Specific labeling of outer membrane vesicles with antibiotic-conjugated probe reveals early bacterial infections in blood.

Bacterial outer membrane vesicles (OMVs) are nano-sized structures derived from the outer membrane of Gram-negative bacteria, which have emerged as key players in host-pathogen interactions, yet their potential as biomarkers remains largely unexplored due to the difficulty of identification in complex biological samples. Here we show an approach for detecting and quantifying bacterial OMVs in blood using a Polymyxin B-fluorescein probe (PmBF), which targets bacterial lipopolysaccharides (LPS). The probe selectively labels OMVs, enabling their differentiation from host extracellular vesicles and quantitative analysis using nano-flow cytometry. In male mouse models of pneumonia, we observe elevated serum PmBF EVs as early as 6 h post-infection, preceding positive blood cultures. In clinical samples, PmBF EVs show superior performance for diagnosing bacterial infections and differentiate them from virus or mycoplasma infections. Our findings highlight circulating PmBF EVs as promising biomarkers of bacterial infections.