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Jundishapur J Microbiol. 2016 September; 9(9):e40137.
Published online 2016 August 27.
doi: 10.5812/jjm.40137.
Research Article
The Effects of Sugars on the Biofilm Formation of Escherichia coli 185p
on Stainless Steel and Polyethylene Terephthalate Surfaces in a
Laboratory Model
Mahdi Khangholi,1and Ailar Jamalli2,*
1Golestan University of Medical Sciences, Gorgan, IR Iran
2Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, IR Iran
*Corresponding author: Ailar Jamalli, Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, IR Iran. Tel: +98-9112692547, E-mail:
a_jamalli@yahoo.com
Received 2016 June 19; Revised 2016 August 13; Accepted 2016 August 21.
Abstract
Background: Bacteria utilize various methods in order to live in protection from adverse environmental conditions. One such
method involves biofilm formation; however, this formation is dependent on many factors. The type and concentration of sub-
stances such as sugars that are present in an environment can be effective facilitators of biofilm formation.
Methods: First, the physico-chemical properties of the bacteria and the target surface were studied via the MATS and contact an-
gle measurement methods. Additionally, adhesion to different surfaces in the presence of various concentrations of sugars was
compared in order to evaluate the effect of these factors on the biofilm formation of Escherichia coli, which represents a major food
contaminant.
Results: Results showed that the presence of sugars has no effect on the bacterial growth rate; all three concentrations of sugars
were hydrophilic and demonstrated a high affinity toward binding to the surfaces.
Conclusions: The impact of sugars and other factors on biofilm formation can vary depending on the type of bacteria present.
Keywords: Biofilm, Adhesion, Sugars, Escherichia coli
1. Background
Bacterial biofilm can cause problems in almost any nat-
ural, industrial, or medical ecosystem. In the food indus-
try, food items can become contaminated by pathogenic
or non-pathogenic microorganisms (1). Pathogenic bacte-
ria can cause disease via the release of secretory toxins in
food; in the case of non-pathogenic microorganisms, con-
tamination can result in economic losses (2).
Biofilm is an accumulation of bacteria that is sur-
rounded by a solution of extracellular matrix proteins and
which causes bacteria to bond to each other and to the sur-
face. This multi-layer structure protects the bacteria from
the host’s immune system responses; moreover, studies
show that extraordinary resistance to various antibiotics
can result from biofilm formation in bacteria. Biofilms
are formed in water systems as well as in items relating to
the medical equipment and food industries (3). Via this
method of growth, bacteria have developed a compelling
means of escalating their own survival (2). This often re-
sults in the increase of bacterial resistance to adverse fac-
tors such as environmental moisture reduction, radiation,
toxins, changes in pH, temperature fluctuations, hydrody-
namic pressure, hydrolytic agents, phages, detergents, and
antibiotics (3). This resistance, in turn, facilitates the trans-
fer of genetic material between bacteria (4,5). Further-
more, the formation of biofilm on devices, equipment, and
medical devices can lead to contamination and the subse-
quent transmission of nosocomial infections (4).
Escherichia coli is often one of the contaminating fac-
tors of various nutritive substances, particularly of dairy
products such as yogurt and of fruit and fruit juices. Cer-
tain concentrations of sucrose sugars, glucose, fructose,
and lactose are present in these products. The question
now is: what effect do these sugars, in their special concen-
trations, have on the antimicrobial activity of the antimi-
crobial material?
To limit or even inhibit biofilm formation (biologi-
cal contamination), we must first understand the mech-
anisms that facilitate a bacteria’s initial attachment to a
cell surface. The physico-chemical interactions involved
in the infection of inanimate surfaces (polymers, metals,
glass) are acid-based and principally constitute Van Der
Waals electrostatic forces (5). Various studies have shown
Copyright © 2016, Ahvaz Jundishapur University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons
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Khangholi M and Jamalli A
that any physical or chemical change in an environment
that surrounds a bacteria can be involved in biofilm forma-
tion. The presence of different sugars in a bacterial envi-
ronment or even the presence of drugs can cause changes
to the physico-chemical properties of a constant surface,
which in turn leads to indirect changes in the formation
of biofilm (6).
