Tween 80

The actives and excipients used in the study were of analytical grade. The miconazole base was purchased from Fagron (Rotterdam, Holland). Isopropyl myristate was purchased from Merck Schuchardt (Merck, Hohembrunn, Germany), Oleic acid vegetable from Merck (Merck KgaH, Darmstadt, Germany), Tween 20 from Sigma-Aldrich (Sigma Aldrich Chimie, L’ lsle D’Abeau Chesnes, France) and Tween 80 was purchased from Carl Roth GmbH + CoKG (Karlsruhe, Germany), Kolliphor P407 was acquired from Sigma (Sigma Aldrich, St. Louis, MO, USA) and propylene glycol was supplied from Sigma Aldrich (Steinheim, Germany). Polyethylene glycol 200 and polyethylene glycol 400 were acquired from Scharlau (Scharlab S.L., Sentmenat, Spain). Di-sodium hydrogen phosphate heptahydrate (Merck, Darmstadt, Germany), Potassium dihydrogen phosphate, and ethanol (Chemical Company, Iași, Romania) were selected to prepare the in vitro release medium. Ultrapure Milli-Q water with a specific resistance of 18.2 MΩ/cm and total organic carbon (TOC) of less than 5 μg/L was generated from a Milly-Q^® Direct 8 Water Purification System (Merck Millipore, Bedford, MA, USA), and used as the aqueous phase.

The screening experiments were performed under static conditions in a polystyrene Petri dish (diameter 90 mm) with a microscope glass coverslip (24 × 60 mm) for the monitoring of cell adhesion. Each dish contained 30 mL of culture media, which was inoculated by T. cutaneum (OD₄₀₀ = 0.20 ± 0.02). The cultivation was performed at 30 °C and 50 rpm for 24 h when the biofilm surface area reached about 50%. Afterwards, the surfactants were added, and their effect was examined after 2 and 16 h of exposition. The concentration range (1, 5, 10, 100, 250, 500 and 1000 mg L⁻¹) including the critical micellar concentration (CMC) was tested to find conditions for analysis under dynamic conditions. For biofilm analysis, the glass slides were carefully removed from the medium and rinsed by a sterile saline solution to remove the loosely attached cells. The effect of surfactants on cell adhesion was determined as described below. For comparison, synthetic surfactants anionic surfactant sodium dodecyl sulphate (SDS, Art. No. 4360.1, Carl Roth, Karlsruhe, Germany) and nonionogenic Tween 80 (Art. No. 9139.1 Carl Roth, Karlsruhe, Germany) were used in addition to the four rhamnolipids. All experiments were performed in triplicate; error bars represent standard deviation.

Spore load was calculated for individuals infected with each microsporidian strain isolated from a population with a 10² (low) starting concentration of microsporidia, hereafter referred to as “low isolate”, used in the survival assay for which a sufficient amount of material extracted from dead larvae taken from the experiment was available (five of the seven lines), as well as the ancestral parasite. Only the low line was used as the intermediate line did not show a consistent change in induced mortality in the survival assay. 10 adults of the Cro1 stock line were allowed to oviposit on 5 g flour containing 10⁵ spores per gram of the relevant isolate for 7 days, following which they were removed and discarded. Offspring were allowed to mature for a further 5 weeks, following which 3 dead larvae from each isolate were taken and ground individually in 1 ml water plus a small amount of Tween® 80 (Carl Roth, Karlsruhe, Germany) in a 2 ml tube (Sarstedt, Nuembrecht, Germany) using a 2000 Geno/Grinder ball mill homogeniser for 15 seconds at a speed of 500 rpm with 2 mm diameter steel balls. Spores present in the homogenised solution were subsequently counted using a “Fast Read 102” disposable counting chamber (Biosigma, EU). This method was used in order to most accurately obtain all the spores contained in a single larva.

The two essential oils used in this study, nutmeg and fir needle essential oil, were obtained from Roth (Roth, Germany). In order to produce the desired materials, emulsions of these oils had to be obtained using Tween 80, produced by Roth (Germany), as well as ethanol and glycerin, produced by Sigma-Aldrich (Darmstadt, Germany). Deionized water was used during the assays. All these reagents were of reactive grade and were used without additional purification. The antimicrobial assessments were performed utilizing Nutrient Broth No 2 and Agar (microbiological grade) Sigma-Aldrich (Germany).

We used the following bacterial species: Streptococcus agalactiae ATCC 12403, Staphylococcus aureus MRSA ATCC 43300, Klebsiella pneumoniae Extended Spectrum β-Lactamase (ESBL) ATCC 700603, Acinetobacter baumanii ATCC19606, and Enterococcus faecium (VRE) DSM17050. Bacteria were cultured at 37 °C/5% CO₂ in liquid THY broth (Todd-Hewitt Broth, Oxoid, Hampshire, UK) supplemented with 0.5% yeast extract (BD, Miami, FA, USA). Pseudomonas aeruginosa PA14 (DSM 19882) was cultivated in liquid Müller–Hinton–Broth (Carl Roth, Karlsruhe, Germany) at 37 °C, under orbital shaking at 160 rpm.
In addition, the employed Mycobacterium tuberculosis ATCC 27294 (Mtb) was grown in 7H9-medium containing 7H9 BBL Middlebrook broth (BD), glycerol (Honeywell, Charlotte, NC, USA) OADC (Oelic Albumin Dextrose Catalase; BD), Tween 80 (Roth, Karlsruhe, Germany), and ddH₂O. The pH was adjusted between 7.2–7.4 and sterile filtration was performed with a 0.2-µm filter membrane (Thermo Scientific^TM Nalgene^TM Rapid-Flow^TM, Thermo Scientific, Bremen, Germany).

M. smegmatis mc² 155 (DSMZ No. 43756) and M. avium subsp. hominissuis strain 104 [16 (link)] were cultured on Middlebrook 7H11 (BD Life Sciences, Heidelberg, Germany) agar plates including 10% OADC supplement (BD Life Sciences) and 0.5% glycerol (Carl Roth GmbH, Karlsruhe, Germany) at 37 °C until colonies were visible. Colonies were transferred from plates to Middlebrook 7H9 broth (BD Life Sciences) supplemented with 10% ADC (BD Life Sciences) and 0.05% Tween-80 (Carl Roth GmbH) and grown at 37 °C until the culture reached an optical density (OD₆₀₀) = 1. From this pre-culture, the main culture was inoculated and adjusted to OD₆₀₀ = 0.1 and cultured again at 37 °C until OD₆₀₀ = 1. Bacteria were harvested by centrifugation, quick-frozen in liquid nitrogen, and kept at −80 °C in PBS containing 10% glycerol until used for infection experiments. For quantification of bacteria, the number of colony-forming units was determined by plating serial dilutions on Middlebrook 7H11 agar plates which were incubated at 37 °C until colonies were visible.