**3. Results and discussion**

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

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

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

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

human patients merits study.

Asdapted from (Rasooly, 2005).

2005).

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 391

described in some detail in the legend of Figure 3. It is likely that related charged polyaromatic compounds, including naturally occurring polyphenolic compounds, may operate by similar mechanisms. The results suggest that Phloxine B has the potential to be used as an antimicrobial agent against *S. aureus* in pathogens and in veterinary and human medicine and against other pathogenic bacteria. Its possible use against atopic dermatitis in

Fig. 1. Antibiotic potency of Phloxine B was determined by agar diffusion assay. Various concentrations of Phloxine B (10, 5, 2.5, 1.25, 0.62 µg) and 5 μg of chloramphenicol (10 units) and 1.5 μg tetracycline (2.5 units) were added to agar plates seeded with *S. aureus* (a), or *B. cereus* (b). Following incubation, the plates were examined for bacterial growth inhibition.

Fig. 2. Dose and time effect of Phloxine B (D&C Red#28) on *S. aureus*. Mid-log phase culture of *S. aureus* incubated with various concentrations of the dye. Turbidity of the media and bacteria viable cell count was measured at 20-minute intervals. Adapted from (Rasooly,

contaminating groundwater, surface water, irrigation water, fruits, and vegetables. They can then disseminate through the food chain and enter the human intestinal tract after the produce or undercooked meat is eaten (Davis & Lederberg, 2001; Walsh, 2003).

We showed that the following natural substances have antibacterial activity against three resistant pathogens including Staphylococcus aureus (ATCC12715): cinnamon oil, oregano oil, thyme oil, carvacrol, (S)-perillaldehyde, 3,4-dihydroxybenzoic acid (β-resorcylic acid), and 3,4-dihydroxyphenethylamine (dopamine) (Friedman et al., 2004a). Exposure of pathogens to a dilution series of the test compounds revealed that oregano oil is the most active substance (Table 1). Activities of the test compounds were in the following approximate order: oregano oil > thyme oil ≈ carvacrol > cinnamon oil > perillaldehyde > dopamine > β-resorcylic acid. The order of susceptibilities of the pathogens to inactivation is: *B. cereus* (vegetative) >> *S. aureus* ≈ *E. coli* >> *B. cereus* (spores). Some of the test substances may be effective against antibiotic-resistant bacteria in foods and feeds and in hospital environments.


Table 1. Antibacterial activities of three plant essential oils (oregano, thyme, cinnamon), two essential oil compounds (carvacrol, perillaldehyde), dopamine, and the phenolic compound β-resorcylic acid) against antibiotic-resistant *S. aureus* (log CFU/ml; average ± SD, n=3). CFU = colony-forming-units or bacterial counts. Superscript letters not in common are not significantly different (p<0.05). Adapted from (Friedman et al. 2004a).

### **3.2 The food dye Phloxine B inactivates** *S. aureus*

The dye Phloxine B has been approved by the Food and Drug Administration (FDA) for human consumption. It is used in food, drugs, and cosmetics. We found that Phloxine B exhibits strong antimicrobial activities against several pathogenic bacteria including *S. aureus* (Rasooly, 2005).

The diffusion assay we used to determine bactericidal effects shows that the activity of Phloxine B is similar to that of the medicinal antibiotics chloramphenicol and tetracycline (Figure 1). Figure 2 shows the dose-dependence of the inactivation in the range 25 to 100 μg/ml. A postulated mechanism of antimicrobial effects of the negatively charged dye is

contaminating groundwater, surface water, irrigation water, fruits, and vegetables. They can then disseminate through the food chain and enter the human intestinal tract after the

We showed that the following natural substances have antibacterial activity against three resistant pathogens including Staphylococcus aureus (ATCC12715): cinnamon oil, oregano oil, thyme oil, carvacrol, (S)-perillaldehyde, 3,4-dihydroxybenzoic acid (β-resorcylic acid), and 3,4-dihydroxyphenethylamine (dopamine) (Friedman et al., 2004a). Exposure of pathogens to a dilution series of the test compounds revealed that oregano oil is the most active substance (Table 1). Activities of the test compounds were in the following approximate order: oregano oil > thyme oil ≈ carvacrol > cinnamon oil > perillaldehyde > dopamine > β-resorcylic acid. The order of susceptibilities of the pathogens to inactivation is: *B. cereus* (vegetative) >> *S. aureus* ≈ *E. coli* >> *B. cereus* (spores). Some of the test substances may be effective against antibiotic-resistant bacteria in foods and feeds and in

