Fig. 1. Schematic diagram displaying the antibacterial testing experimental process. Three coupons of each metal alloy (AHEA, 304 SS, 304 Cu-SS, and Cu) were immersed in 50 mL of the simulated seawater containing an initial bacterial concentration of 105 CFU/mL. Samples of each metal were incubated at 30 °C for one, three, and seven days. Planktonic bacteria and bacterial biofilms attached to the coupon surfaces were removed and serially diluted. 100 μL of each dilution was plated on 2216E agar, and live bacterial cell numbers were calculated after 48 h of incubation at 30 °C.

Fig. 2. (a) XRD patterns of the Al0.4CoCrCuFeNi AHEA; (b) detailed scans of the (111) peak, and (c,d) SEM images showing the microstructure of the Al0.4CoCrCuFeNi AHEA at different magnifications.

Fig. 3. Elemental mapping of Al, Co, Cr, Cu, Fe, and Ni elements in the Al0.4CoCrFeNi AHEA.

Fig. 4. Coupon biofilm and planktonic colonies of (a) P. aeruginosa and (b) B. vietnamensis cultured on 2216E agar plates after incubation for 1, 3, and 7 days at 30 °C. The following dilutions are shown: (1) P. aeruginosa coupon biofilms: 304 SS 1000-fold, 304 Cu-SS 1000-fold, AHEA 10-fold, and Cu 10-fold. 2 Planktonic P. aeruginosa cells: 304 SS (10,000-fold) 304 Cu-SS (10,000-fold), AHEA (10-fold), and Cu (10-fold). (3) B. vietnamensis coupon biofilms: 304 SS (100-fold), 304 Cu-SS (100-fold), AHEA (10-fold), and Cu (10-fold). (4) Planktonic B. vietnamensis cells: 304 SS 1000-fold, 304 Cu-SS 1000-fold, AHEA 10-fold, and Cu 10-fold.

Fig. 5. Live/dead cell staining images of (a) P. aeruginosa biofilms and (b) B. vietnamensis biofilms after incubation with 304 SS, 304 Cu-SS, AHEA, or pure Cu for 1, 3, and 7 days. Green fluorescence represents living cells while red fluorescence represents dead cells.

Fig. 6. Cells and EPS staining images of (a) P. aeruginosa biofilms and (b) B. vietnamensis biofilms after incubation with 304 SS, 304 Cu-SS, AHEA, or pure Cu for 1, 3, and 7 days. Blue fluorescence represents bacterial cells, green fluorescence displays EPS polysaccharides, and red fluorescence displays EPS proteins.

Fig. 7. Concentration of copper ions released from 304 Cu-SS and AHEA after seven days of incubation in the simulated seawater and SEM images: (a) concentration of copper ions in the P. aeruginosa solution, (b) concentration of copper ions in the B. vietnamensis solution, (c) adhesive P. aeruginosa colonies and (d) adhesive B. vietnamensis colonies on the 304 SS, 304 Cu-SS, AHEA, and copper coupons after seven days of exposure.

Fig. 8. Schematic illustration for (a) the antimicrobial mechanism of AHEA, (b) polarization curves of 304 SS, 304 Cu-SS, and AHEA in the simulated seawater and (c) yield stresses and antibacterial rates. The yield stresses of 316/317 Cu-SS were reported in the literatures [58,59]. Antibacterial rates for the antibacterial SS were measured under identical conditions with AHEA in the presence of P. aeruginosa and B. vietnamensis.