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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.

SUBMITTER: Herten M

PROVIDER: S-EPMC5723318 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Herten Monika M Bisdas Theodosios T Knaack Dennis D Becker Karsten K Osada Nani N Torsello Giovanni B GB Idelevich Evgeny A EA

Frontiers in microbiology 20171205

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 gra ...[more]

PMID: 29259580

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Project description:BackgroundOsteomyelitis is a severe and often debilitating disease characterized by inflammatory destruction of bone. Despite treatment, chronic infection often develops which is associated with increased rates of treatment failure, delayed osseous-union, and extremity amputation. Within affected bone, bacteria exist as biofilms, however the impact of biofilms on osteoblasts during disease are unknown. Herein, we evaluated the effect of S. aureus biofilms on osteoblast viability, osteogenic potential, and the expression of the pro-osteoclast factor, receptor activator of NF-kB ligand (RANK-L).MethodsOsteoblasts were exposed to biofilm conditioned media (BCM) from clinical wound isolates of Staphylococcus aureus under normal growth and osteogenic conditions to assess cellular viability and osteoblast differentiation, respectively. Cell viability was evaluated using a live/dead assay and by quantifying total cellular DNA at days 0, 1, 3, 5, and 7. Apoptosis following treatment with BCM was measured by flow-cytometry using the annexin V-FITC/PI apoptosis kit. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and intracellular accumulation of calcium and osteocalcin for up to 21 days following exposure to BCM. Expression of genes involved in osteogenic differentiation and osteoclast regulation, were also evaluated by quantitative real-time PCR.ResultsBCM from clinical strains of S. aureus reduced osteoblast viability which was accompanied by an increase in apoptosis. Osteogenic differentiation was significantly inhibited following treatment with BCM as indicated by decreased alkaline phosphatase activity, decreased intracellular accumulation of calcium and inorganic phosphate, as well as reduced expression of transcription factors and genes involved in bone mineralization in viable cells. Importantly, exposure of osteoblasts to BCM resulted in up-regulated expression of RANK-L and increase in the RANK-L/OPG ratio compared to the untreated controls.ConclusionsTogether these studies suggest that soluble factors produced by S. aureus biofilms may contribute to bone loss during chronic osteomyelitis simultaneously by: (1) reducing osteoblast viability and osteogenic potential thereby limiting new bone growth and (2) promoting bone resorption through increased expression of RANK-L by osteoblasts. To our knowledge these are the first studies to demonstrate the impact of staphylococcal biofilms on osteoblast function, and provide an enhanced understanding of the pathogenic role of staphylococcal biofilms during osteomyelitis.

Dataset Information

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

Publications

Rapid <i>in Vitro</i> Quantification of <i>S. aureus</i> Biofilms on Vascular Graft Surfaces.

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