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Applied and Environmental Microbiology, May 2001, p. 2319-2325, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2319-2325.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Shewanella putrefaciens Adhesion and
Biofilm Formation on Food Processing Surfaces
Dorthe
Bagge,1,*
Mette
Hjelm,1
Charlotte
Johansen,2
Ingrid
Huber,3,
and
Lone
Gram1
Danish Institute for Fisheries Research,
Department of Seafood Research, c/o Technical University of
Denmark, DK-2800 Kgs. Lyngby,1
Novozyme A/S, 1A.1, DK-2800 Bagsværd,2
and Biotechnological Institute, DK-2970
Hørsholm,3 Denmark
Received 29 December 2000/Accepted 28 February 2001
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ABSTRACT |
Laboratory model systems were developed for studying
Shewanella putrefaciens adhesion and biofilm formation
under batch and flow conditions. S. putrefaciens plays a
major role in food spoilage and may cause microbially induced corrosion
on steel surfaces. S. putrefaciens bacteria suspended in
buffer adhered readily to stainless steel surfaces. Maximum numbers of
adherent bacteria per square centimeter were reached in 8 h at
25°C and reflected the cell density in suspension. Numbers of
adhering bacteria from a suspension containing 108 CFU/ml
were much lower in a laminar flow system (modified Robbins device)
(reaching 102 CFU/cm2) than in a batch system
(reaching 107 CFU/cm2), and maximum numbers
were reached after 24 h. When nutrients were supplied, S.
putrefaciens grew in biofilms with layers of bacteria. The rate
of biofilm formation and the thickness of the film were not dependent
on the availability of carbohydrate (lactate or glucose) or on iron
starvation. The number of S. putrefaciens bacteria on
the surface was partly influenced by the presence of other bacteria
(Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the
biofilm. Numbers of bacteria on the surface must be quantified to
evaluate the influence of environmental factors on adhesion and biofilm
formation. We used a combination of fluorescence microscopy
(4',6'-diamidino-2-phenylindole staining and in situ hybridization, for
mixed-culture studies), ultrasonic removal of bacteria from surfaces,
and indirect conductometry and found this combination sufficient to
quantify bacteria on surfaces.
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INTRODUCTION |
The behavior of adherent and
growing bacteria on surfaces has received increasing attention during
recent years. Examples of such behavior include fouling of ship hulls
(5, 23) or contamination of medical devices (28,
36); also, the benefits of floc formation in sludge systems are
being studied (3). Biofilm formation by sulfide-producing
bacteria poses special problems, as corrosion of steel surfaces
(microbially induced corrosion) can take place (6, 25).
The food industry has also realized that adhesion and colonization of
bacteria may cause problems (22). Bacteria colonizing the
processing equipment may be an important source of bacterial contamination, and studies have shown that both spoilage bacteria like
Pseudomonas spp. (29) and pathogenic bacteria
like Listeria monocytogenes may contaminate products
directly from the processing environment (1, 30). Despite
rigorous cleaning and disinfection procedures, pathogenic bacteria and
spoilage microorganisms may be isolated from many types of surfaces in
food processing plants (B. Fonnesbech Vogel, personal communication).
This may at least partly be explained by an increased resistance of
adherent bacteria to adverse conditions like disinfection (21,
32, 41).
Shewanella putrefaciens is a marine, gram-negative bacterium
which is of importance in many areas. In the food industry, it plays a
role as a spoilage bacterium of marine fish, some vacuum-packed meats,
and chicken due to its ability to produce volatile sulfides, amines,
and the fishy-smelling compound trimethylamine. In the environment,
S. putrefaciens participates in the biogeochemical cycling
of metals due to its ability to reduce a variety of compounds [e.g.,
Fe(III) and Mn(IV)] by anaerobic respiration (9, 31). S. putrefaciens is capable of adhering to and forming
biofilms on different surfaces. Thus, Obuekwe et al. (33)
showed that a thick fibrous biofilm was formed on stainless steel
plates. Also, the adherence of S. putrefaciens to stainless
steel surfaces coupled with its ability to produce sulfides and reduce
iron explains its importance in microbially induced corrosion of steel
surfaces (6, 7).
