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in vitro studie van Candida albicans en Streptococcus mutans biofilms

K Honraet (2005)
abstract
The importance and ubiquitous nature of biofilms have become generally recognized in microbiology. These sessile communities of microorganisms imbedded in an extracellular polymeric matrix on a surface are characterized by a high resistance to antimicrobial treatments. Also, their contribution to numerous persistent infections in humans has been well established. In this work, two types of biofilms, i.e. C. albicans on silicone and S. mutans on hydroxyapatite have been studied. Suitable in vitro systems were developed to grow and quantify these biofilms and were subsequently used to evaluate strategies for the prevention of biofilm formation. First, the occurrence of Candida species on tracheoesophageal voice prostheses and their susceptibility to antimycotics have been investigated. C. albicans was the predominant yeast species and the isolates showed a uniform sensitivity to nystatin in low concentration. Oral biofilms are known to harbour a great number of different microorganisms. S. mutans biofilms are representative of dental plaque. Three model systems for in vitro growth of C. albicans and S. mutans biofilms have been evaluated. For the static system, questions could be raised whether “real” mature biofilms were obtained or rather adhering and accumulating planktonic cells. The Modified Robbins Device (MRD) proved to be an excellent system to grow biofilms in parallel under different conditions and to evaluate antibiofilm strategies. The densest biofilms were achieved in the CDC biofilm reactor, which is well suited for monitoring biofilm growth kinetically. The golden standard for quantifying biofilm biomass is plating, although complete detachment of sessile cells from their surfaces is not obvious. In this work, the performance of “surrogate” assays, based on either the measurement of metabolic activity (the XTT and FDA assays) or the staining of a cellular element (the SYTO® 9, SYTOX® Green and PI assays), for quantifying both planktonic cells and biofilms have been evaluated. For C. albicans biofilms, the FDA assay was most useful. The XTT assay, though being the method of choice in the literature for quantifying yeast biomass in biofilms performed less favorably. The SYTO® 9 assay was most suitable for the quantification of S. mutans biofilms. The PI and SYTOX® Green assays were not able to detect dead cells in numbers required to assess antimicrobial effects. Potentially antimycotic silicone was produced by loading it with nystatin before (” admixed”) and after (“impregnated”) curing. HPLC extracts of both types of silicone revealed that significant amounts of nystatin could be released. A disk diffusion test showed that the antimycotic effect of the silicone was correlated with this release. When C. albicans biofilms were grown on the “admixed” silicone, biofilms with reduced biomass were only found in the static system. In the MRDs and the CDC biofilm reactor, no effect of the nystatin on the biofilm development occurred, probably because the continuous flow of medium led to leaching of the antimycotic. These findings suggest a temporary burst effect of the loaded nystatin. S. mutans produces glucosyltransferases (GTF), which catalyze the biosynthesis of extracellular polysaccharides. For the prevention of streptococcal biofilms, plant extracts (PE) were selected on the basis of their relationship to plants with known GTF inhibition properties. First, the antibacterial effect of these PE on S. mutans was examined in order to choose a concentration devoid of it. The microbial contamination of the PE was monitored to estimate their possible interference with the development of the streptococcal biofilms. S. mutans biofilms were grown in the MRDs in the presence and absence (= control) of PE in a sub-MIC concentration and the biomasses, determined in the SYTO® 9 assay were compared. Several extracts inhibited the S. mutans biofilm formation. To examine the inhibition of GTF by the PE, two enzyme assays were developed, one based on the cleavage of p-nitrophenyl-alpha-D-glucopyranoside, the other on the monitoring of the formation of dextran. Based on the results of the biofilm experiments and the second enzyme assay, the PE could be divided into four groups. The first group contained extracts that inhibited both the biofilm formation and the GTF, suggesting a causal relationship between both phenomena. The extracts that exhibited no effect on either the biofilm formation or GTF, fell into the second group. The third group consisted of extracts that had no influence on the GTF activity, although an inhibitory effect on the biofilm development was noticed. The latter was probably due to a non-GTF related mode of action. The fourth group contained PE that only inhibited GTF, but had no influence on the biofilm formation. The concentration of GTF inhibiting substances, unlike in the enzyme assay, in these extracts was probably too low to yield an effect in the biofilm system. In conclusion, this work delivers “tools” for the study of the development and the prevention of yeast and bacterial biofilms. Follow-up research using these tools has pertained to the effect of chemically modified silicone surfaces on C. albicans biofilm formation, the study of gene expression in C. albicans biofilms and the testing of fractionated PE in connection with S. mutans biofilms. Other on–going “spin-off” studies are concerned with the prevention or elimination of S. aureus, P. aeruginosa and Propionibacterium acnes biofilms.
