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MICROBIAL

BIOFILMS
Shilpa.K
Microbiology Tutor
AIMSRC
ORIGIN OF SURVIVAL AND
COMMUNICATION AMONG MICROBES

On earth 3.4 Led to evolution of post


billion years ago The oldest story biofilm planktonic phenotype
of
unity is strength
taught
Microbes were by the Microbes formed
struggling to most minute communities within biofilms
survive, grow and creatures to transfer to
adapt on different environments
Earth

As the earth cooled Moved to multispecies adaptation

SINGLE START TO MINGLE TO FORM BETTER SINGLE


HISTORY
Van Leeuwenhoek credited for the discovery of
microbial biofilms.

Heukelekian and Heller observed the bottle effect


for marine microrganisms.

Zobell observed number of bacteria on surface


was higher than in surrounding medium (ex:seawater)

Jones et al. used scanning & transmission electron


microscopy to examine biofilms
Characklis studied microbial slimes in industrial
water systems and showed that they were highly
resistant to disinfectants like chlorine.

Costerton et al. in 1978 explained the theory of


biofilms.
BIOFILMS
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.
WHEN DO MICROBES DECIDE TO FORM
BIOFILMS
Mainly when they are capable
of recognition of specific or
non-specific attachment sites
on a surface

Nutritional cues

Exposure of planktonic cells to


sub-inhibitory concentrations
of antibiotics
INGREDIENTS TO PREPARE A BIOFILM

Non cellular materials:


( extra cellular polymer) Celular materials:

mineral crystals bacteria


corrosion particles parasites
clay or slit particles fungus
blood components etc virus
polysachharides
proteins
CHARACTERISTICS OF A SURFACE

Liquid surface Solid surface

Non living surface:


Living surface:
Industrial setting,
Leaves, wounds, Hospital setting,
Post surgical sites, Indwelling medical devices,
Marine invertebrates, etc Rocks/boats in sea,
Toothbrush etc
ENVIRONMENTAL AND CULTURAL FACTORS
AFFECTING BIOFILM
ATTACHMENT
EFFICIENCY
NUTRIENT
RESOURCES CYCLIC STAGE

BIOFILM COMMUNITY
GENOTYPIC STRUCTURE AND ANTI EFFECTIVE
FORCES EVOLUTION HOSTILE FORCES

SUBSTRATUM MECHANICAL FACTORS PHYSIOCHEMICAL


AND ENVIRONMENT
SHEAR FORCES
Role of physiochemical properties of a surface:

Microbes attach more rapidly to teflon and other plastics than


to glass or metals.

Conditioning films:

Formed when a material surface is exposed in aqueous


medium and becomes conditioned or coated by polymers
from that medium, the resulting modification will affect the
rate and extent of microbial attachment.
Hydrodynamics:
the flow velocity immediately adjacent to the
substratum/liquid interface is negligible.
higher velocities would be expected to equate
to more rapid attachment or association to the
surface, atleast until velocities become high
enough to exert substantial shear forces on the
attaching cells, resulting in detachment of
these cells.
Characteristics of the aqueous medium:

seasonal effect on bacterial attachment depends on water


temperature.

increase in concentration of cations affects attachment


presumably by reducing the repulsive forces between the
negatively charged bacterial cells & surfaces like glass.

Also it was seen that an increase in nutrient concentration


correlated with increase in the number of attached bacterial
cells.
STEPS IN THE FORMATION OF BIOFILM
STEP 1: Initial / reversible attachment -
binding of 1st colonists

STEP 2: Irreversible attachment


they anchor themselves permanently using pili

STEP 3: Maturation 1
inter-communication through quorum sensing

STEP 4: Maturation 2 / Development


final stage of modification

STEP 5: Dispersion
essential stage for biofilm dispersion and life-cycle

Role of enzymes dispersin B and deoxyribonucleases in step 5 and medical


application of cis-2-decenoic acid
QUORUM SENSING
Is a type of decision making process used by a decentralized
group to coordinate behavior.