Despite the potential of this bacterium to adhere and grow on surfaces
and the growing awareness of adherent bacteria in the food industry,
little is known about the ability of this spoilage bacterium to adhere
to different types of surfaces and its ability to persist in the
seafood processing environment. The current study was undertaken to
develop models to study the ability of the fish spoilage bacterium
S. putrefaciens to adhere and form biofilms on food
processing surfaces. Factors influencing biofilm formation by S. putrefaciens, such as nutrient concentration and accompanying
microflora, were evaluated.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
S.
putrefaciens strain A2 (35) and Pseudomonas
fluorescens strain AH2 (15) were cultured on iron
agar Lyngby (Oxoid CM964) (14) at 25°C.
Adhesion and biofilm formation in batch systems.
Stainless
steel (AISI 316, unpolished) was cut into 10- by 20-mm disks with a
thickness of 1 mm. Sterile steel disks were clamped vertically in a
sterile steel circular rack placed in a beaker. The rack holds up to 20 disks in an arrangement which, when the rack is immersed in culture
medium, allows the free circulation of liquid. For adhesion studies of
S. putrefaciens, strain A2 was precultured in tryptone soya
broth (TSB; Oxoid CM129) for 24 h with agitation at 25°C. The
bacteria were harvested by centrifugation at 3,000 × g for 10 min and resuspended in phosphate-buffered saline
(PBS; 0.8% NaCl, 0.02% KCl, 0.144%
Na2HPO4, 0.024%
NaH2PO4; pH 7.4). The
sterile rack containing the disks was immersed in dilute (1:7) TSB for
30 min, allowing a conditioning film to be formed on the steel disks.
The rack containing the disks was transferred to a new sterile beaker
containing S. putrefaciens cells suspended in PBS at
different concentrations. Adhesion was allowed to take place on both
sides of the disks at room temperature under slow-stirring (250-rpm) conditions.
The biofilm formation of S. putrefaciens on stainless steel
was investigated in the batch system described above, except that growth medium (1:5 TSB or 1:7 TSB) was added instead of buffer, allowing the bacteria to proliferate. S. putrefaciens was
inoculated at an initial level of 103 CFU/ml. A
biofilm was allowed to develop on both sides of the stainless steel
disks at room temperature under slow-stirring (250-rpm) conditions. The
effect of iron excess or limitation was studied by addition of 100 µM
FeCl3 or 200 µM ethylenediamine di(o-hydroxyphenylacetic acid) (EDDHA; Sigma E-4135), and
the effect of extra carbohydrates was studied by the addition of 1% glucose. To study the effect of the presence of other bacteria, S. putrefaciens was coinoculated with a P. fluorescens strain, AH2, that was precultured in TSB at 25°C for
24 h. Different initial ratios of the two organisms were studied,
covering starting concentrations of
103:103 and
105:103 of S. putrefaciens and P. fluorescens, respectively.
Adhesion and biofilm formation in flow systems.
The adhesion
of S. putrefaciens to stainless steel disks was investigated
in a flow model system using a modified Robbins device (MRD) (Tyler
Research Corporation, Edmonton, Alberta, Canada). S. putrefaciens was precultured, and bacteria were harvested as described above. A suspension of 108 CFU of
S. putrefaciens/ml of PBS (pH 7.4) was circulated in the MRD
at a flow rate of 10 ml/min (equaling 0.0062 m/s). The formation of a
biofilm by S. putrefaciens in the MRD was investigated with different food processing surfaces, stainless steel and polypropylene. S. putrefaciens was precultured in TSB for 24 h at
25°C and then inoculated in TSB diluted 1:7 with sterile water. The
MRD was sterilized, and a conditioning film was allowed to develop by circulating TSB diluted 1:7 for 30 min, after which the MRD was inoculated for 3 h with a circulating suspension of approximately 106 CFU/ml in TSB diluted 1:7. Thereafter,
sterile TSB diluted 1:7 was continuously supplied at a flow rate of 0.5 ml/min (equaling 0.00031 m/s).