Compound 0 (control) 66.7 6.67 1.34 0.067 Oregano oil 4.6 ± 0.081 <1.5 3.4 ± 0.12c 4.6 ± 0.07ab 4.6 ± 0.12b Thyme oil 4.6 ± 0.04 <1.5 4.5 ± 0.04b 4.6 ± 0.09b 4.6 ± 0.05b Cinnamon oil 4.6 ± 0.12 2.6 ± 0.58 4.6 ± 0.02b 4.6 ± 0.07b 4.7 ± 0.11ab Carvacrol 4.9 ± 0.12 0 ± 0 4.8 ± 0.21a 4.8 ± 0.04a 4.8 ± 0.12a Perillaldehyde 4.7 ± 0.05 4.1 ± 0.1 4.6 ± 0.11b 4.6 ± 0.12b 4.8 ± 0.02a

 0 (control) 3330 670 330 67 Dopamine 4.6 ± 0.07 3.2 ± 0.33 4.5 ± 0.03 4.5 ± 0.11 4.6 ± 0.12

Control 6000 5330 4670 3330

β-Resorcylic acid 4.8 ± 0.06 <1.5 <1.5 <1.5 4.6 ± 0.03 Table 1. Antibacterial activities of three plant essential oils (oregano, thyme, cinnamon), two essential oil compounds (carvacrol, perillaldehyde), dopamine, and the phenolic compound β-resorcylic acid) against antibiotic-resistant *S. aureus* (log CFU/ml; average ± SD, n=3). CFU = colony-forming-units or bacterial counts. Superscript letters not in common are not

The dye Phloxine B has been approved by the Food and Drug Administration (FDA) for human consumption. It is used in food, drugs, and cosmetics. We found that Phloxine B exhibits strong antimicrobial activities against several pathogenic bacteria including *S.* 

The diffusion assay we used to determine bactericidal effects shows that the activity of Phloxine B is similar to that of the medicinal antibiotics chloramphenicol and tetracycline (Figure 1). Figure 2 shows the dose-dependence of the inactivation in the range 25 to 100 μg/ml. A postulated mechanism of antimicrobial effects of the negatively charged dye is

significantly different (p<0.05). Adapted from (Friedman et al. 2004a).

**3.2 The food dye Phloxine B inactivates** *S. aureus* 

*Concentration in well (μg/ml)* 

*Concentration in well (μg/ml)* 

*Concentration in well (μg/ml)* 

produce or undercooked meat is eaten (Davis & Lederberg, 2001; Walsh, 2003).

hospital environments.

*aureus* (Rasooly, 2005).

described in some detail in the legend of Figure 3. It is likely that related charged polyaromatic compounds, including naturally occurring polyphenolic compounds, may operate by similar mechanisms. The results suggest that Phloxine B has the potential to be used as an antimicrobial agent against *S. aureus* in pathogens and in veterinary and human medicine and against other pathogenic bacteria. Its possible use against atopic dermatitis in human patients merits study.

Fig. 1. Antibiotic potency of Phloxine B was determined by agar diffusion assay. Various concentrations of Phloxine B (10, 5, 2.5, 1.25, 0.62 µg) and 5 μg of chloramphenicol (10 units) and 1.5 μg tetracycline (2.5 units) were added to agar plates seeded with *S. aureus* (a), or *B. cereus* (b). Following incubation, the plates were examined for bacterial growth inhibition. Asdapted from (Rasooly, 2005).

Fig. 2. Dose and time effect of Phloxine B (D&C Red#28) on *S. aureus*. Mid-log phase culture of *S. aureus* incubated with various concentrations of the dye. Turbidity of the media and bacteria viable cell count was measured at 20-minute intervals. Adapted from (Rasooly, 2005).

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

**for vaccine and therapy** 

not yet realized.

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 393

**3.3 Inhibition of SEA Release from** *S. aureus* **via autoinduction of virulence as a target** 

With the increase in antibiotic resistance among staphylococci, there is an urgent need to develop vaccines to control bacterial infections. Although there are currently several vaccines at different stages of clinical development (Broughan et al., 2011), it seems that the development of a practical vaccine for which immunological responses can be monitored, thus enabling the prediction of its effectiveness in humans, is quite a challenging objective,

We believe that following novel approach we proposed merits further study (Balaban et al., 1998). The suggested approach is to interfere directly with bacterial virulence by interfering with transduction that leads to the production of toxins. This approach offers the possibility

Antibiotic therapy against *S. aureus* is an important component of treatment for atopic dermatitis. However, the emergence of methicillin-resistant *S. aureus* (MRSA) presents new therapeutic challenges that suggest the need to develop new antimicrobial drugs and vaccines as an important objective. As mentioned earlier, the pathogenic effects of *S. aureus* are largely due to the production of bacterial toxins, especially SEA, which is regulated by the RNA molecule, RNAIII. The *S. aureus* protein called RAP activates RNAIII, and a peptide called RIP produced by non-pathogenic bacteria inhibits RNAIII. We discovered that mice vaccinated with RAP or treated with purified or synthetic RIP were protected

