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. It 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
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. It 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
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 X 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.
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, liS 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
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
CFU jml 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 Ff 510,
barrier filter LP 520). Bacterial cells in ten randomly chosen fields from each chip
were counted, and the average number of 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
Ao-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.
152 R. Dewanti, A.C.L. Wong I Int. 1. Food Microbiology 26 (1995) 147-164
1 0 ~ - - - - - - - - - - - - - - - - - - - -
8
CII
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,
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).
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.
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 lOOC 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
CFU Iml 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
CFU Iml after 2 days, while adherent bacteria were 10
6
CFU I 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.
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.
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 < 0.025).
o
o
5.3 [0.85] c
8.7 [0.20) c,d
d Significantly different from MSM-0.04% glucose grown planktonic cells (p < 0.025).
158 R. Dewanti, A.C.L. Wong / Int. 1. Food Microbiology 26 (1995) 147-164
Table 3
Effect of media transfer on E. coli 0157:H7 biofilm
Transfer of biofilm
From TSB to MSM
From TSB to TSB
From MSM to TSB
From MSM to MSM
Before transfer
CFU/cm
2
1.2 X 10
4
1.7 X 10
4
4.7x10
5
8.9x10
5
Nature of
biofilm
S
S
DE
DE
After transfer
CFU/cm
2
Nature of
biofilm
7.6x10
4
ME
2.0xl0
4
S
9.8x10
4
E
6.3 x 10
5
DE
S = sparse single cells with no apparent extracellular matrix; ME = medium density of cells with
extracellular matrix; DE = dense clusters of cells with extracellular matrix; E = extracellular matrix
only; very few cells observed.
3.7. Media transfer studies
Our results indicated that TSB supported poor initial attachment and promoted
detachment of biofilm cells. The opposite effects were observed with MSM-0.04%
glucose. To determine if a change in nutrient conditions would alter the nature of
biofilms established in a different growth medium, a series of media transfer
studies were conducted. The results are shown in Table 3. When biofilms devel-
oped on chips from a 2 days TSB culture were transferred to MSM-0.04% glucose
for 4 days, an increase in the adherent cell population was observed. The biofilm
bacteria after the transfer were similar to those established in MSM-0.04%
glucose, i.e. smaller size cells with thicker ECM. The biofilm density was less than
that developed in MSM-0.04% glucose only for 2 days. Transferring chips from
TSB to TSB caused no change in the number of adherent bacteria. As observed
previously, very few single cells were seen under SEM on the SS chips.
When biofilms on SS chips from a 2 days MSM-0.04% glucose culture were
transferred to TSB for 4 days, there was a decrease in the number of adherent
bacteria. After the transfer, it was observed with SEM that the ECM was present,
but very few bacterial cells remained.
4. Discussion
Escherichia coli 0157:H7 has been found to adhere to rat intestine in vitro
(Sajjan and Forstner, 1990) as well as to INT 407 tissue culture cells (Junkins and
Doyle, 1992), but its ability to interact with inert surfaces has not been reported. In
this study, it was demonstrated that the bacteria were able to attach and subse-
quently form biofilms on SS when grown under certain culture conditions.
Biofilm formation occurs in several phases: substratum conditioning, where
organic molecules are deposited in the surface from the liquid phase, reversible
attachment of bacteria, irreversible attachment, bacterial growth and extracellular
matrix production, and detachment (Characklis, 1990). It was found that initial
attachment, detachment and characteristics of biofilms of E. coli 0157:H7 were
affected by the nutrient status of the medium in which the biofilm was developed.
R. Dewanti, A.C.L. Wong / Int. J. Food Microbiology 26 (1995) 147-164 159
In complex media, biofilms developed faster and higher numbers of adherent
cells were recovered when the apparent growth rate of planktonic cells was slower.
Biofilms developed in TSB appeared as single cells without significant amounts of
ECM, and decreased numbers of adherent cells were observed during prolonged
incubation. Detachment studies showed that adherent bacteria developed in TSB
dissociated easily from SS by mild agitation in PBS. These observations suggested
that the association between E. coli 0157:H7 and the SS surface was not strong
enough to maintain the biofilm population. Weak association of bacteria on SS
may have resulted in detachment of some cells during SEM preparation which
involved many steps such as fixation, dehydration and coating (Chang and Rittman,
1986). Biofilm development occurred faster when nutrient availability in the
medium was lower. In addition, significant ECM production also appeared to be
associated with low nutrient levels. In 1/5 TSB, biofilms with ECM were observed
after 8 days of incubation. In BP, which contained only 0.1% peptone, nutrients
probably were depleted faster and biofilms with ECM were observed at 3 days.
