Métodos de detección de biopelículas

Methods of biofilm detection

Métodos de detección de biopelículas

Methods of biofilm detection

Iwona B. Beech

University of Portsmouth, UK

Iwona B. Beech

University of Portsmouth, UK

Bacteria prefer to live in communitiesBacteria prefer to live in communities

1 µm

Multispecies biofilmMultispecies biofilm Single species biofilmSingle species biofilm

a

b

Morning Glory Pool in Yellowstone National Park

BIOPELÍCULAS

Intercambiadores de calor

Tuberías

Placa dental

Lentes de contacto

Cateteres

Implantes

BIOPELÍCULAS

BIOFILMS ON HISTORIC STRUCTURES

FRACTURES

Cracking of marbleFuente de Los Leones de

La AlhambraPIGMENTATION

Catedral Santiago Compostela

Fuente de Los Leones de La Alhambra

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”.

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”.

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 …

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.

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

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

How to detect biofilms ?How to detect biofilms ?

Early stages of biofilm development :

Too few cells to culture

(QS ?)

Early stages of biofilm development :

Too few cells to culture

(QS ?)

EPIFLUORESCENCE MICROSCOPYEPIFLUORESCENCE MICROSCOPYTitaniumTitanium

62 days 90 days 254 days

174 days 254 days

The lack of growth detectionThe lack of growth detection

AISI 316 stainless steelAISI 316 stainless steel

CopperCopper

Sampling from relevant areas in the system

Correct sample handling

The use of appropriate cultivation techniques

Sampling from relevant areas in the system

Correct sample handling

The use of appropriate cultivation techniques

Biofilm sampling: On surfaces!

���� Scratching, scratching, scratching(razor blades, rubber scrapers etc)

���� Removal of parts of supportmaterial with biofilms ⇒⇒⇒⇒ laboratory

���� Expose test surfaces („coupons“), remove after given time ⇒⇒⇒⇒ laboratory

Constructed: 1509-1512 Sank in the Solent in1545Salvaged in 1982

Constructed:1625-1628Sank on its maiden voyage 1628

A TALE OF DETERIORATION IN THREE SHIPSA TALE OF DETERIORATION IN THREE SHIPS

The Vasa

Salvaged in 1961

The Mary Rose

The Cutty SarkConstructed: 1869A composite ship constructed of wroughtiron and wood. Placed in dry dock in 1954

THE SULFUR PROBLEMTHE SULFUR PROBLEM

Gun shield from the Mary Rose

2000 - Acid detected in the hull of the Vasa in conjunction with yellow

deposits of sulfur at the wood surface.

Similar acidification observed in the Hull of the Mary Rose and

small wooden objects exposed to iron

Yellow sulfur deposits observed in timbers exposed to ironfrom the Cutty Sark

Sample Location Sample

Type

SRB SOB IRB

1 Frame 60 Metal Positive Positive Positive

2 Frame 60 Wood* Positive Positive Positive

3 Frame 61 Water Positive Positive Positive

4 Frame 61 Wood* Positive Positive Positive

5 Frame 61 Corrosion

Product

Positive Positive Positive

6 Main Mast Wood* Positive Positive Positive

7 Frame 68 Swab from

timbers

Positive Positive Positive

8 Frame 68 Intercostal

Wood

Positive Positive Positive

9 Frame

123

Wood* Positive Positive Positive

Sample source, sample type and result from SRB and SOB

testing *Core wood samples taken

CULTURING BACTERIA FROM DRY SHIP TIMBERS

Samples from the Cutty Sark

Samples from the Vasa

Stored Beams and Hull Spray Sediment/ Soil

Extract DNA and RNA samples

cDNA of rRNA molecules

Clone and Sequence

Sequence identity: BLAST searches and Phylogenetic analysis

Primers:(Eubacteria)EUBF933-EUBR1387

RT-PCR

Cloned into TOPO vector Sequenced using M13 universa l primers

16S rDNA

PCR

Sample TD3.6b (C9) – Oak

Acidithiobacillus ferroxidans

selective medium pH 2 with FeSO4

Green – Eubacterial probe EUB338

(GCT GCC TCC CGT AGG AGT)

Red – THIO 820 probe (ACC AAA

CAT CTA GTA TTC ATC) for

A. ferroxidans and T. thiooxidans

1.5µm

1.5µm

FISH for sulfur-oxidising and

Iron-reducing bacteria within

Vasa wood - C9 region

1.5µm 1µm

Sample 050125-UP-HS-17.40 (Oak, pH 2-3, surface)

Extraction in buffer EUB 338 & THIO 820 probes

FUTURE PERSPECTIVES

for the study of biofilms

Spatial and temporal correlation between

microbial presence and chemical processes

- omics

Mass Spectrometry Analysis and Imaging

FUTURE PERSPECTIVES

for the study of biofilms

Spatial and temporal correlation between

microbial presence and chemical processes

- omics

Mass Spectrometry Analysis and Imaging

Analytical Challenges of Biofilm Characterization

Past efforts to probe the chemistry of biofilms have used

traditional and now outdated analytical techniques.

A detailed molecular- level characterization of biofilms is

needed to reach a better understanding of their function and

enable new approaches to their prevention and control.

A rapid method for reliable detection and identification of

medical and environmentally important biofilms is required

and would have enormous economic and health impacts.

Biochemical mass spectrometry is possibly the only

method that can achieve above goals.

BIOCHEMICAL MASS

SPECTROMETRY

BIOCHEMICAL MASS

SPECTROMETRY

PROTEOMICS

METABOLOMICS

PROTEOMICS

METABOLOMICS

SIMS using

Bin+ cluster ion beam

and ToF/MS

Maps of lipids

Red m/z=255&283

Green m/z=892

Blue m/z=771

Pixel size 70x70 µm

Touboul et al. JASMS 2005, 16, 1608-18.

Molecular imaging of rat brain tissue sectionMolecular imaging of rat brain tissue section

12 mm

Chemical Imaging of Biological Materials

using

Electrospray Laser Desorption Ionization

(ELDI)-MS characterization of biofilms, including

proteomics

Chemical Imaging of Biological Materials

using

Electrospray Laser Desorption Ionization

(ELDI)-MS characterization of biofilms, including

proteomics

d4

αd3

d2

d1

EC

SS

A

LB

ISC

SP

Schematic of ELDI imaging ion source

FUTURE PERSPECTIVES

for the study of biofilms

Who is out there, what are they doing and when

are they doing “it” ??

FUTURE PERSPECTIVES

for the study of biofilms

Who is out there, what are they doing and when

are they doing “it” ??