infection control strategies of plasmid-mediated resistance.

OBJECTIVE:This study aimed to characterize the IncHI2 plasmids co-harbouring carbapenemase genes and mcr-9 genes in carbapenem-resistant Enterobacter hormaechei (CREH) isolated from a children's hospital in Suzhou, China. METHODS:Whole-genome sequencing (WGS) was employed to determine the genome sequences of 12 non-duplicate CREH strains. The molecular features of CREH strains and IncHI2 plasmids harbouring carbapenemase genes with or without mcr-9 genes were characterized through genomic analysis. Conjugal transfer assay and plasmid stability testing were used to evaluate the transfer ability and stability of IncHI2 plasmids. RESULTS:The genomic analysis revealed that 12 CREH strains all carried carbapenemase genes blaNDM-1, blaNDM-5 or blaIMP-26. Although the sequence types (STs) of these 12 strains were relatively diverse, most of the strains (11/12) carried IncHI2 and IncHI2A replicons. Further plasmid analysis showed that carbapenemase genes blaIMP-26 and blaNDM-1 could coexist with mcr-9.1 or mcr-9.2 on IncHI2 plasmids. While the backbones of these IncHI2 plasmids were relatively conserved, the accessory modules carrying the carbapenemase and mcr-9 genes exhibited diverse genetic contexts, involving transposon Tn125, Tn1696 and Tn6725, and integron In27-like, In615b, and In837. Conjugal transfer assay and plasmid stability testing confirmed the transferability and stability of IncHI2 plasmids. CONCLUSIONS:This study reports the emergence and diversity of IncHI2 plasmids that co-harbour carbapenemase genes and mcr-9.1/9.2 in E. hormaechei. To our knowledge, this is also the first report of blaNDM-1 and mcr-9.2 being carried on a single plasmid in China. The prevalence of such evolutionarily diverse and conjugative carbapenemase-encoding IncHI2 plasmids warrants close monitoring.

The continuous evolution of NDM variants, some of which exhibit enhanced hydrolytic activity, poses a serious public health threat and necessitates ongoing surveillance. This study aimed to investigate the clinical and molecular characteristics of Escherichia coli strains producing NDM-9 and NDM-13 variants. Six blaNDM-9-carrying and two blaNDM-13-carrying E. coli strains were isolated from six hospitals in China. MALDI-TOF MS and Vitek-2 systems were used for identification and antimicrobial susceptibility testing (AST). Phenotypic detection of carbapenemases was conducted using a combined disc test. Multi-locus sequence typing (MLST) was used to assess clonal relatedness among the eight E. coli isolates. Whole genome sequencing (WGS) and bioinformatics analysis were used to characterize the blaNDM-9 and blaNDM-13-carrying strains. Phylogenetic trees and core SNP comparisons were generated using isolates from this study and publicly available sequences from the GenBank database. Conjugation experiments were performed to evaluate the horizontal transferability of blaNDM genes. Plasmid stability was assessed by serial passage for 10 days without antibiotic selection. All E. coli isolates exhibited a multiple drug resistance phenotype, but remained susceptible to amikacin and tigecycline. The E. coli types were diverse by MLST, including ST1 (n = 2), ST19 (n = 2), ST2 (n = 1), ST664 (n = 2), and one novel ST type. WGS analysis identified 41 and 49 resistance genes and virulence factor genes. The blaNDM-9 genes were carried by IncHI2-type plasmids (n = 4) or IncK2-type plasmids (n = 2), while all blaNDM-13 genes were exclusively carried by IncX3-type plasmids (n = 2). Both variants were associated with Tn125-related transposons. Phylogenetic analysis of core SNPs revealed that 52 E. coli strains (including 44 genome sequences from GenBank) were divided into six clustering clades with SNP differences ranging from 2 to 10,625. Two blaNDM-13-carrying isolates differed by only two SNPs. Conjugation experiments confirmed the successful transfer of blaNDM-9 and blaNDM-13 to E. coli C600, and plasmids remained stable after 10 days of passage. The detailed clinical and molecular characteristics of NDM-9 and NDM-13 producing CR-EC enhance our understanding of the evolution and dissemination of NDM variants. These insights highlight the further comprehensive surveillance to evaluate the public-health risk of NDM variants producing strains.

