InstituteInstitute for for Microbiology, Medical Faculty of Masaryk University Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospitaland St. Anna Faculty Hospital in Brno in Brno

Miroslav Votava, Vladana WoznicováMiroslav Votava, Vladana Woznicová

MICROBIAL BIOFILM – II MICROBIAL BIOFILM – II

Lecture for 2nd-year studentsLecture for 2nd-year studentsMarch 1March 166, 200, 20099

Properties of biofilm – reviewProperties of biofilm – review

Biofilm is a higher and Biofilm is a higher and moremore complexcomplex form of form of microbial growthmicrobial growth

• It utilizes the opportunity of mutual It utilizes the opportunity of mutual cooperationcooperation of of cellscells

• It enables the easier It enables the easier transfer of genestransfer of genes• It is characterized by an It is characterized by an effectiveeffective homeostasishomeostasis• It shows features of a primitive It shows features of a primitive circulation systemcirculation system• It provides a high It provides a high protectionprotection against antimicrobial against antimicrobial

factors factors • It plays an It plays an important part in many significantimportant part in many significant

occasions occasions including medically important including medically important conditionsconditions

Difficult penetrationDifficult penetration

EXTRACELLULAR MATRIX AS THE EXTRACELLULAR MATRIX AS THE PENETRATION BARRIERPENETRATION BARRIER

• difficult difficult diffusion diffusion of the antimicrobialof the antimicrobiallogical explanationlogical explanationnot always demonstrablenot always demonstrable

• influence of surface influence of surface chargechargeseems more importantseems more important

e.g. aminoglycosides (+) combine withe.g. aminoglycosides (+) combine with

alginate (-) of alginate (-) of P. aeruginosaP. aeruginosa

Altered environmentAltered environmentLOCAL ALTERATION IN THE BIOFILM LOCAL ALTERATION IN THE BIOFILM

ENVIRONMENT ENVIRONMENT consumption of Oconsumption of O22 in certain areasin certain areas

increase in increase in osmotic pressureosmotic pressure

accumulation of accumulation of acidic products acidic products of metabolismof metabolism

AFFECTAFFECTSS THE ACTION OF ANTIBIOTIC THE ACTION OF ANTIBIOTICSS

directlydirectly – – suppression of the action of suppression of the action of

antibioticantibioticss chinolones, aminoglycosideschinolones, aminoglycosides

indirectlyindirectly – reducing the growth rate – reducing the growth rate

beta-lactams, glycopeptidesbeta-lactams, glycopeptides

Altered microbial populationAltered microbial population

• common dosage of acommon dosage of a toxic substance kills 99 % microbes toxic substance kills 99 % microbes in the biofilmin the biofilm

• a subpopulation of cells remains a subpopulation of cells remains that is that is highly resistant highly resistant to to the particular substancethe particular substance it is not obvious if the subpopulation existed from it is not obvious if the subpopulation existed from the the

beginning, beginning, or if it has evolved by the action of the toxic or if it has evolved by the action of the toxic substancesubstance

• thethe subpopulationsubpopulation is responsible for the is responsible for the increased increased resistance resistance of biofilmof biofilm

Biofilm and diseaseBiofilm and disease – 1 – 1

Biofilm takes part in the pathogenesis ofBiofilm takes part in the pathogenesis of1.1. chronic infections in generalchronic infections in general2.2. infections of implanted devicesinfections of implanted devices

• the the progressprogress of these infections is of these infections is slowslow

• they are they are withoutwithout distinctive distinctive symptomssymptoms

• acute acute exacerbations exacerbations occur occasionallyoccur occasionally

• the the effect of antibiotic effect of antibiotic therapy is therapy is transitory transitory only only

• after stopping antibiotics after stopping antibiotics infections recurinfections recur (even if (even if

bacteria grown from them appear sensitive bacteria grown from them appear sensitive in vitroin vitro))

Biofilm and diseaseBiofilm and disease – 2 – 2

Chronic infections of natural bodily surfacesChronic infections of natural bodily surfaces

dental caries dental caries (oral streptococci(oral streptococci, , mainlymainly Streptococcus mutansStreptococcus mutans))

periodontitis periodontitis (Gram-negative oral anaerobes)(Gram-negative oral anaerobes)

otitis media otitis media ((Haemophilus influenzaeHaemophilus influenzae))

osteomyelitis osteomyelitis ((Staphylococcus aureusStaphylococcus aureus))

cholecystitis and cholangoitis cholecystitis and cholangoitis (enterobacteria)(enterobacteria)

prostatitis prostatitis ((Escherichia coliEscherichia coli))

subacute bacterial endocarditis subacute bacterial endocarditis (oral streptococci)(oral streptococci)

pneumonia in cystic fibrosispneumonia in cystic fibrosis ( (Pseudomonas aeruginosaPseudomonas aeruginosa))

