Probiotics and biofilms

Probiotics
and
Biofilms
By : Tewdros Tesfa (BSc., MSc.)
Haramaya University
Wondmagegn Demsiss (BSc., MSc.)
Wollo University
Probiotics and biofilms TW

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Outline
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 Probiotics
 Mechanism of action of probiotics
 Introduction About biofilm
 Formation, development and dispersal of biofilms
 Properties of biofilms and Quorum sensing
 Uses and problems of biofilms
 Biofilms and infectious diseases
 Control and removal of biofilms


Objectives
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At the end of this presentation you will be able to:
 Define biofilms and Probiotics
 Understand the mechanism of action of Probiotics
 Describe how biofilms are formed.
 State the role of biofilms in infectious disease.
 Understand the antimicrobial drug resistance
mechanisms of biofilms.
 Know the prevention and control mechanism of
biofilms.

Definition
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 Live microorganisms which when administered in

adequate amounts confer a health benefit on the
host” (FAO/WHO, 2002)
 Are live microorganisms; could be fungi or bacteria
 Food ingredients
 Have beneficial health effects
e.g. Lactobacillus, Bifidobacterium, Saccharomyces
boulardii (yeast)


Definition
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 Prebiotics- dietary substances (non

digestible by human enzymes, non starch
carbohydrates) e.g.
inulin, lactulose, oligofructose
 Stimulate favorable growth or activity of
probiotic bacteria
 Synbiotics (eubiotics)- Products that contain

both probiotics and Prebiotics

Mechanism of action
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Anti cancer effects
Are

shown to reduce concentration of
carcinogenic chemicals (Scavenge
superoxide radicals)
Modify epithelial cell kinetics


Mechanism of action
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Anti diarrheal effect
Lowering

GI pH
Secreting bacteriocins
Reconstruction of normal flora
(especially antibiotics associated and
radiation induced diarrhea)
Competing for adhesion sites

Mechanism of action
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Immunomodulation
 Enhanced

barrier

 Increased

mucus secretion
 Epithelial membrane integrity
 Activation
Bacterial

surface antigens

 Suppression
 Inducing

T-reg cells
 Inducing regulatory cytokines; TGF-β, IL-10
Counteract pro-inflammatory cytokines

Mechanism of action
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Anti-allergy effects
 Reducing

antigenic challenge; by enhanced
membrane integrity ( as most allergic
reactions are thought as a result of large
antigenic insult after barrier disintegrates)
 Modification of allergen receptor


Mechanism of action
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 Stomach and Urogenital health
 Inhibitory
 An

effect of lactic acid for H.pylori

estimated reason for Urogenital health

is maintenance of acidic environment
(lactic acid) and production of other
antibacterial chemicals (e.g. H2O2)


Other roles of Probiotics
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Reduction of hypertension- by fermentation

end products which have inhibitory effect on
angiotensin I conversion
Lowering blood cholesterol level
Improvement in Ca absorption
Production of some vitamins and digestive
enzymes


Questions to be answered
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 Dose of the probiotic bacteria

 For how long should it be taken
 Correlation of specific probiotic species with

its specific action and target


Summary
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Introduction
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 What are biofilms?

 Why should we care about biofilms?
 What makes them special?
 What happens as a result of biofilms?
 How they are formed?
 How they are removed?


Introduction about biofilms
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 The concept of bacterial biofilm was proposed in

1936.
 In natural aquatic environments bacteria are
predominantly not free floating but grow as
multi-species communities attached to submerged
surfaces.
 Over 90% of all bacteria live in biofilms.
 In clinical medicine, many environments provide
optimal conditions for the formation of bacterial
biofilm, such as contact lenses, urinary
catheters, and so on.

Introduction
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What is Biofilm??
 A biofilm is a community of microorganisms, attached to a wet or moist
surface. Microorganisms include
bacteria, yeasts, algae, fungi.
 These microbes make a sticky, slimy, gel-like
substance and surround themselves with it.


Bio film constitutes
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

A biofilm is an aggregate of microorganisms in which

cells are stuck to each other and/or to a surface.
 These adherent cells are frequently embedded within a self-

produced matrix known as extracellular polymeric
substance (EPS).


Biofilm EPS, which is also referred to as "slime," is a polymeric
jumble of DNA, proteins and polysaccharides.


Bio film constitutes
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 Biofilms may form on living or non-living surfaces and can

be prevalent in natural, industrial and hospital settings.
 For example, a pebble in a pond is covered in slimy green

stuff. That slime is a biofilm.
 The slippery layer that builds up inside a waste pipe is a

biofilm.
 The plaque that builds up between the gum and the tooth

is a biofilm.

