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.

INTRODUCTIONBacterial 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.

AIMS & OBJECTIVESThe 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.

MATERIALS & METHODSWe 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 MicroscopyP. 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 AssaysThe 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.

RESULTS

CONCLUSIONS 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

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

Fig 1. Structure of -L-guluronate (G) and -D-manuronate (M); OligoG has at least 90-95% of the monomer residues as G residues.

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.

ACKNOWLEDGEMENTSThis 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.).

S. aureus P. aeruginosaE. coli P. mirabilis

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B. cepaciaB. cenocepacia B. multivorans

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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.

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Motility score 0; no spread from the line of inoculation

Motility score 4; complete spread from the line of inoculation

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Fig 2. Diagrammatic representation of MTA stab culture assay

Zeta Potential Analysis

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.

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).

OligoG treatment of the PAO1 resulted in an increase in negative surface charge (at all observed pH values; p<0.05; Fig 4).

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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.

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.

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Fig 6. Bacteria washed twice (5,500 g, 3 mins); (A) PAO1, (B) PAO1 grown in 10% OligoG.

Size analysis of PAO1 following exposure to OligoG demonstrated that the bacteria-OligoG interactions were not disrupted by exposure to hydrodynamic shear (Fig 6).

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 post-washing and was associated with the observed ”cell-clumping.”

Cell Sizing Analysis with Hydrodynamic Shear

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AFM Imaging

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).

TOPOGRAPHY PHASE AMPLITUDE

Fig. 7. AFM images of (A) PAO1 (4 μm), (B) PAO1 with OligoG (4 μm; post-wash), z scale of 800 nm and (C) PAO1 withOligoG (7μm; post-wash), z scale of 700 nm.

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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 – Plate Assay

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Motility Testing – Stab Assay

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.

Plate assayIso-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 assayMotility 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

REFERENCES1Jiang 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.

RESULTS continued

Email: hillke1@cardiff.ac.uk

School of Dentistry / Ysgol am Deintyddiaeth

CHARACTERISATION OF THE EFFECT OF OLIGOG ON THE BACTERIAL CELL SURFACE OF PSEUDOMONAL BIOFILMS