 *n (%) N Mean size* 

RAP 24 17 (71) 6 96 1 (4) RAP\* 9 7 (78) 2 84 0 (0) CFA 10 3 (30) 5 177 2 (20) Untreated 12 0 (0) 9 370 3 (25)

SD+RIP 4 3 (75) 1 33 0 (0) SD+saline 4 1 (25) 3 39 0 (0)

SD+RIP 8 4 (50) 4 45 0 (0) SD+saline 6 0 (0) 6 100 0 (0)

SD+RIP 10 3 (30) 3 39 4 (40) SD+saline 10 2 (20) 6 160 2 (20) SD+Pep 10 9 (90) 1 56 0 (0) SD+DMSO 9 2 (20) 4 128 3 (22)

Table 2. Vaccination or suppression of *S. aureus* SD infections. Adapted from (Balaban et

*mice No lesions Lesions Death* 

*(mm2) n (%)* 

of transforming a toxin-producing pathogen to a non-pathogenic organism.

against pathologic effects induced by *S. aureus*. (Table 2; Figure 4).

*Treatment group No. of* 

Vaccination with RAP as an antigen

RIP suppression of 8.5 × 107SD

RIP suppression of 1.4 × 108SD

RIP and Pep suppression of 1.4 × 109SD

al., 1998).

Fig. 3. The proposed mechanism of antimicrobial effects of the dye Phloxine B. Phloxine B (a) is ionized in water and (b) becomes negatively charged. The negatively charged anion (b) has a strong affinity for the positively charged cellular components such as proteins. Upon illumination (c), the photosensitized Phloxine B gains the energy associated with this light causing debromination and formation of free radicals and singlet oxygen (**1O2**) (d), which reacts with bacterial biomolecules (e), leading to cell death of the Gram-positive bacteria. The Gram-negative bacteria (f), which have highly negatively charged cell surfaces, repel the Phloxine B. Therefore, there is no binding and no antimicrobial effect between the dye and the Gram-negative bacteria. In the presence of EDTA, cell permeability increases, enabling dye penetration, leading to cell death. Adapted from (Rasooly, 2005).

Fig. 3. The proposed mechanism of antimicrobial effects of the dye Phloxine B. Phloxine B (a) is ionized in water and (b) becomes negatively charged. The negatively charged anion (b) has a strong affinity for the positively charged cellular components such as proteins. Upon illumination (c), the photosensitized Phloxine B gains the energy associated with this light causing debromination and formation of free radicals and singlet oxygen (**1O2**) (d), which reacts with bacterial biomolecules (e), leading to cell death of the Gram-positive bacteria. The Gram-negative bacteria (f), which have highly negatively charged cell surfaces, repel the Phloxine B. Therefore, there is no binding and no antimicrobial effect between the dye and the Gram-negative bacteria. In the presence of EDTA, cell permeability increases, enabling dye penetration, leading to cell death. Adapted from (Rasooly, 2005).

#### **3.3 Inhibition of SEA Release from** *S. aureus* **via autoinduction of virulence as a target for vaccine and therapy**

With the increase in antibiotic resistance among staphylococci, there is an urgent need to develop vaccines to control bacterial infections. Although there are currently several vaccines at different stages of clinical development (Broughan et al., 2011), it seems that the development of a practical vaccine for which immunological responses can be monitored, thus enabling the prediction of its effectiveness in humans, is quite a challenging objective, not yet realized.

We believe that following novel approach we proposed merits further study (Balaban et al., 1998). The suggested approach is to interfere directly with bacterial virulence by interfering with transduction that leads to the production of toxins. This approach offers the possibility of transforming a toxin-producing pathogen to a non-pathogenic organism.

Antibiotic therapy against *S. aureus* is an important component of treatment for atopic dermatitis. However, the emergence of methicillin-resistant *S. aureus* (MRSA) presents new therapeutic challenges that suggest the need to develop new antimicrobial drugs and vaccines as an important objective. As mentioned earlier, the pathogenic effects of *S. aureus* are largely due to the production of bacterial toxins, especially SEA, which is regulated by the RNA molecule, RNAIII. The *S. aureus* protein called RAP activates RNAIII, and a peptide called RIP produced by non-pathogenic bacteria inhibits RNAIII. We discovered that mice vaccinated with RAP or treated with purified or synthetic RIP were protected against pathologic effects induced by *S. aureus*. (Table 2; Figure 4).


Table 2. Vaccination or suppression of *S. aureus* SD infections. Adapted from (Balaban et al., 1998).

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

atopic dermatitis, and because SEA is a representative antigen.

mechanism of this improvement is largely unknown.

food safety and human health is discussed.

from (Rasooly et al., 2010).

of cell proliferation.