It can occur within a single bacterial species as well as between


diverse species.

It serves as a simple communication network.

Many species of bacteria use it to coordinate their gene


expression according to the local density of their population
MOLECULES USED AS SIGNALS
In gram positive bacteria
oligopeptides
In gram negative bacteria
AHL
Also, in both types of bacteria
AI-2
In combination of bacteria and fungi
3-oxo-C12homoserinelactone

These biochemicals diffuse through water channels seen in matrix.

Regulate the transcription of various genes as per the requirement of the


community to produce various proteins, enzymes, etc.

Serves as a major means of communication that allows the sessile organisms


to survive and grow in the presence of unsuitable environmental conditions
and antibiotics or detergents
FUNCTIONING OF BIOFILM
Outermost layer-
Highest concentration of oxygen and nutrients, resembles their panktonic
counterparts.
They slough off and initiate biofilm formation downstream

Second layer-
Organisms here downregulate in their metabolic activity
Although they can clearly utilize the nutrients, exchange genes and have
the potential for multiple drug resistance
The benefit is obtained from their alignment in this layer which depends
upon spatial arrangement, physiologic heterogeneity and non uniformity

Innermost layer-
Attached to the substratum and is the earliest part of the biofilm
Also efficiently downregulate and are least metabolically active
Most persisters are found to be fossilized here
Provides inheritance for future populations that transfer laterally
Short term stress is absorbed elastically and long term stress is
dissipated through viscous flow

Phenomenon of rolling and creeping.

Up and down regulation of various genes is seen in the


attaching cells.

The gene transfer is much enhanced here than in the


planktonic cells.

Biofilm association also provides a mechanism for selecting


and promoting the spread of bacterial resistance to
antimicrobial agents
Native valve
endocarditis

Otitis media Periodontitis


Human
infection
involving
biofilms

Chronic
Cystic
bacterial
fibrosis
prostatitis
Native valve endocarditis
This results from the interaction between vascular
endothelium of the heart valves and bacteria or fungi

Streptococcus spp
Staphylococcus spp
CoNS
E. Coli
Klebsiella spp
Candida species
Aspergillus spp
Organism enter into blood steam through oropharynx, GI tract, genitourinary
tract.

When endothelium is damaged thrombus will develop and fibronectin is


secreted by the endothelial cells and bacteria adhere to the endothelial
cells

Secrete dextran helps in adherence of bacteria to endothelial cells

Within one hour it begin to multiply & develop matrix &


capsule which protected from white blood cells

It may result in the vascular tissue damage


2) OTITIS MEDIA
Disease of middle ear which involves inflammation

Streptococcus pneumoniae

Haemophilus influenzae

Staphylococcus aureus

Staphyloccus epidermidis

Pseudomonas aeruginosa

Moraxella catarhalis
3) CHRONIC BACTERIAL PROSTATITIS
In this prostate gland may become infected by bacteria that
have ascended from the urethra.
Escherichia coli
Pseudomonas aeruginosa
Klebsiella spp
Proteus spp
Serratia spp
Enterococcus faecalis
4)CYSTIC FIBROSIS
It is a chronic disease of lower
respiratory tract.

In this disease, deficiency of water


hinders the movement of mucus &
increase the secretion of mucus & lead to
dehydration and thickening of respiratory
epithelium.

The organism which can causes the


biofilm mainly is
Pseudomonas aeruginosa.
5) PERIODONTITIS
Pellicle develops on the surface of enamel

This pellicle comprises of albumin, lysozome,


glycoprotein , oral flora colonizes on the
surface & secrete the dextrons which helps in
the adherence of bacteria & form biofilm that is
called plaques.

