Accepted Manuscript Pomegranate Extract Exhibits in Vitro Activity Against Clostridium Difficile Sydney M. Finegold, M.D Paula H. Summanen, M.S Karen Corbett, B.S Julia Downes, B.S Susanne M. Henning, Ph.D Zhaoping Li, M.D PII:

S0899-9007(14)00135-X

DOI:

10.1016/j.nut.2014.02.029

Reference:

NUT 9248

To appear in:

Nutrition

Received Date: 27 September 2013 Revised Date:

27 November 2013

Accepted Date: 27 February 2014

Please cite this article as: Finegold SM, Summanen PH, Corbett K, Downes J, Henning SM, Li Z, Pomegranate Extract Exhibits in Vitro Activity Against Clostridium Difficile, Nutrition (2014), doi: 10.1016/ j.nut.2014.02.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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POMEGRANATE EXTRACT EXHIBITS IN VITRO ACTIVITY AGAINST

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CLOSTRIDIUM DIFFICILE Running head: Activity of pomegranate against Clostridium difficile

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Sydney M. Finegold, M.D#1,2,3,4, Paula H. Summanen, M.S.2, Karen Corbett, B.S.2, Julia

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Downes, B.S.2, Susanne M. Henning, Ph.D.5, Zhaoping Li, M.D.2,4,5,

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Infectious Diseases Section1 and Research Service2, VA Medical Center West Los Angeles,

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11301 Wilshire Blvd., Los Angeles, CA 90073; Department of Microbiology, Immunology and

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Molecular Genetics3, Department of Medicine4, and UCLA Center for Human Nutrition5, UCLA

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School of Medicine, 405 Hilgard Ave., Los Angeles, CA 90095

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SMF, ZL, and SMH designed research; PHS, KC, and JD conducted research; SMF, JD and PHS

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analyzed data; SMF, PHS, and SMH wrote the paper.

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Word count: 1071

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Tables/figures: 1 table

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Address correspondence to:

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Sydney Finegold

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Infectious Diseases Section and Research Service

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VA Medical Center WLA

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Los Angeles, CA 90073

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[email protected]

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Phone -- 310 2683678

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Fax – 310 268 3594

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Clostridium difficile is associated with severe infections with significant morbidity and

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mortality [1]. The use of antimicrobials, in particular penicillins, cephalosporins, clindamycin

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and fluoroquinolones, is a major risk factor for C. difficile infection [2]. Resistance of C. difficile

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to the newer fluoroquinolones contributed to the emergence of C. difficile type 027/NAP-1 in

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healthcare centers [3]. Vancomycin and metronidazole currently remain the standard treatments

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of C. difficile infections [4]. The increasing virulence and degree of resistance of enteric

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organisms such as C. difficile highlight the importance of finding and developing new,

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alternative agents with selective activity in the gut.

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Pomegranate juice exceeds the in vitro antioxidant potency of other common commercial

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fruit juices [5]. The most abundant type of polyphenols in pomegranate juice are ellagitannins

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[6]. Punicalagin is unique to pomegranate and is part of a family of ellagitannins. The

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ellagitannins are hydrolyzed to ellagic acid in the gut, and further metabolized by the colon

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microbiota to form urolithin A and B. Recent studies have shown that pomegranate polyphenols

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can alter bacterial populations in mixed cultures while also being metabolized by the bacteria to

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smaller metabolites such as urolithin A [7]. Investigations using pure bacterial in vitro cultures

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have shown that pomegranate by-products and punicalagins significantly inhibited the growth of

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pathogenic Escherichia coli, Pseudomonas aeruginosa, clostridia and Staphyloccocus aureus [8].

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There is no data on the effect of pomegranate on the growth of toxigenic C. difficile. In this

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study, we used a pomegranate extract prepared from the whole fruit (husks, seeds and peels

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remaining after juice production) (POM Wonderful, Inc., Los Angeles, CA), to establish in vitro

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activity of pomegranate against C. difficile.

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The bacteria included in this study were stool isolates from patients in the Greater Los Angeles VA Healthcare Center. Altogether 29 clinical C. difficile isolates were included (Table

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1). Reference strains of Bacteroides fragilis (ATCC 25285) and C. difficile (ATCC 8689) were

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included in each test. Bacteria were identified by 16S rRNA sequence analysis. The restriction

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endonuclease analysis (REA) typing was performed on 23 isolates at the Clostridium difficile

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Microbiology Reference Laboratory VA Hines (Hines, Illinois) [9] (Table 1).

