Microbial Biofilms

By
Prof. Dr. Naeem Ali
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Two Forms of Microbial Growth

*Growth in planktonic form

Isolated microbial cells float freely in a fluid environment

*Growth in biofilm form

More than 99.9% of the bacteria grow in biofilms on a wide variety of surfaces.

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 Examples of biofilm
• Have you ever slipped on a wet stone in a creek?

Certainly – and it was biofilm that you slipped on

• Have you an aquarium and do you clean its walls?

If you do, what you wipe from them is the biofilm

formed by algae

• Do you clean your teeth regularly?

I hope so and by doing this you remove the biofilm

called dental plaque
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 Where are Biofilms Found?
• Biofilms are EVERYWHERE!
– Tooth plaque
– Ships hulls
– Medical Implants (leading cause of
rejection)
– Contact lenses
– Dairy/Petroleum pipelines
– Rock surfaces in streams/geysers
– Clogged drains

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Antone Van Leeuwenhoek
was the first person to
visualize, graphically illustrate,
and label "animalcules"
(bacteria later termed as
biofilm) that he found in
plaque scraped from his own
teeth in the seventeenth
century

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 Biofilm

A community of microorganisms that

forms at the boundary of phases (usually
solid and fluid)

Consortia of microorganisms adherent to

biotic and abiotic surfaces embedded in
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.
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Role of Gene: Recent studies have shown that algD, algU, rpoS, and
the genes controlling polypho-sphokinase synthesis are all
upregulated in biofilm formation and that as many as 45 genes differ
in expression between sessile cells and their planktonic counterparts

The new definition of a biofilm is a microbially derived sessile

community characterized by cells that are irreversibly attached to a
substratum or interface or to each other, are embedded in a matrix of
extracellular polymeric substances that they have produced, and
exhibit an altered phenotype with respect to growth rate and gene
transcription. (Biofilms: Survival mechanisms of clinically relevant microorganisms, Donlan,
Rodney M. Costerton, J. William, Clinical Microbiology Reviews)

Study of biofilms à Biofilmology

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 Biofilm Pros and Cons
qAdvantages q Disadvantages
–Nutrients tend to concentrate n Waste can accumulate to
at surfaces toxic levels inside biofilm
–Protection against predation n Access to oxygen and
and external environment
water can become limited
–Pooling of resources
(enzymes) from varying
bacterial species in biofilm

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 Biofilm Structure
• Biofilm
– micro colonies of different species of microbial cells (+15% by volume)
– matrix material (+85%).
– EPS may vary in chemical and physical properties but it primarily
consists of polysaccharides.
– Some of the polysaccharides are neutral or polyanionic.
– The presence of uronic acids (D-glucuronic, D-galactouronic and
mannuronic) or ketal linked pyruvate confers the anionic
properties.This property helps in the association of divalent cations
such as calcium and magnesium, which have been shown to cross-link
with the polymer strands and provide greater binding force in a
developed biofilm .6

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Protection from Environment
• EPS of biofilm provides certain degree of shelter and homeostasis to
the bacteria residing in biofilm. It largely depend on the nature of
both the agent and the EPS matrix
– roles in structure and function of different biofilm communities.
– physically prevent the access of certain antimicrobial agents into the biofilm
by acting as an anion exchanger.
– Restricts the diffusion of compounds from surroundings into the biofilm.
– Sequester metal ions, cations and toxins and reported to provide protection
from variety of environmental stresses such as pH shift, UV radiation,
osmotic shock and desiccation.

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Biofilm Syntrophism
• Biofilm provides an ideal
environment for the
establishment of syntrophic
relationship.

• Syntrophism is a special case of

symbiosis in which two
metabolically distinct bacteria
depend on each other to utilize
certain substrates, typically for
energy requirements.
• Facilitates interspecies substrate
exchange and removal and
distribution of metabolic
products.

• Syntrophism has been well

studied with regard to
methanogenic degradation
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 Acquisition of New Genetic Trait
Horizontal gene transfer
• Horizontal gene transfer is important for the evolution and genetic diversity of natural microbial
community.
• Acquisition of new genetic trait gives chances to the microbial communities to transcribe the
necessary genes to become the active member of the biofilm communities. This is due to
transcription of different genes by biofilm forming communities and the phenotypic characters
are the expression of a particular genotypic character.
• Acquisition of metabolic traits
– Resistance
– Mineralization and transformation of compounds and relieving biogeocycles
– Pathogen and non pathogenic interactions and avoidance
– Acquiring of more rigidity in terms of spreading infection and pathogenisis

• Transcription of algC regulated by sigma factors of pulmonary isolates (Pseudomonas aeruginosa)

are mucoid due to the synthesis of large amounts of alginate.
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 Penetration of Antimicrobial Agent
• EPS acts as diffusion barrier for these molecules by influencing the rate of
transport of the molecule to the biofilm interior or the reaction of antimicrobial
agents with the matrix material.
– The penetration of ciprofloxacin to the normal sterile surface required 40 sec whereas
penetration into the biofilm containing surfaces took 21 min.