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 395

Atopic dermatitis (eczema) is an inflammatory skin disease that affects 10-20% of children and 1-3% of adults (1-3). Antibiotics that suppress colonization of *S. aureus* are reported to mitigate the severity of atopic dermatitis disease. Most strains of *S. aureus* isolated from atopic skin lesions produce exotoxins with superantigen properties (Leung et al., 1993). It has been reported that staphylococcal superantigens can induce skin inflammation by several different mechanisms (Taskapan & Kumar, 2000). The reason we selected the superantigen for this study is that it is has been demonstrated to be an aggravating factor in

It has been previously reported that consumption of apple condensed tannins from unripe apples improve the symptoms associated with atopic dermatitis (Kojima et al., 2000). The

The main objective of our study was therefore to determine whether the beneficial effect of apple polyphenols is due to binding and inhibition of the superantigen and/or to inhibition

In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and of a commercial apple polyphenol preparation (Apple Poly®) to inhibit the biological activity of SEA. The results are depicted in Figures 5-9. Dilutions of freshly prepared apple juices and of Apple Poly® inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents (Fig. 8). The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after the induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial

Fig. 5. Effect of apple juice on splenocyte proliferation. Apple juice and media used as a control with or without SEA (1 ng/mL) were incubated for 48 h with splenocyte cells followed by determining newly synthesized DNA (A) by cleavage of the peptide GF-AFC and (B) by use of live splenocytes. Error bars (n = 3) represent standard errors. Adapted

Fig. 4. Inhibition of RNAIII by native and synthetic RIP. Increasing amounts of RIP, which was purified on a C18 column or increasing amounts of synthetic peptide (Pep) were added to early exponential wild-type S. aureus and tested for the ability to inhibit RNAIII. Density of RNAIII is shown. p<0.05. Adapted from (Balaban et al., 1998).

Experimentally, we found that when we used increased inoculums of bacteria (1.4 × 108 cells per injection), four of eight animals were protected, and the remaining four developed a lesion that was 55% smaller than in control animals (Table 2). All the control animals (seven out of seven) challenged with SD and saline developed a lesion. When more bacteria were used (1.4 × 10), the synthetic RIP (0.5 mg of Pep) protected animals—90% (9 out of 10) of the animals showed no sign of disease (Table 2). These results suggest that the ratio between RIP and the bacteria is critical and helps determine the success of the host's immune response to eliminate the bacteria.

These observations suggest that targeting autoinducers of virulence or the signal transduction they activate may, therefore, be a unique and useful approach in preventing pathogenesis of toxin-producing *S. aureus* and possibly other toxin-producing pathogenic bacteria.

#### **3.4 Apple polyphenols inhibit T-helper cell proliferation and cytokine production in spleen cells from C57BL/6 female mice**

As mentioned earlier, *S. aureus* is a major bacterial pathogen that causes clinical infection and food-borne illnesses (Dinges et al., 2000). This bacterium produces a group of twentyone known enterotoxins (SEs) that have two separate biological activities: they cause gastroenteritis in the gastrointestinal tract and act as a superantigen on the immune system. Functional enterotoxins bind to the alpha-helical regions of the major histocompatibility complex (MHC) class II molecules outside the peptide-binding groove of the antigen presenting cells (APC), and also to the variable region (Vß) on T-cell receptors. The toxin then forms a bridge between T cells and APCs. This event then initiates the proliferation of a large number (~20%) of T cells that induce the release of cytokines. At high concentrations, cytokines are involved in the etiology (causes) of several known human and animal diseases. These include atopic dermatitis and rheumatoid arthritis in humans (Lin et al., 2011; MacIas et al., 2011; Yeung et al., 2011).

Fig. 4. Inhibition of RNAIII by native and synthetic RIP. Increasing amounts of RIP, which was purified on a C18 column or increasing amounts of synthetic peptide (Pep) were added to early exponential wild-type S. aureus and tested for the ability to inhibit RNAIII. Density

Experimentally, we found that when we used increased inoculums of bacteria (1.4 × 108 cells per injection), four of eight animals were protected, and the remaining four developed a lesion that was 55% smaller than in control animals (Table 2). All the control animals (seven out of seven) challenged with SD and saline developed a lesion. When more bacteria were used (1.4 × 10), the synthetic RIP (0.5 mg of Pep) protected animals—90% (9 out of 10) of the animals showed no sign of disease (Table 2). These results suggest that the ratio between RIP and the bacteria is critical and helps determine the success of the host's immune

These observations suggest that targeting autoinducers of virulence or the signal transduction they activate may, therefore, be a unique and useful approach in preventing pathogenesis of toxin-producing *S. aureus* and possibly other toxin-producing pathogenic

**3.4 Apple polyphenols inhibit T-helper cell proliferation and cytokine production in** 

As mentioned earlier, *S. aureus* is a major bacterial pathogen that causes clinical infection and food-borne illnesses (Dinges et al., 2000). This bacterium produces a group of twentyone known enterotoxins (SEs) that have two separate biological activities: they cause gastroenteritis in the gastrointestinal tract and act as a superantigen on the immune system. Functional enterotoxins bind to the alpha-helical regions of the major histocompatibility complex (MHC) class II molecules outside the peptide-binding groove of the antigen presenting cells (APC), and also to the variable region (Vß) on T-cell receptors. The toxin then forms a bridge between T cells and APCs. This event then initiates the proliferation of a large number (~20%) of T cells that induce the release of cytokines. At high concentrations, cytokines are involved in the etiology (causes) of several known human and animal diseases. These include atopic dermatitis and rheumatoid arthritis in humans (Lin et al.,

of RNAIII is shown. p<0.05. Adapted from (Balaban et al., 1998).

response to eliminate the bacteria.

