dewanti, 2005
TRANSCRIPT
-
8/10/2019 Dewanti, 2005
1/18
International Journal of
ELSEVIER
Food Microbiology
26
(1995) 147-164
Intemational Journal
of
Food
Microbiology
Influence
of
culture conditions on biofilm formation
by
Escherichia coli
0157:H7
Ratih Dewanti, Amy c L Wong
Food Research Institute Department of Food Microbiology and Toxicology
1925
Willow Drive
University
of
WISconsin-Madison Madison WI 53705 USA
Received
22
February
1994;
revision received
27
June
1994;
accepted
14
July 1994
Abstract
Biofilms of Escherichia coli 0157:H7 were developed on stainless steel chips in trypti
case
soy
broth (TSB),
1 5
dilution of TSB,
0.1
Bacto peptone (BP) and a minimal salts
medium (MSM) supplemented with 0.04% of one of the following carbon sources: glucose,
glycerol, lactose, mannose, succinic acid, sodium pyruvate or lactic acid.
t
was found that
biofilms developed faster and a higher number
of
adherent cells (ca. 10
6
CFU cm
2
) were
recovered when the organisms were grown in the low nutrient media. Regardless of the
carbon source, biofilms developed in MSM consisted of shorter bacterial cells and thicker
extracellular matrix (ECM), with glucose as the best substrate for stable biofilm formation.
Fewer bacteria in initial attachment, non-hydrophobicity of bacterial cells, lack of ECM
formation and easy detachment
of
the biofilm bacteria may contribute to poor biofilm
formation in TSB. ECM
is
probably important for the stability
of
biofilms; however, at 10C
and under anaerobic conditions, ECM seems to be unnecessary.
Keywords
Biofilm; Escherichia coli 0157:H7; Culture conditions; Extracellular matrix
1. Introduction
Microbial attachment to surfaces and the development of biofilms are known to
occur in many environments. Biofilms have been studied most extensively in
marine and aquatic environments and medical areas (Characklis and Marshall,
1990 .
Often biofilms in these situations create economic and health problems. For
example, they cause fouling of industrial equipment such
as
heat exchangers (Bott,
Corresponding author. Tel. (608)-263-1168. Fax: (608)-263-1114.
0168-1605/95/ 09.50
1995
Elsevier Science B.V. All rights reserved
SSDI 0168-1605 94)00103-0
-
8/10/2019 Dewanti, 2005
2/18
148
R Dewanti A C L Wong
/
Int. 1. Food Microbiology
26 1995) 147-164
1992) and ship hulls Cooksey and Wigglesworth-Cooksey, 1991), which results in
reduced heat transfer, energy loss, increased fluid frictional resistance and acceler
ated corrosion. Biofilm formation in water distribution systems decreases water
quality and increases health risks Block, 1992). Biofilm accumulation on teeth and
gum, urinary tract, and implanted medical devices such as catheters Sheretz et aI.,
1990) may lead to infections.
Only recently has biofilm formation gained attention in food environments. The
attachment of microorganisms and subsequent development of biofilms in food
processing environments are potential sources of contamination and may lead to
food spoilage
or
transmission of diseases. t has been shown that even with
cleaning and sanitation procedures consistent with good manufacturing practices,
microorganisms can remain on equipment surfaces Maxcy, 1964; Czechowski,
1990;
Mattila
et
aI., 1990). These organisms may survive for prolonged periods
depending on the environmental conditions Maxcy, 1971). Listeria monocytogenes
and other
Listeria
spp. can be isolated frequently from various surfaces in dairy
and meat processing environments Anonymous, 1988; Charlton
et
aI.,
1990;
Nelson, 1990).
