Antibiolm Effect of Trans-Cinnamaldehyde on Uropathogenic

Escherichia coli
Mary Anne Roshni Amalaradjou, Amoolya Narayanan, Sangeetha Ananda Baskaran
and Kumar Venkitanarayanan*
From the Department of Animal Science, University of Connecticut, Storrs and Glastonbury High School (AN), Glastonbury, Connecticut
Abbreviations
and Acronyms
PBS phosphate buffered saline
TC trans-cinnamaldehyde
TSA tryptic soy agar
TSB tryptic soy broth
UPEC uropathogenic
Escherichia coli
UTI urinary tract infection
Submitted for publication October 13, 2009.
* Correspondence: Department of Animal Sci-
ence, Unit-4040, 3636 Horsebarn Hill Rd. Exten-
sion, Storrs, Connecticut 06269 (telephone: 860-
486-0947; FAX: 860-486-4375; e-mail: Kumar.
venkitanarayanan@uconn.edu).
Purpose: Urinary tract infections are the most common hospital acquired infec-
tions in humans, caused primarily by uropathogenic Escherichia coli. Indwelling
urinary catheters for bladder drainage in humans become encrusted with uro-
pathogenic E. coli biolms that are resistant to common antibiotics, resulting in
chronic infections. We studied the efcacy of the cinnamon ingredient trans-
cinnamaldehyde (Sigma) for preventing uropathogenic E. coli biolm. We also
determined the efcacy of trans-cinnamaldehyde as an ingredient in catheter
lock solution to inactivate preformed uropathogenic E. coli biolm.
Materials and Methods: Polystyrene plates and urinary catheters inoculated
with uropathogenic E. coli (5 to 6.0 log cfu) were treated with trans-cinnamalde-
hyde (0%, 0.1%, 0.25% or 0.5%) at 37C. Catheters with uropathogenic E. coli
biolm were also treated with lock solution containing trans-cinnamaldehyde
(0%, 1%, 1.25% or 1.5%). Uropathogenic E. coli biolm on control and trans-
cinnamaldehyde treated plates and catheters was determined on incubation days
0, 1, 3 and 5. Trans-cinnamaldehyde potential cytotoxity, if any, was determined
in HTB-4 bladder epithelial cells (ATCC).
Results: At all concentrations trans-cinnamaldehyde effectively prevented uro-
pathogenic E. coli biolm on plates and catheters. As a constituent in catheter
lock solution, it inactivated uropathogenic E. coli biolm on catheters. Trans-
cinnamaldehyde produced no cytotoxic effects on human bladder epithelial cells
at the tested concentrations.
Conclusions: Results suggest that trans-cinnamaldehyde may be applied as a
catheter surface coating or as an ingredient in catheter lock solution to prevent
urinary tract infection in humans.
Key Words: urinary tract infections, uropathogenic Escherichia coli, urinary
catheterization, biolms, cinnamic aldehyde
CATHETER associated UTI is the most
common hospital and nursing home
acquired human infection, accounting
for an estimated 150 million cases per
year worldwide.
13
UPEC is the pri-
mary bacterium causing UTI in hu-
mans, accounting for 70% to 90% of
these infections.
4
Infected catheters
are colonized by UPEC biolms, in
which bacteria embedded in an exo-
polysaccharide matrix are protected
from antimicrobial agents.
5
Catheter
lock solutions containing antimicro-
bial agents and anticoagulants are
commonly used to maintain catheter
luminal patency. However, antibiotics
are not effective for preventing UTIs
and repeat antibiotic use may lead to
the emergence of antibiotic resistant
pathogens.
6,7
Thus, there is a need for
effective strategies to prevent UPEC
biolm on catheters.
358 www.jurology.com
0022-5347/10/1841-0358/0 Vol. 184, 358-363, July 2010
THE JOURNAL OF UROLOGY