**spleen cells from C57BL/6 female mice** 

2011; MacIas et al., 2011; Yeung et al., 2011).

bacteria.

Atopic dermatitis (eczema) is an inflammatory skin disease that affects 10-20% of children and 1-3% of adults (1-3). Antibiotics that suppress colonization of *S. aureus* are reported to mitigate the severity of atopic dermatitis disease. Most strains of *S. aureus* isolated from atopic skin lesions produce exotoxins with superantigen properties (Leung et al., 1993). It has been reported that staphylococcal superantigens can induce skin inflammation by several different mechanisms (Taskapan & Kumar, 2000). The reason we selected the superantigen for this study is that it is has been demonstrated to be an aggravating factor in atopic dermatitis, and because SEA is a representative antigen.

It has been previously reported that consumption of apple condensed tannins from unripe apples improve the symptoms associated with atopic dermatitis (Kojima et al., 2000). The mechanism of this improvement is largely unknown.

The main objective of our study was therefore to determine whether the beneficial effect of apple polyphenols is due to binding and inhibition of the superantigen and/or to inhibition of cell proliferation.

In the present study, we evaluated the ability of one commercial and two freshly prepared apple juices and of a commercial apple polyphenol preparation (Apple Poly®) to inhibit the biological activity of SEA. The results are depicted in Figures 5-9. Dilutions of freshly prepared apple juices and of Apple Poly® inhibited the biological activity of SEA without any significant cytotoxic effect on the spleen cells. Additional studies with antibody-coated immunomagnetic beads bearing specific antibodies against the toxin revealed that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents (Fig. 8). The results suggest that food-compatible and safe anti-toxin phenolic compounds can be used to inactivate SEA in vitro and possibly also in vivo, even after the induction of T-cell proliferation by long-term exposure to SEA. The significance of the results for microbial food safety and human health is discussed.

Fig. 5. Effect of apple juice on splenocyte proliferation. Apple juice and media used as a control with or without SEA (1 ng/mL) were incubated for 48 h with splenocyte cells followed by determining newly synthesized DNA (A) by cleavage of the peptide GF-AFC and (B) by use of live splenocytes. Error bars (n = 3) represent standard errors. Adapted from (Rasooly et al., 2010).

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

activity of SEA by apple compounds is visually illustrated in Figure 9.

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 397

These observations suggest that Red Delicious juice has an inhibitory effect even after cell proliferation was initiated. We suggest that components of the juice disrupt the connection between antigen presenting cells (APCs) and T cells. Our results also imply that the mechanism by which consumed apple juice or apples may decrease the symptoms associated with atopic dermatitis is via inhibition of proliferation of T cells and the release of cytokines. The postulated mechanism that may govern the inhibition of the biological

Fig. 8. Extraction and elution of SEA from apple juice treated with immunomagnetic beads. Apple juices were spiked with SEA (1 ng/mL) and incubated for 16 h with immunomagnetic beads. The toxin was dissociated from the beads and incubated with spleen cells. This was followed by the determination of newly synthesized DNA. Error bars (n = 3) represent standard errors. These results indicate that SEA added to apple juice appears to be largely

The described findings suggest that apple juices and polyphenol-rich apple skin extracts have the potential to counteract adverse effects in animals and humans induced by SEA, and possibly also by the foodborne pathogen *S. aureus* that produces this virulent toxin. It would be of interest to find out whether or not the inhibited toxin in apple juice is reactivated in the digestive tracts of animals and humans and whether or not phenolic compounds present in other juices can concurrently inhibit the growth of *S. aureus* and other pathogens and the

In summary, our studies with apple juice and apples skin extracts demonstrated that apple polyphenols strongly inhibited superantigen-induced T-cell proliferation and cytokine production. The results also indicate that the low inhibitory action of freshly prepared Red Delicious apple juice is enhanced by added apple polyphenols. Further studies are needed to determine whether or not this combination may protect against animal and human

irreversibly bound to the juice constituents. Adapted from (Rasooly et al. 2010).

toxins produced by the pathogens.

diseases induced by high cytokine levels.