Biofilm development
is
a dynamic process. Bacteria exposed to surfaces attach
readily. Under suitable conditions, organisms that remain irreversibly attached
grow and develop into biofilms, usually embedded in a polymer matrix of microbial
origin Characklis and Marshall, 1990). This matrix
is
generally assumed to be
polysaccharide in nature and
is
often referred to
as
glycocalyx Costerton et
aI.,
1987). Portions
of
biofilms may eventually detach and colonize
other
parts of the
system. Cells in biofilms are generally hardier than their planktonic free-living)
counterparts, and exhibit increased resistance to adverse conditions such as desic
cation Costerton
et
aI., 1987), extreme temperatures Frank and Koffi, 1990) and
the presence
of
antibiotics Nickel et aI., 1985)
or
sanitizers Marrie and Costerton,
1981; Stickler at aI., 1989).
Several food spoilage and pathogenic bacteria have been reported to attach and
form biofilms in vitro on food contact surfaces such as
stainless steel, polystyrene
or
rubber Speers
et
aI.,
1984; Czechowski, 1990). These biofilm bacteria are also
known to be more resistant to cleaners and sanitizers Krysinski et aI., 1992 .
Ronner and Wong 1993) found that attachment surface affected biofilm formation
by
L. monocytogenes
and
Salmonella typhimurium
and also their relative resistance
to sanitizers.
Escherichia coli
0157:H7 was first identified as a pathogen in 1982 and
is
now
recognized as an important cause of foodborne disease Doyle, 1991). The illnesses
caused by this organism can be manifested as hemorrhagic colitis, hemolytic
uremic syndrome HUS) and thrombotic thrombocytopenic
purpura TIP).
Hem
orrhagic colitis
is
the most common syndrome, and
is
typified by severe abdominal
pain and grossly bloody diarrhea. HUS
is
a leading cause of renal failure in
children and patients often require dialysis and blood transfusions. Symptoms of
TIP
are similar but more severe than HUS. Death may result from HUS or TIP.
Outbreaks due to
E.
coli 0157:H7 have been associated primarily with consump
tion of undercooked ground beef. The most recent outbreak involved over 500
-
8/10/2019 Dewanti, 2005
3/18
R. Dewanti A.C.L. Wong / Int. 1. Food Microbiology 26 (1995) 147-164 149
laboratory-confirmed cases and four deaths (CDC,
1993 .
Other infection vehicles
include unpasteurized milk, roast beef, apple cider and person-to-person transmis
sion. A one-year, prospective, population-based study (MacDonald et aI., 1988
indicated that the incidence rate for the organism was
8/100000
person-years,
compared to
21/100000
person-years for
Salmonella
and
7/100000
person-years
for
Shigella.
Hence infection by E
coli
0157:H7 is quite common.
An understanding on how
E coli
0157:H7 can establish and survive in the
processing environment is essential to finding ways to prevent contamination. This
study
is
a first step in delineating conditions under which this organism can attach
and form biofilms, and in characterizing the process. Effects of nutrients, low
temperature and anaerobic conditions on biofilm formation
by
these organisms
were examined.
2 Materials and methods
2.1. Bacteria
Escherichia coli
0157:H7 strain 932, a clinical isolate from a ground beef
associated hemorrhagic colitis outbreak in 1982, was obtained from the Centers for
Disease Control. The strain was maintained in 80 glycerol
at
- 20C and grown
in trypticase soy broth (TSB, Becton Dickinson, Cockeysville, MD) for 16-18 h at
room temperature prior to use.
2.2. Stainless steel chips
Stainless steel (SS) type 304 with a 4 finish, commonly used in food processing
equipment and contact surfaces, was cut into 1 1
cm
chips. The chips were
washed in a hot detergent solution
1
Micro; International Products, Corp.,
Trenton,
NJ
for 1 h, rinsed in distilled water twice and air dried. Cleaned
SS
chips were dry autoclaved at
121C
for
20
min prior to use.
