2.12. In vitro antibiofilm assay
For establishment of biofilms, 5×108 CFU mL-1 of K.pneumoniae cells were added to 96-well plate(100 μL per well)and incubated under stationary conditions at 37 °C for 1 day. After biofilm formation, the medium was discarded and the plates were washed with PBS to remove the planktonic bacteria. Then 200 µL of HMPB(100μg/mL), ofloxacin(10 μg/mL) and OHH NPs with HMPB concentration of 100 μg/mL was added to the plates, respectively. For control group, 200 µL of PBS without nanomaterials was used. All groups were incubated at 37°C for 12 h. For the NIR irradiated group, 808 nm laser at power density of 1 W/cm2 was used to illuminate sample for 5 min. The media in each well was then removed, and the plates were washed carefully with PBS one time. The biofilm in each well was fixed with methanol for 15 min and stained with 0.1% crystal violet for 15 min. After washing with Milli-Q water for three times, ethanol (100 µL per well) was added to solubilize the crystal violet staining. OD560 in each well was read to determine the biofilm formation.
Additionally, to obtain the 3D morphology of the K.pneumoniaebiofilms, the biofilms formed under different treatments was dyed by propidium iodide (PI) for 30 min in the dark. After, the biofilms stained by PI were washed three times with PBS and observed by a confocal laser scanning microscope (FV1200, Olympus). The red fluorescence intensity of 3D images of biofilms was quantified using Image J.