Transcript Slide 1

School of Dentistry / Ysgol am Deintyddiaeth
CHARACTERISATION OF THE EFFECT OF OLIGOG ON THE BACTERIAL CELL
SURFACE OF PSEUDOMONAL BIOFILMS
Powell, Lydia C1, 2; Pritchard, Manon F1, 2; Emanuel, Charlotte 1; Hill, Katja E1; Khan, Saira 1; Wright, Chris J2; Onsøyen, Edvar 3; Myrvold, Rolf 3; Dessen, Arne 3; Thomas, David W1
1Tissue
Engineering and Restorative Dentistry, Cardiff University School of Dentistry, Cardiff, United Kingdom; 2Multidisciplinary Nanotechnology Centre, School of Engineering , Swansea University, Swansea, United Kingdom; 3Algipharma AS, Sandvika, Norway.
INTRODUCTION
RESULTS
RESULTS continued
Bacterial biofilms are an important cause of morbidity and mortality in a range of human
diseases, being associated with an estimated >80% of persistent human chronic infections,
many of which are resistant to treatment.1 Multi-drug resistant (MDR) gram-negative bacterial
biofilms, for example, Pseudomonas aeruginosa and Burkholderia spp. complicate the
treatment of cystic fibrosis.2 The formulation of new anti-biofilm therapies is of utmost
importance. Alginates are natural biopolymers composed of (1-4)-linked α-L-guluronate (G)
and β-D-mannuronate (M) residues in a linear polymer (Fig 1) that are routinely used in the
food and drink industry and medicines, including drug delivery and wound dressings. Bacterial
motility usually involves swimming, swarming and twitching, and is associated with either
flagella or type IV pili. Motility has been strongly implicated in bacterial virulence, playing
important roles in colonisation, attachment, survival and biofilm formation.3 Swarming motility
is responsible for surface motility, and is thought to be important in early stage biofilm
development.3 We have previously shown that OligoG (Fig 1), derived and processed from
alginate, composed of 90-95% G (MW 2600 g mol-1) potentiates the activity of conventional
antibiotics (up to 500-fold) against a range of multi-drug resistant Gram-negative bacteria, and
that OligoG has an ability to modify the rheology and structure of P. aeruginosa biofilms.
Whilst these effects have been extensively characterized, the precise mechanism by which
OligoG interacts with the bacterial surface and induces these changes in biofilms is unknown.
Zeta Potential Analysis
Motility Testing – Plate Assay
Fig 1. Structure of -L-guluronate (G)
and -D-manuronate (M); OligoG has
at least 90-95% of the monomer
residues as G residues.
A
C
B
Fig 3.
Zeta potential
distributions measured in
0.01 M NaCl and pH 7 of
(A) 10% OligoG; (B) PAO1;
(C) PAO1 combined with
10% OligoG (post-wash).
B
-80
-70
-60
-50
-40
-30
-20
-10
0
pH 5
pH 7
Zeta Potential (mV)
A
Zeta Potential (mV)
OligoG treatment resulted in modulation of the bacterial surface charge (Fig 3). A more negative zeta-potential peak was evident after interaction between
OligoG and PAO1 cells post-wash (-57.8 ±2.7 mV). These results demonstrated that OligoG binds to the PAO1 surface, causing it to become more negatively
charged. A similar effect was seen in PAO1 treated with 2% OligoG.
-80
-70
-60
-50
-40
-30
-20
-10
0
pH 9
Fig 4. Zeta potential peak
values of Oligo (10%),
PAO1 or PAO1 combined
with OligoG (post-wash) at
various pH values and (A)
0.001 M NaCl; (B) 0.01 M
NaCl.
pH 5
pH 7
0%
0.2%
0.5%
2%
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6%
A
Fig 8. (A) P. mirabilis
cultures grown in MH
broth with 0%, 0.2%,
0.5%, 2%, 6% and 10%
OligoG and plated on
ISO agar containing no
OligoG; (B) P. mirabilis
cultures grown in MH
broth without OligoG
and plated on ISO agar
containing 0%, 0.2%,
0.5%, 2%, 6% OligoG;
(C)
PAO1
cultures
grown in MH broth with
0%, 0.2%, 0.5%, 2%,
6% and 10% OligoG
and plated on BM2 agar
containing no OligoG;
(D)
PAO1
cultures
grown in MH broth
without OligoG and
plated on BM2 agar
containing 0%, 0.2%,
0.5%, 2%, 6% OligoG.
ISO agar-OligoG
B
ISO agar+OligoG
C
BM2 agar-OligoG
D
pH 9
BM2 agar+OligoG
OligoG treatment of the PAO1 resulted in an increase in negative surface charge (at all observed pH values; p<0.05; Fig 4).
Cell Sizing Analysis
AIMS & OBJECTIVES
The specific aims of the study were:
 To visualise interactions between P. aeruginosa PAO1 and OligoG using atomic force microsopy (AFM).
 To quantify changes induced in bacterial cell surface charge and size induced by OligoG binding.
 To study the resistance of this binding to hydrodynamic shear.
 To determine the anti-biofilm mode of action of OligoG in relation to bacterial motility.
3000
2500
2000
1500
1000
500
3500
3000
Fig 5. Cell size analysis
(size
distribution
by
volume) of PAO1 and
PAO1 treated with 10%
OligoG (A) 0.001 M NaCl
and (B) 0.01 M NaCl.
