OBJECTIVES:To characterise the phylogenetic structure and clinical implications of carbapenemase-aerobactin (iuc) convergence among respiratory Klebsiella pneumoniae isolates. METHODS:We performed whole-genome sequencing of 707 non-duplicate respiratory K. pneumoniae isolates collected at a tertiary hospital in China from 2017 to 2023 and linked the genomic data to blinded clinical adjudication. Isolates were grouped by carbapenemase gene carriage and iuc status. Core-genome SNP phylogenies were constructed for ST11 and ST23, and reference-plasmid mapping to pK2044 was performed in a targeted subset. Within the carbapenemase-positive subset, logistic regression was used to test the incremental contribution of iuc beyond age, Charlson Comorbidity Index, and ICU admission during hospitalisation. All-cause mortality at 14, 28, and 90 days was assessed among infection cases. RESULTS:Overall, 548/707 isolates (77.5%) were adjudicated as infection, including 280 community-acquired pneumonia and 268 hospital-acquired pneumonia cases. Seventy-nine isolates (11.2%) were classified as carb+/iuc+, and 74/79 (93.7%) belonged to ST11. Phylogenetic analysis showed that convergence was concentrated in lineage-restricted ST11 branches, particularly KL64/KL47-associated backgrounds, whereas ST23 formed a more dispersed classical iuc-positive lineage background. Carb-/iuc+ ST23 retained a conserved hypervirulence-associated module and showed high compatibility with pK2044, whereas carb+/iuc+ ST11 showed a more fragmented virulence profile and heterogeneous, incomplete pK2044 coverage. Within the carbapenemase-positive subset, adding iuc to a model including age, Charlson Comorbidity Index, and ICU admission during hospitalisation did not improve infection attribution (adjusted OR 0.93, 95% CI 0.33-2.68; likelihood ratio test P = 0.892; ΔAIC = 1.98). Among infection cases, carb+/iuc+ and carb+/iuc- groups showed broadly similar all-cause mortality at 14, 28, and 90 days. CONCLUSIONS:Carbapenemase-aerobactin convergence among respiratory K. pneumoniae was concentrated in dominant ST11 backgrounds and occurred in distinct lineage-specific virulence contexts. However, within the carbapenemase-positive respiratory subset, iuc added limited value for infection attribution and did not clearly separate short- to intermediate-term mortality beyond host and care-setting factors.

ABSTRACT Acinetobacter baumannii usually causes high-mortality infections as the World Health Organization’s priority pathogen. Herein, we isolated a novel lytic phage pT2784 from hospital wastewater, specifically targeting the emerging Acinetobacter baumannii ST40-KL47 lineage and demonstrated significant antibacterial effects in in vitro and in vivo models. Morphological analysis showed that pT2784 is a myovirus with a head diameter of 54.53 ± 0.27 nm and a tail length of 71.96 ± 0.87 nm. Genomic analysis revealed that pT2784 possessed a 44,335 bp DNA genome with low identity (≤61%) to known phages, supporting its classification into a new genus within Caudoviricetes class. Phage pT2784 displayed an optimal multiplicity of infection of 0.01, a short latent period of approximately 5 min, and a burst size of 193 plaque-forming units (PFU)/cell. It maintained high stability over a broad temperature range (4°C–65°C) and pH range (2–11). Phage-resistant strains were isolated and purified following 24-h co-culture. Whole-genome sequencing identified missense mutations in itrA3 and gtr50, which encoded key enzymes involved in the early-stage pathway of capsule polysaccharide synthesis. Phenotypic analysis confirmed that phage-resistant strains exhibited capsule thinning, impaired phage adsorption, significantly reduced biofilm formation, and attenuated virulence, evidenced by over 80% survival in Galleria mellonella at 72 h, while none survived in the wild-type group by 12 h. Collectively, this study not only presented pT2784 as a promising therapeutic candidate but also elucidated the mechanism by which evolution under phage pressure promoted attenuated virulence in exchange for resistance, offering a crucial theoretical basis for developing phage-based interventions against Acinetobacter baumannii.IMPORTANCEThe rise of antibiotic-resistant Acinetobacter baumannii is a critical global health threat, urgently demanding new treatments. This study introduced a newly discovered phage, pT2784, which exhibited potent and specific lytic activity against Acinetobacter baumannii ST40-KL47. Crucially, we demonstrated that upon the emergence of phage resistance, the bacteria concurrently underwent a significant attenuation of their pathogenic potential. This work not only provided a promising new candidate for phage therapy but also revealed a fundamental trade-off where the path to phage resistance leads to attenuated bacterial virulence, offering a strategic advantage for managing challenging Acinetobacter baumannii infections.