Microscopic evaluation of adhesion and biofilm formation.
The disks were rinsed in 5 ml of sterile PBS, and nonadherent or poorly
attached bacteria were removed by carefully placing the disk (both
sides) on sterile absorbent paper. Care was taken not to swab or rub
the disk at this stage. The amount of attached bacteria was estimated
by fluorescence microscopy after staining with 2 µg of
4',6'-diamidino-2-phenylindole (DAPI; Sigma D-9542) per ml for 5 min.
The surface was examined by direct fluorescence microscopy (Olympus BH2
fluorescence microscope with a 320- to 400-nm excitation filter and a
>420-nm barrier filter or a Zeiss Axioscope 20 microscope [Carl
Zeiss, Brock & Michelsen, Birkerød, Denmark] using a Zeiss
F31-600 filter set [excitation filter, D360/50; beam splitter, 400 dichroic long-pass emission; and barrier filter, D460/50]).
Mixed biofilms of S. putrefaciens and P. fluorescens were developed, and numbers of each organism were
estimated using a specific rRNA-targeted oligonucleotide. The disks
were fixed in 4% paraformaldehyde and hybridized at 46°C with two
rRNA-targeted oligonucleotides using 5 (and 6)-carboxytetramethyl
rhodamine for S. putrefaciens and fluorescein
isothiocyanate (FITC) for P. fluorescens (I. Huber, B. Spanggaard, K. F. Appel, L. Gram, and T. Nielsen, unpublished
data). A minimum of 10 fields were examined for each plate under
a Zeiss Axioscope 20 microscope using the following filter sets: red,
HQ-Cy3; excitation filter, HQ545/30; beam splitter, Q565LP; and barrier
filter, HQ610/75; green, excitation filter, HQ480/40; beam splitter,
Q505LP; and barrier filter, HQ535/50 (for TAMRA and FITC, respectively).
Quantification of attached bacteria by indirect conductometry or
removal by sonication.
Attached bacteria were also enumerated by
indirect conductance measurements (18). The steel disks
with the adherent bacteria were transferred to Malthus glass tubes
containing 3 ml of TSB as a growth medium. Growth of the adherent
bacteria causes development of CO2, which
diffuses into an inner tube, containing 0.5 ml of sterile 0.1 M NaOH.
Electrodes measure the conductance in the NaOH solution and thus the
change in conductance as CO2 dissolves in the
alkali. The time from the start of the measurement until a rapid change
(decrease) in conductance occurs, the so-called detection time, is
inversely related to the initial number of bacteria. The detection time
can be related to the initial number of bacteria by use of a
calibration curve constructed by using a 10-fold dilution series of
bacteria. Calibration curves were constructed for each separate system,
e.g., bacteria suspended in PBS to study attachment or bacteria from
the liquid TSB used to study biofilm formation. Also, calibration
curves were constructed at several time points during an experiment to
evaluate the effect of growth phase on detection times. An estimate of
numbers of adherent S. putrefaciens bacteria from a mixture
also containing P. fluorescens was made using a direct
conductometric method. The disks with a mixed biofilm were transferred
to Malthus glass tubes containing 8 ml of a nutrient broth containing
1 g of trimethylamine oxide (TMAO) per liter (42).
S. putrefaciens reduces the neutral TMAO to ionized
trimethylamine by anaerobic respiration and causes an increase in
conductance (11, 35). Pseudomonads, which do not respire
using TMAO, cause no change in electrical properties of the medium.
To validate the numbers of adherent bacteria derived from the
conductometric measurements and conversion via the standard
curve,
parallel experiments were conducted in which four disks
from an
experiment were used for conductometric measurements and
four disks
were used to enumerate bacteria after removal by sonication.