Fig. 6. Red Delicious apple juice inhibits high SEA concentrations. Three concentrations of SEA were incubated for 48 h with splenocyte cells followed by determining newly synthesized DNA. Error bars (n = 3) represent standard errors. Adapted from (Rasooly et al. 2010).

Fig. 7. Red Delicious apple juice reduces the activity of SEA after 24 or 48 h of incubation. Different amounts of SEA were added to the splenocytes, which were then incubated for 24 or 48 h. This was followed by the addition of Red Delicious apple juice and the determination of biological activity by cleavage of GF-AFC, produced by the live splenocyte cells after (A) 48 h or (B) 72 h. Error bars (n = 3) represent standard errors. Adapted from (Rasooly et al., 2010).

Fig. 6. Red Delicious apple juice inhibits high SEA concentrations. Three concentrations of

synthesized DNA. Error bars (n = 3) represent standard errors. Adapted from (Rasooly et al.

Fig. 7. Red Delicious apple juice reduces the activity of SEA after 24 or 48 h of incubation. Different amounts of SEA were added to the splenocytes, which were then incubated for 24

determination of biological activity by cleavage of GF-AFC, produced by the live splenocyte cells after (A) 48 h or (B) 72 h. Error bars (n = 3) represent standard errors. Adapted from

or 48 h. This was followed by the addition of Red Delicious apple juice and the

SEA were incubated for 48 h with splenocyte cells followed by determining newly

2010).

(Rasooly et al., 2010).

These observations suggest that Red Delicious juice has an inhibitory effect even after cell proliferation was initiated. We suggest that components of the juice disrupt the connection between antigen presenting cells (APCs) and T cells. Our results also imply that the mechanism by which consumed apple juice or apples may decrease the symptoms associated with atopic dermatitis is via inhibition of proliferation of T cells and the release of cytokines. The postulated mechanism that may govern the inhibition of the biological activity of SEA by apple compounds is visually illustrated in Figure 9.

Fig. 8. Extraction and elution of SEA from apple juice treated with immunomagnetic beads. Apple juices were spiked with SEA (1 ng/mL) and incubated for 16 h with immunomagnetic beads. The toxin was dissociated from the beads and incubated with spleen cells. This was followed by the determination of newly synthesized DNA. Error bars (n = 3) represent standard errors. These results indicate that SEA added to apple juice appears to be largely irreversibly bound to the juice constituents. Adapted from (Rasooly et al. 2010).

The described findings suggest that apple juices and polyphenol-rich apple skin extracts have the potential to counteract adverse effects in animals and humans induced by SEA, and possibly also by the foodborne pathogen *S. aureus* that produces this virulent toxin. It would be of interest to find out whether or not the inhibited toxin in apple juice is reactivated in the digestive tracts of animals and humans and whether or not phenolic compounds present in other juices can concurrently inhibit the growth of *S. aureus* and other pathogens and the toxins produced by the pathogens.

In summary, our studies with apple juice and apples skin extracts demonstrated that apple polyphenols strongly inhibited superantigen-induced T-cell proliferation and cytokine production. The results also indicate that the low inhibitory action of freshly prepared Red Delicious apple juice is enhanced by added apple polyphenols. Further studies are needed to determine whether or not this combination may protect against animal and human diseases induced by high cytokine levels.

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

**biological activity of SEA** 

pathogens (Rasooly et al., 2010).

inactivated the pathogens.

Adapted from (Friedman et al., 2011).

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 399

**3.5 The olive compound 4-hydroxytyrosol inactivates both** *S. aureus* **and inhibits the** 

Our observations that dilutions of freshly prepared apple juices and of a commercial apple skin preparation inhibited the biological activity of SEA in a spleen cell assay suggested that other natural plant-derived compounds and plant extracts have the potential to inhibit the growth of foodborne pathogens and the toxicological effects of toxins produced by some

To further demonstrate this possibility, the objectives of another study were to determine whether the pure olive compound hydroxytyrosol (Figure 12) and a commercial olive powder named Hidrox-12 that contains hydroxytyrosol can inactivate *S. aureus* bacteria and inhibit the biological activity of SEA (Friedman et al., 2011). We found that olive ingredients also posses anti-*S. aureus* activity that reduced the counts of the bacteria in a dosedependent manner. With hydroxytyrosol, the viable count of *S. aureus* bacteria after treatment at a concentration of 0.67 mg/ml for 60 min of contact, determined by plating on LB agar media, was decreased by 85%. We also found that dilutions of both test substances

Fig. 11. Effect of hydroxytyrosol on splenocyte proliferation determined by two

independent methods. Different concentrations of the toxin (0, 5, and 200 ng/mL) were exposed to hydroxytyrosol or the control (media) and were then incubated for 48 h with splenocyte cells followed by determining (A) GF-AFC cleavage by live cell protease (a measure of cellular activity) or (B) BrdU incorporation into newly synthesized DNA (a measure of cellular proliferation). Conditions: (A) GF-AFC substrate in intact cells is cleaved by live cell protease releasing the fluorescent AFC, quantified at an excitation wavelength of 355 nm and an emission wavelength of 523 nm. (B) BrdU (5-bromo-2 deoxyyridine)-labeled DNA was determined spectrophotometrically at absorbances of 620 nm and 450 nm. Error bars (n = 3) represent standard errors. Both assays show that hydroxytyrosol inhibited the biological activity of SEA. Error bars represent standard error (SEs), and an asterisk indicates significant differences (P<0.05) between treatments.