Abstract Rapid and accurate pathogen identification is crucial for the clinical management of infectious diseases, particularly sepsis and severe respiratory infections, yet standard clinical workflows remain slow and resource-intensive. Here, we developed an automated, high-throughput imaging platform built on standard, clinically accessible bright-field microscopy, and generated a large dataset comprising 24.9 million label-free bacterial cells across six focal pathogens. Leveraging this resource, we trained a neural network (ESKAPe-ResNet) to identify ESKAPe species at the single-bacterium level. The model achieved >92% accuracy in species-level classification and >82% accuracy in quantifying ESKAPe abundance in mock mixtures, with high specificity against non-ESKAPe bacteria. In clinical validation using sputum, bronchoalveolar lavage fluid and blood samples from patients with respiratory infections and sepsis, the approach correctly identified the dominant ESKAPe pathogen in >78% of samples after minimum broth culture enrichment. The imaging-to-identification pipeline was completed in under 10 minutes, and coupled with brief cultivation, the median time to accurate identification was reduced to 5–6 hours, compared with days for conventional blood culture-based workflows. This work establishes the proof-of-principle for label-free, hardware-minimal rapid pathogen identification, providing a clinically deployable workflow to expedite diagnosis and reduce mortality in severe bacterial infections.

IntroductionGlobally, respiratory infections remain a leading cause of mortality, with treatment efficacy increasingly challenged by antimicrobial resistance. This study aimed to investigate the role of serum metabolites in the prognosis of severe human pneumonia.MethodsUntargeted and targeted serum metabolomics were performed on intensive care unit (ICU) patients. Experimental validation was conducted in a murine bacterial infection model and cellular models. RNA sequencing was used for mechanistic exploration to identify the signaling pathways regulated by the key metabolite.ResultsValeric acid, a short-chain fatty acid, was significantly elevated in survivors compared with non-survivors of severe pneumonia. In the murine Klebsiella pneumoniae model, valeric acid treatment alleviated infection severity, reduced body weight loss, lung inflammation, and bacterial load. Mechanistically, RNA sequencing revealed that valeric acid suppresses IL-17-associated inflammation and upregulates pathways related to mucociliary clearance. We further delineated the underlying mechanism, finding that valeric acid acts as a histone deacetylase (HDAC) inhibitor, specifically targeting HDAC3. This inhibition activates the canonical Wnt/β-catenin signaling pathway, leading to the upregulation of the master transcriptional regulator Foxj1 and subsequent promotion of cilia assembly and function in airway epithelia.DiscussionThe findings establish a protective role for the gut microbiome-derived valeric acid in respiratory infections via the novel HDAC-Wnt-FOXJ1 axis, revealing its potential as a therapeutic agent to improve clinical outcomes.

Objective To compare whether genetically proxied perturbation of major glucose-lowering drug targets is associated with susceptibility to lower respiratory infections and sepsis, with the aim of identifying clinically relevant target-specific differences in people with diabetes. Research Design and Methods We performed a two-sample drug-target Mendelian randomization study using variants proxying insulin signaling, thiazolidinediones, sulfonylureas, metformin-related targets, sodium-glucose cotransporter 2 inhibition, and glucagon-like peptide 1 receptor agonism. Outcomes included bacterial pneumonia, viral pneumonia, respiratory tuberculosis, influenza pneumonia, severe COVID-19, and sepsis from FinnGen and the UK Biobank. Positive-control analyses against type 2 diabetes were used to assess instrument validity before clinical interpretation. Results PPARG/thiazolidinedione proxies showed the clearest adverse pattern, with higher risks of bacterial pneumonia, respiratory tuberculosis, and influenza pneumonia, although estimates were imprecise. In contrast, insulin signaling and selected metformin-related targets were associated with lower risks of bacterial or viral pneumonia. Sulfonylurea proxies showed heterogeneous associations, including higher bacterial pneumonia risk but lower severe COVID-19 susceptibility. Glucagon-like peptide 1 receptor agonist proxies were not associated with infection outcomes. Sodium-glucose cotransporter 2 instruments failed positive-control validation for type 2 diabetes; corresponding infection estimates were therefore considered exploratory. Conclusions Genetically proxied glucose-lowering drug targets showed heterogeneous associations with respiratory infection and sepsis phenotypes. The most consistent adverse signals involved PPARG-related pathways, whereas insulin signaling and selected metformin-related targets showed potentially protective associations for specific outcomes. These findings support a more target-aware view of infection risk in diabetes and provide a rationale for prioritizing infectious outcomes when evaluating glucose-lowering therapies.

