Concepto y estructurade los biopeliculas

Concepto y estructurade los biopeliculas

Iwona B. Beech

University of Portsmouth, UK

Iwona B. Beech

University of Portsmouth, UK

BACTERIAL GROWTH IN NATURAL

AND MAN MADE ENVIRONMENTS

PLANKTONIC CELLS(PESENT IN THE LIQUID PHASE)

SESSILE CELLS(ASSOCIATED WITH SURFACES)

MICROSCOPY IN BIOFILM RESEARCH

MICROSCOPY IN BIOFILM RESEARCH

• LIGHT• LIGHT

• CONFOCAL SCANNING LASER• CONFOCAL SCANNING LASER

• ATOMIC FORCE• ATOMIC FORCE

• (ENVIRONMENTAL) SCANNING ELECTRON

• (ENVIRONMENTAL) SCANNING ELECTRON

Conjunto de microorganismos embebidos en una matrizexopolimérica de origen microbiano que los mantiene unidosjunto a otras sustancias del medio en que se encuentran(Characklis y Marshall, 1990)

CONCEPTO BIOPELÍCULA

FORMACIÓN Y DESARROLLOFORMACIÓN Y DESARROLLO

1.- TRANSPORTE A LA SUPERFICIE

2.- ADHESIÓN INICIAL

3.- CONSOLIDACIÓN DE LA ADHESIÓN O “ATTACHMENT “

4.- COLONIZACIÓN

5.- DESORCIÓN

BIOFILM

MICROBIAL GROWTH AT INTERFACES

Morning Glory Pool in Yellowstone National Park

BIOPELÍCULAS

Intercambiadores de calor

Tuberías

Placa dental

Lentes de contacto

Cateteres

Implantes

BIOPELÍCULAS

Characteristic features of a multilayer biofilmCharacteristic features of a multilayer biofilm

BIOFILMS ON HISTORIC STRUCTURES

FRACTURES

Cracking of marbleFuente de Los Leones de

La AlhambraPIGMENTATION

Catedral Santiago Compostela

Fuente de Los Leones de La Alhambra

BIODETERIORO

Cambio indeseable en las propiedades de un material causado por la acción de un organismo vivo

(Hueck, 1965)

The Mary Rose

• The Mary Rose is the only 16th century warship on d isplay anywhere in the world. Built between 1509 and 1511, she was a firm favourite of King Henry VIII.

• In 1545, while maneuvering to engage a French fleet outside Portsmouth, she unexpectedly went down in 1 4 m of water.

• The wreck was rediscovered in 1971, and salvaged, a long with some 19,000 objects in 1982.

• Approximately one-third of the original hull remain s. Most of the starboard side and parts of the decks had su rvived deeply embedded in soft yielding clay.

• Currently the hull is still in a ‘wet’ state. It is in the final stages of PEG spraying before drying and open publi c exhibition in 2010/2011.

BiofoulingBiofouling

Biofilms in health

Three examples of possible points of entry into the body for infectious biofilms:

catheter

hip replacement

periodontal disease

Biofilms in health

Three examples of possible points of entry into the body for infectious biofilms:

catheter

hip replacement

periodontal disease

STAGES OF BIOFILM FORMATION

SUBSTRATUMConditioning layer

Cellassociation

Biofilmformation

Biofilmsloughing

Reversible

adhesion

Irreversible

adhesionCell

division

Microcolony

formation

Biofilm model based on CSLM imagingBiofilm model based on CSLM imaging

EPIFLUORESCENCE MICROSCOPYEPIFLUORESCENCE MICROSCOPYTitaniumTitanium

62 days 90 days 254 days

174 days 254 days

Attachment of D. alaskensis and D. indonensiensis mixed population to the AISI 316 stainless

steel surface after 4 h (a) and 12 h (b) of exposure.

DNA-DAPI-staining shows in blue all attached cells.

D. indonensiensis cells (arrows) are identified by double staining in green (FITC) and blue

(DAPI). Superposed images of FITC and DAPI (insets) show that D. alaskensis colonises surface

more rapidly than D. indonesiensis. Bar =1µµµµm.

