Glycerol reduces culturability and impairs predatory activity of Bdellovibrio bacteriovorus

Bdellovibrio and like organisms (BALOs) are typically obligate predators of Gram-negative bacteria, including clinically relevant pathogens. Due to their potential use as an alternative approach against multidrug-resistant bacteria, they have been dubbed “living antibiotics”. However, information regarding the effects of glycerol -a compound widely used in laboratories and pharmaceutical industries- on BALOs remains limited. Here, we report that glycerol reduced recoverable plaque-forming units (PFU) of Bdellovibrio bacteriovorus 109 J by 69% at 1% (v/v) after 48 h and by > 99% at concentrations of 5% (v/v) or higher after 72 h at 30°C, whereas no such effect occurs at -70°C. This effect was only observed with glycerol, as other polyalcohols did not induce similar defects at equivalent isoosmolar concentrations. Furthermore, 5% (v/v) glycerol impaired predation, increasing the prey-to-predator ratio by nearly 2 log at days 1 and 4. At the same concentration, 45% of cells showed BacLight staining patterns consistent with membrane damage after three days. Notably, glycerol effects were also observed in other environmental BALO isolates, with PFU counts falling below the limit of detection (LOD) in the most affected isolate. These findings reveal a critical limitation in using glycerol in routine BALOs handling and potential future applications.