This prospective observational translational cohort study will investigate whether extra-pulmonary infection caused by hypervirulent Klebsiella pneumoniae (hvKP) is associated with an increased risk of sepsis-associated acute lung injury (SALI), compared with infection caused by classical Klebsiella pneumoniae (cKP). The study will further examine whether circulating bacterial outer membrane vesicle (OMV) signals and ferroptosis-related biomarker profiles are associated with subsequent SALI development.

Adults with Sepsis-3 and microbiologically confirmed extra-pulmonary K. pneumoniae infection will be enrolled within 6 hours of sepsis recognition. Patients with acute lung injury at enrollment will be excluded from the primary cohort. Blood samples will be collected at enrollment, 24 hours, and 72 hours. Clinical isolates will undergo molecular characterization to classify infections as hvKP or cKP. The primary outcome will be new-onset SALI within 7 days after enrollment. A nested translational substudy will evaluate the effects of patient-isolate-derived OMVs on human pulmonary microvascular endothelial cells.

The study will not alter antimicrobial therapy, source control, respiratory support, fluid management, or any other aspect of routine clinical care.

Sepsis-associated acute lung injury is a major complication of severe extra-pulmonary Gram-negative bacterial infection. Hypervirulent Klebsiella pneumoniae (hvKP) is increasingly recognized as a cause of invasive infection with a high inflammatory burden and a propensity for metastatic spread. Bacterial outer membrane vesicles (OMVs) are biologically active nanoparticles that may transport lipopolysaccharide, virulence-associated molecules, and other inflammatory cargo to distant organs. Experimental evidence suggests that OMV-mediated oxidative stress and ferroptosis-related injury may contribute to pulmonary endothelial dysfunction and acute lung injury; however, the clinical relevance of these mechanisms in patients with extra-pulmonary hvKP sepsis remains unclear.

This prospective multicenter observational translational cohort study will investigate the relationship among molecularly characterized hvKP infection, circulating bacterial OMV-related signals, ferroptosis-related biomarker profiles, and the subsequent development of sepsis-associated acute lung injury. Adults with microbiologically confirmed extra-pulmonary Klebsiella pneumoniae sepsis will be enrolled early after sepsis recognition and followed during the acute phase of illness. Clinical isolates will undergo molecular characterization for prespecified hypervirulence-associated genes and additional strain-level features. Blood specimens collected during routine early follow-up will be processed using standardized procedures for plasma, serum, and peripheral blood mononuclear cell analyses.

The laboratory component will quantify circulating Gram-negative bacterial OMV-related signals in platelet-depleted plasma and evaluate systemic ferroptosis-related biomarker profiles, including lipid peroxidation products, glutathione redox status, iron-related indices, and ferroptosis-associated gene expression. Patient-derived Klebsiella pneumoniae isolates will also be cultured under standardized conditions for OMV isolation and characterization.

A nested translational substudy will compare OMVs derived from representative hvKP and classical Klebsiella pneumoniae isolates. Equal-particle-number OMV preparations will be applied to human pulmonary microvascular endothelial cells to assess lipid peroxidation, ferroptosis-related molecular changes, endothelial barrier integrity, and the in vitro rescue effect of Ferrostatin-1. No investigational intervention will be administered to study participants, and all clinical management will remain at the discretion of the treating physicians.

Inclusion Criteria:

• Age 18-85 years .
• Clinical suspicion of infection with acute organ dysfunction consistent with suspected sepsis at the time of enrollment, as determined by the treating clinical team.
• Presumed extra-pulmonary source of infection at enrollment, including but not limited to hepatobiliary, urinary, intra-abdominal, skin and soft-tissue, vascular catheter-related, or primary bloodstream infection.
• Blood cultures and/or clinically indicated source cultures obtained or ordered as part of routine clinical care.
• Enrollment and first research blood collection completed within 6 hours after initiation of the clinical sepsis evaluation.
• No evidence of acute lung injury at the index time point, according to the protocol-defined criteria.
• Written informed consent obtained from the participant or legally authorized representative, unless an ethics-approved deferred-consent procedure is used.

Exclusion Criteria:

• Suspected or confirmed primary pulmonary infection at enrollment, including community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, or aspiration pneumonia.

Acute lung injury or acute respiratory distress syndrome present at the index time point.

• Acute cardiogenic pulmonary edema, acute decompensated heart failure, or another condition that would preclude reliable adjudication of subsequent non-cardiogenic acute lung injury.
• Recent inhalation injury, near-drowning, major thoracic trauma, or transfusion-related acute lung injury.
• Previous enrollment in this study.
• Inability to obtain informed consent from the participant or legally authorized representative when deferred-consent procedures are not permitted by the local ethics committee.

Consecutively enrolled adults with clinically suspected sepsis caused by a presumed extra-pulmonary infection will be recruited from emergency departments, hospital wards, and intensive care units. Baseline blood samples will be collected during early sepsis evaluation, before culture results or bacterial molecular typing are available.

The primary analytic cohort will include participants who subsequently meet Sepsis-3 criteria, have microbiologically confirmed extra-pulmonary Klebsiella pneumoniae infection, have no acute lung injury at enrollment, and have an available clinical isolate for molecular characterization. Isolates will be classified as hypervirulent Klebsiella pneumoniae, classical Klebsiella pneumoniae, or indeterminate phenotype according to prespecified virulence-gene criteria. Patients with primary pulmonary infection, non-Klebsiella pneumoniae infection, mixed infection, culture-negative sepsis, or non-infectious diagnoses will not enter the primary analysis.

The decision regarding sharing of de-identified individual participant data has not yet been finalized. The research team will evaluate data-sharing feasibility after study completion, taking into account participant privacy, informed consent, institutional ethics requirements, data protection regulations, and the sensitivity of clinical and microbiological data.