Rapid in Vitro Quantification of S. aureus Biofilms on Vascular Graft Surfaces

Abstract

Objectives: Increasing resistance of microorganisms and particularly tolerance of bacterial biofilms against antibiotics require the need for alternative antimicrobial substances. S. aureus is the most frequent pathogen causing vascular graft infections. In order to evaluate the antimicrobial efficacy, quantification of the bacterial biofilms is necessary. Aim of the present study was the validation of an in vitro model for quantification of bacterial biofilm on vascular graft surfaces using three different assays.Methods: Standardized discs of vascular graft material (Dacron or PTFE) or polystyrene (PS) as control surface with 0.25 cm2 surface area were inoculated with 10−3 diluted overnight culture of three biofilm-producing S. aureus isolates (BEB-029, BEB-295, SH1000) in 96-well PS culture plates. After incubation for 4 and 18 h, the biofilm was determined by three different methods: (a) mitochondrial ATP concentration as measure of bacterial viability (ATP), (b) crystal violet staining (Cry), and (c) vital cell count by calculation of colony-forming units (CFU). The experiments were performed three times. Quadruplicates were used for each isolate, time point, and method. In parallel, bacterial biofilms were documented via scanning electron microscopy.Results: All three methods could quantify biofilms on the PS control. Time needed was 0:40, 13:10, and 14:30 h for ATP, Cry, and CFU, respectively. The Cry assay could not be used for vascular graft surfaces due to high unspecific background staining. However, ATP assay and CFU count showed comparable results on vascular graft material and control. The correlations between ATP and CFU assay differed according to the surface and incubation time and were significant only after 4 h on Dacron (BEB-029, p = 0.013) and on PS (BEB-029, p < 0.001). Between ATP and Cry assay on PS, a significant correlation could be detected after 4 h (BEB-295, p = 0.027) and after 18 h (all three strains, p < 0.026). The reproducibility of the ATP-assay presented as inter-assay-variance of 2.1 and as intra-assay variance of 8.1 on polystyrene.Conclusion: The in-vitro model reproducibly quantifies biofilm on standardized vascular graft surfaces with ATP assay as detection system. The ATP assay allows accelerated microbial quantification, however the correlation with the CFU assay may be strain- and surface-dependent.

Activity of Antibiotics against Staphylococcus aureus in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium

“…The second improvement brought to the model consists of following bacterial viability by measuring in parallel metabolic activity and bacterial counts. The detection of fluorescent metabolites as a surrogate for viability is essentially justified by the ease and rapidity of their use in high-throughput assays (41). We complemented it here with CFU counting because we suspected that the metabolization of molecules used as probes could be altered in the dormant but viable bacteria that are present in biofilms (42,43).…”

Quantitative Examination of Antibiotic Susceptibility of <em>Neisseria gonorrhoeae</em> Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

“…The results can either be combined or individually used for examining bacterial survival within aggregates after antibiotic treatment. The ATP utilization assay has been shown to measure accurately viable bacteria in S. aureus biofilms 20,21 . Here, MS11Opa+Pil+ strain was used to examine the role of GC aggregation in antibiotic susceptibility.…”

Biomechanical Stability and Osteogenesis in a Tibial Bone Defect Treated by Autologous Ovine Cord Blood Cells—A Pilot Study

“…Luminescence, produced by the luciferase-catalyzed reaction of luciferin and ATP, was measured using a multilabel plate reader (VICTOR3™ PerkinElmer LAS, Rodgau-Jügesheim, Germany). Additionally, the USSC growth on the biomaterial was documented via SEM analysis as described before [47].…”