Evidence of Correlation between Membrane Phase Transition and Clonogenicity in Dehydrating : A Combined Micro-Raman and AFM Study.

The Gram-negative bacterium is one of the most resilient multidrug-resistant pathogens in hospitals. Among Gram-negative bacteria, it is particularly resistant to dehydration (anhydrobiosis), and this feature allows to persist in hospital environments for long periods, subjected to unfavorable conditions. We leverage the combination of μ-Raman spectroscopy and atomic force microscopy (AFM) to investigate the anhydrobiotic mechanisms in cells by monitoring the membrane (both inner and outer membranes) properties of four strains during a 16-week dehydration period and in response to temperature excursions. We noted that the membranes of remained intact during the dehydration period despite undergoing a liquid-crystal-to-gel-phase transition, accompanied by changes in the mechanical properties of the membrane. This was evident from the AFM images, which showed the morphology of the bacterial cells alongside modifications of their superficial mechanical properties, and from the alteration in the intensity ratio of μ-Raman features linked to the CH and CH symmetric stretching modes. Furthermore, employing a universal power law revealed a significant correlation between this ratio and bacterial fitness across all tested strains. Additionally, we subjected dry to a temperature-dependent experiment, the results of which supported the correlation between the Raman ratio and culturability, demonstrating that the phase transition becomes irreversible when cells undergo different temperature cycles. Besides the relevance to the present study, we argue that μ-Raman can be used as a powerful nondestructive tool to assess the health status of bacterial cells based on membrane properties with a relatively high throughput.