2. Methods
2.1. Bacterial Strains and Culture Conditions
For this study, standard pathogenic E. coli185p strains
were prepared in the UMG laboratory in Lyon, France. LBB
(Luria-Bertani Broth, Merck, Germany) solutions with 30%
glycerol and stored at -80°C were used.
2.2. Bacterial Storage Conditions
In this study, stocks of samples were stored at -80°C,
prepared in the LBB, and kept at -20°C for daily use.
2.3. Determination and Comparison of the Growth Curve of E.
coli in Two Different Sugar Culture Mediums
Evaluation of the growth curves was performed by
measuring the absorbance at 400 nm using a spectropho-
tometer (Spectronic 20 Genesis, USA) and counting the
number of living cells; this was accomplished via the pour
plate method. Experiments were performed in three medi-
ums: an LBB, an LBB medium with 15% sucrose (Sigma),
and an LBB medium with 8 - 7% sucrose and 10% glucose
(Sigma). All experiments were performed in 20°C condi-
tions. The time required to add 1 ml of -20°C stoke to 100 ml
of the culture medium was considered the “zero time”. The
resulting optical density was measured every two hours
(until 54 hours had passed).
2.4. Preparation of Surfaces and Bacterial Suspensions to Test
Adhesion and Biofilm Growth
2.4.1 Solid Surfaces
In this study, two types of surfaces that are regularly
used in the medical and food industries were selected.
The selected surfaces included stainless steel, which is a
medium hydrophilic surface, and polyethylene terephtha-
late (PET), which is a hydrophilic surface.
2.4.2. Preparation and Treatment of the Selected Surfaces
Solid surfaces (3 ×1) were first cleaned in the RBS 35
(Sigma) with agitation for 15 minutes. Then, the surfaces
were rinsed with hot water an additional five times for five
minutes. Finally, the surfaces were washed in sterile water
five times for five minutes. Eventually, the surfaces were
placed in a laminar flow cabinet for drying.
2.5. Determination of the Physico-Chemical Properties
2.5.1. Micro-Organisms
Hydrophobicity and electron donor-acceptor proper-
ties were determined via the microbial adhesion to sol-
vents method (MATS) (7). According to this method, four
types of solvents (Sigma) were used, including chloro-
form, ethyl acetate, hexadecane, and decane. For the pur-
poses of this study, researchers first prepared a bacterial
suspension-delivered OD (Optical Density) at 400 nm to
0.08 (A0). Then, 2.4 ml of room temperature bacterial sus-
pension was added to 0.4 ml of each of the above sol-
vents. The solvents were kept stationary for 15 minutes, af-
ter which time they were subjected to vortex. Finally, the
OD of the aqueous phase was measured at 400 nm (A1). The
adhesion percentage of microorganisms to the above sol-
vents was calculated according to the following formula:
(1)%Adhesion =1−A1
A0×100
2.5.2. Solid Surfaces
Measurements of the physico-chemical properties of
the desired surfaces were completed by calculating the
contact angle. For the purposes of this study, three liq-
uids with specific energetic characteristics were selected,
including distilled water (MilliQ, Millipore), formamid
(Sigma), and D-iodine methane (Sigma). The surface en-
ergy of steel and polyethylene terephthalate (γs), as well
as the accompanying Van der Waals forces (γsLW ), electron
donors (γs-), and electron acceptors (γs+), were calculated
according to the following YOUNG-VAN OSS formula (VAN
OSS, 1988):
(1+ cosθ)γL= 2 γLW
SγLW
L1
2+γ+
Sγ−
L
1
2+γ−
Sγ+
L
1
2
(2)
2.6. Attached Cell-to-Surface Count
2.6.1. Total Flora
In this study, bacteria were kept in a stationary phase
during all tests. Acridine orange was used in order to ob-
serve and evaluate the total amount of flora (living, dead,
and uncultivable bacteria). The surfaces, which had been
contaminated with bacteria and stained with 0.01% Acri-
dine orange for 15 minutes, were placed in a laminar flow
cabinet after being washed with sterile water. The stained
surfaces were observed by an Epi-fluorescence microscope
DMBL Leica EFM (objective ×10).
2Jundishapur J Microbiol. 2016; 9(9):e40137.
