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Anti-virulence effects of aqueous pomegranate peel extract
on E. coli urinary tract infection
Wissam Zam1, Aziz Khaddour2
1 Department of Analytical and Food Chemistry, Faculty of Pharmacy, Al-Andalus University for Medical Sciences, Tartous,
Syrian Arab Republic - E-mail: w.zam@au.edu.sy; 2 Department of Microbiology, Faculty of Pharmacy, Al-Andalus University
for Medical Sciences, Tartous, Syrian Arab Republic
Summary. Urinary tract infections (UTIs) are among the most prevailing infectious diseases and may be clas-
sified as uncomplicated or complicated, depending upon the urinary tract anatomy and physiology. e Gram
negative bacteria of E. coli cause 70-95% of upper and lower UTIs. Pomegranate peels (Punica granatum L.)
are considered byproducts obtained during juice processing and characterized by the significant presence of
polyphenols associated with biological properties such as antimicrobial and antioxidant agents. e aim of
this study was to estimate the antimicrobial and anti-virulence effect of aqueous pomegranate peel extract
against E. coli cultures collected from urinary cultures of the Microbiology Laboratory of Al-Bassel Hospital,
in Syria, 2016. e inhibitory activity was found to be dose and pH dependent with an MIC value of 0.6 mg/
ml and an MBC value of 1.2 mg/ml at the pH of the aqueous extract (3.5). e assay of adhesion carried out
at MIC showed a reduction of up to 80% of the adhesion index accompanied with a reduction in motility
and ornithine decarboxylation as indicated by MIO test. Results indicate that the aqueous pomegranate peel
extract could be an important source of new antimicrobial compounds in order to treat E. coli urinary tract
infections.
Key words: aqueous pomegranate peel extract, antimicrobial effect, anti-virulence effect, E. coli, UTI
Progress in Nutrition 2017; Vol. 19, Supplement 1: 98-104 DOI: 10.23751/pn.v19i1-S.5693 © Mattioli 1885
Original article
Introduction
Urinary Tract Infection (UTI) is defined as the
microbial invasion of any tissues in different parts of
the urinary tract and is the second most common in-
fectious presentation in community medical practice
(1). Individual susceptibility to UTI is complex, de-
pending on several factors such as genetic, biologic,
and behavioral ones. e pathogenic bacteria can ad-
here, grow and resist against host defenses which will
result in colonization and infection of the urinary tract.
Each bacterial species has distinct virulence mecha-
nisms that facilitate UTI (2, 3).
It has been reported in several studies that the
Gram negative bacteria of E. coli cause 70-95% of up-
per and lower UTIs (3). e severity of the infection
depends both on the virulence factors of the infecting
bacteria and on the vulnerability of the host. Up to
95% of UTIs occur in an ascending beginning with
bacterial colonization of the periurethral area followed
by infection of the bladder and may then ascend the
ureters to reach the kidneys (4). If left untreated, the
infection could access the bloodstream and causes bac-
teremia (4).
e uropathogenic E. coli possess adherence
factors called pili or fimbriae, which allow them to
successfully initiate infections and may protect the
bacteria from urinary lavage, increasing their abili-
ty to multiply and invade renal tissue (5). Flagella,
an organelle responsible for bacterial motility, are
Anti-virulence effects of aqueous pomegranate peel extract on E. coli urinary tract infection 99
involved in the interaction of various pathogenic E.
coli strains with epithelial cells (6). e role of fla-
gellummediated motility in the rise of uropathogenic
E. coli to the upper urinary tract and in its diffusion
into the bloodstream as well as in the maintenance
of persistent infection has been well established (7).
A fluoroquinolone for 7-10 days can be recom-
mended as first-line therapy and third-generation
oral cephalosporin could be an alternative (8, 9). Ho-
wever, it has been found that the numbers of fluo-
roquinolone-resistant E. coli have increased in some
parts of the world, thus restricting their use of fluo-
roquinolones (8, 9).
Medicinal plants have always been a good source
to find new remedies for human health problems. Re-
cently, a wide range of these plants have been scree-
ned for antimicrobial property (10).
