Dimethyl Sulfoxide Protects Escherichia coli from Rapid Antimicrobial-Mediated Killing

Abstract

The contribution of reactive oxygen species (ROS) to antimicrobial lethality was examined by treating Escherichia coli with dimethyl sulfoxide (DMSO), an antioxidant solvent frequently used in antimicrobial studies. DMSO inhibited killing by ampicillin, kanamycin, and two quinolones and had little effect on MICs. DMSO-mediated protection correlated with decreased ROS accumulation and provided evidence for ROS-mediated programmed cell death. These data support the contribution of ROS to antimicrobial lethality and suggest caution when using DMSO-dissolved antimicrobials for short-time killing assays. O ne approach to help stem the emergence of new antimicrobial resistance is to kill bacterial pathogens rapidly, thereby quickly reducing bacterial burden and restricting effects of stressinduced mutagenesis (1, 2). Reactive oxygen species (ROS) have been proposed to be key factors in antimicrobial lethality (3-5), and substantial evidence supports this proposition (3-19). However, their role in lethality has been challenged (20, 21). If ROS are indeed integral to antimicrobial-mediated killing, compounds that act as antioxidants and radical scavengers should reduce antimicrobial lethality. We chose to examine this hypothesis using the radical scavenger dimethyl sulfoxide (DMSO) (22, 23), because it is also a popular solvent that is widely used in the pharmaceutical industry and in antimicrobial research due to its (i) low toxicity, (ii) ability to dissolve both organic and inorganic compounds, (iii) ability to remain in a liquid state over a broad temperature range (e.g., from 19°C to 189°C), (iv) ability to enhance cell membrane permeability, and (v) miscibility in water and a wide range of organic solvents. We report here that DMSO interferes with rapid killing of Escherichia coli and Acinetobacter baumannii by members of three antimicrobial classes.E. coli K-12 strains BW25113 and ATCC 25922 and A. baumannii strain ATCC 17978 were grown in Luria-Bertani (LB) broth or on LB agar at 37°C. LB medium, ampicillin, and kanamycin were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Oxolinic acid, ciprofloxacin, and DMSO were acquired from Sigma-Aldrich Co. (St. Louis, MO). Meropenem (Sumitomo Dainippon Pharma Co. Ltd.) was obtained from Zhongshan Hospital Pharmacy. The fluorescent probe carboxy-H 2 DCFDA [5(6)-carboxy-2=,7=-dichlorodihydrofluorescein diacetate] was purchased from Invitrogen (Grand Island, NY). All chemical stock solutions were dissolved in sterile water (except carboxy-H 2 DCFDA, which was dissolved in DMSO) and stored at Ϫ80°C until use. MICs were assayed by broth dilution according to CLSI protocols (24); exponentially growing cultures were diluted to 10 5 CFU/ml for MIC determinations. To measure rapid bacterial killing, exponentially growing cultures at about 5 ϫ 10 8 CFU/ml were treated with antimicrobials, after which they were serially diluted and plated on drug-free agar. Viable colony counts were determined after an overnight incubation at 37°C; percentage survival rates were calculat...

Potential Use of Dimethyl Sulfoxide in Treatment of Infections Caused by Pseudomonas aeruginosa

“…However, possible interactions between DMSO and antibiotics also need to be addressed. It has recently been reported that DMSO inhibits the action of some reactive oxygen species-dependent antibiotics against Escherichia coli (54). In light of this report, we have tested the effect of DMSO on the efficacy of six different antibiotics against P. aeruginosa.…”

“…It has recently been reported that DMSO affects antibiotic susceptibilities in Escherichia coli (54). To test whether DMSO interferes with the effect of conventional antibiotics, we tested the MICs of six antibiotics from different classes for P. aeruginosa in the presence of different concentrations of DMSO.…”

Metabolic Fingerprinting with Fourier-Transform Infrared (FTIR) Spectroscopy: Towards a High-Throughput Screening Assay for Antibiotic Discovery and Mechanism-of-Action Elucidation

“…On the other hand, DMSO can alter cell membrane permeability, which is speculated to explain its inhibitory effect, and can result in an apparent increase in potency, as the entry into the cell by certain antibiotics is facilitated. Ultimately, these findings discourage the use of DMSO as a solvent for antimicrobials, especially in rapid-killing assays [37].…”

DNA double-strand breaks induced by reactive oxygen species promote DNA polymerase IV activity inEscherichia coli

“…The ROS-driven potentiation of killing by both antibiotic treatment and TLD can be abrogated through the addition of ROS mitigators to the culture medium (1,5,12). For example, dimethyl sulfoxide (DMSO) and 2,2’-bipyridine (BiP), both, effectively mitigate the accumulation of antibiotic-induced ROS (5, 17). Using microscopy to quantify ssDNA gaps and DSBs in cells undergoing TLD, Hong and co-workers discovered that thymine starvation initially leads to the accumulation of ssDNA gaps, which are subsequently converted to DSBs in an ROS-dependent process (5).…”