Tryptic soy agar

The 31 A. baumannii isolates were recovered from cryostorage onto prepoured tryptic soy agar (VWR) and incubated aerobically at 37°C for 24 h. A second passage from a single colony of each isolate was performed, and after 24 h, an overloaded 1-μL loop of confluent growth was collected. The cells were added to a 1.5-mL suspension vial containing inert metal cylinders (Bruker IR Biotyper kit) and 50 μL 70% (vol/vol) ethanol and vortexed to obtain a homogenous suspension. Fifty microliters of HiPerSov Chromanorm water (VWR) was added, and each vial was vortexed for 1 min. Quintuple 15-μL spots of each isolate were pipetted onto a 96-spot microtiter plate. Duplicate 12-μL spots of Bruker infrared test standards 1 and 2 (IRTS1 and IRTS2) were included during each run. The standards were prepared according to the manufacturer’s instructions. The microtiter plate was dried above a 37°C hot plate for 30 min, and strain typing was performed using an IR Biotyper and the IR Biotyper software version 2.1.0.195 (Bruker UK).

References strains such as Candida auris DSM 21092 and the Gram-negative Klebsiella pneumoniae ATCC 13883 were used in this work. Both C. auris and K. pneumoniae were maintained in the laboratory on Tryptic Soy Agar and cultivated in Tryptic Soy Broth supplemented or not with 1% w/v glucose, respectively (VWR chemicals).
HaCAT cells (non-tumorigenic human keratinocyte cells) were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA). They were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Sigma Aldrich), supplemented with 10% Fetal Bovine Serum, 1% L-glutamine, and 1% penicillin/streptomycin (Sigma Aldrich) in a humidified incubator at 37 °C and 5% CO₂. Once 70–80% confluency was reached, the cells were detached with Trypsin/EDTA solution (Sigma Aldrich) and cultured into new flasks. The medium was replaced twice a week.

Five Bacillus spp. were used in all the assays. Three species, B. mojavensis strain 3, B. velenzensis strain 2, and B. pumilis GB 34 were originally isolated, identified and stored by Dr. J. W. Kloepper at Auburn University, Auburn, AL. Two species, B. firmus I-1582 and B. amyloliquefaciens QST713, are the active ingredients of Bayer CropScience products, VOTiVO^® and Serenade^®, respectively. The Bacillus spp. were stored in 30% glycerol at -80°C. Prior to utilization, the Bacillus spp. were transferred to tryptic soy agar (VWR, Radnor, PA) plates and incubated at 35°C for 24 hours. The vegetative cells were washed into beakers and standardized. For the greenhouse in planta assay, in vitro assay, split root assay and RT-qPCR, the bacteria were standardized to 1 x 10⁶ CFU/mL. For the qPCR, the bacteria were standardized to 1 x10⁸ CFU/mL.

The S. aureus (NCTC 10788) and P. aeruginosa (NCTC 12924) strains used in this study were obtained from the National Collection of Type Cultures, Porton Down, UK. Both were cultured using tryptic soy agar (VWR Ltd., Leicestershire, UK) and incubated at 32 °C. The fungal pathogen C. albicans (NCPF 3179) was obtained from the National Collection of Pathogen Fungi, Porton Down, UK. This was cultured using sabouraud dextrose agar (VWR Ltd., Leicestershire, UK) and incubated at 32 °C. The Acanthamoeba strains, A. polyphaga (ATCC 30461) and A. castellanii (ATCC 50370), were obtained from the American Type Culture Collection (LGC Standards, Teddington, UK). Trophozoites were maintained in tissue culture flasks in Ac#6 medium at 30 °C in a static incubator, and cysts were produced using Neff’s encystment medium (NEM) in tissue culture flasks at 30 °C in a shaking incubator as previously described [7 (link)].

Biofilms were grown on titanium alloy Ti6Al4V coupons (Biosurface Inc., Bozeman, MT, USA) in order to mimic implant surface characteristics. These coupons are unpolished cylinders measuring 12.7 mm in diameter and 3.175 mm in height. The initial inoculum was prepared from bacteria grown overnight on Tryptic Soy Agar (VWR, Leuven, Belgium) (TSA), suspended in Phosphate Buffer Saline (PBS), adjusted to an optical density at 620 nm of 0.5 (CECIL 2021 spectrophotometer, CECIL, United-Kingdom) and diluted 1:100 in TGN, reaching a bacterial density of ~6.5 log₁₀ CFU/mL. Sterile coupons were incubated for 24 h at 37°C in 12 wells plates containing 2mL of bacterial suspension in TGN per well, under a continuous orbital shaking of 50 rpm in order to induce shear stress. Biofilms reached maturity after 24 h (i.e., no meaningful change in biomass or bacterial counts was observed when prolonging the incubation for 48 h, Supplementary Data Figure 1) and used for testing the treatments.

Four reference strains (PAO1^{48 (link)}, PT629 [MexAB overproducer derivative of PAO1^{49 (link)}], PAO1mexAB^{50 (link)}, PAO509 [PAO1 Δ(mexAB-oprM) Δ(mexCD-oprJ) Δ(mexEF-oprN) Δ(mexJK)^{50 (link)}]) and 67 clinical isolates (Supplementary Data 1) obtained from patients with cystic fibrosis (n = 43), urinary tract infections (n = 10), or hospital-acquired pneumonia (n = 14) were used in this study. Transposon mutants were obtained from the PAO1 library of the University of Washington^{51 (link)}. For specific experiments, additional clinical isolates resistant to colistin^{25 (link)} were also included. Bacteria were inoculated overnight at 37 °C in tryptic soy agar (VWR). A single colony was then added to 10 mL cation-adjusted Mueller-Hinton Broth (CA-MHB; Sigma-Aldrich) and incubated at 37 °C overnight under gentle agitation (130 rpm). CFUs (colony-forming units) were counted on tryptic soy agar.

The reference strains used in this study were obtained from commercially available sources: Ent. faecalis ATCC 6057 and S. aureus ATCC 25923 (American Type Culture Collection (ATCC), Manassas, VA, USA), and were shipped in glass vials.
The reference strains were inoculated with a sterile loop on Tryptic Soy Agar (TSA) (VWR^® International GmbH, Vienna, Austria) and incubated at 37 °C for 24 h. Subsequently, one colony of each bacterial strain was transferred into sterile Tryptic Soy Broth (VWR^® International GmbH, Vienna, Austria) and incubated for 24 h at 37 °C. For further application, the suspended bacterial medium was centrifuged (3000× g) twice with the addition of sterile NaCl (0.5%) solution (5 mL) each, after removing the liquid parts of the samples between the centrifugation steps and at the end of the procedure. The bacterial count was determined using a DensiCheck Plus (bioMerieux^® Austria GmbH, Vienna, Austria) and found to be 10⁹ colony-forming units (CFUs). A dilution series using NaCl (0.5%) solution was performed to reduce the bacterial count from 10⁹ CFUs to 10³ CFUs.