MICROBIAL BIOFILM, FOOD SAFETY, AND FOODBORNE

DISEASE

A Comprehensive Report
Submitted to
Professor Ivy C. Emnace, PhD.
Department of Food Science and
Technology
Visayas State
University Visca,
Baybay City, Leyte

In Partial fulfillment of the course

FTEC 231 – Advanced Food Microbiology

MA. RICKA C.
DUMAS MSFT – 1
 TABLE OF CONTENTS

INTRODUCTION

Definition of Biofilm

Historical Perspective of Biofilm

Composition of Biofilm

The Reason Behind the Formation of Biofilm

FORMATION OF BIOFILM

Development of a Surface Conditioning Film

Movement of Microorganisms into close proximity with

the surface Adhesion (Reverse and Irreversible

Adhesions of Microbes) Growth and Division of the

Organisms

Biofilm Cell Detachment/Dispersal

SIGNIFICANCE OF BIOFILM IN THE FOOD

INDUSTRY

MICROORGANISMS RESPONSIBLE FOR BIOFILM

FORMATION BENEFICIAL ASPECTS OF BIOFILM

BIOFILM CONTROL

CONCLUSION

REFERENCES
INTRODUCTION

Definition of Biofilm

A biofilm formation can be defined as a microbial community

characterized by its adhesion to a solid surface and by the production of an
extracellular polymeric matrix in which the associated microorganisms are
embedded. This matrix also provides shelter to the inner cells and could
contribute to absorbing nearby nutrients as well as adhesion to the surface
(Alvarez, et al., 2019). In addition, Wilder and Charaklis 1989 describes
biofilm as relatively indefinable microbial community randomly distributed
in a shaped matrix or glycocalyx. The formation of the biofilm is a social
behavior generally coordinated through cell-to-cell communication or
also called as “Quorum-sensing Systems”.

These formations are usually seen on surfaces that is immersed in

natural aqueous environment. Hence, these formations could commonly be
seen in the water systems, medical devices, kitchen, processing area that
involves high relative humidity and high moisture content. Biofilms,
particularly in water systems, may be highly complex while others such as
those on medical devices may be simpler, and composed of single, coccoid
or rod-shaped organisms.

The definition of a biofilm must be kept general and thus may be

redefined as “microbial cells immobilized in a matrix of extracellular
polymers acting as an independent functioning ecosystem,
homeostatically regulated” (Percival et al., 2009)

Historical Perspective of Biofilm

Anton Van Leeuwenhoek has first observed

“animalcules” swarming on living and dead
animals. Due to his continuous curiosity on these
tiny organisms, he sampled his own tartar and
discovered these “animalcules”. Despite after
meticulous cleansing, there were remaining
opaque deposits
isolated between his teeth (Chandki et al., 2011). These deposits contained
a mat of various forms of “animalcules” that we know now as dental plaque.
This was the first documented evidence of the existence of microbial
biofilms.

The so-called "bottle effect" in marine

microorganisms was not discovered until
1940, following van Leuwenhoek's
pioneering study (Heukelekian and Heller
1940). This demonstrated that when bacteria
were adhered to a surface, their growth was
greatly accelerated. When Zobell discovered
in 1943 that the quantity of bacteria on
surfaces was higher than that of the
surrounding saltwater, it significantly
advanced our understanding of biofilms.
Based on his research, Zobell also proposed
that bacterial adhesion was a two-step
process that started with reversible
adhesion and ended with irreversible
adherence. Jones et al. (1969) investigated
biofilms on trickling filters in a wastewater
treatment plant using scanning and
transmission electron microscopy.

This research demonstrated that a wide range of distinct bacteria

made up biofilms and that polysaccharides made up the majority of the
matrix material, or EPS. At this time, electron microscopy was a valuable
tool in the study of biofilms since it could provide details about the
structure of the biofilms as well as if EPS was present. When Characklis
studied microbial slimes in industrial water in 1973, he discovered that biofilms
were extremely resilient to the antibacterial actions of chlorine and also
quite persistent (Characklis 1973).

