Among pathogenic bacteria, Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa were the six leading causes for the deaths associated with antibiotic resistance in 2019. Although new treatment options are urgently needed, the precise identification of the bacterial species remains pivotal for an accurate diagnosis and effective treatment. Clinically, mass spectrometry is used to distinguish these bacteria based on their protein mass pattern at the genus and species level. Herein, we report an alternative approach to identify these bacteria using the nitroreductase-based "turn-on" fluorescent probes (ETH1-NO₂ and ETH2-NO₂), with potential visual indicators for the six individual bacteria species. The limits of detection (LODs) of the probes for NTRs are 0.562 (ETH1-NO₂) and 0.153 μg/mL (ETH2-NO₂), respectively. They respond effectively to both Gram-positive and Gram-negative bacteria, with the lowest LOD at 1.2 × 10⁶ CFU/mL for E. coli. In particular, different bacteria show noticeable difference in the apparent color of ETH1-NO₂ samples, allowing possible identification of these bacteria visually. In addition, ETH1-NO₂ also has potential applications in bacterial fluorescence imaging. Thus, our study provides an alternative approach for bacteria identification and new reagents for bacteria imaging.

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