Pomegranate peels (Punica granatum) are con-
sidered wastes or byproduct obtained through jui-
ce processing (11). It is characterized by significant
presence of ellagitannins and polyphenols, gallic acid
and ellagic acid (12) as well as flavonoids-associated
with biological properties such antimicrobial agents
(13).
Various extracts prepared from pomegranate fru-
it peels were evaluated for their antimicrobial activity
against some food-borne pathogens using several me-
thods (14-17). It was found that 80% methanolic ex-
tract of peels was a potent inhibitor for Listeria mono-
cytogenes, Yersinia enterocolitica, Klebsiella pneumonia,
Proteus vulgaris, Bacillus subtilis, Staphyllococcus aureus
and Escherichia coli (14-17). Alam Khan and Hanee
had shown that Ethanolic extract of pomegranate pe-
els has lowest MIC against E. coli, P. aeruginosa and
S. aureus compared to MICs of methanolic and hot
water extracts (18). e inhibitory zones of all the
three extracts were greater than that of the standard
antibiotic Tetracycline (18). In contrast Nuamsetti et
al. found that the hot water extract of the peels was
most potent against E. coli compared to 95% ethanol
and acetone extracts (19).
e objective of this study was to explore the ef-
ficacy of using aqueous pomegranate peel extract to
reduce pathogenicity of E. coli responsible for UTI
and attempt to find a safety method to solve the
problem of multi-drug resistance pathogen.
Materials and Methods
Pomegranate peel extract
Fresh pomegranates were collected from Syrian
markets. ey were cleaned with water and dried with
a cloth. e peels were manually separated, dried for a
few days in an open air shade and then powdered in a
blender. e moisture content was determined by us-
ing a moisture analyzer balance.
1 g of dried and ground peel was placed in a ther-
mostatic water bath shaker with 100 ml of DI water at
50°C for 20 min. e liquid extract was centrifuged at
2000 rpm for 10 min and the supernatant was trans-
ferred to a 100 ml flask. DI water was added to make
the final volume 100 ml (20).
Microbial cultures
Cultures of E. coli were provided from urinary
culture collections of the Microbiology Laboratory
of Al-Bassel Hospital, in Syria, 2016. en, bacteria
were incubated at 37±0.1°C for 24 h by injection into
Nutrient Broth. A standardized suspension of E. coli
was prepared by suspending colonies from overnight
culture in peptone to obtain 1.5x108 CFU/ml.
Determination of MIC and MBC
e MIC of aqueous pomegranate peel extract was
evaluated using the microdilution broth method ac-
cording to National Committee for Clinical Laboratory
Standards, 2003. Geometric dilutions ranging from 0.2,
0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mg/mL of the pomegranate
extracts were prepared. e standardized suspension of
E. coli was tested in tubes against the varying concentra-
tions of aqueous pomegranate peel extract. e tubes
were incubated for 24h at 37°C and the growth of the
pathogen was detected using spectrophotometer at 600
nm. Concentration in the tube showing no turbidity
was considered as MIC. Aliquots of 100 µl from each
transparent tube showing no turbidity were separately
cultured on Eosin Methylene Blue agar (EMB) plates.
After 24 h of incubation at 37°C, the concentration of
antibacterial agent in the tube that showed no bacterial
growth was recorded as MBC (21).
W. Zam, A. Khaddour
100
Adhesion assay
Collection of uroepithelial cells
e in vitro adherence of E. coli to uroepithelial
cells was studied according to the method of Suzanne
et al. (22). Uroepithelial cells were obtained from fresh
urine collected over a 24-h period from normal healthy
women with no history of urinary or vaginal infections
and who are not taking contraceptive or antimicrobial
agents. e urine was immediately centrifuged at 4000
rpm for 15 minutes, the supernatant was discarded and
the uroepithelial cells were harvested by washing the
sediment three times with 5ml of phosphate buffer
saline (pH 5). e number of cells was calculated by
direct light microscopy and an epithelial cell count of
2x105 cells/ml was obtained by re-suspending a suit-
able number of the epithelial cell in phosphate buffer
saline pH 5.
