Assessing carbonylation in Pseudomonas aeruginosa clinical isolates infected by bacteriophages using flow cytometry

Phage therapy is emerging as an alternative therapy for difficult-to-treat bacterial infections. Flow cytometry (FCM) can rapidly screen for infective phages, reducing the 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. Here, we aimed to assess carbonylation for predicting the bacterial response to phage infection. We hypothesised that the extent of protein carbonylation corresponds to bacterial phage susceptibility.

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. The cascade blue hydrazine fluorescence intensities obtained from the FCM analyses were used to construct polar plots illustrating both the extent of carbonylation and the heterogeneity of isolates exhibiting carbonylation.

Increased carbonylation was observed at 2 h 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. A phasor approach was adopted to estimate when the isolate would exhibit decreased carbonylation, and our estimations were accurate in 11 of 14 isolates assessed (accuracy: 78.6%).

Compared to traditional methods requiring overnight incubation, assessing carbonylation by FCM is a more rapid method to determine phage infectivity. As carbonylation often causes irreversible loss of function in proteins, we postulate that the onset of subsequent decreased carbonylation indicates bacterial attempts to survive by degrading carbonylated proteins.