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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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Quantitative assessment of biofilm development by clinical pneumococcal isolates. Fig. 2A. Biofilm development by clinical isolates at days 1, 3 and 6 of culture on polystyrene plates as shown by viable adherent cells (CFUs/cm2). Points represent an average of three duplicate wells per time point in two independent experiments. Error bars represent SD. Fig. 2B. Biofilm development (initial attachment) assayed by crystal violet absorbance comparing 6 clinical pneumococcal isolates on polystyrene over 24 hours. Bars show average triplicate samples of 5 independent experiments. Bars represent SD. Fig. 2C. COMSTAT assessment of pneumococcal biofilm development after 6 days of culture. Two parameters of surface attached pneumococci are shown: maximum thickness (biofilm towers) (left axis) and biomass (biofilm volume) (right axis). Bars represent an average of 3–5 images taken from duplicate plates in 2 independent experiments (minimum n = 12). Error bars represent standard error of the mean. Fig. 2D. Biofilm forming index (BFI) of the 6 pneumococcal clinical strains (serotype in parentheses) combining statistical analyses from the widely used biofilm assays: CFU/cm2, CV assay and COMSTAT analysis. The index ranks each strain according to biofilm formation.
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Figure 2: Quantitative assessment of biofilm development by clinical pneumococcal isolates. Fig. 2A. Biofilm development by clinical isolates at days 1, 3 and 6 of culture on polystyrene plates as shown by viable adherent cells (CFUs/cm2). Points represent an average of three duplicate wells per time point in two independent experiments. Error bars represent SD. Fig. 2B. Biofilm development (initial attachment) assayed by crystal violet absorbance comparing 6 clinical pneumococcal isolates on polystyrene over 24 hours. Bars show average triplicate samples of 5 independent experiments. Bars represent SD. Fig. 2C. COMSTAT assessment of pneumococcal biofilm development after 6 days of culture. Two parameters of surface attached pneumococci are shown: maximum thickness (biofilm towers) (left axis) and biomass (biofilm volume) (right axis). Bars represent an average of 3–5 images taken from duplicate plates in 2 independent experiments (minimum n = 12). Error bars represent standard error of the mean. Fig. 2D. Biofilm forming index (BFI) of the 6 pneumococcal clinical strains (serotype in parentheses) combining statistical analyses from the widely used biofilm assays: CFU/cm2, CV assay and COMSTAT analysis. The index ranks each strain according to biofilm formation.

Mentions: To better assess biofilm development quantitatively, CFUs of attached pneumococci were enumerated at 3 time points to assess the kinetics of biofilm development. Viable adherent pneumococci increased over time in all strains. Attached viable pneumococci were present for all clinical isolates at day 1 ranging from 3.1(log10) CFUs/cm2 for BS75 up to 4.9(log10) for BS72 (Fig. 2A). BS75 biofilms contained significantly fewer culturable cells and BS72 biofilms contained a significantly greater number of cells than all of the other 5 strains at day 1, confirming CLSM observation. By day 6, there was a significant difference in the number of adherent cells among the clinical isolates; BS69 and BS75 showed the most viable attached pneumococci with an average CFU/cm2 of 6 and 5.7, respectively. BS72 averaged 5.4 log10 CFUs/cm2, however the number of CFUs/cm2 for this strain was not statistically different from BS68, BS71 or BS73 biofilms, which demonstrated average viable attached pneumococcus on the order of 5.4, 5.0 and 5.5 log10 CFUs/cm2, respectively. Although the kinetics of surface attachment varied among clinical pneumococcal isolates, viable adherent cells increased over 6 days in all strains.


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)

Quantitative assessment of biofilm development by clinical pneumococcal isolates. Fig. 2A. Biofilm development by clinical isolates at days 1, 3 and 6 of culture on polystyrene plates as shown by viable adherent cells (CFUs/cm2). Points represent an average of three duplicate wells per time point in two independent experiments. Error bars represent SD. Fig. 2B. Biofilm development (initial attachment) assayed by crystal violet absorbance comparing 6 clinical pneumococcal isolates on polystyrene over 24 hours. Bars show average triplicate samples of 5 independent experiments. