Antibacterial Effects of Plant-Derived Extracts on Methicillin-Resistant Staphylococcus aureus

Abstract

Natural chemicals have been reported to have antibacterial effects against a variety of bacteria. The present study evaluated the antibacterial effects of commercially available grape-seed extract (GSE), pomegranate polyphenols (PP), and lab-prepared cranberry proanthocyanidins (C-PAC) against two strains of methicillin-resistant Staphylococcus aureus (MRSA). GSE, PP, and C-PAC at concentrations of 2 mg/mL, 10 mg/mL, or controls were mixed with equal volumes of overnight cultures of MRSA at ∼6 log₁₀ colony-forming units (CFU)/mL and incubated for 0, 1, 2, 8, and 24 h at 37°C. Treatments were neutralized/stopped using tryptic soy broth containing 3% beef extract. Serial dilutions of the treated MRSA strains and controls were spread-plated on trypticase soy agar and incubated for 24–48 h at 37°C and colonies were counted. Among the three tested agents, GSE at 1 and 5 mg/mL was found to be most effective against MRSA, resulting in a 2.9–4.0 log₁₀ CFU/mL reduction of both strains after 2 h at 37°C. PP at 1 and 5 mg/mL was found to cause 1.1–2.3 log₁₀ CFU/mL reduction, while C-PAC at 1 mg/mL caused <1 log₁₀ CFU/mL reduction of the two MRSA strains after 2 h at 37°C. All three extracts at the tested concentrations decreased the two MRSA strains to undetectable levels within 24 h, with the exception of 1 mg/mL PP for strain 33591. Scanning electron microscopy of MRSA after 2 h of treatment showed that GSE and PP caused bacterial cell wall alteration, with negligible effect observed by C-PAC treatment. However, the in vivo activity and clinical safety applications of GSE, PP, and C-PAC need to be evaluated before suggestion for use as a treatment/control measure.

Introduction

M ethicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen-causing nosocomial infections and presents a significant threat to public health. The U.S. Centers for Disease Control and Prevention (CDC) reported that, in 2007, MRSA were responsible for 94,360 serious infections and were associated with 18,650 deaths in 2005 in the United States alone (CDC, 2007). Therapeutic options for MRSA infections are currently very limited due to the fact that most MRSA strains are resistant to commonly used antibiotics such as lactams, macrolides, aminoglycosides, and fluoroquinolones (Schentag et al., 1998; Tacconelli et al., 2008). Vancomycin and other glycopeptide antibiotics are presently used for treatment of MRSA infections. However, glycopeptides frequently cause side effects, and emergencies related to vancomycin-resistant S. aureus infections have been reported (Molling et al., 2010; Tascini et al., 2011). Therefore, alternative treatments are needed to prevent and control the spread of MRSA infections.

Various plant extracts have been reported to have antimicrobial activities (Becerril et al., 2007; Goni et al., 2009; Scalbert, 1991; Taguri et al., 2004). Some plant polyphenols are known to be effective against MRSA strains (Al-Habib et al., 2010; Hatano et al., 2005; Lin et al., 2008). Recently, Al-Habib et al. (2010) found that grape seed extract (GSE) at 3 mg/mL completely inhibited all 43 tested MRSA strains by causing disruption of the bacterial cell wall (Al-Habib et al., 2010). Gould et al. (2009) showed that pomegranate rind extract alone had limited effects on MRSA, but when combined with copper (Cu) ions caused a 4 log₁₀ colony-forming units (CFU)/mL reduction in clinical isolates of MRSA (Gould et al., 2009). Green tea extracts have also been shown to have antibacterial activity against MRSA (Cho et al., 2008). To the best of our knowledge, the effect of lab-prepared cranberry proanthocyanidins (C-PAC) on MRSA has not yet been reported in literature, though some reports on the effects of cranberry juice and cranberry extracts on S. aureus have been published (Magarinos et al., 2008; Qiu and Wu, 2007).

The purpose of the present study was to compare the effect of three plant-derived extracts, namely, commercially available GSE, commercially available pomegranate polyphenols (PP), and C-PAC, on two MRSA strains. The anti-MRSA activities of GSE, PP, or C-PAC at 1 and 5 mg/mL were evaluated after 0, 1, 2, 8, and 24 h of incubation at 37°C. The MRSA titers after treatment were determined by spread plating and compared to water-treated controls. To gain insights on the antibacterial mechanisms of GSE, PP, and C-PAC, scanning electron microscopy (SEM) was employed to determine any structural changes associated with the treatments.

Methods

Bacteria and culture conditions

MRSA (strain ATCC 33591 and ATCC 33593) were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The stock cultures were grown on Baird-Parker agar plates at 37°C for 2 days and maintained at 4°C. A typical single colony of each strain was inoculated into trypticase soy broth (TSB; Becton, Dickinson and Company, Sparks, MD) and incubated at 37°C for 24 h. The cultures were transferred once a day, and an overnight culture was used to determine the anti-MRSA effects of GSE, PP, and C-PAC.

Anti-staphylococcal activity of GSE, PP, and C-PAC

Gravinol-S (GSE) was obtained as a gift from OptiPure^® Chemco Industries (Los Angeles, CA). PP was provided as a gift from POM Wonderful (Los Angeles, CA). C-PAC was obtained from Dr. Amy Howell, Rutgers University (Chatsworth, NJ). The above extracts were dissolved in ethanol, filter sterilized, and diluted in sterile distilled deionized water to 10 and 2 mg/mL, respectively (with the final concentration of ethanol at 5%).

For the bacterial treatment, overnight cultures of MRSA were diluted in phosphate-buffered saline (PBS, pH 7.4), and 0.5 mL of diluted MRSA at titers of ∼6 log₁₀ CFU/mL was mixed with equal volumes of GSE or PP at 10 and 2 mg/mL and C-PAC at 2 mg/mL. Water, 2.5% ethanol (final concentration), and PBS at pH 6.0 were used as controls. The pH of the resulting bacterial-GSE mixture or bacteria–C-PAC mixture was pH 7.4, whereas that of bacterial-PP mixture was ∼pH 6.0–6.5. After incubation times of 0, 1, 2, 8, and 24 h, the treatments were neutralized/stopped by 10-fold dilution in TSB containing 3% beef extract. The treatment and controls were then serially diluted in PBS and 0.1 mL of each dilution was spread-plated on tryptic soy agar (TSA) and incubated at 37°C for 24–48 h. Colonies were counted, and recovered bacterial titers were expressed as log₁₀ CFU/mL.

SEM observation

SEM was conducted at the Advanced Microscopy and Imaging Center at the University of Tennessee at Knoxville by Dr. John Dunlap to determine any structural changes in MRSA after treatment with GSE, PP, or C-PAC. MRSA at a titer of ∼7 log₁₀ CFU/mL was mixed with an equal volume of sterile water, GSE, PP, or C-PAC, each at 2 mg/mL, and incubated at 37°C for 2 h. The bacteria were fixed with 3% glutaraldehyde in PBS for 1 h, followed by fixation in 2% osmium tetroxide for 1 h. A drop of the bacterial suspension was added to silicon chips, dehydrated in degraded ethanol, and finally subjected to critical point drying. The dried samples were sputter-coated with carbon and viewed by SEM at an accelerating voltage of 2 kV as described elsewhere (Su and D'Souza, 2012).

Statistical analysis

All the treatments were replicated three times, and each treatment was evaluated in duplicate for microbiological analysis. Statistical analysis was carried out using analysis of variance (ANOVA) with SAS software (version 9.2, SAS Institute, Cary, NC) and Tukey's test on a completely randomized design with six sets of data under each treatment condition.

Results and Discussion

There are reports in the literature on the anti-MRSA effects of GSE and PP (Al-Habib et al., 2010; Braga et al., 2005; Gould et al., 2009; Reddy et al., 2007). However, to the best of our knowledge, a comparison between the anti-MRSA activity of GSE and other plant-derived extracts is currently not available. As can be seen in Table 1, all three tested extracts showed anti-MRSA effects after 24 h of treatment. Within 2 h, GSE was found to be the most effective among the three tested plant-derived extracts against MRSA, followed by PP, and then C-PAC.

Table 1.

Reduction (in log₁₀ CFU/mL) of Methicillin-Resistant Staphylococcus aureus (MRSA) at Initial Titers of ∼6-loglog₁₀ CFU/mL After Treatment with Grape Seed Extract (GSE), Pomegranate Polyphenols (PP), and Cranberry Proanthocyanidins (C-PAC) for up to 24 h at 37°C

(a)
Incubation time (h)	Water	2.5% EtOH	pH 6.0	1 mg/mL GSE	5 mg/mL GSE	1 mg/mL PP	5 mg/mL PP	1 mg/mL C-PAC
0	0.00±0.05 A	0.04±0.05 A	0.03±0.09 A	0.16±0.05 A	0.21±0.07 A	0.22±0.09 A	0.08±0.04 A	0.12±0.08 A
1	0.07±0.10 A	0.07±0.09 A	0.19±0.13 AB	1.98±0.13 B	1.35±0.04 B	1.04±0.06 B	0.51±0.08 B	0.70±0.09 B
2	0.00±0.04 A	0.10±0.10 A	0.35±0.10 B	3.97±0.14 C	3.49±0.11 C	1.68±0.08 C	2.31±0.20 C	0.87±0.11 B
8	0.08±0.06 A	0.27±0.18 A	0.76±0.03 C	6.59±0.00 D	6.59±0.00 D	2.81±0.13 D	6.59±0.00 D	3.36±0.23 C
24	0.06±0.07 A	0.64±0.04 B	1.65±0.05 D	6.59±0.00 D	6.59±0.00 D	4.07±0.34 E	6.59±0.00 D	6.59±0.00 D

(b)
Incubation time (h)	Water	2.5% EtOH	pH 6.0	1 mg/mL GSE	5 mg/mL GSE	1 mg/mL PP	5 mg/mL PP	1 mg/mL C-PAC
0	0.00±0.06 A	0.10±0.12 A	0.07±0.12 A	0.10±0.10 A	0.15±0.07 A	0.14±0.04 A	0.10±0.11 A	0.00±0.15 A
1	0.10±0.16 A	0.07±0.07 A	0.14±0.09 A	1.66±0.04 B	1.36±0.28 B	0.55±0.13 B	0.16±0.08 A	0.38±0.12 B
2	0.02±0.06 A	0.18±0.11 A	0.24±0.09 A	3.08±0.19 C	2.89±0.22 C	1.70±0.13 C	1.14±0.09 B	0.71±0.06 C
8	0.00±0.07 A	0.33±0.16 A	1.49±0.14 B	6.45±0.00 D	6.45±0.00 D	3.74±0.14 D	3.35±0.20 C	6.45±0.00 D
24	0.25±0.09 AB	0.75±0.06 B	2.26±0.12 C	6.45±0.00 D	6.45±0.00 D	6.45±0.00 E	6.45±0.00 D	6.45±0.00 D

(a) Strain American Type Culture Collection (ATCC) 33591.

(b) Strain ATCC 33593.

Different letters when compared within each column indicate significant differences (p<0.05).

For MRSA strain ATCC 33591, the titers were reduced by 1.98, 3.97, and 6.59 log₁₀ CFU/mL after incubation with 1 mg/mL GSE for 1, 2, and 8 h, respectively. Thus, the effect of GSE against this strain of MRSA was found to be time-dependent, where longer contact time resulted in greater reduction. When GSE at 5 mg/mL was used, titer reduction of MRSA was 1.35, 3.49, and 6.59 log₁₀ CFU/mL after incubation at 37°C for 1, 2, and 8 h, respectively. Increasing the concentration of GSE did not appear to cause an increase in the titer reduction. PP at 1 mg/mL reduced MRSA strain ATCC 33591 by 1.04, 1.68, and 2.81 log₁₀ CFU/mL after 1, 2, and 8 h, respectively; and 5 mg/mL PP reduced this MRSA strain by 0.51, 2.31, and 6.59 log₁₀ CFU/mL, respectively. For PP, MRSA titer reduction was found to be time dependent as well as dose dependent. C-PAC at 1 mg/mL caused 0.70, 0.87, and 3.36 log₁₀ CFU/mL reduction of MRSA strain ATCC 33591 after 1, 2, and 8 h at 37°C, respectively. All three extracts at the tested concentrations were found to decrease MRSA to undetectable levels after 24 h at 37°C, with the exception that 1 mg/mL PP caused ∼4.07 log₁₀ CFU/mL reduction.

For MRSA strain ATCC 33593, GSE at 1 and 5 mg/mL reduced MRSA strain ATCC 33593 by 1.4–1.7 and 2.9–3.1 log₁₀ CFU/mL, respectively, after 1 and 2 h at 37°C. GSE at both concentrations reduced MRSA to undetectable levels after 8 h. PP at 1 and 5 mg/mL reduced MRSA strain ATCC 33593 by 0.2–0.6, 1.1–1.7, and 3.4–3.7 log₁₀ CFU/mL, respectively, after 1, 2, and 8 h at 37°C, respectively. PP at 1 and 5 mg/mL required 24 h to reduce MRSA strain 33593. C-PAC at 1 mg/mL caused <1 log₁₀ CFU/mL reduction after 2 h, but reduced MRSA to undetectable levels after 8 h. Treatment for 1 h with GSE, PP, and C-PAC at 1 mg/mL caused less reduction in strain 33593 than in strain 33591. According to ATCC (the source of these strains), MRSA strain 33593 is methicillin and gentamicin resistant, while strain 33591 is reported to be only methicillin resistant. This could account for strain 33593 being more resistant to treatment with GSE, PP, and C-PAC at 1 mg/mL than strain 33591.

To further understand the mechanism of action of the tested plant extracts against MRSA, each strain treated with GSE, PP, or C-PAC at 1 mg/mL for 2 h at 37°C was viewed using SEM. Figures 1 and 2 show the SEM micrographs of MRSA strain 33591 and 33593 treated with water, 1 mg/mL GSE, 1 mg/mL PP, and 1 mg/mL C-PAC, respectively. As can be observed, MRSA cells treated with water showed an oval morphology with smooth surfaces around 400–600 nm in size. While GSE-treated MRSA seemed to have the same morphology and size as the water-treated control, the surface appeared to be rough and disrupted. PP-treated MRSA showed “amorphous droplets” at the surface that were up to 100 nm in size. C-PAC–treated MRSA showed smoother surfaces than GSE-treated cells and did not show the “amorphous droplets or blobs” on the surface. Both MRSA strains showed the same changes in characteristics compared to the control samples. Similar structural changes were also reported by Al-Habib (2010) on their GSE-treated MRSA strains using clinical isolates from hospitals in Kuwait.

FIG. 1.

Scanning electron microscopy (SEM) micrographs of methicillin-resistant Staphylococcus aureus (MRSA) strain American Type Culture Collection (ATCC) 33591 treated with water (a), 1 mg/mL grape seed extract (GSE) (b), 1 mg/mL pomegranate polyphenols (PP) (c), and 1 mg/mL cranberry proanthocyanidins (C-PAC) (d) for 2 h at 37°C.

FIG. 2.

Scanning electron microscopy (SEM) micrographs of methicillin-resistant Staphylococcus aureus (MRSA) strain American Type Culture Collection (ATCC) 33593 treated with water (a), 1 mg/mL grape seed extract (GSE) (b), 1 mg/mL pomegranate polyphenols (PP) (c), and 1 mg/mL cranberry proanthocyanidins (C-PAC) (d) for 2 h at 37°C.

GSE contains >95% flavonol, of which 82% are oligomeric proanthocyanidins and 12% are highly active monomeric proanthocyanidins (OptiPure^® Chemco Industries, Los Angeles, CA). Al-Habib et al. (2010) tested the effect of GSE against 43 tested clinical strains of MRSA and found that the minimum inhibitory concentration (MIC) was 20.7 μg/mL GSE flavonoid content for all strains. In this study, MRSA strains after treatment with 0.5 and 1 mg/mL GSE were reduced by 40–50% and 60–70%, respectively.

There are also reports on the effect of pomegranate extracts against MRSA (Braga et al., 2005; Gould et al., 2009; Reddy et al., 2007). PP contains hydrolysable tannins (ellagitannins), such as oligomers and punicalagin/punicalin, with small amounts of ellagic acid and anthocyanins (delphinidin, cyanidins, and pelargonidin) and their glycosides (Aviram et al., 2008). Gould et al. (2009) studied the antibacterial activity of pomegranate rind extract (PRE) for 2 h at room temperature and showed ∼1 log₁₀ CFU/mL reduction in MRSA and methicillin-sensitive S. aureus (MSSA) strains, with titer reductions slightly less than reported here in this study. The difference could be due to the difference in incubation temperatures; our study was at 37°C, and higher temperature is known to result in higher reduction. The authors also reported that pomegranate extracts in combination with cupric sulphate caused ∼4 log₁₀ CFU/mL reduction in MRSA and MSSA strains. Other researchers such as Braga et al. (2005) confirmed the inhibitory effect of pomegranate extract on both the antibiotic-sensitive and antibiotic-resistant isolates of S. aureus. By using the checkerboard method, they found that pomegranate extract at 0.1–2% (v/v) dramatically enhanced the activity of chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, achieving synergistic effects (synergy occurs at a fractional inhibitory concentration [FIC] index ≤0.5) of 65.5%, 38.1%, 71.4%, 70%, and 72.7%, respectively, which offers an alternative combinatory method for the use of these antibiotics and plant extracts.

Currently, there are few reports available on the effect of C-PAC against S. aureus. Cranberry juice and cranberry concentrates have been shown to have antibacterial effect against S. aureus (Magarinos et al., 2008; Qiu and Wu, 2007). Qiu and Wu (2007) studied the effect of cranberry concentrate on the activity of S. aureus at 21°C and 4°C and found cranberry concentrate suppressed S. aureus growth by 2.3–4.1 log₁₀ CFU/mL at 7°C and by 5.5–8.0 log₁₀ CFU/mL at 21°C after 7 days. Magarinos et al. (2008) also concluded that cranberry juice had inhibitory effect on S. aureus. However, it is unknown if these strains were methicillin-resistant or methicillin-sensitive.

Several antibacterial mechanisms of plant-derived extracts have been proposed, such as disruption of cytoplasmic membrane, inhibition of extracellular microbial enzymes, and changes in microbial metabolism (Al-Habib et al., 2010; Puupponen-Pimia et al., 2005). Previous electron microscopy results showed that GSE-treated MRSA reduced residual cellular content with partial disintegration of the bacterial cell surfaces (Al-Habib et al., 2010). Wu et al. (2008) showed that cranberry extracts caused cell wall damage of S. aureus and impaired cells were seen surrounded by lysate material (Wu et al., 2008). Our SEM results showed cell surface roughening after GSE treatment and formation of “blobs” on cell surface by PP treatment, which indicate that the cell surface was affected by these treatments.

Currently, information on the toxicity of C-PAC is not available. However, the safety of GSE and PP has been studied in animal models. It has been shown that oral administration of GSE to rats at levels of up to 1.5 g/kg or PP at 0.6 g/kg for a period of 90 days was not mutagenic and did not induce any significant toxicological effects (Patel et al., 2008; Wren et al., 2002; Yamakoshi et al., 2002). GSE at up to 0.2 mg/mL and PP at up to 0.4 mg/mL on three animal cell lines, RAW 264.7, a macrophage cell line, Crandell Reese feline kidney cells (CRFK), and fetal rhesus monkey kidney cells (FRhK4) did not show observable cytotoxic effects (Su and D'Souza, 2011; Su et al., 2010).

Conclusion

The three studied plant-derived extracts, GSE, PP, and C-PAC, were shown to have anti-MRSA activities after 24 h at 37°C. Among the three plant extracts tested, GSE at 1 and 5 mg/mL was found to be the most fast acting agent which caused 3–4 log₁₀ CFU/mL reduction in MRSA titers within 2 h, while PP at 1 and 5 mg/mL reduced MRSA titers by 1.1–2.3 log₁₀ CFU/mL after 2 h at 37°C. C-PAC showed <1 log₁₀ CFU/mL reduction under similar treatment conditions. The SEM images of GSE- and PP-treated MRSA strains showed effects against the bacterial cell wall. Since GSE, PP, and C-PAC are effective in reducing MRSA titers, further research should focus on exploring preventive and/or therapeutic formulations that show maximal antibacterial effects for MRSA treatment. However, comprehensive clinical studies are needed to determine the safety of these plant-derived extracts.

Footnotes

Acknowledgments

We gratefully acknowledge the assistance provided by Dr. John Dunlap (UT-Knoxville) for the SEM analysis. Funding for this research that was provided by the Tennessee Agricultural Experiment Station (TEN 00391) is gratefully acknowledged.

Disclosure Statement

No competing financial interests exist.

References

Al-Habib

, Al-Saleh

, Safer

, Afzal

. Bactericidal effect of grape seed extract on methicillin-resistant Staphylococcus aureus (MRSA) J Toxicol Sci, 2010; 35:357–364.

Aviram

, Fuhrman

, Rosenblat

et al. Pomegranate juice polyphenols decreases oxidative stress, low-density lipoprotein atherogenic modifications and atherosclerosis. Free Radic Res, 2002; 36:72–73.

Becerril

, Gomez-Lus

, Goni

, Lopez

, Nerin

. Combination of analytical and microbiological techniques to study the antimicrobial activity of a new active food packaging containing cinnamon or oregano against E. coli and S. aureus. Anal Bioanal Chem, 2007; 388:1003–1011.

Braga

, Leite

AAM

, Xavier

KGS

et al. Synergic interaction between pomegranate extract and antibiotics against Staphylococcus aureus. Can J Microbiol, 2005; 51:541–547.

[CDC] Centers for Disease Control and Prevention. MRSA: Methicillin-resistant Staphylococcus aureus in healthcare settings. Atlanta: CDC, 2007.

Cho

, Schiller

, Oh

. Antibacterial effects of green tea polyphenols on clinical isolates of methicillin-resistant Staphylococcus aureus. Curr Microbiol, 2008; 57:542–546.

Cote

, Caillet

, Doyon

, Dussault

, Sylvain

, Lacroix

. Antimicrobial effect of cranberry juice and extracts. Food Control, 2010; 22:1413–1418.

Goni

, Lopez

, Sanchez

, Gomez-Lus

, Becerril

, Nerin

. Antimicrobial activity in the vapour phase of a combination of cinnamon and clove essential oils. Food Chem, 2009; 116:982–989.

Gould

SWJ

, Fielder

, Kelly

, Naughton

. Anti-microbial activities of pomegranate rind extracts: Enhancement by cupric sulphate against clinical isolates of S. aureus, MRSA and PVL positive CA-MSSA. BMC Complement Altern Med, 2009; 9:23–35.

10.

Hatano

, Kusuda

, Inada

et al. Effects of tannins and related polyphenols on methicillin-resistant Staphylococcus aureus. Phytochemistry, 2005; 66:2047–2055.

