BiofilmsBiofilms

Microbial BiochemistryMicrobial Biochemistry

Definition of a BiofilmDefinition of a Biofilm

BiofilmsBiofilms are communities of are communities of microorganisms in a matrix that joins them microorganisms in a matrix that joins them together and to living or inert substrates together and to living or inert substrates

BiofilmsBiofilms are surface-attached communities are surface-attached communities of bacteria, encased in an extracellular of bacteria, encased in an extracellular matrix of secreted proteins, matrix of secreted proteins, carbohydrates, and/or DNA, that assume carbohydrates, and/or DNA, that assume phenotypes distinct from those of phenotypes distinct from those of planktonic cells planktonic cells

Formation of biofilms in Formation of biofilms in naturenature

Biofilms offer their Biofilms offer their member cells member cells several benefitsseveral benefits

Biofilms are Biofilms are diverse from their diverse from their formation on teeth formation on teeth as plaques and as plaques and submerged rocks in submerged rocks in a streama stream

BiofilmsBiofilms

BIOFILMS BIOFILMS may form:may form:

On solid substratums in contact with On solid substratums in contact with moisturemoisture

On soft tissue surfaces in living On soft tissue surfaces in living organisms organisms 

At liquid-air interfaces.  At liquid-air interfaces. 

Study of biofilmsStudy of biofilms

Formation of multicellular Formation of multicellular communities depends on the communities depends on the production of extracellular production of extracellular substances( matrix)substances( matrix)

Diversity in the formation of the Diversity in the formation of the matrixmatrix

Pathogens that have been Pathogens that have been studied for the formation of studied for the formation of

biofilmsbiofilms Staphylococcus aureusStaphylococcus aureus Staphylococcus mutansStaphylococcus mutans Salmonella typhiSalmonella typhi Enterococcus faecalisEnterococcus faecalis Pseudomonas aeruginosaPseudomonas aeruginosa

Biofilms – Now a Universal Biofilms – Now a Universal FeatureFeature

Now scientists believe biofilm Now scientists believe biofilm formation is a universal feature of all formation is a universal feature of all bacteriabacteria

Biofilm characteristicsBiofilm characteristics

Submerged biofilms seems to form Submerged biofilms seems to form columns and mushroom like projections columns and mushroom like projections that are separated by water-filled channelsthat are separated by water-filled channels

Floating biofilms form a skin or pellicle at Floating biofilms form a skin or pellicle at the air- liquid interface – shows the air- liquid interface – shows organization of cells with the matrix at the organization of cells with the matrix at the outsideoutside

Films that form on the surface of solid Films that form on the surface of solid media such as agar or other surfacesmedia such as agar or other surfaces

Steps in biofilm Steps in biofilm formationformation

Initiation of biofilm formation – Initiation of biofilm formation – interaction of cells with a surface or interaction of cells with a surface or with each otherwith each other

Films aggregateFilms aggregate Then the cells form an extracellular Then the cells form an extracellular

matrixmatrix Structure of biofilms are dramatically Structure of biofilms are dramatically

different due to the specific organisms different due to the specific organisms in the film and environmental in the film and environmental conditionsconditions

Figure from: Kolter, R. and R. Losick. Figure from: Kolter, R. and R. Losick. 1998. One for all and all for one. 1998. One for all and all for one.

Science 280:226-227Science 280:226-227..

Steps in Biofilm Steps in Biofilm FormationFormation

ExopolysaccharidesExopolysaccharides

ExopolysaccharidesExopolysaccharides In the glycoclyx In the glycoclyx

contribute to contribute to biofilm formationbiofilm formation

MatrixMatrix

Key components of Key components of the matrix are the matrix are extracellular extracellular polysaccharides polysaccharides and proteinsand proteins

Dead cells have Dead cells have also been identified also been identified in biofilmsin biofilms

Extracellular DNA Extracellular DNA is also importantis also important

Biofilm formationBiofilm formation

Matrix constituentsMatrix constituents

PolysaccharidesPolysaccharides

Carbohydrates significantly impact Carbohydrates significantly impact bacterial virulencebacterial virulence