Printed in U.S.A.
2010 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC. DOI:10.1016/j.juro.2010.03.006
Various plant derived polyphenols serve as di-
etary constituents and active components in a num-
ber of herbal and traditional medicines.
8
More than
5,000 plant polyphenols have been identied, of
which several show a wide spectrum of biological
effects, including anti-inammatory, antimicrobial
and anticarcinogenic properties.
9
Since plant anti-
microbials contain different functional groups, their
antimicrobial activity is attributable to multiple
mechanisms.
10
Thus, unlike antibiotics, the poten-
tial for bacteria to develop resistance to plant anti-
microbials is relatively smaller.
11
TC, the principal component of cinnamon oil,
10
is a food grade molecule classied as generally
recognized as safe by the Food and Drug Admin-
istration. Although TC antibacterial activity was
reported,
12,13
to our knowledge its efcacy for kill-
ing UPEC biolm has not been determined. We
investigated TC efcacy for preventing UPEC bio-
lm and killing preformed UPEC biolm for poten-
tial application as an antimicrobial coating on uri-
nary catheters. We also determined the antibiolm
effect of TC on UPEC as an ingredient in catheter
lock solution.
MATERIALS AND METHODS
Bacterial Culture
We studied the TC antibiolm effect in 2 cystitis UPEC
isolates, namely strains ATCC 700415 and UTI 89. TC
antibiolm efcacy was also determined in Proteus mira-
bilis (ATCC 29906) on urinary catheters. Isolates were
separately cultured in 10 ml TSB (Difco) at 37C for 24
hours. The culture was sedimented by centrifugation at
3,600 gravity for 12 minutes at 4C, washed, resus-
pended in PBS (pH 7.2) and used as inoculum. Inoculum
bacterial count was determined on TSA plates (Difco) with
incubation at 37C for 24 hours.
TC Prevention of UPEC Biolm
On polystyrene plates. Sterile 96-well polystyrene Costar
tissue culture plates were inoculated with 200 l UPEC(about
5.0 log cfu), followed by adding 0%, 0.1%, 0.25% or 0.5% TC,
respectively. The plates were incubated at 37C and biolm
bacterial populations were counted.
14
On incubation days 0, 1,
3 and 5 the wells were washed 3 times with 200 l sterile
PBS to remove unattached bacteria. Biolm UPEC counts
were determined by scraping the microtiter plates with a
sterile steel spatula, serial dilution in PBS and plating on
TSA. Three replicate wells were included and the assay
was done 3 times.
On urinary catheters. TC efcacy for preventing UPEC
and P. mirabilis biolm on urinary catheter was deter-
mined according to the method of Trautner et al.
15
Latex
12Fr Foley urinary tract catheters (At Home Medical)
were cut into 3 cm pieces. Each piece was sealed at 1 end,
lled with 1 ml bacterial culture (about 5.0 log cfu) and
sealed at the other end. The piece was incubated at 37C
for 30 minutes to facilitate bacterial attachment to the
catheter luminal surface. At the end of incubation the
catheter piece was washed with sterile PBS to remove
unattached cells and transferred to sterile tubes contain-
ing 10 ml TSB with 0% (control), 0.1%, 0.25% or 0.5% TC.
The pieces were incubated statically at 37C for 5 days to
facilitate biolm formation. On incubation days 0, 1, 3 and
5 biolm was assessed by counting bacteria after dislodg-
ing the biolm from the catheter surface.
15
Three catheter
pieces were included per treatment and the experiment
was repeated 3 times.
UPEC Biolm Inactivation on Polystyrene
We determined the TC antibiolm effect on UPEC by
microtiter plate assay.
16
Briey, UPEC strains were sep-
arately grown overnight in TSB, sedimented, washed as
described and diluted 1:40 in TSB. Sterile 96-well polysty-
rene Costar tissue culture plates were inoculated with 200
l of bacterial suspension (about 6.0 log cfu) and incu-
bated at 37C for 24 hours. After biolm formation 0%, 1%,
1.25% or 1.5% TC was tested with an exposure time of 0,
1, 3 and 5 days. After TC exposure the wells were washed
3 times with sterile PBS, dried at room temperature and
stained with 1% crystal violet for 15 minutes. After rins-
ing 3 times with sterile distilled water and subsequent
destaining with 95% ethanol adherent biolm absorbance
was measured at 570 nm in Model 550 a microplate reader
(Bio-Rad). Uninoculated wells containing TSB served as
blanks. Five replicate wells were included per treatment
and the assay was repeated 3 times.
TC Antibiolm Effect
As catheter lock solution constituent. We determined
TC efcacy for inactivating preformed UPEC and P. mira-
bilis biolm on urinary catheters when used as a constit-
uent in catheter lock solution.
17
Three cm pieces of latex
12Fr Foley urinary tract catheters were inoculated with
bacteria, as described. The pieces were incubated at 37C
for 5 days to facilitate biolm formation on the catheter
luminal surface. After 5 days the pieces were washed with
sterile saline to remove unattached cells, sealed at 1 end,