It wasn't until 1978 that the first accurate examination of biofilms

as a whole was acknowledged, as research revealed that many bacteria
lived their whole lives in surface- attached, sessile communities. Costerton
et al. (1978) did research on dental plaque and sessile communities in
mountain streams allowed them to hypothesize the ways in which
microorganisms.

Similar approaches have been taken in the research of biofilms in

industrial, ecological, and medicinal contexts since the 1970s. Early
research on biofilms mostly focused on the
composition, particularly on the polymer matrix, or "glycocalyx," which was
supposed to retain and concentrate the digestive enzymes that the bacteria
generated, boosting the cells' metabolic efficiency. This glycocalyx also
served as an ionic exchange matrix, capturing nutrients that were then
taken up by incredibly effective permeases and transferred into cells,
according to research by Costerton and Geesey (1979).

In 1981 (Costerton et al. 1981), glycocalyx was characterised as a

hydrated polyanionic polysaccharide matrix produced by polymerases
affixed to the lipopolysaccharide component of the bacterial cell wall.
Additionally, the glycocalyx provides a physical/chemical barrier that offers
partial protection against antibacterial agents.

Since biofilms form under diverse conditions, and may be composed of

single or multiple species, the structures of various biofilms will necessarily
have distinct features. In 1998, important advances to our understanding of
the development and behaviour of biofilms were made, when studies that
combined molecular genetic approaches with confocal laser scanning
microscopy (CLSM) emerged.

Bacteria are remarkably adept at surviving “feast and famine”, and

also adjusting their needs to accommodate highly diverse environments.
The capacity to form and maintain biofilms is key to these adaptations.

The Composition of Biofilm

Extracellular polymeric secretions (EPS) form a matrix around and

shield the microbial cells that make up a biofilm. Polysaccharides, proteins,
lipids, and extracellular DNA (eDNA) are the usual components of the EPS
matrix.

Extracellular enzymes (eEnzymes) are found within the extracellular

matrix (EPS) as depicted in the picture. Small DNA fragments known as
plasmids, which contain certain genes, are also present. Plasmids and e-
Enzymes are shielded from destruction by the biofilm. And lastly, bacteria
release signal molecules for quorum sensing, a chemical communication
process that will be covered later. Although a biofilm can grow from a few to
hundreds of micrometers above a surface, it has several innate adaptations
that planktonic cells lack. A significant portion of biofilm, up to 97%, is made
up of water, which is also what allows nutrients to move through the matrix
of the biofilm.
 The Reason Behind the Formation of Biofilm

There are several potential benefits obtained by a microbe in a biofilm

as opposed to a planktonic one. These include the production of copious
amounts of extracellular polymers, phenotypic changes in colony
morphology, increased expression of beneficial genes, plasmid transfer-
acquired antibiotic resistance genes, improved nutrient access, and closer
cell proximity that promotes mutualistic or synergistic associations and
protection (Costerton et al. 1987; Costerton and Lappin Scott 1989).

Safeguard them against environmental

stressors including dehydration and UV rays.
These adaptations rely heavily on the ability to
create and sustain biofilms. Since attachment
gives protection and some degree of control
over the nutritional environment, it is both
beneficial and possibly essential for microbes to
survive in the natural world.

They concluded that cells respond to adverse conditions by

modifications to the cell envelope that not only enhance survival, but also
change the adhesive properties of the cell environmental stresses.

STAGES FOR THE FORMATION OF BIOFILM

Biofilms can develop on many different surfaces, such as living

tissues found in natural aquatic systems, medical devices that are
implanted within, and pipes in industrial or potable water systems. These
biofilms, which release toxic and hazardous chemicals that are contained in
the biofilm matrix, can be benign or pathogenic. Biofilm formation is a
phenomena that affects most moist surfaces, plant roots, and almost all
living things. It can happen in both natural and artificial environments
and under a variety of circumstances. Because biofilm infections are
resistant to being eliminated by antimicrobials and host defense
mechanisms, they are extremely difficult to eradicate once they have
been established.
 The process of biofilm formation is complex, but generally
recognized as consisting of five stages (Palmer and White 1997):

1. Development of a surface conditioning

2. Movement of microorganisms into close proximity with the surface
3. Adhesion (reversible and irreversible adhesion of microbes to the
conditioned surface)
4. Growth and division of the organisms with the colonization of the
surface, microcolony formation and biofilm formation; phenotype and
genotype changes
5. Biofilm cell detachment/dispersal

Stage 1. Development of a Surface Conditioning Film

In the natural world, bacteria attach themselves to a conditioning film

that is known to form on most substrata rather than directly to a
substratum. The intricate makeup of the conditioning film causes a chemical
alteration of the initial surface, which in turn affects the degree and rate of
microbial attachment (Mittelman 1996).

