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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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Capsule expression during biofilm growth conditions in selected strains. Fig. 5A. cpsA, the first gene in the pneumococcal capsule operon is expressed in each isolate over 100-fold relative to R6, an unencapsulated strain (hatched bars), but is downregulated when pneumococcal strains are grown as biofilms (solid bars). Fig. 5B. Immunostaining with anti-capsule specific antibody labeled with a secondary Texas red anti-rabbit antibody, shows that pneumococci express type specific capsule in the biofilm despite cpsA downregulation. Scale bar = 10 μm.
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Figure 5: Capsule expression during biofilm growth conditions in selected strains. Fig. 5A. cpsA, the first gene in the pneumococcal capsule operon is expressed in each isolate over 100-fold relative to R6, an unencapsulated strain (hatched bars), but is downregulated when pneumococcal strains are grown as biofilms (solid bars). Fig. 5B. Immunostaining with anti-capsule specific antibody labeled with a secondary Texas red anti-rabbit antibody, shows that pneumococci express type specific capsule in the biofilm despite cpsA downregulation. Scale bar = 10 μm.

Mentions: To investigate whether pneumococci in biofilms were encapsulated we examined cpsA expression under planktonic and biofilm growth conditions in 4 of the isolates (2 high ranked and 2 low ranked BFI strains) to see if capsule production was modulated during biofilm development. (All strains grown under planktonic conditions were confirmed to be positive for capsule by the Quellung (agglutination) reaction.) All planktonic grown encapsulated clinical isolates expressed more cpsA (~100 fold) than the unencapsulated R6 strain (Fig. 5A). However, expression of cpsA was downregulated in the biofilm relative to planktonic growth conditions in all strains, regardless of serotype or BFI. Expression of cpsA was higher overall in strains with a high BFI (BS69 and BS72) compared to low ranked strains (BS71 and BS73) with the relative fold reduction 6.3, 7.1, 10 and 7.7, respectively, indicating that BS71 exhibited the most extensive downregulation. In situ examination of biofilm-grown isolates using immunofluorescence with type-specific capsule (Fig. 5B) indicated that capsule-specific antibody binding was brightest in biofilm towers suggesting that pneumococci attached to the surface have a reduced amount of capsule. Taken together these results suggest that capsule expression undergoes complex modulation during biofilm growth.


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)

Capsule expression during biofilm growth conditions in selected strains. Fig. 5A. cpsA, the first gene in the pneumococcal capsule operon is expressed in each isolate over 100-fold relative to R6, an unencapsulated strain (hatched bars), but is downregulated when pneumococcal strains are grown as biofilms (solid bars). Fig. 5B. Immunostaining with anti-capsule specific antibody labeled with a secondary Texas red anti-rabbit antibody, shows that pneumococci express type specific capsule in the biofilm despite cpsA downregulation. Scale bar = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Capsule expression during biofilm growth conditions in selected strains. Fig. 5A. cpsA, the first gene in the pneumococcal capsule operon is expressed in each isolate over 100-fold relative to R6, an unencapsulated strain (hatched bars), but is downregulated when pneumococcal strains are grown as biofilms (solid bars). Fig. 5B. Immunostaining with anti-capsule specific antibody labeled with a secondary Texas red anti-rabbit antibody, shows that pneumococci express type specific capsule in the biofilm despite cpsA downregulation. Scale bar = 10 μm.
Mentions: To investigate whether pneumococci in biofilms were encapsulated we examined cpsA expression under planktonic and biofilm growth conditions in 4 of the isolates (2 high ranked and 2 low ranked BFI strains) to see if capsule production was modulated during biofilm development. (All strains grown under planktonic conditions were confirmed to be positive for capsule by the Quellung (agglutination) reaction.) All planktonic grown encapsulated clinical isolates expressed more cpsA (~100 fold) than the unencapsulated R6 strain (Fig. 5A). However, expression of cpsA was downregulated in the biofilm relative to planktonic growth conditions in all strains, regardless of serotype or BFI. Expression of cpsA was higher overall in strains with a high BFI (BS69 and BS72) compared to low ranked strains (BS71 and BS73) with the relative fold reduction 6.3, 7.1, 10 and 7.7, respectively, indicating that BS71 exhibited the most extensive downregulation. In situ examination of biofilm-grown isolates using immunofluorescence with type-specific capsule (Fig. 5B) indicated that capsule-specific antibody binding was brightest in biofilm towers suggesting that pneumococci attached to the surface have a reduced amount of capsule. Taken together these results suggest that capsule expression undergoes complex modulation during biofilm growth.

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