lled with 1 ml sterile normal saline (control), or saline
containing 1%, 1.25% or 1. 5% TC, sealed at the other end
and incubated at 37C. On incubation days 0, 1, 3 and 5
biolm was assessed by counting bacteria after dislodging
the biolm from the catheter surface, as described. Three
catheter pieces were included per treatment and the ex-
periment was repeated 3 times.
In presence of urine. Inoculated urinary catheter pieces
were incubated at 37C for 30 minutes as described to
facilitate UPEC attachment on the catheter luminal sur-
face. At the end of incubation the pieces were washed with
sterile PBS to remove unattached cells and transferred to
sterile tubes containing 10 ml lter sterilized synthetic
human urine (Wards Natural Science, Rochester, New
York), supplemented with 0% (control), 0.1%, 0.25% or
0.50% TC. The pieces were incubated statically at 37C for
5 days to facilitate biolm formation. On incubation days
0, 1, 3 and 5 biolmformation was determined by counting
bacteria after dislodging the biolm from the catheter
surface, as described.
TRANS-CINNAMALDEHYDE ANTIBIOFILM EFFECT ON ESCHERICHIA COLI 359
Cell Culture
Monolayers of HTB-4 cells derived from T24 human blad-
der carcinoma cells were grown at 37C in McCoys 5A
medium (ATCC) supplemented with 1.5 mM L-glutamine
and 10% heat inactivated fetal calf serum (Sigma) in 5%
CO
2
and 95% air. Cell viability before cytotoxicity assay
was conrmed by excluding the vital dye trypan blue.
18
Cytotoxicity Assay
The potential cytotoxic effect of TC on HTB-4 cells was
determined after incubating exponentially growing cells
with and without TC (1%, 1.25% or 1.5%) by MTT assay.
19
Control and TC treated cell morphology was also in-
spected for microscopically detectable alterations, such as
monolayer loss, rounding, shrinking of cells, granulations
and vacuolation in the cytoplasm.
Confocal Microscopy
To obtain depth selective information on the 3-dimen-
sional structure of the UPEC biolm we performed in situ
confocal laser scanning microscopy.
20
UPEC biolms were
grown at 37C in TSB on a Lab-Tek 8-chambered No. 1
borosilicate cover glass system. The coverslips were
treated with TC (1.5%). Living and dead cells were imaged
after staining with 2.5 M SYTO and 5 M propidium
iodide (Molecular Probes). Control biolms unexposed to
TC were also imaged to view the normal UPEC biolm
architecture. Three coverslips were imaged under each
condition tested. Samples were examined under a True
Confocal Scanner SP2 microscope (Leica) using the wa-
ter immersion lens. A krypton-argon mixed gas laser with
a PMT2 lter served as the excitation source. For each
sample 4 randomly selected elds of view were analyzed.
For each eld a series of images were taken in the z-
direction beginning at the outer biolm surface and con-
tinuing at 0.2 m intervals.
Statistical Analysis
A completely randomized factorial design was used for the
study. The model included treatment concentration and
time as the major effects. For each treatment and control
data on the independent replicate trials were pooled and
analyzed using the SAS proc mixed subroutine. The
least signicant difference test was used to determine
signicant differences at p 0.05 due to treatment con-
centration and time on bacterial counts.
RESULTS
Since TC was equally effective against the 2 UPEC
isolates, only ATCC 700415 isolate results are
shown. TC was highly effective for preventing UPEC
biolm formation on polystyrene plates (g. 1, A). At
incubation day 5 control plates with 0% TC showed
a fully formed biolm containing about 7.0 log cfu
UPEC while 0.25% and 0.50% TC completely inhib-
ited biolm formation (p 0.05). TC at 0.5% pre-
vented biolm after 3 days and 0.25% TC prevented it
after 5. As observed on polystyrene, TC also prevented
biolm on urinary catheters (g. 1, B). At 24 hours
about 7.0 log cfu/ml UPECwere recovered fromcontrol
biolms but UPEC was inactivated by greater than 4
log on 0.5%TCtreated catheters. At 5 days UPECwas
not detected on 0.25%TCtreated catheters but control
biolms had 7.0 log cfu/ml UPEC even after 5 days. In
the presence of urine TC was equally effective for
inactivating UPEC and preventing biolm formation
(data not shown).
As a lock solution constituent, all TC concentra-
tions inactivated UPEC biolm on polystyrene
plates (g. 2, A). At 24 hours a fully formed biolm
was recovered from control plates but none was de-
tected on plates treated with 1.25% and 1.5% TC. At
3 days 1% TC also completely eliminated the biolm
(p 0.05). Control plates showed a fully developed
biolm even after 5 days. As observed on polysty-
rene, all TC concentrations effectively inactivated
the biolm on urinary catheters also (g. 2, B). At 24
hours 1.5% TC completely eliminated the biolm on
catheters. Biolm counts on control catheters re-
mained at 6.0 log cfu/ml throughout the 5-day ex-
periment but at 5 days UPEC was not detected on all
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
time (days)
U
P
E
C

