**2. Experimental procedures**

The following experimental methods were used to create the data shown in the Tables and Figures.

### **2.1 Antibacterial activities of plant compounds against antibiotic-resistant** *S. aureus*

The following procedure was adapted from Friedman et al. (2004b). Stock suspensions, 1 mg/mL, of cinnamon oil, oregano oil, thyme oil, carvacrol, and perillaldehyde were prepared by vigorous vortexing in phosphate-saline buffer at pH 7. Dilutions from stock solutions were revortexed, to ensure even mixing, before being further diluted or added to the cell suspension in the microtiter plate. The two solids (β-resorcylic acid and dopamine) formed clear solutions in the buffer. Each test substance was prepared fresh before use. Individual wells of a microtiter plate (Becton Dickinson, Oxford, UK) were filled with 100 ml of the test substance in phosphate-saline buffer and 50 ml of the culture. Triplicate wells were used for each concentration, with phosphate-saline used in the blank. The plates were shaken and then incubated at 37 °C for 1 h. The number of organisms remaining viable was then determined by a Nutrient Agar Spread Plate Technique on a 10-ml aliquot removed from each well. Plates were incubated overnight at 37 °C prior to counting.

#### **2.2 Antibacterial activity of Phloxine B against** *S. aureus*

The following procedure was adapted from Rasooly (2005). Bacteria (200 μL) grown in BHI broth were removed during the logarithmic growth phase (OD of 0.3 at 600 nm) and spread over the surface of a BHI agar plate with a bent glass rod. Various concentrations of Phloxine B (10, 5, 2.5, 1.25 and 0.62 μg) and 5 μg of chloramphenicol (10 units) and 1.5 μg of tetracycline (2.5 units) were added to the wells of these plates as antibiotic standards. Plates were then incubated for 16 h at 37 °C in the light and in the dark. The potency of Phloxine B was determined by measuring the diameter of the inhibition zone made compared to zone produced by the standard antibiotics.

#### **2.3 Antibacterial activity of 4-hydroxytyrosol and Hidrox-12 against** *S. aureus*

The procedure was adapted from Friedman et al. (2011). *S. aureus* bacteria were grown overnight at 37 °C under aerobic conditions maintained by shaking at 200 rpm in Lucia-Bertani agar (LB) medium. The effect of 4-hydroxytyrosol and Hidrox-12 on bacterial growth was studied by addition of a bacterial suspension (~104 diluted from ~108 bacteria/mL) to various concentrations of 4-hydroxytyrosol (range: 0.71-0.022 mg/mL) and Hidrox-12 suspension (range: 1.29-0.040 mg/mL) in phosphate buffer pH 7.0. After 60 minutes of incubation at 37 °C, 200 rpm, each dose/bacterial suspension was diluted 1/10 and then 20 μL of each dose/bacterial suspension were dropped at the top of a square LB plate with grids. The plates were tilted and the drops ran down the plate. The plates were dried (~10-15 min) and then incubated overnight 37 °C. Each dose was sampled in quadruplicate with control values ~100 colony forming units (CFUs).

#### **2.4 Prophylactic vaccination of mice with** *S. aureus*

388 Atopic Dermatitis – Disease Etiology and Clinical Management

2008), phenolic compounds (Rúa et al., 2010), licochalcone A (Qiu et al., 2010), essential oils (de Souza et al., 2010; Friedman et al., 2004a; Nuñez et al., 2007; Parsaeimehr et al., 2010; Qiu

The objective of our research effort is to discover food-compatible ways to inhibit or inactivate both the pathogen and the toxin. In this chapter we will briefly summarize our

The following experimental methods were used to create the data shown in the Tables and

**2.1 Antibacterial activities of plant compounds against antibiotic-resistant** *S. aureus* The following procedure was adapted from Friedman et al. (2004b). Stock suspensions, 1 mg/mL, of cinnamon oil, oregano oil, thyme oil, carvacrol, and perillaldehyde were prepared by vigorous vortexing in phosphate-saline buffer at pH 7. Dilutions from stock solutions were revortexed, to ensure even mixing, before being further diluted or added to the cell suspension in the microtiter plate. The two solids (β-resorcylic acid and dopamine) formed clear solutions in the buffer. Each test substance was prepared fresh before use. Individual wells of a microtiter plate (Becton Dickinson, Oxford, UK) were filled with 100 ml of the test substance in phosphate-saline buffer and 50 ml of the culture. Triplicate wells were used for each concentration, with phosphate-saline used in the blank. The plates were shaken and then incubated at 37 °C for 1 h. The number of organisms remaining viable was then determined by a Nutrient Agar Spread Plate Technique on a 10-ml aliquot removed

