MICROBIAL BIOFILMS

Shilpa.KMicrobiology TutorAIMSRC

ORIGIN OF SURVIVAL AND COMMUNICATION AMONG MICROBES

On earth 3.4 billion years ago

Microbes were struggling to

survive, grow and adapt

As the earth cooled Moved to multispecies adaptation

Microbes formed communities within biofilms

to transfer to different environments

Led to evolution of post biofilm planktonic phenotypeThe oldest story

of “unity is strength”

taught by the

most minute creatures

on Earth

SINGLE START TO MINGLE TO FORM BETTER SINGLE

Van Leeuwenhoek credited for the discovery of microbial biofilms.

Heukelekian and Heller observed the “bottle effect” for marine microrganisms.

Zobell observed number of bacteria on surface was higher than in surrounding medium (ex:seawater)

Jones et al. used scanning & transmission electron microscopy to examine biofilms

HISTORY

• Characklis studied microbial slimes in industrial water systems and showed that they were highly resistant to disinfectants like chlorine.

• Costerton et al. in 1978 explained the “theory of biofilms”.

BIOFILMS

Biofilm is an assemblage of microbial cells

that is irreversibly associated (not removed by gentle rinsing) with a surface and enclosed in a

matrix of primarily polysaccharide material.

WHEN DO MICROBES DECIDE TO FORM BIOFILMS

• Mainly when they are capable of recognition of specific or non-specific attachment sites on a surface

• Nutritional cues

• Exposure of planktonic cells to sub-inhibitory concentrations of antibiotics

INGREDIENTS TO PREPARE A BIOFILM

Celular materials:

bacteria parasites fungus virus

Non cellular materials: ( extra cellular polymer)

mineral crystals corrosion particles clay or slit particles blood components etc polysachharides proteins

CHARACTERISTICS OF A SURFACE

Liquid surface Solid surface

Living surface:

Leaves, wounds, Post surgical sites,

Marine invertebrates, etc

Non living surface:

Industrial setting,Hospital setting,

Indwelling medical devices,Rocks/boats in sea,

Toothbrush etc

ENVIRONMENTAL AND CULTURAL FACTORS AFFECTING BIOFILM

BIOFILM COMMUNITY STRUCTURE AND

EVOLUTION

ATTACHMENT EFFICIENCY

CYCLIC STAGE

ANTI EFFECTIVE HOSTILE FORCES

PHYSIOCHEMICALENVIRONMENT

MECHANICAL FACTORS AND

SHEAR FORCES

SUBSTRATUM

GENOTYPIC FORCES

NUTRIENT RESOURCES

Role of physiochemical properties of a surface:

Microbes attach more rapidly to teflon and other plastics than to glass or metals.

Conditioning films:

Formed when a material surface is exposed in aqueous medium and becomes conditioned or coated by polymers from that medium, the resulting modification will affect the rate and extent of microbial attachment.

Hydrodynamics: the flow velocity immediately adjacent to the

substratum/liquid interface is negligible. higher velocities would be expected to equate

to more rapid attachment or association to the surface, atleast until velocities become high enough to exert substantial shear forces on the attaching cells, resulting in detachment of these cells.

Characteristics of the aqueous medium:

seasonal effect on bacterial attachment – depends on water temperature.

increase in concentration of cations affects attachment presumably by reducing the repulsive forces between the negatively charged bacterial cells & surfaces like glass.

Also it was seen that an increase in nutrient concentration correlated with increase in the number of attached bacterial cells.

STEPS IN THE FORMATION OF BIOFILM

• STEP 1: Initial / reversible attachment - binding of 1st colonists

• STEP 2: Irreversible attachment – they anchor themselves permanently using pili

• STEP 3: Maturation 1 – inter-communication through quorum sensing

• STEP 4: Maturation 2 / Development – final stage of modification

• STEP 5: Dispersion – essential stage for biofilm dispersion and life-cycle

• Role of enzymes dispersin B and deoxyribonucleases in step 5 and medical application of cis-2-decenoic acid

QUORUM SENSING• Is a type of decision making process used by a decentralized

group to coordinate behavior.