Khangholi M and Jamalli A
2.6.2. Viable Cultivable Flora
First, contaminated samples were placed in 6 ml of
sterile saline. Sonication was performed at 35°C for two
minutes in order to count the number of attached viable
bacteria to surfaces (Ultrasonic 250 Jamestown, NY, USA, 40
kHz). Then, the samples were shaken mechanically for 30
seconds using a rotator. Finally, the serial dilution method
was used to accomplish the bacterial count. It should be
noted that bacteria were cultured in an LBB, and 24 hours’
incubation at 30°C was performed.
2.7. Statistical Analyses
The data were analyzed according to the GLM proce-
dures found in the SAS software version 8.0 (SAS Institute
Inc., Cary, NC) and the SPSS 16.
3. Results
3.1. Effects of Carbohydrates on the Bacterial Growth Curve
An empirical model was used to calculate the genera-
tion time and the lag phase following the measurement of
optical density and the counting of bacterial numbers by
serial dilution in three mediums with different concentra-
tions of glucose.
6
6.5
7
7.5
8
8.5
9
9.5
0 10 20 30 40 50 60
Temps,h
log 10, UFC/mL
Growth Rate = 0,33h-1
Lag Phase = 0
N0 = 3,16*106
Nmax = 1*109
Time g = 2,11
Figure 1. Growth Curve of E. coli 185p at 20°C in LBB (Blue Spot, Data; RedLine, Model)
6
6.5
7
7.5
8
8.5
9
9.5
0 10 20 3 0 4 0 50 6 0
Temps, h
Growth Rate = 0,32h-1
Lag Phase = 1,51h
N0 = 2,7*106
Nmax = 303*108
log 10, UFC/mL
Figure 2. Growth Curve of E. coli 185p in LBB + 15% Sucrose at 20°C (Blue Spot, Data;
Red Line, Model)
The results indicate that the growth rate of bacteria in
LBB is 0.33 ±0.007 h-1 (P value = 0.147), the growth rate of
bacteria in LBB + 15% sucrose is 0.32 ±0.4 hours-1 (P value =
0 10 2 0 3 0 4 0 50 60
Temps, h
Growth Rate = 0,33h-1
Lag Phase = 1,67h
N0 = 1,5*106
Nmax = 3,6*108
Time g = 2h
log 10, UFC/mL
9.5
9
8.5
8
7.5
7
6.5
6
Figure 3. Growth Curve of E. coli 185p in LBB + 7% Sucrose + 10% Glucose at 20°C (Blue
Spot, Data; Red Line, Model)
0.0983), and the growth rate of bacteria in LBB + 7% sucrose
+ 10% glucose is 0.33 ±0.007 hours-1 (P value = 0.128). These
results suggest that the presence of these sugars does not
affect the bacterial growth rate (Figures 1,2and 3).
The lag phase in LBB was equal to zero (P value =
0.0001). However, the lag phase was 1.51 ±0.38 hours
for the cultured bacteria in LBB + 15% sucrose (P value =
0.0063) and 1.67 ±0.12 (P value = 0.0013) for the cultivated
bacteria in LBB + 7% sucrose + 10% glucose. This indicates
that the above sugars serve to increase the length of the sta-
tionary phase.
Eventually, the generation time of these bacteria in dif-
ferent mediums was measured. The results of these mea-
surements indicate that the generation time was 2.11 hours
for cultured bacteria in LBB, 2.12 hours for cultured bacte-
ria in LBB + 15% sucrose, and 2 hours for cultured bacteria
in LBB + 7% sucrose + 10% glucose (P value < 0.05).
3.2. Physicochemical Properties of the Bacterial Cell Surface
Experiments were performed during the stationary
phase of bacterial growth. Hydrophilic-hydrophobic prop-
erties of E. coli 189p were determined via the MATS method
following growth in each of the selected mediums. The ten-
dency percentages (affinity%) of the bacterial cells to each
of used solvents are provided below in Table 1.
