Syto9 stain

For measurement of PSY2 expression, quantitative real-time PCR (qRT-PCR) was carried out using a LightCycler 480 system (Roche) using SYTO 9 stain (ThermoFisher Scientific). The reaction mixture comprised a total volume of 20 μl, containing 2 μl 4× diluted cDNA, 0.3 μl R Taq (Takara Bio), 2 μl 10× PCR buffer, 2 μl 10 mM dNTPs, 0.5 μl 10 pmol primers, and 0.5 μl SYTO 9. The primer sequences were as follows: 5′-AGACAGAGGTGGAATTTTGGGTCT-3′ (forward) and 5′-CAAATTCCCCGGAAGCACA-3′ (reverse). The real-time PCR involved 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 20 s. Three replicate reactions were performed. Actin served as an internal control.

To confirm that the bleach treatment that we employed to isolate candidate endophytic bacteria from duckweed can effectively remove most if not all surface-associated bacteria, fluorescent microcopy was performed on Lemna minor strain 370-DWC112 inoculated with DAB 1A (Supplementary Figure 1). Plant tissue was treated with 0.3% (v/v) sodium hypochlorite for 2 min using the method described for isolation of bacteria, which left only the meristem to retain chlorophyll. The tissue was placed in a microcentrifuge tube with 100 μL of 6 μM Syto 9 stain (ThermoFisher Scientific, Waltham, MA). The Syto 9 stain was removed after 3 min and the tissue was washed twice with 200 μL of sterile water. The tissue was then placed on a microscope slide and observed using an Olympus FSX100 epifluorescence microscope (460–495 nm excitation/510–550 nm emission). To image the surface of the plant tissue, the 10x objective lens was first used to focus on the guard cells of the stomates before switching to higher magnification objectives to resolve stained bacteria on the same focal plane.

Biofilms were examined by SEM and CLSM (Djeribi et al., 2012 (link)). For SEM examination, biofilms were developed at 37 °C for 24 h in polystyrene coverslips placed into a 24-well plate with MHB containing bacterial cells (OD_600nm 0.01) added. After fixed in 3.7% formaldehyde for 40 min at room temperature and then rinsed twice with PBS, further fixation was performed with 1% osmium tetroxide for 15 min at room temperature and then rinsed twice with PBS. The samples were then dehydrated by passing them through different concentrations of ethanol: 35%, 50%, 70%, 80%, and 95%, each for 5 min, followed by 100% ethanol twice for 10 min each time. The samples were then dried at 60 °C for 24 h. After being coated with gold-palladium (via sputter coating), the samples were examined under a bioscanning electron microscope (Hitachi, S-4700, Type II).
For CLSM examination, biofilms formation in polystyrene coverslips and fixation in 3.7% formaldehyde were the same as mentioned above. Then the samples were stained with a prepared 100 nM solution of Syto-9 stain (Invitrogen, Carlsbad, CA) for 30 min (Luo et al., 2015b (link)). Syto-9 stained biofilms were excited with a 488 nm solid-state laser, and fluorescence was captured between 500–550 nm. The images of biofilms were rendered and assembled using appropriate computational software (ZEISS LSM780).

Three representative isolates were chosen for imaging. They included a bap alone isolate, a bap + bla_OXA−23 positive isolate, and a bap + bla_OXA−23 negative isolate (note, no bla_OXA−23 alone were detected in our study) of A. baumannii (isolates DMC0256, DMC0420, and DMC0551, respectively). A modified method that was developed by Rao and colleagues was used (Rao et al., 2011 (link)). Biofilms were developed overnight at 37°C in polystyrene 4-well chamber slide system (Lab-Tek, Naperville, IL) containing 2 mL of R2A media per well. The media was aspirated from each well and stained with prepared 10 μM solution of Syto-9 stain (Invitrogen, Carlsbad, CA) for 30 min. Subsequently, the biofilms were washed with PBS (pH 7.4) three times. A Leica SPE (Leica, Buffalo Grove, IL) confocal laser scanning microscope (CLSM) was used to examine the stained biofilms. Syto-9 stained biofilms were excited with a 488 nm solid-state laser and fluorescence was captured between 500–550 nM with a 40 × 1.25 NA objective lens. Gain and offset were calibrated using the Leica look-up-table and kept constant for experiments. Following biofilm imaging, biofilms were rendered using IMARIS version 7.3.1 (Bitplane, Zurich, Switzerland) imaging software. Captured renderings were assembled in CORELDRAW v. X4 (Corel, Mountain View, CA).

Biofilms formed in presence of the potentially active extracts were visualized by epifluorescence microscopy. For this analysis, S. aureus biofilms were grown as described above but in a 6-well microplate (Falcon, TC-treated, polystyrene) and with a total volume of 6.0 mL (3.0 mL of S. aureus bacterial suspension prepared in BB 2X (10² CFU/mL) + 3.0 mL of tested extract or 3.0 mL of SDW for the biofilm growth control).
After 24 h of incubation at 37 °C and in order to evaluate the potential effect of extracts on S. aureus biofilm (cells number and matrix), 1.0 mL of SYPRO Ruby stain (Invitrogen^TM, FilmTracer^TM, SYPRO^TM Ruby biofilm matrix stain) was added after discarding wells content. This stain binds to most classes of proteins including glycoproteins, lipoproteins, phosphoproteins and fibrillar proteins. After 30 min of incubation in dark at room temperature, wells were carefully washed twice with 1.0 mL of SDW. Six mL of SDW were then added supplemented with 1.0 µL of Syto9 stain (5 mM, Invitrogen^TM, ThermoFisher Scientific, llkirch, France) for cell visualization.
Microscopic observations were made with Zeiss—Axiotech microscope using a 20 ×/0.50 (Zeiss, EC Plan-Neofluar) objective and equipped with an HXP 120 C light source. Images were acquired with a digital camera (Zeiss AxioCam ICm 1) and then the set of photos was processed with ZEN software.