Outer Surface-Labeled Bacteria as Live Sensors Accurately Quantitating Interfacial pH: A Smart Technique for Antimicrobial Resistance.

The techniques to quantitatively monitor environmental factors surrounding the bacterial outer surface rather than the host's subcellular regions (e.g., lysosomes) should be the key to evaluate bacterial immune escape behavior. We report wild Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) labeled with a fluorescent resonance energy transfer probe, 4SR-L-BDP, on their outer surfaces as smart live sensors to quantify interfacial pH. The dual emission of 4SR-L-BDP affords high sensitivity to pH change in a ratiometric way in the pH range of 4-8 with high precision. Notably, 4SR-L-BDP possesses an anchoring group to fix on the bacterial surface for sensing the microenvironment encountered. Super-resolution imaging clearly demonstrates the specific labeling of bacterial membranes. These live sensors are applied in two-channel ratiometric imaging to dynamically visualize and quantify their interfacial pH changes during infection of macrophages. It is found that the interfacial pH of MRSA is lower by 0.2 units compared to that of SA. Such small but critical difference in pH reflects MRSA's ability to adapt to microenvironmental pH inside macrophages. These labeled bacteria as live sensors are also proven to be practically applicable in mice models with immune deficiency and immune activation.