In vitro assay
One ml of bacterial suspension was mixed with
one ml of epithelial cell suspension. e mixture was
incubated in shaking water bath at 37°C for 3 hours.
en it was washed three times and a portion of the
final cell suspension was placed on a slide, air dried,
methanol fixed and stained with Giemsa stain (10%)
for 30 minutes and examined under light microsco-
py (X100). e average number of adhering bacteria
per cell was obtained from an examination of 50 cells.
Each test was performed in triplicate.
E. coli was grown for 36 h at 37°C in bacterio-
logical peptone with the addition of pomegranate
peel aqueous extract, at the minimum inhibitory con-
centration. en the incubated bacterial suspension
was placed in contact with the cells and incubated
at 37°C for 3 h. Finally, they were washed with PBS
to remove any bacteria that had not adhered, the
cells were then air dried, methanol fixed and stained
with Giemsa stain (10%) for 30 minutes, then they
were observed at the microscope (X100). e average
number of adhering bacteria per cell was obtained
from an examination of 50 cells. Each test was per-
formed in triplicate.
Motility Assay
A sterile needle was used to pick a well-isolated
colony of E. coil before and after treatment with aque-
ous pomegranate peel extract at MIC and stabbed into
the MIO medium to within 1 cm of the bottom of the
tube. Tubes were incubated at 35°C for 18 hours.
pH effect
E. coli strains grow in a broad pH range of 4.4–
10.0, with an optimum pH of 6–7 (23). In order to
clarify the acidic properties influence of aqueous pome-
granate peel extract (pH value 3.5) on E. coli growth,
strains were grown in three different pH ranges. e
MIC and MBC of strains was evaluated using the mi-
crodilution broth method according to National Com-
mittee for Clinical Laboratory Standards, 2003 (24).
e pH of aqueous pomegranate peel extract was
adjusted to 7 using sodium carbonate and a citric acid
buffer was prepared at pH 3.5. Geometric dilutions
ranging from 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 mg/mL of
the aqueous pomegranate peel extract (pH=3.5) and
the modified extract (pH=7) were prepared. e stan-
dardized suspension of E. coli was tested in tubes
against the above varying concentrations and varying
dilutions of citric acid buffer. e tubes were incubated
for 24h at 37°C and the growth of the pathogen was
detected using spectrophotometer at 600 nm. Concen-
tration in the tube showing no turbidity was consid-
ered as MIC. Aliquots of 100 µl from each transparent
tube showing no turbidity were separately cultured on
Eosin Methylene Blue agar (EMB) plates. After 24 h
of incubation at 37°C, the concentration of antibacte-
rial agent in the tube that showed no bacterial growth
was recorded as MBC (21).
Results and Discussion
Determination of MIC and MBC
In general, the extent of the inhibitory effects of
the pomegranate extracts could be attributed to their
polyphenol content. ese compounds are very abun-
dant in aqueous pomegranate peel extract as reported
by our previous work (20) and their effects on bacterial
metabolism are identified by the effect of tannins, such
as punicalagin, on bacterial membrane, because they
can pass through cell walls and bind to their surface
which prevents their normal activity (25). Punicalagin
and gallic acid also showed antibacterial efficacy against
Anti-virulence effects of aqueous pomegranate peel extract on E. coli urinary tract infection 101
methicillin resistant Staphylococcus aureus strains, Cory-
nebacterium, Streptococcus, Bacillus subtilis, Shigella, Sal-
monella, Escherichia and Vibrio species (26, 27).
As shown in Figure 1, results indicated a signifi-
cant effect of pomegranate peel extract on decreasing
the bacterial growth at concentration starting from 0.2
mg/ml. MIC value for aqueous pomegranate extract
was 0.6 mg/ml, whereas MBC value was 1.2 mg/ml.
In different recent studies, MIC varied from
0.19–25 mg/ml against several strains of E. coli (16,
28). e variation in MIC can be related to the differ-
ences in the amount of antibacterial substances (such
as tannins and phenolic substances) among pomegran-
ate cultivars and genotypes. Minor differences in labo-
ratory techniques and the strain of E. coli species used
in the experiments run by researchers may also be in-
volved in the variation of the reported results.
