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.
ACCEPTED MANUSCRIPT
1
POMEGRANATE EXTRACT EXHIBITS IN VITRO ACTIVITY AGAINST
2
CLOSTRIDIUM DIFFICILE Running head: Activity of pomegranate against Clostridium difficile
4
Sydney M. Finegold, M.D#1,2,3,4, Paula H. Summanen, M.S.2, Karen Corbett, B.S.2, Julia
5
Downes, B.S.2, Susanne M. Henning, Ph.D.5, Zhaoping Li, M.D.2,4,5,
6
Infectious Diseases Section1 and Research Service2, VA Medical Center West Los Angeles,
7
11301 Wilshire Blvd., Los Angeles, CA 90073; Department of Microbiology, Immunology and
8
Molecular Genetics3, Department of Medicine4, and UCLA Center for Human Nutrition5, UCLA
9
School of Medicine, 405 Hilgard Ave., Los Angeles, CA 90095
M AN U
SC
RI PT
3
10
SMF, ZL, and SMH designed research; PHS, KC, and JD conducted research; SMF, JD and PHS
12
analyzed data; SMF, PHS, and SMH wrote the paper.
13
Word count: 1071
14
Tables/figures: 1 table
15
TE D
11
Address correspondence to:
17
Sydney Finegold
18
Infectious Diseases Section and Research Service
19
VA Medical Center WLA
20
Los Angeles, CA 90073
21
[email protected] 22
Phone -- 310 2683678
23
Fax – 310 268 3594
AC C
EP
16
24 1
ACCEPTED MANUSCRIPT
Clostridium difficile is associated with severe infections with significant morbidity and
26
mortality [1]. The use of antimicrobials, in particular penicillins, cephalosporins, clindamycin
27
and fluoroquinolones, is a major risk factor for C. difficile infection [2]. Resistance of C. difficile
28
to the newer fluoroquinolones contributed to the emergence of C. difficile type 027/NAP-1 in
29
healthcare centers [3]. Vancomycin and metronidazole currently remain the standard treatments
30
of C. difficile infections [4]. The increasing virulence and degree of resistance of enteric
31
organisms such as C. difficile highlight the importance of finding and developing new,
32
alternative agents with selective activity in the gut.
SC
Pomegranate juice exceeds the in vitro antioxidant potency of other common commercial
M AN U
33
RI PT
25
fruit juices [5]. The most abundant type of polyphenols in pomegranate juice are ellagitannins
35
[6]. Punicalagin is unique to pomegranate and is part of a family of ellagitannins. The
36
ellagitannins are hydrolyzed to ellagic acid in the gut, and further metabolized by the colon
37
microbiota to form urolithin A and B. Recent studies have shown that pomegranate polyphenols
38
can alter bacterial populations in mixed cultures while also being metabolized by the bacteria to
39
smaller metabolites such as urolithin A [7]. Investigations using pure bacterial in vitro cultures
40
have shown that pomegranate by-products and punicalagins significantly inhibited the growth of
41
pathogenic Escherichia coli, Pseudomonas aeruginosa, clostridia and Staphyloccocus aureus [8].
42
There is no data on the effect of pomegranate on the growth of toxigenic C. difficile. In this
43
study, we used a pomegranate extract prepared from the whole fruit (husks, seeds and peels
44
remaining after juice production) (POM Wonderful, Inc., Los Angeles, CA), to establish in vitro
45
activity of pomegranate against C. difficile.
46 47
AC C
EP
TE D
34
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
2
ACCEPTED MANUSCRIPT
1). Reference strains of Bacteroides fragilis (ATCC 25285) and C. difficile (ATCC 8689) were
49
included in each test. Bacteria were identified by 16S rRNA sequence analysis. The restriction
50
endonuclease analysis (REA) typing was performed on 23 isolates at the Clostridium difficile
51
Microbiology Reference Laboratory VA Hines (Hines, Illinois) [9] (Table 1).
