Biofilms and polymicrobial

Infections

Mustafa Jaradat
Beginning of Microbes
 Bacteria first appeared on
earth about 3.6 billion years
ago, long before the
appearance of Homo sapiens
around 100,000 years ago..
Van Leeuwenhoek was the
first person to visualize,
graphically illustrate, and
label "animalcules"
(bacteria) that he found in
plaque scraped from his own
teeth.
Biofilm constitutes
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 of extracellular polymeric substance
(EPS). Biofilm EPS, which is also referred to as
"slime," is a polymeric jumble of DNA, proteins and
polysaccharides.
Biofilm is a complex substance.
 A biofilm is a complex
aggregation of
microorganisms growing on
a solid substrate. Biofilms
are characterized by
structural heterogeneity,
genetic diversity, complex
community interactions, and
an extracellular matrix of
polymeric substances.
Biofilms found in Nature everywhere where is
there is moisture
More properly known as biofilm, slime cities thrive
wherever there is water - in the kitchen, on contact
lenses, in the gut linings of animals. When the urban
sprawl is extensive, bio films can be seen with the
naked eye, coating the inside of water pipes or
dangling slippery and green from plumbing."
(Coghlan 1996)
Biofilm supports the Bacterial growth
Biofilm are a common mode of bacterial growth in
nature and their presence has an enormous impact
on many aspects of our lives, such as sewage
treatment, corrosion of materials, food
contamination during processing, pipe collapse,
plant-microorganisms interaction in the biosphere,
the formation of dental plaque, the development of
chronic infections in live tissue (mastitis, Otitis,
pneumonia, urinary infections, osteomyelitis) or
problems related to medical implants.
Formation of Biofilms
Biofilms may form on
living or non-living
surfaces, and represent a
prevalent mode of
microbial life in natural,
industrial and hospital
settings
Biofilms increases Antibiotic resitance
With
microorganisms are highly
resistant to antimicrobial
treatment and are
tenaciously bound to the
surface
Mechanisims of Biofilm formation
 Formation of a biofilm begins
with the attachment of free-
floating microorganisms to a
surface. These first colonists
adhere to the surface initially
through weak, reversible 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
Factors Influencing Rate and Extent of
Biofilm Formation
Indwelling medical device when 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
Technology understands Biofilms
better…
 Technological progress in
microscopy, molecular
genetics and genome
analysis has significantly
advanced our understanding
of the structural and
molecular aspects of
biofilms, especially of
extensively studied model
organisms such as
Pseudomonas aeruginosa.
Steps in Biofilm Development
 Biofilm development can be
divided into several key
steps including attachment,
micro colony formation,
biofilm maturation and
dispersion; and in each step
bacteria may recruit different
components and molecules
including flagella, type IV
pili, DNA and exo
polysaccharides.
Stages of biofilm development.
Steps in Biofilm formation
Bacteria associated with Biofilms
differ
Bacteria living in a biofilm can have significantly
different properties from free-floating bacteria, as the
dense and protected environment of the film allows
them to cooperate and interact in various ways. One
benefit of this environment is increased resistance to
detergents and antibiotics, as the dense extracellular
matrix and the outer layer of cells protect the interior
of the community.
Biofilms major cause of Nosocomial
infections
Microbial biofilms,
which often are formed
by antimicrobial-
resistant organisms, are
responsible for 65% of
infections treated in the
developed world.
Biofilms a Great threat to Implants

A significant number of people are affected by biofilm

infections which develop on medical devices
implanted in the body such as catheters (tubes used to
conduct fluids in or out of the body), artificial joints,
and mechanical heart valves. When implanted material
becomes colonized by microorganisms, a slow
developing but persistent infection results.
 Biofilms a Grwoing concern in Modern
Medicine

Prosthetic device infections, such as those involving

artificial heart valves, intravascular catheters, or
prosthetic joints, are prime examples of biofilm-
associated infections. With the increasing use of such
devices in the modern practice of medicine, the
prevalence of these infections is expected to increase.
 Dental plaque
Dental plaque is a
yellowish biofilm that
build up on the teeth. If
not removed regularly, it
can lead to dental caries.
Dental plaques
The formation of dental
plaque biofilms includes
a series of steps that
begins with the initial
colonization of the
pellicle and ends with
the complex formation
of a mature
biofilm.
Formation of Dental
Biofilms

