0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

UMD.1 – Anti-staphylococcal activities of A.Actinomycetemcomitans dispersin B
The amount of antibiotic adsorbed on the exposed disk surfaces will be
expressed in g per unit area of polymer surface. The antibacterial activity of
antibiotic-coated polymers will be assessed by a modified Kirby-Bauer assay.
Polymeric disks will be placed on Petri plates containing Muller-Hinton agar
and seeded with 108 CFU/ml of the specific bacterial strain. After incubation
at 37°C for 18 h, the diameter of the zone of inhibition around the antibiotic
loaded polymeric disks will be measured.
Expected results: Dispersin B and Vancomycin are expected to adsorb to the
tested polymeric materials with different avidities. Dispersin B should
increase Vancomycin desorption from polymeric disks when tested in the
Kirby-Bauer assay because of its ability to promote antibiotic killing in the
area around the disk.
AIM 2: How efficiently do Dispersin B- and Vancomycin-loaded polyurethanes
resist S. aureus and S. epidermidis colonization and biofilm formation?
The ability of novel polyurethanes (PEUA, PEUED, PEUEA) to resist S.
aureus and S. epidermidis colonization and biofilm formation will be measured
in vitro. We plan to test (a) unloaded polymers, (b) polymers loaded with
Dispersin B or Vancomycin alone, (c) polymers loaded with Dispersin B plus
Vancomycin. Biomaterial colonization and biofilm formation will be assessed
by CFU counting and microscopy.
Previous studies showed that polyurethanes loaded with Dispersin B and
cefamandole nafate resist staphylococcal colonization and biofilm formation
[Donelli et al. 2007]. The aim of this experiment is to identify which polymers
and agents most efficiently resist staphylococcal colonization and biofilm formation.
Polymers loaded with Dispersin B alone or in combination with Vancomycin
will be tested as follows. Polymeric disks will be immersed for 24 h
in 2.5 ml of bacterial suspension (0.5 McFarland unit) grown in TSB. Two
strains of S. aureus and two strains of S. epidermidis will be tested [Donelli et
al. 2007]. Polymer disks will be collected, washed twice with PBS to remove
loosely adherent cells, sonicated for 5 min, placed in test tubes containing 10
ml of Ringer’s solution, and mixed by vortex agitation for 10 sec to detach
biofilm cells. CFUs will be enumerated by dilution plating.
Biofilm growth on unloaded and loaded polymers will also be assessed by
fluorescence microscopy and SEM as previously described [Donelli et al.
2007]. Briefly, for fluorescence microscopy, biofilms cultured on polymeric
materials coated onto glass coverslips will be stained with LIVE/DEAD®
BacLight reagent (Bacterial Viability Kit L7007, Molecular Probes) and
observed under a Nikon Optiphot fluorescence microscope. For SEM, samples
will be fixed with glutaraldehyde, postfixed in OsO4, dehydrated, critical
point dried and gold coated as previously described [Donelli et al. 2007]. To
evaluate biofilm surface area coverage, image analysis will be performed by
using ImageJ software [http://rsb.info.nih.gov/ij/].
To evaluate the possible synergistic activity of dispersin B and Vancomycin,
unloaded and loaded polymer disks will be incubated for 24 h with a
0.5 McFarland bacterial broth culture. Polymers will then be rinsed twice
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