Transcript Architecture of biofilm I

Institute for Microbiology, Medical Faculty of Masaryk University
and St. Anna Faculty Hospital in Brno
Miroslav Votava
MICROBIAL BIOFILM – II
The 6th lecture for 2nd-year students of Dentistry
March 28, 2012
Definition of biofilm – revision
Microbial biofilm is a community of
microorganisms that
• forms at the boundary of phases
(usually of the solid and fluid phase)
• sticks to inert as well as to live
surfaces
• is surrounded by an extracellular
matter, in which a complex system of
channels may form
Development of the biofilm
– revision
Development of biofilm = cyclic process
1. Attraction of planktonic cells to a surface
2. Adhesion of planktonic cells to the surface
3. Aggregation of cells and the development
of colonies – quorum-sensing phenomenon
4. Accumulation of exopolysaccharide
matrix (slime) – development of typical architecture
5. Dispersal of cells from the surface of
biofilm
Quorum sensing – revision
During division individual cells emit chemical
signals
After reaching a particular number of cells
(quorum) the elevated concentration of signals
causes the change of cellular properties:
- switching off some so far functioning genes
(e.g. a gene for the production of flagellin)
- expression of other genes, and from this
ensuing
- production of new molecules (in particular
exopolysaccharides)
Architecture of biofilm I – revision
It depends above all on the concentration of
nutrients
• <10 mg/L (mountain streams, lakes, open sea):
heterogeneous mosaic (a thin layer + columns of
microcolonies)
• 10-1000 mg/L (majority of our rivers and ponds):
complex system with channels (created by
mushroom-like, partially merging microcolonies)
• 1000 mg/L (in the environment of macroorganism):
compact biofilm (almost without traces of channels)
Architecture of biofilm II – revision
Low concentrations of nutrients (0.1 – 10 mg/L – mountain streams,
lakes, open sea)
Heterogeneous mosaic = thin layer of individual cells above which
columned microcolonies rise here and there
Architecture of biofilm III – revision
Medium concentration of nutrients (10 – 1000 mg/L – eutrophic water
environment)
System with channels = mushroom-shaped microcolonies partially
merging together, interwoven with water channels
Architecture of biofilm IV – revision
Architecture of biofilm V – revision
High concentrations of nutrients (>1000 mg/L – in the macroorganism)
compact biofilm = closely interconnected numerous microcolonies almost
without traces of possible channels
a) polymicrobial = e.g. dental plaque, normal microflora of mucous
membranes
Architecture of biofilm VI – revision
High concentrations of nutrients (>1000 mg/L – in the macroorganism)
compact biofilm = closely interconnected numerous microcolonies almost
without traces of possible channels
b) monomicrobial = e.g. chronic osteomyelitis
biofilm on inert surfaces of medical devices
Properties of microbes in biofilm –
revision
The properties of microbes growing in the
biofilm form are fundamentally different
from the properties of microbes growing in
the planktonic form; the microbes in biofilm
– express different genes
– produce different products
(extracellular matrix  flagella)
– enjoy a higher degree of protection
Properties of biofilm – summary
& revision
• Biofilm is a higher and more complex form of
microbial growth
• It utilizes the opportunity of mutual cooperation of
cells
• It enables the easier transfer of genes
• It is characterized by an effective homeostasis
• It shows features of a primitive circulation system
• It provides a high protection against antimicrobial
factors
• It plays an important part in many significant
occasions including medically relevant conditions
Importance of biofilm for the life
of microorganisms – I
More favourable environment for the life of
microorganisms
Possibility of effective cooperation and specialization of
cells
because of stable mutual position of cells of different
species in the intercellular matrix and corresponding
presence of various enzymes
Considerably easier transfer of genes
Effective homeostasis
Inside the aerobically established biofilm anaerobic
places may occur
Primitive circulation system
brings in and carries away nutrients, waste products as
well as signal molecules
Importance of biofilm for the life
of microorganisms – II
Protection against harmful influences
in environment: against amoebae, phages,
dessication, washing away,
toxic substances
in macroorganism: against phagocytes,
washing away,