The rinsed
disks were placed in 5 ml of PBS, and bacteria were
removed from the
surface by two treatments for 10 s each using
an MSE Soniprep 150 ultrasonic disintegrator (Sanoy, Integrated
Services, TCP Inc.) at 27 kHz. The disks were rinsed with 5 ml
of PBS into the same tube. Colony
counts were determined by 10-fold
serial dilution and plating onto iron
agar Lyngby (Oxoid CM964).
The tubes were placed on ice before
sonication, and the temperature
did not exceed 22°C during
treatment.
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RESULTS |
Calibration curves relating detection times to colony counts.
The detection times, as expected, decreased with increasing initial
counts of bacteria. Comparing initial CFU per Malthus cell to detection
times gave, for all systems studied, a linear relationship. The
calibration curves were similar as S. putrefaciens in the
batch system gave rise to statistically identical curves independently
of being grown in 1:7 TSB or suspended in PBS (data not shown).
Calibration curves made from bacteria released from the biofilm flow
system had a significantly lower intersect with the y axis
(Fig. 1), indicating a shorter lag phase
than for bacteria taken from the batch system. Identical curves were
obtained for the indirect measurement and the direct measurement (data
not shown). All CFU per square centimeter reported below were derived from the appropriate calibration curve. As the curves cover a range of
1 × 101 to 5 × 106 CFU/Malthus cell, higher or lower counts are
derived by extrapolation of the standard curve. The bacteria used to
develop the calibration curves, although taken from suspensions in
which surfaces and adhernt bacteria are immersed, are not surface-bound
bacteria.
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FIG. 1.
Comparison of CFU of S. putrefaciens per
milliliter determined by colony counts with conductometric detection
times (duplicate samples) determined by indirect measurements for
adhesion ( ) and biofilm formation ( ) in flow systems.
Measurements were made at 25°C. Ninety-five percent confidence
intervals are shown.
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To verify that the conductance detection times did reflect the numbers
of surface-associated bacteria, we compared CFU per
square centimeter
as derived from the calibration curve with the
number of bacteria from
a parallel set of stainless steel disks
from which bacteria were
removed by sonication and enumerated
by plate counts. Excellent
agreement was obtained between the
numbers determined by the two
methods (Fig.
2). We used two 10-s
treatments, which had no effect on the viability of cells and
were as
effective as longer treatments (e.g., two 30-s treatments).
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FIG. 2.
Colony counts per square centimeter of stainless steel
disks as determined by use of conductance measurements and
transformation by the standard curve (open bars) or by use of
ultrasound removal of cells and subsequent enumeration by plate counts
(hatched bars). Error bars are standard deviations of quadruplicate
determinations.
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Adhesion of S. putrefaciens in batch systems.
S. putrefaciens adhered readily to stainless steel disks,
increasing from 102 CFU/cm2
immediately after immersion to 105
CFU/cm2 after 8 h of incubation (Fig.
3a). The adhesion was facilitated by the
formation of an initial conditioning film of TSB, as a significantly
lower level of bacteria adhered to disks on which no conditioning film
had been formed (Fig. 3a). The number of bacteria adhering reflected
the level of bacteria in suspension (Fig. 3b). Within 8 h, the
adhesion reached a stationary state corresponding to the number of CFU
per milliliter in the suspension. The number of bacteria in suspension
did not increase during this 8-h period; however, growth did occur when
the adhesion experiment was extended for 24 h (data not shown).
The number of adherent S. putrefaciens bacteria was not
systematically influenced by the presence of P. fluorescens
(Fig. 4).
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FIG. 3.
Adhesion of S. putrefaciens suspended in
PBS to stainless steel. (a) 105 CFU/ml adhering to
conditioned () or nonconditioned surfaces (). (b) Adhesion to
conditioned surfaces from suspensions with 105 ( ),
107 (), or 109 () CFU/ml. All experiments
were performed at 25°C with slow stirring. Counts are derived from
standard curves. Error bars are standard deviations of duplicate
samples.
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FIG. 4.