Why Do Bacteria Make Biofilms?
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Biofilms protect the pathogenic

bacteria, making them hard to kill.
Hiding in biofilms, bacteria can spread
throughout the body.
Large, sticky biofilms resist the attack
of the immune system and antibiotics.


Formation and Development of Biofilms
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• Biofilm formation is not a random process

 Biofilms start as just a few bugs forming a

thin layer.
 They can develop into complex, three
dimensional structures housing millions of
individual bacterium.
 Like miniature cities, they have
towers, columns, bridges and
channels for the flow of nutrients.

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 What is the use of understanding

the….?
formation,
composition, and
characteristics of the biofilm assists in its
control and for developing treatment
strategies of biofilm diseases.
 The greater our understanding of the
processes involved in biofilm formation, the
greater the chance of developing remedies.

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 The pattern of biofilm formation can be

divided into three phases:
I. Attachment of bacteria to a solid surface;
II. Formation of micro colonies on the
surface; and
III. Formation of the mature biofilms


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 Neighbors in the biofilm work together to

build their niche environment.
 Some bugs use their bodies to build
support structures, arches, columns.
 Others form the foundation and are good at
sticking onto the host surface.
 Some make the sticky goo or slime that
protects them.

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 Formation of a biofilm begins with the attachment of

free-floating microorganisms to a solid surface.
 These first colonists adhere to the surface initially
through weak, reversible adhesion via van der Waals
forces.
 If the colonists are not immediately separated from
the surface, they can anchor themselves more
permanently using cell adhesion structures such as
pili.


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 Once a bacterium attaches to a surface, it activates a

whole different set of genes that gives the bacterium
different characteristics from those that it had as a
free-floating organism.
 The first colonists facilitate the arrival of other cells by
providing more diverse adhesion sites and beginning to
build the matrix that holds the biofilm together.
 Some species are not able to attach to a surface on their
own but are often 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.

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 Once colonization has begun, the biofilm

grows through a combination of cell
division and recruitment of new
bacteria.
 Biofilm doubling times are rapid in early
development and slower in more mature
biofilms.
 A second wave of bacterial colonizers adheres
to bacteria that are already attached to the
surface.

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 The bacteria cluster together to form

sessile, mushroom-shaped micro colonies that
are attached to the surface at a narrow base.
 Co aggregation is the ability of new bacterial
colonizers to adhere to the attached cells.
 The result of co aggregation is the formation
of a complex array of different bacteria linked
to one another. This is what we call it mature
biofilm
 Their Own Little World!!!!

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Microbes form a biofilm in response to many
factors, which may include
 cellular recognition of specific or nonspecific attachment sites on a surface,
 nutritional cues, or
 in some cases, by exposure of planktonic
cells to sub-inhibitory concentrations of
antibiotics.

Dispersal of biofilms
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 Dispersal of cells (bacteria) 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.


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Quorum sensing
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 To work together efficiently, the bugs need to talk to one

another. They do so by chemical signals. Bacteria produce
diffusible extracellular signalling molecules ( AHLs and
oligopeptides)
 Bacteria are often considered as simple unicellular
organisms, but research has recently shown that many
bacteria possess ability to communicate with one another
and to organize to communal groups.
 to monitor their own population density and to
coordinate expression of specific sets of genes in
response to the cell density.
 This type of cell-density-dependent gene regulation is
termed Quorum sensing.

Properties and structure of biofilms
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 Biofilms are surface-attached microbial

communities with characteristic architecture and
phenotypic and biochemical properties distinct from
their planktonic counterparts.
 One of the best-known of these biofilm specific
properties is the development of antibiotic
resistance
 Biofilms are characterized by
• structural heterogeneity, genetic diversity, complex
community interactions, and an extracellular matrix
of polymeric substances.

properties cont…
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The structure of a biofilm community comprises
bacterial
A. Micro colonies,
B. An extracellular slime layer,
C. Fluid channels, and
D. A primitive communication system.


properties cont…
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A. Each micro colony is a tiny, independent community
containing thousands of compatible bacteria.
 Bacteria in the center --strict anaerobic environment, while
other bacteria at the edges -aerobic environment.
 Thus, the biofilm structure provides a range of customized
living environments (with differing PHs, nutrient
availability, and oxygen concentrations) within which
bacteria with different physiological needs can survive.
B. The extracellular slime layer is a protective barrier
that surrounds the mushroom shaped bacterial microcolonies.

properties cont…
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 The slime layer protects the bacterial micro colonies

from antibiotics, antimicrobials, and host defense
mechanisms.
C. A series of fluid channels penetrates the extracellular
slime layer.
 These fluid channels provide nutrients and oxygen for
the bacterial micro colonies and facilitate movement of
bacterial metabolites, waste products, and enzymes
within the biofilm structure.
D. Each bacterial micro colony uses chemical signals to
create a primitive communication system used to
communicate with other bacterial micro colonies.

