Maxq 4450

Gram(+) Staphylococcus aureus (ATCC 25923) and Gram(–) Escherichia coli (ATCC 25922) were grown in 20 mL medium containing 30 g L⁻¹ Todd-Hewitt-Bouillon (Roth) and 3 g L⁻¹ yeast extract (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 37 °C overnight under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA). Pathogenic yeast Candida albicans (Mya 273) was grown in 20 mL medium containing 32 g L⁻¹ Peptone Casein (Sigma-Aldrich), 20 g L⁻¹ yeast extract (Sigma-Aldrich), 5 g L⁻¹ sodium chloride (VWR International, Vienna, Austria) and 5 mM sodium hydroxide (Fluka Analytical, Munich, Germany) at 37 °C overnight under constant agitation at 200 rpm on a shaking incubator (MaxQ 4450, Thermo Scientific, Marietta, OH, USA).

S. aureus was prepared for inoculation as previously published.^{31 (link),42 (link),61 (link)} Of note, Xen36 can be isolated from contaminants due to possession of a kanamycin resistance selection marker on its lux operon. Therefore, 200 mg·mL⁻¹ kanamycin (Sigma–Aldrich, St. Louis, MO) was added to all cultures to ensure sample purity. Xen36 was streaked onto tryptic soy agar plates (tryptic soy broth (TSB) plus 1.5% bacto agar, BD Biosciences, San Jose, CA) and cultured at 37 °C overnight. Next, single colonies of S. aureus were individually grown in TSB and again cultured overnight at 37 °C in a shaking incubator (240 r·min⁻¹) (MaxQ 4450, ThermoFisher, Grand Island, NY). After a 2-h subculture of a 1:50 dilution of the resultant culture, mid-logarithmic phase bacteria were attained. Finally, using a centrifuge, bacterial cells were pelleted, re-suspended, and washed in phosphate-buffered saline (PBS). Bacterial inoculums (5 × 10¹, 1 × 10², 1 × 10³, and 1 × 10⁴ CFUs in 2 μL PBS) were approximated by measuring the absorbance at 600 nm [A600, Biomate 3 (Thermo)].

The preparation of Aib2-Oxm solutions was carried out in a class 2 cabinet using sterile glass vials. Aib2-Oxm was dissolved at 10-20 mg mL^-1 in sterile NaOH (4.3 mM) and 0.09% saline solution (diluted from the 0.9% saline solution) to achieve a pH between 7.0 and 7.3. The solution was passed through a 0.22 µm pore size membrane, and peptide concentration was measured before diluting the solution to 10 mg mL^-1 using 0.09% saline. The solution was then incubated at 37°C for 3 to 6 days with orbital shaking (200 rpm, ThermoScientific MaxQ 4450 benchtop orbital shaker) and then 2–3 days without agitation. Thereafter, 1 mg mL^-1 of fibrillar Aib2-Oxm was incubated with 10 mg mL^-1 free peptide in 0.09% for 1 week at RT without agitation. Finally, the 10 mg mL^-1 fibrillar Oxm solution was diluted to varying concentration between 1 to 4 mg mL^-1 using sterile 0.09% saline, incubated at RT for 2–9 days before being stored at 4°C. The conversion yield was assessed by measuring the concentration of the remaining free peptide. The fibrillar material was separated from the free peptide in solution after centrifugation of an aliquot of the solution for 30 min at 16 200 x g and filtering the supernatant through a 50 kDa molecular weight cut-off membrane.