Live dead baclight kit

Bacterial colonies, grown overnight on blood agar plates, were inoculated into 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) to obtain a turbidity of 0.5 ± 0.1 McFarland (McF) corresponding approximately to 1 × 10⁸ colony-forming units (CFU)/ml. Samples were diluted 1:1000 and resuspended in 1 ml of brain heart infusion broth (BHI) in a μ-Slide, 8 well glass bottom chamber slides (Ibidi, Germany). The bacterial suspension was incubated at 37 °C for 24 h to allow biofilm formation. Afterwards, the medium was removed and biofilms were washed with 0.45% saline solution. The samples were stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), according to supplier specifications. Biofilm samples were analyzed using a Zeiss LSM5 Pascal Laser Scan Microscope (Zeiss, Oberkochen, Germany) as described previously [36 (link)].

Colonies of L. crispatus P17631 and L. paracasei I1688, cultivated overnight on blood agar plates, were used to prepare a bacterial suspension. Specifically, the colonies were suspended in 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) until they reached a turbidity of 2.5 ± 0.3 on the McFarland scale, equating to roughly 1 × 10⁸ CFU/ml. This suspension was then diluted at a ratio of 1:1000 and transferred into 1 ml of BHI within μ-Slide, 8-well glass bottom chamber slides (Ibidi, Germany). The bacterial mixture was incubated at 37 °C over 48 h for biofilm development. After this period, the medium was discarded, and the samples were rinsed with 0.45% saline solution. Biofilm cells were then stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), following the manufacturer's guidelines, and examined with an Apotome system (Zeiss, Oberkochen, Germany) connected to an Axio Observer inverted fluorescence microscope (Zeiss). Data were analyzed with the ZEN 3.2 (blue edition) software (Zeiss).

LIVE/DEAD BacLight kit (Life Technologies, Paisley, UK) was used to measure membrane integrity. Harvested cells were reconstituted with saline and 3 µl of the dye mixture (1.5 µl of SYTO9 (3.34 mM) and 1.5 µl of propidium iodine (20 mM)) was added to each 1 ml of bacterial suspension and mixed. Tubes were incubated for 15 minutes in the dark with occasional shaking and fixed with 20% paraformaldehyde (PFA) and kept at 4°C. Specimens were viewed on an Olympus BX60 microscope fitted with an Andor Ultrahigh-resolution CCD setup. A ×20 oil immersion lens was used to obtain a 200 µm field width. Excitation and emission filters were 480/520 nm and 515/560 nm respectively.

S. aureus colonies, grown overnight on blood agar plates, were used to inoculate 3 ml of 0.45% saline solution (Air Life, Carefusion, CA, USA) to obtain a turbidity of 0.5 ± 0.1 McFarland (McF) corresponding approximately to 1 × 10⁸ colony-forming units (CFU)/ml. Samples were diluted 1:1000 and resuspended in 3 ml of BHI containing sterile polystyrene slices of 1 cm². Bacterial suspension was incubated at 37 °C for 22 hours to allow biofilm formation onto polystyrene slices. Subsequently, the medium was removed and the polystyrene slices were washed in 0.45% saline solution. The samples were stained using the LIVE/DEAD BacLight kit (Life Technologies, New York, NY, USA), according to supplier specifications. Images were collected by a Zeiss LSM 510 Meta confocal laser scanning microscope equipped with a 20X and 60X/1.23 NA oil immersion objective (Carl Zeiss, Jena, Germany). Data were analyzed with the LSM 510 R. 3.2 META image analysis software (Carl Zeiss, Jena, Germany).

To measure the bactericidal activity of histone H4, R. solanacearum GMI1000 cells were grown overnight in CPG, pelleted, washed, and adjusted to an OD₆₀₀ of 1.5 with sterile water. Recombinant human histone H4 (New England Biolabs, Ipswitch, MA) was added to a final concentration of 10, 20, 50 or 100 μg/ml and incubated for 3 h at 28°C. Bacterial viability was then measured using the LIVE/DEAD Baclight kit (Life Technologies) according to the manufacturer’s protocol. The percentage of live cells was determined by plotting the SYTO9/PI fluorescence ratio against a standard curve based on known mixtures of live and dead cells. The experiment was repeated twice, each with three technical replicates.

The survival of bacteria in hemolymph was examined using the LIVE/DEAD BacLight kit (Molecular Probes). The hemolymph was collected as described above and cytospun on polylysine glass. The staining procedure was performed according to the manufacturer’s instructions. Bacteria were visualized by confocal laser scanning microscopy (CLSM) using a Zeiss LSM 880 confocal system equipped with 100× oil immersion objectives. The acquired Z-stack images from five different fields of view were analyzed using Zeiss ZEN microscopy software and Fiji software. The quantification of bacteria in the hemolymph of G. mellonella was calculated as an area of fluorescence spot intensity using ImageJ software.

The acylase from Aspergillus melleus (specific activity of 0.25 U mg⁻¹), Luria Bertani broth (LB)—for acylase QQ activity evaluation on C. violaceum, Mueller Hinton broth (MHB)—for determining minimal inhibitory/bactericidal concentrations, TSB—for biofilm inhibition tests and cetrimide selective agar for the culturing and enumeration of P. aeruginosa were purchased from Sigma-Aldrich, Madrid, Spain. The L-α-phosphatidylethanolamine (PE) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The Live/Dead^® BacLight kit™ (Molecular probes L7012) and AlamarBlue™ reagent—for bacterial and human cells viability tests, respectively, were obtained from Invitrogen, Life Technologies Corporation (Sant Cugat del Vallès, Spain). The P. aeruginosa ATCC 10145, human foreskin fibroblasts (BJ-5ta cell line) and keratinocytes (HaCaT cell line) were obtained from the American Type Culture Collection (ATCC LGC Standards, Barcelona Spain). The Live/Dead^® kit for mammalian cells viability was obtained from Thermo Fischer Scientific (Sant Cugat del Vallès, Spain). All of the other reagents were purchased from Sigma-Aldrich (Spain), unless otherwise specified. Ultrapure MilliQ water with a resistivity of 18.2 MΩ cm was used in all of the experiments.