SEM images of biofilmsSEM images of biofilmsTitaniumTitaniumTitanium

26 days

Smooth Rough

90 days

285 days

AISI 316 stainless steelAISI 316 stainless steel

CopperCopper

BIOFILM MATRIXBIOFILM MATRIX

BACTERIAL EXTRACELLULAR POLYMERIC SUBSTANCES

EPS

facilitate irreversible cell adhesion to a substratum

form the biofilm matrix

BACTERIAL EXTRACELLULAR POLYMERIC SUBSTANCES

EPS

facilitate irreversible cell adhesion to a substratum

form the biofilm matrix

EPSEPSEPSEPSEPSEPSEPSEPSEPSEPSEPSEPS

Marine biofilm on the surface of carbon steelMarine biofilm on the surface of carbon steel

ESEM imageESEM image

Diferencia entre las condiciones externas y las propias de la biopelícula:

MICROAMBIENTE

PROPIEDADES DE LAS BIOPELÍCULAS

Adhesion a superficies

Protección frente a condiciones externas

Varios tipos de microorganismos

Concentracion de nutrientes

Transporte de sustancias

Gradiente de O2

Cambios de pH

Agregación de células

Reconocimiento celular

Retención de agua

Quorum Sensing in Bacteria

Quorum Sensing in Bacteria

Bacteria prefer to live in communitiesBacteria prefer to live in communities

1 µm

Multispecies biofilmMultispecies biofilm Single species biofilmSingle species biofilm

Quorum sensing (QS) is the ability

of bacteria to communicate and coordinate behavior

via signaling molecules.

QS is regulated by population density of the same species and the presence

of other species

Quorum sensing (QS) is the ability

of bacteria to communicate and coordinate behavior

via signaling molecules.

QS is regulated by population density of the same species and the presence

of other species

Bacterial cells “talk” to each other …Bacterial cells “talk” to each other …

http://www.che.caltech.edu/groups/fha/quorum.html

Vibrio fischeri

Quorum sensing was first discovered in a marine luminescent bacterium, Vibrio fischeri, which is a

facultative symbiont of marine animals.

Vibrio fischeri

Quorum sensing was first discovered in a marine luminescent bacterium, Vibrio fischeri, which is a

facultative symbiont of marine animals.The jelly-fish lounge (Image: J Nicholson and K Takayama)The jelly-fish lounge (Image: J Nicholson and K Takayama)

3-Oxohexanoyl homoserine lactone (AHL) is produced by LuxI and recognized by LuxRin Vibrio fischeri

3-Oxohexanoyl homoserine lactone (AHL) is produced by LuxI and recognized by LuxRin Vibrio fischeri

Bacteria are sensitive to the presence of

“neighbors”

Bacteria are sensitive to the presence of

“neighbors”

It was discovered that when Vibrio fischeri cells

were solitary, they did not luminesce.

Only when many cells came together, in places

such as the gut of a fish, did the luminescence

“turned on”.

This is makes sense, as there is no advantage

for a single, isolated bacterium to produce

light.

It was discovered that when Vibrio fischeri cells

were solitary, they did not luminesce.

Only when many cells came together, in places

such as the gut of a fish, did the luminescence

“turned on”.

This is makes sense, as there is no advantage

for a single, isolated bacterium to produce

light.

Each bacterium produces autoinducer molecules into its environment. Different species typically produces

different compounds.

The autoinducers are sometimes referred to as

pheromones or AI-1.

Each bacterium has a receptor for its own AI-1.

When only a few other bacteria of the same kind are in

the vicinity, the concentration of the inducer in the

surrounding medium is very low.

Each bacterium produces autoinducer molecules into its environment. Different species typically produces

different compounds.

The autoinducers are sometimes referred to as

pheromones or AI-1.

Each bacterium has a receptor for its own AI-1.

When only a few other bacteria of the same kind are in

the vicinity, the concentration of the inducer in the

surrounding medium is very low.

The General Principle of Intra-Species QSThe General Principle of Intra-Species QS

The purpose of quorum sensing is to coordinate certain behaviour or actions between bacteria,

based on their local density.

QS can occur within a single bacterial species

(as well as between disparate species)

and regulates a range of different processes, essentially serving as a communication network.

The purpose of quorum sensing is to coordinate certain behaviour or actions between bacteria,

based on their local density.

QS can occur within a single bacterial species

(as well as between disparate species)

and regulates a range of different processes, essentially serving as a communication network.

Why intra-species quorum sensing?Why intra-species quorum sensing?

