Biocidal efficacy of copper alloys against pathogenic enterococci involves degradation of genomic and plasmid DNAs

Abstract

The increasing incidence of nosocomial infections caused by glycopeptide-resistant enterococci is a global concern. Enterococcal species are also difficult to eradicate with existing cleaning regimens; they can survive for long periods on surfaces, thus contributing to cases of reinfection and spread of antibiotic-resistant strains. We have investigated the potential use of copper alloys as bactericidal surfaces. Clinical isolates of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium were inoculated onto copper alloy and stainless steel surfaces. Samples were assessed for the presence of viable cells by conventional culture, detection of actively respiring cells, and assessment of cell membrane integrity. Both species survived for up to several weeks on stainless steel. However, no viable cells were detected on any alloys following exposure for 1 h at an inoculum concentration of <or=10(4) CFU/cm(2). Analysis of genomic and plasmid DNA from bacterial cells recovered from metal surfaces indicates substantial disintegration of the DNA following exposure to copper surfaces that is not evident in cells recovered from stainless steel. The DNA fragmentation is so extensive, and coupled with the rapid cell death which occurs on copper surfaces, that it suggests that mutation is less likely to occur. It is therefore highly unlikely that genetic information can be transferred to receptive organisms recontaminating the same area. A combination of effective cleaning regimens and contact surfaces containing copper could be useful not only to prevent the spread of viable pathogenic enterococci but also to mitigate against the occurrence of potential resistance to copper, biocides, or antibiotics and the spread of genetic determinants of resistance to other species.

FIG. 1.

Survival of vancomycin-resistant E. faecium NCTC 12202 (graph 1) and clinical isolates 1 (graph 2), 2 (graph 3), 3 (graph 4), 4 (graph 5), and 5 (graph 6) on stainless steel, pure copper, and copper alloys (S30400 [•], C28000 [○], C75200 [▾], C26000 [▵], C70600 [▪], C51000 [□], and copper C11000 [⧫]) at 22°C.

FIG. 2.

Survival of vancomycin-resistant E. faecalis ATCC 51299 (graph 1), E. faecalis clinical isolates 1 (graph 2) and 2 (graph 3), E. gallinarum (graph 4), and vancomycin-sensitive E. faecalis NCTC775 (graph 5) on stainless steel, pure copper, and copper alloys (S30400 [•], C28000 [○], C75200 [▾], C26000 [▵], C70600 [▪], C51000 [□], and copper C11000 [⧫]) at 22°C.

FIG. 3.

Survival of vancomycin-resistant E. faecalis (ATCC 51299) (•) and E. faecium (NCTC 12202) (○) on stainless steel at 22°C.

FIG. 4.

Effect of inoculum concentration on survival of vancomycin-resistant E. faecium (NCTC 12202) on stainless steel, pure copper, and copper alloys (S30400 [•], C75200 [○], C26000 [▾], C70600 [▵], C5100 [▪], and C11000 [□]) at 22°C. Inoculum concentrations tested: 10⁵ CFU/cm² (graph 1), 10⁴ CFU/cm² (graph 2), and 10³ CFU/cm² (graph 3).

FIG. 5.

(A) Assessment of viability of E. faecalis (ATCC 51299) on copper (a) and stainless steel (b) surfaces using the redox dye CTC (positive for respiring cells) and SYTO 9 (total cell count, regardless of viability) (row 1) or BacLight (row 2) to detect bacterial membrane integrity. (B) Effects of inoculum concentration and cell stacking on the susceptibility of alloy C26000 to inhibit respiration. The alloy was inoculated with VRE strain E. faecium NCTC 12202 at inoculum concentrations of 5 ×10⁷ CFU/cm² (images 1, 3, and 5) and 5 × 10⁶ CFU/cm² (images 2, 4, and 6) for 2 h. Images 1 and 2 were captured using EDIC microscopy. Circled in image 1 are areas where bacterial cells at the higher inoculum concentration may not be in direct contact with the metal surface, as they are stacked on top of each other. At the lower inoculum concentrations (image 2), the cells are spread in small clumps and are all exposed to the alloy directly (this spreading of individual cells is also clearly visible in epifluorescence image 4). Epifluorescence images 3 and 4 represent SYTO 9 total cell staining. CTC-positive staining of respiring cells is present at the higher (image 5) but not at the lower (image 6) inoculum concentration. Bars, 10 μm.

FIG. 6.

Agarose gel electrophoresis of purified enterococcal DNA. (A) Purified genomic DNA of E. faecium NCTC 12202. Lane 2, cells not exposed to metal surfaces; lane 3, cells exposed to stainless steel for 2 h; lane 4, cells exposed to copper for 2 h; lane 5, cells exposed to copper for 4 h. (B) Purified genomic DNA of clinical isolates E. faecium 5 (lanes 7 and 8) and E. faecalis 2 (lanes 9 and 10) exposed to stainless steel (lanes 7 and 9) or copper (lanes 8 and 10) for 2 h at 22°C. (C) Purified plasmid DNA of E. faecium NCTC 12202 not exposed to metal (lane 11) or exposed to stainless steel (lane 12) or copper (lane 13) for 2 h at 22°C. Control lanes are Bioline Hyperladder I (lanes 1) and Hyperladder II (lanes 6). Genomic DNA was purified using the Qiagen DNeasy Blood and Tissue kit (2% agarose) and the Qiaprep Spin Miniprep kit for plasmid DNA (0.9% agarose).

FIG. 7.

Analysis of genomic DNA of E. faecalis ATCC 51299 in situ by DNA fragmentation assay following 2 h of exposure to copper (image 1) or steel (image 2). Images 3 and 4 show bacterial cells not exposed to metal surfaces and are of live and dead (heat-killed) cells, respectively, which were then used in the DNA fragmentation assay. Loops of DNA are visible in all of the samples except that in image 1, suggesting that exposure to copper has resulted in disintegration of the bacterial DNA into fragments too small to be visualized even by the sensitive nucleic acid stain SYBR Gold used for this assay.

FIG. 8.

Analysis of genomic DNA of clinical isolates E. faecalis 2 (images 1 and 2) and E. faecium 5 (images 3 and 4) in situ by DNA fragmentation assay following a 1-h exposure to copper (images 1 and 3) or steel (images 2 and 4). No DNA loops are visible on cells exposed to copper, whereas DNA fragments are visible emanating from cells isolated from stainless steel surfaces.