• It can occur within a single bacterial species as well as between diverse species.

• It serves as a simple communication network.

• Many species of bacteria use it to coordinate their gene expression according to the local density of their population

MOLECULES USED AS SIGNALS• In gram positive bacteria oligopeptides• In gram negative bacteria AHL• Also, in both types of bacteria AI-2• In combination of bacteria and fungi 3-oxo-C12homoserinelactone

These biochemicals diffuse through water channels seen in matrix.

Regulate the transcription of various genes as per the requirement of the community to produce various proteins, enzymes, etc.

Serves as a major means of communication that allows the sessile organisms to survive and grow in the presence of unsuitable environmental conditions and antibiotics or detergents

FUNCTIONING OF BIOFILM Outermost layer-• Highest concentration of oxygen and nutrients, resembles their panktonic

counterparts.• They slough off and initiate biofilm formation downstream

Second layer-• Organisms here downregulate in their metabolic activity• Although they can clearly utilize the nutrients, exchange genes and have

the potential for multiple drug resistance• The benefit is obtained from their alignment in this layer which depends

upon spatial arrangement, physiologic heterogeneity and non uniformity

Innermost layer-• Attached to the substratum and is the earliest part of the biofilm• Also efficiently downregulate and are least metabolically active• Most persisters are found to be fossilized here• Provides inheritance for future populations that transfer laterally

Short term stress is absorbed elastically and long term stress is dissipated through viscous flow

Phenomenon of rolling and creeping.

Up and down regulation of various genes is seen in the attaching cells.

The gene transfer is much enhanced here than in the planktonic cells.

Biofilm association also provides a mechanism for selecting and promoting the spread of bacterial resistance to antimicrobial agents

Human infection involving biofilms

Native valve endocarditis

Periodontitis

Cystic fibrosisChronic

bacterial prostatitis

Otitis media

Native valve endocarditisThis results from the interaction between vascular

endothelium of the heart valves and bacteria or fungi

Streptococcus spp

Staphylococcus spp

CoNS

E. Coli

Klebsiella spp

Candida species

Aspergillus spp

Organism enter into blood steam through oropharynx, GI tract, genitourinary tract.

When endothelium is damaged thrombus will develop and fibronectin is secreted by the endothelial cells and bacteria adhere to the endothelial

cells

Secrete dextran helps in adherence of bacteria to endothelial cells

Within one hour it begin to multiply & develop matrix & capsule which protected from white blood cells

It may result in the vascular tissue damage

2) OTITIS MEDIA•Disease of middle ear which involves inflammation

Streptococcus pneumoniae

Haemophilus influenzae

Staphylococcus aureus

Staphyloccus epidermidis

Pseudomonas aeruginosa

Moraxella catarhalis

3) CHRONIC BACTERIAL PROSTATITIS • In this prostate gland may become infected by bacteria that

have ascended from the urethra.

Escherichia coli

Pseudomonas aeruginosa

Klebsiella spp

Proteus spp

Serratia spp

Enterococcus faecalis

4)CYSTIC FIBROSIS • It is a chronic disease of lower

respiratory tract.

• In this disease, deficiency of water

hinders the movement of mucus &

increase the secretion of mucus & lead to

dehydration and thickening of respiratory

epithelium.

• The organism which can causes the

biofilm mainly is

Pseudomonas aeruginosa.

5) PERIODONTITIS • Pellicle develops on the surface of enamel

• This pellicle comprises of albumin, lysozome,

glycoprotein , oral flora colonizes on the

surface & secrete the dextrons which helps in

the adherence of bacteria & form biofilm that

is called plaques.

• Fusobacterium spp.,

Eubacterium spp.,

Peptostreptococcus spp.,

Haemophilus aphrophilus.