52
RI PT
48
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
54
equivalent [GAE] for the highest concentration. Minimum inhibitory concentrations (MICs)
55
were determined by the Clinical and Laboratory Standards Institute (CLSI)-approved Wadsworth
56
agar dilution technique [11]. The MIC testing was done twice. A suspension of colonies taken
57
from 48 hr culture on Brucella blood agar plates was used to prepare Brucella broth tubes with a
58
density equal to that of a 0.5 McFarland standard, and achieving a final inoculum of 105 colony
59
forming units (CFU)/spot when applied to the pomegranate extract-containing plates using a
60
Steers replicator. The MIC testing was done on Brucella agar (Anaerobe Systems, Morgan Hill,
61
CA) plates supplemented with 0.001% vitamin K1 and 5% laked sheep blood containing 2-fold
62
serial dilutions of pomegranate extract (6.25 µg/ml to 400 µg/ml). Plates were incubated in an
63
anaerobic chamber for 48 hours at 37oC. MICs were defined as the lowest concentration
64
resulting in no growth or a marked change in the appearance of growth as compared to the
65
control plate, as described in the CLSI protocol.
M AN U
TE D
EP
AC C
66
SC
53
The C. difficile strains typed as BI group are the highly virulent strains described by the
67
CDC as NAP-1 (Table 1). They resemble REA types BI 6, BI 8, and BI 17, three subtypes of
68
group BI that have been implicated in multiple outbreaks in the US and Canada [3]. Eight of the
69
strains belonged to a single REA group, group N, and appeared to be identical to one another.
70
Type BI 27 was described as a new BI type, and was at least 90% related to the other BI types.
3
ACCEPTED MANUSCRIPT
71
Two strains belonged to types K and J which have been implicated in outbreaks from the 1980's
72
to the present. All the C. difficile strains tested had MICs at 12.5-25 µg/ml GAE level range
73
(Table 1). To our knowledge, our study presents the first data of antimicrobial in vitro activity for
RI PT
74
pomegranate extract against toxigenic C. difficile. Despite increasing interest by researchers,
76
industry and consumers towards the beneficial health properties of pomegranate, the impact of
77
pomegranate products on human gut microbiota has not been adequately studied. A recent study
78
in mice showed that oral administration of a pomegranate peel extract rich in polyphenols has
79
prebiotic effect and is able to modulate the gut microbiota in favor of bifidobacteria [12].
80
Previous studies by our group have demonstrated that pomegranate extract is well-tolerated and
81
without significant gastrointestinal or systemic toxicity after ingesting two 1000 mg capsules per
82
day [13]. We also showed no difference in pharmacokinetics of plasma ellagic acid metabolites
83
between pomegranate juice and extract [14]. Pomegranate ellagitannin metabolites such as
84
urolithins have been found to be formed in an in vitro fermentation system with human fecal
85
material [7]. Upon absorption in the human intestine they are further glucuronidated and
86
methylated and are present in human plasma and excreted in urine for up to 48 hours [15]. Future
87
studies of these metabolites may reveal whether they contribute to the antimicrobial activity of
88
pomegranate extract. Future studies utilizing an animal model will be required to determine the
89
anti-C. difficile activity of pomegranate extract in vivo. In summary, our results suggest the
90
possible utility of pomegranate in the management and/or prevention of C. difficile disease or
91
colonization.
AC C
EP
TE D
M AN U
SC
75
92 93
4
ACCEPTED MANUSCRIPT
94 95 96
Acknowledgements We thank Dr. Dale Gerding at the Clostridium difficile Microbiology Reference Laboratory VA Hines for the REA testing of the isolates.
AC C
EP
TE D
M AN U
SC
RI PT
97
5
ACCEPTED MANUSCRIPT
99 100
SC
M AN U
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
TE D
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
AC C
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
RI PT
Table 1. In vitro activity of pomegranate extract against C. difficile
EP
98
6
ACCEPTED MANUSCRIPT
102 103 104 105
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
RI PT
101
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
109
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
TE D
112
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
AC C
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
ACCEPTED MANUSCRIPT
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.
AC C
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.
8
ACCEPTED MANUSCRIPT
145
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.
AC C
EP
TE D
M AN U
SC
RI PT
146
9