When many bacteria of the same kind are present, the

concentration of the autoinducer increases above a critical

threshold.

In response, the bacteria start to synthesis more autoinducer.

This forms a positive feedback loop.

The receptor becomes fully activated, and this induces the up-

regulation of specific genes.

For example, activation of luciferase (lux gene) in V. fishcheri

causes light emission, and activation of genes in other bacteria

causes pathogenicity.

When many bacteria of the same kind are present, the

concentration of the autoinducer increases above a critical

threshold.

In response, the bacteria start to synthesis more autoinducer.

This forms a positive feedback loop.

The receptor becomes fully activated, and this induces the up-

regulation of specific genes.

For example, activation of luciferase (lux gene) in V. fishcheri

causes light emission, and activation of genes in other bacteria

causes pathogenicity.

http://www.che.caltech.edu/groups/fha/quorum.html

Quorum sensing makes cells able to react to high cell densities

Quorum sensing makes cells able to react to high cell densities

Different species usually have different autoinducers

(Quorum Pheromones)

Gram-negative Gram-positive

Different species usually have different autoinducers

(Quorum Pheromones)

Gram-negative Gram-positive

Science (2006) 311: 1113-1116

Homoserine lactones (AHL) PeptidesHomoserine lactones (AHL) Peptides

Cell communicationin Gram-negative and Gram-positive bacteria

Cell communicationin Gram-negative and Gram-positive bacteria

www.nottingham.ac.uk/quorum/

The model luminous bacterium Vibrio harveyi

produce two different autoinducers, called AI-1 and AI-2,

• each autoinducer is detected by its own sensor

protein.

• Both sensors transmit information to a shared

integrator protein to control the output, light emission.

• An analogous mechanism operate in V. cholerae to

control virulence.

The model luminous bacterium Vibrio harveyi

produce two different autoinducers, called AI-1 and AI-2,

• each autoinducer is detected by its own sensor

protein.

• Both sensors transmit information to a shared

integrator protein to control the output, light emission.

• An analogous mechanism operate in V. cholerae to

control virulence.

•V. harveyi and V. cholerae use the AI-1 quorum sensing circuit for intra-species communication and the AI-2 quorum sensing circuit for inter-speciescommunication

The Al-2 autoinducer appears to serve as a 'universal' signal for inter-species communication.

The chemical identity of AI-2 remained unknown until recently …

•V. harveyi and V. cholerae use the AI-1 quorum sensing circuit for intra-species communication and the AI-2 quorum sensing circuit for inter-speciescommunication

The Al-2 autoinducer appears to serve as a 'universal' signal for inter-species communication.

The chemical identity of AI-2 remained unknown until recently …

The AI-2 autoinducerThe AI-2 autoinducer

Inter-species communicationInter-species communication

To investigate the mechanism of AI-2 signaling, Bessler et al. constructed mutants and cloned the gene responsible for AI-2 production from several bacteria.

In each case the gene was highly homologous, and they named it luxS. Homologues of luxS and AI-2 production are widespread in the bacterial world, suggesting that communication via an AI-2 signal response system is a common mechanism that bacteria employ for inter-species interaction in natural environments.

To investigate the mechanism of AI-2 signaling, Bessler et al. constructed mutants and cloned the gene responsible for AI-2 production from several bacteria.

In each case the gene was highly homologous, and they named it luxS. Homologues of luxS and AI-2 production are widespread in the bacterial world, suggesting that communication via an AI-2 signal response system is a common mechanism that bacteria employ for inter-species interaction in natural environments.

What is AI-2?What is AI-2?

The chemical identity of AI-2 was obtained in 2002 by solving the crystal structure of the V. harveyi sensor protein in complex with its AI-2 molecule1.

[1] Chen, X., Schauder, S., Potier, N., Van Dorssel aer, A., Pelczer, I.,

Bassler, B. L., and Hughson, F. M. (2002). Nature 415, 545-549

The chemical identity of AI-2 was obtained in 2002 by solving the crystal structure of the V. harveyi sensor protein in complex with its AI-2 molecule1.

[1] Chen, X., Schauder, S., Potier, N., Van Dorssel aer, A., Pelczer, I.,

Bassler, B. L., and Hughson, F. M. (2002). Nature 415, 545-549

The V. harveyi AI-2 was found to

be a furanosylborate diester. Finding boron in the active

molecule was surprising because

boron, while widely available in

nature, has almost no known role

in biology.

