Ruthenium red

According to the previous method, ruthenium red staining assessed EPS production (Adnan et al., 2020 (link)). Cell suspensions (10⁶ CFU/ml) of E. coli O157:H7 and different concentrations of Ech were cultured in a 96-well plate for 24 h at 37°C. The plate was washed with PBS (pH = 7.2), stained with 0.01% ruthenium red (Yuanye, Shanghai, China), and then incubated at 37°C for 1 h. 0.01% ruthenium red was used to fill the wells without biofilm was used as blank. 0.01% ruthenium red was used to fill the wells with biofilm and without Ech was used as a positive control. The absorbance was performed at 450 nm using Multiskan Go Reader after the liquid carrying the residual stain was transferred to new 96-well plates (Thermo Fisher Scientific, USA). EPS inhibition was calculated as follows formula (Adnan et al., 2020 (link)):
Whereas:
AB = absorbance of the blank.
AS = absorbance of the sample.
AP = absorbance of the positive control.

The EPS production was quantitatively estimated by ruthenium red Staining [78 (link)]. Cell suspensions (100 µL) of E. coli (10⁶ CFU/mL) and different concentrations of Gin were cultured for 24 h at 37 °C. Afterward, the cells were washed with PBS (pH = 7.2). In each well, biofilm cells were stained with 0.01% ruthenium red (Yuanye, Shanghai, China) by incubation at 37 °C for 60 min. ruthenium red was served as a blank. Following that, the solution containing the remaining stain was retransferred onto a new 96-well plate, and the absorbance at 450 nm was measured. EPS inhibition was calculated as: $$EPS \mathrm{inhibition}\left(\%\right)=\frac{\mathrm{AS}-\mathrm{AP}}{\mathrm{AB}-\mathrm{AP}}\times 100$$
where:

AB = absorbance of blank

AS = absorbance of sample

AP = absorbance of positive control

Clinical strains of K. pneumoniae were kindly provided by Professor Du Xiangdang of Henan Agricultural University (Zhengzhou, China), and all strains were cultured under conditions in MHB medium (LA, HuanKai Microbial, Guangdong, China). Vibrio harveyi BB170 (V. harveyi BB170) and V. harveyi BB152 were gifts from Researcher Han Xiangan (Shanghai Veterinary Research Institute, Shanghai, China, Chinese Academy of Agricultural Sciences, Beijing, China).
FLD and ruthenium red were purchased from Shanghai Yuanye Bio-technology Co., Ltd. (Shanghai, China), and it was dissolved in distilled water. A LIVE/DEAD BacLight bacterial viability kit was purchased from Invitrogen. TIANcombi DNA Lyse & Det PCR Kit was purchased from Tiangen Biotech. G. mellonella were purchased from Tianjin Huiyude Biotech Company. Alamar Blue (AB) was purchased from Thermo Fisher Scientific (Shanghai, China), and 0.1% crystal violet was purchased from Beijing Solaibao Biological Technology Co., Ltd. (Beijing, China) RNAiso Plus kit (Takara Bio Inc., Beijing, China), TB Green^® Premix Ex Taq^TM Ⅱ (Tli RNaseH Plus), and PeimerScript^TM reagent kit with gDNA Eraser were purchased from Takara Bio Inc.