Ultrafast and Multiplexed Bacteriophage Susceptibility Testing by Surface Plasmon Resonance and Phase Imaging of Immobilized Phage Microarrays

Abstract

In the context of bacteriophage (phage) therapy, there is an urgent need for a method permitting multiplexed, parallel phage susceptibility testing (PST) prior to the formulation of personalized phage cocktails for administration to patients suffering from antimicrobial-resistant bacterial infections. Methods based on surface plasmon resonance imaging (SPRi) and phase imaging were demonstrated as candidates for very rapid (<2 h) PST in the broth phase. Biosensing layers com-posed of arrays of phages 44AHJD, P68, and gh-1 were covalently immobilized on the surface of an SPRi prism and exposed to liquid culture of either Pseudomonas putida or methicillin-resistant Staphylococcus aureus (i.e., either the phages’ host or non-host bacteria). Monitoring of reflectivity reveals susceptibility of the challenge bacteria to the immobilized phage strains. Investigation of phase imaging of lytic replication of gh-1 demonstrates PST at the single-cell scale, without requiring phage immobilization. SPRi sensorgrams show that on-target regions increase in reflectivity more slowly, stabilizing later and to a lower level compared to off-target regions. Phage susceptibility can be revealed in as little as 30 min in both the SPRi and phase imaging methods.