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Depiction of the dynamic nature of a biofilm community.

USE AND PROBLEMS OF BIOFILMS
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Benefits
 Help remove pathogens and reduce the amount of organic
matter in the water, and
 Eliminate petroleum oil from contaminated oceans or
marine systems.
Problems
 They are associated with persistent infections and
 They are notorious for their pipe-clogging and corroding properties, a costly irritation in industrial
settings.
 Undesirable taste and odor issues in the water industry;
 Pipe corrosion within oil and gas industries and souring of
oil in pipelines and storage facilities.
 Can make sanitation difficult in food preparation areas.

Biofilms and infectious diseases
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• Biofilms have been found to be involved in a

wide variety of microbial infections in the
body.
 It has taken longer to make a clear connection
of biofilms to persistent infections in native
tissue.
 According to the CDC, 65% of all infections in
developed countries are caused by biofilms
 Biofilms are produced by most if not all
pathogens.

Biofilms and cont…
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 A well known example of a microbial biofilm in human

disease is dental plaque, which is the most known
and studied biofilm (secreting acids that destroy teeth
and gums).
 Pseudomonas aeruginosa, causing an incurable
infection in cystic fibrosis patients (Singh et al. 2000),
 and Staphylococcus aureus and Staphylococcus
epidermidis, infecting indwelling devices (Mack et al.
2004), are probably the best-known biofilm-producing
organisms.

40

 Biofilms are responsible for a significant portion of

acute infections.
 A classic case is that of Legionnaire’s disease, an acute
respiratory infection resulting from aspiration of
clumps of Legionella biofilms detached from air and
water heating/cooling systems.
 Many food borne pathogens such as E. coli
0157:H7, Listeria monocytogenes, Yersinia
enterocolitica, Salmonella spp. and Camphylobacter
jejuni can form either single-species or multi-species
biofilms on food surfaces and equipment

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Teeth with Dental Caries
The term dental caries refers to the destruction, or necrosis, of teeth usually by
bacterial action resulting in a condition commonly known as tooth decay.
.

Examples of diseases associated with
biofilm
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 Otitis media (middle ear infections)
 Periodontal disease
 Gingivitis
 Dental Caries
 Chronic bacterial prostatitis
 Endocarditis
 Sinusitis
 UTI
 Infections in cystic fibrosis

 Infections of all known indwelling devices such as

catheters, orthopedic prostheses, and heart valves;


Anti-microbial resistance of Biofilm
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 Bacteria within biofilms are up to 1000 times more

resistant to antimicrobials than the same bacteria in
suspension.
 Extracellular matrix of the biofilm is a passive
barrier for antibiotics
 To protect the cells against the antibiotics
tobramycin and gentamicin P. aeruginosa produces
only in biofilms a periplasmic cyclic glucan which
complexes antibiotics turning them into harmless
compounds.

Anti-microbial resistance cont…
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 In biofilms up to 20% of all genes are differently expressed.
 Bacteria in biofilms grow slower and with the reduced

metabolic activity they are less prone against most
antibiotics.
 Preliminary evidence suggests that different bacteria within
a biofilm can trade genes -- possibly including Abx
resistance genes
 The infections associated with biofilms may appear to
respond to systemic antibiotics because planktonic cells
respond and symptoms are reduced, but the persistence
of adherent cells leads to recurrent episodes of
infection.

Three hypotheses for mechanisms of antibiotic
resistance in biofilms
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Prevention and control of biofilm
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 An effective mechanical control mechanism is

biofilm detachment.
 Interfere with the bacterial cell to cell
communication
 A novel alternative to antibiotics are Probiotics.
 A unique spray made from stabilized enzymes
that can readily dissolve the biofilms.


Conclusion and recommendation
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 Biofilms, once considered odd curiosity, are now one of the

hottest topics in microbiology. Biofilms occur everywhere:
dental plaque is one the most common biofilms that
decay teeth,
 other biofilms can clog water pipes, others can
contaminate
 almost any medical device inserted into the
body, ranging from contact lenses to catheters and
artificial hearts.
 Microbiologists have traditionally researched only freefloating, individual bacteria growing in laboratory
cultures.“Microbiologists have been barking up the wrong
tree since the time of Pasteur,” says Costerton.

Probiotics and biofilms

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