complement,
antibodies,
antibiotics
Resistance of biofilm towards
toxic substances
MICROBES IN THE BIOFILM FORM ARE ALWAYS
MORE RESISTANT THAN IN THE PLANKTONIC
FORM
• Higher resistance applies also to disinfectants and
antibiotics
• Differences in sensitivity sometimes amount up to 3 orders
• General mechanism of the higher resistance is not known
• In each microbe-antimicrobial combination the mechanism
can be different
Possible causes of higher
resistance of biofilm
1. More difficult penetration of toxic matter
through the biofilm
2. Character of environment in the biofilm is
altered
3. Also the microbial population in the biofilm is
altered
Ad 1. Difficult penetration
EXTRACELLULAR MATRIX SERVES AS THE
PENETRATION BARRIER
•
difficult diffusion of the antimicrobial:
logical explanation
not always demonstrable
•
influence of surface charge seems more important
e.g. aminoglycosides (+) combine with
alginate (-) of P. aeruginosa
Ad 2. Altered environment
LOCAL ALTERATION IN THE BIOFILM
ENVIRONMENT
(consumption of O2 in certain areas,
increase in osmotic pressure,
accumulation of acidic products of metabolism)
AFFECTS THE ACTION OF ANTIBIOTICS
directly – suppression of the action of
antibiotics
chinolones, aminoglycosides
indirectly – reducing the growth rate of microbes
beta-lactams, glycopeptides
Ad 3. Altered microbial
population
• common dosage of a toxic substance kills 99 % microbes
in the biofilm
• a subpopulation of cells remains that is highly resistant to
the particular substance
it is not obvious if the subpopulation existed from the
beginning,
or if it has evolved by the action of the toxic
substance
• the subpopulation is responsible for the increased
resistance of biofilm
Dental plaque as a biofilm
Dental plaque = adhesive layer at the tooth
surface composed of microbes + their
products + components from the saliva
Localization: 1. supragingival plaque
2. subgingival, a) adherent
b) nonadherent
Composition: in all places Actinomyces spp.
Ad 1. yellow, green & purple
complex
Ad 2. red & orange complex
Microbial composition of
supragingival dental plaque
Higher No. of G+ rods & fibers and G+ cocci
(actinomycetes and streptococci)
Overall bacteria of yellow, green & purple complex
Yellow: Streptococcus mutans, S. oralis, S.
sanguis
Green: Eikenella corrodens, Capnocytophaga
sputigena, C. gingivalis, C. ochracea,
Aggregatibacter actinomycetemcomitans
Purple: Veillonela parvula, Actinomyces
odontolyticus
Microbial composition of
subgingival dental plaque
Subgingival, a) adherent
narrow layer at the tooth root
analogy of the supragingival plaque (mainly the green &
purple complex)
Subgingival, b) nonadherent
between the adherent plaque and the gum
above all motile G– anaerobes: red & orange complex
Red: Porphyromonas gingivalis, Tannerella forsythia,
Treponema denticola
Orange: Prevotella intermedia, P. nigrescens,
Peptostreptococcus micros, Fusobacterium nucleatum
ssp. nucl., ssp. vincentii, ssp. polymoprphum, F.
periodontium
Biofilm and disease – 1
Biofilm takes part in the pathogenesis of
1. chronic infections in general
2. infections of implanted devices
• the progress of these infections is slow
• they are without distinctive symptoms
• acute exacerbations occur occasionally
• the effect of antibiotic therapy is transitory only
• after stopping antibiotics infections recur (even if
bacteria grown from them appear sensitive in vitro)
Biofilm and disease – 2
Chronic infections of natural bodily surfaces
dental caries (oral streptococci, mainly Streptococcus mutans)
periodontitis (Gram-negative oral anaerobes)
otitis media (Haemophilus influenzae)
osteomyelitis (Staphylococcus aureus)
cholecystitis and cholangoitis (enterobacteria)
prostatitis (Escherichia coli)
subacute bacterial endocarditis (oral streptococci)
pneumonia in cystic fibrosis (Pseudomonas aeruginosa)
Biofilm and disease – 3
Chronic infections of artificial surfaces
central venous catheters (coagul. neg. staphylococci, candidae)
prosthetic heart valves (Staph. aureus, Staph. epidermidis)
joint prostheses (Staphylococcus aureus, Staph. epidermidis)
surgical sutures (Staphylococcus aureus, Staph. epidermidis)
vascular grafts (Gram-positive cocci)
endotracheal tubes (various bacteria and yeasts)
intrauterine contraceptive devices (Actinomyces israelii)
urinary catheters (E. coli or others, mainly Gram-negative rods)
contact lenses (Pseudomonas aeruginosa, Gram-positive cocci)
Problems with biofilm outside the
medicine
•
•
•
•
•
•
•
Soiling of surfaces