2.3. Biofilm development
Escherichia coli
0157:H7 (approximately
10
7
CFU) was inoculated into
50 ml
of
growth medium in 125-ml Erlenmeyer flasks and four SS chips were placed in each
flask. Incubation was
at
room temperature (22-25C) with mild agitation (70-90
rpm) on a rotary shaker (Labline, Melrose Park, IL). At specified times, duplicate
SS
chips were removed and rinsed in sterile distilled water. Adherent cells on the
SS surfaces were removed
by
scraping with a Teflon spatula, followed by swabbing
with a calcium alginate swab (Frank and Koffi, 1990). The cells were dispersed and
serially diluted in 0.01 M phosphate buffered saline (PBS), then surface plated on
trypticase soy agar (TSA, Becton Dickinson) for enumeration. The other two SS
chips were processed for scanning electron microscopy (SEM). Planktonic cells, i.e.
-
8/10/2019 Dewanti, 2005
4/18
150
R
Dewanti, A.C.L. Wong
/
Int. 1. Food Microbiology
26 (1995)
147-164
bacteria suspended in the culture medium, were enumerated as above. Planktonic
cells from certain growth conditions were also observed under SEM.
Media used in this study were: TSB, l i TSB, 0.1 Bacto peptone (BP, Difco)
and a minimal salts medium (MSM) containing 7 g KH
2
P0
4
, 3 g K
2
HP0
4
, 1 g
(NH
4
)2 S04
0.1
g
MgS0
4
and 1 mg of yeast extract per liter (Camper et al.,
1991).
The MSM was supplemented with the following carbon sources: 0.01-1.0 D-glu
cose, 0.04-1.0 D-mannose, 0.04 D-lactose, 0.04 glycerol, 0.04 sodium
pyruvate, 0.04 succinic acid or 0.04 lactic acid. The initial
pH
of all media
ranged from 6.8 to
7.1.
Carbon sources were filter sterilized using a 0.2
JLm
cellulose acetate filter unit (Coming Inc., NY) before addition to the MSM. All
media were sterilized at 121C, 15 psi for 20 min.
Biofilm formation in MSM-0.04 glucose was also examined at
10C
and under
anaerobic conditions. For anaerobic incubation, 125-ml Erlenmeyer flasks contain
ing bacterial cells and the SS chips were degassed with a vacuum pump. Air was
replaced with a mixture of 80 nitrogen, 10 carbon dioxide and 10 hydrogen
and the flasks were incubated at 22-25C.
2 4
Scanning electron microscopy SEM)
Biofilms from all growth conditions were prepared for SEM using a fixation
method described
by
Birdsell et al. (1975) with minor modifications. The chips
were rinsed twice in sterile distilled water and placed in
0.1
w
Iv
concanavalin
A (con A; Sigma Co., St. Louis, MO) in 0.1 M phosphate buffered saline
containing 0.1 CaCl
2
and 0.1 MgCl
2
(PBS-CM, pH 7.2) for 20 min. The SS
chips were then washed in PBS-CM twice and fixed with 1 glutaraldehyde
(Sigma) in 0.2 M cacodylate buffer (Sigma) overnight at
4C.
A second method of fixation (Fassel et al., 1992) employing ruthenium red (RR,
Sigma) was also used for biofilms developed in TSB and MSM-0.04 glucose.
Ruthenium red is a stain specific for acidic polysaccharide (Luft, 1971). Briefly,
rinsed SS chips were placed in a pre-fixation solution containing 0.15 RR in 0.1
M cacodylate buffer for 1 h. The chips were rinsed in a wash buffer 0.1 M
cacodylate buffer, pH 7.0-7.3), then fixed in 2 glutaraldehyde in 0.1 M cacody
late buffer containing 0.05 RR. After 2 h of fixation, SS chips were rinsed in
wash buffer and placed in a post-fixation solution containing 2 osmium tetroxide
in 0.2 M cacodylate buffer for 2.5
h.
The chips were then rinsed five times in wash
buffer.
Stainless steel chips fixed with either method were dehydrated twice
5
min
each time) in a graded ethanol series of
35, 50, 70, 85, 95
and 100 ethanol.
