The antibacterial activity of copper surfaces was evaluated by overlying suspensions of S. enterica and C. jejuni on copper surfaces, stainless steel, and a synthetic polymer. Standardized bacterial suspensions, 3 × 106 cells per ml, were deposited over the surfaces and bacterial concentrations counted at 0, 2, 4 and 8 hours. The experiments were performed at 10°C and 25°C, and each assay was carried out in duplicate. Results were calculated as the average of five independent assays for each strain and temperature.
S. enterica was grown on xylose lysine deoxycholate (XLD) agar and Brain Heart Infusion broth (BHI). C. jejuni was grown on Skirrow agar (Columbia agar base supplemented with 7% horse blood), and Exeter base broth. All media were acquired from Oxoid.
S. enterica (serovar enteritidis) and C. jejuni utilized in this study were isolated from chicken and identified by standard biochemical tests and PCR (15, 16). E. coli was isolated from a healthy individual, all strains were maintained frozen (-20°C).
Metallic (electrolytic purity) copper sheets 0.5 mm thickness was used to evaluate copper antibacterial activity. Stainless steel sheets (0.5 mm) and polyformaldehide (formalite) obtained locally were used as controls.
S. enterica was inoculated in BHI broth from a single colony in a XLD plate and incubated at 37°C overnight, 0.5 ml was then taken and added to 25 ml of fresh broth, which was incubated with shaking at 37°C for 90 minutes. The absorbance at 600 nm of the bacterial growth was measured, and the culture was adjusted to approximately 3 × 106 bacterial cells/ml by dilution in BHI broth. C. jejuni was seeded on a Skirrow selective plate, incubated in a reduced oxygen atmosphere in a gas jar with a gas pack (Campylobacter Gas generating Kit BR60) at 42°C for 18 hours. The bacterial growth was harvested in Exeter broth and also adjusted to 3 × 106 bacterial cells/ml as described for S. enterica.
Viability assays on copper and control surfaces
Bacterial suspensions were overlaid on sterile copper and control surfaces, the viable counts were performed at 0, 2, 4 and 8 hours. Copper, stainless steel, and polyformaldehide sheet circles (8.5 cm diameter) were kept in Petri dishes during the experiments, which are maintained in a closed plastic box in humid conditions (with wet paper towels). 1.5 ml of the bacterial suspensions was overlaid on the center of each circle. Aliquots, 100 μl, were successively taken in duplicate for each of the conditions assayed, and diluted in order to obtain suitable plate counts. 100 μl of each dilution were inoculated on appropriate agar plates for each strain. Plate Count Agar was used for S. enterica, and incubated at 37°C for 24 hours. C. jejuni was plated onto Skirrow agar plates and incubated at 42°C for 40 hours in microaerophilic conditions. The colonies were counted, and the results were expressed as colony forming units per ml of sample (CFU/ml).
Determination of copper content in meat
In order to determine if copper would adsorb to exposed meats, chicken and pork pieces 2 × 2 cm (4–5 g) were placed for 0, 10, 20, 30, 40 and 50 minutes on copper sheets at room temperature (22–26°C). A set of copper sheets was used several times in these assays. A total of 30 samples of chicken and 30 samples of pork were analyzed. Prior to the exposure to the copper surfaces, all meat pieces were kept for few seconds in saline solution (0.85%) to simulate wet conditions commonly found in the processing of these products. The copper content in the meat pieces was determined by atomic absorption spectrophotometry in duplicate samples, according to the AOAC procedure . To take into account a potential effect of the bacterial load on the amount of copper detected in the exposed meat, the same procedure was performed in a subset of 3 samples of chicken and pork, which were previously contaminated by immersion for 5 minutes in a suspension of E. coli (104 CFU/ml in saline solution).
The data were analyzed using the statistical software Statistica (Anova/Manova for the statistical parameters of the averages, and the Scheffe test of multiple comparisons for the statistical significance). Each point of the results represents the average of five independent assays, for each time of exposure to copper.