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Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates.

Hall-Stoodley L, Nistico L, Sambanthamoorthy K, Dice B, Nguyen D, Mershon WJ, Johnson C, Hu FZ, Stoodley P, Ehrlich GD, Post JC - BMC Microbiol. (2008)

Bottom Line: Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin.Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon.All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, PA 15212, USA. lstoodle@wpahs.org

ABSTRACT

Background: Streptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro.

Results: Multiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains.

Conclusion: All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.

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DNA staining of the pneumococcal EPS matrix and disruption with DNase. Fig. 4A. Biofilms stained with PicoGreen, a dsDNA stain, and Syto 59 shows that S. pneumoniae biofilms treated with 1000 μg ml-1 Pulmozyme (+) were substantially reduced compared to untreated biofilms (-) in both high and low BFI strains. Scale bar = 30 μm. Figure 4B. Quantification of reduction in biofilm volume (total biomass) measured by COMSTAT upon treatment with different concentrations of Pulmozyme: Untreated controls □; 1 μg ml-1 Pulmozyme ▩; 100 μg ml-1 Pulmozyme ⊟; 1000 μg ml-1 Pulmozyme ■. Bars show average of PicoGreen signal, which stains extracellular dsDNA, and Syto59, which stains intracellular nucleic acid. (Error bars represent standard error of the mean; n = 10; 5 randomly chosen microscopic fields from duplicate experiments. *Significantly different from untreated controls (P values < 0.05); ** (P values < 0.01). Statistical comparisons were made using one-way analysis of variance (ANOVA) (Excel 2000, Microsoft).
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Figure 4: DNA staining of the pneumococcal EPS matrix and disruption with DNase. Fig. 4A. Biofilms stained with PicoGreen, a dsDNA stain, and Syto 59 shows that S. pneumoniae biofilms treated with 1000 μg ml-1 Pulmozyme (+) were substantially reduced compared to untreated biofilms (-) in both high and low BFI strains. Scale bar = 30 μm. Figure 4B. Quantification of reduction in biofilm volume (total biomass) measured by COMSTAT upon treatment with different concentrations of Pulmozyme: Untreated controls □; 1 μg ml-1 Pulmozyme ▩; 100 μg ml-1 Pulmozyme ⊟; 1000 μg ml-1 Pulmozyme ■. Bars show average of PicoGreen signal, which stains extracellular dsDNA, and Syto59, which stains intracellular nucleic acid. (Error bars represent standard error of the mean; n = 10; 5 randomly chosen microscopic fields from duplicate experiments. *Significantly different from untreated controls (P values < 0.05); ** (P values < 0.01). Statistical comparisons were made using one-way analysis of variance (ANOVA) (Excel 2000, Microsoft).

Mentions: To further examine the composition of the pneumococcal biofilm matrix, biofilms were stained with PicoGreen and the nucleic acid dye, Syto 59. Figure 4A indicates that dsDNA is present extracellularly in the biofilm matrix for strains BS69 and BS73. To confirm the presence of extracellular DNA, when pneumococcal biofilms were treated with Pulmozyme® (recombinant human DNase I) a significant loss of cells and biomass was observed (Fig. 4B). Biofilm degradation following Pulmozyme® treatments was quantified using COMSTAT and results demonstrate that the biomass and average thickness of biofilms of all strains were significantly reduced by DNase treatment in a dose responsive manner (Fig. 4B). This was true for all strains regardless of their BFI, with the highest concentration of DNase resulting in reductions of over 90% in the average thickness of all strains except BS75 (Table 2). However, maximum thickness was less affected by DNase treatment in all of the strains, suggesting that the EPS matrix in biofilm towers consisted of non-DNA components.


Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates.

Hall-Stoodley L, Nistico L, Sambanthamoorthy K, Dice B, Nguyen D, Mershon WJ, Johnson C, Hu FZ, Stoodley P, Ehrlich GD, Post JC - BMC Microbiol. (2008)

DNA staining of the pneumococcal EPS matrix and disruption with DNase. Fig. 4A. Biofilms stained with PicoGreen, a dsDNA stain, and Syto 59 shows that S. pneumoniae biofilms treated with 1000 μg ml-1 Pulmozyme (+) were substantially reduced compared to untreated biofilms (-) in both high and low BFI strains. Scale bar = 30 μm. Figure 4B. Quantification of reduction in biofilm volume (total biomass) measured by COMSTAT upon treatment with different concentrations of Pulmozyme: Untreated controls □; 1 μg ml-1 Pulmozyme ▩; 100 μg ml-1 Pulmozyme ⊟; 1000 μg ml-1 Pulmozyme ■. Bars show average of PicoGreen signal, which stains extracellular dsDNA, and Syto59, which stains intracellular nucleic acid. (Error bars represent standard error of the mean; n = 10; 5 randomly chosen microscopic fields from duplicate experiments. *Significantly different from untreated controls (P values < 0.05); ** (P values < 0.01). Statistical comparisons were made using one-way analysis of variance (ANOVA) (Excel 2000, Microsoft).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2600794&req=5

Figure 4: DNA staining of the pneumococcal EPS matrix and disruption with DNase. Fig. 4A. Biofilms stained with PicoGreen, a dsDNA stain, and Syto 59 shows that S. pneumoniae biofilms treated with 1000 μg ml-1 Pulmozyme (+) were substantially reduced compared to untreated biofilms (-) in both high and low BFI strains. Scale bar = 30 μm. Figure 4B. Quantification of reduction in biofilm volume (total biomass) measured by COMSTAT upon treatment with different concentrations of Pulmozyme: Untreated controls □; 1 μg ml-1 Pulmozyme ▩; 100 μg ml-1 Pulmozyme ⊟; 1000 μg ml-1 Pulmozyme ■. Bars show average of PicoGreen signal, which stains extracellular dsDNA, and Syto59, which stains intracellular nucleic acid. (Error bars represent standard error of the mean; n = 10; 5 randomly chosen microscopic fields from duplicate experiments. *Significantly different from untreated controls (P values < 0.05); ** (P values < 0.01). Statistical comparisons were made using one-way analysis of variance (ANOVA) (Excel 2000, Microsoft).
Mentions: To further examine the composition of the pneumococcal biofilm matrix, biofilms were stained with PicoGreen and the nucleic acid dye, Syto 59. Figure 4A indicates that dsDNA is present extracellularly in the biofilm matrix for strains BS69 and BS73. To confirm the presence of extracellular DNA, when pneumococcal biofilms were treated with Pulmozyme® (recombinant human DNase I) a significant loss of cells and biomass was observed (Fig. 4B). Biofilm degradation following Pulmozyme® treatments was quantified using COMSTAT and results demonstrate that the biomass and average thickness of biofilms of all strains were significantly reduced by DNase treatment in a dose responsive manner (Fig. 4B). This was true for all strains regardless of their BFI, with the highest concentration of DNase resulting in reductions of over 90% in the average thickness of all strains except BS75 (Table 2). However, maximum thickness was less affected by DNase treatment in all of the strains, suggesting that the EPS matrix in biofilm towers consisted of non-DNA components.

Bottom Line: Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin.Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon.All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, PA 15212, USA. lstoodle@wpahs.org

ABSTRACT

Background: Streptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro.

Results: Multiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains.

Conclusion: All pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.

Show MeSH
Related in: MedlinePlus