Evaluación de la carbonilación en aislados clínicos de Pseudomonas aeruginosa infectados por bacteriófagos mediante...

Phage therapy is emerging as alternative therapy for difficult-to-treat bacterial infections. Flow cytometry (FCM) can screen for infective phages rapidly, reducing time taken for screening infective phages to deliver a timely and effective phage-based therapy. Phage infection increases reactive oxygen species in bacteria, which cause oxidative damage, such as protein carbonylation - an irreversible addition of carbonyls to amino acids, causing loss-of-function in proteins. We aim to assess carbonylation for predicting bacterial response to phage infection. We hypothesise that the extent of protein carbonylation corresponds to the susceptibility of bacteria to phage.

Fourteen Pseudomonas aeruginosa clinical isolates were exposed to bacteriophage at a bacteriophage:bacteria ratio of 100:1. Bacteria were then stained with cascade-blue hydrazine, which labels carbonylation, prior to FCM. Fluorescence intensities of cascade-blue hydrazine from FCM analyses were used to construct polar plots, illustrating both the extent of carbonylation and heterogeneity of isolates exhibiting carbonylation.

Increased carbonylation was observed at 2 hours-post-phage-infection (hppi), in clinical isolates determined to be phage-susceptible by double agar overlay assay. FCM further revealed decreased carbonylation in several isolates at 4-hppi from 2-hppi. Phasor approach was adopted to estimate when the isolate would exhibit decreased carbonylation. Our estimations are hitherto accurate in 11 of 14 isolates assessed (accuracy: 78.6%).

Compared to traditional methods requiring overnight incubation, assessing carbonylation by FCM is more rapid at determining phage infectivity. As carbonylation often cause irreversible loss-of-functions in proteins, we postulate the onset of subsequent, decreased carbonylation indicates bacterial attempt to survive by degrading carbonylated proteins.