Biofilm and diseaseBiofilm and disease – 3 – 3

Chronic infections of artificial surfacesChronic infections of artificial surfaces

central venous catheters central venous catheters (coagul. neg. staphylococci, candidae)(coagul. neg. staphylococci, candidae)

prosthetic heart valves prosthetic heart valves ((StaphStaph. . aureusaureus, , StaphStaph. . epidermidisepidermidis))

joint prostheses joint prostheses ((StaphylococcusStaphylococcus aureusaureus, , StaphStaph. . epidermidisepidermidis))

surgical sutures surgical sutures ((StaphylococcusStaphylococcus aureusaureus, , StaphStaph. . epidermidisepidermidis))

vascular grafts vascular grafts (Gram-positive cocci)(Gram-positive cocci)

endotracheal tubes endotracheal tubes (various bacteria and yeasts)(various bacteria and yeasts)

intrauterine contraceptive devices intrauterine contraceptive devices ((Actinomyces israeliiActinomyces israelii))

urinary catheters urinary catheters ((E. coliE. coli or others, mainly Gram-negative rods) or others, mainly Gram-negative rods)

contact lenses contact lenses ((Pseudomonas aeruginosaPseudomonas aeruginosa, Gram-positive cocci), Gram-positive cocci)

Problems with biofilm outside the Problems with biofilm outside the medicinemedicine

• Soiling Soiling of surfacesof surfaces• Increase in turbulenceIncrease in turbulence of flowing fluid of flowing fluid • Narrowing Narrowing the lumen up to the lumen up to blockingblocking the tube the tube• Corrosion Corrosion of pipelines, fuel tanks in aircraftsof pipelines, fuel tanks in aircrafts• BlackeningBlackening of fluids by reduced metals of fluids by reduced metals • Insulating layer Insulating layer in heat exchangersin heat exchangers• Resistance Resistance of ship hull during passageof ship hull during passage and many othersand many others

Possibilities of affecting the Possibilities of affecting the biofilm – I biofilm – I

Prevention of the biofilm developmentPrevention of the biofilm development

Now:Now: modifying themodifying the surfacesurface of biomaterials (change of of biomaterials (change of charge)charge) impregnation impregnation of biomaterials of biomaterials with antimicrobialswith antimicrobials (antibiotics, antiseptics)(antibiotics, antiseptics)

In future:In future: interferenceinterference with quorum-sensing with quorum-sensing signalssignals inhibition inhibition of of extracellular matrixextracellular matrix production production inhibition inhibition of highly resistant of highly resistant persistors persistors developmentdevelopment

cece se signály typu se signály typu quorum-sensingquorum-sensing inhibiceinhibice tvorby tvorby mimobuněčné hmotymimobuněčné hmoty inhibiceinhibice vzniku vysoce odolných vzniku vysoce odolných perzistorůperzistorů

Possibilities of affecting the Possibilities of affecting the biofilm – IIbiofilm – II

Disrupting the already present biofilmDisrupting the already present biofilm

Now:Now: high concentration of an antimicrobial – so-called high concentration of an antimicrobial – so-called antibiotic plug in a venous catheterantibiotic plug in a venous catheter combination of antimicrobials with different combination of antimicrobials with different mechanisms of action mechanisms of action disruption of extracellular matrix – e.g. with enzymes disruption of extracellular matrix – e.g. with enzymes

(polysaccharide lyases) (polysaccharide lyases)

In future:In future: use of molecules causing the autodestruction of use of molecules causing the autodestruction of biofilmbiofilm

Detection of biofilm – 1 Detection of biofilm – 1

Phenotypic methodsPhenotypic methods

• staining staining of biofilm on the inner wall of a vessel of biofilm on the inner wall of a vessel (test tube, well in microplate)(test tube, well in microplate)= Christensen method= Christensen methoduniversal for most microbesuniversal for most microbes

• character of colonies on agar with character of colonies on agar with Congo redCongo red for staphylococci onlyfor staphylococci onlynegativenegative – colonies – colonies redred, glossy, glossypositivepositive – colonies – colonies blackblack, rough, rough

Inoculum: 0.5 McFarland scale; culture: Sabouraud broth Inoculum: 0.5 McFarland scale; culture: Sabouraud broth with 8 % glucose, 48 hrs, 37 °Cwith 8 % glucose, 48 hrs, 37 °C

PS = polystyrene, S = glassPS = polystyrene, S = glass

PSPS

SS

BiofilmBiofilm ++ BiofilmBiofilm ──

Biofilm production on glass and Biofilm production on glass and on hardened polystyreneon hardened polystyrene

Positive production of slime on Positive production of slime on agar with Congo redagar with Congo red

Black colonies of a biofilm-positive staphylococcus strainBlack colonies of a biofilm-positive staphylococcus strain

Detection of biofilmDetection of biofilm – 2 – 2

Genotypic methodsGenotypic methods

• e.g. proof of a e.g. proof of a gene gene set called set called icaica--operonoperon responsible for the production responsible for the production of intercellular adhesin inof intercellular adhesin in Staphylococcus epidermidisStaphylococcus epidermidis

Slime and Slime and ica-operonica-operon in in staphylococcistaphylococci

isolated from blood and skinisolated from blood and skin

0

10

20

30

40

50

60

ica-operon slime

blood

skin

Clinical importance of biofilm Clinical importance of biofilm detectiondetection

• Biofilm = marker of clinical importance of Biofilm = marker of clinical importance of the strainthe strain

Is the strain isolated from blood culture clinically Is the strain isolated from blood culture clinically relevant?relevant?

