- A biofilm is a thin but robust layer of mucilage adhering to a solid surface and containing a
community of bacteria and other microorganisms.
- A biofilm is an assemblage of microbial cells that is irreversibly associated (not removed by
gentle rinsing) with a surface and enclosed in a matrix of primarily polysaccharide material.

-A biofilm is an aggregate of microorganisms in which cells adhere to each other on a surface.
-These cells are frequently embedded within a self-produced matrix of extracellular polymeric
substance (EPS). The EPS matrix can constitute up to 90% of the biofilm biomass.
-Biofilm EPS, also referred to as slime, is a polymeric conglomeration composed of
extracellular DNA, proteins, and polysaccharides.
-Biofilms may form on a wide variety of surfaces, including living tissues, indwelling medical
devices, industrial or potable water system piping, or natural aquatic systems.
-Non-cellular materials such as mineral crystals, corrosion particles, clay or silt particles, or
blood components, depending on the environment in which the biofilm has developed, may
also be found in the biofilm matrix.
-Microbes form a biofilm in response to many factors, including cellular recognition of specific
or non-specific attachment sites on a surface, nutritional cues, or exposure of planktonic cells
to sub-inhibitory concentrations of antibiotics.

Properties of Biofilms
A biofilm confers certain properties to bacteria that are not seen in the planktonic state.
1) Differential gene expression
-Biofilm-associated organisms differ from their planktonic (freely suspended) counterparts
with respect to the genes that are transcribed and therefore carrying out different activities.
-When genes are activated to make chemical products (amino acids and proteins), they are
said to be upregulated; when the genes are de-activated, they are downregulated. Not all of
the genes in a cell are activated to make proteins all of the time.
-When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in
behaviour in which large suites of genes are differentially regulated.
2) Another important characteristic of biofilm associated bacteria is the gene transfer.
3) A major advantage is the protection that biofilm provides to the colonizing species from
- competing micro-organisms,
- host defence mechanisms &
- potentially toxic substances like lethal chemicals or antibiotics.
All these are implications of differential gene expression and gene transfer.
4) Biofilms also facilitate processing and uptake of nutrients, cross feeding and removal of
potentially harmful metabolic products through the voids or water channels between the
micro-colonies, acting as a primitive circulatory system.
5) Quorum sensing
-An important characteristic seen in Biofilm-associated bacteria is Quorum sensing, or cell
density mediated gene expression. This involves the regulation of expression of specific genes
through the accumulation of signaling compounds that mediate intercellular communication.

-Quorum sensing is a system of stimuli and response correlated to population density. Many
species of bacteria use quorum sensing to coordinate gene expression according to the
density of their local population.
-It's a type of cell-to-cell communication system.
-Some of the best-known examples of quorum sensing come from studies of bacteria. Bacteria
use quorum sensing to coordinate certain behaviors such as biofilm formation, antimicrobial
peptide synthesis, regulation of virulence, symbiosis, transfer of conjugative plasmids,
sporulation etc.
-Bacterial gene expression is what is actually regulated by quorum sensing.
- A variety of different molecules are used as signals in quorum sensing.
Common classes of signaling molecules are
> Oligopeptides in Gram-positive bacteria and their action is species-specific,
> N-acyl homoserine lactones (AHL) (AI-1 signaling molecule) in Gram-negative bacteria and
these are also species specific, and
> Autoinducer-2 (AI-2) (are furanosyl borate diesters in nature) in both Gram-negative and
Gram-positive bacteria.
-The enzyme responsible for their synthesis of AI signalling molecules is encoded by the luxS
gene. These AI systems play important roles in bacterial biofilm formation.
-The mechanism of signaling system mediated by chemical autoinducer molecules produced
by bacteria is as follows: When these molecules reach a critical threshold concentration, a
signal transduction cascade is triggered. The autoinducer molecules bind to the appropriate
transcription regulator(s) when the bacterial population reaches the quorum level (that is, the
signal concentration reaches a threshold concentration sufficient to facilitate binding to the
receptor). Binding of the autoinducers is followed by activation or repression of target genes.
-Quorum sensing can occur within a single bacterial species as well as between diverse
species, and can regulate a host of different processes.