Dehydration was completed in a Tousimis Sam Dri 7808 critical point dryer using
carbon dioxide as the transition medium. The chips were mounted on SEM
specimen stubs with silver paint and coated with gold-palladium alloy using a
Polaron E-5000M vacuum evaporator (Bio Rad, Richmond, CA). Biofilms were
viewed with a Hitachi S 570 scanning electron microscope.
Planktonic cells from selected growth conditions were also viewed under SEM.
Approximately 10
ml of culture was filtered using a 0.2
JLm
cellulose acetate
-
8/10/2019 Dewanti, 2005
5/18
R
Dewanti, A.C.L. Wong
/
Int.
1.
Food Microbiology
26
J995)
147 164
151
membrane (Corning). The membrane was treated with con A, fixed in glutaralde
hyde in cacodylate buffer and dehydrated for SEM observation.
2 5
Attachment studies
The ability of E
coli
0157:H7 to attach to
SS
surfaces in TSB or MSM-0.04
glucose was compared. Overnight cultures grown in TSB were centrifuged at
5000 X g for 10 min and the cell pellets washed in PBS. The cells were suspended
in PBS and the appropriate amount to achieve 10
7
CFUjml was inoculated into
100 ml of TSB or MSM-0.04 glucose in a 1-1 beaker. Each beaker contained
three
SS
chips. After 1 h at room temperature, the SS chips were rinsed twice in
distilled water, and stained with 0.026 acridine orange (Sigma) for 5 min. The
chips were rinsed
five
times in distilled water and bacteria were enumerated using
the oil immersion objective 100 x and a 10 X ocular lens on a Carl Zeiss
Standard Microscope equipped for epifluorescence with an HB 050 mercury light
source and the Zeiss 09 filter combination (excitor AP 450-490, reflector F f 510,
barrier filter LP 520). Bacterial cells in ten randomly chosen fields from each chip
were counted, and the average number
o
attached cells per field was determined.
The microscopic field was measured using a stage micrometer, and attached cells
per
cm
2
were determined (Pusch et aI., 1984).
2. 6.
Detachment studies
Biofilms of E coli 0157:H7 on SS chips developed in TSB or MSM-0.04
glucose for 2 days were rinsed twice in distilled water and placed in 20 ml PBS in
100 X 15
mm
Petri dishes. The dishes were shaken at
70
rpm on a rotary shaker for
30
min. Cells remaining on the chips were scraped and swabbed for enumeration
by
plate count.
2.
7.
Hydrophobicity determination
The surface hydrophobicity of planktonic and biofilm cells developed in TSB
and MSM-0.04 glucose was examined using the bacterial adhesion to hydrocar
bons (BATH) assay (Rosenberg,
1980).
This assay measures the distribution of
cells between an aqueous and a hydrophobic phase. Briefly, bacterial cells were
collected, washed and suspended in PBS to achieve an optical density at
600 nm
(O.D.
600
of 0.4-0.6 using a Spectronic 20 (Milton Roy Co.). Three milliliters of
bacterial suspension were placed in a clean 10 X 13 mm test tube. Hexadecane
(150
JLI
Sigma) was added to the cell suspension and mixed twice for
10
s each
time with a 5-s interval. The suspension was allowed to separate for 10 min and the
A o A
O.D'
600
was read. Bacterial adhesion to hexadecane was determined as
=
Ao
X 100 , where Ao is the initial O.D'
600
of the bacterial suspension and A is
O.D'
600
of the suspension after mixing with hexadecane.
-
8/10/2019 Dewanti, 2005
6/18
152
R Dewanti, A.C.L. Wong I Int. 1. Food Microbiology 26 (1995) 147-164
1 0 ~ - - - - - - - - - - - - - - - - - - - -
8
II
j
4
2
2 4 6 8 10
Incubation
Time d)
Fig.
1.