Fusobacterium spp.,

Eubacterium spp.,

Peptostreptococcus spp.,

Haemophilus aphrophilus.
BIOFILM ON MEDICAL DEVICES
Central
venous
catheter

Prosthetic Intrauterine
heart valve device

Indwelling
Medical
devices

Urinary
catheter Artificial hip
prosthesis

Artificial
voice
prosthesis
MEDICAL DEVICES AND COMMON ORGANISM
WHICH CAUSES BIOFILM
MEDICAL DEVICES ORGANISM

Central venous CoNS, Staphylococcus aureus, Enterococcus faecalis,


catheter Klebsiella pneumoniae, Pseudomonas spp., Candida
albicans
Prosthetic heart Viridans Streptococci, Staphylococcus aureus,
valves Enterococci
Urinary catheter Staphylococcus epidermidis, E.coli, Klebsiella, Proteus
mirabilis
Intrauterine device Staphylococcus epidermidis, Staphylococcus aureus,
Micrococcus spp, Enteroccocci spp, Group B
Streptococci, Candida albicans
Artificial voice Candida albicans, Streptococcus spp, Staphylococcus
prosthesis epidermidis.
1) PROSTHETIC HEART VALVES

The surgical implantation of


prosthetic valve leading to
accumulation of platelets and
fibrin at the suture site and
device & there is a formation
of biofilm.
2) CENTRAL VENOUS CATHETER
When catheter is introduced into
the blood vessels the skin colonizer
may migrate along inner lumen
from the skin insertion site.

When these organism get contact


with blood they form thrombus this
lead to formation of biofilm
3)URINARY CATHETER
Organisms form biofilm when
the catheter is inserted into the
urethra.

This organism may alter the pH


and form substance like
ammonia & minerals form
biofilm
4)CONTACT LENSES
Bacteria adhere readily to the
lenses like
Staphylococcus aureus,
Pseudomonas aeroginosa,
Staphylococcus epidermidis
E.coli, Streptococcus pyogenes,
Serratia marcescence , Candida
albicans, Acanthameoba ,
Fusarium.
COMMON IMPLANT INFECTION
DETECTION, MEASUREMENT AND
CONTROL OF BIOFILMS

Microscopic Examination
Quantitative detection
Qualitative detection
Molecular detection
Microscopy
Electron Microscopy
For examination & characterisation of biofilms on medical
devices & human infections.
SEM- has been used for early investigative studies.
Limitation dehydration leads to
significant sample distortion & artifacts.
TEM- With the use of ruthenium red stain helps identify
nature of extracellular fibers & their association with cells.
Confocal Laser Scanning Microscopy
High resolution optical images with depth selectivity
3D reconstruction of the image.
Specimen stained with fluorescent stains which can be
used to probe specific cellular functions. Eg: Nucleic acid
stains- DAP 1,AO,Syto 9 etc.
Other stains for probing cell viability- Propidium iodide ,
5-cyano-2,3-ditoyl tetrazolium chloride.
Epifluorescence microscopy.
Fluorescent antisera.
Qualitative methods
Tube method
Colony inoculated into 10 ml TSB

Incubate at 37Cfor 24 hours

Decant tubes, wash with phosphate buffer saline

Dried & stained with 0.1% crystal violet


RESULTS:
Positive - visible film on the wall & bottom of the tube

Negative Ring formation at the liquid interface

Grading based on biofilm formation

0 : Absent

1 : Weak

2 : Moderate

3 : Strong
A- High

B- Non Biofilm Producer

C- Moderate

A B C
Quantitative methods
Roll plate method
Tissue culture plate method
Congo red method
Calgary biofilm device
Roll plate method:
Used in case of central venous catheter.

Disadvantage:
Cannot detect cells on inner surface of he device.
Cannot detect more than 1000 cfu.

Can be improved by sonication and vortexing.


Tissue culture plate method:
Used to detect ability of an organism to form biofilm.

24 hr old isolate inoculated onto TSB with 1% glucose.


Incubated at 37 degree for 24 h.

Diluted 1:100 in fresh medium.

0.2 ml aliquots filled into sterile polystyrene tissue culture


plates

Incubate at 37 degree for 18-24 hours.


Contents of each well removed by gentle tapping & washed
with 0.2% phosphate buffer.

Biofilms formed are fixed by sodium acetate and stained with


0.1% crystal violet for 5 -10 minutes.

