Polystyrene tube

The 1 day, standardized PFGE protocol (Han et al., 2013 (link)) was used for all CRKP isolates during the study periods. Cell suspensions were placed in polystyrene tubes (Falcon; 12 × 75 mm), and their optical densities were adjusted to 3.8–4.0 by a Densimat photometer (BioMérieux, Marcy l'Etoile, France). Slices of CRKP agarose plugs were digested using 50 U of XbaI (TaKaRa Bio, Dalian, China) per slice for 4 h at 37°C, and electrophoresis was performed using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was conducted with a switch time of 6 to 36 s for 18.5 h, and images were captured using a Gel Doc 2000 system (Bio-Rad) and converted to TIFF files which were analyzed by BioNumerics version 5.1 software (Applied Maths, Kortrijk, Belgium). A similarity analysis of the PFGE patterns was performed by calculating the Dice coefficients (S_D) and clustering was performed using the unweighted-pair group method with average linkages (UPGMA).

We used the 1-day, standardized PFGE protocol for K. pneumoniae [35 (link)]. Cell suspensions were placed in polystyrene tubes (Falcon; 12 × 75 mm), and their optical densities were adjusted to 3.8–4.0 using a Densimat photometer (BioMérieux, Marcy l’Etoile, France). Slices of K. pneumoniae agarose plugs were digested using 50 U of XbaI (Takara) per slice for 4 h at 37°C, and electrophoresis was performed using a CHEF-DRIII system (Bio-Rad Laboratories, Hercules, CA, USA). Electrophoresis was conducted with a switch time of 6 s to 36 s for 18.5 h, and images were captured using a Gel Doc 2000 system (Bio-Rad) and converted to TIFF files. The TIFF files were analyzed using BioNumerics version 5.1 software (Applied Maths, Kortrijk, Belgium). A similarity analysis of the PFGE patterns was performed by calculating the Dice coefficients (SD) [36 (link)] and clustering was performed using the unweighted-pair group method with average linkages (UPGMA).

P. aeruginosa and S. aureus were grown in 15 mL polystyrene tubes (Falcon) containing 5 mL of LB or TSB medium, incubated at 37 °C for 48 or 24 h respectively, in static conditions, in the presence of EPS B3-15 (300 µg/mL) or PBS as control. To obtain the RNA, 2 mL of each culture was centrifuged (8000 rpm × 10 min) and the bacterial cells were suspended in Trizol Reagent (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Residual DNA was removed by digestion for 30 min at 37 °C with 1 U of RNase-free DNase (Promega Corporation, Madison, WI, USA). The reaction was stopped by adding 1 μL of RQ1 DNase stop solution and the tubes were incubated at 65 °C for 10 min. RNA samples were quantitatively analyzed using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A total of 300 ng of RNA was used for complementary DNA (cDNA) synthesis. The reverse transcription (RT) was carried out in a 20 µL reaction mixture, containing 1 × reaction buffer, 0.5 mM dNTP, 20 pmol primers, 3 mM MgCl₂, 20 U RNase inhibitor, and 200 U Improm II reverse transcriptase (Promega Corporation, USA). The reaction was performed using an initial incubation at 25 °C for 5 min, followed by incubation at 37 °C for 60 min and finally at 70 °C for 15 min. After reverse transcription, the cDNA of each sample was directly analyzed in Real-Time PCR.