Adhesion of S. putrefaciens to stainless
steel as monoculture or mixed culture with P.
fluorescens. Bacteria were suspended in PBS at 25°C. Symbols:
, S. putrefaciens (106 CFU/ml); ,
S. putrefaciens plus P. fluorescens
(106:106 CFU/ml); , S.
putrefaciens plus P. fluorescens
(106:104 CFU/ml). Counts are derived from
standard curves in TMAO broth. Error bars are standard deviations of
duplicate samples.
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Microscopic examination of the mixed adhered cultures using in situ
hybridization showed an organized colonization of the
surface. Cells of
S. putrefaciens were evenly distributed, whereas
cells of
P. fluorescens clustered together (Fig.
5).
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FIG. 5.
Fluorescence microscopy of mixed adhesion of S.
putrefaciens and P. fluorescens to stainless
steel. The bacteria are visualized by 16S rRNA oligonucleotide probes
with TAMRA (red) for S. putrefaciens and FITC (green)
for P. fluorescens. Bacteria were suspended in PBS at
25°C.
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Adhesion of S. putrefaciens in flow systems.
The kinetics of adhesion was very different in the flow system from
that in the batch system. Despite a high number of bacteria (108 CFU/ml) in the circulating suspension,
bacteria adhered slowly, reaching approximately
102 CFU/cm2 after 30 h
and remaining at this level for the rest of the experimental period. No
difference was seen in numbers of adherent bacteria, depending on
preconditioning of the steel surface (Fig.
6). Initially, when low numbers adhered,
the standard deviation was large. The low number is based on
extrapolation from the calibration curve, and this could, in part, be a
reason for the large deviation.
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FIG. 6.
Adhesion of S. putrefaciens to stainless
steel in a flow system (MRD). With () or without () a
conditioning film, bacteria were suspended in PBS at 108
CFU/ml, and the suspension was recirculated at 25°C. Counts are
derived from standard curves. Error bars are standard deviations of
triplicate samples.
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Formation of S. putrefaciens biofilms in batch
systems.
The effects of nutrient and iron limitation, excess
glucose or iron, and an associated microflora on biofilm formation were investigated by culturing the bacteria in 1:7 diluted TSB. S. putrefaciens strain A2 formed a multilayered biofilm on the
stainless steel disks, reaching 106 to
107 CFU/cm2 in 1 to 2 days
(Fig. 7 and
8). The addition of glucose or iron had
no effect on biofilm formation in the batch system. Addition of the
iron chelator EDDHA caused slower growth; however, the biofilm
formation as a function of cell growth in the medium was similar to
iron-rich culture conditions (Fig. 7). The addition of a competing
organism (P. fluorescens) decreased the number of S. putrefaciens bacteria on the surface between 25 and 100 h
compared to the numbers adhering from a monoculture of S. putrefaciens (Fig. 9). As in the
adhesion experiment, P. fluorescens clustered whereas
S. putrefaciens bacteria were evenly distributed.
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FIG. 7.
Biofilm formation of S. putrefaciens on
stainless steel. S. putrefaciens was grown in 1:7 TSB
with no addition  , 100 µM FeCl3  , 200 µM EDDHA
 , 1% glucose  , 1% lactate  , or 1% lactate plus 100 µM
FeCl3  . All experiments were carried out at 25°C.
Counts are derived from standard curves. Error bars are standard
deviations of duplicate samples.
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FIG. 8.
DAPI staining of a biofilm of S.
putrefaciens on stainless steel. The bacteria were grown in 1:7
TSB at 25°C.
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FIG. 9.
Biofilm formation of S. putrefaciens on
stainless steel as monoculture or mixed culture with P.
fluorescens. Bacteria were grown in 1:7 TSB at 25°C. Symbols:
, S. putrefaciens (105 CFU/ml); ,
S. putrefaciens plus P. fluorescens
(105:105 CFU/ml); , S.
putrefaciens plus P. fluorescens
(105:103 CFU/ml). Counts are derived from
standard curves. Error bars are standard deviations of duplicate
samples.
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Formation of S. putrefaciens biofilms in flow
systems.
S. putrefaciens also formed biofilms on
stainless steel in a flow system. As for the adhesion process, a longer
time was required to reach a steady state (Fig.