The structure and nature of chemical groups on the
surface of bacterial cells determines its physicochemical
properties (8). These characteristics are not fixed; rather,
they are dependent on the physical and chemical factors
that surround the bacterial cell, even in different culture
mediums (9). Escherichia coli 189p is super-hydrophilic in
the LBB medium because its affinity with apolar solvents is
low (decane 0.86% et hexadecane 0.0%). Differences in the
affinity of E. coli 189p in this medium following the addition
of chloroform and hexadecane indicate the electron donor
properties of this bacteria. Similarly, the electron donor
properties of the bacteria cultured in LBB + 15% sucrose
were also demonstrated. However, its low affinity with ap-
olar hydrophobic solvents (decane and hexadecane) rep-
Jundishapur J Microbiol. 2016; 9(9):e40137. 3
Khangholi M and Jamalli A
Table1. Percentage of Affinity to Solvents
Medium CH HD D AE
Aa38.23 ±4.22 0 ±0 0.64 ±0.86 0.38 ±0.52
Bb33.46 ±11.84 0 ±0 0.89 ±0.77 2.34 ±2.74
Cc64.02 ±7.76 0.63 ±0.98 2.80 ±2.80 37.97±5.45
Abbreviations: AE, Acetate Ethyl; CH, Chloroform; D, Decane; HD, Hexadecane
aLBB
bLBB + 15% Sucrose
cLBB + 7% Sucrose + 10% Glucose
resents the hydrophilic properties of the bacterial surface.
Nonetheless, the bacteria demonstrated “Lewis acid” prop-
erties (revealing a great affinity with chloroform [64.02]
and also with ethyl acetate [34.97] ) when cultured in an
LBB + 7% sucrose + 10% glucose medium. Conversely, the
bacteria displayed “Lewis base” properties when cultured
in the two other mediums. It is noteworthy that the elec-
tron donor property was stronger than the electron accep-
tor property. In this medium, E. coli 189p had a hydrophilic
property, such as in the two previous mediums (P value <
0.05).
3.3. Physico-Chemical Properties of the Selected Surfaces
Contact angle measurements of the selected surfaces
were performed for each of the three selected liquids (as
shown in Table 2).
Table 2. Values of the Measured Contact Angles (°) of the Different Liquids to the
Surface of the Steel and the PET
Steel PET
θDistilled water 74.13 ±1.23 76.18 ±2.42
θdiodomethane 52.2 ±0.81 53.6 ±1.20
θFormamide 50.65 ±2.40 33.13 ±1.36
The results indicate that steel is hydrophilic (the con-
tact angle with water was 74.13°), which is associated with
a “Lewis base” (γ-= 7.9 mJ/m2) (P value = 0.0004). Addition-
ally, PET has hydrophilic properties (the contact angle with
water was 76.18°), which also indicates that it has “Lewis
base” properties (γ-= 7 mJ/m2) (P value = 0.009).
3.4. Adhesion and Bacterial Biofilm Analysis
The number of viable bacteria attached to both studied
surfaces (steel and PET) was counted after 3 hours and after
24 hours. An Epi-fluorescent microscope was used to view
the total number of bacteria attached to the surfaces (liv-
ing or non-living). Cultivable live bacteria were cultured in
the LBB and were reported according to log10 (cells/cm2).
3.5. Total Flora
Multiple shots taken by microscope Epi-fluorescence
indicate the number of bacteria attached to the surface
and reveal levels of biofilm formation. The bacterial per-
centages found on the surface of each material were calcu-
lated and are presented in Figure 4.
These bacteria can be homogeneously spread upon the
surface (as was the case for the cultured bacteria in LBB and
in LBB + 15% sucrose) or perched on the target’s surface in
a grid-like fashion (as occurred with the cultured bacteria
in LBB + 7% sucrose + 10% glucose) (Figure 4).
3.6. Viable Cultivable Flora
The results of the tests for adhesion and biofilm forma-
tion on the steel and polyethylene terephthalate surfaces
taken at 3 hours and at 24 hours are shown below (Table 3).
Hydrophilic bacteria bind more strongly to hy-
drophilic surfaces, and bacteria with hydrophobic prop-
erties bind more potently to hydrophobic surfaces (10).