Stages of Biofilm Formation
There are five stages of biofilm development.
Stage 1: Initial attachment:
-Planktonic (free floating) bacteria adhere to the biomaterial surface.
-These first colonists initially form a weak, reversible adhesion to the surface via van der
Waals forces.
Stage 2: Irreversible Attachment:
-If the colonists are not immediately separated from the surface, they can anchor themselves
more permanently using cell adhesion structures such as pili.
-Some species are not able to attach to a surface on their own but are able to anchor
themselves to the matrix or directly to earlier colonists. It is during this colonization that the
cells are able to communicate via quorum sensing using such products as AHL.
-Cells aggregate, form micro colonies and excrete extracellular polymeric substances (EPS),
i.e. slime. The attachment becomes irreversible.
Once colonization has begun, the biofilm grows through a combination of cell division and
Stage 3: Maturation 1:
-A biofilm is formed.
-It matures and cells form multi-layered clusters.
Stage 4: Maturation 2:
-Three-dimensional growth and further maturation of the biofilm, providing protection against

host defense mechanisms and antibiotics.
Stage 5: Dipersion:
-The biofilm reaches a critical mass and disperses planktonic bacteria, ready to colonize other
-Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle.
Dispersal enables biofilms to spread and colonize new surfaces.
-Enzymes that degrade the biofilm extracellular matrix, such as dispersin B and
deoxyribonuclease, may play a role in biofilm dispersal.
-Biofilm matrix-degrading enzymes may be useful as anti-biofilm agents.
-Recent evidence has shown that one fatty acid messenger, cis-2-decenoic acid, is capable of
inducing dispersion and inhibiting growth of biofilm colonies. Secreted by Pseudomonas
aeruginosa, this compound induces cyclo heteromorphic cells in several species of bacteria
and the yeast Candida albicans. Nitric oxide has also been shown to trigger the dispersal of
biofilms of several bacteria species at sub-toxic concentrations, so it shows potential for use in
the treatment of patients that suffer from chronic infections caused by biofilms.

Fig Stage 1: initial attachment; stage 2: irreversible attachment; stage 3: maturation I; stage
4: maturation II; stage 5: dispersion.
Each stage of development in the diagram is of a developing Pseudomonas aeruginosa

Host Defence Mechanism & Antibiotic response w.r.t Biofilm
Biofilm communities of microbes are different from their planktonic counterparts in very
important ways.
First, when microbes live as a community, they become much less susceptible to antibiotics,
even if highly susceptible as individual cells. Thus, when microorganisms form a community,
they are protected against a variety of antibiotics that clinicians commonly prescribe for their

Second, and more the focus of this perspective, these communities of microorganisms resist
attack and killing by the host immune system.
Organisms in a Biofilm are 1000-1500 times more resistant to antibiotics than in their
planktonic state.
The mechanisms of this increased resistance differ from species to species, antibiotic to
antibiotic and for biofilms growing in different habitats.
This antibiotic resistance in bacteria is thought to be affected by their nutritional status,
growth rate, temperature, pH and prior exposure to sub-effective concentrations of
antimicrobial agents.
Bacteria in biofilms resist antibiotics via several mechanisms such as
- The slower rate of growth of bacterial species in a biofilm, which makes them less
susceptible to bactericidal antibiotics.
-Decreased penetration or diffusion of antibiotics through biofilm matrix.
-‘Super-resistant’ bacteria have been identified within a biofilm, which have multidrugresistance pumps that can extrude antimicrobial agents from the cell. Since these pumps
place the antibiotics outside the outer membrane, the process offers protection against
antibiotics that target cell wall synthesis.
- Starvation or stress responses
- Genetic switches that turn susceptible planktonic cells into antibiotic-resistant persisters;
involves quorum sensing.
- In addition, biofilms increase the opportunity for gene transfer between bacteria and may be
significant for the transfer of resistance genes to associated susceptible bacteria. Gene
transfer can also onvert a previously avirulent strain into a virulent pathogen.
The mechanisms that enable bacteria in biofilms to resist host defenses are less well
characterized, but include
(i) Limited penetration of leukocytes and their bactericidal products into the bio- film.
(ii) global response regulators and quorum sensing activities that increase resistance to
(iii)-Decreased ability of leukocytes to engulf biofilm bacteria (i.e. decreased phagocytosis).
-Antibodies and other serum or salivary proteins may fail to penetrate into the biofilm. Cells
within the biofilm remain hidden from antibody and complement factor recognition, and thus
from subsequent white blood cell phagocytosis.
- Since phagocytes are unable to effectively engulf a bacterium growing within a complex
polysaccharide matrix attached to a solid surface. This may result in phagocytes releasing
large amounts of pro-inflammatory enzymes and cytokines, leading to inflammation and
destruction of nearby tissues
(iv) genetic switches that increase resistance of bacterial cells in biofilms to the immune
system, and
(v) suppression of leukocyte activity through effector regulation.

