Cellular Adenine Nucleotide Levels as a Read‐out for Microbial Viability using Bioluminescence‐Based Strategies
Authors: Goueli Said , Hsiao Kevin , Mondal Subhanjan 
Published: June 2021

Monitoring the efficacy of bacterial killing using biocides is critical in minimizing health hazards and harmful effects of bacterial growth. Biocides manage to kill bacteria through different mechanisms , these include oxidizing biocides (bromine and chlorine) and non‐oxidizing organic chemicals (glutaraldehyde)which dissolve bacterial cell wall and lytic biocides such as cationic quaternary ammonium/amine compounds (QACs) charge.

To monitor the efficacy of biocide treatment, culturing and susceptibility testing have been the dominant method to assess the effect of treatment on killing bacteria by inducing cell death. The inability to produce colony‐forming units (CFU) on an appropriate solid medium is a criterion for defining dead cells and consequently, viable but non‐culturable bacterial cells which cannot grow or recover by forming colonies are not counted. Furthermore, it takes days and possibly weeks to assess the effect of treatment on plate counts.

One indicator of the energetic status of living cells is the adenylate pool where there is a positive correlation between the growth potential or functional capacity of cells and its energy charge (EC), and measurements of adenylate charge provide a framework for estimating metabolic potentials of naturally occurring microbial populations. Spores usually have very low EC (0.1) while values of 0.8 and higher are found in metabolically active epilimnion while it is lower than 0.6 in deep water samples. It is also documented that there is a reciprocal correlation between AMP and ATP in different parts of the biofilm, where AMP concentration is the predominant nucleotide especially in the deeper parts of the biofilm and decreases towards the surface while ATP increases. The transcription factor σB which controls the general stress regulon of B subtilis with over 200 genes that confer multiple stress resistance on the bacterium is activated by limited phosphate concentration in the stationary phase or simply by phosphate starvation. Under these conditions, a rapid drop in ATP concentration coincident with cessation of growth was observed and the decline in ATP precedes the induction of σB by about 20 minutes Thus, modulation of energy status of the cell has significant consequences to cellular metabolism, cellular growth and cell death. Towards this goal, we developed a homogenous and bioluminescent (BL) strategy to monitor these cellular adenylate metabolites using robust HTS formatted and easy to use protocol. The assay is very sensitive, and it was validated using HPLC for quantification of adenine nucleotides. Upon treatment of bacteria with low conc. of various biocides, a significant change in adenine nucleotide profile was observed within 5 minutes. The changes in adenine nucleotides ratio upon biocide treatment show distinct differences between Gram negative vs. Gram positive bacteria which warrants further investigation. Such investigations will help in understanding bacterial persistence and dormancy upon various biocides and environmental stress and lead to more effective management of biofouling.

Support or Funding Information

Promega Corp supported this research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.