3.2 The antibacterial property and antibacterial mechanisms of the CDs
To evaluate the antibacterial activity of the CDs, E. coli, S. aureus P. aeruginosa , S. typhimurium , and V. parahaemolyticus strains were utilized as the model bacteria. The antibacterial property of the CDs for each bacterium was tested in the dark using the spread plate method. As shown inFigure S4 , the number of colonies decreased drastically with the increase in the concentration of the CD. A broad-spectrum antibacterial capacity of the CDs was therefore demonstrated.
Due to the electrostatic action, the positively charged CDs may complex with the membrane upon adsorption on bacteria, which alters the fluidity of the lipid bilayer and destroy the membrane structure for antibacterial purposes.[31] ONPG was used to assess the permeability of the cytoplasmic membranes ofE. coli and S. aureus . It is recognized that when the cytoplasmic membrane is disrupted, β -galactosidase is released from the cell and catalyzes the hydrolysis of ONPG to produce the yellow o-nitrophenol.[32] As shown in Figure 3a , the absorbance at 420 nm increased gradually with the increase in the CD concentration and incubation time, although the rate of increase slowed down after around 30 min. This indicates that the permeability of the cytoplasmic membranes of E. coli was positively correlated with the concentration of the CDs and incubation time. Similar results were attained for the S. aureus group (Figure 3b ). Noteworthy, higher concentrations of the CD were required for S. aureus to cause a consequence of cytoplasmic membrane damage comparable to that of E. coli . This result can be ascribed to the thicker cytoderm of S. aureus that is composed of peptidoglycan, in comparison with that of E. coli .[33] The above results suggest that the CDs disrupt the bacterial cytoplasmic membrane.
To directly visualize the disrupted bacterial structure by the CDs, the morphological change in E. coli and S. aureus were studied using electron microscopies. As shown in the SEM images, E. coliin the control group possessed a smooth surface with an intact cell wall and a well-stereo-shaped body, while the bacterial cell walls treated with the CDs became rough and displayed obvious wrinkles and damage (Figure 3c ). The TEM images further confirmed the disruption ofE. coli structure caused by the CDs (Figure 3d ).E. coli treated with the CDs demonstrated significant morphological change, and the intracellular content appeared to oozed out. Similar phenomena were observed for S. aureus . Both the SEM and TEM characterizations reveal that S. aureus treated with the CDs had severe surface structural damage and clear leakage of intracellular content. The above result indicates that the disruption of the membrane structure by the CDs may be responsible for the bacteria leakage and the bacteria-killing effect (Figure 3e ).