Use of Resazurin To Rapidly Enumerate Bdellovibrio and Like Organisms and Evaluate Their Activities

Abstract

ABSTRACT A method to rapidly quantify predatory bacterial cell populations using resazurin reduction to resorufin and its resulting fluorescence kinetics (dF/dt) are described. The reliability of this method to measure the predatory populations was demonstrated with the type strain, Bdellovibrio bacteriovorus HD100, as well as B. bacteriovorus 109J and two natural isolates, Halobacteriovorax strains JA-1 and JA-3, with clear correlation when densities were between 107 and 109 PFU/ml. Resazurin was also used to evaluate how B. bacteriovorus HD100 and Halobacteriovorax strain JA-1 respond to harmful conditions, i.e., exposure to sodium dodecyl sulfate (SDS), with both the dF/dt and PFU/ml indicating Halobacteriovorax strain JA-1 is more sensitive to this surfactant. Tests were also performed using media of different osmolalities, with the dF/dt values matching the 24-h predatory activities reasonably well. Finally, this method was successfully applied in near real-time analyses of predator-prey dynamics and, when coupled with SDS, was capable of differentiating between the predatory and prey populations. All of these tests serve to prove this method is (i) very rapid, needing only 15 min from start to finish; (ii) very reliable with different predatory bacterial species; and (iii) very versatile as it can be easily adapted to measure predatory numbers and activities in a range of experiments. IMPORTANCE Bdellovibrio and like organisms are predatory bacteria that are capable of attacking, killing, and consuming many bacterial pathogens, including multidrug-resistant strains. These qualities have led to them being labeled as “living antibiotics.” Research work with these remarkable strains, however, has been hampered by long growth times needed to quantify the predatory populations through plaque assays, which typically take 4 days to develop. Here, we describe a fluorescence-based method using the conversion of resazurin (low fluorescence) to resorufin (high fluorescence) after it is reduced by the predators’ NADH. Not only do we show that the fluorescence correlates strongly with the predatory concentration and that we can use it to evaluate if the predators are viable, but the entire procedure from start to finish takes only 15 min, drastically reducing the time researchers need to quantify the predatory numbers. Employing this technique will greatly advance research related to predatory bacteria and their potential applications.

Keywords

• bdellovibrios
• predatory bacteria
• resazurin
• fluorescence assays