The previous tests showed that both surfaces chosen for
this project are hydrophilic. However, the studied bacte-
ria demonstrated hydrophilic properties; therefore, it is
expected that the cultured bacteria in each of the three
mediums would bind to both target surfaces and form
biofilms. Viable cells cultured in LBB count for E. coli, which
has strong hydrophilic properties that are indicative of
its strong bond with steel (6.76 ±0.30 log10) (P value =
0.0031) and polyethylene terephthalate (6.69 ±0.53 log10)
(P value = 0.068). Similarly, for the bacteria cultured in LBB
+ 15% sucrose, a number of bacteria attached to the steel
(6.77 ±0.028 log10) (P value = 0.0090) and to PET (6.38 ±
0.0 log10) (P value = 0.012). Finally, the number of bacteria
that attached to the steel for the cultured bacteria in LBB +
10% glucose + 7% sucrose was 6.29 ±0.45 log10.
4. Discussion
In the food industry, microorganisms are capable of
causing biofilms to form on wet surfaces. The adhesion
4Jundishapur J Microbiol. 2016; 9(9):e40137.
Khangholi M and Jamalli A
Surface Steel PET
Contact Time 3h 24h 3h 24h
LBB
LBB+15% Sac
LBB+7% Sac+10% Glu
Figure 4. Observations from the Epi-fluorescence microscope (objective ×10) of E. coli 185p in various media (stainless steel and PET). The cells were stained with acridine
orange.
Table3. Results of Adhesion Tests for Contact Bacteria/Support at 3 and 24 Hours
Medium Steel 3, H, (UFC/cm2) Steel 24, H, (UFC/cm2) PET 3, H, (UFC/cm2) PET 24, H, (UFC/cm2)
LBB 1.7 ×1066.59 ×1069.75 ×1057.03 ×106
LBB + 15% sucrose 1.35 ×1065.99 ×1065.55 ×1052.40 ×106
LBB + 7% sucrose + 10% glucose 8.07 ×1052.54 ×1051×1061.89 ×106
of food spoiling bacteria and pathogens to surfaces that
come into contact with food could be potential sources
of contamination and disease transmission (11). Physico-
chemical conditions (temperature, pH, sugar, and salt
compounds) are effective facilitators of biofilm formation.
The above conditions cause changes to bacterial cell wall
components, surface physicochemical properties such as
hydrophobicity, and electron donor and acceptor proper-
ties (12).
The selected surfaces have many applications to the
food and medical industries, and the selected bacteria are
one of the most significant causes of corruption and dis-
ease for the food industry. Sugar’s effect on the bacterial
growth curve was examined via the creation of an adapta-
tion phase (stationary phase), which was higher for the cul-
tured bacteria in the LBB + 10% glucose + 7% sucrose solu-
tion than for the cultured bacteria in the LBB + 15% sucrose
solution. The bacterial growth rate was the same in all
three mediums. Results of the study of the physicochemi-
cal properties of bacteria in different candied mediums in-
dicate that the examined bacteria is extremely hydrophilic
in the LBB and LBB + 15% sucrose solutions. Moreover, the
bacteria cultured in the LBB + 7% sucrose + 10% glucose so-
lution had hydrophilic properties but also demonstrated a
“Lewis acid-base” property.
The results of measuring the contact angles of the sur-
Jundishapur J Microbiol. 2016; 9(9):e40137. 5
Khangholi M and Jamalli A
faces (steel and polyethylene terephthalate) indicate the
hydrophilic surface properties of the desired surfaces. The
percent of surface coverage by E. coli 185p was analyzed by
fluorescent microscopy and image J programing; results
indicate the formation of biofilms on both surfaces in all
three mediums. Biofilm formation was expected in all con-
ditions, since inanimate coats of the bacterial cell surface
have hydrophilic properties, as indicated by the results.
Additionally, because steel had a stronger hydrophilic sur-
face than polyethylene terephthalate, more biofilm was
formed on that surface. However, results of the measure-
ment and counting of participating biofilm bacteria re-
vealed an increase in the number of bacteria at 24 hours as
compared to the measurements taken at 3 hours; this re-
sult seems reasonable, as more bacteria attach to surfaces
with the passage of time. In general, the amount of biofilm
formation and the adhesion ability of bacteria in the LBB
+ 7% sucrose + 10% glucose solution was less significant
than that found in the cultured bacteria of LBB and LBB +
15% sucrose. Because the above medium can contain Lewis
acid-base properties of bacteria, the bacteria in the envi-
ronment no longer demonstrates open Luiz properties; in-
deed, these bacteria contain Lewis base properties in the
two other mediums. A study conducted by Jackson et al.
on the effect of glucose on the biofilm formation of E. coli
showed that glucose inhibits the biofilm formation that is
mediated by catabolic suppression systems (CRP) (13).
