Biofilm in bacteria
• MUDr. Černohorská Lenka, PhD.
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•Department of Microbiology Masaryk University Medical School and St. Anna´s Faculty Hospital,
Brno, Czechia
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* Who can form biofilm (bacteria, viruses, yeasts, 1species, more species, multibacterial
community)
* Where is the biofilm (almost on solid surfaces)
* How do microbes form biofilm (difficult mechanism)
* Why do they form biofilm (guard against various circumstances)
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Who can form a biofilm?
•P. aeruginosa
•S. coagulase negative
•K. pneumoniae
•E. coli
•S. aureus
•E. faecalis
•A. baumanii
•almost all bacteria, yeasts and fungi
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Where do we find biofilm?
* Catheters (bloodstream..)
* Contact lens
* Teeth implantates
* Medical devices
* Water tubes
* Plaque on teeth, higher consumation of sugars + minimum teeth brushing are the cofactors in
dental caries formation
* Stones in river – physiological!
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Stages of biofilm development
* Direct contact of a planktonic bacteria  with a surface              +
* Attachment to this surface
* Adhesion, growth, and aggregation of cells into microcolonies
* Production of polymeric matrix
* Formation of three-dimensional structure known as a biofilm
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Biofilm formation in time
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•                      aviable on www.medmicro.info
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Main importance of biofilm formation
•Bacteria harbored inside are protected against:
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v antibiotic action
v host´s immune response
v disinfection
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The inefficiency of antibiotics may be due to:
* Polyanionic charge of sessile cells
* Decreased bacterial growth
* Diffusion barrier of glycocalyx
* Reaction with biofilm matrix
* Formation of protected phenotypes
* Mechanism of intercellular signalling
* Host´s immune response mechanisms…
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Biofilm detection
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vIn test tubes or in plate via Christensen1
•      cultivation, washing of planktonic bacteria + staining of adhering bacteria – blue colour on
whole tube wall not only a ring or sediment – it is a biofilm!
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vDetection in tubes stained with alcian blue
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vEl. microscopy, SEM, TEM
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vSpecial cultivators and phenotypic methods
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vGenotypic methods (PCR, FISH) etc.
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Biofilm susceptibility testing
vCultivation of wild-type bacteria v “microtitrate“ plate in broth
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vWashing of planktonic bacteria +  adding of dilluted antibiotic (combination of ATB) and
cultivation
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vMBEC (minumum biofilm eradication concentration) was determined
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vMBEC was compared with MIC (minimum inhibitory concentration)
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vSynergy testing of 2 antibiotics: FBIC (fractionate biofilm inhibitory concentration) was
determined in special laboratories
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vClinical relations
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 MIC vs. MBEC values
vAbbreviations: ams - ampicillin/sulbactam, ch - chloramphenicol, ery - erytromycin, te -
tetracyclin, cli - clindamycin, tei - teicoplanin, van - vancomycin, ofl - ofloxacin
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vPlanktonic bacteria (MIC)
vBiofilm-forming bacteria (MBIC)

Antibiotic susceptibility of
S. aureus no. 351
v* susceptible according to conventional MIC evaluation. MBIC  4x - 256x higher than MIC (mg/l)
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v PEN OXA AMS CMP TET COT ERY CLI  CIP  GEN  TEI  VAN
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vGrowth control
v Determination of MBEC

Results + eradication
* MBIC values are often higher than break point for tested antibiotics (bacteria are resistant) +
also higher than MIC
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* 1 ATB does not work? Use ATB combination - synergy testing can be also used for multiresistant
planktonic bacteria (with modifications)
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* Resistance to ATB combinations? Explantation of a biofilm focus (removing of catheters,
joint/teeth  implantates)
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* Prevention: catheters coated with antimicrobial. substances - minocyclin and rifampicin, washing,
hygiene…
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1 – Microscopy of oral biofilm
* Gramm staining - G +, G- bacteria, host cells (epitelium, leukocyte etc.)
* Alcian blue stains polysaccharide material
Alcian 02

v2 – Teeth brushing and oral biofilm
detekce plaku - erytrosin
v   Volunteer gum tablette with colour staining the teeth plaque. Tablette stay in oral cavity for
2 min. Draw the plaque

* Classic cultivation in broth: We detect only bacteria in planktonic form. Bacteria  in biofilm
wonť leave the catheter wall
* Semiquantitative method: We detect bacteria on surface of a catheter and can semiquantitative
count them, but we can not detect bacteria inside the lumen
* Sonification: destroys biofilm on catheter surface as a well as on catheter inside. Inoculation
of specific sample volume enable to determinate  quantity of microbes
3- Catheters

4 – Presence of saccharides in food and teeth plaque formation
* Look at various amount of saccharides in food and biofilm formation speed in cariogennous
Streptococcus mutans. What´s happen?
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•Higher amounts of saccharides in food + higher length of staying in oral cavity-higher plaque….!

* MIC - minimum inhibitory
•   concentration of ATB  (planktonic form)
* MBEC - the lowest concentration of antibiotic, where is the eradication of a biofilm possible
(any living cells, pH without change - row stay red) (for biofilm forming bacteria).
5 – MIC versus MBEC