2500
2000
1500
1000
500
0
0
pH 5
pH 7
pH 5
pH 9
pH 7
Swarming motility of P. mirabilis (Fig 8A and B) and P. aeruginosa (Fig 8C and D) was inhibited in a dose
dependant manner with increasing concentrations of OligoG (0% to 6%), however, this effect was only evident in
the presence of OligoG. The effect of OligoG was diminished when the bacteria exposed to OligoG in broth culture
were then subsequently plated onto agar containing no OligoG (Fig 8A and C).
Motility Testing – Stab Assay
pH 9
S. aureus
E. coli
P. aeruginosa
P. mirabilis
B. cenocepacia
B. cepacia
B. multivorans
Cell size analysis showed a 2 -fold increase in bacterial size of OligoG -treated cells (Fig 5). This change was maintained in PAO1 combined with OligoG postwashing and was associated with the observed ”cell-clumping.”
MATERIALS & METHODS
We studied the interaction of P. aeruginosa (PAO1) and OligoG on cell surface structure (using atomic
force microscopy; AFM), surface charge and cellular assembly (using zeta-potential and sizing analysis)
and cell motility.
Atomic Force Microscopy
P. aeruginosa (PAO1) was grown (37˚C; 24 h) in Mueller-Hinton broth (MH) and washed twice (5,500 g, 3
mins). For samples combined with OligoG, PAO1 was added to 0.5% OligoG for 20 mins. Combined
samples were then centrifuged at 2,500 g for 6 mins to remove excess OligoG. A Dimension 3100 AFM
(Bruker) was used to achieve AFM images, using tapping mode operation in air and a scan speed of 0.8
Hz. Samples were dried on 0.01% poly-L-lysine coated mica slides for imaging.
Zeta-potential and cell sizing
A Nano Series Zetasizer (Malvern Instruments) was used for sizing (employing dynamic light scattering)
and zeta-potential (utilising Smoluchowski’s model) measurements.4 For samples combined with OligoG,
PAO1 was added to 2% and 10% OligoG for 20 mins (zeta potential and cell sizing respectively).
Clinically relevant electrolyte solutions of 0.01 M, 0.001 M NaCl at pH 5, 7 or 9 were used for measuring
size and zeta-potential. To analyse the strength of bacterial-OligoG interactions, PAO1 was grown in MH
with 10% OligoG for 24 h, where the bacterial samples were exposed to hydrodynamic shear
(centrifugation at 5,500 g for 3 mins).
Motility Assays
The ability of OligoG (at concentrations <10%) to affect bacterial motility was studied by incorporating
OligoG either into Isosensitest agar plates or into motility test agar (MTA) “stab” inoculations containing a
redox indicator and observing bacterial spread of P. aeruginosa (PAO1) and Proteus mirabilis (NSM6)
across/through the agar. Overnight cultures of P. aeruginosa (PAO1), P. mirabilis (NSM6), B. cenocepacia
(LMG 16656), B. cepacia (BCC 0001), B. multivorans (BCC 0011), S. aureus (NCTC 6571; negative
control) and E. coli (NCTC 10418; positive control) were grown in tryptone soya broth (TSB) at 37˚C.
Cultures were diluted 1 in 100 in Mueller-Hinton Broth (MHB) supplemented with 0%, 0.2%, 0.5%, 2%,
6% and 10% OligoG and incubated for 18 h at 37˚C.
Plate assay
Iso-sensitest agar (ISO) and Basal medium 2
(BM2)5 were prepared containing 0%, 0.2%,
0.5%, 2%, 6% OligoG. Plates were inoculated
with 10 µl of MHB cultures and incubated at
37˚C for 23 h. Distance of bacterial spread was
recorded at 2, 5, 7, 13 and 23 h.
Stab culture assay
Motility
test
agar
(MTA;
MAST)
was
supplemented with 0%, 0.2%, 0.5%, 2%, 6%
OligoG and 5 ml aseptically pipetted into bijou
tubes (Fig 2). MTA was stab inoculated with
prepared bacterial cultures.
Tubes were
incubated at 37˚C for 24 h. Motility appeared as
a red/pink diffuse lateral spread throughout the
agar. Motility was scored from 0 to 4; (0, no
growth beyond the inoculation track, non-motile;
4, growth throughout MTA, motile).6
B
3500
Cell Size (nm)
Cell Size (nm)
A
Inoculating needle
Cell Sizing Analysis with Hydrodynamic Shear
A
B
Size analysis of PAO1 following exposure to OligoG demonstrated that the bacteria-OligoG interactions were not disrupted by exposure to hydrodynamic shear
(Fig 6).
AFM Imaging
TOPOGRAPHY
PHASE
B
Triphenyl
tetrazolium
choride
AMPLITUDE
Fig. 7. AFM images of (A)
PAO1 (4 μm), (B) PAO1
with OligoG (4 μm; postwash), z scale of 800 nm
and (C) PAO1 with
OligoG (7μm; post-wash),
z scale of 700 nm.
6%
0%
6%
0%
6%
0%
6%
0%
6%
0%
6%
MTA demonstrated that 6% OligoG was almost completely able to inhibit motility of the normally motile E. coli, P.
aeruginosa and P. mirabilis (Fig 9). MTA also demonstrated that OligoG (6%) was able to inhibit motility of the
important cystic fibrosis pathogens B. cenocepacia, B. cepacia, and B. multivorans (Fig 9). Negative control (S.
aureus) indicated no bacterial motility with or without OligoG as expected.

