P19 - Bacterias

Vesicle-mediated PDC β-lactamase release from cystic fibrosis pathogens: a functional and expansion microscopy study

Hita, Francisco ¹ - Cassanelli, Martin ² - Tribelli, Paula ³ - Smania, Andrea ⁴ - Vila, Alejandro ^1,5 - López, Carolina ¹

1) Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Santa Fe, Argentina.
2) Hospital General de Niños “Pedro de Elizalde”, Buenos Aires, Argentina.
3) Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina.
4) Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET), Departamento de Química Biológica Ranwel Caputto, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.
5) Universidad Nacional de Rosario (UNR), Rosario, Santa Fe, Argentina.
Contacto: hita@ibr-conicet.gov.ar

Pseudomonas aeruginosa is an opportunistic pathogen and a major cause of chronic respiratory infections in cystic fibrosis (CF) patients. Its persistence in the CF lung is frequently accompanied by co-infection with other microbes, such as Staphylococcus aureus, a combination known to accelerate disease progression. Both pathogens exhibit remarkable adaptability and resistance to antibiotics, complicating long-term treatments. In P. aeruginosa, a key resistance mechanism is the production of β-lactamases, including the class C enzyme PDC.
Here, we investigated the extracellular release of PDC-1 via membrane vesicles (MVs) and its potential role in community-level antibiotic resistance. Upon exposure to β-lactams such as penicillin (PenG) and cefoxitin (FOX), we detected PDC-1 in MVs isolated from P. aeruginosa PAO1. Nitrocefin assays confirmed that vesicle-associated PDC-1 remains catalytically active. However, these MVs failed to protect β-lactam-susceptible P. aeruginosa and E. coli strains from FOX or ceftazidime. Notably, incubation with these MVs caused growth defects in E. coli, suggesting a toxic effect of the vesicles under these conditions. These results prompt us to study naturally occurring PDC variants with enhanced catalytic efficiency or higher expression levels, since they may have a greater impact on cross-species interactions and antibiotic resistance in polymicrobial infections.
To further investigate MV composition and bacterial interactions, we employed expansion microscopy (ExM), enabling nanoscale visualization of vesicle cargo and strain-specific envelope proteins. Preliminary ExM data confirm the presence of PDC-1 specifically in PenG/FOX-induced MVs. Moving forward, ExM will allow us to distinguish vesicles from co-cultured P. aeruginosa and S. aureus, and to characterize their content in both mono- and polymicrobial settings.
Our current findings underscore the limitations and complexities of vesicle-mediated resistance, suggesting that extracellular protection may depend on specific vesicle cargo, β-lactamase variants, expression levels, and vesicle dynamics. This work will advance our understanding of microbial interactions and antibiotic efficacy in polymicrobial infections.

Palabras clave: Β-LACTAM RESISTANCE, PSEUDOMONAS AERUGINOSA, Β-LACTAMASE PDC, BACTERIAL MEMBRANE VESICLES, EXPANSION MICROSCOPY