Synergistic effects of oxidative and acid stress on bacterial membranes of Escherichia coli and Staphylococcus simulans

Oxidative stress in combination with acid stress has been shown to inactivate a wide spectrum of microorganisms, including multi-resistant bacteria. This occurs e.g. in phagolysosomes or during treatment by cold atmospheric pressure plasmas (CAP) and possibly depends on the cell membrane. We therefore explored the effects of CAP-generated reactive oxygen and nitrogen species (RONS) on bacterial growth inhibition and membranes in neutral and acidic suspensions. We observed that growth inhibition was most efficient when bacteria were treated by a mix of short and long-lived RONS in an acidic environment. Membrane packing was affected mainly upon contact with short-lived RONS, while also acidity strongly modulated packing. Under these conditions, Gram-negative bacteria displayed large potassium release while SYTOX Green influx remained marginal. Growth inhibition of Gram-negative bacteria correlated well with outer membrane (OM) permeabilization that occurred upon contact with short and/or long-lived RONS in synergy with acidity. In Gram-positive bacteria, CAP impaired membrane potential possibly through pore formation upon contact with short-lived RONS while formation of membrane protein hydroperoxides was probably involved in these effects. In summary, our study provides a wide perspective on understanding inactivation mechanisms of bacteria by RONS in combination with acidity.