**Food Compounds Inhibit** *Staphylococcus Aureus* **Bacteria and the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis**

Reuven Rasooly and Mendel Friedman

*Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture, Albany, California USA*

#### **1. Introduction**

386 Atopic Dermatitis – Disease Etiology and Clinical Management

[106] Food and Agriculture Organization, World Health Organization. The Food and

[108] Kukkonen AK, Kuitunen M, Savilahti E, Pelkonen A, Malmberg P, Mäkelä M. Airway

[109] Baken KA, Ezendam J, Gremmer ER, de Klerk A, Pennings JL, Matthee B, et al.

autoimmunity and gene expression. Int J Food Microbiol. 2006; 112(1): 8-18. [110] Okitsu-Negishi S, Nakano I, Suzukim K, Hashira S, Abe T, Yoshino K. The induction of

[111] Prescott SL, Björkstén B. Probiotics for the prevention or treatment of allergic diseases.

[112] Chapman CM, Gibson GR, Rowland I.Health benefits of probiotics: are mixtures more

[113] Van Huynegem K, Loos M, Steidler L. Immunomodulation by genetically engineered

[114] Egervärn M, Roos S, Lindmark H. Identification and characterization of antibiotic

[115] Despande GC, Rao SC, Keil AD, Patole SK. Evidence based guidelines for use of

[116] Elias J, Bozzo P, Einarson A. Are probiotics safe for use during pregnancy and

[117] Marko Kalliomäki, Jean-Michel Antoine, Udo Herz, Ger T. Rijkers, Jerry M. Wells,

[118] Shida K, Nanno M, Nagata S. Flexible cytokine production by macrophages and T cells

resistance genes in Lactobacillus reuteri and Lactobacillus plantarum. J Appl

Annick Mercenier. Guidance for Substantiating the Evidence for Beneficial Effects of Probiotics: Prevention and Management of Allergic Diseases by Probiotics. J.

in response to probiotic bacteria: a possible mechanism by which probiotics exert multifunctional immune regulatory activities. Gut Microbes. 2011; 2(2): 109-14.

the evaluation of probiotics in food. FAO/WHO Report No. 4-30-2002. [107] Salminen S, von Wright A, Morelli L et al. Demonstration of safety of probiotics: a

review. Int J Food Microbiol 1998; 44:93–106.

J Allergy Clin Immunol. 2007; 120(2): 255–262.

lactic acid bacteria. Front Biosci. 2009; 14: 4825-35.

effective than single strains? Eur J Nutr. 2011; 50(1): 1-17.

probiotics in preterm neonates. BMC Med. 2011; 9(1): 92.

lactation? Can Fam Physician. 2011; 57(3): 299-301.

Immunopathol. 1996; 78: 30-40.

Microbiol. 2009; 107(5): 1658-68.

Nutr. 2010; 140: 713S–721S.

2011; 22(2): 249-51. doi: 10.1111/j.1399-3038.2010.01079.x

Agriculture Organization of the United Nations and the World Health Organization Joint FAO/WHO Working Group report on drafting guidelines for

inflammation in probiotic-treated children at 5 years. Pediatr Allergy Immunol.

Evaluation of immunomodulation by Lactobacillus casei Shirota: immune function,

cardioangitis by Lactobacillus casei cell wall in mice: I. The cytokine production from murine macrophages by Lactobacillus casei cell wall extract. Clin. Immunol.

> Enterotoxigenic *S. aureus* is a major bacterial pathogen that develops multi-drug resistance to antibiotics (Pereira et al., 2009; Pu et al., 2011; Rhee & Woo, 2010). The enterotoxigenic bacteria and secreted toxins have been reported to cause clinical infections, and it has also been reported that they contaminate a broad variety of foods, including breaded chicken products (Pepe et al., 2006), canned mushrooms (Anderson et al., 1996), cheeses (Ertas et al., 2010; Ostyn et al., 2010; Rosengren et al., 2010), raw milk (Fusco et al., 2011; Heidinger et al., 2009), pork meat (Wallin-Carlquist et al., 2010), and other foods (Balaban & Rasooly, 2000) as well contaminating handles of shopping carts (Mizumachi et al., 2011) causing many foodborne illnesses in the United States each year (Shinefield & Ruff, 2009). Staphylococcal food poisoning is due to the absorption from the digestive tract into the circulation of the enterotoxins preformed in food A toxin level of <1 µg may induce symptoms of food poisoning. This level is reached with a bacterial population of >105 CFU/g food (Stewart, 2005).

> *S. aureus* produces the virulent staphylococcal enterotoxin A (SEA), a single chain protein that consists of 233 amino acid residues with a molecular weight of 27,078 Da. It has been estimated that staphylococcal enterotoxin A (SEA) secreted by the bacteria is associated with 78% of staphylococcal outbreaks (Vernozy-Rozand et al., 2004). Heat used to eliminate the pathogenic bacteria may not eliminate toxins already formed (Margosch et al., 2005; Pepe et al., 2006).

> Staphylococcus enterotoxins (SEs) exhibit two separate biological activities: they cause gastroenteritis in the gastrointestinal tract and they act as a superantigen on the immune system. Previous research has shown that emetic activities and superantigenic activities of SEs are related (Shinefield & Ruff, 2009; Stewart, 2005).

> Because SEA is present in contaminated foods and exerts adverse effects on the gastrointestinal tract, there is a need to find food-compatible safe conditions to inactivate it. Efforts to inhibit the toxin or its release from *S. aureus* include the use of electrolyzed water (Suzuki et al., 2002), high pressure and heat (Margosch et al., 2005), radiation and pulsed electric fields (Walkling-Ribeiro et al., 2008), condensed tannins (Choi et al., 2007) and other plant extracts (Carlos et al., 2010; Ifesan & Voravuthlkunchai, 2009), peptides (Wang et al.,

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

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

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

significant difference.

**3. Results and discussion** 

*aureus*

quadruplicate with control values ~100 colony forming units (CFUs).

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 389

dried (~10-15 min) and then incubated overnight 37 °C. Each dose was sampled in

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

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

Here, we briefly describe our studies designed to demonstrate the potential of bioactive food ingredients to inhibit growth of *S. aureus* bacteria and to inactivate SEA produced by these bacteria. The results suggest that it may possible to reduce the toxic potential of these

**3.1 Naturally occurring compounds inactivate antibiotic-resistant** *Staphylococcus* 

Antibiotic resistant microorganisms often arise from the administration of sub-therapeutic levels of antibiotics in animal feeds. They are present in the animal waste, often

and the enzyme cleavage GFA-AFC assays which work only with live cells.

toxin-producing organisms with the aid of edible food ingredients.

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

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