The following procedure was adapted from Rasooly (2005). Bacteria (200 μL) grown in BHI broth were removed during the logarithmic growth phase (OD of 0.3 at 600 nm) and spread over the surface of a BHI agar plate with a bent glass rod. Various concentrations of Phloxine B (10, 5, 2.5, 1.25 and 0.62 μg) and 5 μg of chloramphenicol (10 units) and 1.5 μg of tetracycline (2.5 units) were added to the wells of these plates as antibiotic standards. Plates were then incubated for 16 h at 37 °C in the light and in the dark. The potency of Phloxine B was determined by measuring the diameter of the inhibition zone made compared to zone

**2.3 Antibacterial activity of 4-hydroxytyrosol and Hidrox-12 against** *S. aureus*

The procedure was adapted from Friedman et al. (2011). *S. aureus* bacteria were grown overnight at 37 °C under aerobic conditions maintained by shaking at 200 rpm in Lucia-Bertani agar (LB) medium. The effect of 4-hydroxytyrosol and Hidrox-12 on bacterial growth was studied by addition of a bacterial suspension (~104 diluted from ~108 bacteria/mL) to various concentrations of 4-hydroxytyrosol (range: 0.71-0.022 mg/mL) and Hidrox-12 suspension (range: 1.29-0.040 mg/mL) in phosphate buffer pH 7.0. After 60 minutes of incubation at 37 °C, 200 rpm, each dose/bacterial suspension was diluted 1/10 and then 20 μL of each dose/bacterial suspension were dropped at the top of a square LB plate with grids. The plates were tilted and the drops ran down the plate. The plates were

from each well. Plates were incubated overnight at 37 °C prior to counting.

**2.2 Antibacterial activity of Phloxine B against** *S. aureus*

produced by the standard antibiotics.

et al., 2011), and toxin-specific antibodies (Larkin et al., 2010).

**2. Experimental procedures** 

Figures.

finding in these two areas that are also relevant to atopic dermatitis.

The following procedure was adapted from Balaban et al. (1998). RAP (regulatory RNAIII activating protein, 10 μg) was injected with CFA (completer Freund's adjuvant) on first injection, followed with ICFA (incomplete Freund's adjuvant) on second and third injections subcutaneously into 4-week old immunocompetent hairless male mice on days 0, 7, and 21. Control mice were either injected with the adjuvant alone or not injected. Vaccinated and control mice were challenged on day 31 with 1.24 x 108 CFU of wild-type Smith diffuse strain (SD) of *S. aureus* subcutaneously together with 1 mg of Cytodex beads to induce local infection. The size of the lesion was measured daily. Fisher's exact probability test was used to compare proportions of mice developing lesions and mice developing RAP antibodies among the RAP vaccinated, CFA controls, and untreated control groups. Among animals that developed lesions after challenge with *S. aureus*, the size of the lesions was compared by single-factor analysis of variance. Post-hoc testing was done by Fisher's protected least significant difference.

### **2.5 Staphylococcus enterotoxin (SEA) activity assays**

The procedure was adapted from Friedman et al. (2011). Spleen cells were placed in 96-well plates (1 X 106/mL, 0.2 mL) in Russ-10 medium and treated with various concentrations of SEA following incubation at 37 °C in a 5% CO2 incubator. After incubation at various time points, cell proliferation was measured by adding bromodeoxyuridine (5-bromo-2 deoxyuridine, BrdU)-labeled DNA to each well 4 h before fixation as described by the instructions of the manufacturer. Spectroscopic measurements were made at 620 and 450 nm. A second measure of inhibition activity of SEA activity was determined by an enzyme cleavage assay. Briefly, the glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC) substrate (50 μL) was added to the wells. After mixing and incubation for 30 min at 37°C, this substrate enters intact cells. The live cell protease then cleaves GF-AFC, releasing AFC which generates a fluorescent signal. The resulting fluorescence was measured by a fluorescence plate reader (excitation at 355 nm and emission at 523 nm). We used 5 and 200 ng/mL of the toxin because at (a) < 5 ng/mL we did not detect cell proliferation induced by the toxin; and (b) at ~200 ng/mL proliferation of spleen cells reach a maximum. Results are expressed as representative data from triplicate wells from two different methods, BRDU and the enzyme cleavage GFA-AFC assays which work only with live cells.
