Our investigations on extracellular biochemical events to find readily and sensitively detectable/measurable molecular targets for developing easier, simpler, and quicker diagnostic methods and tools for bacterial pathogens led to the observation that bacteria grown in the presence of glucose produced a compound capable of quenching fluorescein. Under the experimental conditions, among various sugars, glucose was found to induce maximum amount of the quencher when Escherichia coli was grown in presence of 50 mM glucose in rarified LB. The release of quencher closely following bacterial growth significantly from fourth hour after moderate inoculation. This fluorescein-quencher was purified using TLC and HPLC and identified using GC-MS as 13-docosenamide or erucamide, originally known as plant lipid, is a neuroactive compound in human and animals. Fluorescence and UV-absorption spectral analysis showed that the compound formed stable adduct with fluorescein in the ground state. Commercial 13-docosonamide enabled quantitation of the compound produced in micromolar quantities during glucose utilization from the medium. Twenty-seven different commonly encountered bacteria, pathogens or otherwise, could produce the quencher. A simple microplate-based growth monitoring method was developed exploiting quenching as an easily and readily measurable signal, either using a reader or an imager. While 13-docosenamide release by bacteria may be relevant in host-bacteria interactions, especially when growing under conditions that provide glucose, the new approach with inexpensive reagents can provide a new antibiogram technique.
Keywords: 13-docosenamide; Adduct formation; Eurcamide; Growth monitoring; Molecular targets; New antibiogram technique; Quencher; Quenching; Rarified LB.