The Use of a Mouse Model in the Study of the Mechanisms of Staphylococcus Aureus Biofilm Formation in Vivo

Staphylococcus aureus is one of the leading causes of both healthcare-associated and communityacquired infections. We previously demonstrated that vancomycin treatment enhanced biofilm formation of vancomycin-resistant S. aureus (VRSA) through increased acquisition of extracellular DNA (eDNA) via a cidA-mediated autolysis mechanism. We recently found that this enhancement was even stronger in an environment that had a higher glucose concentration such in a diabetic environment. The capacity to form biofilms was evaluated through a catheter-associated biofilm assay in VRSA-infected diabetic mice upon vancomycin treatment. A 10- and 1000-fold increase in biofilm-bound bacterial colony forming units was observed in samples from diabetic mice without and with vancomycin treatment, respectively, compared to 表 CM02 共 2 頁 第2 頁 healthy mice. Those formed biofilms have dissimilar conformations under scanning electron microscope upon different treatments suggesting that those biofilms may have different compositions. The classical view of biofilms is mostly based on in vitro observations. Our knowledge of the developmental processes of medical biofilms is largely derived from surface growth in vitro systems. Therefore, we are interested in studying the molecular basis of the biofilm formation in vivo, particularly in diabetic mice infected by drug-resistant S. aureus upon improper antibiotic treatment. We focus on analyzing above four different biofilms (healthy/diabetic mice with/without vancomycin treatment) using a proteomic approach for the identification and comparison of the expression of proteins among those biofilms. We first attempted to analyze expression profile of proteins extracted from in vivo (as above) and in vitro biofilms using a 1-D gel electrophoresis assay. A significant difference of the protein profiles between in vivo and in vitro biofilms was observed, as well as variations observed sample by sample. Several host proteins were identified by MALDI-TOF and albumin and hemoglobin were found among all the in vivo biofilms. Preliminary results indicate that biofilm formation was significantly increased in the presence of albumin even in a PIA (polysaccharide intercellular adhesion encoded by ica operon) deficient strain in vitro. eDNA was essential for the formation of albumin-based biofilm and expression of PIA was suppressed upon albumin treatment suggesting that host factors may alter the expression of bacterial biofilm-associated genes. In this proposal, we try to further investigate the role of albumin in biofilm formation in vivo. The cidA, ica and cidA/ica mutant strains, together with biofilm non-producing clinical isolates will be employed to investigate their biofilm forming capacity in vivo. Expression level of bacterial biofilm-associated genes upon albumin treatment will be determined by qRT-PCR. Similar strategy will be used for studying the role of hemoglobin in biofilm development. Proteome of different in vivo biofilms will be analyzed through a 2-D gel-based technique and the iTRAQ analysis. Candid proteins identified from proteomic analysis will be subjected to functional assay for investigating the relatedness to biofilm formation in vivo. Function of bacterial proteins of interest will be studied by generating various mutant strains followed by in vivo biofilm formation assay. Function of murine proteins will be investigated by injecting respective monoclonal antibody or inhibitors to mice. We anticipate this study can get more insight into the molecular basis of the mechanisms for in vivo biofilm formation and promote the identification of new therapeutic strategies to treat infectious diseases in patients with diabetes.

Project ID:PC10507-0244
External Project ID:MOST105-2320-B182-024