Bacteria have capsular Bacteria have capsular polysaccharides and polysaccharides and exopolysaccharidesexopolysaccharides

The polysaccharides are not soluble The polysaccharides are not soluble and do not disassociate with the and do not disassociate with the bacterial cellsbacterial cells

PolysaccahridesPolysaccahrides Many bacteria have been found to produce Many bacteria have been found to produce

cellulosecellulose This is a novel finding in the case of This is a novel finding in the case of Salmonella Salmonella

typhimuriumtyphimurium and and E. coliE. coli The bacterium The bacterium Gluconacetobacter xylinusGluconacetobacter xylinus has has

been recognized as a cellulose producerbeen recognized as a cellulose producer Many other bacteria have genes homologous to Many other bacteria have genes homologous to

the bcs, bacterial cellulose synthesis genesthe bcs, bacterial cellulose synthesis genes Vibrio choleraVibrio cholera does not appear to have a gene does not appear to have a gene

which encodes a cellulose – but the bacterium which encodes a cellulose – but the bacterium has two domains homologous to has two domains homologous to GluconeacetobacterGluconeacetobacter..

Biofilm Genetics - initiationBiofilm Genetics - initiation

Staphylococcal polysaccharide Staphylococcal polysaccharide intercellular adhesin( PIA)intercellular adhesin( PIA)

PIA – poly-N-acetyl glucosamine (PNAG) PIA – poly-N-acetyl glucosamine (PNAG) polymerpolymer

PIA polymers present in PIA polymers present in E. coliE. coli The pga gene is involved with the The pga gene is involved with the

formation of biofilmsformation of biofilms This gene is similar to the staphylococcal This gene is similar to the staphylococcal

gene icagene ica Now Now Yersinia pestisYersinia pestis has been shown to has been shown to

make a gene homologous to thismake a gene homologous to this

Bap ProteinBap Protein

The biofilm associated proteinThe biofilm associated protein Structurally similiar to the surface Structurally similiar to the surface

proteins proteins

Esp of Esp of Enterococcus faecalisEnterococcus faecalis

mus20 of mus20 of Pseudomonas aeruginosaPseudomonas aeruginosa

sty2875 of sty2875 of Salmonella typhiSalmonella typhi

Pseudomonas Pseudomonas aeruginosaaeruginosa

Pseudomonas Pseudomonas strains were thought to strains were thought to produce alginateproduce alginate

However alg mutants did not produce However alg mutants did not produce changes in the biofilm formationchanges in the biofilm formation

Two new polymers have been found in Two new polymers have been found in PseudomonasPseudomonas polymers polymers

pelA-G gene produces a glucose rich pelA-G gene produces a glucose rich polymerpolymer

pslA-O genes produce a mannose rich pslA-O genes produce a mannose rich polymerpolymer

Population density and Population density and polysaccharide productionpolysaccharide production

The connection between quorum The connection between quorum sensing and biofilm architecturesensing and biofilm architecture

Biofilm thickness seems to affect Biofilm thickness seems to affect communicationcommunication

Quorum sensing mechanism is not Quorum sensing mechanism is not clearly defined but seems to be clearly defined but seems to be essential in the formation of the film essential in the formation of the film and its channelsand its channels

Vibrio choleraVibrio cholera

Major extracellular polysaccharide in Major extracellular polysaccharide in the biofilm are VPS and the genes, vpsthe biofilm are VPS and the genes, vps

VPS is negatively regulated by hapR. VPS is negatively regulated by hapR. Hap R mutants produce rugose Hap R mutants produce rugose colonies and narrow channels in the colonies and narrow channels in the biofilm architecturebiofilm architecture

hapR gene encodes a transcription hapR gene encodes a transcription factorfactor

hapR is repressed by LuxOhapR is repressed by LuxO

Conjugative piliConjugative pili

Conjugative pili greatly accelerates Conjugative pili greatly accelerates initial adhesion and biofilm initial adhesion and biofilm development by development by E. coliE. coli