The V. harveyi AI-2 was found to

be a furanosylborate diester. Finding boron in the active

molecule was surprising because

boron, while widely available in

nature, has almost no known role

in biology.

Chemical Identity of AI-2Chemical Identity of AI-2

AI-2 as recognized by V. harveyiAI-2 as recognized by V. harveyi

X-ray crystal structure of the V. harveyi

AI-2 / sensor protein complex

X-ray crystal structure of the V. harveyi

AI-2 / sensor protein complex

X-ray crystallographic electron density of AI-2 (blue), with the

boron atom shown in yellow. Protein side chains of the AI-2

sensor protein LuxP hydrogen bond (red dashed lines) to the

furanosyl borate diester ligand.

X-ray crystallographic electron density of AI-2 (blue), with the

boron atom shown in yellow. Protein side chains of the AI-2

sensor protein LuxP hydrogen bond (red dashed lines) to the

furanosyl borate diester ligand.

http://www.che.caltech.edu/groups/fha/quorum.html

Quorum sensing makes cells able to react to high cell densities

Quorum sensing makes cells able to react to high cell densities

Different species usually have different autoinducers

(Quorum Pheromones)

Gram-negative Gram-positive

Different species usually have different autoinducers

(Quorum Pheromones)

Gram-negative Gram-positive

Science (2006) 311: 1113-1116

Homoserine lactones (AHL) Pep tidesHomoserine lactones (AHL) Pep tides

Cell communicationin gram-negative and gram-positive bacteria

Cell communicationin gram-negative and gram-positive bacteria

www.nottingham.ac.uk/quorum/

Modern work has shown that there are two different autoinducers in

Vibrio harveyi

Modern work has shown that there are two different autoinducers in

Vibrio harveyi

Bonnie Bassler et al. have shown that

• the model luminous bacterium Vibrio harveyi produce two different autoinducers, called AI-1 and AI-2 ,

• each autoinducer is detected by its own sensor protein.

• Both sensors transmit information to a shared integrator protein to control the output, light emission.

• An analogous mechanism operate in V. cholerae to control virulence.

Bonnie Bassler et al. have shown that

• the model luminous bacterium Vibrio harveyi produce two different autoinducers, called AI-1 and AI-2 ,

• each autoinducer is detected by its own sensor protein.

• Both sensors transmit information to a shared integrator protein to control the output, light emission.

• An analogous mechanism operate in V. cholerae to control virulence.

•V. harveyi and V. cholerae use the AI-1 quorum sensing circuit for intra-species communication and the AI-2 quorum sensing circuit for inter-species communication

The Al-2 autoinducer appears to serve as a 'universal' signal for inter-species communication.

The chemical identity of AI-2 remained unknown until recently …

•V. harveyi and V. cholerae use the AI-1 quorum sensing circuit for intra-species communication and the AI-2 quorum sensing circuit for inter-species communication

The Al-2 autoinducer appears to serve as a 'universal' signal for inter-species communication.

The chemical identity of AI-2 remained unknown until recently …

The AI-2 autoinducerThe AI-2 autoinducer

Inter-species communicationInter-species communication

To investigate the mechanism of AI-2 signaling, Bessler et al. constructed mutants and cloned the gene responsible for AI-2 production from several bacteria. In each case the gene was highly homologous, and they named it luxS. Homologues of luxS and AI-2 production are widespread in the bacterial world, suggesting that communication via an AI-2 signal response system is a common mechanism that bacteria employ for inter-species interaction in natural environments.

To investigate the mechanism of AI-2 signaling, Bessler et al. constructed mutants and cloned the gene responsible for AI-2 production from several bacteria. In each case the gene was highly homologous, and they named it luxS. Homologues of luxS and AI-2 production are widespread in the bacterial world, suggesting that communication via an AI-2 signal response system is a common mechanism that bacteria employ for inter-species interaction in natural environments.

So what is AI-2?So what is AI-2?

The chemical identity of AI-2 was obtained in 2002 by solving the crystal structure of the V. harveyi sensor protein in complex with its AI-2 molecule 1. [1] Chen, X., Schauder, S., Potier, N., Van Dorssel aer, A., Pelczer, I., Bassler, B. L., and Hughson, F. M.