• Biofilm mode of growth associated advantages to the microbial community in the

following ways:
– (a) as the growth is restricted all the energy is used up by the bacteria in making the EPS that
will give protection to the microbial community.
• P. fluoroscens produces exopolysaccharide lyase that degrades the biofilm associated
exopolysaccharides for consumption and release cells from biofilm scaffold to seek more favorable
environment
– (b) as the growth is restricted, bacteria will remain in dormant stages that will give protection
to the microbial community against antibiotics because most of the antibiotics are active
against the growth phase of the bacteria.
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Scope and Applications of Biofilms

Medical Industrial

Biofilms

Food and
Environmental
Agricultural
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The CDC (Center for Disease Control) estimate that over 65% of
Nosocomial (hospital-acquired) infections are caused by biofilms.

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Biofilm – A Nuisance

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 MICROORGANISMS ASSOCIATED WITH BIOFILM ON INDWELLING DEVICES

No. Medical devices Causative organism

1 Urinary catheter, Intra uterine devices, prosthetic heart valves, central Coagulase -negative
venous catheter Staphylococci

2 Central venous catheter, urinary catheter Klebsiella pneumoniae

3 Artificial voice prosthesis, central venous catheter, intra uterine devices Candida albicans

4 Artificial hip prosthesis, prosthetic heart valve, urinary catheter Enterococcus spp.

5 Contact lenses P. aeruginosa, E. coli, S. aureus, S.

epidermidis Proteus sp., Serratia,
Candida sp.

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Biofilm and Pathogenesis
• Streptococcus, Staphylococcus, Pneumococci, Candida, Aspergillus and some Gram negative
bacteria
Native Valve
Endocarditis • These organisms mainly enter into the blood stream via oropharynx, gastrointestinal
tract and genitourinary tract. Biofilm formed by microbe’s damages valve tissue.

Chronic
• E. coli, P. aeruginosa, and species of Klebsiella, Proteus, Serratia, Bacteroides etc.
Bacterial
Prostatitis

• A chronic disease of the lower respiratory tract.

Cystic • S. aureus, H. influenzae, P. aeruginosa
Fibrosis

• The main microbe associated with periodontitis is Porphyromonas gingivalis.

• Other organisms are Fusabacterium nucleatum, Peptostreptococcus micros, Eubacterium
Periodo timidum, E. brachy, Pseudomonas anerobicus, etc
ntitis 20
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Food borne pathogens and the spoilage organisms in Biofilm

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Impact of Biofilm on Deterioration of Water Quality

The quality of water, when it flows through the distribution system, is adversely affected by
several factors i.e. type of piping material, temperature, type of disinfectants, resistance of bacteria
to disinfectants, etc.

Dissolved organic compounds in finished drinking water is responsible for

• Enhanced chlorine demand
• Trihalomethane production
• Bacterial colonization of water distribution systems – Bacteria enter from the external source
by number of ways such as open reservoirs, breakage due to the new pipeline construction
that may disturb the existing distribution system.

• Increases resistance to disinfection i.e. E.coli is 2400X more resistant to chlorine when in
attached mode
• Increases frictional resistance of fluids
• Causes taste and odor problems e.g. due to H2S production.
• May cause regrowth of certain bacteria by the use of biodegradable compounds.
• May lead to growth of pathogenic bacteria e.g. Legionella
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 Industrial Applications of Biofilms

• Bioremediation: Bacterial degradation of polluted environments

• Biofiltration: Selective removal of chemical species from solution
• Biobarriers: Protection of objects using extremely rugged
glycocalyx produced by biofilms
• Bioreactors: Production of compounds using engineered biofilms

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 Biofilm – A boon

On-Site Urban - Municipal Rural - Municipal

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Industrial
 Factors Affecting Biofilm Formation
• Biofilm may be formed on a wide variety of surfaces
– living tissues, indwelling medical devices, industrial or
portable water system piping or natural water system piping.
• The water system biofilm is highly complex. contains
corrosion products, clay material, freshwater diatoms
and filamentous bacteria.
• The biofilm on the medical devices composed of a single
coccid organism and the associated extracellular
polymeric substances matrix.
• Different factors affecting the formation of biofilm
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1.

Substratum Effect

•Surface roughness
•Higher surface free energy à
greater wettability of surfaces
àmore hydrophilic i.e. stainless
steel and glass
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2. Conditioning Film
Ø Solid surfaces which have been exposed in an aqueous medium become conditioned or coated
with polymers from the medium which affects the rate and extent of microbial attachment.
Ø Surface energy of the suspending medium may affect hydrodynamic interactions of the microbial
cells with surfaces by altering the substratum effects.
Ø Surface is converted to hydrophilic by cleaning with alkali or strong acid (4M nitric acid) of
stainless steel surfaces.
Ø Once the stainless steel is exposed to air or water, it is passivated by the formation of a chromium
oxide layer.
Ø Organic soil adheres to the oxide layer, producing a conditioned substratum to which bacteria
adhere.
Ø Acquired pellicle” which develops on tooth enamel surfaces in oral cavity; consist of;
Ø albumin, lysozyme, glycoprotein, phosphoproteins, lipids and gingival crevice fluid.
Bacteria, from oral cavity, colonize pellicle-conditioned surfaces within hours of exposure
to these surfaces.
Ø Host-produced conditioning films such as blood, saliva, tears, urine, intravascular fluid and
respiratory secretions influence the attachment of bacteria to biomaterials.
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3. Hydrodynamics
• Biofilm response is altered in flow conditions.
• Biofilms grown under laminar flow are found to be
patchy and consist of rough cell aggregates separated
by interstitial voids.
• Biofilms grown under turbulent flow cells are also
patchy but are elongated “streamers” that oscillate in
the bulk fluid.
• Association of cells with the surface also depends on
cell size and cell motility.