Gram negative bacteria have Gram negative bacteria have adhesins at the tip of its fimbriaeadhesins at the tip of its fimbriae

E. coli E. coli responds to levels of nutrients responds to levels of nutrients and osmolarityand osmolarity

AdhesinsAdhesins

Adhesins are Adhesins are molecules that are molecules that are attached to attached to bacterial fimbriaebacterial fimbriae

Staphylococcus aureusStaphylococcus aureus

E. Coli AdhesinsE. Coli Adhesins

AdhesinsAdhesinsBacteriumBacterium AdhesinsAdhesins ReceptorReceptor AttachmentAttachment DiseaseDisease

Vibrio choleraVibrio cholera N-N-methylphenylalanimethylphenylalaninene

Fucose and Fucose and MannoseMannose

pilipili Intestinal Intestinal epitheliumepithelium

CholeraCholera

Bordetella Bordetella pertussispertussis

Fimbriae Fimbriae ("filamentous ("filamentous hemagglutinin")hemagglutinin")

Galactose on Galactose on sulfated glycolipidssulfated glycolipids

pilipili Respiratory Respiratory epithelium epithelium

Whooping Whooping coughcough

Streptococcus Streptococcus pyogenespyogenes

Protein FProtein F amino amino terminus of terminus of fibronectinfibronectin

Pharyngeal Pharyngeal epitheliumepithelium

Sore throatSore throat

E. coliE. coli Type 1 FimbriaeType 1 Fimbriae Species Species specific specific carboydratcarboydrateses

Intestinal Intestinal epitheliumepithelium

DiarrheaDiarrhea

Adhesins and vaccinesAdhesins and vaccines

Bacterial Cell Bacterial Cell CharacteristicsCharacteristics

The phenotypes of the cells include:The phenotypes of the cells include: a slower growth ratea slower growth rate increased antibiotic resistanceincreased antibiotic resistance elevated frequency of lateral gene elevated frequency of lateral gene

transfer transfer

Quorum sensingQuorum sensing Is controlled by at least two different Is controlled by at least two different

quorum sensing signalsquorum sensing signals Acyl-homoserine lactone CAI-1( cholera Acyl-homoserine lactone CAI-1( cholera

autoinducer 1) appears to play a significant autoinducer 1) appears to play a significant role in biofilm formationrole in biofilm formation

Under low cell density CAI levels are lose Under low cell density CAI levels are lose enough to permit LuxO mediated repression enough to permit LuxO mediated repression of hapR resulting in VPS productionof hapR resulting in VPS production

This bacterium appears to initiate This bacterium appears to initiate production of an extracellular matrix under production of an extracellular matrix under conditions of low population density conditions of low population density presumably before the establishment of a presumably before the establishment of a multicellular communitymulticellular community

Signaling in biofilmsSignaling in biofilms

Signaling and biofilmsSignaling and biofilms

Gram NegativeGram Negative

Gram PositiveGram Positive

Antibiotic ResistanceAntibiotic Resistance

In the center of the biofilm the In the center of the biofilm the bacteria have greater antibiotic bacteria have greater antibiotic resistanceresistance

Opsonisation of antibioticsOpsonisation of antibiotics To resistance to phagocytosisTo resistance to phagocytosis

E. faecalisE. faecalis

E. faecalisE. faecalis biofilms on dental root biofilms on dental root canals, urethral catheters, uretheral canals, urethral catheters, uretheral stents ,and heart valves have been stents ,and heart valves have been observed. observed.

While it is not clear that the ability of While it is not clear that the ability of E. faecalisE. faecalis to form biofilms is to form biofilms is essential for virulence, it appears essential for virulence, it appears that a majority of clinical isolates do that a majority of clinical isolates do possess the ability to form a biofilm possess the ability to form a biofilm in vitro in vitro

Dental plaqueDental plaque

Genome – genome Genome – genome interactions:bacterial communities in interactions:bacterial communities in initial dental plaque – Paul initial dental plaque – Paul Kolenbrander et al – Trends in Kolenbrander et al – Trends in Microbiology. January 2005.Microbiology. January 2005.

