Source of bacterial isolates and identification
All isolates used in this study were isolated from routine clinical specimens submitted to the University of Liverpool Veterinary Teaching Hospital, Leahurst, Wirral, UK. All isolates were identified morphologically and biochemically by standard laboratory procedure.
Outer membrane protein assay
Mueller-Hinton broth (MHB – Laboratory M, Bury, UK) and Mueller-Hinton agar (MHA – Laboratory M, Bury, UK) were used throughout. Poloxamer F127 was obtained from Univar (Essex, UK). All other chemicals and reagents were obtained from BDH (Poole, UK), Bio Rad (Hemel Hempstead, UK) or Sigma (Poole, UK).
Poloxamer hydrogels (biofilm phenotype induction)
Poloxamer F127 was incorporated into MHB at a concentration of up to 30% which was then refrigerated overnight (4°C). The dissolved poloxamer was then autoclaved and returned to the fridge. The liquefied poloxamer was then poured into Petri dishes in 20 ml volumes. Dishes were incubated overnight at 35°C before inoculation.
Biofilm cultures of all bacteria were prepared by inoculating a 96 well microtitre plate (Nunclon®, Scientific Laboratory Supplies, Manchester, UK) with MHB containing a mid-log phase culture. A Nunc-TSP pin-lid (SLS, Manchester, UK) with 96 pegs was then placed onto the plastic microtitre plate so that the pins inserted into each well of the plate, which provided a surface for bacterial attachment. The wells, containing MHB, were inoculated with approximately 108 of the test bacteria (based upon McFarlane standards) and placed onto a rocker at 37°C. The pegs were colonized then for 24 h. After 24 hours the biofilm was determined by breaking several pegs from various points on the lid. The removed pegs were placed in microfuge tubes, washed in sterile saline (to remove planktonic cells) and biofilm cells were then harvested by sonicating in an ultrasonic water bath for 5 minutes at an amplitude of 50 Hz.
Preparation and analysis of cell envelopes
The preparation and analysis of cell envelopes were conducted according to the methods of Gilbert et al., . In brief, cell suspensions harvested from MH broth cultures, poloxamer hydrogels and biofilm cultures were centrifuged at 10 000 g for 10 minutes at 15°C (Biofuge 13R, Heraus Sepatech, Fisher Scientific, Loughborough, UK). The resultant pellets were resuspended in 500 μl sterile physiological saline and placed in 1.5 ml Eppendorf tubes and sonicated in the water bath for 1 minute at 4°C. N-laurylsarcosine (10% w/v) was added to give a final concentration of 2% w/v. The samples were resonicated for 30 seconds and centrifuged (10 000 g, 1 hour) at 4°C. Pellets were resuspended in Laemmli sample buffer (Bio Rad, Hemel Hempstead, UK) and mercaptoethanol, 5% w/v, and heated for 5 minutes at 100°C. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was conducted with a 15% gel and molecular weight standards (2.5–200 kDa, Invitrogen, Paisley, UK), using sample volumes containing 10 μg protein. Gels were then stained with Coomassie Brilliant Blue G250 (BDH, Poole, UK) for 2 hours and then destained for 45 minutes. Molecular weights were analysed using the Gene Snap computer package (SynGene Bio-Imaging System, Cambridge, UK).
Antimicrobial suceptibility test
Twenty-eight bacterial organisms were evaluated in this study and included; Acinetobacter sp, Actinobacillus equuli, Aeromonas hydrophilia, Bacillus sp, Bordetella bronchiseptica, Corynebacterium sp., Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Klebsiella sp, Listeria sp, Micrococcus sp, Morganella morganii, Nocardia asteroides, Proteus sp, Pseudomonas aeruginosa, Rhodococcus equi and Staphylococcus sp. Also, three standard bacterial strains were used, namely: Escherichia coli NCIMB 12210, Pseudomonas aeruginosa NCIMB 12469 and Staphylococcus aureus NCIMB 12702.
Antibiotic suceptibility testing
Susceptibilities to various antibiotics were determined by modified Kirby-Bauer disk diffusion methods according to the Clinical Laboratory Standards Institute  on both agar and 30% Poloxamer hydrogels. In brief, colonies from an overnight culture of a bacterial isolate were suspended in sterile physiological saline until the density of the test suspension matched the turbidity standard which was the equivalent of a bacterial concentration of 3.0 × 108/ml (McFarland Standard, BioMérieux, Marcy l'Étoile, France). MH agar and poloxamer gel plates were inoculated with 1 ml of bacterial suspension. The suspension was spread over the surface of the agar plates using a sterile 1 ml syringe and swilled around the surface of the poloxamer gel plates to ensure complete coverage. Plates were left for 5 minutes before excess fluid was removed using a sterile pipette. Sterile forceps were used to place the antimicrobial discs on the plates. The antimicrobial discs were then placed on both a MH agar and poloxamer gel plate, in duplicate for each bacteria. Plates were repeated in duplicate for each bacterial organism. Discs were evenly spaced approximately 15 mm from the edge of the plate. Each disc was gently pressed to ensure even contact with the surface of the medium. After overnight incubation at 35°C, plates were removed from the incubator. The diameter of the zone of clearance around each antimicrobial disc was measured with callipers, together with additional light enhancement, and recorded in millimetres. For discs with high efficacy for which the zone could not be measured, Non- Measurable (NM) was recorded. As the poloxamer gel formation is temperature dependent (liquid below 15°C), and readily reversible, whilst recording zones of inhibition the temperature of the Petri dishes were kept at constant at 25°C.
The following antibiotic impregnated discs were used: amoxicillin/clavulanic acid (30 μg), ampicillin/sulbactam (20 μg and 30 μg), ciprofloxacin (5 μg), clindamycin (10 μg), erythromycin (15 μg and 30 μg), imipenem (10 μg),; levofloxacin (5 μg), meropenem (10 μg), penicillin G (5 u); all from Oxoid (Oxoid Ltd; Basingstoke, Hampshire, England).
Antibiotic diffusion investigation
This investigation was carried out in order to compare the diffusion rates of different antibiotics through MH agar and 30% Poloxamer gel (biofilm model). Different antibiotics with different molecular weights (MW) were chosen for this study. These included Ciprofloxacin (5 μg – MW 331.34), Doxycycline Hydrochloride (15 μg – MW 512.94), Gentamicin (15 μg – MW 653.21), Levofloxacin (5 μg – MW 361.37) and Meropenem (10 μg – MW 356.37). In separate experiments an antibiotic disk was placed in the centre of a Petri dish containing MHA or 30% poloxamer. Three 13 mm sterile filter paper disks (Whatman, UK) were placed next to the antibiotic disks in every Petri dish at various distances away from the antibiotic disk. The concept behind this is that over a 24 hour period the known antibiotic will diffuse through the agar or poloxamer gel and become impregnated into the filter disks placed at known distances from the central antibiotic disk. The newly impregnated filter disks was then be removed and their efficacy against a named organism, in this case E.coli, would be investigated using a zone of inhibition test (ZOI), according to NCCLS guidelines  on agar. Where a zone of clearing was detected around the newly impregnanted disc it would indicate that the antibiotic has diffused to that distance. This was repeated in triplicate.