-In order to reduce the toxicity caused by high concentrations of antiseptics inclusion
compounds have been developed for use of more effective antiseptic agents.
-These compounds offer advantages in effectiveness, long-term activity, and low effective
antimicrobial concentrations.
-The key component of the inclusion compounds is the cyclodextrin, which creates a

hydrophobic cavity that surrounds the drug.
-Cyclodextrins (CDs) are cyclic non-reducing, non-hygroscopic, water-soluble oligosaccharides,
most commonly formed by six, seven and eight glucopyranose units, denominated -CD, -CD
and -CD respectively.
-CDs can improve the therapeutic efficacy of poorly water-soluble drugs, enhance the physical
and chemical stability, and also protect the guest molecules from degradation in the
gastrointestinal tract. ]

Biofilms associated with indwelling medical devices
-Microbial biofilms can develop on or within indwelling medical devices.
e.g., contact lenses,
intrauterine devices, urinary catheters
mechanical heart valves, pacemakers,
needleless connectors,
endotracheal tubes, tympanostomy tubes,
peritoneal dialysis catheters, central venous catheters,
prosthetic joints,
voice prostheses etc.
-Biofilms on indwelling medical devices may be composed of gram-positive or gram-negative
bacteria or yeasts. Bacteria commonly isolated from these devices include
the gram-positive : Enterococcus faecalis, Staphylococcus aureus, Staphylococcus
epidermidis, and Streptococcus viridans;
the gram-negative Escherichia coli, Klebsiella pneumoniae, Proteus
mirabilis, and Pseudomonas aeruginosa.
-These organisms may originate from the skin of patients or health-care workers, tap water to
which entry ports are exposed, or other sources in the environment.
-Biofilms may be composed of a single species or multiple species, depending on the device
and its duration of use in the patient.
Factors Influencing Rate and Extent of Biofilm Formation
-When an indwelling medical device is contaminated with microorganisms, several variables
determine whether a biofilm develops.
-First the microorganisms must adhere to the exposed surfaces of the device long enough to
become irreversibly attached.
-The rate of cell attachment depends on
>the number and types of cells in the liquid to which the device is exposed,
>the flow rate of liquid through the device, and
>the physicochemical characteristics of the surface.
>Components in the liquid may alter the surface properties and also affect rate of
-Once these cells irreversibly attach and produce extracellular polysaccharides to develop a
biofilm, rate of growth is influenced by
>flow rate,
>nutrient composition of the medium,
>antimicrobial-drug concentration, and
>ambient temperature.
Some examples of biofilms on indwelling devices

1) Central Venous Catheter Biofilms:
- A central venous catheter is a thin, flexible tube that is inserted into a vein, usually below the right collarbone,
and guided (threaded) into a large vein above the right side of the heart called the superior vena cava. It is used
to give intravenous fluids, blood transfusions, chemotherapy, and other drugs. The catheter is also used for taking
blood samples. It may stay in place for weeks or months and helps avoid the need for repeated needle sticks

These long, flexible catheters empty out in or near the heart, allowing the catheter to give the
needed treatment within seconds.
-Virtually all indwelling central venous catheters are colonized by microorganisms embedded
in a biofilm matrix.
-The organisms most commonly isolated from catheter biofilms are
>Staphylococcus epidermidis,
>S. aureus,
>Candida albicans,
>P. aeruginosa,
>K. pneumonia and
>Enterococcus faecalis .