Found on the enamel of teethFound on the enamel of teeth Epithelial cells of the oral mucosaEpithelial cells of the oral mucosa Participate in coaggregation which Participate in coaggregation which

occurs between different species of occurs between different species of bacteriabacteria

Dental Plaque - Dental Plaque - colonizationcolonization

Plaque smearPlaque smear 400 different 400 different bacterial species bacterial species can be found in can be found in plaqueplaque

10101010bacteria/mg High levels of Ca+

+ and P Matrix forms with

cells

DefinitionDefinition

Supra gingival – Supra gingival – above gumsabove gums

Subgingival –below Subgingival –below the gumsthe gums

Also related to the Also related to the tooth surfacetooth surface

Primary ColonizersPrimary Colonizers

The pellicle-coated tooth surface is The pellicle-coated tooth surface is colonized by Gram-positive bacteria such colonized by Gram-positive bacteria such as as Streptococcus sanguis, Streptococcus Streptococcus sanguis, Streptococcus mutansmutans, and , and Actinomyces viscosusActinomyces viscosus

These organisms are examples of the These organisms are examples of the "primary colonizers" of dental plaque. "primary colonizers" of dental plaque. Bacterial surface molecules interact with Bacterial surface molecules interact with components of the dental pellicle to components of the dental pellicle to enable the bacteria to attach or adhere to enable the bacteria to attach or adhere to the pellicle-coated tooth surface. the pellicle-coated tooth surface.

Primary colonizationPrimary colonization For example, specific protein molecules found as For example, specific protein molecules found as

part of the bacterial fimbria (hair-like protein part of the bacterial fimbria (hair-like protein extensions from the bacterial cell surface) on extensions from the bacterial cell surface) on both both Streptococcus sanguisStreptococcus sanguis and and Actinomyces Actinomyces viscosusviscosus interact with specific proteins of the interact with specific proteins of the pellicle (the proline-rich proteins) with a "lock and pellicle (the proline-rich proteins) with a "lock and key" mechanismkey" mechanism

This results in the bacteria firmly sticking to the This results in the bacteria firmly sticking to the pellicle-coating on the tooth surface pellicle-coating on the tooth surface (Mergenhagen et al. 1987). Within a short time (Mergenhagen et al. 1987). Within a short time after cleaning a tooth, these Gram-positive after cleaning a tooth, these Gram-positive species may be found on the tooth surface. species may be found on the tooth surface.

Mechanisms for plaque Mechanisms for plaque formationformation

two distinct mechanisms: 1 ) the two distinct mechanisms: 1 ) the multiplication of bacteria already multiplication of bacteria already attached to the tooth surface, and 2) attached to the tooth surface, and 2) the subsequent attachment and the subsequent attachment and multiplication of new bacterial multiplication of new bacterial species to cells of bacteria already species to cells of bacteria already present in the plaque mass. present in the plaque mass.

MechanismsMechanisms Two distinct Two distinct

mechanisms: mechanisms: 1 ) the multiplication 1 ) the multiplication

of bacteria already of bacteria already attached to the tooth attached to the tooth surfacesurface

2) the subsequent 2) the subsequent attachment and attachment and multiplication of new multiplication of new bacterial species to bacterial species to cells of bacteria cells of bacteria already present in the already present in the plaque mass. plaque mass.

Complexity increasesComplexity increases

The secondary The secondary colonizers include colonizers include Gram-negative Gram-negative species such as species such as Fusobacterium Fusobacterium nucleatum, nucleatum, Prevotella Prevotella intermediaintermedia, and , and CapnocytophagaCapnocytophaga species. species.

