Action of ciprofloxacin on planktonic bacteria and biofilm of Proteus mirabilis

Abstract

ABSTRACTProteus mirabilis can persist in biofilms, with the bacteria in this state tending to resist antibiotic therapy. Until now, the relationship between the action of ciprofloxacin and the production of reactive oxygen species (ROS) has not been studied in planktonic and biofilmic P. mirabilis. Our results show that ciprofloxacin stimulates the production of ROS in planktonic P. mirabilis, but that the increase in ROS was observed in sensitive strains (n = 4) only in the absence of the extracellular matrix (ECM). This augmentation of ROS was principally intracellular, invoking an increase in intracellular superoxide dismutase (SOD). ROS were assayed by chemiluminescence (CL) and SOD by inhibition of reduction of nitroblue tetrazolium in the presence of methionine, riboflavin and light. The antibiotic-resistant strains (n = 4) did not suffer oxidative stress and exhibited a higher antioxidant capacity than antibiotic-sensitive ones, as indicated by tripyridyltriazine assay. Both types of bacterial strain showed a reduction in antioxidant capacity in the presence of ciprofloxacin, and only the resistant bacteria returned to normal count levels within 5 min of introduction of antibiotic. Ciprofloxacin stimulated ROS more than it did nitric oxide (NO) in planktonic bacteria, as determined by Griess's reaction. Proteus mirabilis biofilms treated with ciprofloxacin did not suffer any increase in ROS but there was an increase in NO and the ratio of intracellular ROS:NO decreased to 25%. Biofilms of P. mirabilis were neither stressed nor inhibited by 40 µg ciprofloxacin/ml, a dose higher than the minimum inhibitory concentration (i.e. supra MIC). Both resistant and sensitive strains maintained the number of viable bacteria in biofilms incubated with supra MIC ciprofloxacin at concentrations that stressed and reduced substantially the number of colony-forming units of planktonic bacteria per millilitre. These results contribute to understanding of the differences between biofilmic and planktonic bacteria, with respect to susceptibility to oxidative stress caused by ciprofloxacin and also the antioxidant effect of ECM.