Mupirocin

Tryptic soy broth (TSB), Luria broth (LB), D-glucose, phosphate-buffered saline (PBS), Dulbecco’s PBS (DPBS), Keratinocyte-SFM medium, DMEM (high glucose, GlutaMAX™ Supplement, pyruvate), and Ham’s F-12 Nutrient Mix were purchased from ThermoFisher Scientific (Waltham, MA). DermaLife K Keratinocyte Complete Medium with LifeFactors was obtained from Lifeline Cell Technology (Oceanside, CA). CnT-Prime 3D Barrier Medium was purchased from CELLnTEC Advanced Cell Systems AG (Zurich, Switzerland). Gentamicin, lysostaphin trimethoprim, sucrose, glycerol, mupirocin, neutral-buffered formalin solution (10%), and various supplements for skin culture media including hydrocortisone, isoproterenol, bovine insulin, selenious acid, L-serine, L-carnitine, bovine serum albumin (BSA), palmitic acid, linoleic acid, and arachidonic acid were obtained from Sigma-Aldrich (St. Louis, MI).

Total microbial enumeration was performed using plate count [12 (link)] as it gives only viable bacteria. Standard total plate count (TPC) was done using TOS propionate agar (TPA) with mupirocin (Sigma-Aldrich (St. Louise, Missouri, USA)), under anaerobic conditions in triplicate, using the standard aseptic culture procedure of serial dilution with sterile PBS. The total probiotic population in the fermentation mixture was expressed as colony-forming units (CFUs). Breast-fed infants contain in gut mostly probiotic species. In addition, TPA (TOS propionate agar) were specifically designed and prepared for a selective enumeration of presumptive Bifidobacteria sp. It contains highly purified “galacto-oligosaccharides”, which are one of the most excellent bifidobacterial growth substances. Therefore, we enumerated TPC as an indicator of total probiotics [11 (link)].

Depending on the number of available faecal pellets, samples were re-suspended in either 450 or 900 μl of sterile phosphate buffer saline (Sigma-Aldrich, UK) and used to produce tenfold serial dilutions (neat - 10^-4). The samples were then vortexed for 30 s and mixed using on a shaker at 1600 rpm. An aliquot of each dilution (100 μl) was plated onto Brain Heart Infusion (BHI) (Oxoid, UK) agar supplemented with mupirocin (50 mg/l) (Sigma-Aldrich, UK), l-cysteine hydrochloride monohydrate (50 mg/l) (Sigma-Aldrich, UK) and sodium iodoacetate (7.5 mg/l) (Sigma-Aldrich, UK) and incubated in an anaerobic cabinet for 48–72 h (Baker Ruskinn, UK). Three colonies from each dilution were randomly selected and streaked to purity on BHI agar supplemented with l-cysteine hydrochloride monohydrate (50 mg/l). Pure cultures were stored in cryogenic tubes at −80 °C.

Six variants of the treatment of skin-excision wounds were used: pure collagen scaffold (control, C); collagen scaffold enriched with empty poly(lactic acid-co-glycolic acid) (PLGA)-poly(vinyl alcohol) (PVA) nanoparticles (NPs) (NP); collagen scaffold with fish oil alone (FO); collagen scaffold with antibiotic mupirocin alone (MUP); collagen scaffold with PLGA-PVA NPs with entrapped fish oil (NP/FO); and collagen scaffold with PLGA-PVA NPs with entrapped mupirocin (NP/MUP). For our study, all FO treatments used commercial cod liver oil (oleum jecoris aselli) characterized by fatty acid content as % of the sum of total fatty acids as follows: 14:0 4.6, 16:0 11.1, 17:0 1.0, 18:0 2.9, 16:1 10.4, 18:1 19.5, 20:1 15.8, 18:2n-6 3.3, 18:3n-6 0.8, 20:2n-6 1.0, 20:4n-6 1.0, 18:3n-3 1.6, 20:5n-3 10.4, 22:5n-3 1.9 and 22:6n-3 13.6. MUP treatments used Sigma-Aldrich mupirocin (>92% powder; St. Louis, MO, USA).

Faeces was collected from healthy (i.e., had not received any antibiotics/probiotics prior to sampling), full-term breast-fed infants in accordance with protocols laid out by the National Research Ethics Service approved UEA/QIB Biorepository (Licence no: 11208) and Quadram Institute Bioscience Ethics Committee (see Table S1). Infant faeces were isolated on RCM (Oxoid, Hampshire, UK) supplemented with mupirocin and l-cysteine (0.05 mg/mL each, Sigma-Aldrich, Dorset, UK). Bacterial isolates were randomly selected from agar plates, and all subsequent Bifidobacterium and Lactobacillus strains were grown at 37 °C in either RCM, de Man Rogosa and Sharpe (MRS) media, or modified MRS (mMRS) with specified carbohydrates in an anaerobic chamber (Don Whitley Scientific, Bingley, UK) containing 5% CO₂, 10% hydrogen, 85% nitrogen gas.