Antimicrobial-resistant pathogens pose an existential threat to modern medicine, yet the evolutionary forces driving their adaptation in healthcare systems remain largely unexplored. We revealed that hospital network architecture functions as a primary selective pressure, driving pathogen evolution through infrastructure-dependent virulence-transmission trade-offs. Phylogenomic analysis of 5,023 Acinetobacter baumannii isolates across China's centralized healthcare system identifies two co-existing evolutionary strategies: a virulence-optimized clade (ESL2.4) that spread slowly (20.4 km per year) in low-connectivity hospitals, and a transmission-optimized clade (ESL2.5) that disseminate rapidly (65.2 km per year) through mega-city healthcare hubs, likely attributed to its capsule conversion and increased upper respiratory colonization. Comparative analysis with European A. baumannii populations demonstrates that healthcare connectivity, not geography, governs pathogen distribution through convergent genomic adaptations. Our simulation suggests competitive asymmetries of the two clades, following the ecotype principle and enabling stable coexistence. The COVID-19 pandemic provided a natural experiment validating these mechanisms: outpatient restrictions reduced transmission-optimized lineage spread by 89%, while virulence-optimized lineage persisted through inpatient networks. These findings establish healthcare infrastructure as a critical evolutionary driver with immediate implications for predicting and controlling antimicrobial resistance emergence across diverse healthcare systems. ### Competing Interest Statement The authors have declared no competing interest. National Natural Science Foundation of China, 82530102, 32170003, 32370099 Provincial-level Talent Program for National Center of Technology Innovation for Biopharmaceuticals, NCTIB2024JS0101 Natural Science Foundation of Jiangsu Province, BK20243008 Suzhou Top-Notch Talent Groups, ZXD2022003 Shenzhen Medical Research Fund, B2403009 Sichuan Provincial Healthcare and Health Promotion Association, KY2024SJ0018

Klebsiella pneumoniae is an important opportunistic pathogen in both humans and animals. Controlling it has become increasingly difficult due to the rapid spread of antimicrobial resistance. In this study, we isolated and characterized a novel lytic bacteriophage, vB_Kp_Z1, and evaluated its therapeutic efficacy against K1-serotype K. pneumoniae. Host range analysis showed that vB_Kp_Z1 was strictly specific to K1 strains, as confirmed across multiple prevalent capsular types. The in vivo efficacy of vB_Kp_Z1 was assessed using intraperitoneal infection models in mice. Two hypervirulent K1 strains were used: a pigeon-derived strain (KP1897) and a human clinical strain (KP177). Phage treatment significantly improved survival compared with phosphate-buffered saline-treated controls. It provided complete protection in KP1897-infected mice and achieved an 87.5% survival rate in KP177-infected mice. In addition, phage administration markedly reduced bacterial loads in the blood, liver, and lungs, indicating effective control of systemic dissemination. These findings demonstrate that vB_Kp_Z1 is a K1-specific bacteriophage with therapeutic potential against hypervirulent K. pneumoniae, including strains from different host species.

AIMS:The worldwide spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) has posted a global threat. Treatment options for CRKP are limited, especially for strains producing New Delhi metallo-β-lactamase (NDM). The emergence of the siderophore antibiotic cefiderocol has brought hope; however, recent clinical and research data show that NDM-producing K. pneumoniae have a high rate of resistance to cefiderocol, and the reason remains unclear. METHODS:This study focused on the dynamic changes in K.pneumoniae under host nutritional immunity and cefiderocol stress. It identified one of the main reasons for the strong cefiderocol resistance in NDM-producing strains. Further studies found that the JNMCOFLA_01041 gene plays an important role in this process and is crucial for cefiderocol resistance and virulence in K.pneumoniae. RESULTS:Under host nutritional immunity and cefiderocol stress, K.pneumoniae is exposed to increasing oxidative stress. This oxidative stress results in a dynamic pattern of rapid increases in intracellular Zn²⁺ concentrations. This autonomous changes in Zn²⁺ levels in response to cefiderocol challenge can rapidly increase NDM enzyme activity and enhance the MIC, which may be one of the main reasons for the high cefiderocol minimal inhibit concentration (MIC) in NDM-producing strains. Further study identified a key gene, JNMCOFLA_01041, for Zn²⁺ and hemin absorption in K. pneumoniae. The Zn²⁺ acquisition function plays an important role in oxidative stress resistance, NDM activity, metabolism, and even pathogenicity. CONCLUSIONS:Taken together, our research highlights the impact of dynamic changes in bacterial metal ion concentrations on bacterial survival and drug resistance under complex host conditions. It emphasizes that, while using antimicrobial drugs, monitoring and regulating changes in the bacterial internal environment under pathological conditions is crucial for maximizing anti-infective efficacy. Furthermore, JNMCOFLA_01041 could be a target of novel drug and vaccine development.