L
o
g

C
F
U
/
m
l
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
time (days)
U
P
E
C

(
l
o
g

C
F
U
/
c
a
t
h
e
t
e
r
p
i
e
c
e
)
A B
Figure 1. UPEC biolm prevention on microtiter plates (A) and Foley catheters (B) by 0% (diamonds), 0.1% (squares), 0.25% (circles)
and 0.5% (triangles) TC on incubation days 0, 1, 3 and 5.
TRANS-CINNAMALDEHYDE ANTIBIOFILM EFFECT ON ESCHERICHIA COLI 360
TC treated catheters (negative by enrichment).
Since P. mirabilis represents another important uro-
pathogen
2
in addition to UPEC, the antibiolm ef-
fect of TC on P. mirabilis ATCC strain 29906 was
also investigated. Results indicated that TC was
equally effective for P. mirabilis and UPEC biolms
on catheters (g. 3).
Confocal images of control biolmwith no added TC
revealed dense biolmwith an average thickness of 14
m (maximum 18), seen as living cells stained green
by SYTOdye (g. 4, A and C). The image of TCtreated
samples showed patchy breaks in biolm due to cell
loss and organizational disruption (g. 4, B), seen as
dead cells stained red by propidium iodide. Average
TC treated biolm thickness was 3 m (maximum 5).
MTT assay revealed that TC did not result in any
decreased HTB-4 cell viability compared to that of
controls (data not shown). Microscopy of TC treated
HTB-4 cells did not reveal any detectable morpho-
logical change compared to controls.
DISCUSSION
Catheter associated UTIs are a major challenge to
the health care industry. Several preventive mea-
sures and treatment strategies have been re-
searched to control catheter related infection, in-
cluding catheter antimicrobial coating,
21
insertion
site disinfection
22
and antimicrobial catheter lock
solution.
23
A few approaches have had some success
but catheter related UTI remains a concern world-
wide, highlighting the necessity of effective ap-
proaches to control UPEC. Approaches should be
0
10
20
30
40
50
60
70
80
90
100
0 1 3 5
time (days)
A
b
s
o
r
b
a
n
c
e

a
t

5
7
0
n
m

(
%
)
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6
time (days)
U
P
E
C

(
l
o
g

C
F
U
/
c
a
t
h
e
t
e
r

p
i
e
c
e
)
A B
Figure 2. Preformed UPEC biolm inactivation on microtiter plates (A) and Foley catheters (B) by 0% (black bars and diamonds), 1%
(hatched bars and squares), 1.25% (dashed bar and circles) and 1.5% (dotted bar and triangles) TC on incubation days 0, 1, 3 and 5.
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
time (days)
P
r
o
t
e
u
s

m
i
r
a
b
i
l
i
s

(
l
o
g

C
F
U
/
c
a
t
h
e
t
e
r

p
i
e
c
e
)
0
1
2
3
4
5
6
7
0 1 2 3 4 5 6
time (days)
P
r
o
t
e
u
s
m
i
r
a
b
i
l
i
s