Please use this url to cite or link to this publication:
author
promoter
H Nelis
organization
year
type
dissertation
DOI
1854/3929
UGent publication?
yes
classification
D1
id
471547
handle
http://hdl.handle.net/1854/LU-471547
alternative location
http://lib.ugent.be/fulltxt/RUG01/000/941/603/RUG01-000941603_2010_0001_AC.pdf
date created
2006-01-26 10:10:47
date last changed
2009-01-27 13:56:08
@phdthesis{471547,
  abstract     = {The importance and ubiquitous nature of biofilms have become generally recognized in microbiology. These sessile communities of microorganisms imbedded in an extracellular polymeric matrix on a surface are characterized by a high resistance to antimicrobial treatments. Also, their contribution to numerous persistent infections in humans has been well established. In this work, two types of biofilms, i.e. C. albicans on silicone and S. mutans on hydroxyapatite have been studied. Suitable in vitro systems were developed to grow and quantify these biofilms and were subsequently used to evaluate strategies for the prevention of biofilm formation. First, the occurrence of Candida species on tracheoesophageal voice prostheses and their susceptibility to antimycotics have been investigated. C. albicans was the predominant yeast species and the isolates showed a uniform sensitivity to nystatin in low concentration. Oral biofilms are known to harbour a great number of different microorganisms. S. mutans biofilms are representative of dental plaque. Three model systems for in vitro growth of C. albicans and S. mutans biofilms have been evaluated. For the static system, questions could be raised whether {\textquotedblleft}real{\textquotedblright} mature biofilms were obtained or rather adhering and accumulating planktonic cells. The Modified Robbins Device (MRD) proved to be an excellent system to grow biofilms in parallel under different conditions and to evaluate antibiofilm strategies. The densest biofilms were achieved in the CDC biofilm reactor, which is well suited for monitoring biofilm growth kinetically. The golden standard for quantifying biofilm biomass is plating, although complete detachment of sessile cells from their surfaces is not obvious. In this work, the performance of {\textquotedblleft}surrogate{\textquotedblright} assays, based on either the measurement of metabolic activity (the XTT and FDA assays) or the staining of a cellular element (the SYTO{\textregistered} 9, SYTOX{\textregistered} Green and PI assays), for quantifying both planktonic cells and biofilms have been evaluated. For C. albicans biofilms, the FDA assay was most useful. The XTT assay, though being the method of choice in the literature for quantifying yeast biomass in biofilms performed less favorably. The SYTO{\textregistered} 9 assay was most suitable for the quantification of S. mutans biofilms. The PI and SYTOX{\textregistered} Green assays were not able to detect dead cells in numbers required to assess antimicrobial effects. Potentially antimycotic silicone was produced by loading it with nystatin before ({\textquotedblright} admixed{\textquotedblright}) and after ({\textquotedblleft}impregnated{\textquotedblright}) curing. HPLC extracts of both types of silicone revealed that significant amounts of nystatin could be released. A disk diffusion test showed that the antimycotic effect of the silicone was correlated with this release. When C. albicans biofilms were grown on the {\textquotedblleft}admixed{\textquotedblright} silicone, biofilms with reduced biomass were only found in the static system. In the MRDs and the CDC biofilm reactor, no effect of the nystatin on the biofilm development occurred, probably because the continuous flow of medium led to leaching of the antimycotic. These findings suggest a temporary burst effect of the loaded nystatin. S. mutans produces glucosyltransferases (GTF), which catalyze the biosynthesis of extracellular polysaccharides. For the prevention of streptococcal biofilms, plant extracts (PE) were selected on the basis of their relationship to plants with known GTF inhibition properties. First, the antibacterial effect of these PE on S. mutans was examined in order to choose a concentration devoid of it. The microbial contamination of the PE was monitored to estimate their possible interference with the development of the streptococcal biofilms. S. mutans biofilms were grown in the MRDs in the presence and absence (= control) of PE in a sub-MIC concentration and the biomasses, determined in the SYTO{\textregistered} 9 assay were compared. Several extracts inhibited the S. mutans biofilm formation. To examine the inhibition of GTF by the PE, two enzyme assays were developed, one based on the cleavage of p-nitrophenyl-alpha-D-glucopyranoside, the other on the monitoring of the formation of dextran. Based on the results of the biofilm experiments and the second enzyme assay, the PE could be divided into four groups. The first group contained extracts that inhibited both the biofilm formation and the GTF, suggesting a causal relationship between both phenomena. The extracts that exhibited no effect on either the biofilm formation or GTF, fell into the second group. The third group consisted of extracts that had no influence on the GTF activity, although an inhibitory effect on the biofilm development was noticed. The latter was probably due to a non-GTF related mode of action. The fourth group contained PE that only inhibited GTF, but had no influence on the biofilm formation. The concentration of GTF inhibiting substances, unlike in the enzyme assay, in these extracts was probably too low to yield an effect in the biofilm system. In conclusion, this work delivers {\textquotedblleft}tools{\textquotedblright} for the study of the development and the prevention of yeast and bacterial biofilms. Follow-up research using these tools has pertained to the effect of chemically modified silicone surfaces on C. albicans biofilm formation, the study of gene expression in C. albicans biofilms and the testing of fractionated PE in connection with S. mutans biofilms. Other on--going {\textquotedblleft}spin-off{\textquotedblright} studies are concerned with the prevention or elimination of S. aureus, P. aeruginosa and Propionibacterium acnes biofilms.},
  author       = {Honraet, K},
  school       = {Ghent University},
  title        = {in vitro studie van Candida albicans en Streptococcus mutans biofilms},
  url          = {http://dx.doi.org/1854/3929},
  year         = {2005},
}

Chicago
Honraet, K. 2005. “In Vitro Studie Van Candida Albicans En Streptococcus Mutans Biofilms.”
APA
Honraet, K. (2005). in vitro studie van Candida albicans en Streptococcus mutans biofilms.
Vancouver
1.
Honraet K. in vitro studie van Candida albicans en Streptococcus mutans biofilms. 2005.
MLA
Honraet, K. “In Vitro Studie Van Candida Albicans En Streptococcus Mutans Biofilms.” 2005 : n. pag. Print.