Growth and biofilm formation
by
E
coli
0157:H7
in
TSB,
1 5
TSB, and BP. Open symbols
represent growth
Jog
CFU
1m )
in
TSB
0);
1 5TSB
.c.);
and BP
D),
and filled symbols are biofilms
Jog CFU cm
2
)
in TSB (e);
1 5TSB (.&);
and BP . ) .
2 8 Media transfer studies
Duplicate chips from cultures grown in TSB and MSM-0.04 glucose for 2 days
were collected. The chips were rinsed twice
in
distilled water to remove non-ad
herent cells, placed into Erlenmeyer flasks with either MSM-0.04 glucose or TSB
and incubated for four additional days. Planktonic and adherent cells were
enumerated
by
plate count and biofilms were observed under SEM.
2.9.
Data analysis
Results are presented as the means from duplicate experiments. Data were
analysed using the Student's two-tailed t-test.
3. Results
3 1
Growth
and
biofilm development in complex media
Studies were conducted initially in complex media containing different levels of
nutrients. Growth and biofilm development by E
coli
0157:H7 on SS in TSB, 1/5
TSB and BP are shown in Fig.
1
The organisms grew well in TSB, reaching a
maximum number of approximately 10
9
CFU/ml at 1 day, and remained at the
same level for up to 7 days. The number of adherent bacteria on
SS
surfaces was
maximum (approximately
10
4
CFU/cm
2
)
at
1-2
days, decreased to
10
3
CFU/cm
2
by 7 days and remained constant until
14
days (data not shown). Growth of
planktonic cells in 1/5 TSB was similar to that in TSB, but the number of bacteria
recovered from SS chips increased with incubation time and reached a maximum
of
10
5
CFU/cm
2
at 8 days. A longer lag phase and slower growth of planktonic
cells in BP were observed when compared to that in TSB or
1/5
TSB. However,
-
8/10/2019 Dewanti, 2005
7/18
R Dewanti
A C L
Wong
/
Int. 1 Food Microbiology 26 1995)
147 164
153
Fig.
2
Scanning electron microscopy of E. coli 0157:H7 in complex media: A) biofilm bacteria in TSB
at 2
d;
B) planktonic bacteria in TSB at 2 d; C) biofilm bacteria
in
1/5 TSB at 8
d;
D) biofilm
bacteria
in
BP at 3 days. Bar 1 JLm
the biofilm population developed faster and reached a maximum of 10
6
CFU/cm
2
at
5 days. Extensive clumping of planktonic cells was observed in BP.
With SEM, dramatic differences were observed in biofilms developed on SS
with the three media. Biofilms formed in TSB consisted of sparse single cells
adhering to
SS
without apparent extracellular matrix ECM) when fixed with either
the con A-glutaraldehyde Fig. 2a) or the ruthenium red method. All SEM
photographs presented in this paper were obtained
by
fixing planktonic or adher
ent
bacteria with con A and glutaraldehyde.
The
biofilms appeared the same with
up to 14 days incubation. Planktonic cells of E coli 0157:H7 grown in TSB,
however, showed extensive ECM Fig. 2b).
-
8/10/2019 Dewanti, 2005
8/18
154 R Dewanti,
A C L
Wong / Int. J
Food
Microbiology 26 (1995) 147-164
In
1 5
TSB, biofilms containing sparse single cells were observed at 3 days. At 8
days, ECM development
was
observed and biofilms consisting of clusters
of
bacterial cells distributed on the SS surface with ECM connecting cell to cell or
cell to
SS
were seen (Fig.
2c).
In BP, biofilms with ECM were first observed at 3
days. The biofilm distribution on the SS was less uniform than in 1 5 TSB, but
ECM appeared similar (Fig. 2d).