(
l
o
g

C
F
U
/
c
a
t
h
e
t
e
r

p
i
e
c
e
)
A
B
Figure 3. P. mirabilis biolm prevention (A) and inactivation (B) on catheters pretreated with 0% (diamonds), 0.1% (squares), 0.25%
(circles) and 0.5% (triangles) TC (A), and on catheters with preformed P. mirabilis biolm treated with lock solution containing 0%
(diamonds), 1% (squares), 1.25% (circles) and 1.5% (triangles) TC (B) on incubation days 0, 1, 3 and 5.
TRANS-CINNAMALDEHYDE ANTIBIOFILM EFFECT ON ESCHERICHIA COLI 361
safe and capable of preventing UPEC biolm and
inactivating existing biolm on catheters.
TC effectively prevented UPEC biolm on urinary
catheters and effectively inactivated preformed bio-
lm when used as an antimicrobial constituent in
catheter lock solution. TC was effective against
UPEC biolm on 2 surfaces, namely polystyrene and
latex. In clinical settings indwelling urinary cathe-
ters with UPEC biolm are exposed to urine and TC
antimicrobial activity may potentially be compro-
mised by urine composition due to a number of or-
ganic molecules. Thus, we studied TC antibiolm
activity in synthetic urine. However, results indi-
cated that TC retained its antibiolm activity
against UPEC on catheters in the presence of urine.
The exceptional inhibitory effect of TC on UPEC
may be attributable to its multiple antimicrobial
mechanisms. A critical property of essential oils or
their components, including TC, is hydrophobicity,
which helps to target the lipid containing bacterial
cell membrane and mitochondria.
24
This makes
these membranes more permeable, leading to the
leakage of ions and other cell contents. TC is also
believed to kill bacteria by inhibiting energy gener-
ation and glucose uptake.
25
Another mechanism by
which cinnamon oil and its components kill micro-
organisms is their inhibitory effect on enzymes such
as amino acid decarboxylases.
26
Results also revealed that TC did not have any
deleterious effect on bladder epithelial cells, sug-
gesting that it may be safe to use the natural mole-
cule on urinary catheters or in catheter lock solu-
tion. In conclusion, these results indicate that TC
may potentially be a safe, effective antimicrobial
agent to prevent and inactivate UPEC biolms on
catheters.
ACKNOWLEDGMENTS
Dr. Sheryl S. Justice, The Research Institute at Na-
tionwide Childrens Hospital, Columbus, Ohio, pro-
vided UPEC isolate UPI 89. Dr. Carol Norris, De-
partment of Molecular and Cell Biology, University
of Connecticut, assisted with confocal microscopy.
REFERENCES
1. Stamm WE and Norrby SR: Urinary tract infec-
tions: disease panorama and challenges. J Infect
Dis, suppl., 2001; 183: S1.
2. Jacobsen SM, Stickler DJ, Mobley HLT et al:
Complicated catheter-associated urinary tract in-
fections due to Escherichia coli and Proteus mira-
bilis. Clin Microbiol Rev 2008; 21: 26.
3. Saint S and Chenoweth CE: Biolms and cathe-
ter-associated urinary tract infections. Infect Dis
Clin North Am 2003; 17: 411.
4. Hancock V, Ferrires L and Klemm P: Biolm
formation by asymptomatic and virulent urinary
tract infectious Escherichia coli strains. FEMS
Microbiol Lett 2007; 267: 30.
5. Denstedt JD, Wollin TA and Reid G: Biomaterials
used in urology: current issues of biocompatibility,
infection, and encrustation. J Endourol 1998; 12: 493.
6. Manges AR, Johnson JR, Foxman B et al: Wide-
spread distribution of urinary tract infections
caused by a multidrug-resistant Escherichia coli
clonal group. N Engl J Med 2001; 345: 1007.
A B
20 m 20 m
5 m
5 m
C D
Figure 4. Confocal microscopy reveals UPEC in biolm on coverslips without (A and C, green areas) and with (B and D, red areas) TC