-Colonization of these devices can occur rapidly (within 24 hours) and may be a function of
host-produced conditioning films (platelets, plasma, and tissue proteins).
- The extent and location of biofilm formation depended on the duration of catheterization:
>short-term (<10 days) catheters had greater biofilm formation on the external surface.
>long-term catheters (30 days) had more biofilm formation on the catheter inner lumen.
- The nature of the fluid administered through central venous catheters may affect microbial
growth: gram-positive organisms (S. epidermidis, S. aureus) did not grow well in intravenous
fluids, whereas the gram-negative aquatic organisms (e.g., P. aeruginosa, Klebsiella spp.,
Enterobacter spp., Serratia spp., and Pantoea sp.) sustained growth.
- Several studies have examined the effect of various types of antimicrobial treatment in
controlling biofilm formation on these devices.

>addition of sodium metabisulfite to the dextrose-heparin flush of the left atrial catheter
eliminated microbial colonization of these catheters.
>catheters impregnated with minocycline and rifampin were less likely to be colonized than
those impregnated with chlorhexidine and silver sulfadiazine.
>catheters coated with a cationic surfactant (tridodecylmethylammonium chloride), were less
likely to become contaminated and develop biofilms than were untreated catheters.
>There are also other several ways to control biofilms on central venous catheters, including
using aseptic technique during implantation, using topical antibiotics, minimizing the duration
of catheterization, using an in-line filter for intravenous fluids, coating the inner lumen of the
catheter with an antimicrobial agent.
2) Mechanical Heart Valve Biofilms:
-An artificial heart valve is a device implanted in the heart of a patient with
valvular heart disease. When one of the four heart valvesmalfunctions, the medical choice
is to replace the natural valve with an artificial valve. This requires open-heart surgery.
- Microorganisms may attach and develop biofilms on components of mechanical heart valves
and surrounding tissues of the heart, leading to a condition known as prosthetic valve

-The primary organisms responsible for this condition are S. epidermidis, S. aureus,
Streptococcus spp., gram-negative bacilli, diphtheroids, enterococci, and Candida spp.
-These organisms may originate from the skin, other indwelling devices such as central
venous catheters, or dental work. The identity of the causative microorganism is related to its
source: whether the contaminating organism originated at the time of surgery (early
endocarditis, usually caused by S. epidermidis), from an invasive procedure such as dental
work (Streptococcus spp.), or from an indwelling device (a variety of organisms).
-Implantation of the mechanical heart valve causes tissue damage, and circulating platelets
and fibrin tend to accumulate where the valve has been attached. Microorganisms also have a
greater tendency to colonize these locations. The resulting biofilms more commonly develop
on the tissue surrounding the prosthesis or the sewing cuff fabric used to attach the device to
the tissue than on the valve itself.
-Antimicrobial agents are usually administered during valve replacement.
3) Urinary Catheter Biofilms:
-In urinary catheterization a latex, polyurethane, or silicone tube known as a urinary catheter
is inserted into a patient's bladder via the urethra. Catheterization allows the patient's urine

to drain freely from the bladder for collection.

-Urinary catheters are tubular latex or silicone devices, which when inserted may readily
acquire biofilms on the inner or outer surfaces.
-The organisms commonly contaminating these devices and developing biofilms are S.
epidermidis, Enterococcus faecalis, E. coli, Proteus mirabilis, P. aeruginosa, K. pneumoniae,
and other gram-negative organisms.
-The longer the urinary catheter remains in place, the greater the tendency of these
organisms to develop biofilms and result in urinary tract infections.
- Adhesion to catheter materials was dependent on the hydrophobicity of both the organisms
and the surfaces; catheters displaying both hydrophobic and hydrophilic regions allowed
colonization of the widest variety of organisms.
-Divalent cations (calcium and magnesium) and increase in urinary pH and ionic strength all
resulted in an increase in bacterial attachment.
- Several strategies have been attempted to control urinary catheter biofilms: antimicrobial
ointments and lubricants, bladder instillation or irrigation, antimicrobial agents in collection
bags, impregnation of the catheter with antimicrobial agents such as silver oxide, or use of
systemic antibiotics. Most such strategies have been ineffective, although silver-impregnated
catheters delayed onset of bacteriuria for up to 4 days.
4) Ocular Prostheses:
-Ocular prostheses include intraocular lenses, ocular explants, and contact lenses.
-Conjunctivitis and keratitis are the two principal infections that occur in association with
-The causative bacteria are primarily S. aureus, streptococci, and Pseudomonas species.
Improperly maintained cleansing solutions are an important source of pseudomonads.
Fungi may also cause lens-related infection.
A rare but devastating infection with the free-living fresh water protozoan Acanthamoeba sp.
has been reported in association with lens use. This protozoan contaminates the lens when
proper sterility of the cleansing solution is not maintained.
-Treatment is usually readily accomplished by removal of the lens and topical application of
antibiotics for several days.
- The most effective preventive measure against contact lens-associated infection is strict
adherence to the manufacturer's guidelines for wearing and cleaning the lens.