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The total phenolics content of the pomegranate extract was determined by using the Folin-Ciocalteau colorimetric method [10] and adjusted to achieve 400 µg/ml gallic acid

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equivalent [GAE] for the highest concentration. Minimum inhibitory concentrations (MICs)

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were determined by the Clinical and Laboratory Standards Institute (CLSI)-approved Wadsworth

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agar dilution technique [11]. The MIC testing was done twice. A suspension of colonies taken

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from 48 hr culture on Brucella blood agar plates was used to prepare Brucella broth tubes with a

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density equal to that of a 0.5 McFarland standard, and achieving a final inoculum of 105 colony

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forming units (CFU)/spot when applied to the pomegranate extract-containing plates using a

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Steers replicator. The MIC testing was done on Brucella agar (Anaerobe Systems, Morgan Hill,

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CA) plates supplemented with 0.001% vitamin K1 and 5% laked sheep blood containing 2-fold

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serial dilutions of pomegranate extract (6.25 µg/ml to 400 µg/ml). Plates were incubated in an

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anaerobic chamber for 48 hours at 37oC. MICs were defined as the lowest concentration

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resulting in no growth or a marked change in the appearance of growth as compared to the

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control plate, as described in the CLSI protocol.

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The C. difficile strains typed as BI group are the highly virulent strains described by the

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CDC as NAP-1 (Table 1). They resemble REA types BI 6, BI 8, and BI 17, three subtypes of

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group BI that have been implicated in multiple outbreaks in the US and Canada [3]. Eight of the

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strains belonged to a single REA group, group N, and appeared to be identical to one another.

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Type BI 27 was described as a new BI type, and was at least 90% related to the other BI types.

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Two strains belonged to types K and J which have been implicated in outbreaks from the 1980's

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to the present. All the C. difficile strains tested had MICs at 12.5-25 µg/ml GAE level range

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(Table 1). To our knowledge, our study presents the first data of antimicrobial in vitro activity for

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pomegranate extract against toxigenic C. difficile. Despite increasing interest by researchers,

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industry and consumers towards the beneficial health properties of pomegranate, the impact of

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pomegranate products on human gut microbiota has not been adequately studied. A recent study

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in mice showed that oral administration of a pomegranate peel extract rich in polyphenols has

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prebiotic effect and is able to modulate the gut microbiota in favor of bifidobacteria [12].

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Previous studies by our group have demonstrated that pomegranate extract is well-tolerated and

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without significant gastrointestinal or systemic toxicity after ingesting two 1000 mg capsules per

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day [13]. We also showed no difference in pharmacokinetics of plasma ellagic acid metabolites

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between pomegranate juice and extract [14]. Pomegranate ellagitannin metabolites such as

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urolithins have been found to be formed in an in vitro fermentation system with human fecal

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material [7]. Upon absorption in the human intestine they are further glucuronidated and

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methylated and are present in human plasma and excreted in urine for up to 48 hours [15]. Future

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studies of these metabolites may reveal whether they contribute to the antimicrobial activity of

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pomegranate extract. Future studies utilizing an animal model will be required to determine the

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anti-C. difficile activity of pomegranate extract in vivo. In summary, our results suggest the

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possible utility of pomegranate in the management and/or prevention of C. difficile disease or

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colonization.

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Acknowledgements We thank Dr. Dale Gerding at the Clostridium difficile Microbiology Reference Laboratory VA Hines for the REA testing of the isolates.

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MIC (µg/ml GAE) 25 25 12.5 12.5 25 25 25 25 12.5 12.5 25 25 25 12.5 12.5 25 12.5 12.5 25 25 25 12.5 25 25 25 12.5 12.5 12.5 25 25 >400

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REA type n/a n/a n/a n/a n/a n/a n/a K type N group J type BI group N group N group BI group BI group BI group N group BI group N group BI group BI group BI group BI group BI group N group BI group N group BI group New BI 27 N group B. fragilis

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C. difficile strain ATCC 9689 14150 14186 14959 14974 17450 18566 18667 18670 18671 18742 18743 18744 18745 18746 18748 18750 18757 18758 18759 18780 18783 18787 18789 18790 18791 18793 18794 18795 18799 ATCC 25285

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Table 1. In vitro activity of pomegranate extract against C. difficile

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References 1. Bartlett, J. G. and T. M. Perl. 2005. The new Clostridium difficile--what does it mean? N.Engl.J Med 353:2503-2505. 2. Bartlett, J. G. and D. N. Gerding. 2008. Clinical recognition and diagnosis of Clostridium

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difficile infection. Clin.Infect.Dis. 46 Suppl 1:S12-S18.