 Additionally, through the

growth process of the plaque
biofilm, the microbial
composition changes from
one that is primarily gram-
positive and streptococcus-
rich to a structure filled with
gram-negative anaerobes in
its more mature state.
Cell-cell signaling (ex. quorum sensing), and
communication with different bacteria enhance
Biofilm formation
 Biofilms everywhere
 They're everywhere: on your
shower curtain, on medical
devices implanted in
patients, on rocks in rivers
and streams, and in your
nose. With the sheer number
of different organisms a
biofilm may contain makes it
a challenge to study,
 CDC – on Biofilms

Biofilms form on the surface of catheter lines and

contact lenses. They grow on pacemakers, heart valve
replacements, artificial joints and other surgical
implants. The CDC (Centers for Disease Control)
estimate that over 65% of Nosocomial (hospital-
acquired) infections are caused by biofilms.
Biofilms interfere with Antibiotic Therapy

 Bacteria growing in a
biofilm are highly resistant
to antibiotics, up to 1,000
times more resistant than the
same bacteria not growing in
a biofilm. Standard
antibiotic therapy is often
useless and the only recourse
may be to remove the
contaminated implant.
 Biofilm and Antibiotic resistance
 A key property of biofilms is
that individual
microorganisms are bound
together by a polymeric
substance excreted by the
microorganisms.. This
protective encapsulation is
believed to play a role in
some antibiotic-resistant
infection.
Bacterial resitance and Biofilms

Another area of great importance from a public health

perspective is the role of biofilms in antimicrobial-
drug resistance. Bacteria within biofilms are
intrinsically more resistant to antimicrobial agents
than planktonic cells because of the diminished rates
of mass transport of antimicrobial molecules to the
biofilm associated cells or because biofilm cells differ
physiologically from planktonic cells
Biofilms in Cystic fibrosis
Biofilms are involved in
numerous diseases. In
cystic fibrosis patients
have Pseudomonas
infections that often
result in antibiotic
resistant biofilms.
 Endocarditis and Biofilms

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 endocarditis.
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.
 Eye and Biofilms

 The presence of bacterial

biofilms has been
demonstrated on many
medical devices including
intravenous catheters, as
well as materials relevant to
the eye such as contact
lenses, scleral buckles,
suture material, and
intraocular lenses. Many
ocular infections often occur
when such prosthetic devices
come in contact with or are
implanted in the eye.
Biofilms and Contact lenses
 Bacterial biofilm formation
on contact lenses and contact
lens storage cases may be a
risk factor in contact lens-
associated corneal
infections. Studies have
shown that contamination of
lens cases by bacteria, fungi,
and amoebae is common
with 20% to 80% of lens
wearers having a
contaminated lens case.
 Urinary catheters and Biofilms

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.
Biofilms and indwelling medical devices

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; and the gram-negative Escherichia coli,
Klebsiella pneumoniae, Proteus mirabilis, and
Pseudomonas aeruginosa.
 Indwelling catheters and Biofilms*
Central venous catheters, the reference method for
quantification of biofilms on catheter tips is the
roll-plate technique, in which the tip of the catheter
is removed and rolled over the surface of a
nonselective medium. Quantification of the biofilm
depends on the number of organisms recovered by
contact with the agar surface. Biofilm-associated
cells on the inner lumen of the device are not
detected with this method, which has low
diagnostic sensitivity and low predictive value for
catheter-related bacteraemia.
 Indwelling catheters and Biofilms*
In addition, this method cannot detect more than 1,000
colony-forming units (CFU) per tip. A method that
used sonication plus vortexing as a means of
quantifying biofilms on catheter tips showed that a
level of 104 CFU per tip is predictive of catheter-
related septicaemia
* Biofilms and Device-Associated Infections
Rodney M. Donlan Centers for Disease Control and Prevention Atlanta, Georgia,
USA
Antibiotic therapy alone may not cure ?
 Antimicrobial agents are
administered during valve
replacement and whenever
the patient has dental work
to prevent initial attachment
by killing all
microorganisms introduced
into the bloodstream. As
with biofilms on other
indwelling devices,
relatively few patients can
be cured of a biofilm
infection by antibiotic
therapy alone
 Biofilms a concern in Antimicrobial Therapy