A study performed by Yang et al. on the effects of sug-
ars and antimicrobial substances on oral microbial biofilm
formation demonstrated that sucrose increases biofilm
formation more significantly than does glucose, fructose,
galactose, and lactose (14). In 2011, a study conducted by
Michu et al. on the effects of glucose and salt on the
biofilm formation of staphylococcus epidermidis on sur-
faces of stainless steel found that while the presence of
salt can increase biofilm formation, the presence of salt
and glucose strongly increases biofilm formation (15). Xu
et al. examined biofilm formation in different salt con-
centrations (10 - 0%) of Listeria monocytogenes,Staphylococ-
cus aureus,Shigella boydii, and Salmonella typhimurium. Re-
sults showed that increases in the concentration of salt
reduces biofilm formation; however, a significant reduc-
tion was observed in the 2% concentration. One reason for
this decrease in biofilm formation involves the reduced hy-
drophobicity of bacterial cells in such conditions (16).
A study performed Bonaventura et al. on the effect of
temperature on the biofilm formation of 44 strains of Lis-
teria monocytogenes on different food contact surfaces re-
vealed that biofilm formation on glass surfaces is greater
than that seen on polystyrene surfaces or on stainless steel
at 4°C, 12°C, and 22°C (17). A study conducted by Giaouris et
al. was carried out using the bead vortexing and conduc-
tance measurements methods; this study examined the ef-
fects of temperature (5°C, 20°C, and 37°C) and pH (4.5, 5.5,
6.5, and 7.4) on the biofilm formation of Salmonella enter-
ica enteritidis PT4 on of stainless steel surfaces. Results in-
dicated that most of the biofilm formed after 6 days at 20°C
and that at this temperature, the amount of biofilm forma-
tion depended on the pH value after the seventh day (18).
A study performed by Pan et al. on the effects of glucose
(at a concentration of 0.25% to 10.0% wt/vol), salt (0.5 to
7%), and temperature (22.5°C, 30°C, and 37°C) on the biofilm
formation of 36 strains of Listeria monocytogenes found
that 97% of strains (35 strains) formed thicker biofilms in
mediums containing glucose (1% to 10%), as compared with
glucose-free mediums, at all three temperatures. Addition-
ally, most strains formed more biofilms in the 2 - 5% salt
solutions. It is possible that glucose, salt, and tempera-
ture have a synergistic effect on biofilm formation (19). In
a study conducted by Chai et al. on the effect of galac-
tose metabolism on the biofilm formation of Bacillus sub-
tilis, it was found that galactose metabolism genes play a
major role in biofilm formation and the development of a
polysaccharide matrix (EPS) (20).
Changes in the nature and bacterial surroundings of
an environment lead to changes in bacterial cell surfaces
and biofilm formation (1,21). In this study, concentrations
of selected sugars (equivalent to the concentration of sug-
ars in fruit yogurt) somewhat reduced the level of bac-
terial attachment and biofilm formation on two surfaces
(polyethylene terephthalate for the packaging of fruit yo-
gurt, and stainless steel for the storage of yogurt); this can
help to prevent the attachment of bacteria as well as its
pathogenesis. However, the types of microorganisms that
are able to grow in these special foods are often variable;
therefore, the extrinsic and intrinsic factors of each food
and of each bacteria must be considered.
Footnotes
Authors’ Contribution: Ailar Jamalli developed the orig-
inal idea and the protocols. The design and conduct of this
study was performed by Mahdi Khangholi.
Financial Disclosure: The funding organizations are pub-
lic institutions and had no role in the design and conduct
of the study, the collection, management, and analysis of
the data, or the preparation, review, and approval of the
manuscript.
Funding/Support: Funding for this project was provided
by the laboratory sciences research center. This study was
supported by the Golestan University of Medical Sciences
and the laboratory sciences research center.
6Jundishapur J Microbiol. 2016; 9(9):e40137.
Khangholi M and Jamalli A
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