(2002). Nature 415, 545-549

The chemical identity of AI-2 was obtained in 2002 by solving the crystal structure of the V. harveyi sensor protein in complex with its AI-2 molecule 1. [1] Chen, X., Schauder, S., Potier, N., Van Dorssel aer, A., Pelczer, I., Bassler, B. L., and Hughson, F. M.

(2002). Nature 415, 545-549

X-ray crystal structure of the V. harveyiAI-2 / sensor protein complexX-ray crystal structure of the V. harveyiAI-2 / sensor protein complex

X-ray crystallographic electron density of AI-2 (blue), with the boron atom shown in yellow. Protein side chains of the AI-2 sensor protein LuxP

hydrogen bond (red dashed lines) to the furanosyl borate diester ligand. [1] Chen, X., Schauder, S., Potier, N., Van Dorsselaer, A., Pelczer, I.,

Bassler, B. L., and Hughson, F. M. (2002). Nature 415, 545-549

X-ray crystallographic electron density of AI-2 (blue), with the boron atom shown in yellow. Protein side chains of the AI-2 sensor protein LuxP

hydrogen bond (red dashed lines) to the furanosyl borate diester ligand. [1] Chen, X., Schauder, S., Potier, N., Van Dorsselaer, A., Pelczer, I.,

Bassler, B. L., and Hughson, F. M. (2002). Nature 415, 545-549

The V. harveyi AI-2 was found to be a furanosylboratediester. Finding boron in the active molecule was surprising because boron, while widely available in nature has almost no known role in biology.

The V. harveyi AI-2 was found to be a furanosylboratediester. Finding boron in the active molecule was surprising because boron, while widely available in nature has almost no known role in biology.

Chemical Identity of AI-2Chemical Identity of AI-2

AI-2 as recognized by V. harveyiAI-2 as recognized by V. harveyi

V. fischeri, a symbiont, glows when signal molecules

from its own kind reach critical levels.

In contrast, free-living V. harveyi require sufficient

amounts of two autoinducers — both the species-

specific AI-1 and the universal AI-2, to activate their

luminescence genes.

V. harveyi mutant strains respond to only one signal or

the other. Mutants were used to show that one system

tells the bacteria how many of its own species are in the

area; the other tells how many other types of bacteria

are around.

V. fischeri, a symbiont, glows when signal molecules

from its own kind reach critical levels.

In contrast, free-living V. harveyi require sufficient

amounts of two autoinducers — both the species-

specific AI-1 and the universal AI-2, to activate their

luminescence genes.

V. harveyi mutant strains respond to only one signal or

the other. Mutants were used to show that one system

tells the bacteria how many of its own species are in the

area; the other tells how many other types of bacteria

are around.

Different QS strategies …Different QS strategies …

In a petri dish, the arrow contains a mutant form of V. harveyi.

On the left is a patch of E. coli that causes intestinal infections; on the right is Salmonella; in the middle, above and below the stem of the arrow, is a lab strain of non-pathogenic E. coli.

In the dark (bottom photo), V. harveyiglows in the presence of the two pathogenic bacteria but not the harmless one.

In a petri dish, the arrow contains a mutant form of V. harveyi.

On the left is a patch of E. coli that causes intestinal infections; on the right is Salmonella; in the middle, above and below the stem of the arrow, is a lab strain of non-pathogenic E. coli.

In the dark (bottom photo), V. harveyiglows in the presence of the two pathogenic bacteria but not the harmless one.

http://www.princeton.edu/pr/pwb/99/0329/bacterial.htm

Crystal structure of the S. typhimurim AI-2 receptor, LsrB (Miller, S.T. et al.) Crystal structure of the S. typhimurim AI-2 receptor, LsrB (Miller, S.T. et al.)

Different species of bacteria recognize different forms of AI-2.

S. typhimurim recognize a chemically distinct adduct of DPD as AI-2.

The reason for this complexity is not known. It may allow different species to “interpret” the signal in different ways.

Different species of bacteria recognize different forms of AI-2.

S. typhimurim recognize a chemically distinct adduct of DPD as AI-2.

The reason for this complexity is not known. It may allow different species to “interpret” the signal in different ways.