Antimicrobial resistance represents a significant global health threat, demanding alternative treatments beyond traditional antibiotics. Phage therapy has resurged as a promising solution to address this challenge. This manuscript offers an in-depth examination of phage applications in clinical settings, encompassing the treatment of multidrug-resistant infections, prevention of hospital-acquired infections, and development of phage-based vaccines. Advanced strategies are explored, including phage-antibiotic synergy, biomaterial-enhanced delivery systems to improve phage stability, and the rational design of engineered phages to expand host range and optimize lytic efficacy. Additionally, the application of genetic engineering to broaden phage host ranges and convert temperate phages into lytic variants is discussed. In hospital infection prevention, phages demonstrate substantial potential, such as eliminating bacterial biofilms on medical devices, disinfecting environmental surfaces, and controlling waterborne pathogens in hospital water systems. Furthermore, phages offer a versatile platform for vaccine development, facilitating efficient antigen display and nucleic acid delivery. Despite progress, challenges persist in pharmacokinetics, standardized production, and regulatory approval. This review synthesizes recent preclinical and clinical developments, emphasizing the transformative potential of phage-based therapies while acknowledging the barriers to their clinical implementation.

Introduction: Hospital-acquired pneumonia (HAP) is a major type of hospital-acquired infection with substantial morbidity. Large-scale epidemiological data on HAP in China remain limited. This multi-center retrospective study investigates the epidemiology, etiology, and risk factors of HAP to support clinical management. Methods: From January to December 2019, 543 patients from 37 tertiary hospitals meeting the enrollment criteria (who meet the diagnosis of HAP according to the Guidelines for the Diagnosis and Treatment of HAP in Adults in China and have clear clinical outcomes) were included. Patient data were retrospectively analyzed. Results: The study recorded 15.8% overall mortality. Multivariate analysis identified independent risk factors including secondary ventilator-associated pneumonia, admission to the intensive care unit, positive airway secretion culture, liver dysfunction, heart failure, cancer, long-term catheter indwelling, sequential organ failure assessment (SOFA) score >2 and quick SOFA (qSOFA) score >1 (p < 0.05). The top four pathogens of HAP were Acinetobacter baumannii (24.89%), Klebsiella pneumoniae (21.57%), Pseudomonas aeruginosa (13.73%), and Staphylococcus aureus (5.88%). Conclusion: Given the high mortality rate associated with HAP, prevention and early diagnostic assessment of HAP during hospitalization are paramount for these patients. The main pathogens of HAP are Gram-negative bacteria, which frequently exhibit substantial antimicrobial resistance. Therefore, antibiotics should be selected carefully.

