Bactericidal antibiotics increase hydroxyphenyl fluorescein signal by altering cell morphology.

It was recently proposed that for bactericidal antibiotics a common killing mechanism contributes to lethality involving indirect stimulation of hydroxyl radical (OH•) formation. Flow cytometric detection of OH• by hydroxyphenyl fluorescein (HPF) probe oxidation was used to support this hypothesis. Here we show that increased HPF signals in antibiotics-exposed bacterial cells are explained by fluorescence associated with increased cell size, and do not reflect reactive oxygen species (ROS) concentration. Independently of antibiotics, increased fluorescence was seen for elongated cells expressing the oxidative insensitive green fluorescent protein (GFP). Although our data question the role of ROS in lethality of antibiotics other research approaches point to important interplays between basic bacterial metabolism and antibiotic susceptibility. To underpin such relationships, methods for detecting bacterial metabolites at a cellular level are needed.