Inhibitors of Reactive Oxygen Species Accumulation Delay and/or Reduce the Lethality of Several Antistaphylococcal Agents

Abstract

bPerturbation of hydroxyl radical accumulation by subinhibitory concentrations of 2,2=-bipyridyl plus thiourea protects Escherichia coli from being killed by 3 lethal antimicrobial classes. Here, we show that 2,2=-bipyridyl plus thiourea delays and/or reduces antimicrobial killing of Staphylococcus aureus by daptomycin, moxifloxacin, and oxacillin. While the protective effect of 2,2=-bipyridyl plus thiourea varied among strains and compounds, the data support the hypothesis that hydroxyl radical enhances antimicrobial lethality.T he growing problem of antimicrobial resistance has become increasingly difficult to counter by developing new agents for two reasons. First, most large pharmaceutical companies have shelved their antimicrobial development programs due to financial, regulatory, and legal considerations (9, 10). Second, expectations that novel genomic/proteomic approaches would quickly produce many new antimicrobials may not be realistic, since no antimicrobial has materialized from such approaches, despite more than 10 years of effort (8). As an alternative to finding new antimicrobial classes, we have been seeking ways to enhance the efficacy of existing agents. Recent studies indicate that reactive oxygen species (ROS) contribute to antimicrobial-mediated killing of Escherichia coli for three important drug classes (5, 11). If the lethal action of many antimicrobials is stimulated by ROS with many pathogens, it may be possible to develop broad-spectrum, small-molecule enhancers of multiple antimicrobial classes by identifying novel, bacterium-specific targets within oxidative stress pathways. Since work to date has focused primarily on E. coli (only norfloxacin has shown a correlation between killing and hydroxyl radical surge with Staphylococcus aureus [5]), the relevance of ROS-mediated lethality to antimicrobial treatment of Gram-positive bacteria is largely unknown. Below, we describe the effects of inhibitors of ROS accumulation on the ability of three agents to kill two strains of Staphylococcus aureus under laboratory conditions. Two methicillin-susceptible S. aureus strains (RN450 [7] and ATCC 25923) were grown at 37°C on Mueller-Hinton agar or in Mueller-Hinton broth with shaking at 250 rpm. Bacterial growth was measured by diluting an overnight culture into fresh MuellerHinton broth by 100-fold, followed by turbidity determinations at 600 nm with a spectrophotometer. The MIC and minimal bactericidal concentration (MBC) were determined by broth microdilution according to CLSI guidelines (1) for daptomycin (Cubist Pharmaceuticals, Lexington, MA), moxifloxacin (Bayer AG, West Haven, CT), and oxacillin (Sigma, St. Louis, MO) in the presence or absence of subinhibitory concentrations (one-half of the MIC) of 2,2=-bipyridyl plus thiourea, two compounds that interfere with antimicrobial-mediated hydroxyl radical accumulation (5, 11). Concentration-kill curves and killing kinetics were measured by incubating exponentially growing cells with daptomycin, moxifloxacin, or oxacillin in the presence or ab...

Role of Autofluorescence in Flow Cytometric Analysis of Escherichia coli Treated with Bactericidal Antibiotics

“…Autofluorescence can interfere with specific fluorescent probes in flow cytometric analysis of E. coli. The fluorescent dye HPF was used in combination with flow cytometry to detect druginduced hydroxyl radicals (8,(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). HPF is a fluorescein derivate that is used to selectively detect highly reactive oxygen species such as hydroxyl radicals.…”

“…Other studies used the same or similar approaches to measure hydroxyl radical formation with HPF in bacterial cells via flow cytometry (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36). Dwyer et al used HPF fluorescence measurements to show that gyrase inhibitors induce an oxidative damage cellular death pathway in E. coli after treatment with norfloxacin or the natural peptide toxin, CcdB (27).…”

Prevalence and antibiotic susceptibility of bacteria from acute and chronic wounds in Malaysian subjects

“…This may largely be due to the frequent usage of systemic antibiotics in the management of chronic wounds. Bacteria became more tolerant of antibiotics and ROS may be involved in this situation [53]. Systemic antibiotics should be avoided if clinical signs of infections are absent.…”

Vitamin D and respiratory infections

“…Evolution and adaptation of microorganisms, possible through reactive oxygen species [63,64], forced by the selective pressure caused by the indiscriminate use of antibiotics, has led to a significant decrease in the effectiveness and activity of these drugs through the phenomenon of antimicrobial resistance [65].…”