Indwelling Medical devices

Central venous

catheter

Intrauterine device

Artificial hip prosthesis

Artificial voice

prosthesis

Urinary catheter

Prosthetic heart valve

BIOFILM ON MEDICAL DEVICES

MEDICAL DEVICES ORGANISM

Central venous catheter

CoNS, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas spp., Candida

albicansProsthetic heart

valvesViridans Streptococci, Staphylococcus aureus,

Enterococci

Urinary catheter Staphylococcus epidermidis, E.coli, Klebsiella, Proteus mirabilis

Intrauterine device Staphylococcus epidermidis, Staphylococcus aureus, Micrococcus spp, Enteroccocci spp, Group B

Streptococci, Candida albicansArtificial voice

prosthesisCandida albicans, Streptococcus spp, Staphylococcus

epidermidis.

MEDICAL DEVICES AND COMMON ORGANISM WHICH CAUSES BIOFILM

1) PROSTHETIC HEART VALVES

The surgical implantation of

prosthetic valve leading to

accumulation of platelets and

fibrin at the suture site and

device & there is a formation

of biofilm.

2) CENTRAL VENOUS CATHETER

•When catheter is introduced into

the blood vessels the skin colonizer

may migrate along inner lumen

from the skin insertion site.

•When these organism get contact

with blood they form thrombus this

lead to formation of biofilm

3)URINARY CATHETER • Organisms form biofilm when

the catheter is inserted into the

urethra.

•This organism may alter the pH

and form substance like

ammonia & minerals form

biofilm

4)CONTACT LENSES

• Bacteria adhere readily to the

lenses like

• Staphylococcus aureus,

Pseudomonas aeroginosa,

Staphylococcus epidermidis

E.coli, Streptococcus pyogenes,

Serratia marcescence , Candida

albicans, Acanthameoba ,

Fusarium.

COMMON IMPLANT INFECTION

DETECTION, MEASUREMENT AND CONTROL OF BIOFILMS

Microscopic ExaminationQuantitative detectionQualitative detectionMolecular detection

Microscopy…• Electron Microscopy For examination & characterisation of biofilms on medical

devices & human infections. SEM- has been used for early investigative studies.Limitation – dehydration leads to significant sample distortion & artifacts. TEM- With the use of ruthenium red stain helps identify

nature of extracellular fibers & their association with cells.

• Confocal Laser Scanning Microscopy High resolution optical images with depth selectivity 3D reconstruction of the image. Specimen stained with fluorescent stains which can be used to

probe specific cellular functions. Eg: Nucleic acid stains- DAP 1,AO,Syto 9 etc.

Other stains for probing cell viability- Propidium iodide , 5-cyano-2,3-ditoyl tetrazolium chloride.

Epifluorescence microscopy.Fluorescent antisera.

Qualitative methods

Decant tubes, wash with phosphate buffer saline

Incubate at 37֯Cfor 24 hours

Colony inoculated into 10 ml TSB

Dried & stained with 0.1% crystal violet

•Tube method

Positive - visible film on the wall & bottom of the tube

Negative – Ring formation at the liquid interface

Grading based on biofilm formation

0 : Absent

1 : Weak

2 : Moderate

3 : Strong

RESULTS:

A B C

A- High

B- Non Biofilm Producer

C- Moderate

Quantitative methods• Roll plate method• Tissue culture plate method• Congo red method• Calgary biofilm device

• Roll plate method: Used in case of central venous catheter. Disadvantage: Cannot detect cells on inner surface of he device.Cannot detect more than 1000 cfu.

Can be improved by sonication and vortexing.

• Tissue culture plate method:Used to detect ability of an organism to form biofilm.

Incubate at 37 degree for 18-24 hours.

0.2 ml aliquots filled into sterile polystyrene tissue culture plates

Diluted 1:100 in fresh medium.

24 hr old isolate inoculated onto TSB with 1% glucose. Incubated at 37 degree for 24 h.

Contents of each well removed by gentle tapping & washed with 0.2% phosphate buffer.

Excess stains are washed with deionised water & plates are kept for drying.

Biofilms formed are fixed by sodium acetate and stained with 0.1% crystal violet for 5 -10 minutes.

