The world-wide threat of antimicrobial resistance demands continued development of innovative pharmaceutical drugs capable of selectively targeting microbial pathogens by either crossing or increasing the permeability of the bacteria membrane. For example, macrolides form a general class of antibiotic compounds which have been effectively used in clinical treatments for well over 50 years. There has been considerable interest in characterizing macrolide-membrane interactions over the last decade. In the interest of efficiently optimizing antimicrobial compounds which target the bacteria membrane, it is desirable to develop methodologies capable of monitoring changes to membrane properties in living bacteria. We have previously demonstrated the utility of second-harmonic light scattering (SHS), as a real-time and surface-sensitive technique, for monitoring chemically induced changes to membrane permeability in living bacteria. Here, we present time-resolved SHS to quantify azithromycin (AZM)-induced changes to bacterial membrane permeability in colloidal suspensions of living Escherichia (E.) coli. Variations in membrane properties of E. coli were monitored through changes in the adsorption and transport rates of an SH-active molecule, malachite green (MG). Our results revealed that regardless of the AZM concentration, instantaneous treatment with AZM has no significant effect on the bacteria membrane permeability. However, 1 hour pretreatment with sub-minimum inhibitory concentrations (MIC) of AZM induced an order-of-magnitude enhancement in the permeability of both the outer membrane and, through facilitation of a new transport mechanism, the cytoplasmic membrane of the bacteria as well. Of significance, there was a dual decay behavior (i.e., fast and slow) observed for cytoplasmic membrane transport events following 1 h exposure to either 75 μM (0.5×MIC) or 150 μM (1×MIC) AZM. Further, the relative efficiency of the fast transport process was observed to increase with AZM concentration in which the fast decay process accounted for 13% of the 75 μΜ AZM case and increased to 62% for 150 μΜ AZM. This suggests that accumulation of increasing concentrations of AZM creates an efficient secondary transport route across the bacteria cytoplasmic membrane. This study illustrates SHS as a novel tool for monitoring antimicrobial-induced changes to membrane properties in living bacteria.

Monitoring macrolide-induced changes to membrane properties of living bacteria by using second-harmonic light scattering

256th National Meeting (2018)

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