It has been demonstrated that complex polysaccharides,

glycoproteins, and humic substances make up the conditioning layer in
both aquatic and terrestrial settings (Chamberlain 1992; Marshall et al.
1971; Baier 1980; Rittle et al. 1990). Comparatively, the complexity of
human host conditioning films can also depend on the conditioning site.
 One well-researched aspect of "biofilmology" is the contribution of
dental plaque to oral illness. Accordingly, a proteinaceous "pellicle" made up
of albumin, glycoproteins, lipids, lysozyme, phosphoproteins, and other
substances found in saliva and gingival crevicular fluid conditions the
enamel of teeth (Marsh 1995).

Another essential factor in the production of biofilms is the surface

topography that a microbial cell adheres to. Generally speaking,
bacterial adhesion rises with surface roughness (Characklis et al. 1990a).
This could be due to a number of factors, chief among them being that the
microorganisms are protected from the effects of shear pressures by the
shelter. Rough surfaces improve mass transfer coefficients and enable cells
to "anchor" to micro-irregularities, where they are better shielded from
potential desorption, in addition to expanding the available interfacial area
(Characklis et al. 1990b).

Apart from surface roughness, the physical and chemical

characteristics of the surface influence the adherence of bacteria, as
they adhere more quickly to non-polar, hydrophobic surfaces like Teflon
and other plastics, as opposed to hydrophilic materials like metals or
glass (Pringle and Fletcher 1983; Bendinger et al. 1993; Percival and
Thomas 2009).

Stage 2. Movement of Microorganisms into close proximity with the surface

Nutrients and microbial cells are typically transported to a surface via

a variety of well- known fluid dynamic processes including microbial motility.
These include gravity impacts (differ entail settling, sedimentation;
Characklis 1981), mass transport, and thermal processes (Brownian motion,
molecular diffusion).
 Laminar and turbulent flow are the two
primary flow regimes found in fluid transport
pipes. Laminar flow, which is characterized by
parallel smooth flow patterns with little to no
lateral mixing and the quickest flow in the
center, is clearly seen in the bloodstream and
urinary system (Fletcher and Marshall 1982;
Lappin-Scott et al. 1993).

Microorganisms and nutrients follow a

straight course and stay in a stable position
determined by the flow rate during laminar
flow (Lappin-Scott et al. 1993). Contrarily,
turbulent flow is haphazard and
unpredictable,
which eventually increases microbial adhesion (Percival et al. 1999) and the
mixing of bacteria and nutrients (Characklis et al. 1990a, b). This turbulent
flow increases the likelihood of adhesion by pushing or driving bacteria to
within close proximity to the surface due to random and unpredictable flow.
The likelihood of adhesion is known to be increased by a bacterium's
motility (Fletcher 1977; Marmur and Ruckenstein 1986).

Sufficient potential energy to

defeat any known repulsive interactions
acting between the bacterial surface. As
a result, cells with appendages like
fimbriae and pili overcome repulsion and
approach the substratum with a
decreased radius from the cell to the
surface. When the synthesis of
exopolymeric molecules begins, these
cells adhere to the substratum and
constitute irreversible cellular adhesion.
Adhesins are expressed in nonmotile
bacteria, which enhances adhesion to
both the surface and other bacteria.
Flagella and pili are essential for motile
bacteria's initial surface adhesion.
 Stage 3. Adhesion (Reverse and Irreversible Adhesions of Microbes)

Adhesion of the microorganism typically occurs as previously

mentioned, following surface conditioning and transport of the bacteria into
an area near the substratum surface. This process was first described in
1943 (Zobell 1943), and it involves two steps that involve both reversible
and irreversible processes.