Trial designAcute Respiratory Distress Syndrome (ARDS) remains a life-threatening critical illness with high mortality and limited specific therapies. This open-label, multicenter Phase I clinical trial aimed to evaluate the safety, tolerability, and preliminary efficacy of allogeneic human umbilical cord mesenchymal stem cells (hUC-MSCs, BC-U001) in patients with mild-to-moderate ARDS. A total of 12 eligible patients were enrolled into three dose groups following a “3 + 3” dose-escalation design from 2019 to 2024.MethodsAll patients received standard ARDS care plus a single intravenous infusion of BC-U001, with 28-day follow-up to assess safety and efficacy as well as exploratory immunological indicators (immunoglobulins, inflammatory cytokines, lymphocyte subsets).ResultsBaseline characteristics were balanced across groups except for more severe baseline lung injury in high dose patients (P = 0.027). No dose-limiting toxicity, treatment-related severe adverse events (SAE), or Suspected Unexpected Serious Adverse Reactions (SUSAR) were observed across all dose groups. The overall 28-day all-cause mortality rate was 8.3% (1/12), with 0% mortality among 5 COVID-19-related ARDS patients. Efficacy analysis showed significant improvements in the middle dose group, including a marked increase in PaO2/FiO2 (+100.07 mmHg, P < 0.001) and PaO2 (+21.88 mmHg, P = 0.002), as well as significant reductions in Lung Injury Score (LIS), Sequential Organ Failure Assessment (SOFA), and Acute Physiology and Chronic Health Evaluation (APACHE) II scores, without dose-dependent effects observed. Exploratory analyses preliminarily revealed that hUC-MSCs modulated the inflammatory response and restored immune balance.ConclusionsThese findings demonstrate that hUC-MSCs are safe and well-tolerated in mild-to-moderate ARDS patients, with the middle dose showing promising therapeutic effects. This trial provides critical data to support the design of future large-scale, randomized controlled trials to confirm the efficacy of hUC-MSCs for ARDS.

BACKGROUND:Ceftazidime/avibactam (CZA) resistance (CZAr) poses critical challenges for the treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. The early identification of high-risk populations is essential for controlling nosocomial transmission and guiding empirical therapy. This study aimed to systematically focus on the correlation between the clinical characteristics of patients and the microbial features of CZArCRKP. METHODS:We conducted a retrospective cohort study (January 2020-May 2023) of 97 patients with CRKP infection. Antimicrobial susceptibility testing and whole-genome sequencing (WGS) were used to analyze resistance phenotype-genotype correlations. Survival outcomes were evaluated using Kaplan-Meier analysis. RESULTS:CZArCRKP primarily caused lower respiratory (72.16 %) and urinary tract (16.49 %) infections, predominantly affecting elderly patients with comorbidities (81.4 %) or undergoing invasive procedures (63.9 %). Chronic renal failure combined with platelet counts > 149× 10⁹/L post-infection strongly predicted patients with CZArCRKP in pulmonary infections. NDM-1 gene carriage and OmpK36 mutations were associated with CZA resistance. CZA-treated patients demonstrated lower 14-day mortality rates. CONCLUSION:We established the first integrated host-pathogen risk model for CZArCRKP and identified chronic renal failure and platelet count as key clinical predictors. NDM-1/OmpK36 mutations represents a clear mechanism of resistance. CZA use may improve the survival of selected patients with CRKP. These findings advance the surveillance and therapeutic strategies for multidrug-resistant infections.

Background/Objectives: Meropenem pharmacokinetic variability in sepsis often leads to suboptimal exposure and therapeutic failure. Existing covariates like creatinine clearance (CLcr) only partially explain this variability. This study evaluated pyridoxic acid (PDA), an endogenous biomarker of OAT1/3 transporters, as a novel covariate to quantify active tubular secretion and explore pharmacokinetic/pharmacodynamic (PK/PD) linkages with clinical outcomes. Methods: A population PK (PopPK) model was constructed using data from a prospective septic cohort (n = 28). Subsequent exposure-response analysis was conducted in an expanded cohort (n = 49), and Monte Carlo simulations were utilized to evaluate various dosing regimens. Results: The PopPK analysis suggested that PDA may complement CLcr in characterizing meropenem clearance variability. While CLcr explained 10.7% of inter-individual variability (IIV) in clearance, the inclusion of PDA explained an additional 13.7%, reducing total IIV from 50.8% to 26.4%. Achieving a stringent target of 100%fT > 4MIC was significantly associated with a rapid decline in procalcitonin (p = 0.027), establishing a key PD endpoint. Simulations demonstrated that standard dosing (1 g q8h, 1 h infusion) is insufficient for patients with normal or augmented renal function. Target attainment was highly dependent on PDA levels. Conclusions: PDA is a valuable translational biomarker for OAT-mediated clearance. To achieve 100%fT > 4MIC, we recommend (i) 1 g q8h with 3 h infusion for patients with low CLcr and high PDA levels (MIC = 0.5 mg/L), and (ii) an intensified regimen of 2 g q8h with 3 h infusion for patients with normal CLcr and low PDA levels or high resistance risk (MIC ≥ 2 mg/L).

