Fluorescent probe-based detection of outer membrane damage of Gram-negative bacteria.

Infections caused by drug-resistant bacteria, particularly Gram-negative species, represent one of the most significant global public health challenges. The outer membrane (OM) is a crucial target for the development of drugs against Gram-negative bacteria. For the confirmation of the mechanism of OM-targeted drugs, the evaluation of OM damage is necessary. In this study, we optimized the method for detecting OM damage employing N-phenyl-1-naphthylamine (NPN) and ethidium bromide (EtBr) as probes in Escherichia coli (E. coli), including the bacterial loading, probe concentration, and incubation time. In addition, three OM-targeted compounds with distinctive mechanisms: polymyxin B, ACHN-975, and IMB-0042, were used to investigate the advantages and problems of two fluorescent probes. The compound fluorescence and quenching effect on the probes were detected. It was found that IMB-0042 caused fluorescence quenching on NPN. The treatment time of compounds with different OM damage mechanisms had a significant impact on the detection. For polymyxin B-treated E. coli cells, which directly disrupts OM, significant fluorescence changes were observed with both probes at short (30 min) and long durations (1-5 h). In contrast, compounds ACHN-975 and IMB-0042, which inhibit OM biosynthesis, showed detectable fluorescence only at long durations. In summary, this study presents a detailed scheme for the detection of OM damage induced by different antibiotics based on NPN and EtBr.