Extensive biofilm formation was observed in MSM-0.04% glucose at 2 days.
Biofilms developed in this medium also appeared different, consisting of shorter
cells and thicker ECM as compared to those formed in complex media. Reduction
in bacterial size during starvation conditions in Vibrio has been reported and is
considered a survival strategy of some bacteria (Humphrey et aI., 1983; Kjelleberg
et aI., 1983). Following size reduction, bacterial membrane fatty acid composition
changed, resulting in more hydrophobic cells which were more adhesive (Kjelle-
berg and Hermansson, 1984; Kjelleberg et aI., 1987). Marine vibrios starved for as
little as 5 h have been shown to attach better to siliconized glass (Dawson, 1981).
In MSM-0.04%, the E. coli 0157:H7 cells are probably nutrient stressed. They
became more hydrophobic than cells grown in TSB (Table 2), which may partially
account for the aggregation of planktonic cells in MSM-0.04% glucose. The
increase in cell mass would in turn facilitate sedimentation onto the SS surfaces
and result in a higher degree of initial attachment. Subsequent development of
ECM helped stabilize the biofilm.
Response of E. coli 0157:H7 biofilm cells to nutrient conditions was further
supported by results of the media transfer studies. About 80% of adherent bacteria
present in the biofilm developed in MSM-0.04% glucose dissociated after being
transferred to TSB (Table 3), probably in response to the availability of nutrients
in TSB. In contrast, adherent bacteria transferred from TSB to MSM-0.04%
glucose developed into biofilms with extensive ECM.
Our results indicated that increasing the glucose concentration from 0.04% to
0.1 and 1 % did not have any significant effect on the number of adherent bacteria
as quantitated by plate count. With SEM, however, it was observed that biofilm
populations in MSM-0.1% or MSM-1.0% glucose were less dense than that in
MSM-0.04% (not shown). A higher proportion of the biofilm bacteria in MSM-
0.04% glucose may have been dead by 2 days. Marshall et aI. (1971) suggested that
attachment of Pseudomonas was favored when the glucose concentration was 0.7%
and reduced when 1.4 or 2.1 % glucose was present. A different result was reported
by Delaquis et aI. (1989) who showed that glucose depletion resulted in detach-
160 R. Dewanti, A.C.L. Wong/Int. 1. Food Microbiology 26 (1995) 147-164
ment of Pseudomonas fluorescens from glass surface. They reported that using
0.015, 0.03 and 0.08% glucose in a minimal medium reduced the biomass of
attached cells when glucose was depleted in the medium after 5, 6 and 9 h,
respectively. On the other hand, the biomass of adherent cells remained constant
for up to 24 h when 1 % of glucose was used. Use of different organisms and
shorter periods of attachment may partially account for different results in these
studies.
Substitution of glucose with lactose, glycerol, succinic acid or sodium pyruvate
did not affect the number of planktonic or adherent cells significantly. In contrast,
use of 0.04% mannose or 0.04% lactic acid decreased the number of adherent cells
significantly (p < 0.025). Regardless of the carbon source, short cells and thick
ECM were observed in these biofilms, suggesting that nutrient limitation was
maintained in MSM supplemented with carbon sources other than glucose. With
SEM, biofilm populations did not appear to be as dense as those developed in
MSM-0.04% glucose, suggesting that glucose was probably a better substrate for
stable biofilm formation.
Extracellular polymers have been reported as a means of bacterial adherence to
solid surfaces (Marshall et aI., 1971). The composition of these polymers is usually
not known, but it is generally believed to contain polysaccharide (Fletcher and
Floodgate, 1973). Our results showed that biofilms developed in 1/5 TSB, BP and
MSM possessed ECM connecting cell-to-cell and cell to SS surface. Biofilms
developed in TSB showed very little ECM and only single cells were observed by
SEM. Lack of extensive biofilms in TSB could be partially due to the low number
of adherent cells and/or lack of ECM. We used two methods of biofilm fixation
for SEM viewing. One method utilizes con-A which is specific for a-D-mannosyl
and a-D-glucosyl residues. The other utilizes ruthenium red which is specific for
acidic polysaccharide. Both methods gave similar results with biofilms developed in
TSB or MSM-0.04% glucose. This suggested that the ECM in E. coli 0157:H7
biofilms contained a polysaccharide component and that ECM plays a role in
stability of the biofilms.
