By Dorthe Bagge, Mette Hjelm, Charlotte Johansen, Ingrid Huber, and Lone Gram

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Shewanella putrefaciens Adhesion and Biofilm
Formation on Food Processing Surfaces

• By Dorthe Bagge, Mette Hjelm, Charlotte Johansen,
  Ingrid Huber, and Lone Gram
• Applied and Environmental Microbiology, May 2001
• p. 2319-2325, Vol. 67, No. 5

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Adherence and colonization of bacteria on the
surface

• Fouling of ship hulls
• Contamination of medical devices
• Corrosion of steel surfaces
• Contamination in food industry by spoilage
  bacteria

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Shewanella putrefaciens

• Marine
• Gram negative bacteria
• Plays a role as a spoilage bacterium of marine
  fish, vacuum-packed meats and chicken
• Capable of producing volatile sulfides, amines
  and fishy-smelling trimethylamine

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Shewanella putrefaciens

• Participates in biogeochemical cycling of metals
• Capable of reducing Fe (III) and Mn (IV) by
  anaerobic respiration
• Capable of adhering to and forming biofilms on
  different surfaces, including stainless steel
  plates
• Capable of producing sulfides and reducing iron
  causing corrosion of steel surfaces

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Objectives

• To study the ability of fish spoilage bacterium
  S. putrefaciens to adhere and form biofilm on
  food processing surface
• To evaluate factors influencing biofilm formation
  such as nutrient concentration and accompanying
  microflora

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Materials and methods

• S. putrefaciens strain A2 and P. fluorescens
  strain AH2 cultured on iron agar Lyngby at 25 C
• Adhesion in batch system
• 10 x 20 mm stainless steel disks, 1 mm thick
  clamped vertically in a sterile steel circular
  rack placed in a beaker
• S. putrefaciens strain A2 were precultured in TSB
  for 24 hour at 25 C

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• Bacteria were harvested by centrifugation and
  resuspended in phosphate-buffered saline (PBS)
• Sterile rack was immersed in dilute (17) TSB for
  30 minutes to form conditioning film
• The rack then transferred to a new sterile beaker
  containing S. putrefaciens suspended in PBS at
  different concentration
• Allowed adhesion to take place at room temperature

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Biofilm formation in batch system

• Growth medium (15 TSB or 17 TSB) was added
  instead of buffer for proliferation of bacteria
• S. putrefaciens was inoculated at 103 CFU/ml
• Allowed biofilm formation to take place at room
  temperature
• Also study the effect of iron excess or
  limitation and the effect of the presence of
  other bacteria (P. fluorescens)

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Adhesion and biofilm formation in flow system

• Using a modified Robbins device (MRD)
• A suspension of 108 CFU of
• S. putrefaciens /ml of PBS was circulated in MRD
  at a flow rate of 10 ml/min
• Biofilm formation was investigated with food
  processing surfaces, stainless steel and
  polypropylene

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Microscopic evaluation of adhesion and biofilm
formation

• Disk were rinsed in 5 ml sterile PBS to remove
  nonadherent bacteria
• Using fluorescence microscopy to estimate number
  of attached bacteria
• Mixed biofilms of S. putrefaciens and P.
  fluorescens were estimated using a specific
  rRNA-targeted oligonucleotide

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Quantification of attached bacteria

• Enumeration of attached bacteria by indirect
  conductance measurement
• Measure the detection time which is inversely
  related to the initial number of bacteria
• Used direct conductometric method to estimate
  numbers of adherent S. putrefaciens and P.
  fluorescens from a mixture
• Using sonication to validate number of adherent
  bacteria

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Calibration curves relating detection time to
colony counts

• Calibration curves for biofilm flow system had a
  lower intersect compared to adhesion
• Detection time decreased with increasing initial
  counts of bacteria
• Conduction time reflected the numbers of surface
  associated bacteria

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Adhesion of S. putrefaciens suspended in PBS to
stainless steel in batch system

• S. putrefaciens increase from 102 CFU/ml to 105
  CFU/ml after 8 h of incubation
• Adhesion was facilitated by the formation of an
  initial conditioning film on TSB
• The number of bacteria adhering reflected the
  level of bacteria in suspension
• Reached stationary state in 8 hours

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Adhesion of S. putrefaciens to stainless steel as
monoculture or mixed culture with P. fluorescens

• Adhesion not systematically influenced by the
  presence of
• P . fluorescens

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Microscopic examination of the mixed culture of
S. putrefaciens and P. fluorescens
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Adhesion of S. putrefaciens in flow system (MRD)

• High number of bacteria in the circulating
  suspension (108 CFU/ml)
• Bacteria adhered slowly, 102 CFU/cm2 after 30
  hour!
• No effect on preconditioning surface

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Biofilm formation of S. putrefaciens in batch
system

• 106 107 CFU/cm2 in 1-2 days
• Glucose, lactate or iron had no effect on biofilm
  formation
• Iron chelator EDDHA caused slower growth

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Biofilm formation of S. putrefaciens on stainless
steel as monoculture or mixed culture with P.
fluorescens

• Addition of competing organism (P. fluorescens)
  decreased the number of S. putrefaciens between
  25 h and 100 h

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Biofilm formation of S. putrefaciens in flow
system on different surfaces

• Surface type influenced biofilm formation
• Lower number of bacteria adhering to
  polypropylene than stainless steel

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Other spoilage bacteria

• L. monocytogenes can also attach to stainless
  steel
• L. monocytogenes reached the maximum attached
  number in 3 hours, whereas S. putrefaciens need
  almost 8 hours

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Conditioning film vs nonconditioning film

• Salmonella enterica serovar Typhimurium adhesion
  is facilitated by an initial organic layer (a
  conditioning film)
• Pseudomonas fragi and L. monocytogenes are found
  in nonconditioned surface

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Reducing the adhering bacteria on stainless steel
surface

• Increasing the flow across the surface
• Increasing the shear stress (esp. for P.
  fluorescens)
• Some food components, e.g. milk proteins may
  decrease attachment of bacteria

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Conclusion

• Fish spoilage bacterium S. putrefaciens is able
  to attach and form biofilm on food processing
  surface
• Maximum number of adhering bacteria per square
  centimeter were reached in 8 h at 25 C and
  reflected the cell density in suspension
• Number of adhering bacteria were much lower in
  laminar flow system than in batch system

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• The rate of biofilm formation and the thickness
  of the film not dependent on carbohydrate
  (lactate and glucose) or on iron starvation
• The number of S. putrefaciens bacteria on the
  surface was partly influenced by the presence of
  other bacteria (P. fluorescens) that reduced the
  number of S. putrefaciens in the biofilm