treatment. SYTO (A and C) and propidium iodide (B and D) staining. Scale bars indicate 20 (A and B) and 5 (C and D) m.
TRANS-CINNAMALDEHYDE ANTIBIOFILM EFFECT ON ESCHERICHIA COLI 362
7. Nicolle LE: A practical guide to antimicrobial
management of complicated urinary tract infec-
tion. Drugs Aging 2001; 18: 243.
8. Wollenweber E: Occurrence of avonoid agly-
cones in medicinal plants. Prog Clin Biol Res
1988; 280: 45.
9. Beretz A, Anton R and Stoclet JC: Flavonoid
compounds are potent inhibitors of cyclic AMP
phosphodiesterase. Experientia 1977; 34: 1054.
10. Burt S: Essential oils: their antibacterial proper-
ties and potential applications in foodsa re-
view. Int J Food Microbiol 2004; 94: 223.
11. Ohno T, Kita M, Yamaoka Y et al: Antimicrobial
activity of essential oils against Helicobacter py-
lori. Helicobacter. 2003; 8: 207.
12. Bowles BL, Sackitey SK and Williams AC: Inhib-
itory effects of avor compounds on Staphylococ-
cus aureus WRRC B124. J Food Safety 1995; 15:
337.
13. Helander IM, Alakomi HL, Latva-Kala K et al:
Characterization of the action of selected essen-
tial oil components on gram-negative bacteria. J
Agricult Food Chem 1998; 46: 3590.
14. Djordjevic D, Wiedmann M and McLandsborough
LA: Microtiter plate assay for assessment of
Listeria monocytogenes biolm formation. Appl
Environ Microbiol 2002; 68: 2950.
15. Trautner BW, Hull RA and Darouiche RO: Esche-
richia coli 83972 inhibits catheter adherence by a
broad spectrum of uropathogens. Urology 2003;
61: 1059.
16. Ayebah B, Hung YC, Kim C et al: Efcacy of
electrolyzed water in the inactivation of plank-
tonic and biolm Listeria monocytogenes in the
presence of organic matter. J Food Prot 2006; 69:
2143.
17. Shah A, Mond J and Walsh S: Lysostaphin-
coated catheters eradicate Staphylococccus au-
reus challenge and block surface colonization.
Antimicrob Agents Chemother 2004; 48: 2704.
18. Pazos P, Fortaner S and Prieto P: Long-term in
vitro toxicity models: comparisons between a
ow-cell bioreactor, a static-cell bioreactor and
static cell cultures. Altern Lab Anim 2002; 30:
515.
19. Mosmann T: Rapid colorimetric assay for cel-
lular growth and survival: application to prolif-
eration and cytotoxicity assays. J Immunol Met
1983; 65: 55.
20. Chae MS and Schraft H: Comparative evaluation
of adhesion and biolm formation of different
Listeria monocytogenes strains. Int J Food
Microbiol 2000; 62: 103.
21. Saint S, Veenstra DL, Sullivan SD et al: The
potential clinical and economic benets of silver
alloy urinary catheters in preventing urinary tract
infection. Arch Intern Med 2000; 160: 2670.
22. Maki DG, Ringer M and Alvarado CJ: Prospective
randomized trial of povidone-iodine, alcohol, and
chlorhexidine for prevention of infection associ-
ated with central venous and arterial catheters.
Lancet 1991; 338: 339.
23. Shah CB, Mittelman MW and Costerton JW:
Antimicrobial activity of a novel catheter lock
solution. Antimicrob Agents Chemother 2002; 46:
1674.
24. Sikkema J, de Bont JA and Poolman B: Interac-
tions of cyclic hydrocarbons with biological mem-
branes. J Biol Chem 1994; 269: 8022.
25. Gill AO and Holley RA: Disruption of Escherichia
coli, Listeria monocytogenes and Lactobacillus
sakei cellular membranes by plant oil aromatics.
Int J Food Microbiol 2006; 108: 1.
26. Wendakoon CN and Sakaguchi M: Inhibition of
amino acid decarboxylase activity of Enterobacter
aerogenes by active components in spices. J
Food Prot 1995; 58: 280.
TRANS-CINNAMALDEHYDE ANTIBIOFILM EFFECT ON ESCHERICHIA COLI 363