10). Surface type influenced the biofilm formation, as there was a lower number of bacteria adhering to
polypropylene than to stainless steel (Fig. 10).
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FIG. 10.
Biofilm formation of S. putrefaciens in
flow systems on different surfaces, stainless steel () and
polypropylene (). Bacteria were grown in continuously freshened 1:7
TSB at 25°C. Counts are derived from standard curves. Error bars are
standard deviations of triplicate samples.
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DISCUSSION |
S. putrefaciens colonizes food and is an important food
spoilage bacterium. Its biofilm formation has been investigated due to
its role in microbially induced corrosion caused by production of
hydrogen sulfide and reduction of iron(III) (6, 33). We demonstrated in this study that S. putrefaciens readily
adheres to inert food processing surfaces (stainless steel and
polypropylene), and if nutrients are supplied, multilayered biofilms
are formed (Fig. 8). The adhesion of S. putrefaciens to
surfaces occurred rapidly. Similar kinetics has been reported for the
attachment of L. monocytogenes to stainless steel and
buna-N-rubber (39). L. monocytogenes reached
maximum attached numbers (104
CFU/cm2 from a suspension of
107 CFU/ml) in approximately 3 h, whereas
the number of adherent S. putrefaciens bacteria increased
for almost 8 h. Thus, during a normal shift (8 to 12 h) in a
food processing plant, there is ample time for the organism to attach
to surfaces.
In most environments, including the food processing industry, a layer
of organic material (a conditioning film) will rapidly be formed on
surfaces. We found that the adhesion of S. putrefaciens to
stainless steel was facilitated by a conditioning film of TSB (Fig.
3a). Hood and Zottola (17) found that, for some bacteria like Salmonella enterica serovar Typhimurium, adhesion to
stainless steel was similarly facilitated by an initial organic layer,
whereas higher numbers of both Pseudomonas fragi and
L. monocytogenes bacteria were found on nonconditioned
surfaces. Some food components, e.g., milk proteins, may actually
decrease attachment of bacteria (2). In theory, the thin
layer of organic material on the surface may also have allowed the
bacteria to multiply; however, application of rRNA probes revealed a
very low signal after 2 days, indicating that no growth took place
(unpublished data).
Increasing the flow across the surface dramatically reduced the number
of S. putrefaciens bacteria adhering to stainless steel (Fig. 6) even at the very low flow (0.0062 m/s) used. Also, Duddridge et al. (10) reported that increasing the shear stress
significantly reduced the number of P. fluorescens bacteria
attaching to a stainless steel surface. Reaching the maximum number of
S. putrefaciens bacteria under flow conditions took longer
(30 h) than under batch conditions. Harkes et al. (16)
found that Escherichia coli already after 3 h under
flow conditions had reached a maximum level of approximately 8 × 105 CFU/cm2. Formation of a
conditioning film had no effect on the adhesion; however, this could be
due to a washout of the film before adhesion commenced.
The simultaneous attachment of P. fluorescens had no effect
on the adhesion of S. putrefaciens (Fig. 4). In a study
determining the simultaneous attachment of a range of bacteria,
McEldowney and Fletcher (27) found that, in most cases,
the bacteria had no effect on each other. However, in some cases
bacteria decreased the attachment of others, whereas in one case, the
attachment of a Staphylocccus sp. strain was increased by
the simultaneous presence of an Acinetobacter strain.
