Toward a Molecular Understanding of the Antibacterial Mechanism of Copper-Bearing Titanium Alloys against Staphylococcus aureus

Abstract

The antibacterial mechanism of the Cu-containing materials has not been fully understood although such understanding is crucial for the sustained clinical use of Cu-containing antibacterial materials such as bone implants. The aim of this study is to investigate the molecular mechanisms by which the Gram-positive Staphylococcus aureus is inactivated through Cu-bearing titanium alloys (Ti6Al4V5Cu). Cu ions released from the alloys are found to contribute to lethal damage of bacteria. They destroy the permeability of the bacterial membranes, resulting in the leakage of reducing sugars and proteins from the cells. They also promote the generation of bacteria-killing reactive oxygen species (ROS). The ROS production is confirmed by several assays including fluorescent staining of intracellular oxidative stress, detection of respiratory chain activity, and measurement of the levels of lipid peroxidation, catalase, and glutathione. Furthermore, the released Cu ions show obvious genetic toxicity by interfering the replication of nuc (species-specific) and 16SrRNA genes, but with no effect on the genome integrity. All of these effects lead to the antibacterial effect of Ti6Al4V5Cu. Collectively, our work reconciles the conflicting antibacterial mechanisms of Cu-bearing metallic materials or nanoparticles reported in the literature and highlights the potential use of Ti6Al4V5Cu alloys in inhibiting bacterial infections.

Keywords: Staphylococcus aureus; antibacterial mechanism; copper; titanium alloys.

Figure 1

Schematic illustration of the general idea for this study and the possible anti-bacterial mechanisms on the surface of Ti6Al4V5Cu implants. Ti6Al4V5Cu releases Cu ions. The released Cu ions (1) accumulate in the cell membrane affecting membrane permeability; (2) disrupt the activity of respiratory chain; (3) enter bacterial cells to generate ROS; and (4) disturb the gene replication of S.aureus

Figure 2

Microstructures (A, B) and XRD patterns (C) of Ti6Al4V and Ti6Al4V5Cu alloys. (B) exhibited metallurgical structures of the annealed Ti6Al4V5Cu alloys, showing that equiaxed α phase (indicated by the red arrow) was distributed in the matrix of β phase. The typical biphasic microstructure of Ti6Al4V5Cu alloys (B) was similar to that of Ti6Al4V alloys (A). (C) exhibited the XRD patterns of Ti6Al4V5Cu and Ti6Al4V alloys. The two profiles were similar and reflection peaks belonging to α and β phase could be clearly indexed. However, an intermetallic phase Ti2Cu was found apparently in the matrix of Ti6Al4V5Cu alloy compared to Ti6Al4V alloy.

Figure 3

Cumulative concentration of Cu ions released from Ti6Al4V5Cu alloys and the average release rates. The data indicated that the release of Cu ions was continuous from Ti6Al4V5Cu alloy in saline and the accumulated concentration was increased with time in 24 h, but not in a linear trend. At the beginning, Cu ions kept a high releasing rate, however, the rate slowed down with time. It was these Cu ions released in the early stage that played the antibacterial role.

Figure 4

The role of Ti6Al4V5Cu in inhibiting the viability of S.aureus. The live/dead bacterial cells were stained at 6 h, 12 h and 24 h by LIVE/DEAD Baclight assay, and representative images after cell staining were observed by fluorescence microscopy. Live cells were dyed by SYTO9 and showed green fluorescence, whereas dead cells were dyed by PI and showed red fluorescence. The number of live bacteria in Ti6Al4V group with green fluorescence increased significantly in comparison to the Ti6Al4V5Cu group. However, numbers of live cells decreased remarkably in Ti6Al4V5Cu group, almost all bacteria died (red fluorescence) at 24 h.

Figure 5

Effects of Ti6Al4V5Cu on the membrane leakage of proteins and reducing sugars. (A) Leakage of proteins from S.aureus treated with Ti6Al4V5Cu using BCA assay. The leakage amount of proteins was increased obviously after 12 h co-culturing (p < 0.01). (B) Leakage of reducing sugars from S.aureus co-cultured with Ti6Al4V5Cu. The highest amount of leakage was reached on 24 h, at a level of 97±8.02 % more than the blank control group (p < 0.01). All data represent the mean ± standard deviation of three independent experiments. *: compared to the blank control group, #: compared to the Ti6Al4V group. One symbol, p < 0.05; Two symbols, p < 0.01.

Figure 6

The role of Ti6Al4V5Cu in ROS generation of S.aureus. (A) Representative micro-photographs of Ti6Al4V5Cu-induced ROS generation in S.aureus adhered on samples. ROS was studied using 2,7-DCFH-DA dye, and images were snapped under fluorescence microscope. The increasing fluorescence value indicated that continuous Cu ions released from Ti6Al4V5Cu accelerated the oxidative damage to S.aureus. (B) ROS fluorescence intensities in the suspended bacteria co-cultured with Ti6Al4V5Cu. ROS generation induced by Cu ions from Ti6Al4V5Cu was elevated remarkably and significantly higher than that from the blank control group and Ti6Al4V group (p < 0.01). All data represent the mean ± standard deviation of three independent experiments. *: compared to the blank control group, #: compared to the Ti6Al4V group. One symbol, p < 0.05; Two symbols, p < 0.01

Figure 7

Effects of Ti6Al4V5Cu on the oxidative stress markers in S.aureus. (A) Activity of respiratory chain dehydrogenases analyzed by catalytic reaction using INT reagent. (B) Activity of catalase (CAT) measured by CAT activity kit. Both the two enzyme activity was inhibited in S.aureus treated with Ti6Al4V5Cu obviously with the extension of the culture tim, and reached the lowest level at 24 h. (C) Glutathione (GSH) levels were reduced and (D) Lipid peroxidation (LPO) levels were enhanced significantly in comparison to blank control and Ti6Al4V group (p < 0.01). All data represent the mean ± standard deviation of three independent experiments. *: compared to the blank control group, #: compared to the Ti6Al4V group. One symbol, p < 0.05; Two symbols, p < 0.01.

Figure 8

Effects of Ti6Al4V5Cu on the genotoxicity in S.aureus. (A) Determination of genomic integrity in S.aureus at 24 h. The image of genome DNA obtained by agarose gel electrophoresis and ethidium bromide staining indicated that there were no damages of genomic integrity. Lane M, 1.5 kb DNA ladder; Lane1, Blank control group; Lane 2, Ti6Al4V group; Lane 3, Ti6Al4V5Cu group. (B) Detection of 16srRNA and (C) nuc expression in S.aureus using EMA-PCR method. 16SrRNA and nuc gene expression levels of the bacteria co-cultured with Ti6Al4V5Cu samples decreased constantly within 24 h, and reached the lowest level on 24 h. Lane M, 2000bp DNA ladder; Lane1, 4, 7, Blank control group at 6 h, 12 h and 24 h, respectively; Lane 2, 5, 8, Ti6Al4V group at 6 h, 12 h and 24 h, respectively; Lane 3, 6, 9, Ti6Al4V5Cu group at 6 h, 12 h and 24 h, respectively. Error bars represent standard deviations of the average. All data represent the mean ± standard deviation of three independent experiments. *: compared to the blank control group, #: compared to the Ti6Al4V group. One symbol, p < 0.05; Two symbols, p < 0.01.