Bioorthogonal probes for L-form conversion visualization and insights into antimicrobial resistance

Cell wall-deficient bacteria (CWDB) are key contributors to antimicrobial resistance (AMR), enabling persistent infections by evading antibiotics through their transition to L-form states. Therefore, molecular tools for detecting L-form conversion and AMR mechanisms are crucial for developing novel strategies against bacterial infections. Herein, we present the development of small-sized, peptidoglycan-specific fluorogenic probes employing a two-step bioorthogonal strategy that enables real-time visualization of CWDB formation. Tz-FL-S rapidly reacts with the novel d-alanine derivative TCO-d-Ala at a rate of (2.61 ± 0.07) × 10³ M^-1 s^-1, resulting in a 4.9-fold increase in fluorescence intensity. This platform exhibited excellent labeling of peptidoglycan in both Gram-positive and Gram-negative bacteria (signal-to-noise ratio: 15 to 305), effectively capturing the transition from N-form to L-form. Furthermore, we investigated the impact of 14 kinds of antibiotics on L-form conversion and found 13 of them induced CWDB. Besides, we explored the relationship between L-form conversion and AMR. This research enhances our understanding of bacterial adaptations and resistance mechanisms, paving the way for innovative strategies to combat drug-resistant infections.