Post on 23-Dec-2015
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Rzhepishevska & Ramstedt, unpublished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
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
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
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
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