Excess stains are washed with deionised water & plates are
kept for drying.

OD values determined by micro ELISA auto reader at 570nm

Mean OD values Adherence Biofilm formation


<0.120 Non Non/weak
0.120-0.240 Moderately Moderate
>0.240 Strong High
High

Moderate

Non
Congo Red agar method.

Media used- BHIA with 5% sucrose and Congo red


indicator.
A fresh isolate of pathogenic strain is inoculated and
incubated for 24 h at 37 degree.

Results-
Positive : Black colony with dry crystalline
consistency
Negative : Pink colored colony
Intermediate : Dark colony without dry crystalline
consistency
A B
Screening of biofilm producers by Congo red agar medium
A) S. epidermidis ATCC 35984 (black colonies)
B) S. epidermidis ATCC 12228 (pink colonies)
Calgary Biofilm Device:

Recently discovered
A device that can detect biofilms as in tissue culture
plate along with Antimicrobial Susceptibility testing.
Widely accepted method.
Peg

Microtitre
plate
Common methods used by clinical microbiologists for recovery &
measurement of clinically relevant biofilms on medical devices.
METHOD BASIC PROTOCOL ADVANTAGE LIMITATION

Roll plate Roll the catheter tip Easy to use Examines only outer
over surface of BA surface . Inaccurate

Vortex then viable Catheter section in Measures intraluminal Recovery efficiency


count. PBS is vortexed then & extraluminal unknown.
cultured on different biofilm.
media.

Sonicate , vortex, then Catheter section in Measures intraluminal Recovery efficiency


viable count. TSB , sonicate , vortex & extraluminal unknown.
& culture on BA. biofilm.

Sonicate, vortex, Catheter section in Recovery efficiency Measures intraluminal


homogenise, then PBS/vortex repeatedly determined. biofilms only.
viable count. then homogenise &
culture on BA.
Acridine orange direct Following roll plate, Allows direct Method doesnt allow
staining. catheter section is examination of quantification.
stained with AO catheter.

Endoluminal brush Brush is introduced Allows examination of Effect of procedure on


into the implanted indwelling catheter. patient & recovery
catheter,removed, efficiency unknown
placed in PBS,
sonicated & plated.
Apparatuses used for growing & testing Biofilms

APPARATUS ORGANISMS FLOW SUBSTRATUM METHOD FOR


TESTED DYNAMICS REMOVING &
QUANTIFYING
BIOFILM
Modified Robbins Pseudomonas Batch/mixing Silastic disks Method of removal
device pseudomallei not given; viable
count

Calgary biofilm device P. aeruginosa, S. Batch/mixing Plastic pegs Sonicate peg, then
aureus, E. coli viable count

Disk reactor Gram-negative Batch/mixing Teflon coupons Sonicate, vortex,


bacteria homogenize,
then viable or direct
count
CDC biofilm reactor Gram-negative Continuous/open Needleless Sonicate, vortex,
bacteria system Connectors. (plastic) homogenize,
then viable or direct
count
Perfused biofilm Candida albicans Continuous/open Cellulose-acetate Shake in sterile water,
fermentor system filters then viable count .

Model bladder Gram negative Continuous/open Urinary catheters Direct examination by


bacteria system SEM or
TEMa or by chemical
MOLECULAR METHODS

FISH

PCR

RT-PCR

RFLP

RAPD
ANTIMICROBIAL SUSCEPTIBILITY
TESTING
Determination of MIC Standard method for
AST.
Calgary Biofilm Device is most accepted
method used against E.coli, S.aureus ,
P.aeruginosa.
Modified Robbins Device is also used
BIOFILMS & CLINICAL DECISION
MAKING
Collect paired blood samples.
Repeated negative results for samples may
not imply absence of biofilms.
The coagulase-negative staphylococci spp
dilemma.
TREATMENT
Issues: Drug resistance due to

Impaired penetration of antibiotics.