Adhesion assay
e adhesion assay carried out on the E. coli treat-
ed with aqueous pomegranate peel extract at MIC
showed a reduction of up to 80% of the adhesion in-
dex. e count of adhering bacteria was carried out
manually, both for controls and treated strains (Fig-
ure 2). About 112.4±5.7 of E. coli bacteria adhered on
the assayed epithelial cell before treatment, while only
23.6±3.7 of E. coli adhered to the epithelial cells after
treatment with aqueous pomegranate peel extract.
ese results were in accordance with different
previous studies (29, 30) where aqueous pomegran-
ate peel extract worked as an anti-adhesive because of
large amounts of saponins, alkaloids, and polyphenols
(31-33).
Motility assay
A positive motility test is indicated by a diffuse
cloud of growth away from the line of inoculation,
whereas ornithine decarboxylation is indicated by a
purple color in the medium. A negative ornithine reac-
tion produces a yellow color at the bottom of the tube.
Results indicate a reduction in E. coli motility in
tubes treated with aqueous pomegranate peel extract
accompanied with a reduction in ornithine decarbox-
ylation as in Figure 3.
Activity of ornithine decarboxylase results in pro-
duction of polyamines such as putrescine and spermi-
dine which play an important role in biofilm formation
and so in cellular adherence of E. coli (34).
Recently, it was shown that antibiotics such as
fluoroquinolones, aminoglycosides and cephalosporins
are able to induce oxidative stress, a substantial con-
tributor to cell death by the damage of protein and
DNA (35). Polyamines help E. coli to survive with
stress conditions, such as oxidative radicals (36) and
low pH (37) which eventually results in a considerable
increase in cell viability, growth recovery and antibiotic
resistance.
Figure 1. E. coli growth on EMB before (a) and after (b1, b2 and
b3) treatment with aqueous pomegranate peel extract.
Figure 2. E. coli adherent to a cell on a control slide. b: E. coli
adherent to cells after treatment with aqueous pomegranate peel
extract at the concentration of 1.0 mg/ml. e lower number of
adherent bacteria can be clearly seen.
W. Zam, A. Khaddour
102
Based on these results, an attractive idea is that
of potentiation of antibiotic effects in the course of
treatment of E. coli urinary tract infectious diseases by
lowering polyamine synthesis in the patient by the use
of aqueous pomegranate peel extract.
pH effect
Results showed that the use of citric acid buffer
at different concentrations could inhibit the E. coli
growth with no bactericidal properties. e current
results indicate that the acidic property would not be
a key factor for influencing the survival of E. coli and
the aqueous pomegranate peel extract is emerging its
antimicrobial properties due to its considerable poly-
phenol content as reported in several recent articles
(38-40).
e aqueous pomegranate peel extract adjusted
to pH=7 showed no bacteriostatic effect on E. coli.
is could be explained by the effect of polyphenol
oxidase (PPO). PPO is the main enzyme involved in
the oxidation of phenolic compounds and its activity is
pH dependent (41). is reaction is called enzymatic
browning and occurs readily when the pH is between
5 and 7, while the activity of PPO is irreversibly inhib-
ited at pH less than 3.5.
Conclusion
e aqueous pomegranate peel extract exhibited
bacteriostatic, bactericidal and anti-virulence activities
against urinary tract infectious E. coli. e use of the
extract caused a reduction in the adhesion index ac-
companied with a reduction in motility and ornithine
decarboxylation of the E. coli strains. e presence of
phytochemicals including phenols, tannins and flavo-
noids may be responsible for these activities.
Further studies are required to identify and isolate
the active compounds present in the pomegranate’s
peel which exhibits the antimicrobial effect and also to
confirm these effects in vivo. e synergy between the
extract active compounds and drug should be atten-
tively studied which will probably solve the problem
of multiple drug resistance, toxicity and overdose since
when they combine a little concentration of two agents
is required.
Acknowledgment
e authors thank the scientific and training department
in Al-Bassel Hospital, Tartous, for providing E. coli cultures.
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Correspondence:
Wissam Zam
Department of Analytical and Food Chemistry,
Faculty of Pharmacy, Al-Andalus University for
Medical Sciences, Tartous, Syrian Arab Republic
E-mail: w.zam@au.edu.sy