3 2 Growth and biofilm development in minimal salts media MSM)
Studies in complex media suggested that biofilms may be formed more readily
under lower nutrient conditions. A minimal salts medium was used to explore this
hypothesis. Addition
of
0.01 glucose to MSM was necessary for growth of these
organisms. In
MSM-O.01
glucose,
10
4
CFU
cm
2
adherent cells were recovered
from SS at 2 days. Only sparse single cells were observed with SEM. The bacteria
were smaller in size and contained thicker ECM compared to those in 1 5 TSB or
BP. Increasing the glucose concentration to 0.04 resulted in significant changes
in growth and biofilm formation. Extensive clumping of planktonic cells occurred,
as in the case with BP. Although planktonic growth in this medium after 2 days
was
similar to that in
1 5
TSB, the number of adherent bacteria was 2
10gIO
CFU cm
2
higher. Biofilms at 2 days consisted of relatively shorter cells with
thicker ECM similar to that seen in MSM-0.01 glucose, but a lot more adherent
bacteria were observed (Fig. 3a). Biofilms fixed with either method yielded the
same results. Planktonic cells grown in MSM-0.04 glucose also appeared shorter
but possessed ECM similar to that associated with TSB-grown planktonic cells
(Fig. 3b). Further increase in glucose concentration to 0.1 and 1 did not
significantly alter the number planktonic and adherent bacteria. However, these
biofilms were less dense as compared to those developed in MSM-0.04 glucose.
Fig.
3.
Scanning electron microscopy
of
E coli 0157:H7 in MSM-O.04 glucose at 2 d: (A) biofilm,
arrow indicates thick ECM; (B) planktonic cells. Bar 1 /Lm
-
8/10/2019 Dewanti, 2005
9/18
R
Dewanti A.C.L. Wong/Int 1. Food Microbiology 26 (1995) 147-164
Table 1
Effect of carbon source in MSM on growth and biofilm formation by E coli
0157:H7
Carbon source
Planktonic bacteria
( )
a
Adherent bacteria ( )
b
(0.04 in MSM)
Glucose 100 100
Sodium pyruvate
100 85
Glycerol
88
66
Lactose 107 47
Succinic acid 164
42
Lactic acid 96
30 c
Mannose 85
7.4 c
a Average of duplicate cultures, cell counts expressed ad of MSM-glucose control.
b Average of four replicate chips, cell counts expressed as
of
MSM-glucose control.
C
Significantly different from MSM-O.04 glucose
p
0.025).
155
Growth and biofilm formation in MSM with different carbon sources were
compared to those in MSM-0.04 glucose (Table
1).
Use of 0.04 lactose,
glycerol, succinic acid or sodium pyruvate yielded planktonic and biofilm popula
tions similar in number and appearance to those in MSM-0.04 glucose. However,
biofilms were less than those in MSM-0.04 glucose. Significant decreases
p
0.025) in bacterial adherence were observed when MSM was supplemented with
0.04 mannose
or
lactic acid. Increasing the amounts of mannose to
0.1
or 1 ,
however, resulted in biofilms similar to those in MSM-0.04 glucose (data not
shown).
3.3. Growth and biofilrn formation at lOO
and
under anaerobic conditions
Low
temperatures are generally maintained in meat or other food processing
environments. In hard to clean locations, such as gaskets, valves, and dead ends,
some degree of anaerobiosis may be maintained. Biofilms have been shown to
develop in gaskets of dairy environments (Czechowski,
1990 .
In addition, it has
been shown in our laboratory that
S.
typhimurium
attached better to
SS
under
anaerobic conditions (unpublished results).
The potential for E coli 0157:H7 to grow and develop biofilm in MSM-0.04
glucose at 10C or anaerobically was examined. Compared to growth at 25C, the
organisms grew slowly at 10C. Bacterial adherence was slow initially, but the
number of adherent cells was similar to that at
25C by
8 days (Fig. 4 . The number
of planktonic cells reached a maximum number of
10
8
CFUIml at 6 days and
biofilm bacteria reached 10
6
CFU
Icm
2
at 8 days. Biofilms developed at
10C
were
different from those developed at 25C. They consisted of normal size single cells
adhering to
SS
with very little ECM (Fig. 5).