Is it Is it not not a contaminant?a contaminant?

• Detection of biofilm can bring valuable Detection of biofilm can bring valuable clinical informationclinical information

How to proceed in further treatment?How to proceed in further treatment? Which antibiotics should be used Which antibiotics should be used for for destroying thedestroying the

biofilm?biofilm?Will the common dosage suffice?Will the common dosage suffice?

MIC, MBC and MBECMIC, MBC and MBEC

• MIC = minimal inhibition concentrationMIC = minimal inhibition concentrationthe lowest concentration of an antimicrobial capable of the lowest concentration of an antimicrobial capable of stopping the growth of the tested microbial strainstopping the growth of the tested microbial strain

• MBC = minimal bactericidal concentrationMBC = minimal bactericidal concentrationthe lowest concentration of an antimicrobial capable of the lowest concentration of an antimicrobial capable of killing the examined strainkilling the examined strain

• MBEC = minimal biofilm eradicating concentrationMBEC = minimal biofilm eradicating concentrationthe lowest concentration of an antimicrobial capable of the lowest concentration of an antimicrobial capable of killing the strain growing as a biofilmkilling the strain growing as a biofilm

Determination ofDetermination of MBECMBEC – I – I

• On U-type On U-type microtitration platesmicrotitration plates with a 96-pin with a 96-pin lid (so-called „hedgehog“)lid (so-called „hedgehog“)

• Biofilm of the examined strain Biofilm of the examined strain is grown on is grown on the pins the pins

• The accumulated biofilm is The accumulated biofilm is treated with treated with antimicrobialsantimicrobials in different concentrations in different concentrations

• The treated biofilm is The treated biofilm is broken up with broken up with ultrasoundultrasound

• The subsequent cell suspensions are The subsequent cell suspensions are cultured and cultured and survivingsurviving cells are searched forcells are searched for

Determination of MBEC – II Determination of MBEC – II

BIOFILM EXPOSED BIOFILM EXPOSED TO ANTIBIOTICSTO ANTIBIOTICS

Biofilm grown on pins of the „hedgehog“Biofilm grown on pins of the „hedgehog“

Detection of Detection of viable viable bacteriabacteria after the after the influence of ATBinfluence of ATB

PEN OXA AMS CMP TET COT ERY CLI CIP GEN TEI VAN

The concentration of ATB decreases from above The concentration of ATB decreases from above downdown

Live microbes betray themselves by Live microbes betray themselves by yellowingyellowing the medium in the detection microplatethe medium in the detection microplate

Microbes killed: the medium remains Microbes killed: the medium remains carminecarmine

ultrasoundultrasound

Biofilm and healthBiofilm and health

In the body the biofilm plays even In the body the biofilm plays even a beneficial rolea beneficial role::

• OOur mucosae are coated with the ur mucosae are coated with the biofilm of biofilm of normal microbial flora normal microbial flora

• which provides them with relatively efficient which provides them with relatively efficient protection againstprotection against pathogens pathogens gaining thegaining the

footholdfoothold

SummarySummary

• Biofilm is the Biofilm is the natural way ofnatural way of microbial microbial growthgrowth• It is a It is a microbial community microbial community placed placed in a structured in a structured

intercellular matterintercellular matter• It sticks firmly to It sticks firmly to solid surfacessolid surfaces• Its Its structure structure depends on the depends on the amount of nutrients amount of nutrients

in the environmentin the environment• It is It is more advantageous more advantageous for microbes for microbes bothboth

metabolically as well as a protectionmetabolically as well as a protection against against adverse conditionsadverse conditions

• Microbes in the biofilm have Microbes in the biofilm have different propertiesdifferent properties• Biofilm Biofilm brings problems brings problems in many fieldsin many fields• Getting rid Getting rid of biofilm is very of biofilm is very difficultdifficult

Recommended reading materialRecommended reading material Paul de Kruif: Microbe HuntersPaul de Kruif: Microbe Hunters

Paul de Kruif: Men against DeathPaul de Kruif: Men against Death

Axel Munthe: The Story of San MicheleAxel Munthe: The Story of San Michele

Sinclair Lewis: Sinclair Lewis: ArrowsmithArrowsmith

André Maurois: La vie de Sir Alexander FlemingAndré Maurois: La vie de Sir Alexander FlemingCould you kindly supply me with another work inCould you kindly supply me with another work in

connection with microbes or at least medicine?connection with microbes or at least medicine?

Please mail me your suggestions at:Please mail me your suggestions at:

mvotava@med.muni.cz

Thank you for your attentionThank you for your attention