3.4 Effect of the CDs on biofilms
Biofilms can provide physical barriers for bacteria, and enhance antibiotic resistance, making it even more difficult to kill.[3] We further employed the CDs in a trial of biofilm elimination using S. aureus and S. typhimurium as the model bacteria, which are both foodborne pathogens that can produce biofilms.[44] They have been recognized as an important food safety issue causing huge economic losses in the food industry.[45] The crystal violet-staining method was used to assess the extent of damage to the biofilms by the CDs,[46] in which crystal violet binds to lipid molecules in the biofilms via hydrogen bonding and electrostatic interaction. The occurrence of the purple solution shown inFigure 5 is attributed to the alcohol-induced disruption of the interactions, and subsequently the shedding of the crystal violet from the biofilms. The absorbance of the solution at 590 nm gradually decreases with increasing amount of the CD in the concentration range of 1‒50 mg/mL, consistent with the lighter color of the solution (Figure 5a ). This indicates that the CDs could destroy theS. aureus biofilms in a concentration-dependent manner over a range of concentrations. Though the absorbance of the CD-treated biofilm system decreases no matter under light or in the dark, there is a photo-enhancement effect on the disruption of the biofilm by the CDs. When the concentration of the CD was 10 mg/mL, the extent of damage for the S. aureus biofilm was enhanced by 22% with the photo-activated CDs. The absorbance remains almost unchanged when the CD concentration reaches 50 mg/mL, indicative of a saturation effect for the concentration of the CDs. Under the condition, the S. aureusbiofilm was disrupted in a percentage of as high as 83.2%. Similar results were attained for the S. typhimurium group (Figure 5b ). Notably, when the concentration of the CDs was 10 mg/mL, there was a strong photosensitization of the CDs, boosting the damage of the S. typhimurium biofilm by about 17.8% compared with that in the dark.
By treating bacteria with the CDs at early stages, we also discovered that the CDs could inhibit biofilm formation. Figure 5c shows the absorbance at 590 nm decreases with increasing the CD concentration in the range of 1‒20 mg/mL. Further, there is still a photo-enhancement effect on the inhibition of biofilm formation by the CDs. The CDs could enhance the inhibition to the formation of S. aureus biofilm by 34.5% under irradiation at the concentration of 15 mg/mL. The photoactivated inhibition was nearly saturated at the concentration of 20 mg/mL, whereas a much higher one (30 mg/mL) was required to achieve a similar consequence in the dark, agreeing very well with the digital photo results. Interestingly, the CDs could inhibit S. aureusbiofilm formation in an extent of about 95.9% under irradiation at a concentration of 20 mg/mL. We observed similar results for the S. typhimurium group (Figure 5d ). It is further inferred that the CD at a concentration of 15 mg/mL could inhibit S. typhimuriumbiofilm formation in a rate of approximately 97.5% under irradiation. We therefore suggest that the CDs afford high potential in both effective disruption and inhibition of biofilm (Figure 5e ). On the other hand, the inhibition of biofilm by the CDs is likely more effective than the disruption. This result can be ascribed to the ease of killing bacteria at the early stage of biofilm formation, in which no compact extracellular polymeric substances around the bacteria are produced yet.[47] Furthermore, the CDs are featured with low cytotoxicity. A minor decrease in cell viability (~92%) was observed after incubation for 24 h at a CD concentration of 100 mg/mL (Figure S7 ), indicative of a good balance of high antibacterial activity and low toxicity.