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.
Highlights
Bacterial biofilms are naturally occurring in dental plaque (Sanz et al, 2017), their presence is mainly associated to critical medical conditions such as infections of the upper respiratory or urogenital tract, peritonitis, and on implanted medical devices, which are increasingly used (Lynch and Robertson, 2008; Romling et al, 2014)
Bacterial colonization of the vascular graft surface is associated with bacterial biofilm production, the accumulation of extracellular polymer substance matrix (EPS), consisting of polysaccharides, proteins, glycolipids, and bacterial DNA (Becker et al, 2014)
This study evaluated an in vitro model for rapid quantification of bacterial biofilm adherence on vascular graft material with an adenosine triphosphate (ATP) assay
Summary
Bacterial biofilms are naturally occurring in dental plaque (Sanz et al, 2017), their presence is mainly associated to critical medical conditions such as infections of the upper respiratory or urogenital tract, peritonitis, and on implanted medical devices, which are increasingly used (Lynch and Robertson, 2008; Romling et al, 2014).Vascular grafts are used in aortic reconstructions and for lack of autologous vein material in peripheral bypass operations. Despite highest hygienic precautionary measures during surgery, vascular graft infections (VGIs) occur in 2–4% of cases and are associated with high morbidity and mortality rates (10–75%) (Hepp, 2009; Young et al, 2012). Bacterial colonization of the vascular graft surface is associated with bacterial biofilm production, the accumulation of extracellular polymer substance matrix (EPS), consisting of polysaccharides, proteins, glycolipids, and bacterial DNA (Becker et al, 2014). The EPS shields the bacteria by preventing the penetration of macromolecules like antibiotics and inflammatory cells of the immune defense into the biofilm matrix as well as by displaying a diffusion barrier for molecules with antimicrobial properties (Olsen, 2015). Subpopulations can become metabolic inactive and form dormant persister cells. Since some antibiotics are only effective for metabolic active bacteria, the resting bacteria may be insensitive toward antibiotics (Lewis, 2007)
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