Reduced growth rate of bacteria

Altered micro environment

Altered gene expression

Reduced biofilm specific phenotype


Treatment is based on MIC & MBEC results.
Fluoroquinolones , aminoglycosides &
cephalosporins for gram negative bacteria.
Drugs like vancomycin for gram positive bacteria.
Triazoles, lipid formulations of amphotericin B &
echinocandins for Candida species.
Photodynamic therapy
Ultrasonic wave therapy.
Immune modulation by use of azithromycin & low
dose doxycycline .
Antimicrobials like, silver & tobramycin.
CONTROL AND PREVENTION
Common disinfectants used Iodophor, hypochlorite,
acid anion, peroxyacetic acid, quaternary ammonium
compounds & sanitisers.
On medical devices-
Catheters impregnated with minocycline and rifampicin
Catheters coated with a cationic surfactant
Attachable subcutaneous cuffs containing silver ions
inserted after local application of polyantibiotic ointment
Control using phages
Control through enzymatic detergents (Food industry)
Control through microbial interactions/metabolite
molecules.
FUTURE RESEARCH PERSPECTIVES
More reliable methods for detection & measurement of
biofilms should be developed.

Elucidation of the genes specifically expressed by biofilm-


associated organisms

Evaluation of various control strategies .

Studies related to the effect of probiotics on biofilms.


REFERENCES
Biofilm Formation in Uropathogenic Escherichia coli Strains: Relationship With
Prostatitis, Urovirulence Factors and Antimicrobial Resistance - S. M. Soto,* A.
Smithson, J. A. Martinez, J. P. Horcajada, J. Mensa and J. Vila
MINIMUM INHIBITORY CONCENTRATION (MIC) VERSUS MINIMUM BIOFILM
ELIMINATING CONCENTRATION (MBEC) IN EVALUATION OF ANTIBIOTIC SENSITIVITY OF
GRAM-NEGATIVE BACILLI CAUSING PERITONITIS. Peritoneal Dialysis International, Vol.
24, pp. 6567
Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a
halogenated furanone compound. Microbiology (2002), 148, 87102.
Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. CLINICAL
MICROBIOLOGY REVIEWS, Apr. 2002, p. 167193
Microbial Biofilms: from Ecology to Molecular Genetics MARY ELLEN DAVEY AND
GEORGE A. OTOOLE* Department of Microbiology, Dartmouth Medical School,
Hanover, New Hampshire 03755
Cooperation and conflict in microbial biofilms Joao B. Xavier and Kevin R. Foster*
Center for Systems Biology, Harvard University, Bauer Laboratory, 7 Divinity Avenue,
Cambridge, MA 02138 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1982, p.
11%-1204 Vol. 44, No. 5
Biofilms: discovery of a new mechanism of virus propagation
Biofilm-control strategies based on enzymic disruption of the extracellular
polymeric substance
matrix a modelling study
JOURNAL OF BACTERIOLOGY, 10.1128/JB.183.18.53855394.Sept. 2001, p.
53855394 Vol. 183, No. 18, American Society for Microbiology.
Biofilm Formation by the Fungal Pathogen Candida albicans: Development,
Architecture, and Drug Resistance Rev Iberoam Micol 2002; 19: 139-143 139
Medical importance of biofilms in Candida infections L. Julia Douglas
Biofilms and Device-Associated Infections Rodney M. Donlan
Centers JOURNAL OF CLINICAL MICROBIOLOGY, June 1999, p. 17711776
Vol. 37, No. 6 American Society for Microbiology. All Rights Reserved.
The Calgary Biofilm Device: New Technology for Rapid Determination of
Antibiotic Susceptibilities of Bacterial Biofilmsfor Disease Control and
Prevention Atlanta, Georgia, USA
Biofilms: Microbial Life on Surfaces Rodney M. Donlan*
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Factors Regulating Microbial Biofilm Development in a System with Slowly
Flowing SeawaterKARSTEN PEDERSEN Paris, December 21, 2009
Bacteriologists had it wrong for the past
300 years- Bacteria dont live alone
They grow best when each one does its
own thing. Together!!!

Thank you!!!!