OligoG binds irreversibly to the bacterial cell surface of PAO1 and this binding modifies the surface
structure and charge, as well as biofilm assembly and bacterial cell motility.

The previous finding that OligoG exhibits activity against a number of non-motile bacterial species
indicates, however, that other mechanisms are undoubtedly involved.7

This inhibition of motility may be significant in both preventing biofilm formation and in disruption of
the biofilm structure by preventing macromolecule nutrient delivery through the biofilm. OligoG may
also impair further colonisation.

These physical, surface-charge and structural effects may, in part, explain the observed action of
OligoG on bacterial assembly, biofilm formation and antibiotic potentiation that has been previously
described.7
REFERENCES
1Jiang
C
Motility score 4;
complete spread
from the line of
inoculation
Bacterial
succinate
dehydrogenase
0%
CONCLUSIONS
Bacteria
Motility Test Agar
6%
Fig 9. Motility test agar (MTA) supplemented with 0% or 6% OligoG, inoculated with S. aureus (negative control), E. coli (positive control), P.
aeruginosa, P. mirabilis, B. cenocepacia, B. cepacia or B. multivorans.
A
Non-motile
Motility score 0;
no spread from
the line of
inoculation
0%
Fig 6. Bacteria washed
twice (5,500 g, 3 mins);
(A) PAO1, (B) PAO1 grown
in 10% OligoG.
et al. (2011) PloS One 6:e18514; 2Son et al. (2007) Infect Immun 75:53313-5324; 3Shrout et al. (2006)
Mol Microbiol 62:1264-1277; 4Klodzinska et al. (2010) Electrophoresis 31:1590-1596; 5Köhler et al. (2000) J
Bacteriol 182:5990-5996; 6Murinda et al. (2002) J Clin Microbiol 40:4685-4690; 7Khan et al. (2012) Antimicrob
Ag Chemother 56:5134-5141.
ACKNOWLEDGEMENTS
This work was funded by AlgiPharma AS and the authors gratefully
acknowledge funding from the Cystic Fibrosis Foundation and the Faculty of
Dental Surgery of the Royal College of Surgeons of England (C.E.).
Motile
Fig 2. Diagrammatic representation of MTA stab culture assay
AFM revealed uniform binding of OligoG to the cell surface of PAO1 (Fig 7B). Exposure to hydrodynamic shear before imaging had no effect, indicating the
strength of the interaction between PAO1 and OligoG (Fig 7B). AFM images revealed OligoG induced aggregation & clumping of PAO1 (Fig 7C).