Tertiary colonizersTertiary colonizers

After one week of plaque accumulation, After one week of plaque accumulation, other Gram-negative species may also other Gram-negative species may also be present in plaque. These species be present in plaque. These species represent what is considered to be the represent what is considered to be the "tertiary colonizers", and include "tertiary colonizers", and include Porphyromonas gingivalis, Porphyromonas gingivalis, Campylobacter rectus, Eikenella Campylobacter rectus, Eikenella corrodens, Actinobacillus corrodens, Actinobacillus actinomycetemcomitansactinomycetemcomitans, and the oral , and the oral spirochetes (spirochetes (TreponemaTreponema species). species).

CharacteristicsCharacteristics The structural characteristics of dental plaque in The structural characteristics of dental plaque in

this time period reveal complex patterns of this time period reveal complex patterns of bacterial cells of cocci, rods, fusiform, filaments, bacterial cells of cocci, rods, fusiform, filaments, and spirochetes. and spirochetes.

In particular, specific associations of different In particular, specific associations of different bacterial forms have been observed. For example, bacterial forms have been observed. For example, the adherence of cocci to filaments results in a the adherence of cocci to filaments results in a typical form referred to as "test-tube brushes" or typical form referred to as "test-tube brushes" or "corn-cob" arrays "corn-cob" arrays

these structures can be seen in The structural these structures can be seen in The structural interactions of the bacteria probably are a interactions of the bacteria probably are a reflection of the complex metabolic interactions reflection of the complex metabolic interactions that are known to occur between different plaque that are known to occur between different plaque microorganisms. microorganisms.

SELECTED BACTERIAL SPECIES FOUND IN DENTAL PLAQUE

Facultative Anaerobic

Gram-PositiveStreptococcus mutansStreptococcus sanguisActinomyces viscosus

Gram-negative

ActinobacillusactinomycetemcomitansCapnocytophypa species

Eikenella corrodens

Porphyromonas gingivalisFusobacterium nucleatum

Prevotella intermediaBacteroides forsythusCampylobacter rectus

SpirochetesTreponema denticola

(Other Treponema species)

CoaggregationCoaggregation

Express components that mediate Express components that mediate cell to cell bindingcell to cell binding

One cell type in a coaggregation One cell type in a coaggregation partnerships, one cell type expresses partnerships, one cell type expresses a heat-inactivated protease sensitive a heat-inactivated protease sensitive surface adhesionsurface adhesion

The other partner expresses a The other partner expresses a complementary heat stable proteincomplementary heat stable protein

Biofilm BiochemistryBiofilm Biochemistry The production of succinic acid from The production of succinic acid from

CampylobacterCampylobacter species that is known to be used species that is known to be used as a growth factor by as a growth factor by Porphyromonas gingivalisPorphyromonas gingivalis. . StreptococcusStreptococcus and and ActinomycesActinomyces species produce species produce formate, which may then be used by formate, which may then be used by CampylobacterCampylobacter species. species.

FusobacteriumFusobacterium species produce both thiamine species produce both thiamine and isobutyrate that may be used by spirochetes and isobutyrate that may be used by spirochetes to support their growth. The metabolic and to support their growth. The metabolic and structural interactions between different plaque structural interactions between different plaque microorganisms are a reflection of the incredible microorganisms are a reflection of the incredible complexity of this ecological niche. complexity of this ecological niche.

Genes and BiofilmsGenes and BiofilmsNovember 2005November 2005

Biologist Alejandro Toledo Arana has Biologist Alejandro Toledo Arana has identified two genes that regulate identified two genes that regulate the formation of biofilms in the formation of biofilms in Staphylococcus aureusStaphylococcus aureus

Chronic InfectionsChronic Infections

The study has been boosted on The study has been boosted on discovering their relation to chronic discovering their relation to chronic infections associated to medical infections associated to medical implantsimplants

These include those tissues involving These include those tissues involving infections of the middle ear, of the infections of the middle ear, of the prostate gland, pneumonia in prostate gland, pneumonia in patients with cystic fibrosis, patients with cystic fibrosis, osteomyelitis osteomyelitis

