Investigator: R. Lane Smith, PhD Project Category: Arthritis - 2000 Background: Biofilm producing organisms contribute to colonization of orthopaedic implants and the associated antibiotic resistance of the adherent biofilm producing organisms increases morbidity of surgical procedures that use internal hardware. A number approaches advanced to address this problem involve use of bacterial prophylaxis. However, application of excess antibiotics exhibiting limited efficacy against biofilm producing organisms may be counter-productive and only produce increased selection pressure for drug resistance. Hypothesis: New antibiotics are currently being developed and tested for activity against biofilm producing organisms. The hypothesis being tested in this study is that the new antibiotics that function through inhibition of bacterial metabolism will demonstrate efficacy in the presence of biofilm deposition due to none cell wall dependent bacteriostatic mechanisms. Objective: The purpose of this feasibility study is to develop an in vitro protocol to establish evidence that Linezolid, an oxazolidinone synthetic antibacterial agent, exhibits inhibitory effects on Staphylococcal biofilm producing organisms. The intention of this feasibility study will be to provide a foundation for an in vivo analysis of antibacterial activity against biofilm producers localized on orthopaedic biomaterials. Procedure: The in vitro experimental protocol will follow two approaches. The first approach addresses the problem of prevention of bacterial colonization of an implanted device. This type of infection often follows a nominal bacterial inoculum during fracture fixation for traumatic injury where the wound margins were compromised during injury. The second approach address the potential for eradication of an established infection where the organism has already colonized an implant device and where removal of the device might be counter indicated or be a choice of last resort. The deposition of bacterial biofilm in the presence of antibacterial agent will determined using methods developed in our laboratory. For quantification of toluidine blue staining, bacterial extracellular matrix will be reacted with the following solutions: Carnoy's fixative, toluidine blue (0.1%) and sodium hydroxide (0.2N) as previously described. In this procedure, one milliliter of each solution will be added to each test sample. Carnoy's and toluidine blue are each allowed to stand for half an hour before being removed. After exposure to toluidine blue, the excess stain will be removed by washing two times, each with three milliliters of saline. Sodium hydroxide is then added to each tube and the biofilm is solubilized by heating for one hour at 85oC. The optical density of toluidine blue was determined by using wavelength of 590 nm. All experiments will be performed in triplicate or quadruplicate, repeated and the data expressed as the mean and standard error of the mean. Statistical analysis will be carried out using analysis of variance with correction for multiple comparison testing. Funding Source: Pharmacia and Upjohn |
