Precision glide

Approximately the same number of S. bullata pupae were collected as described above. Pupae were subsequently rinsed in sterile millipore water to remove small particulates. They were then crushed by hand through a 100 µm nylon net (EMD Millipore, Merck Millipore, Billerica, MA, USA) and the filtrate was collected in a sterile 250 ml glass beaker. Nylon powder-free non-sterile gloves were worn during this extraction. The filtrate was centrifuged at 4 °C (25,000xG) for 10 min to separate the sediment, protein, and lipid layers. Using a 22 gauge needle (BD PrecisionGlide; Becton, Dickinson and Company, Franklin Lakes, NJ), the protein layer was transferred to a sterile beaker. Schneider’s Drosophila media was added to the protein extract to triple the volume and, using a reusable vacuum filtration apparatus (Nalgene; Thermo Fisher scientific Incorporated, Waltham, MA, USA), the resulting mixture was passed through filters with gradually smaller pore sizes (11, 6, 2.5, 0.8, and 0.45 µm). A 0.22 µm syringe filter (Costar; Corning Incorporated, Corning, NY, USA) was used to remove bacteria. The final product was stored at 4 °C until use (Fig. 1). The following is a step-wise protocol for the production of NRMv2.

A culture of A. baumannii strain 6077/12 was grown to mid-exponential phase in a volume of 500 mL and was infected with 1 mL of previously obtained phage suspension [titer 10⁹ plaque forming units (PFUs)/mL]. After overnight incubation, the lysate was centrifuged in Sorvall RC3B centrifuge at 13,689.2 × g for 30 min in rotor GS3 to pellet bacterial debris. The supernatant was collected and treated with DNaseI and RNase A (Thermo Fisher) for 2 h at 37°C. Afterward, NaCl and polyethylene glycol (PEG 8000; Sigma) were added (to obtain 1 M and 10% final concentrations, respectively), stirred, and incubated overnight at +4°C to precipitate the phages. The precipitate was centrifuged at 13,689.2 × g for 30 min at +4°C (Sorvall RC3B centrifuge, rotor GS3), and the pellet was resuspended in 4 mL of SM buffer. CsCl was added to reach 0.75 g/mL density. The samples were ultracentrifuged at 131,980.8 × g for 24 h in Beckman Coulter Optima L-80 XP ultracentrifuge in rotor SW55 Ti, after which the distinct phage-containing bluish band was visible at the midpoint of the tube. The phages were extracted from tube by using a 22-gauge needle (PrecisionGlide™; Becton Dickinson, Dubline, Ireland) and dialyzed overnight against 2 L of SM buffer at +4°C. Purified phages were filtered through 0.22-μm filters (Filtropur S 0.45; Sarstedt) and used in subsequent experiments.

To create the solution, 5% of 5 and 20 UMF MH were, separately, dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP, Oakwood Chemical) through a 20-minute sonication in a RT water bath (Branson 200 Ultrasonic Cleaner, Branson Ultrasonics). Lyophilized SF (5%) was then dissolved overnight in the MH-containing solution using a mechanical shaker. The solution was loaded into a 5 mL syringe tipped with a blunted 18-gauge needle (PrecisionGlide, Becton Dickinson). Electrospinning was completed using a syringe pump (78-01001, Fisher Scientific) set at 3 mL/hr. A high-voltage DC power supply (CZE1000PN30, Spellman High Voltage Electronics Corp.) provided a voltage of 23 kV onto a spinning (400 rpm) rectangular (0.5 cm × 2.5 cm × 9 cm) stainless steel mandrel using a working distance of 16.5 cm. All ES scaffolds were removed from the mandrel and stored at −20°C in a desiccation chamber. Three of each ES scaffold were used for all characterization and testing.

Five percent SF electrospun (ES) scaffolds were fabricated, with and without 5% MH. For the ES scaffolds that included honey, 5% MH (Manuka Honey, Medical Grade 12+, Ndal Laboratories, Monterey, CA, USA) was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFP, Oakwood Chemical, West Columbia, SC, USA) using a 20 min water bath sonication (Branson 200 Ultrasonic Cleaner, Branson Ultrasonics, St. Louis MO, USA). Following dissolution, 5% lyophilized SF was placed in the solution and allowed to dissolve overnight under mechanical agitation. The following day, the solution was loaded in a 5 mL syringe fixed with a blunt-tipped 18 gauge needle (PrecisionGlide, Becton Dickinson, Franklin Lakes, NJ, USA). The solution was then electrospun under the following parameters: voltage of 23 kV, working distance of 16.5 cm, and extrusion rate of 3 mL/h. The fibers were collected on a translating, rotating (400 rpm) rectangular (0.5 cm × 2.5 cm × 9 cm) stainless steel mandrel. Following removal, all ES scaffolds were crosslinked using methanol vapor and stored at room temperature (RT) in a desiccation chamber. Note that for the ES scaffolds that did not include honey, the initial sonication step was excluded from the solution preparation.