Article Figures & Data
Figures
- Fig. 1.
Survival of vancomycin-resistant E. faecalis ATCC 51299 (A) and E. faecium NCTC 12202 (B) on copper surfaces with the wet fomite inoculum, with the addition of EDTA prolonging the protective effect of Tiron in E. faecalis ATCC 51299 bacteria exposed to copper surfaces (C). (A and B) Approximately 107 CFU in 20 μl was inoculated onto 1-cm2 coupons in PBS (●) or PBS supplemented with EDTA (○), BCS (▾), d-mannitol (▵), Tiron (■), SOD (□), catalase (♦), and sucrose (♢) at 22°C. (C) Approximately 107 CFU in 20 μl was inoculated onto 1-cm2 coupons in PBS (● and ▾) or PBS supplemented with Tiron (○ and ▵). Cells were removed from coupons and assessed for culturability as described in the text. The addition of EDTA at 65 min of contact did not prolong survival if cells had been inoculated in PBS (▾) but did so in cells inoculated in Tiron (▵).
- Fig. 2.
Survival of enterococcal clinical isolates on copper surfaces in the presence of chelators of Cu(I) (BCS) and Cu(II) ions (EDTA) with the wet fomite inoculum. Approximately 107 CFU of E. faecalis (A) or E. faecium (B), resuspended in 20 μl of 20 mM EDTA or 20 mM BCS, was inoculated onto 1-cm2 copper coupons. Cells were removed and assessed for culturability after 2 and 3 h of contact with the surface at 22°C. Strains used were as follows: ATCC 51299 in EDTA (black bars) or BCS (white bars), wound swab isolate 1 in EDTA (dark gray bars) or BCS (light gray bars), and fecal isolate 2 in EDTA (white spotted bars) or BCS (black spotted bars) (A) and NCTC 12202 in EDTA (black bars) or BCS (white bars), ascitic fluid isolate 1 in EDTA (dark gray bars) or BCS (light gray bars), swab isolate 2 in EDTA (white spotted bars) or BCS (black spotted bars), blood culture isolate 3 in EDTA (white diagonal bars) or BCS (gray diagonal bars), gastric aspirate isolate 4 in EDTA (white crossed bars) or BCS (gray crossed bars), and isolate from central venous catheter tip 5 in EDTA (white horizontal striped bars) or BCS (gray horizontal striped bars) (B). (For ease of observation, time zero results are not shown for each graph.) No viable cells were present in PBS alone at these time points.
- Fig. 3.
Survival of vancomycin-resistant E. faecalis ATCC 51299 (A and C) and E. faecium NCTC 12202 (B and D) on copper (A and B) and copper alloy C26000 (C and D) surfaces with the dry inoculum. Approximately 107 CFU in 1 μl was inoculated onto 1-cm2 coupons in PBS (black bars) or PBS supplemented with EDTA (white bars), BCS (dark gray bars), d-mannitol (light gray bars), Tiron (white spotted bars), SOD (black spotted bars), catalase (white diagonal striped bars), and sucrose (gray diagonal striped bars) at 22°C (time zero results not shown).
- Fig. 4.
Protection of bacterial genomic DNA by chelators and ROS quenchers determined by use of a DNA fragmentation assay with the wet and dry inocula. Approximately 107 bacterial cells (in 20 μl [wet] or 1 μl [dry]) were inoculated onto copper coupons in PBS (control) or PBS supplemented with chelators or ROS quenchers and incubated at 22°C for 2 h (wet) or 10 min (dry). The cells were removed from the coupons, and genomic DNA was analyzed with a DNA fragmentation assay. For cells that had been inoculated onto coupons in PBS or PBS supplemented with mannitol or SOD, very little DNA was visible, presumably because the fragments were too small to be visualized and had diffused away from the body of the cell. However, in the presence of EDTA, BCS, and Tiron, intact DNA was visible, suggesting that they exerted a protective effect on the nucleic acid. On stainless steel all supplements demonstrated intact genomic DNA (not shown for the dry inoculum). The bar represents 10 μm.
- Fig. 5.
Rapid breakdown of bacterial DNA on copper (A) and alloy (C26000) (B) surfaces compared to stainless steel (C) occurs as part of the killing mechanism in enterococci. Approximately 107 CFU in 1 μl was inoculated onto 1-cm2 coupons, and cells were removed at the time points indicated (minutes of contact) with glass beads and PBS-EDTA (20 mM) as described in the text. Cells were pooled and stained with SYTO 9 (5 μM) to detect double-stranded DNA and observed by using epifluorescence microscopy. Clumping and disintegration of DNA (reduced staining) were seen at 0 to 30 s of contact with copper surfaces and at 30 s to 1 min of contact with alloy but not on stainless steel. Results for E. faecalis ATCC 51299 are shown. Bar, 10 μM.
- Fig. 6.
Protection of bacterial respiratory pathways by chelators and ROS quenchers on copper surfaces with the wet (A) or dry (B) inoculum. Approximately 107 bacterial cells (in 20 μl) were inoculated onto copper coupons in PBS or PBS supplemented with chelators or ROS quenchers and incubated at 22°C for 2 h. Cells were stained in situ with the redox stain CTC to detect actively respiring cells (fluorescing red) and a nonspecific bacterial stain, SYTO 9, for total cell numbers (green fluorescence DNA stain). In the presence of EDTA, BCS, or Tiron, respiring cells are visible, and the bright staining with SYTO 9 suggests that the DNA is intact. However, no respiring cells were visible in PBS or PBS supplemented with mannitol, catalase, or SOD (not shown). Respiring cells were detected on stainless steel for all supplements tested (not shown). For the dry inoculum (1 μl), cells were removed from coupons as described in the text, and at the times indicated, for culture; pooled; and stained with CTC for 1 h before transferring them onto a microscope slide. Cells were actively respiring at 10 min on stainless steel, but very few respiring cells were visible following 1 min of contact with copper surfaces. Bar, 10 μM.
- Fig. 7.
Detection of changes in membrane potential changes in situ in E. faecalis ATCC 51299 cells exposed to copper (for the times indicated) and stainless steel (only the 10-min time point is illustrated) surfaces. Approximately 107 bacterial cells (in 1 μl) that had been stained with rhodamine 123 were inoculated onto metal coupons for 10 min. Images were recorded every minute and were observed by using epifluorescence microscopy. Bright staining indicates that membranes are not compromised until after 8 min of contact; i.e., many cells were already dead, as indicated by the culture results. The ionophore CCCP was used as a negative control to depolarize the membrane and eliminate rhodamine 123 staining (not shown). Bar, 10 μM.