Under anaerobic conditions, the number of planktonic cells reached
10
9
CFUIml after 2 days, while adherent bacteria were
10
6
CFUI cm
2
Biofilms
developed under these conditions appeared similar to those at 10C when viewed
under SEM, except for the shorter bacterial size resembling those incubated
aerobically.
-
8/10/2019 Dewanti, 2005
10/18
156
R. Dewanti, A C L Wong I Int. 1. Food Microbiology
26 (1995) 147-164
10 . - - - - - - - - - - - - - - - - - - - - - .
8
2 4 6 8
Incubation
Time d)
Fig. 4. Growth
and
biofilm formation by
E coli 0157:H7
in MSM-O.04 glucose. Open symbols
represent
growth (log
CFU
1m )
at
room
temperature
0)
and at
lOoC
(l .).
Filled symbols
are
biofilms
(log
CFU/cm
2
at
room
temperature
e)
and
at lOoC
. ) .
3 4
Attachment studies
Our studies indicated that fewer cells (10
4
CFU/cm
2
) were recovered from SS
surfaces after 2 days of growth in TSB compared to MSM-0.04 glucose
10
6
Fig. 5. Scanning electron microscopy of E coli
0157:H7
biofilm developed in MSM-O.04 glucose
at
lOoC for 8 days. Bar 1 110m
-
8/10/2019 Dewanti, 2005
11/18
R
Dewanti A.C.L. Wong/Int. 1. Food Microbiology 26 (1995) 147-164
157
CFU
/cm
2
,
even though the planktonic populations were similar (approximately
10
9
CFU/m ) (Figs. 1 and 4). Bacterial attachment is an initial step in biofilm
formation. The lower number of adherent cells in TSB could be partially due to
poor initial attachment to the
SS
surfaces. To examine this,
SS
chips were exposed
to
E. coli
0157:H7 suspended in TSB
or
MSM-0.04 glucose for 1 h. The results
indicated that bacterial attachment in TSB
3 X 10
4
CFU/cm
2
) was significantly
lower p < 0.025) than that in MSM-0.04 glucose (3.2
x 10
5
CFU
/cm
2
).
3.5. Detachment studies
Biofilms developed in MSM-0.04 glucose consisted of clusters
of
cells con
nected to each other and to the
SS
surface by ECM (Fig. 3a). In contrast, biofilms
developed in TSB consisted of sparsely scattered single cells.
In
addition, the
biofilm population in TSB decreased with time more rapidly (Fig.
1 .
This could be
due to the formation of a less stable biofilm which detached easily from the SS
surface.
The stability
of
biofilms developed in the two media was examined. Two-day old
biofilms on chips were shaken in PBS at 70 rpm for 30 min. Biofilms developed in
MSM-0.04 glucose adhered more strongly with only 11 5% of cells detached
while 97 2.5 of cells on SS developed in TSB dissociated during the treatment.
3.6. Hydrophobicity determination
Hydrophobic forces have been shown to play a role in bacterial attachment
(Lachica, 1990). Growth of bacteria in BP and in MSM-0.04 glucose showed
aggregation of cells which may be caused by changes in surface hydrophobicity. We
examined if there were measurable differences in surface hydrophobicity of plank
tonic and biofilm cells grown in TSB and MSM-0.04 glucose. Results are
presented in Table 2. A significant increase in hydrophobicity p < 0.025) was
observed when E
coli
0157:H7 was grown in MSM-0.04 glucose. Adherent
bacteria developed in MSM-0.04 glucose were also significantly more hydropho
bic when compared to planktonic cells from the same medium.
Table 2
Percent adhesion of planktonic and biofilm
E
coli
0157:H7
to hexadecane
Culture medium Type of cells adhesion to hexadecane a
[SD]b
TSB
TSB
MSM-0.04 glucose
MSM-0.04 glucose
Planktonic
Biofilm
Planktonic
Biofilm
a Average of six replicate samples.
b
[SDl, standard deviation.
c Significantly different from TSB grown cells
p