11.

Lin

, Chin

, Hou

, Lee

. The effects of antibiotics combined with natural polyphenols against clinical methicillin-resistant Staphylococcus aureus (MRSA) Planta Med, 2008; 74:840–846.

12.

Magarinos

HLE

, Sahr

, Selaive

SDC

, Costa

, Figuerola

, Pizarro

. In vitro inhibitory effect of cranberry (Vaccinium macrocarpum Ait.) juice on pathogenic microorganisms. Appl Biochem Microbiol, 2008; 44:300–304.

13.

Molling

, Neander

, Soderquist

, Sundman

. Combined automated screening for detection of methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococci (VRE) in broth enriched clinical samples. J Mol Diagn, 2010; 12:884–884.

14.

Patel

, Dadhaniya

, Hingorani

, Soni

. Safety assessment of pomegranate fruit extract: Acute and subchronic toxicity studies. Food Chem Toxicol, 2008; 46:2728–2735.

15.

Puupponen-Pimia

, Nohynek

, Alakomi

, Oksman-Caldentey

. Bioactive berry compounds—Novel tools against human pathogens. Appl Microbiol Biotechnol, 2005; 67:8–18.

16.

Qiu

, Wu

VCH

. Evaluation of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus in ground beef with cranberry concentrate by thin agar layer method. J Rapid Methods Autom Microbiol, 2007; 15:282–294.

17.

Reddy

, Gupta

, Jacob

, Khan

, Ferreira

. Antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from Punica granatum L. Planta Med, 2007; 73:461–467.

18.

Scalbert

. Antimicrobial properties of tannins. Phytochemistry, 1991; 30:3875–3883.

19.

Schentag

, Hyatt

, Carr

et al. Genesis of methicillin-resistant Staphylococcus aureus (MRSA), how treatment of MRSA infections was selected for vancomycin-resistant Enterococcus faecium, and the importance of antibiotic management and infection control. Clin Infect Dis, 1998; 26:1204–1214.

20.

, D'Souza

. Grape seed extract for the control of human enteric viruses. Appl Environ Microbiol, 2011; 77:3982–3987.

21.

, D'Souza

. Reduction of Salmonella Typhimurium and Listeria monocytogenes on produce by trisodium phosphate. LWT Food Sci Tech, 2012; 45:221–225.

22.

, Sangster

, D'Souza

. In vitro effects of pomegranate juice and pomegranate polyphenols on foodborne viral surrogates. Foodborne Pathog Dis, 2010; 7:1473–1479.

23.

Tacconelli

, De Angelis

, Cataldo

, Pozzi

, Cauda

. Does antibiotic exposure increase the risk of methicillin-resistant Staphylococcus aureus (MRSA) isolation? A systematic review and meta-analysis. J Antimicrob Chemother, 2008; 61:26–38.

24.

Taguri

, Tanaka

, Kouno

. Antimicrobial activity of 10 different plant polyphenols against bacteria causing food-borne disease. Biol Pharm Bull, 2004; 27:1965–1969.

25.

Tascini

, Di Paolo

, Polillo

et al. Case report of a successful treatment of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia and MRSA/vancomycin-resistant Enterococcus faecium cholecystitis by daptomycin. Antimicrob Agents Chemother, 2011; 55:2458–2459.

26.

Wren

, Cleary

, Frantz

, Melton

, Norris

. 90-day oral toxicity study of a grape seed extract (IH636) in rats. J Agric Food Chem, 2002; 50:2180–2192.

27.

VCH

, Qiu

, Bushway

, Harper

. Antibacterial effects of American cranberry (Vaccinium macrocarpon) concentrate on foodborne pathogens. LWT Food Sci Technol, 2008; 41:1834–1841.

28.

Yamakoshi

, Saito

, Kataoka

, Kikuchi

. Safety evaluation of proanthocyanidin-rich extract from grape seeds. Food Chem Toxicol, 2002; 40:599–607.