Numbers of S. putrefaciens reached their maximum in the
biofilm stage after approximately 1 to 2 days in 1:7 TSB. Dewanti and
Wong (8) found that, depending on the nutrient medium, the
biofilm formation of E. coli reached a stationary level in 2, 8, and 5 days for TSB, 1:5 TSB, and Bacto Peptone, respectively. Further, they found that biofilm development occurred faster in Bacto
Peptone and reached a higher level of attached bacteria. Similarly, Kim
and Frank (20) showed that the attachment of L. monocytogenes was higher in D10 (low-nutrient medium) than in TSB,
and only a modification of D10, either by increase of ammonium chloride
or by reduction of iron, affected the attachment of L. monocytogenes. Both modifications showed a decrease in the attachment. McEldowney and Fletcher (26) showed that
changing the carbon source or the level of nitrogen resulted in
variations in the level of attachment of different bacteria. Therefore,
for some bacteria the nutrient level has been shown to affect the adhesion of bacteria as well as the biofilm formation
The biofilm formation of S. putrefaciens was not affected by
the concentration of glucose. Similarly, Kim and Frank
(20) found that glucose had no consistent effect on
biofilm formation of L. monocytogenes. In contrast, the
structure and thickness of a mixed-species biofilm were influenced by
addition of glucose (40). We speculated that lactate,
which is readily metabolized by S. putrefaciens, would be a
better carbohydrate source and thus would result in exopolysaccharide
production and thicken the biofilm; however, adding lactate had no
effect on biofilm formation.
Caccavo et al. (3) reported that flocs and/or
biofilms of Shewanella algae where influenced by iron(III)
as the addition of Fe(III) resulted in deflocculation. As S. putrefaciens also is a prominent Fe(III) reducer (9,
31), we evaluated the effect on biofilms of limiting iron or
adding a surplus. Neither had, in our study, any effect on the number
of bacteria adhering or on the kinetics of biofilm formation.
A bacterium will only in very selective niches exist as a pure culture.
Most commonly, the organism will be part of a community containing
several species. The fluorescent pseudomonads and S. putrefaciens will be the most common members of the microbial community in a range of chilled, proteinaceous foods (4, 13, 14), and we therefore monitored the adhesion and biofilm
formation of S. putrefaciens when coinoculated with P. fluorescens. The lower numbers of S. putrefaciens
bacteria adhering when grown with P. fluorescens could be
caused by the antagonistic capability of P. fluorescens
(15). In contrast, numbers of L. monocytogenes in a biofilm became higher and persisted for longer time when P. fragi was also present (37). This was believed to be
due to the extracellular matrix produced by P. fragi, which
embedded and protected the listeriae.
Many approaches have been used for quantifying bacteria on
nontransparent surfaces. If cell numbers are high, fluorescence microscopy is useful, although a detailed quantification is difficult when bacteria are arranged in clusters or layers. Some authors detach
bacteria from the surface by ultrasound, by mixing, or by swabbing
(38) and quantify these by standard plate counts thereafter. Ultrasonic removal of cells has been used by several authors (24, 34), and we found that this procedure for
quantification was in excellent agreement with counts obtained using
Malthus detection times converted via a standard curve. Similarly,
Flint et al. (12) used the Malthus method to determine the
number of thermophilic streptococci on stainless steel, and Johnston and Jones (19) used impedance measurements to examine the
number of adherent Proteus mirabilis, Staphylococcus aureus,
and P. aeruginosa bacteria in a disinfection experiment.
Also, the conductometric method may, by substrate manipulations, be
used for indicative (e.g., TMAO-reducing) or selective
quantification of specific bacteria.
In conclusion, we have found that the fish spoilage bacterium S. putrefaciens is able to attach and form biofilms on food processing surfaces. In contrast to our expectations, nutrient conditions did not affect biofilm formation, whereas the presence of
P. fluorescens reduced numbers of adhering S. putrefaciens bacteria.
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ACKNOWLEDGMENT |
The study was supported by a grant from The Danish Agency for
Trading and Industry of the Danish Ministry of Commerce.
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FOOTNOTES |
*
Corresponding author. Mailing address: Danish Institute
for Fisheries Research, Department of Seafood Research, Søltofts
Plads, c/o Technical University of Denmark, Bldg. 221, DK-2800 Kgs.
Lyngby, Denmark. Phone: 45 45 25 49 28. Fax: 45 45 88 47 74. E-mail:
dob{at}dfu.min.dk.
Present address: GeneScan Europe AG, Mikrobiologie, D-79108
Freiburg im Breisgau, Germany.
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Applied and Environmental Microbiology, May 2001, p. 2319-2325, Vol. 67, No. 5
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.5.2319-2325.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