Two independent cell assays (BrdU incorporation into newly synthesized DNA and glycylphenylalanyl-aminofluorocoumarin (GF-AFC) proteolysis) demonstrated that the olive compound also inhibits the biological activity of SEA. The described findings suggest that olive compounds have the potential to counteract adverse effects induced by SEA and by

Fig. 9. A schematic representation of cellular events that lead to the inhibition of SEAinduced cell proliferation by apple juice. The individual steps in this scheme involve the (A) the formation of a bridge between APCs and T cells which results in induction of T-cell proliferation and (B) inhibition of T-cell proliferation by added pure apple juice that disrupts the connection between APCs and T cells. The net beneficial result of these events is the prevention of release and the consequent adverse effects induced by cytokines described in the Introduction. Abbreviations: MHC, major histocompatibility complex; TCR, T-cell receptor. Adapted from (Rasooly et al., 2010).

Fig. 10. Structure of the olive compound, hydroxytyrosol that inhibits *S. aureus* and SEA.

Fig. 9. A schematic representation of cellular events that lead to the inhibition of SEAinduced cell proliferation by apple juice. The individual steps in this scheme involve the (A) the formation of a bridge between APCs and T cells which results in induction of T-cell proliferation and (B) inhibition of T-cell proliferation by added pure apple juice that

receptor. Adapted from (Rasooly et al., 2010).

disrupts the connection between APCs and T cells. The net beneficial result of these events is the prevention of release and the consequent adverse effects induced by cytokines described in the Introduction. Abbreviations: MHC, major histocompatibility complex; TCR, T-cell

Fig. 10. Structure of the olive compound, hydroxytyrosol that inhibits *S. aureus* and SEA.

#### **3.5 The olive compound 4-hydroxytyrosol inactivates both** *S. aureus* **and inhibits the biological activity of SEA**

Our observations that dilutions of freshly prepared apple juices and of a commercial apple skin preparation inhibited the biological activity of SEA in a spleen cell assay suggested that other natural plant-derived compounds and plant extracts have the potential to inhibit the growth of foodborne pathogens and the toxicological effects of toxins produced by some pathogens (Rasooly et al., 2010).

To further demonstrate this possibility, the objectives of another study were to determine whether the pure olive compound hydroxytyrosol (Figure 12) and a commercial olive powder named Hidrox-12 that contains hydroxytyrosol can inactivate *S. aureus* bacteria and inhibit the biological activity of SEA (Friedman et al., 2011). We found that olive ingredients also posses anti-*S. aureus* activity that reduced the counts of the bacteria in a dosedependent manner. With hydroxytyrosol, the viable count of *S. aureus* bacteria after treatment at a concentration of 0.67 mg/ml for 60 min of contact, determined by plating on LB agar media, was decreased by 85%. We also found that dilutions of both test substances inactivated the pathogens.

Fig. 11. Effect of hydroxytyrosol on splenocyte proliferation determined by two independent methods. Different concentrations of the toxin (0, 5, and 200 ng/mL) were exposed to hydroxytyrosol or the control (media) and were then incubated for 48 h with splenocyte cells followed by determining (A) GF-AFC cleavage by live cell protease (a measure of cellular activity) or (B) BrdU incorporation into newly synthesized DNA (a measure of cellular proliferation). Conditions: (A) GF-AFC substrate in intact cells is cleaved by live cell protease releasing the fluorescent AFC, quantified at an excitation wavelength of 355 nm and an emission wavelength of 523 nm. (B) BrdU (5-bromo-2 deoxyyridine)-labeled DNA was determined spectrophotometrically at absorbances of 620 nm and 450 nm. Error bars (n = 3) represent standard errors. Both assays show that hydroxytyrosol inhibited the biological activity of SEA. Error bars represent standard error (SEs), and an asterisk indicates significant differences (P<0.05) between treatments. Adapted from (Friedman et al., 2011).

Two independent cell assays (BrdU incorporation into newly synthesized DNA and glycylphenylalanyl-aminofluorocoumarin (GF-AFC) proteolysis) demonstrated that the olive compound also inhibits the biological activity of SEA. The described findings suggest that olive compounds have the potential to counteract adverse effects induced by SEA and by

Food Compounds Inhibit *Staphylococcus Aureus* Bacteria and

et al., 2008; Sirk et al., 2011).

for facilitating the preparation of this chapter.