Antibiotic resistance in Antibiotic resistance in biofilmsbiofilms

In patients such as those that suffer from In patients such as those that suffer from Cystic fibrosis and infections of Cystic fibrosis and infections of Pseudomonas, there is a weighted response Pseudomonas, there is a weighted response in the CD4 T helper cells( Th1/Th2)in the CD4 T helper cells( Th1/Th2)

Th1 might improve prognosis for elimination Th1 might improve prognosis for elimination and management of infections because they and management of infections because they are associated with an influx of phagocytes are associated with an influx of phagocytes and the ingestion of sessile bacteria.and the ingestion of sessile bacteria.

They also ingest biofilm fragmentsThey also ingest biofilm fragments

Survival Strategies of Infectious BiofilmsSurvival Strategies of Infectious BiofilmsC.A. Fux, J. W.Costerton et al C.A. Fux, J. W.Costerton et al Trends in MicrobiologyTrends in Microbiology

There is a growing concern for antibiotic There is a growing concern for antibiotic resistance in bacteria growing in surface-resistance in bacteria growing in surface-adherent biofilmsadherent biofilms

Many antibiotic assays for susceptibility Many antibiotic assays for susceptibility and resistance are based upon planktonic and resistance are based upon planktonic or free cells rather than attachedor free cells rather than attached

Chronic and device related infections go Chronic and device related infections go unresolved even when the organisms do unresolved even when the organisms do indeed test for antibiotic sensitivityindeed test for antibiotic sensitivity

Biofilm characteristicsBiofilm characteristics

Top to bottom gradient of decreasing Top to bottom gradient of decreasing antibiotic susceptibilityantibiotic susceptibility

The gradient originates in the surface The gradient originates in the surface layers of the biofilms where there is layers of the biofilms where there is complete consumption of oxygen and complete consumption of oxygen and glucoseglucose

There are patches of antibiotic resistance There are patches of antibiotic resistance at the surfaceat the surface

Proximity of cells lead to horizontal Proximity of cells lead to horizontal transfer of genes for resistancetransfer of genes for resistance

Biofilms and AntibioticsBiofilms and Antibiotics

The diffusion of antibiotics in biofilms The diffusion of antibiotics in biofilms has been studiedhas been studied

Beta lactamase producing bacteria Beta lactamase producing bacteria increase enzyme production in increase enzyme production in response to antibiotic treatmentresponse to antibiotic treatment

The enzyme accumulates in the The enzyme accumulates in the matrix of the biofilm thereby matrix of the biofilm thereby inactivating the antibioticinactivating the antibiotic

Mar OperonMar Operon

Multiple Antibiotic ResistanceMultiple Antibiotic Resistance operon operon ((MarMar) is chromosomal, and encodes for ) is chromosomal, and encodes for permease proteins (AcrB) which actively permease proteins (AcrB) which actively export a wide range of xenobiotics from export a wide range of xenobiotics from bacterial cells. bacterial cells.

MarMar is widely distributed. Recent reports is widely distributed. Recent reports show that show that MarMar can be regulated not only by can be regulated not only by exposure to sub-MIC levels of antibiotic, but exposure to sub-MIC levels of antibiotic, but also through slow growth rate, the stringent also through slow growth rate, the stringent response and a number of other unrelated response and a number of other unrelated stimuli. stimuli.

Mar IIMar II

It is not regulated through It is not regulated through homoserinelactone but does appear to be homoserinelactone but does appear to be part of a global regulatory system that part of a global regulatory system that also controls exopolymer biosynthesis. also controls exopolymer biosynthesis.

This operon is of major interesdt since it is This operon is of major interesdt since it is likely that this would be switched on in likely that this would be switched on in biofilms and might be a major factor in the biofilms and might be a major factor in the high level antibiotic resistance observed in high level antibiotic resistance observed in biofilms. biofilms.