Gram-negative bacteria pose a significant threat to global health due to their high prevalence, multidrug resistance, and association with various infections. Zinc, an essential trace element, plays a dual role in host–microbe interactions, being crucial for both host immune function and bacterial metabolism. Maintaining optimal zinc homeostasis is essential for effective host defense and microbial survival. We comprehensively examine the multifaceted roles of zinc in host–microbe interactions, particularly focusing on its implications for the pathogenicity of Gram-negative bacteria and host immunity. As part of nutritional immunity, hosts have evolved zinc-mediated antimicrobial strategies that manipulate metal availability at the host–pathogen interface, including zinc limitation that restricts bacterial access to this essential nutrient and zinc intoxication that exposes invading bacteria to cytotoxic zinc concentrations. In Gram-negative bacteria, zinc homeostasis, maintained through zinc transporters and efflux systems, critically governs bacterial virulence, biofilm formation, and survival under host-imposed nutritional immunity. By integrating current advances, this review highlights zinc homeostasis as a central determinant of host–pathogen interactions and a promising target for combating multidrug-resistant Gram-negative infections. Understanding these complex zinc-mediated mechanisms provides new perspectives for developing metal-targeted therapeutic strategies.

Objective: To identify risk factors for progression to sepsis in patients with non-ventilator hospital-acquired pneumonia (NV-HAP) and to develop a practical nomogram for individualized risk assessment in this population. Methods: We retrospectively screened 408 hospitalized patients with hospital-acquired pneumonia at Peking University Third Hospital between January 2017 and December 2021. After excluding patients with an unclear diagnosis date or missing critical variables required for SOFA score calculation, 368 eligible patients with NV-HAP were included and randomly divided into a training cohort (n = 260) and an internal validation cohort (n = 108). An independent temporal validation cohort of 68 patients admitted between January 2022 and December 2022 at the same center was further used for temporal validation. Univariable and multivariable logistic regression analyses with backward stepwise selection were performed in the training cohort to identify predictors associated with progression to sepsis. A nomogram was then constructed based on the final model and evaluated by discrimination, calibration, and decision curve analysis. Results: A total of 368 patients were included in the model development dataset. The final multivariable model retained six predictors: male sex (OR = 2.393, 95% CI: 1.333-4.296), diabetes (OR = 2.205, 95% CI: 1.126-4.319), coagulation dysfunction (OR = 3.327, 95% CI: 1.726-6.413), PaO2/FiO2 (OR = 0.955 per 10-unit increase, 95% CI: 0.912-1.001), platelet count (OR = 0.900 per 10 × 109/L increase, 95% CI: 0.853-0.949), and bilirubin (OR = 1.176 per 1 μmol/L increase, 95% CI: 1.100-1.258). The nomogram showed acceptable performance, with an apparent C-index of 0.809 and a bootstrap-corrected C-index of 0.792 in the training cohort. The C-index was 0.750 (95% CI: 0.658-0.841) in the internal validation cohort and 0.754 (95% CI: 0.639-0.870) in the temporal validation cohort. Calibration analysis showed acceptable agreement between predicted and observed probabilities, and decision curve analysis indicated a positive net clinical benefit across clinically relevant threshold probabilities. Conclusions: In patients with NV-HAP, male sex, diabetes, coagulation dysfunction, lower PaO2/FiO2, lower platelet count, and higher bilirubin were associated with progression to sepsis. The developed nomogram showed acceptable discrimination, calibration, and clinical utility, and may serve as a practical tool for early individualized risk stratification in patients with NV-HAP.