Although ECM was not evident in biofilms developed in TSB, it was observed in
planktonic cells grown in this medium. This suggested that the ECM associated
with planktonic cells did not play a major role in biofilm formation in TSB.
Wrangstadh (1990) showed that Pseudomonas sp. strain S9 produced a peripheral
extracellular polysaccharide which reduced adhesiveness in bacterial cells. This
extracellular polysaccharide was produced transiently and caused bacterial detach-
ment from hydrophobic surfaces. It is possible that ECM observed in TSB grown
planktonic cells has properties similar to that observed by Wrangstadh.
ECM observed in biofilms developed in MSM-0.04% glucose appeared thicker
than that in complex media, suggesting that the ECM produced in MSM-0.04%
glucose might be different in quantity and/or quality from that produced in
complex media and from those associated with planktonic cells. Additionally, ECM
of planktonic cells developed in TSB or MSM-0.04% glucose appeared similar.
Christensen et aI. (1985) reported that Pseudomonas sp strain NCMB 2021
produced two types of extracellular polysaccharides. One was believed to assist
R. Dewanti, A.C.L. Wong / Int. 1. Food Microbiology 26 (1995) 147-164 161
initial attachment and the other was produced after attachment and resulted in
biofilm accumulation. In this study, ECM produced in MSM-0.04% glucose proba-
bly supported irreversible adherence between bacteria and SS surface leading to
stable biofilm formation. The number of adherent bacteria in this medium re-
mained relatively constant for up to 7 days and agitation in PBS did not cause
bacteria to detach easily from SS chips.
Bacterial attachment is one of the initial steps in biofilm formation, and is
affected by many factors including menstruum and surface composition. Our
results indicated that the number of bacteria attached to SS after 1 h in TSB was
significantly lower than that in MSM-0.04% glucose. This could be partly at-
tributed to the fact that TSB contains more proteins than MSM-0.04% glucose.
Adsorption of protein to inert surfaces has been shown in some instance to
increase bacterial attachment (Marshall, 1979). On the other hand, Fletcher (1975)
reported that bovine serum albumin, gelatin, fibrinogen and pepsin impaired
Pseudomonas attachment to polystyrene, and Helke et al. (1993) observed that milk
proteins decreased attachment of L. monocytogenes and S. typhimurium to SS.
It was interesting to note that under anaerobic conditions E. coli 0157:H7
developed biofilms that contained dense single cells without significant amounts of
ECM. Junkins and Doyle (1992) demonstrated decreased production of extracellu-
lar polysaccharide (EPS) by E. coli 0157:H7 when incubated at room temperature
anaerobically. This EPS was associated with the ability of E. coli 0157:H7 to
attach to INT 407 tissue culture cells.
Growth of E. coli 0157:H7 at lOoC has been reported (Hao and Bracket, 1993),
while Jones et al. (1987) showed unique outer membrane proteins synthesized by
E. coli grown at lOe. Our studies also showed that biofilms developed at lOOC or
anaerobically were very stable and did not detach easily (not shown). Other
mechanisms such as alterations in the bacterial cell surface, presumably due to the
presence of certain new outer membrane proteins as reported by Jones et al.
(1987), may have helped stabilize the attached cells without the requirement of
ECM.
This study demonstrated that E. coli 0157:H7 can adhere and form biofilm to a
surface commonly used in food processing environments. The process is affected
by nutrient conditions and environmental factors such as temperature and atmo-
sphere. Further studies are being pursued to study the physiology of biofilm
formation by this pathogen under conditions found in meat processing environ-
ments and to assess methods for biofilm removal and/or inactivation so that risk
of contamination by this pathogen can be minimized.
Acknowledgements
This work was supported by contributions to the Food Research Institute, and
by the Inter University Center of Food and Nutrition, Bogor Agricultural Univer-
sity, Indonesia.
162 R. Dewanti, A.C.L. Wong / Int. 1. Food Microbiology 26 (1995) 147-164
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