EndocarditisEndocarditis Biofilm of bacteria + Biofilm of bacteria +

host components on host components on valve = valve = vegetationvegetation

Requires prior valve Requires prior valve injury injury

200X increase in 200X increase in antibiotic resistance antibiotic resistance

Rabbit model: block Rabbit model: block biofilm formation --> biofilm formation --> acute virulent infection acute virulent infection

Infectious Kidney StonesInfectious Kidney Stones

15-20% involve urinary 15-20% involve urinary tract infection tract infection

Bacterium --> biofilm Bacterium --> biofilm --> mineralization --> mineralization

Causative organisms Causative organisms have urease have urease

urea --> NH4 + H2CO3 urea --> NH4 + H2CO3 Biofilm concentrates Biofilm concentrates

urease --> crystal urease --> crystal formation formation

  

CFCF Mutation in chloride Mutation in chloride

channel in epithelial channel in epithelial cells cells

1st stage: intermittent 1st stage: intermittent infections infections

2nd stage: permanent 2nd stage: permanent infection with infection with Pseudomonas Pseudomonas aeruginosaaeruginosa

Mucoid type - Mucoid type - overproduce alginate overproduce alginate

Antibiotic resistance Antibiotic resistance   

Microarrays Microarrays Used to assess the Used to assess the

genes present in genes present in different stages of different stages of biofilm formationbiofilm formation

In Staphylococcal In Staphylococcal biofilms the same biofilms the same genes are active genes are active the sar A the sar A staphylococcal staphylococcal accessory regulator accessory regulator and the ica ADBC and the ica ADBC regulatorregulator

MicroarraysMicroarrays One of the best One of the best

ways to evaluate ways to evaluate gene expressiongene expression

DNA chips are used DNA chips are used for a solid supportfor a solid support

These are made of These are made of silicon or glasssilicon or glass

They have DNA They have DNA attached in orderly attached in orderly arraysarrays

Microarrays( 1)Microarrays( 1)

The DNA is deliverd to specific The DNA is deliverd to specific position on the chip using tiny pins to position on the chip using tiny pins to apply a solutionapply a solution

The spots are treated and dried in The spots are treated and dried in order to bind the DNAorder to bind the DNA

Usually cDNAUsually cDNA cDNA is prepared from mRNAcDNA is prepared from mRNA These pieces of DNA are usually 500-These pieces of DNA are usually 500-

5000 nucleotides long5000 nucleotides long

Commercial chips( II)Commercial chips( II) Oligonucleotides about 25 bases in length can Oligonucleotides about 25 bases in length can

be synthesized and placed directly on the chipbe synthesized and placed directly on the chip The chip is 1.3 cm on a side and can have over The chip is 1.3 cm on a side and can have over

200,000 addressable positions200,000 addressable positions The probes are often expressed sequence The probes are often expressed sequence

tags( ESTs)tags( ESTs) The nucleic acids to be analyzed are isolated The nucleic acids to be analyzed are isolated

and labeled with fluorescent reporter groupsand labeled with fluorescent reporter groups The DNA or target nucleotides are incubated The DNA or target nucleotides are incubated

with the fluorescent groups and then washedwith the fluorescent groups and then washed The chip is scanned with lasersThe chip is scanned with lasers

Preparation of Preparation of MicroarraysMicroarrays

Interpretation of Interpretation of microarraysmicroarrays

Agents for the destruction Agents for the destruction of biofilms( Industrial of biofilms( Industrial

biocides)biocides) (alexidine, chlorhexidine, (alexidine, chlorhexidine,

polyhexamethylene biguanides)polyhexamethylene biguanides), , monophenylethersmonophenylethers (phenoxyethanol) (phenoxyethanol) and quaternary amonium compoundsand quaternary amonium compounds (cetrimide, benzalkoniums)(cetrimide, benzalkoniums) and and have demonstrated biochemical bases have demonstrated biochemical bases for the activities and associated for the activities and associated mammalian cell toxicities of thiol mammalian cell toxicities of thiol interactive agentsinteractive agents (bronopol, (bronopol, isothiazolones)isothiazolones). .