Bloodstream infections (BSIs) are leading causes of sepsis-related mortality. Although metagenomic next-generation sequencing (mNGS) enables culture-independent pathogen detection, its clinical utility in plasma is limited by the overwhelming abundance of host cell-free DNA (cfDNA) and labor-intensive manual workflows. A plasma host DNA depletion mNGS (HD-mNGS) assay was developed which integrated nucleosome-targeted host DNA depletion with automated DNA extraction and library preparation. Analytical performance was evaluated through limit of detection, linearity, precision, and contamination control. Clinical performance was assessed in a cohort of 107 patients with suspected BSI and benchmarked against blood culture (BC), conventional microbiological testing (CMT), and standard mNGS without host depletion, using a composite clinical reference standard. Nucleosome depletion markedly reduced host DNA background by an average of 66-fold, consequently enriching microbial reads by approximately 46.73-fold. The automated HD-mNGS assay exhibited robust analytical sensitivity, with limits of detection (LoD) ranging from 9.1 to 38 genome equivalents (GE) /mL for bacteria and fungi, and from 283 to 321 GE/mL for viruses and excellent linearity across tested concentrations (R2 = 0.915–0.989). Furthermore, the automated workflow maintained strong quantitative correlation with manual protocols while significantly reducing common skin and environmental contaminants by 71.7

OBJECTIVES:Hypervirulent ceftazidime/avibactam (CAZ/AVI)-resistant Klebsiella pneumoniae (Kp) has emerged; however, its dynamic within-host evolution and competitive features are uncharacterized. This study aimed to clarify the systematic microevolution characteristics of the rapid transformation of blaKPC variants during long-term infection. METHODS:Thirty-nine Kp strains were isolated from a single patient with severe recurrent osteomyelitis during a 2-year period. Whole-genome sequencing and in vitro evolution assay was performed. Microbiological characteristics were examined through antimicrobial susceptibility testing, plasmid stability, growth curve, in vitro competition and Galleria mellonella larvae lethality assays. RESULTS:Among all the clinical Kp isolates, 37 were carbapenem-resistant Kp (CRKP), including 25 CAZ-/AVI-resistant Kp. All isolates belonged to the ST11-K47. During in vivo evolution, the blaKPC variant and its amplification emerged. Twenty-four isolates (24/39, 61.5%) harboured a novel blaKPC variant, blaKPC-144. All five Kp isolates carried blaKPC-2 in 2021. Surprisingly, 24 blaKPC-144-harbouring isolates (70.6%, 24/34) and 10 blaKPC-2-harboring isolates were identified in 2023, indicating rapid changing of blaKPC. Kp4 carried two copies of blaKPC-2, and Kp10-1 exhibited a 1.94-fold increase in the blaKPC-144 copy number. Similarly, in vitro, the blaKPC copy number increased upon exposure to low CAZ/AVI concentrations. However, at higher concentrations (4/1 mg/L), the blaKPC copy number increased significantly, and blaKPC mutations emerged simultaneously. The competition assay indicated that the blaKPC-144-harboring isolates exhibited a superior competitive capacity. CONCLUSIONS:The blaKPC amplification and mutation emerged simultaneously or sequentially during in vivo and in vitro evolution. Kp isolates harbouring blaKPC-144, conferring resistance to CAZ/AVI, exhibited a competitive advantage, promoting the rapid replacement of blaKPC-2.

Pyogenic liver abscess (PLA) is a serious intra-abdominal infection with high morbidity and mortality. Current diagnostic methods are time-consuming, inefficient and high-cost. Rapid identification/assessment of PLA and early recognition of bacterial liver abscess (BLA) improve prognosis. Iron metabolism disorders play a critical role in infections, but their role in PLA (especially ferritin) remains unclear. Notably, dietary nutrition, as a modulator of iron metabolism/immune function, may influence PLA progression, with limited research. In this study, we collected clinical data of 125 PLA patients from the Medical Information Mart for Intensive Care IV (MIMIC-IV) database to identify rapid, low-cost severity biomarkers, and verified them by an independent clinical cohort of 36 patients from Peking University Third Hospital. We analyzed the correlation between iron metabolism and disease severity, explored independent risk factors, and evaluated diagnostic efficiency via receiver operating characteristic (ROC) curves. Iron metabolism disorders were observed in patients with PLA and correlated strongly with liver dysfunction and systemic inflammation. Ferritin was associated with disease severity and served as an independent prognostic marker, with optimal cut-offs of 390 ng/mL for predicting severe PLA and 748 ng/mL for identifying BLA. This study identifies ferritin as a promising multifaceted biomarker for PLA which not only reflects disease severity but also aids in the early identification of bacterial etiology. Additionally, the findings provide a basis for dietary nutritional interventions in PLA management.