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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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Related in: MedlinePlus

CLSM images of biofilm development by clinical isolates of S. pneumoniae stained with BacLight after 6 days of culture showing viable (green fluorescence) and nonviable (red fluorescence) pneumococci within the biofilms. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Sideviews (YZ – left and XZ – bottom) are saggital sections of the biofilm. Scale bar = 30 μm.
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Figure 1: CLSM images of biofilm development by clinical isolates of S. pneumoniae stained with BacLight after 6 days of culture showing viable (green fluorescence) and nonviable (red fluorescence) pneumococci within the biofilms. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Sideviews (YZ – left and XZ – bottom) are saggital sections of the biofilm. Scale bar = 30 μm.

Mentions: Pneumococcal biofilm development was examined in situ over time for each strain using the BacLight Kit and CLSM. All isolates developed biofilms over the 6 day period. However, there was considerable variability in both the extent and kinetics of biofilm development by the isolates, and in the number of viable and nonviable cells (Fig. 1). After 24 hours all 6 pneumococcal isolates had formed heterogeneous biofilms consisting of individual cells, small chains and small clusters of lancet-shaped cells with strain BS72 exhibiting the greatest number of attached cells and a complex biofilm architecture at this time point consisting of small clusters and towers (data not shown). By day 3, strains exhibited a range of ultrastructural characteristics from individual cells and small microcolonies stippled across the surface (BS68, BS71 and BS73) to clusters of bacteria in large towers attached to the substratum (BS69, BS72 and BS75) (data not shown). Strains BS69, BS72 and BS75 developed the most extensive biofilm architecture by day 6 of culture, growing in tall towers of viable cells up to 25 μm in height over the surface (Fig. 1). Time lapse CLSM imaging through the thickness of biofilm towers in real time showed that the towers were free to oscillate indicating that pneumococcal biofilms were dynamic in the fluid (see movie at ). In contrast, strains BS68, BS71 and BS73 exhibited smaller cell clusters (5–10 μm), fewer towers and less extensive surface coverage and ultrastructure. Nevertheless, biofilms formed by these strains exhibited numerous microcolonies of viable cells attached on the surface.


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)

CLSM images of biofilm development by clinical isolates of S. pneumoniae stained with BacLight after 6 days of culture showing viable (green fluorescence) and nonviable (red fluorescence) pneumococci within the biofilms. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Sideviews (YZ – left and XZ – bottom) are saggital sections of the biofilm. Scale bar = 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: CLSM images of biofilm development by clinical isolates of S. pneumoniae stained with BacLight after 6 days of culture showing viable (green fluorescence) and nonviable (red fluorescence) pneumococci within the biofilms. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Sideviews (YZ – left and XZ – bottom) are saggital sections of the biofilm. Scale bar = 30 μm.
Mentions: Pneumococcal biofilm development was examined in situ over time for each strain using the BacLight Kit and CLSM. All isolates developed biofilms over the 6 day period. However, there was considerable variability in both the extent and kinetics of biofilm development by the isolates, and in the number of viable and nonviable cells (Fig. 1). After 24 hours all 6 pneumococcal isolates had formed heterogeneous biofilms consisting of individual cells, small chains and small clusters of lancet-shaped cells with strain BS72 exhibiting the greatest number of attached cells and a complex biofilm architecture at this time point consisting of small clusters and towers (data not shown). By day 3, strains exhibited a range of ultrastructural characteristics from individual cells and small microcolonies stippled across the surface (BS68, BS71 and BS73) to clusters of bacteria in large towers attached to the substratum (BS69, BS72 and BS75) (data not shown). Strains BS69, BS72 and BS75 developed the most extensive biofilm architecture by day 6 of culture, growing in tall towers of viable cells up to 25 μm in height over the surface (Fig. 1). Time lapse CLSM imaging through the thickness of biofilm towers in real time showed that the towers were free to oscillate indicating that pneumococcal biofilms were dynamic in the fluid (see movie at ). In contrast, strains BS68, BS71 and BS73 exhibited smaller cell clusters (5–10 μm), fewer towers and less extensive surface coverage and ultrastructure. Nevertheless, biofilms formed by these strains exhibited numerous microcolonies of viable cells attached on the surface.

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