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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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Investigation of EPS matrix and antibiotic resistance in pneumococcal biofilms. 3A Scanning electron microscopy of pneumococcal biofilms on polystyrene by BS69 (a high BFI strain) and BS73 (a low BFI strain) showing cluster morphology and evidence of extracellular matrix. Extracellular material can be seen on higher magnification in both clinical isolates, however more matrix material is visible with BS69 compared with BS73. Areas of extracellular material can be seen tethering S. pneumoniae cells to the surface (arrows). Scale bar = 10 μm and 2 μm. Fig. 3B. Strain variability in EPS distribution of pneumococcal biofilms by different clinical isolates demonstrated by lectin binding. Lectin (green fluorescence) and Syto 59 (red fluorescence) indicate binding of probes to carbohydrate or nucleic acid, respectively. Yellow indicates co-localization of the two probes. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Scale bar = 10 μm. Fig. 3C. Pneumococcal biofilms treated with azithromycin. High ranked BFI strains (BS69 and BS72) show large cell clusters still viable with the BacLight LIVE/DEAD stain after 24 hours of antibiotic treatment. Low ranked BFI isolates (BS71 and BS73) on the other hand, show only a few viable attached cells (Scale bar = 8 μm.).
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Figure 3: Investigation of EPS matrix and antibiotic resistance in pneumococcal biofilms. 3A Scanning electron microscopy of pneumococcal biofilms on polystyrene by BS69 (a high BFI strain) and BS73 (a low BFI strain) showing cluster morphology and evidence of extracellular matrix. Extracellular material can be seen on higher magnification in both clinical isolates, however more matrix material is visible with BS69 compared with BS73. Areas of extracellular material can be seen tethering S. pneumoniae cells to the surface (arrows). Scale bar = 10 μm and 2 μm. Fig. 3B. Strain variability in EPS distribution of pneumococcal biofilms by different clinical isolates demonstrated by lectin binding. Lectin (green fluorescence) and Syto 59 (red fluorescence) indicate binding of probes to carbohydrate or nucleic acid, respectively. Yellow indicates co-localization of the two probes. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Scale bar = 10 μm. Fig. 3C. Pneumococcal biofilms treated with azithromycin. High ranked BFI strains (BS69 and BS72) show large cell clusters still viable with the BacLight LIVE/DEAD stain after 24 hours of antibiotic treatment. Low ranked BFI isolates (BS71 and BS73) on the other hand, show only a few viable attached cells (Scale bar = 8 μm.).

Mentions: BS69 and BS73 were further examined to assess a high and a low ranked BFI isolate, using high resolution SEM. Figure 3A shows more widespread surface coverage by the high ranked BFI isolate, BS69 compared with BS73 and higher resolution images of the isolates showed BS69 cells surrounded by extensive extracellular material, anchoring pneumococcal cells to the surface. In contrast, SEM showed numerous punctate microcolonies attached over the surface in the low ranked BFI biofilm isolate, BS73. Thus, SEM results confirmed that these two isolates differed in the number of attached cells, but also in the extent of extracellular material attached to 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)

Investigation of EPS matrix and antibiotic resistance in pneumococcal biofilms. 3A Scanning electron microscopy of pneumococcal biofilms on polystyrene by BS69 (a high BFI strain) and BS73 (a low BFI strain) showing cluster morphology and evidence of extracellular matrix. Extracellular material can be seen on higher magnification in both clinical isolates, however more matrix material is visible with BS69 compared with BS73. Areas of extracellular material can be seen tethering S. pneumoniae cells to the surface (arrows). Scale bar = 10 μm and 2 μm. Fig. 3B. Strain variability in EPS distribution of pneumococcal biofilms by different clinical isolates demonstrated by lectin binding. Lectin (green fluorescence) and Syto 59 (red fluorescence) indicate binding of probes to carbohydrate or nucleic acid, respectively. Yellow indicates co-localization of the two probes. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Scale bar = 10 μm. Fig. 3C. Pneumococcal biofilms treated with azithromycin. High ranked BFI strains (BS69 and BS72) show large cell clusters still viable with the BacLight LIVE/DEAD stain after 24 hours of antibiotic treatment. Low ranked BFI isolates (BS71 and BS73) on the other hand, show only a few viable attached cells (Scale bar = 8 μm.).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Investigation of EPS matrix and antibiotic resistance in pneumococcal biofilms. 3A Scanning electron microscopy of pneumococcal biofilms on polystyrene by BS69 (a high BFI strain) and BS73 (a low BFI strain) showing cluster morphology and evidence of extracellular matrix. Extracellular material can be seen on higher magnification in both clinical isolates, however more matrix material is visible with BS69 compared with BS73. Areas of extracellular material can be seen tethering S. pneumoniae cells to the surface (arrows). Scale bar = 10 μm and 2 μm. Fig. 3B. Strain variability in EPS distribution of pneumococcal biofilms by different clinical isolates demonstrated by lectin binding. Lectin (green fluorescence) and Syto 59 (red fluorescence) indicate binding of probes to carbohydrate or nucleic acid, respectively. Yellow indicates co-localization of the two probes. Images are maximum projections or reconstructed confocal stacks consisting of a series of x-y sections. Scale bar = 10 μm. Fig. 3C. Pneumococcal biofilms treated with azithromycin. High ranked BFI strains (BS69 and BS72) show large cell clusters still viable with the BacLight LIVE/DEAD stain after 24 hours of antibiotic treatment. Low ranked BFI isolates (BS71 and BS73) on the other hand, show only a few viable attached cells (Scale bar = 8 μm.).
Mentions: BS69 and BS73 were further examined to assess a high and a low ranked BFI isolate, using high resolution SEM. Figure 3A shows more widespread surface coverage by the high ranked BFI isolate, BS69 compared with BS73 and higher resolution images of the isolates showed BS69 cells surrounded by extensive extracellular material, anchoring pneumococcal cells to the surface. In contrast, SEM showed numerous punctate microcolonies attached over the surface in the low ranked BFI biofilm isolate, BS73. Thus, SEM results confirmed that these two isolates differed in the number of attached cells, but also in the extent of extracellular material attached to 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