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4.

Characteristics of Aqueous
Medium
• pH
• Nutrient levels
• Ionic strength
• Temperature
• Concentration of several cations
such as sodium, calcium,
Magnesium,Aluminium, ferric ions
• Season

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5. Horizontal Gene Transfer (HGT)

•HGT is important for the evolution and genetic diversity of natural

microbial communities.
•Transfer of conjugative transposons from Bacillus subtilis strain
(which confers resistance to tetracycline) within a biofilm resulted in
Streptococcus species resistant to tetracycline
•Transfer of TOL plasmid, which carry the genes for the degradation of
toluene and the benzyl alcohol has occurred in biofilm community
growing on benzyl alcohol as the sole carbon and the energy source.
•Virus-mediated gene transduction is another mode of gene transfer
in biofilm associated microbial communities.

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6. Quorum Sensing (QS)

• The process by which bacteria coordinate their behaviors in a density

dependent manner, using signaling molecules.

• QS has recently been demonstrated to play a role in cell attachment.

• Three types of molecules :

• 1. Acyl-homoserine lactones (AHLs) – mediate QS in Gram negative

bacteria

• 2. Autoinducer peptides (AIPs) – present in both Gram positive and negative

bacteria

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6. Quorum sensing
• P. aeruginosa mutants that do not produce acyl-HSL form biofilms
in which the cells are closely packed together and easily disrupted
by sodium dodecyl sulfate.
• Several QS system mutant bacteria show the heavily reduced
pathogenicity.
• Different types of signals may alter distribution of specific bacterial
species in the biofilm, alter protein expression in neighboring cells,
introduce new genetic trait in neighboring cells and incorporate
bacteria in biofilm.

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 Detection/Characterization Methods
• Analytical techniques for
monitoring biofilms follow two
main strategies:
– Indirect detection of organisms by analysis
of waste and/or metabolism byproducts
• Isolated growth, followed by
analysis of headspace gas or
growing media by a variety of
methods (GC/MS, ICP, HPLC,
etc.)
– Direct detection of organisms
• Microscopy techniques
• Detection of proteins or DNA
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 Detection/Characterization Methods
• Fluorescent in-situ hybridization (FISH) and 16-23S rRNA hybridization with
CLSM are used to observe microstructure and metabolism of biofilm.
• The FISH method was used to confirm decrease in the viability of cells as the
biofilm ages.
• The use of CLSM and epifluorescence microscopy requires the organisms in
biofilms to be stained with fluorescent stains to probe specific cellular functions.
– For example, nucleic acid stain such as DAPI (4¢6¢-diamidino-2- phenyl indole), acridine
orange, and Syto9 will stain the DNA and RNA of all cells regardless of their viability.

• The most commonly used procedure for measurement of biofilm is the viable
plate count method.
– The resuspended and dispersed biofilm cells are plated onto a solid medium,
incubated and counted.
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Fluorescent Probes

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Current objectives on Biofilm research
• Development of improved imaging of biofilms in situ
• Development of improved relevant in vitro and in vivo
models of biofilms under specific in vivo conditions such as
flow rate, nutrient content, and temperature etc
• Development of better probes (genetic, metabolic, and
immunological) for real- time analysis
• Studies of quorum sensing/signaling molecules

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Current objectives on Biofilm research
• Further characterization of biofilm-specific gene expression
• Studies of the exchange of genetic material within biofilms
• Studies of organic contaminants on substrata, and their
influence on biofilm structure
• Development of novel approaches to control pathogenic
bacteria by, for example, devising strategies to favour growth
of non-pathogenic microorganisms in biofilm communities;

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*Current objectives on Biofilm research

• Development of the methodology for the prevention and control of biofilms

from catheters, water unit lines, and other clinically important solid surfaces

• Development of mathematical models and computer simulations of

biofilms

• Specific interactions among bacterial strains of multispecies biofilms

• Effect of individual surface properties on biofilm microbial densities

• Biofouling control strategies

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Detection as Biomarker for Extraterrestrial Life
• Candidates for study:
–Eurpoa: One of Jupiter’s
moons believed to have liquid
water beneath icy surface.
–Mars: Bacteria shown to
grow on simulated Mars soil
http://nssdc.gsfc.nasa.gov/image/planetary/jupiter/europa_close.jpg

and environmental
conditions.

http://antwrp.gsfc.nasa.gov/apod/ap010718.html

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