Both irreversible and permanent bonding of microorganisms to a

surface are referred to as reversible adhesion (Rittman 1989). In
reversible adhesion, bacteria still exhibit Brownian motion and are easily
removed by fluid shear forces, such as simply washing the bacteria off the
surface.

Stronger pressures, like scouring or scraping, are needed for the

hydrophobic contact, dipole-dipole interactions, hydrogen, ionic, and
covalent bonding, and other short-range forces involved in irreversible
adhesion (Marchall et al., 1971).

The separation between the surface and the microbe. A

microorganism and a surface undergo particular and electrostatic
interactions when the distance between the two is smaller than 1.5 nm.
Hydrophobic amino acid residues are abundant in fibrillae (Rosenberg and
Kjelleberg 1986). This feature might influence the hydrophobicity of a cell
surface and, consequently, its ability to bind to a surface. These fimbriae
most likely have the ability to get past the first barrier of electrostatic
repulsion that separates the cell from the substratum (Corpe 1980; Bullitt
and Makowski 1995).
Stage 4. Growth and Division of the Organisms
 The adsorption of macromolecules and attachment of microbial cells
to a substratum are only the initial stages of biofilm development. These
are followed by microbial growth, development of microcolonies and
recruitment of additional microorganisms.

As attachment of microorganisms occurs, the colonising bacteria

grow with the production and accumulation of extracellular polymers. The
microorganisms eventually become embedded in this hydrated polymeric
matrix and immobilised. As a result, the cells are dependent upon substrate
flux from the liquid phase and/or exchange of nutrients with neighboring
cells in the biofilm.

An important feature is that the microorganisms are immobilized in

relatively close proximity to one another. Additional microorganisms may
be located within or on top of the biofilm matrix. Specific functional types
of microorganisms may, through their activities, create conditions that
favour other complementary functional groups of microorganisms. This
leads to the establishment of spatially separated, but interactive, functional
groups of organisms, which exchange metabolites at group boundaries
achieving physiological cooperation (Blenkinsopp and Costerton 1991).

Generally, when the biofilm reaches a thickness of 10–25 conditions at

its base become anaerobic and indicate that the biofilm is approaching a
state of maturity, with high species diversity and stability (Hamilton
1987).

Overall, the development of a biofilm is generally governed by a

number ofparameters including ambient and system temperatures,
which are in turn related to season, day length, climate and wind
velocity; hydrodynamic conditions (shear forces, friction drag and mass
transfer); nutrient availability (concentration, reactivity, antimicrobial
properties); surface roughness, hydrophobicity and electro chemical
characteristics of the surface; pH (an approximately neutral pH of the water
is optimal for the growth of most biofilm-forming bacteria); the presence of
particulate matter (which can become entrapped in the developing biofilm
and thus provide additional attachment sites); and the effectiveness of
biofilm control measures. Of these four major influencing factors are the
surface or interface properties, hydrodynamics, nutrients and biofilm
consortia (Stoodley et al. 1997).

Stage 5. Biofilm Cell Detachment/Dispersal

 As the biofilm ages, the attached bacteria, in order to survive and
colonize new niches, must be able to detach and disperse from the biofilm.
The bacteria from the biofilm, mainly the daughter cells get detached
individually or are sloughed off. The released bacteria may be transported to
newer locations and again restart the biofilm process (Marshall, 1992). High
level of cellular interaction and competitive behaviour arising because of
resource availability as biofilm matures.

Detachment can occur as a result of low nutrient conditions indicating

a survival mechanism. The ability of microorganisms to disperse from
surfaces is of concern in food processing plants where products must be
protected from microbial contaminants. In microbiological terms,
detachment from surfaces may at first be seen to be a disadvantage to the
biofilm. However, it has been found that biofilms with greater detachment
rates have larger fractions of active bacteria.

Therefore, detachment is not just important for promoting genetic

diversity, but also for escaping unfavourable habitats aiding in the
development of new niches. Chemotaxis protein BdlA (polypeptide chains)
activate in response to environmental cues provides biofilm dispersion
signal and elevates c-di-GMP levels and protease ClpP, which is required for
P. aeruginosa biofilms to disperse.

