528 Modern Food Microbiology
where nutrients are brought in and toxic by-products are carried out. Microbial cells in liquids that are
not in a biofilm are in a planktonic (free-floating) state.
From the standpoint of food safety and spoilage, biofilms are important because of their accumulation
on foods, food utensils, and surfaces; and because of the difficulty of their removal. While under natural
conditions, biofilms tend to be composed of mixed cultures, pure culture systems are often used in
laboratory studies. Some of the solid surfaces employed to study foodborne bacteria include floor
sealant, glass slides, nylon, polycarbonate, polypropylene, rubber, stainless steel, and Teflon. Glass
and stainless steel are widely used. From some of the many studies that have been reported in food
environments, the following summaries can be made:
1. Although biofilm formation by single cultures in rich media (e.g., tryptic soy broth) may be
evident after 24 hours when appropriate growth temperatures are used, 3–4 days or more are
necessary for maximum development. On glass slides in a culture medium for 3 days at 24
◦ C,
L. monocytogenes grew to about 6–7 log 10 /cm
21 .
2. Not all strains of the same species are equally capable of initiating biofilm formation,
74 and
surface attachment and biofilm development are different processes.
63
3. Microorganisms in biofilms may exhibit different physiologic reactions than planktonic forms,
and the biofilm may contain cells in the viable but nonculturable state.
18,22
4. Microorganisms in biofilms are considerably more resistant to removal by commonly used
cleaning and sanitizing agents, and cleaners and sanitizers used in combination appear to be
more effective in removing biofilm growth.
1,77
5. The attachment of a given pathogen to surfaces may be aided by the formation of a mixed-culture
biofilm,
17,68,88 and an example is presented below.
In a biofilm with L. monocytogenes and a Flavobacterium sp. growing together on stainless steel,
the attachment of L. monocytogenes was increased and persisted longer in mixed culture than when it
grew alone.
15 In addition, sublethally injured L. monocytogenes cells increased significantly in mixed
culture.
Shewanella putrefaciens readily formed biofilms on inert food processing surfaces, and when
nutrients were supplied, multilayered structures were formed.
7 Three strains of L. monocytogenes,
each from a human outbreak, produced unique “honeycomb” structured biofilms on stainless steel
coupons, and strain Scott A was the most conspicuous.
71 A laboratory strain did not form a biofilm. In
another study, the L. monocytogenes strains that produced the most extra polymeric substance (EPS)
produced a three-dimensional biofilm structure in contrast to control strains.
14 Biofilm formation could
not be correlated with serotypes. Employing a strain of Pseudomonas aeruginosa, extracellular DNA
was found to be essential for biofilm formation using a flow-chamber system.
103 Although the source
of DNA was not determined, the application of DNase I dissolved the biofilm, suggesting that DNA
was an integral part of the biofilm structure.
As noted above, cells in the viable but nonculturable (VBNC) state can form biofilms, and the
VBNC cells of Enterococcus faecalis adhered to Caco-2 and Girardi heart cells, but at a reduced
capacity compared to controls.
82
The inhibition of biofilm formation by Bacillus subtilis has been demonstrated using furanone ([5Z ]4-brome-5-[bromomethyhlene]-3-butyl-2[5H ]-furanone), and it inhibited both growth and swarming
motility of B. subtilis.
83 It was originally isolated from a marine alga. For more on biofilms, see
references 26, 45, and 109.
where nutrients are brought in and toxic by-products are carried out. Microbial cells in liquids that are
not in a biofilm are in a planktonic (free-floating) state.
From the standpoint of food safety and spoilage, biofilms are important because of their accumulation
on foods, food utensils, and surfaces; and because of the difficulty of their removal. While under natural
conditions, biofilms tend to be composed of mixed cultures, pure culture systems are often used in
laboratory studies. Some of the solid surfaces employed to study foodborne bacteria include floor
sealant, glass slides, nylon, polycarbonate, polypropylene, rubber, stainless steel, and Teflon. Glass
and stainless steel are widely used. From some of the many studies that have been reported in food
environments, the following summaries can be made:
1. Although biofilm formation by single cultures in rich media (e.g., tryptic soy broth) may be
evident after 24 hours when appropriate growth temperatures are used, 3–4 days or more are
necessary for maximum development. On glass slides in a culture medium for 3 days at 24
◦ C,
L. monocytogenes grew to about 6–7 log 10 /cm
21 .
2. Not all strains of the same species are equally capable of initiating biofilm formation,
74 and
surface attachment and biofilm development are different processes.
63
3. Microorganisms in biofilms may exhibit different physiologic reactions than planktonic forms,
and the biofilm may contain cells in the viable but nonculturable state.
18,22
4. Microorganisms in biofilms are considerably more resistant to removal by commonly used
cleaning and sanitizing agents, and cleaners and sanitizers used in combination appear to be
more effective in removing biofilm growth.
1,77
5. The attachment of a given pathogen to surfaces may be aided by the formation of a mixed-culture
biofilm,
17,68,88 and an example is presented below.
In a biofilm with L. monocytogenes and a Flavobacterium sp. growing together on stainless steel,
the attachment of L. monocytogenes was increased and persisted longer in mixed culture than when it
grew alone.
15 In addition, sublethally injured L. monocytogenes cells increased significantly in mixed
culture.
Shewanella putrefaciens readily formed biofilms on inert food processing surfaces, and when
nutrients were supplied, multilayered structures were formed.
7 Three strains of L. monocytogenes,
each from a human outbreak, produced unique “honeycomb” structured biofilms on stainless steel
coupons, and strain Scott A was the most conspicuous.
71 A laboratory strain did not form a biofilm. In
another study, the L. monocytogenes strains that produced the most extra polymeric substance (EPS)
produced a three-dimensional biofilm structure in contrast to control strains.
14 Biofilm formation could
not be correlated with serotypes. Employing a strain of Pseudomonas aeruginosa, extracellular DNA
was found to be essential for biofilm formation using a flow-chamber system.
103 Although the source
of DNA was not determined, the application of DNase I dissolved the biofilm, suggesting that DNA
was an integral part of the biofilm structure.
As noted above, cells in the viable but nonculturable (VBNC) state can form biofilms, and the
VBNC cells of Enterococcus faecalis adhered to Caco-2 and Girardi heart cells, but at a reduced
capacity compared to controls.
82
The inhibition of biofilm formation by Bacillus subtilis has been demonstrated using furanone ([5Z ]4-brome-5-[bromomethyhlene]-3-butyl-2[5H ]-furanone), and it inhibited both growth and swarming
motility of B. subtilis.
83 It was originally isolated from a marine alga. For more on biofilms, see
references 26, 45, and 109.