Variability of AI-2 among speciesVariability of AI-2 among species

Quorum sensing and biofilmsQuorum sensing and biofilms

In the cartoon above, various species of bacteria are represented by different colors. Bacteria can produce chemical signals ("talk") and other bacteria can respond to them ("listen") in a process commonly known as cell-cell communication or cell-cell signaling. This communication can result in coordinated behavior of microbial populations. Courtesy, MSU-CBE.

In the cartoon above, various species of bacteria are represented by different colors. Bacteria can produce chemical signals ("talk") and other bacteria can respond to them ("listen") in a process commonly known as cell-cell communication or cell-cell signaling. This communication can result in coordinated behavior of microbial populations. Courtesy, MSU-CBE.

Although planktonic cells secrete chemical signals (HSLs, for homoserine lactones), the low concentration of signal molecules does not change genetic expression. Biofilm cells are held together in dense populations, so the secreted HSLs attain higher concentrations. HSL molecules then re-cross the cell membranes and trigger changes in genetic activity.

Although planktonic cells secrete chemical signals (HSLs, for homoserine lactones), the low concentration of signal molecules does not change genetic expression. Biofilm cells are held together in dense populations, so the secreted HSLs attain higher concentrations. HSL molecules then re-cross the cell membranes and trigger changes in genetic activity.

• Unattached cells that approach a surface may attach. Attachment

involves specific functions.

• Attached cells will proliferate on a surface and use specific

functions to actively move into microcolonies.

• The high-density microcolonies differentiate into mature biofilms

by a 3OC12-HSL-dependent mechanism.

• Unattached cells that approach a surface may attach. Attachment

involves specific functions.

• Attached cells will proliferate on a surface and use specific

functions to actively move into microcolonies.

• The high-density microcolonies differentiate into mature biofilms

by a 3OC12-HSL-dependent mechanism.

Acyl-homoserine lactone QS are required to form mature biofilms of Gram-negative bacteria

Acyl-homoserine lactone QS are required to form mature biofilms of Gram-negative bacteria

Diagram of the P. aeruginosa biofilm-maturation pathway.

Scanning confocal microscope images of a mature P. aeruginosa wild-type biofilm (Upper) and a quorum-sensing mutant biofilm (Lower). In this case the quorum-sensing mutant was a lasR, rhlRdouble mutant. The perspective is from above the biofilm on a glass surface. The glass surface is red, and the green is from the green fluorescent protein encoded by the gfp gene in the recombinant P. aeruginosa. The wild-type biofilm consists of thick microcolonies. The immature mutant biofilm appears thinner, and more of the glass surface is exposed. With the lasR, rhlR mutant shown here (but not with lasI, rhlI mutants) zones of clearing around microcolony towers are often observed. Other experiments have shown that these zones are filled with extracellular polysaccharide (M.R.P., unpublished data).

Scanning confocal microscope images of a mature P. aeruginosa wild-type biofilm (Upper) and a quorum-sensing mutant biofilm (Lower). In this case the quorum-sensing mutant was a lasR, rhlRdouble mutant. The perspective is from above the biofilm on a glass surface. The glass surface is red, and the green is from the green fluorescent protein encoded by the gfp gene in the recombinant P. aeruginosa. The wild-type biofilm consists of thick microcolonies. The immature mutant biofilm appears thinner, and more of the glass surface is exposed. With the lasR, rhlR mutant shown here (but not with lasI, rhlI mutants) zones of clearing around microcolony towers are often observed. Other experiments have shown that these zones are filled with extracellular polysaccharide (M.R.P., unpublished data).

Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: A signaling mechanism involved in associations with higher organisms; Matthew R. Parsek* and E. Peter GreenbergAcyl-homoserine lactone quorum sensing in Gram-negative bacteria: A signaling mechanism involved in associations with higher organisms; Matthew R. Parsek* and E. Peter Greenberg

http://www.asm.org/news/index.asp?bid=24596

Virulence gene expression is induced in these cells, which then colonize the

intestinal epithelium. Subsequent growth to high cell density represses

virulence factor expression, and induces the expression of factors aiding

detachment, such as Hap protease. Bacteria are shed from the host, possibly

as biofilms, and the biofilm structure may enhance V. cholerae persistence in

the environment, or infectivity for new hosts.

