1. The 6th lecture for 2nd-year students
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
March 26, 2012

2. 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

3. 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

4. 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)

5. 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)
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)

6. 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

7. 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

8. Architecture of biofilm IV – revision

9. 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
polymicrobial = e.g. dental plaque, normal microflora of mucous membranes

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. Possible causes of higher resistance of biofilm
More difficult penetration of toxic matter through the biofilm
Character of environment in the biofilm is altered
Also the microbial population in the biofilm is altered

17. 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

18. 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

19. 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

20. Biofilm takes part in the pathogenesis of
Biofilm and disease – 1
Biofilm takes part in the pathogenesis of
chronic infections in general
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)

21. Chronic infections of natural bodily surfaces
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)

22. Chronic infections of artificial surfaces
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)

23. 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

24. 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
ce se signály typu quorum-sensing
inhibice tvorby mimobuněčné hmoty
inhibice vzniku vysoce odolných perzistorů

25. 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

26. 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

27. Biofilm production on glass and on hardened polystyrenePS PS S S
Inoculum: 0.5 McFarland scale; culture: Sabouraud broth with 8 % glucose, 48 hrs, 37 °C PS = polystyrene, S = glass

28. Positive production of slime on agar with Congo red
Black colonies of a biofilm-positive staphylococcus strain

29. Detection of biofilm – 2 Genotypic methods
e.g. proof of a gene set called ica- operon responsible for the production of intercellular adhesin in Staphylococcus epidermidis

30. Slime and ica-operon in staphylococci isolated from blood and skin

31. 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?

32. 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

33. 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

34. 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 carmine

35. In the body the biofilm plays even a beneficial role:
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

36. 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

37. 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
Thank you for your attention