3. McDonald, L. C., G. E. Killgore, A. Thompson, R. C. Owens, Jr., S. V. Kazakova, S. P.

107

Sambol, et al. 2005. An epidemic, toxin gene-variant strain of Clostridium difficile.

108

N.Engl.J.Med. 353:2433-2441.

110 111

4. Higa, J. T. and C. P. Kelly. 2013. New drugs and strategies for management of Clostridium

M AN U

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SC

106

difficile colitis. J Intensive Care Med. 2013 Feb 7. [Epub ahead of print] 5. Seeram, N. P., M. Aviram, Y. Zhang, S. M. Henning, L. Feng, M. Dreher, and D. Heber. 2008. Comparison of antioxidant potency of commonly consumed polyphenol-rich

113

beverages in the United States. J Agric.Food Chem. 56:1415-1422.

114

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6. Gil, M. I., F. A. Tomas-Barberan, B. Hess-Pierce, D. M. Holcroft, and A. A. Kader. 2000. Antioxidant activity of pomegranate juice and its relationship with phenolic composition

116

and processing. J Agric.Food Chem. 48:4581-4589.

EP

115

7. Bialonska, D., P. Ramnani, S. G. Kasimsetty, K. R. Muntha, G. R. Gibson, and D. Ferreira.

118

2010. The influence of pomegranate by-product and punicalagins on selected groups of

119

human intestinal microbiota. Int.J Food Microbiol. 140:175-182.

120

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117

8. Bialonska, D., S. G. Kasimsetty, K. K. Schrader, and D. Ferreira. 2009. The effect of

121

pomegranate (Punica granatum L.) byproducts and ellagitannins on the growth of human

122

gut bacteria. J Agric.Food Chem. 57:8344-8349.

7

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123

9. Killgore, G., A. Thompson, S. Johnson, J. Brazier, E. Kuijper, J. Pepin, et al. 2008. Comparison of seven techniques for typing international epidemic strains of Clostridium

125

difficile: restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-

126

ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat

127

analysis, amplified fragment length polymorphism, and surface layer protein A gene

128

sequence typing. J Clin.Microbiol. 46:431-437.

131

significance. Adv.Food Res.Suppl 1:1-261.

SC

130

10. Singleton, V. L. and P. Esau. 1969. Phenolic substances in grapes and wine, and their

11. Hecht, D. W., D. M. Citron, J. Dzink-Fox, W. W. Gregory, N. V. Jacobus, S. G. Jenkins, et

M AN U

129

RI PT

124

132

al. 2012. Methods for antimicrobial susceptibility testing of anaerobic bacteria; approved

133

standard - eighth edition. CLSI M11.

12. Neyrinck, A. M., V. F. Van Hee, L. B. Bindels, B. F. De, P. D. Cani, and N. M. Delzenne.

135

2013. Polyphenol-rich extract of pomegranate peel alleviates tissue inflammation and

136

hypercholesterolaemia in high-fat diet-induced obese mice: potential implication of the gut

137

microbiota. Br.J Nutr 109:802-809.

13. Heber, D., N. P. Seeram, H. Wyatt, S. M. Henning, Y. Zhang, L. G. Ogden, et al. 2007.

EP

138

TE D

134

Safety and antioxidant activity of a pomegranate ellagitannin-enriched polyphenol dietary

140

supplement in overweight individuals with increased waist size. J.Agric.Food Chem.

141

55:10050-10054.

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139

142

14. Seeram, N. P., Y. Zhang, R. McKeever, S. M. Henning, R. P. Lee, M. A. Suchard, et al.

143

2008. Pomegranate juice and extracts provide similar levels of plasma and urinary

144

ellagitannin metabolites in human subjects. J.Med.Food 11:390-394.

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15.

Seeram, N. P., S. M. Henning, Y. Zhang, M. Suchard, Z. Li, and D. Heber. 2006. Pomegranate juice ellagitannin metabolites are present in human plasma and some persist

147

in urine for up to 48 hours. J Nutr 136:2481-2485.

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Pomegranate extract exhibits in vitro activity against Clostridium difficile.

To determine the possible utility of pomegranate extract in the management or prevention of Clostridium difficile infections or colonization...
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