OD values determined by micro ELISA auto reader at 570nm

Mean OD values Adherence Biofilm formation

<0.120 Non Non/weak

0.120-0.240 Moderately Moderate

>0.240 Strong High

High

Moderate

Non

• Congo Red agar method.

Media used- BHIA with 5% sucrose and Congo red indicator. A fresh isolate of pathogenic strain is inoculated and incubated for 24 h at 37 degree.

Results- Positive : Black colony with dry crystalline consistency Negative : Pink colored colony Intermediate : Dark colony without dry crystalline consistency

Screening of biofilm producers by Congo red agar medium A) S. epidermidis ATCC 35984 (black colonies)B) S. epidermidis ATCC 12228 (pink colonies)

A B

• Calgary Biofilm Device:

Recently discovered

A device that can detect biofilms as in tissue culture

plate along with Antimicrobial Susceptibility testing. Widely accepted method.

Peg

Microtitre plate

Common methods used by clinical microbiologists for recovery & measurement of clinically relevant biofilms on medical devices.

METHOD BASIC PROTOCOL ADVANTAGE LIMITATION

Roll plate Roll the catheter tip over surface of BA

Easy to use Examines only outer surface . Inaccurate

Vortex then viable count.

Catheter section in PBS is vortexed then cultured on different

media.

Measures intraluminal & extraluminal

biofilm.

Recovery efficiency unknown.

Sonicate , vortex, then viable count.

Catheter section in TSB , sonicate , vortex

& culture on BA.

Measures intraluminal & extraluminal

biofilm.

Recovery efficiency unknown.

Sonicate, vortex, homogenise, then

viable count.

Catheter section in PBS/vortex repeatedly

then homogenise & culture on BA.

Recovery efficiency determined.

Measures intraluminal biofilms only.

Acridine orange direct staining.

Following roll plate, catheter section is

stained with AO

Allows direct examination of

catheter.

Method doesn’t allow quantification.

Endoluminal brush Brush is introduced into the implanted catheter,removed,

placed in PBS, sonicated & plated.

Allows examination of indwelling catheter.

Effect of procedure on patient & recovery efficiency unknown

Apparatuses used for growing & testing Biofilms

APPARATUS ORGANISMS TESTED

FLOW DYNAMICS

SUBSTRATUM METHOD FOR REMOVING & QUANTIFYING

BIOFILM

Modified Robbins device

Pseudomonas pseudomallei

Batch/mixing Silastic disks Method of removal not given; viable count

Calgary biofilm device P. aeruginosa, S. aureus, E. coli

Batch/mixing Plastic pegs Sonicate peg, then viable count

Disk reactor Gram-negative bacteria

Batch/mixing Teflon coupons Sonicate, vortex, homogenize,

then viable or direct count

CDC biofilm reactor Gram-negative bacteria

Continuous/open system

NeedlelessConnectors. (plastic)

Sonicate, vortex, homogenize,

then viable or direct count

Perfused biofilmfermentor

Candida albicans Continuous/opensystem

Cellulose-acetate filters

Shake in sterile water, then viable count .

Model bladder Gram negative bacteria

Continuous/open system

Urinary catheters Direct examination by SEM or

TEMa or by chemicalanalysis

MOLECULAR METHODS

• FISH

• PCR

• RT-PCR

• RFLP

• RAPD

ANTIMICROBIAL SUSCEPTIBILITY TESTING

• Determination of MIC – Standard method for AST.

• Calgary Biofilm Device is most accepted method used against E.coli, S.aureus , P.aeruginosa.

• Modified Robbins Device is also used

BIOFILMS & CLINICAL DECISION MAKING

• Collect paired blood samples.• Repeated negative results for samples may not

imply absence of biofilms.• The coagulase-negative staphylococci spp

dilemma.

TREATMENT• Issues: Drug resistance due to

Impaired penetration of antibiotics.

