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.