SIGNIFICANCE OF BIOFILM IN THE FOOD INDUSTRY

It is known that bacteria are remarkably capable of adjusting their

needs for survival in environments. Among the microbial characteristics
enabling these adaptations one of the most important is the ability of the
microorganism to form biofilms, since it facilitates adaptation to harsh
environmental conditions.

The presence of biofilms is common in food industry and

represents a concern because bacteria can adhere to almost any type of
surface, such as plastic, metal, glass, soil particles, wood and food
products (Anderson et al., 2012). Attachment of bacteria to food
products or to product contact surfaces leads to economic losses and to
a higher risk of occurrence of bacterial foodborne diseases. For example,
contamination of equipment with
biofilms was a contributing factor to 59 % of food-borne disease outbreaks
investigated in France (Midelet & Carpentier 2004).

Bacterial in biofilms confer survival advantages to its members since

they are protected from environmental stress such as ultraviolet light,
dehydration or treatment with antimicrobial and sanitizing agents, which
makes their elimination a huge challenge (Anderson et al., 2012).

Occurrence biofilms in food processing plants represents a reservoir of

microorganisms and serves as a potential source of contamination of raw
materials and processed products and may lead to food spoilage,
economical losses, reduced shelf life of products or transmission of
pathogens.

First report published on foodborne bacterial biofilm described the

adhesive properties of Salmonella sp. (Duguid et al., 1966) followed with
Listeria monocytogenes, Yersinia enterocolitica, Campylobacter jejuni,
Staphylococcus spp. and Escherichia coli O157:H7 (Bryers, 1933)

MICROORGANISMS RESPONSIBLE FOR BIOFILM FORMATION

L. monocytogenes is commonly found in food processing environment,

and it has been isolated from both meat and dairy processing plants. This
microorganism can adhere rapidly and firmly to inert surfaces and may
survive in the sessile form for a long period of time (Consterton et al., 1995).
For example, Unnerstad et al. (1996) found the same clone of L.
monocytogenes persisting in a dairy plant for 7 years and Miettinen et al,
(Miettinen et al., 1999) demonstrated that isolates of L. monocytogenes
PFGE type II had also survived in an ice cream plant for at least 7 years.

Staphylococcus aureus is one of the most frequent foodborne

pathogens in the food industry (Meira et al., 2013). Rode et al., (2007)
studied biofilm formation by both food-related and clinical S. aureus strains
grown under different stress conditions (temperature, sodium chloride,
glucose and ethanol), and those authors observed that some ingredients,
food such as sodium chloride and glucose, could promote biofilm formation
by S. aureus.
 E. coli O157:H7 is known to produce exopolysaccharides (EPS) and
to form biofilm on food-contact surfaces and equipment used in beef
processing plant (Bodur et al., 2012). Recently, Mendonça et al. (2012) also
demonstrated that E. coliO157:H7 had the potential to form biofilm on
different surfaces commonly used by food industry while Dourou et al.
(2011).

Served that E. coli O157:H7 attachment occurred not only at

temperatures of beef slaughterhouses areas (15 °C), but also during cold
storage (4 °C) temperatures, indicating more effective sanitation programs
are necessary

BENEFICIAL ASPECTS OF BIOFILM

On the other hand, microbes in biofilms can also be beneficial to food

industry and may be used for biotechnological applications (Demirci et al.,
1997). It helps the production of fermented food and in the waste treatment.

Biofilms are essential elements in the production of fermented foods;

for example, during the production of vinegar, the acetic acid bacteria are
grown on wood chips and the attachment of bacteria promote a more
efficient conversion of substrate to acid (Steir, 2005).

According to Gamarra et al., (531) Aspergillus niger biofilms may be

used for the commercial productionof cellulases since biofilms formed on
polyester cloth yielded 70 % more cellulase activity than freely suspended
mycelial culture. These enzymes are of importance due to their potential
industrial applications for the processing of food, textile, laundry, pulp and
paper. Morikawa, 2006 highlighted good aspects related to biofilm formation
by B. subtilis and other bacilli due to the control of infection caused by plant
pathogens, the reduction of steel corrosion, and the exploitation of novel
compounds.

