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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

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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

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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.

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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

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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.

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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,

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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).

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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

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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.

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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

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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