Membrane lipid peroxidation in copper alloy-mediated contact killing of Escherichia coli

Abstract

Copper alloy surfaces are passive antimicrobial sanitizing agents that kill bacteria, fungi, and some viruses. Studies of the mechanism of contact killing in Escherichia coli implicate the membrane as the target, yet the specific component and underlying biochemistry remain unknown. This study explores the hypothesis that nonenzymatic peroxidation of membrane phospholipids is responsible for copper alloy-mediated surface killing. Lipid peroxidation was monitored with the thiobarbituric acid-reactive substances (TBARS) assay. Survival, TBARS levels, and DNA degradation were followed in cells exposed to copper alloy surfaces containing 60 to 99.90% copper or in medium containing CuSO(4). In all cases, TBARS levels increased with copper exposure levels. Cells exposed to the highest copper content alloys, C11000 and C24000, exhibited novel characteristics. TBARS increased immediately at a very rapid rate but peaked at about 30 min. This peak was associated with the period of most rapid killing, loss in membrane integrity, and DNA degradation. DNA degradation is not the primary cause of copper-mediated surface killing. Cells exposed to the 60% copper alloy for 60 min had fully intact genomic DNA but no viable cells. In a fabR mutant strain with increased levels of unsaturated fatty acids, sensitivity to copper alloy surface-mediated killing increased, TBARS levels peaked earlier, and genomic DNA degradation occurred sooner than in the isogenic parental strain. Taken together, these results suggest that copper alloy surface-mediated killing of E. coli is triggered by nonenzymatic oxidative damage of membrane phospholipids that ultimately results in the loss of membrane integrity and cell death.

Fig 1

E. coli survival and lipid peroxidation following exposure to different CuSO₄ concentrations in liquid medium. E. coli strain ATCC 23724 was grown at 37°C in LB medium to mid-log phase (OD₆₀₀, 0.3). At time zero, CuSO₄ was added to the culture to the indicated concentration, and incubation continued for the course of the experiment. (A) At the indicated time, cells were harvested by centrifugation from 100 ml of culture and resuspended in 200 μl of 0.85% NaCl. Survival, reported as the number of CFU, was determined by diluting culture samples in 0.85% NaCl and plating on LB agar. The error bars indicate standard deviations from three independent cultures for which titers were determined in duplicate. (B) CuSO₄ was added to the cultures to the indicated final concentration and harvested 60 min after the addition of CuSO₄, as described for panel A. The control (0) is the sample taken at time zero, prior to the addition of CuSO₄. Membrane lipid peroxidation products were determined as TBARS and are reported as nmoles MDA equivalents/10⁹ cells (see Materials and Methods). The error bars indicate standard deviations from three independent cultures assayed in duplicate.

Fig 2

E. coli survival and lipid peroxidation on copper-zinc alloy surfaces containing different copper concentrations. E. coli strain ATCC 23724 was grown in LB medium to mid-log phase (OD₆₀₀, 0.3), harvested by centrifugation from 100 ml of culture, and resuspended in 0.85% NaCl to a final volume of 500 μl. Samples (100 μl) of concentrated cells were spread over the surface of metal coupons of 304 stainless steel (S30400), 99.90% copper (C11000), and copper-zinc alloys C24000, C26000, and C28000, containing 80%, 70%, 60% copper, respectively (with the remainder zinc). (A) Following the indicated time of exposure, the cells were washed from the coupon surface with 100 μl of 0.85% NaCl, and samples were taken to determine survival titers, as described for Fig. 1A. (B) Results of the TBARS assay on cells exposed and recovered from the different alloys at the indicated times, as described for panel A. The results represent at least two independent trials. The legend applies to both panels A and B.

Fig 3

DNA degradation following copper surface exposure. E. coli strain ATCC 23724 was grown, harvested, and resuspended in 0.85% NaCl and exposed to coupon surfaces of the indicated alloy as described for Fig. 2. At the indicated times, the cells were washed from the coupon surface with 100 μl of 0.85% NaCl, total DNA was isolated using the Promega Wizard SV genomic DNA purification system, and the DNA was size separated in a 1% agarose gel. The size markers (M) are HindIII-digested λ phage DNA.

Fig 4

Microscopic assay of membrane integrity following exposure to a 99.90% copper surface. E. coli strain ATCC 23724 was grown, harvested, and exposed to 99.90% copper (C11000) coupons, as described for Fig. 2. At the indicated times of exposure, the cells were washed from the coupon surface with 100 μl of 0.85% NaCl and prepared for the Live/Dead BacLight assay as described by the manufacturer (Invitrogen). Cells were observed using a Zeiss fluorescece Axioscope and an AxoCam ICm1 camera. The panels, from left to right, were viewed with phase-contrast illumination, a FITC filter (green), and a rhodamine filter (Rho; red). Total magnification used was 1,000× with oil immersion.

Fig 5

Increased relative unsaturated fatty acid levels correlate with increased E. coli killing, lipid peroxidation, and DNA degradation following exposure to a 99.90% copper surface. E. coli strains PDJ1 (recD::Tn10) and MWF1 (fabR::kan recD::Tn10) were grown to mid-log phase (OD₆₀₀, 0.3) in M9 minimal medium supplemented with 0.4% glucose, 0.01% methionine, 0.0005% thiamine, and 25 μg/ml kanamycin or 20 μg/ml tetracycline, respectively. Cells were exposed to 99.90% copper (C11000) coupons, as described for Fig. 2. UFA:SFA is the ratio of unsaturated to saturated fatty acids as reported by Zhang et al. (60). Survival, TBARS production, and DNA degradation were assayed as described for Fig. 2 and 4.