Biofilm reactors were also applied to promote effective microbial growth

for the treatment of sewage, industrial waste streams or contaminated
groundwater (Feng et al., 2013). It is very important to control harmful
biofilm formation, but beneficial biofilms formed by industrial bacteria may
contribute for the development of new biotechnological processes.
BIOFILM CONTROL

Microorganisms within biofilms are able up to 1,000-fold more

resistant to disinfectants and biocides than planktonic counterparts and this
characteristic may be associated with aspects related to the architecture
and physiology of biofilms, such as reduced diffusion, anaerobic growth,
physiological changes due to reduced growth rates and the production of
enzymes that degrade antimicrobial substances (Hoiby et al., 2010).
Because of those factors, the most common disinfectants used by food
industries including: acidic compounds, aldehyde- based biocides, caustic
products; chlorine, hydrogen peroxide, iodine, isothiazolinones, ozone,
peracetic acid, phenolics, biguanidines, surfactants halogens, and
quaternary ammonium compounds is not enough to remove the biofilms
(Nilsson et al., 2011).

Chemical Treatment

As a biofilm treatment, a variety of concentration- and time-dependent

chemical sanitizers can be used. Sanitization is the process of reducing
bacteria populations to levels that are safe for humans (Schmidt, 2012).
Food processing equipment must be sanitized in order to prevent cross
contamination between batches of food. (Bayoumi et al., 2012). Chlorine-
based sanitizers are the most extensively utilized in the food sector,
however some microorganisms have developed resistance to chlorine
treatments. Chlorine resistance, for example, was linked to the cellulose
synthesis phenotype in Salmonella enterica. The environmental stress
factors encountered in food processing factories influenced this genotype
(Yang et al., 2016). Although gaseous ClO2 has been shown to be more
efficient against Bacillus cereus endospores found in biofilms on food steel
surfaces, aqueous ClO2 is the most extensively used sanitizer in the food
industry (Nam et al., 2014). Aqueous ClO2 was more efficient than NaOCl
(sodium hypochlorite, generally employed at 50–1,000 ppm) in the case of
Escherichia coli O157:H7 biofilms, especially when a drying step followed
the surface treatment in the food factory. Furthermore, the ClO2 treatment
made Escherichia cells more sensitive to other stresses, such as drying
 Steel Coatings

Biofilms' poor reactivity to traditional control strategies emphasizes

the urgent need for new antibacterial and antibiofilm agents.
Nanotechnology agents are one possible method.
Nanoparticles (NPs) are distinguished from their bulk chemical
counterparts by their distinct characteristics. One of these properties is
their huge surface area to volume ratio, which results in a greater number of
functional sites and can increase the impact of NPs on a microorganism.
Most bacterial antibiotic resistance mechanisms are irrelevant when dealing
with NPs since their antibacterial effects are mediated mostly by direct
contact with the bacterial cell wall and do not need penetration (Beyth et al.,
2015). As a result, NPs are less likely than standard antibiotics to cause
bacterial resistance.

Coating a stainless-steel surface with the modified material Ni-P-

polytetrafluoroethylene is one example. In comparison to the control
stainless steel surface, this chemical was able to minimize biofilm
development by Geobacillus stearothermophilus and Bacillus
licheniformis by two orders of magnitude. It also worked well to keep milk
from accumulating on the same area (Jindal et al., 2016).

CONCLUSION

Within the complex architecture of microbial biofilms, pathogens

proliferate and disseminate in surface-adhered form. Microbes that hide in
extracellular polymeric secretions (EPS) and form an organized, multilayered
structure have several advantages, including immunity to harmful
substances, protection from temperature, oxygen, and pH changes, and
protection from host-derived toxins. The complex aggregation of
biopolymers originating from microorganisms known as the extracellular
polymer matrix (EPS) is responsible for ensuring both cell survival and
proliferation by supplying all necessary microenvironmental conditions. The
main elements of extracellular polymeric substances (EPS) matrixes are
hydrolytic enzymes, proteins, lipids, exopolysaccharides, and extracellular
DNA. These components differ according on the kind of microbe that resides
in them.
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