0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

SPECIFIC AIMS
UMD.1 – Anti-staphylococcal activities of A.Actinomycetemcomitans dispersin B
Staphylococcus aureus and Staphylococcus epidermidis are major human
pathogens of increasing importance due to the dissemination of antibioticresistant
strains. Staphylococci form biofilms on implanted medical devices
which often lead to bacteremia, acute sepsis and death. Biofilms are difficult
to eradicate because of their inherent resistance to killing by antimicrobial
agents. Our long-range goal is to develop novel therapies that can disperse
biofilms and kill biofilm-embedded bacterial cells. Previous studies showed
that biofilm-embedded staphylococci produce PNAG (poly-Nacetylglucosamine),
a sticky exopolysaccharide that mediates biofilm formation on
biomaterials. Our laboratory discovered a PNAG-degrading enzyme (Dispersin
B) that exhibits anti-biofilm activity against staphylococci and other
PNAG-producing bacteria.
Our central hypothesis is that PNAG plays a key role in staphylococcal
biofilm cohesion and biofilm resistance, and that dispersin B will be a clinically
useful agent for treating and preventing staphylococcal biofilm infections.
The goal of the present proposal is to demonstrate that dispersin B acts
synergistically with antibiotics to detach and kill S. aureus and S. epidermidis
biofilms in vitro. To accomplish our goal, we propose two specific aims:
AIM 1 is to measure the ability of dispersin B to sensitize S. aureus and S. epidermidis
biofilms to antibiotic killing in a 96-well microtiter plate assay and in
a simulated in vitro catheter lock assay. The antibiotics that we plan to test
include vancomycin, tigecycline and daptomycin. The ability of each antibiotic
to kill enzyme-treated biofilms will be compared to its ability to kill
mock-treated biofilms. The outcomes will be CFU/well values for the
microplate assay and CFU/catheter segment for the catheter lock assay, as
determined by dilution plating. In addition, the ability of Dispersin B to sensitize
S. aureus and S. epidermidis planktonic cells to antibiotic killing will also
be measured.
AIM 2 is to measure the ability of Dispersin B to inhibit S. aureus and S. epidermidis
biofilm formation and increase antibiotic sensitivity when coated
onto polyurethane disks. The ability of Dispersin B and vancomycin (or tigecycline
daptomycin) to adsorb to a variety of functionalized polyurethanes
and to render them resistant to bacterial colonization will be tested. The
outcomes will be CFU/unit area of polymer as determined by dilution plating,
and biofilm morphology as determined by fluorescence microscopy and
SEM.
The proposed research is innovative because it investigates a novel
enzyme-based anti-biofilm strategy that targets and destabilizes the biofilm
matrix. Since Dispersin B does not kill bacteria or inhibit their growth, Dispersin
B-based strategies may be less prone to the development of resistance
than strategies utilizing conventional antibiotics. The results of the proposed
studies are expected to identify the role of PNAG in biofilm cohesion and
biofilm resistance in vitro, and to demonstrate the feasibility of using Dispersin
B as an anti-biofilm agent in vivo.
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