Immune checkpoint inhibitors (ICIs) targeted PD-1/PD-L1 axis generate immune-related adverse events such as myocarditis, limiting their clinical application. Herein, we tried to explore the potential mechanism of ICIs-induced myocarditis. We performed single-cell RNA sequencing of heart tissues and peripheral blood mononuclear cells (PBMC) collected from mice with or without a relative low dose of PD-1/PD-L1 inhibitor (BMS-1) treatment. Compared with PBS treatment, BMS-1 treatment increased T, B, NK cells, and neutrophils but decreased macrophages in the heart. Four T cell subclusters in the heart were identified, including Treg, LEF1+CD4+ T, CCL5+CD8+ T, and STMN1+CD8+ T cells. The BMS-1-heart exhibited increased CCL5+CD8+ T cells depicted by elevated Nkg7 and Ccl5 gene expression compared with the PBS-heart. The number of macrophages declined but revealed inflammatory activity in the BMS-1-heart. Interestingly, CCR5, a receptor for CCL5 expressed in both CCR2- resident and CCR2+ recruit macrophages in the heart, was upregulated by the BMS-1 treatment. In addition, fibroblasts, not endothelial cells, showed an inflammatory activation state. Last, we identified increased CCL5+CD8+ T cells in the BMS-1-PBMC. Immunofluorescence staining also confirmed significantly elevated CCL5+CD8+ T cells in the BMS-1-heart than that of PBS-heart. BMS-1 seems to recruit circulating CCL5+CD8+ T cells to the heart, which further interact with CCR5+ macrophages, resulting in fibroblast activation. The CCL5/CCR5 axis and circulating CCL5+CD8+ T cells may be potential therapeutic/diagnostic strategies for ICIs-induced myocarditis.

Linezolid-resistant Clostridioides difficile, conferred by the acquisition of cfr-like genes, has been reported in Europe and America. However, the emergence of linezolid resistance in C. difficile in the Asia-Pacific region and its impacts on C. difficile pathogenicity remain unclear. In this study, 881 C. difficile isolates from the Asia-Pacific region were screened for cfr-like genes. Whole genome sequencing was performed on 16 cfr-like gene-positive isolates from four countries. Thirteen isolates possessed cfr(B), which was located within Tn6218, while three isolates possessed cfr(C), which was located within the integrative and conjugative elements (ICE) F548 and DA275. Fourteen (87.5%, 14/16) of the cfr-like gene-positive isolates were resistant to linezolid. In comparison to the two isolates susceptible to linezolid, these 14 isolates exhibited significantly higher mRNA expression levels of cfr(B) and cfr(C), along with significantly higher bacterial density at 12 h. Conversely, they demonstrated reduced abilities for sporulation and biofilm formation. After the cfr(B) gene was knocked down by the CRISPR interference, the C. difficile strain presented lower bacterial density at 12 h, higher toxin production and stronger sporulation and biofilm formation abilities. Our findings reveal the emergence of cfr-like genes C. difficile isolates in the Asia-Pacific region, highlighting that cfr-like genes not only mediate linezolid resistance but also contribute to regulating pathogenic potential. Linezolid resistance in CDI should be closely monitored in specific patients.

Antibiotic resistance is arguably one of the greatest threats to global health today. The worldwide emergence of multidrug-resistant and hypervirulent Klebsiella pneumoniae underscores the urgent need for alternative treatments. Bacteriophages (phages) are considered one of the most promising alternatives to address this crisis. In this review, we summarize current knowledge of phage–host interactions and highlight recent advances in phage therapy against K. pneumoniae, including phage cocktails, antibiotic combination therapy, and treatments based on phage-derived proteins. Despite their tremendous therapeutic potential, significant challenges remain. We therefore also discuss strategies to optimize phage research and recent innovations in the field.

Antimicrobial resistance (AMR) constitutes one of the most severe and pressing threats to global public health, food security, and environmental integrity. This review synthesizes current evidence across interconnected One Health domains—humans, animals, food, and the environment—to delineate the scope, mechanisms, and drivers of AMR transmission. Our analysis reveals three principal findings. First, the scope of AMR is alarmingly extensive, with antibiotic-resistant bacteria (ARB) and genes (ARGs) now pervasive across all four ecological compartments, transcending traditional clinical boundaries. Second, this widespread distribution is critically facilitated by horizontal gene transfer mechanisms, particularly via mobile genetic elements such as plasmids, which enable ARGs to disseminate rapidly between diverse bacterial populations across different ecosystems. Third, we identify multiple interconnected drivers that actively promote this cross-ecosystem spread, encompassing both evolutionary and transmission drivers. By characterizing these critical transmission pathways and underlying drivers, this review provides an integrated framework to identify critical transmission risks and inform integrated strategies for mitigating antimicrobial resistance across One Health domains.