--------------------------------------------------------------------------------------------------------------------------------------------Some typical examples of Biofilms by Bacteria
1)Biofilm formation by Helicobacter pylori
>Introduction :
-Helicobacter pylori is a
bacterium that colonizes the human gastrointestinal tract, primarily the stomach.
-Helicobacter pylori is one of the most common causes of bacterial infection in humans.

- Three basic forms of existence of H. pylori have been distinguished: a spiral form (culture,
virulent, contagious), a coccoid form (non-cultivated, less virulent) and a degenerative
coccoid form. The spiral form is the most common form involved in colonization of the human
-Some studies demonstrated that this microorganism has biofilm forming ability in the
environment and on human gastric mucosa epithelium as well as on in vitro abiotic surfaces.
-In the environment, H. pylori could be embedded in drinking water biofilms through water
distribution system in developed and developing countries. Thus, drinking water may serve as
a reservoir for H. pylori infection.
-In the human stomach, H. pylori forms biofilms on the surface of gastric mucosa, suggesting
one possible explanation for eradication therapy failure.

> Biofilm formation:
- The basic mechanism of biofilm formation is same as explained earlier (the five step
[The initial attachment is driven by hydrophobic or electrostatic interactions as well as specific
bacterial surface molecules. The next step is multiplication of the bacteria and formation of
microcolonies with EPS surrounding the microcolonies. In the third step (maturation step), the
biofilm forms thick and mushroom-like or tower-like structures with increasing numbers of
bacteria. Subsequently, the enlarged biofilm shows focal dissolution and liberates planktonic
bacterial cells which can spread to other locations.]
- Environmental biofilm of Helicobacter pylori : Many studies have shown that H. pylori rods
are viable in the environment, particularly in water, where they exist in the coccoid form or in
the form of bacterial biofilm. It does not appear that H. pylori forms biofilms at locations which
are relatively stressful conditions such as less than optimal temperatures and nutrient
- Helicobacter pylori biofilm formation in the gastrointestinal tract : Biofilm produced by H.
pylori is formed in a similar manner as in other bacteria. However, specialized virulence
factors, which allow H. pylori to escape the immune system of the host and colonize the
human gastric environment, in particular also facilitate the formation of a biofilm by these
These bacilli produce large amounts of extracellular exopolysaccharides (EPS), which protect
bacteria against pH changes, allowing them to process the transmission and colonization.
Thanks to matrix composed of exopolysaccharides, biofilm is flexible and very durable.
Like other bacteria, H. pylori also has a gene luxS whose expression results in the production
of autoinductors – particles signalling in the quorum sensing system, which is responsible for

