Skapandet av "oslemmiga" ytor

Olena Rzhepishevska, Umeå Universitet

Vilka är vi?

Madeleine Ramstedt,kemist, docent

Shoghik Hakobyan, kemist, doktorand &Olena Rzhepishevska, mikrobiolog, forskare

What’s biofilm?

Kaneko, Y., J Clin Invest , 2007

Rzhepishevska & Ramstedt, unpublished

Deffinition: complex communities of microorganisms attached to a surface or interface enclosed in an exsopolysacchraride matrix of microbial and host origin to produce a spatially organized three dimentional structure (Costerton et al. 1995)

Biofilm is a natural way to exist for microorganisms

The biofilm on pyritic sediments, Richmond mine, CaliforniaEdwards et al., Science, 2000

Bacteria appear red around the vessel wall. Red blood cells within the lumen appear pink, and DAPI stained host cell nuclei appear blue. Schaber et al. Infect Immun. 2007

Importance of biofilms

Biofilms

Industry

Public health

ClinicalPharmathuticalProcessingShipping

Water FoodDistribution pipes Process surfacesDrinking water Home

Prosthesis & Biomaterials

OrthopaedicsCathetersContact lensesShuntsPins/staplesInfusion lines

Tissue

GutUrinary tractLungsBoneHeart

Dental

TeethGumsTongueImplants

Modified from Jass, Surman, Walker, 2003

Biofilms are responsible for approximately 80% of all microbial infections, and cause 100,000 deaths annually in the USA alone.

Surfaces and bacteria - catheters

Most common infectionsPseudomonas aeruginosaStaphylococcus aureus

Biofilms are responsible for approximately 80% of all microbial infectionsand cause 100,000 deaths annually in the USA alone.

Steps in biofilm formation

Plan

cton

ic c

ells

Reversible Irreversible Microcolonies Macrocolonies (mashroom bodies)attachment

monolayer

Pseudomonas aeruginosa

Staphylococcus aureus & Staphylococcus epidermidis

Primary attachment

Accumulative growth

Red tails are single protein molecules

Modified from Caiazza & O’Toole, 2004

Mack et al. 2004

Steps in biofilm formation

Vibrio cholereaVan Dellen et al.,JBact., 2008

Environment/host (VPS)

Host (TCP)

Sea water (Ca2+)

Reversible (transient)attachment

Irreversible(permanent)attachment

Monolayer

Planctonic cells

TCP - toxin-coregulated pilusVPS - Vibrio polysaccharide

Biofilm, CLSM, 1:1 mixture of yellow fluorescent P. aeruginosa PAO1 wt & cyan fluorescent P. aeruginosa pilA, Klausen et al, 2003

Flagella and pili are needed to build biofilm

Flagella Pili

Matrix - the slimeExopolysaccharides (EPS) - physical & chemical protectionAlginate – P.aeruginosa; colanic acid – E.coli

Rhamnolipids - support mashroom structures

Proteins -attachment, protection from host

DNA -support structure , antibiotic resistance

DNAse treatment of S.aureus biofilm, Mann et al, 2009

Day 2pretreatment

Day 3post-treatment

Mono-rhamnolipid Di-rhamnolipid

Communication is everywhere – quorum sensing

homoserine lactone

N-(3-oxohexanoyl) homoserine lactone, Vibrio fischeri & a shining squid

Our contribution to the field

1. The surface charge of anti-bacterial coatings alters motility and biofilm architecture Rzhepishevska O, Hakobyan S, Ruhal R, Gautrot J, Barbero D, Ramstedt M RSC Biomaterials Science 2013 March

2. The antibacterial activity of Ga3+ is influenced by ligand complexation as well as the bacterial carbon source Rzhepishevska O, Ekstrand-Hammarström B, Popp M, Björn E, Bucht A, Sjöstedt A, Antti H, Ramstedt M Antimicrobial Agents & Chemotherapy 2011 Dec

3. The Gallium-saliciliden acylhydrazide complex shows synergistic anti-biofilm effect and inhibits toxinProduction by Pseudomonas aeruginosaJournal of Inorganic Biochemistry 2014Rzhepishevska O, Hakobyan S, Ekstrand-Hammarström B, Nygren Y, Karlsson T, Bucht A, Elofsson M,Boily JF, Ramstedt M

or

POLIMER BRUSH WITHPOSITIVE CHARGE

POLIMER BRUSH WITHNEGATIVE CHARGE

Polymer brushes vs bacteria

Polymer brushes vs bacteria

Surface charge:

METAC+27mV

SPM-35mV

MEDSAH-16mV

POEGMA-2mV

PMMAneutral

MEDSAHPMMA POEGMA SPM

PUM

P

Flow breakers

Bubble traps

Waste

Flow chamber

Fresh medium

Polymer brushes vs bacteria- confocal microscopy

Biofilm, name of the polymer brush, and its charge in mV

Structure of polymer

METAC+27mV

POEGMA-2mV

PMMAneutral

n O

O

O m

H

n

ON

O

+C l-

n

O

O

GLASS

SPM-35mV

MEDSAH-16mV

n

O

O

S -O 3 K +

n

O

O

N S+

-O3

NO MODIFICATION

glass pmma poegma spm medsahmetac 0

100200300400500600700800900

1000

Fluo

resc

ence

inte

nsity

, 515

nm

BIOFILM AFTER 3 DAYS

Polymer brushes vs bacteria- confocal microscopy

Polymer brushes vs bacteria - motility

?

GaNO3 Torbamycin

Y. Kaneko, 2007

Red–dead; green-alive

Antibiotics & gallium

Penicillin – blocks the key enzyme inbacterial cell wall synthesis

Bacteria produce beta-lactamaseto break penicillin

Streptomycine –sabotages bacterialprotein synthesis

Bacteria mutate in a ribosomalproteins

Fe3+ Fe2+

Iron

Hemoproteins-respiration

Enzyme active center- metabolic reactions

Signaling-regulation of gene expression

Iron has many functions in a bacterial cell

Ga3+

Gallium

Hemoproteins-respiration

Enzyme active center- metabolic reactions

Signaling-regulation of gene expression

Y. Kaneko, 2007Bernstein, L. R. 1998

Gallium

Bacteria +Ga citrate

Bacteria, no Ga

Bacteria+Ga DFO

No bacteria

Gallium protects cells from bacteria

OH

OHO

NNH

NH2

O

O

H2O H2OH2O

Ga

Gallium against bacterial toxins

OH

OHO

NNH

NH2

O

O

H2O H2OH2O

Ga

OH

OHO

NNH

NH2

O

O

H2O H2OH2O

Ga

OH

OHO

NNH

NH2

O

O

H2O H2OH2O

Ga

Ongoing work

Conclusions

Biofilm is a natural way of living for bacteria

Biofilms are both good and bad

On certain surfaces biofilms grow better that on other

There are special methods to fight biofilms and these methods are improving

Tack!!