With the rapid increase in the number of implant operations, the incidence of bone infections has increased. Methicillin-resistant Staphylococcus aureus (S. aureus) and other emerging fully drug-resistant strains make the management of bone infections even more challenging. Bone infections are mainly caused by S. aureus and require extensive surgical intervention and long-term antibiotic therapy. The host autophagy response is critical to the elimination of S. aureus infections. In this study, we demonstrate that a circular RNA (circRNA), circSyk, is a potential biological target for the treatment of S. aureus-induced bone infection. Most importantly, S. aureus regulates circSyk to block autophagy and promote bone destruction via the circSyk/miR-5106/Sik3 axis in a nonclassical pathway, which is involved in the S. aureus infection process through a competitive endogenous RNA network. In summary, this study proposes a novel perspective on the immune escape of S. aureus in bone infections, based on circRNA.

ObjectivesCarbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a significant public health challenge. This study investigated the molecular epidemiology, antimicrobial resistance patterns, clinical characteristics, and risk factors of CR-hvKP infection in Huaian, China.MethodsWe retrospectively studied patients infected with carbapenem-resistant K. pneumoniae (CRKP) between November 2022 and September 2024. Whole-genome sequencing was used to detect carbapenemase, virulence, capsular serotype-related genes, and plasmid types in 374 CRKP isolates.ResultsAmong them, 57.49% (215/374) strains met the criteria for CR-hvKP. The most common type was blaKPC-2-producing ST11(98.60%, 212/215), whereas K64 (56.74%, 122/215) and KL25 (39.53%, 85/215) were the main capsular serotypes. The CR-hvKP strains showed significantly higher resistance to the tested antibiotics, except for ceftazidime/avibactam and colistin. Resistance rates of CR-hvKP to the three tested antibiotics (minocycline, cotrimoxazole, and amikacin) were higher than those of CRnon-hvKP. Phylogenetic analysis based on whole-genome single-nucleotide polymorphisms divided the 251 isolates into four independent branches, with branch 2 being the most prevalent, indicating high clonality among the strains. Multivariate analysis showed diabetes [odds ratio (OR) = 3.771] and surgery (OR =2.042) to be independent variables associated with CR-hvKP infection.ConclusionsNotably, the ST11 lineage carrying blaKPC-2 has emerged as a dominant high-risk clone in Huaian. Given the wide distribution of these novel CR-hvKP isolates, global monitoring and stricter control measures should be implemented to prevent their further spread in hospital settings.

The emergence of metallo-beta-lactamase gene variants, such as blaIMP-26, has posed a significant challenge to bacterial infection treatment, drawing considerable attention in public health. This study aims to investigate the resistance mechanism of blaIMP-26-carrying clinical Enterobacter xiangfangensis in China. blaIMP-26-harboring carbapenem-resistant Enterobacteriaceae (CRE) isolates were collected from a multicenter study across China. The blaIMP-26 was identified by polymerase chain reaction (PCR). Multi-locus sequence typing (MLST) and phylogenetic analysis were conducted to investigate the genetic evolutionary characterization of Enterobacteriaceae carrying blaIMP-26. The genomic contexts of these strains were explored by whole genome sequencing, while conjugation and plasmid stability assays were conducted to assess the transferability and maintenance of the resistance plasmids. Furthermore, the antibiotic susceptibility of IMP-26 to β-lactams was evaluated through antimicrobial susceptibility testing, enzyme kinetic analysis, and molecular docking. Five blaIMP-26-carrying strains were collected, and all were multidrug-resistant Enterobacter xiangfangensis. blaIMP-26 was located either on IncHI2/2A or the novel IncpKPC-CAV1321 plasmid, both exhibiting high and stable self-transfer frequency. In addition, blaIMP-26 was identified within a novel class 1 integron In437, or the classical integron In837. Antimicrobial susceptibility testing and enzyme kinetic assay further showed that IMP-26 could mediate high levels of resistance to common carbapenem and cephalosporin antibiotics. This study characterized the genomic and clinical characteristics of blaIMP-26-carrying clinical Enterobacter xiangfangensis in China. The high carbapenem resistance and transmission capacity of blaIMP-26 highlight the need for enhanced surveillance and preventive measures to contain the spread of blaIMP-26.IMPORTANCEOur research has led to the documentation of a novel IncpKPC-CAV1321 plasmid and the discovery of a novel integron In437, both carrying the blaIMP-26 gene. A comprehensive analysis of the carbapenem resistance levels and enzymatic kinetics exhibited by IMP-26 revealed that IMP-26 could mediate high levels of resistance to common carbapenem and cephalosporin antibiotics. Our findings underscore the critical need for enhanced surveillance and preventive measures to curtail the dissemination of blaIMP-26.