Microbial biofilms may pose a public health

problem for persons requiring indwelling medical
devices. The microorganisms in biofilms are
difficult or impossible to treat with antimicrobial
agents; detachment from the device may result in
infection. Although medical devices may differ
widely in design and use characteristics, specific
factors determine susceptibility of a device to
microbial contamination and biofilm formation.
Biofilms need higher concentration of Antibiotics

Biofilms are remarkably difficult to treat with

antimicrobials. Antimicrobials may be readily
inactivated or fail to penetrate into the biofilm. In
addition, bacteria within biofilms have increased (up to
1000-fold higher) resistance to antimicrobial
compounds, even though these same bacteria are
sensitive to these agents if grown under planktonic
conditions.
 Biofilms help Gene transfer

Biofilms increase the

opportunity for gene transfer
between/among bacteria..
Gene transfer can convert a
previous avirulent
commensal organism into a
highly virulent pathogen.
Biofilms –Quorum sensing
 Certain species of bacteria
communicate with each
other within the biofilm. As
their density increases, the
organisms secrete low
molecular weight molecules
that signal when the
population has reached a
critical threshold. This
process, called quorum
sensing, is responsible for
the expression of virulence
factors.
Quorum sensing helps the survival of pathogens
Biofilms contribute for new
phenotypes
Bacteria express new, and sometimes more virulent
phenotypes when growing within a biofilm. Such
phenotypes may not have been detected in the past
because the organisms were grown on rich nutrient
media under planktonic conditions. The growth
conditions are quite different particularly in the depths
of biofilms,
Biofilms protect from Immune responses

Bacteria embedded within biofilms are resistant to

both immunological and non- specific defence
mechanisms of the body. Contact with a solid surface
triggers the expression of a panel of bacterial enzymes
which catalyze the formation of sticky polysaccharides
that promote colonization and protection.
Biofilms – Protect from Phagocytosis
Phagocytes are unable to effectively engulf a
bacterium growing within a complex polysaccharide
matrix attached to a solid surface. This causes the
phagocyte to release large amounts of pro-
inflammatory enzymes and cytokines, leading to
inflammation and destruction of nearby tissues.
Current objectives on Biofilm
research
o Development of improved imaging of biofilms in
situ. o
Development of improved clinically relevant in
vitro and in vivo models of biofilms under specific
in vivo conditions such as flow rate, nutrient
content, and temperature.
o Development of better probes (genetic,
metabolic, and immunological) for real- time
analysis; o
Studies of quorum sensing/signaling molecules.
Current objectives on Biofilm research
o Further characterization of biofilm-specific gene
expression.
o Studies of the exchange of genetic material within
biofilms.
o Studies of organic contaminants on substrata, and
their influence on biofilm structure.
o Development of novel approaches
to control pathogenic bacteria by, for example,
devising strategies to favour growth of non-
pathogenic microorganisms in biofilm communities.
 Current objectives on Biofilm research

 o Studies of pathogenic
mechanisms of microbes
growing in biofilms;
o Elucidation of
mechanisms of resistance of
biofilms to antimicrobial
agents; o
Studies of host immune
responses, both innate and
adaptive to biofilms;
Current objectives on Biofilm research
 In studies of infectious lung disease in cystic
fibrosis; o
Studies on the potential of diagnostic procedures
such as Bronchoalveloar lavage and bronchoscopy
to disturb local biofilm flora and inoculate distant
locations; o Development
of mathematical models and computer simulations
of biofilms; o
Development of the methodology for the
prevention and control of biofilms from catheters,
water unit lines, and other clinically important
solid surfaces;.
Searching for alternatives – Tissue
engineering
Role of biofilms in multiple pathologies and the
difficulty in resolving these pathologies speaks to
the importance of developing means of replacing
or enhancing the therapies already in use. The use
of synthetic materials in the body ranges from
catheters to mesh to stents to heart valves and
beyond. Until the development of viable and
practical tissue engineering, then number and types
of applications in which synthetic materials are
used will continue to increase.
 Emerging Methods
 Several researchers are
finding solutions for the cure
of Biofilms , yet it is
experimental, with advances
in molecular biology better
model treatments can be
identified to reduce the
problem of Biofilm
interference in Antibiotic
therapy.
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