Vol.280, No. 5362, pp. 438-440

*Microbiology,* Vol.61, No. 1, pp. 1-10

*Vaccines,* Vol.10, No. 5, pp. 695-708

*Pathogenesis,* Vol.42, No. 5-6, pp. 215-224

Washington DC

*Protection,* Vol.59, No. 12, pp. 1292-1299

**5. Acknowledgment** 

**6. References** 

the Toxicity of Staphylococcus Enterotoxin A (SEA) Associated with Atopic Dermatitis 401

The described studies are part of a broader effort, the specific objective of which is to transform toxic proteins to nontoxic, digestible proteins in foods. For example, apple and other polyphenols present in numerous plant foods such as teas, sweet potatoes, and jujube fruits and seeds contain electron-rich aromatic structures and ionizable phenolic OH groups. These structures can in theory change the toxin via non-covalent binding to the toxin and/or by altering the distribution of ionic charges via H-bonding between OH groups and ionizable groups of the protein. We have no direct evidence for this theory, but note that in molecular simulation studies, we observed multiple hydrogen-bonding interactions between polyphenolic tea catechins and cell membranes that may result in anti-bactericidal effects due to disruption of the cell membranes followed by cell death (Sirk et al., 2009; Sirk

We thank our colleagues whose names appear on the cited publications and Carol E. Levin

Anderson, J. E., Beelman, R. R., & Doores, S. (1996). Persistence of serological and biological

Balaban, N., Goldkorn, T., Nhan, R. T., Dang, L. B., Scott, S., Ridgley, R. M., Rasooly, A.,

Balaban, N., & Rasooly, A. (2000). Staphylococcal enterotoxins. *International Journal of Food* 

Broughan, J., Anderson, R., & Anderson, A. S. (2011). Strategies for and advances in the

Carlos, L. A., Amaral, K. A. S., Vieira, I. J. C., Mathias, L., Braz-Filho, R., Samarão, S. S., &

Choi, O., Yahiro, K., Morinaga, N., Miyazaki, M., & Noda, M. (2007). Inhibitory effects of

Davis, J. R., & Lederberg, J. (Eds.). (2001). *Emerging infectious diseases from the global to local* 

de Souza, E. L., de Barros, J. C., de Oliveira, C. E. V., & da Conceição, M. L. (2010). Influence

Dinges, M. M., Orwin, P. M., & Schlievert, P. M. (2000). Exotoxins of *Staphylococcus aureus*.

*Brazilian Journal of Microbiology,* Vol.41, No. 3, pp. 612-620

*Journal of Food Microbiology,* Vol.137, No. 2-3, pp. 308-311

*Clinical Microbiology Reviews,* Vol.13, No. 1, pp. 16-34

activities of staphylococcal enterotoxin A in canned mushrooms. *Journal of Food* 

Wright, S. C., Larrick, J. W., Rasooly, R., & Carlson, J. R. (1998). Autoinducer of virulence as a target for vaccine and therapy against *Staphylococcus aureus*. *Science,* 

development of *Staphylococcus aureus* prophylactic vaccines. *Expert Review of* 

Vieira-da-Motta, O. (2010). *Rauvolfia grandiflora* (apocynaceae) extract interferes with staphylococcal density, enterotoxin production and antimicrobial activity.

various plant polyphenols on the toxicity of Staphylococcal α-toxin. *Microbial* 

*perspective: workshop summary*, National Academy of Sciences, ISBN 0-309-07184-4,

of *Origanum vulgare* L. essential oil on enterotoxin production, membrane permeability and surface characteristics of *Staphylococcus aureus*. *International* 

the foodborne pathogen *S. aureus* that produces this virulent toxin. To our knowledge, this is the first report that demonstrated with the aid of bactericidal and cell assays that the edible olive compound hydroxytyrosol can inactivate both *S. aureus* bacteria and SEA. The results suggest that food-compatible and safe anti-toxin olive compounds merit further study designed to demonstrate their potential to treat atopic dermatitis.

It would be of interest to extend these studies to the inactivation of other pathogens and toxins such as *E. coli* and Shiga toxin *in vitro* and in contaminated food such as meat, milk, and leafy greens.

Fig. 12. (A) Antimicrobial activity of hydroxytyrosol against *S. aureus*. Conditions: bacteria were incubated with different concentrations of hydroxytyrosol. After incubation for 60 min, cells were plated and bacteria counted. (B) Antimicrobial activity of Hidrox-12 against *S. aureus*. Conditions: similar to those used for hydroxytyrosol. Error bars represent standard errors (n = 4). Both the pure olive compound and the olive extract inhibited the bacteria. The extent of inhibition by the extract was approximately equivalent to its content (12%) of hydroxytyrosol. Adapted from (Friedman et al. 2011).