communication between the bacteria forming a biofilm. It has been demonstrated that a
mutation in the gene luxS, causing a reduction in its expression, causes inhibition of biofilm
formation in most bacteria. In contrast, in H. pylori mutation in the gene luxS does not reduce
the adhesion properties, and does not affect the formation of aggregates and the
development of the spatial structure of the biofilm.
The effect of mutations in a cagE gene was double and even a fourfold increase in biofilm
formation as compared to wild strains.
H. pylori, by producing the enzyme mucinase, is able to induce degradation of the oligomeric
structure of mucin. This mechanism facilitates the penetration of bacteria through the
protective layer and determine the tropism of the bacteria to gastric epithelial cells.
After entering the lumen of the stomach, bacteria must cope with the acidic gastric pH and
cross the barrier of the mucus layer to reach the epithelial cells of the stomach – the aim of
their attack. Movement, chemotaxis, adhesion processes conditioning secretion of adhesion
molecules, as well as the ability to produce urease and VacA toxin, are the crucial skills
conditioning the survival of H. pylori in the infection site.
The H. pylori cell wall contains lipopolysaccharide (LPS), which can induce a local and
systemic inflammatory response in mammalian cells. Despite the low toxicity it has been
demonstrated that H. pylori LPS has immunomodulatory capacity and activates immune cells
to produce proinflammatory cytokines. Lipopolysaccharide disrupting the homeostasis of the
stomach environment, inhibiting the synthesis and secretion of mucin and facilitating
adhesion contributes to efficient biofilm structures .
>Quorum sensing:
The luxS gene is the only known quorum-sensing gene present in the sequenced H. pylori
genome. Several reports indicated that H. pylori produces extracellular signaling molecules
related to AI-2, and production of AI-2 is dependent on luxS function.
>Resistance of Helicobacter pylori biofilm to drugs:
-Biofilm formed by H. pylori protects against the human immune system and facilitates the
survival of these bacteria in the difficult environment of the gastric mucosa. It seems that it
also provides high resistance to antimicrobial agents such as antibiotics and disinfectants.
-In the treatment of infections caused by H. pylori a three-component therapy is indicated,
including receiving two of three antibiotics (amoxicillin and clarithromycin or metronidazole)
and a proton pump inhibitor (PPI) (omeprazole, pantoprazole, lansoprazole). However, in 10%
to 20% of cases this therapy is ineffective. The main cause of treatment failure of H. pylori
infection is primary or acquired resistance of the bacteria to one of the used antibiotics. There
are reports that H. pylori eradication failure is related not only to the mechanisms of
resistance to medication, but also to other factors, both from the bacteria and from the host.
>What is Denatal Plaque ? :
Dental plaque is a structurally- and functionally-organized community og micro-organisms
present as biofilm found on a tooth surface. The microbes are embedded in a matrix of
polymers of host and bacterial origin.
>Dental Plaque formation:
Dental plaque forms via an ordered sequence of events, resulting in a structurally- and
functionally-organized, species-rich microbial community.
1. Adsorption of Host and Bacterial Molecules to the Tooth Surface
-Pellicle formation = Within minutes of tooth eruption or a cleaning, pellicle formation begins,
which can be defined as a thin coat of salivary proteins.
-Conditioning film formation = The pellicle acts like an adhesive by sticking to the tooth

surface and encouraging a conditioning film of bacteria to attach to the pellicle.
2. Passive Transport of Oral Bacteria to the Tooth Surface
-Reversible adhesion = Following pellicle formation, there is passive transport of oral bacteria
to the tooth surface. The conditioning film directly influences the initial microbial colonization,
and continues to adsorb bacteria to the tooth surface. Reversible adhesion involving weak
long-range physico-chemical interactions between the cell surface and the pellicle is
-Irreversible adhesion = Reversible adhesion then leads to a much stronger, irreversible
attachment involving short-range interactions between specific molecules on the bacterial
cells and the complementary receptor proteins on the pellicle surface occur. Eg - adhesinreceptor mediated attachment.
3. Co-adhesion resulting in attachment of secondary colonizers to already attached
-The co-adhesion of the later colonizers to the already present biofilm build up the biofilm to
create a more diverse environment, which includes the development of unusual morphological
structures like corn-cobs and rosettes. The many interactions between these diverse bacterial
species begin to create a number of synergistic and antagonistic biochemical interactions.
4. Multiplication of the Attached Microorganisms
-Multiplication of bacterial cells and biofilm formation that includes the synthesis of
exopolysaccharides, that consists of soluble and insoluble glucans, fructans, and
heteropolymers, occurs. This matrix is one of the key structural aspects of the plaque biofilm.
5. On occasion, detachment is seen.
>Microorganisms involved in biofilm formation:
-Through the growth process of the plaque biofilm, the microbial composition changes from
one that is primarily gram-positive and streptococcus-rich in eary stages to a structure filled
with gram-negative anaerobes in its more mature state. This is because as the biofilm
thickens and becomes more mature, these anaerobic bacteria can live deeper within the
biofilm, to further protect them from the oxygen-rich environment within the oral cavity.
- Plaque is natural and contributes (like the resident microflora of all other sites in the body) to
the normal development of the physiology and defenses of the host.
3)Pseudomonas aeruginosa biofilm

Additional imp things (do from mam’s notes)1)Treatment of biofilms infections.
2)Factors foavouring bifilm formation.
3) Teeth and dental plaques; Dental caries.