Increase in turbulence of flowing fluid
Narrowing the lumen up to blocking the tube
Corrosion of pipelines, fuel tanks in aircrafts
Blackening of fluids by reduced metals
Insulating layer in heat exchangers
Resistance of ship hull during passage
and many others
Possibilities of affecting the
biofilm – I
Prevention of the biofilm development
Now: modifying the surface of biomaterials (change of
charge)
impregnation of biomaterials with antimicrobials
(antibiotics, antiseptics)
In future: interference with quorum-sensing signals
inhibition of extracellular matrix production
inhibition of highly resistant persistors development
Possibilities of affecting the
biofilm – II
Disrupting the already present biofilm
Now: high concentration of an antimicrobial – so-called
antibiotic plug in a venous catheter
combination of antimicrobials with different
mechanisms of action
disruption of extracellular matrix – e.g. with enzymes
(polysaccharide lyases)
In future: use of molecules causing the autodestruction of
biofilm
Detection of biofilm – 1
Phenotypic methods
• staining of biofilm on the inner wall of a vessel
(test tube, well in microplate)
= Christensen method
universal for most microbes
• character of colonies on agar with Congo red
for staphylococci only
negative – colonies red, glossy
positive – colonies black, rough
Biofilm production on glass and
on hardened polystyrene
Biofilm ─
Biofilm +
PS
PS
S
S
Inoculum: 0.5 McFarland scale; culture: Sabouraud broth
with 8 % glucose, 48 hrs, 37 °C
PS = polystyrene, S = glass
Positive production of slime on
agar with Congo red
Black colonies of a biofilm-positive staphylococcus strain
Detection of biofilm – 2
Genotypic methods
• e.g. proof of a gene set called icaoperon responsible for the production
of intercellular adhesin in
Staphylococcus epidermidis
Slime and ica-operon in
staphylococci
isolated from blood and skin
60
50
40
blood
skin
30
20
10
0
ica-operon
slime
Clinical importance of biofilm
detection
• Biofilm = marker of clinical importance of
the strain
Is the strain isolated from blood culture clinically
relevant?
Is it not a contaminant?
• Detection of biofilm can bring valuable
clinical information
How to proceed in further treatment?
Which antibiotics should be used for destroying the
biofilm?
Will the common dosage suffice?
MIC, MBC and MBEC
• MIC = minimal inhibition concentration
the lowest concentration of an antimicrobial capable of
stopping the growth of the tested microbial strain
• MBC = minimal bactericidal concentration
the lowest concentration of an antimicrobial capable of
killing the examined strain
• MBEC = minimal biofilm eradicating concentration
the lowest concentration of an antimicrobial capable of
killing the strain growing as a biofilm
Determination of MBEC – I
• On U-type microtitration plates with a 96-pin
lid (so-called „hedgehog“)
• Biofilm of the examined strain is grown on
the pins
• The accumulated biofilm is treated with
antimicrobials in different concentrations
• The treated biofilm is broken up with
ultrasound
• The subsequent cell suspensions are
cultured and surviving cells are searched for
Determination of MBEC – II
Detection of viable
bacteria after the
influence of ATB
PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN
Biofilm grown on pins of the „hedgehog“
BIOFILM EXPOSED
TO ANTIBIOTICS
ultrasound
The concentration of ATB decreases from above
down
Live microbes betray themselves by yellowing
the medium in the detection microplate
Microbes killed: the medium remains red
Biofilm and health
In the body the biofilm plays even
a beneficial role:
• Our mucosae are coated with the biofilm of
normal microbial flora
• which provides them with relatively efficient
protection against pathogens gaining the
foothold
Summary
• Biofilm is the natural way of microbial growth
• It is a microbial community placed in a structured
intercellular matter
• It sticks firmly to solid surfaces
• Its structure depends on the amount of nutrients
in the environment
• It is more advantageous for microbes both
metabolically as well as a protection against
adverse conditions
• Microbes in the biofilm have different properties
• Biofilm brings problems in many fields
• Getting rid of biofilm is very difficult
Recommended reading material
Paul de Kruif: Microbe Hunters
Paul de Kruif: Men against Death
Axel Munthe: The Story of San Michele
Sinclair Lewis: Arrowsmith
André Maurois: La vie de Sir Alexander
Fleming
Hans Zinsser: Rats, Lice, and History
[email protected]
Thank you for your attention