In Vitro Testing of Antimicrobial Activity of Bone Cement

Abstract

The purpose of this study was to establish a reliable and cost-effective microplate proliferation assay for in vitro antimicrobial testing of bone cement samples. Cement samples devoid of antimicrobial agents, loaded with 2% gentamicin or with different concentrations of high-porosity silver, were incubated in a 96-well microplate with several staphylococcal, Pseudomonas aeruginosa, and Enterococcus faecium isolates exhibiting different susceptibilities to gentamicin. After being rinsed, the samples were brought into a soy medium in which adherent cells on the cement surface either were killed by the antimicrobial surface or started to release clonal counterparts. The medium was monitored in real time by recording a time proliferation curve for each well. Microplate testing revealed no antibacterial effect of plain bone cement. The antibacterial activity of gentamicin-loaded bone cement was shown by the microplate test to depend on the gentamicin susceptibilities of the strains. The effect of high-porosity silver was dose dependent. Bactericidal activity against all tested strains was found for bone cement loaded with 1% high-porosity silver. The accuracy of this new proliferation assay was shown by the high correlation between the types of proliferation curves and antibiotic susceptibility. In contrast to routine agar diffusion testing, it assesses the dynamic response of microorganisms to antimicrobial agents in biomaterials and allows high-throughput screening and detection of antimicrobial properties of poorly water-soluble compounds like silver.Infections in total joint arthroplasty are devastating situations (19), and many strategies have been undertaken to reduce infection rates, including the use of helmet aspirator suits (22), laminar airflow (5, 10, 18), and prophylactic intravenous antibiotics (9, 13, 15). Loading polymethylmethacrylate (PMMA) bone cement with antibiotics to reduce infection rates has also been postulated in the literature (6,16,24,26).As in all other fields of medicine, new ideas in orthopedics should be evaluated stepwise, with in vitro and subsequent in vivo investigations of antimicrobial properties occurring before clinical trials. Therefore, testing the antimicrobial activity of bone cement with new antibiotics or other antimicrobial agents should begin with in vitro studies. Agar diffusion testing has been the standard method for many years for in vitro assessment of antibiotic-loaded bone cements (17,23,27,28). However, the assessment by agar diffusion testing of the anti-infective activities of some antimicrobial agents requires accurately defined standard conditions (20). For example, large molecules, such as vancomycin and teicoplanin, harbor a reduced diffusion capacity and their susceptibilities are difficult to determine exactly. Other anti-infective agents, e.g., fosfomycin, are able to interact with components of the culture medium in agar plates, resulting in reduced activity. Therefore, adequate culture media are required (20). New anti-infective agents like microdis...

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The response of Pseudomonas aeruginosa biofilm to the presence of a glass polyalkenoate cement formulated from a silver containing glass

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