Reduced growth rate of bacteria

Altered micro environment

Altered gene expression

Reduced biofilm specific phenotype

•Treatment is based on MIC & MBEC results.•Fluoroquinolones , aminoglycosides & cephalosporins for gram negative bacteria.• Drugs like vancomycin for gram positive bacteria.• Triazoles, lipid formulations of amphotericin B & echinocandins for Candida species.•Photodynamic therapy•Ultrasonic wave therapy.•Immune modulation by use of azithromycin & low dose doxycycline .•Antimicrobials like, silver & tobramycin.

CONTROL AND PREVENTION• Common disinfectants used – Iodophor, hypochlorite, acid

anion, peroxyacetic acid, quaternary ammonium compounds & sanitisers.

• On medical devices- Catheters impregnated with minocycline and rifampicin Catheters coated with a cationic surfactant Attachable subcutaneous cuffs containing silver ions inserted

after local application of polyantibiotic ointment• Control using phages• Control through enzymatic detergents (Food industry)• Control through microbial interactions/metabolite molecules.

FUTURE RESEARCH PERSPECTIVES

• More reliable methods for detection & measurement of

biofilms should be developed.

• Elucidation of the genes specifically expressed by biofilm-

associated organisms

• Evaluation of various control strategies .

• Studies related to the effect of probiotics on biofilms.

REFERENCES• Biofilm Formation in Uropathogenic Escherichia coli Strains: Relationship With

Prostatitis, Urovirulence Factors and Antimicrobial Resistance - S. M. Soto,* A. Smithson, J. A. Martinez, J. P. Horcajada, J. Mensa and J. Vila

• MINIMUM INHIBITORY CONCENTRATION (MIC) VERSUS MINIMUM BIOFILM ELIMINATING CONCENTRATION (MBEC) IN EVALUATION OF ANTIBIOTIC SENSITIVITY OF GRAM-NEGATIVE BACILLI CAUSING PERITONITIS. Peritoneal Dialysis International, Vol. 24, pp. 65–67

• Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology (2002), 148, 87–102.

• Biofilms: Survival Mechanisms of Clinically Relevant Microorganisms. CLINICAL MICROBIOLOGY REVIEWS, Apr. 2002, p. 167–193

• Microbial Biofilms: from Ecology to Molecular Genetics MARY ELLEN DAVEY AND GEORGE A. O’TOOLE* Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755

• Cooperation and conflict in microbial biofilms Joao B. Xavier and Kevin R. Foster*• Center for Systems Biology, Harvard University, Bauer Laboratory, 7 Divinity Avenue,

Cambridge, MA 02138 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1982, p. 11%-1204 Vol. 44, No. 5

• Biofilms: discovery of a new mechanism of virus propagation• Biofilm-control strategies based on enzymic disruption of the extracellular

polymeric substance matrix – a modelling study• JOURNAL OF BACTERIOLOGY, 10.1128/JB.183.18.5385–5394.Sept. 2001, p. 5385–

5394 Vol. 183, No. 18, American Society for Microbiology. • Biofilm Formation by the Fungal Pathogen Candida albicans: Development,

Architecture, and Drug Resistance Rev Iberoam Micol 2002; 19: 139-143 139• Medical importance of biofilms in Candida infections L. Julia Douglas• Biofilms and Device-Associated Infections Rodney M. Donlan• Centers JOURNAL OF CLINICAL MICROBIOLOGY, June 1999, p. 1771–1776 Vol. 37,

No. 6 American Society for Microbiology. All Rights Reserved.• The Calgary Biofilm Device: New Technology for Rapid Determination of Antibiotic

Susceptibilities of Bacterial Biofilmsfor Disease Control and Prevention Atlanta, Georgia, USA

• Biofilms: Microbial Life on Surfaces Rodney M. Donlan**Centers for Disease Control and Prevention, Atlanta, Georgia, USA.

• Factors Regulating Microbial Biofilm Development in a System with Slowly Flowing SeawaterKARSTEN PEDERSEN Paris, December 21, 2009

• “Bacteriologists had it wrong for the past 300 years- Bacteria don’t live alone… They grow best when each one does its own thing…. Together!!! “

Thank you!!!!