BIOFILMS

Baby mize
1613852
M.sc.

Shinu
Asst professor
MMIMSR

AEROBIC
CONDITION

ANEAROBIC
CONDITION

ACID PRODUCE AT
THE SURFACE

ACID PRODUCE
THROUGH OUT THE
TUBE

OXIDATIVE

FERMENTATIVE

HISTORY
1684 -

Van leewenhoek
Dental plaque

1940

Heukelekian & Heller

Bottle-Effect

Zobell - adhesions
1969 Jones different
1943

organisms
Matrix
composed of
polysaccharide
1973 charaklis
antimicrobial
resistant

FORMATION OF
BIOFILMS
Biofilms may form on living or
non-living surfaces
MO have 2 forms SESSILE &
PLANKTONIC
BIOFILM can be simple or
complex
Simple MONOSPECIES
Complex MULTISPECIES OR
CO- BIOFILMS (MC)

DEFINITION
BIOFILM - assemblage of bacterial cell -irreversibly associated
with the surface and enclosed in matrix of Extracellular Polymeric
Substance (EPS).

SURFACES

Rough surface
decrease shear force
increase surface area
Hydrophobic surface
Non polar
more on Teflon than
glass or metals

ENVIRONMENTAL FACTORS
pH

NUTRIENTS

O2 , CO2

Good
biofil
m

TEMPERATU
RE

STEPS IN BIOFILM
DEVELOPEMENT

STAGE 1- REVERSIBLE
BINDING
Takes seconds to initiate
Conditioning film ( Loef & Neihof
1975)
Nutrient rich environment
adherence
Vander wall forces
Reversible some cells detach from
substratum

STAGE 2..IRREVERSIBLE
BINDING

Minutes after stage 1

Adhesion molecules , pilli
, fimbriae
Cell aggregates
EPS is produced

EXTRACELLULAR POLYMERIC
SUBSTANCE (EPS)

Glycocalyx , slime
layer
Highly hydrated
DNA , PROTEINS &
POLYSACCHARIDES
Linear or branched
molecules

BIOFILM QUORUM SENSING

CELL to CELL
communication
Responsible for the
expression of virulence
factors
GRAM POSITIVE
oligopeptides or protein
GRAM NEGATIVE Nacyl homoseriene
lactone

STAGE 3
Maturation I stage
Thickness increase to
more then 10m
Base becomes
anaerobic

STAGE 4

Maturation II
stage
Thickness
increase to more
then 100m

STUCTURE OF BIOFILM
OUTERMOST LAYER
Exposed to high concentration
of nutrients and oxygen
Most active organisms present
Aligned closely
May slough off to colonize
new biofilm
SECOUND LAYER
Not very active
Exchange gene
material Multiple drug
resistance

INNERMOS
T
Earliest part of biofilm least active
Attached to substratum
Per sister cells
slow growing
analogous to bacterial spores
maintain gene pool
Highly resistant to environment
stress & antimicrobial agents

STAGE 5
Cells displace & spread
to colonize new
surfaces
Enzymes that help in
dispersion by degrading
EPS are
DISPERSIN B
DEOXYRIBONUCLEASE
CIS 2 DECENOIC
ACID- pseudomonas

PATHOGENICITY
Colonization
Phagocytosis
Gene transfer
Antibiotic resistance
Metastasis

COLONIZATI
ON

BIOFILMS PROTECTS
FROM PHAGOCYTOSIS
Phagocytes are unable to
effectively engulf a bacterium
due to sticky EPS
This cause phagocytes to release
large amounts of proinflammatory enzymes and
cytokines , leading to
inflammation and destruction of
nearby tissues.

BIOFILMS
HELP GENE
TRANSFER
Gene transfer can
convert a previous
virulent commensals
organism into a highly
virulent pathogen.
Close proximity
Transformation &
conjugation

INTERFERE IN
ANTIBIOTIC
THERAPY

stant than planktonic cells

- not size exclusion

Bacteria growing in
a biofilm are highly
resistant to
antibiotics , up to
1,000 times more
resistant
EPS
GENE TRANSFER
- pH changes (more acidic)
- O2 changes (anaerobic)

- not size exclusio

S. epidermidis treated
with rifampin

BIOFILMS IN CYSTIC
FIBROSIS

In cystic fbrosis
patients have
Pseudomonas
infections that
often results in
antibiotic resistant
bioflms.

CATHETERS AND
BIOFILMS
Inner and outer surfaces
The organism commonly
contaminating are
S. epidermidis
E. faecalis
E. coli
P. mirabilis
p. aeruginosa
K. pneumoniae
Longer duration of
catheter leads to UTI

BIOFILMS AND
CONTACT
LENCES

Contact lenses
Contact lens
storage cases
Risk factor in
contact lens
associated corneal
infections

INTRAUTERINE DEVICES

Candida albicans
Enterococcus
Staphylococcus aureus
Coagulase negative
staphylococcus

Artifcial hip prosthesis

Coagulase negative
staphylococccus
Enterococcus
Pseudomonas aeruginosa
Staphylococcus aureus

CDC ( centers for disease

control )

65% of nosocomial
infections are caused
by biofilms

LAB DIAGNOSIS OF BIOFILM

DIAGNOSTIC

IMAGING
CONVENTIONAL
MICROBIOLOGY
BROTH CULTURE
MAKIS METHODE
QUANTITATIVE

CULTURE
VORTEXING

ULTRASONIFICATIO
N

STAINING
MICROSCOPY
TUBE ADHERENCE
METHOD
MODIFIED CONGO RED
AGAR METHOD
TISSUE CULTURE PLATE
METHOD
RADIO LABELLING
INSITU HYBRIDISATION

TUBE ADHERENCE METHOD

Inoculate on BHI broth (2% sucrose )
incubate ( 24 hours at 37C)

Wash sediment with PBS (pH 7.3) and dried

Stained with 0.1% crystal violet

Washed with distilled water three times

Tubes dried and kept inverted position

OBSERVE FOR BIOFILM FORMATION

MODIFIED CONGO RED AGAR

METHOD

Inoculate on Modified congo red

agar and incubate for 24 to 48
hours at 37c

Black coloured colonies with dry

crystalline consistency interpreted
as positive biofilm producing strain

TISSUE CULTURE PLATE

METHOD
Inoculated on BHI broth (2% sucrose) incubated 18hours at
37 C , diluted 1 in 100 with fresh medium

wells filled 200l aliquots of diluted cultures

TCP incubated for 24 hours at 37C

washed with 0.2ml of phosphate buffer saline , fixed

Biofilms formed stained with crystal violet for 1 minute

Excess stained washed with deionized water and plate kept

for drying

OD observed in ELISA

Mean OD values

Bioflm
formation

<0.120

Non / weak

0.120 0.240

Moderate

> 0.240

High

TREATMENT
Antibiotics
Removal of
indwelling medical
devices
Using bacteriophage
Inhibition of quorum
sensing

PROPHYLAXIS
Alloplastic materials has to be replace
Removal of indwelling medical device
Using bacteriophage
Inhibition of quorum sensing
Careful choice of antibiotics and early
therapy may lead to eradication of
condition
Aseptic insertion of catheter
Avoid insertion of catheter if possible
Shorten the duration of catheterization

THANK YOU