Abstract
Bacterial resistance to antibiotics poses a great clinical challenge in fighting serious infectious diseases due to complicated resistant mechanisms and time-consuming testing methods. Chemical reaction-directed covalent labeling of resistance-associated bacterial proteins in the context of a complicated environment offers great opportunity for the in-depth understanding of the biological basis conferring drug resistance, and for the development of effective diagnostic approaches. In the present study, three fluorogenic reagents LRBL1-3 for resistant bacteria labeling have been designed and prepared on the basis of fluorescence resonance energy transfer (FRET). The hydrolyzed probes could act as reactive electrophiles to attach the enzyme, β-lactamase, and thus facilitated the covalent labeling of drug resistant bacterial strains. SDS electrophoresis and MALDI-TOF mass spectrometry characterization confirmed that these probes were sensitive and specific to β-lactamase and could therefore serve for covalent and localized fluorescence labeling of the enzyme structure. Moreover, this β-lactamase-induced covalent labeling provides quantitative analysis of the resistant bacterial population (down to 5%) by high resolution flow cytometry, and allows single-cell detection and direct observation of bacterial enzyme activity in resistant pathogenic species. This approach offers great promise for clinical investigations and microbiological research.
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