Single-Bacterium Biosensing: A Rapid Electrochemical Strategy to Differentiate Antibacterial Efficacy and Mechanism

The timely identification of drug-resistant pathogens and rapid differentiation of antibacterial mechanisms are critical in combating the escalating crisis of antimicrobial resistance. However, traditional broth culturing methods are inherently time-consuming and lack single-cell sensitivity. In this study, we reported a highly responsive biosensing platform based on single-entity impact electrochemistry (SIE) to rapidly differentiate bacterial activity at the single bacterium level. This method utilized p-benzoquinone (BQ) and BQ-K3Fe(CN)6 as redox mediators that were continuously reduced by the bacterial respiratory chain. Upon the collision of an individual bacterium with the ultramicroelectrode, these mediators were re-oxidized, generating transient current spikes that serve as a direct indicator of cellular viability. By tracking these distinct electrochemical signals, we successfully discriminated the responses of Escherichia coli to three antibacterial agents. Specifically, the heavy metal stress induced by Pb2+ exposure resulted in a progressive decline in collision spike amplitude, with the total transferred charge decreasing from (7.51 ± 0.48) × 10-2 pC to (2.50 ± 0.14) × 10-2 pC per bacterium. The bacteriostatic effect of doxycycline yielded negligible immediate signal change due to its protein synthesis inhibition mechanism. In contrast, the membrane-disrupting effect of polymyxin B triggered a rapid cessation of collision signals alongside an increase in total current amplitude due to the leakage of intracellular redox contents. Based on these unique electrochemical signatures, we successfully identified polymyxin B-resistant E. coli mutants and achieved rapid validation of alternative antibiotics against this drug-resistant strain. This work expanded the analytical capabilities of SIE, offering a highly efficient platform for bacterial phenotypic screening and drug resistance monitoring.