Virulence gene expression is induced in these cells, which then colonize the

intestinal epithelium. Subsequent growth to high cell density represses

virulence factor expression, and induces the expression of factors aiding

detachment, such as Hap protease. Bacteria are shed from the host, possibly

as biofilms, and the biofilm structure may enhance V. cholerae persistence in

the environment, or infectivity for new hosts.

Proposed roles of quorum sensing and biofilm formation inthe life cycle of Vibrio cholerae.

Upon ingestion, the biofilm structure

protects V. cholerae cells from acid

shock in the gastric environment. After

passing through the stomach, individual

cells that escape the biofilm experience

conditions of low cell density.

Proposed roles of quorum sensing and biofilm formation inthe life cycle of Vibrio cholerae.

Upon ingestion, the biofilm structure

protects V. cholerae cells from acid

shock in the gastric environment. After

passing through the stomach, individual

cells that escape the biofilm experience

conditions of low cell density.

First glimpses of the complexity of bacterial communications

“It is a jungle out there”

First glimpses of the complexity of bacterial communications

“It is a jungle out there”

Bacteria manipulate AI-2 molecules for their own benefit:

Some hide their own signals to deceive competing species

Others cleave their neighbors' AHLs

Pseudomonas aeruginosa listens in on other microbes and turns on its own virulence programs only when in a large, protective group (Bassler).

Bacteria manipulate AI-2 molecules for their own benefit:

Some hide their own signals to deceive competing species

Others cleave their neighbors' AHLs

Pseudomonas aeruginosa listens in on other microbes and turns on its own virulence programs only when in a large, protective group (Bassler).

From the games bacteria play …From the games bacteria play …

Enzymes involved in AI-2 production and detection are potential targets for novel antimicrobial drugs.

In particular, molecules that are structurally related to AI-2 have many potential uses.

Enzymes involved in AI-2 production and detection are potential targets for novel antimicrobial drugs.

In particular, molecules that are structurally related to AI-2 have many potential uses.

Furanone is an anti-biofilm compound from the seaweed Delisea pulchra that does not affect the growth of Gram-negative strains, inhibits AI-2 quorum sensing in Gram-negative strains.

Furanone is an anti-biofilm compound from the seaweed Delisea pulchra that does not affect the growth of Gram-negative strains, inhibits AI-2 quorum sensing in Gram-negative strains.

Structure of ( 5Z)-4-bromo-5-(bromomethylene)-3-butyl-2( 5H)-furanoneStructure of ( 5Z)-4-bromo-5-(bromomethylene)-3-butyl-2( 5H)-furanone

… to the games people play… to the games people play

http://cheweb.tamu.edu/orgs/groups/wood/research.html

Inhibition of E. coli biofilm swarming (quorum sensing phenomenon) using furanone

Inhibition of E. coli biofilm swarming (quorum sensing phenomenon) using furanone

FORMACIÓN Y DESARROLLO

4.- COLONIZACIÓN- Desarrollo de la arquitectura intrínseca de la biopelícula; formación de canales y poros y redistribución de las bacterias

- Comunicación célula-célula, que favorece la maduración: “Quorum sensing”(Homoserina Lactonas)

5.- DESORCIÓN- Desprendimiento de células o de porciones de la biopelícula

- Existe regulación:

· Incremento en la concentración de una molécula inductora responsable de la liberación de enzimas que degradan la matriz polimérica

· Densidad celular también puede ser responsable de la liberación de estas enzimas

Who is talking? PROCESAMIENTO DE MUESTRAS POR MICROBIOLOGÍA MOLECULAR

Who is talking? PROCESAMIENTO DE MUESTRAS POR MICROBIOLOGÍA MOLECULAR

Toma de muestras de biopelicula

Extracción de ADN

Amplificación por PCR del ADN

341F (5'-CCT ACG GGA GGC AGC AG-3') y con cola –GC (Muyzer cols, 1993)

907R (5’CCG TCA ATT CCT TTG AGT TT-3’) (Muyzer y cols, 1995)

531R (5’-TAC CGC GGC TGC TGG CAC-3’) (Muyzer y cols, 1995)

Inserto

Análisis por DGGEClonación de fragmento de ADN

amplificado

Aislamiento de clones y extracción

del ADN insertado en el plásmido

Análisis de los clones por DGGE

Secuenciación de ADN

Análisis de secuencias y

relaciones filogenéticas

Bases de datos:

NCBI y EMBL