Efficacy of copper alloys to reduce infectivity of wet fomite contamination with MNV at 4°C (A) and 37°C (B).

<p>Approximately 5×10⁴ pfu were applied to test surfaces that had been acclimatised to required temperature (copper (•), phosphor bronze (95% copper) (○), copper nickel (▾), cartridge brass (70% copper) (Δ), nickel silver (65% copper) (▪), stainless steel (□)) in 20 µL (‘wet’ inoculum). Virus was removed and assessed for infectivity using plaque assay.</p

Composition of metals used in the study.

a<p>Unified Numbering System.</p

Destruction of entire MNV genome occurs on copper.

<p>MNV (PEG concentrate) was exposed to copper (lane 1), cartridge brass (lane 2) or stainless steel (lane 3) for 2 hours. Viral RNA was purified using Qiagen mini prep viral RNA kit and fragments separated on non-denaturing 1% agarose gel electrophoresis and visualised in UV light box. Viral RNA has degraded on copper, less on brass and not at all on stainless steel (see control RNA S2 Supplementary Information). Lanes 4, 5 and 6 are PEG precipitation of uninfected cells (mock) applied to stainless steel, brass and copper respectively. Virus added to all surfaces and removed immediately was similar to lane 1 although some reduction in intensity on copper was visible (not shown). DNA ladder is Bioline hyperladder I (HL1 1 Kb)</p

Comparison between inactivation rates of MNV in wet fomite (A) and dry touch (B) contamination on copper surfaces.

<p>Inactivation rates were calculated for various contact times of MNV exposed to test surfaces as described in the text (from the results generated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075017#pone-0075017-g001" target="_blank">Figure 1</a>). (copper (white bars), phosphor bronze (95% copper) (forward diagonal striped bars), copper nickel (89% copper) (backward diagonal striped bars), cartridge brass (70% copper) (cross hatch bars), nickel silver (65% copper) (horizontal striped bars) and stainless steel (vertical striped bars)). Error bars represent ± SD and data are from multiple experiments.</p

Inactivation of MNV on copper surfaces in the presence of quenchers D-mannitol or Tiron (A) or chelators EDTA or BCS (B) and to remove hydroxyl radical or superoxide, copper II or Cu I, respectively.

<p>Approximately 5×10⁴ pfu MNV was inoculated onto metal surfaces in the presence of chelators or quenchers of reactive oxygen species and assessed for infectious virus using plaque assay as described in text. The results were compared to those obtained without chelators or quenchers to ascertain if there was a protective effect. No quenchers or chelators present is represented by white bars; D-mannitol (A) or EDTA (B) represented by diagonal striped bars; Tiron (A) or BCS (B) represented by cross hatched bars. No significant reduction of infectivity occurred in the presence of any quenchers or chelators on stainless steel surfaces (even though D-mannitol has been reported to interfere with HSV replication) (C and D respectively). Error bars represent ± SD and data are from multiple experiments.</p

Extended depth of field composite EDIC images of silicone catheter sections following long term exposure to <i>P</i>. <i>mirabilis</i>.

<p>a. 12 days exposure, a single struvite crystal on exposed silicone catheter (where the diffuse crystalline material has detached) with motile <i>P</i>. <i>mirabilis</i> on the crystal and on the uncolonised surface. b—d. struvite crystals embedded in diffuse crystalline material. b. 15 days exposure. c. 18 days exposure. d. 20 days exposure. (Magnification a x 1000, bar = 10 μm; b—d x 500, bar = 20 μm).</p

Representative EDIC images of biofilm development on hydrogel latex catheter sections.

<p>a. 3 days exposure showing thick microcrystalline layer. b. 9 days exposure showing development of a three-dimensional, diffuse crystalline structure, extending out from the surface. (Magnification x 1000, bar = 10 μm).</p

Representative EDIC images showing crystalline biofilm development on all silicone catheter sections exposed to <i>P</i>. <i>mirabilis</i>.

<p>a. 24 h exposure showing multi-layered appearance and highly reflective, motile <i>P</i>. <i>mirabilis</i>. b. 3 days exposure showing development of a microcrystalline layer attaching to the initial conditioning film below. c. an individual struvite crystal formed after 3 days exposure. d. after 4 days exposure, copious amounts of diffuse crystalline material (apatite) formed, creating a thick three-dimensional structure. e. at the same time (and over the remaining time course), large, rod-shaped crystal embedded in diffuse crystalline material formed, extending in length to 10 mm. (Magnification a—d x 1000, bar = 10 μm; e x 500, bar = 20 μm).</p

EDIC images of unused catheter sections showing surface topography.

<p>a. All silicone catheter. b. Hydrogel latex catheter. (Magnification x 1000, bar = 10 μm).</p

Schematic representation of the four stages of crystalline biofilm development by <i>P</i>. <i>mirabilis</i>.

<p>This shows the development from initial conditioning film to complex crystalline biofilm with <i>P</i>. <i>mirabilis</i> found throughout the structure.</p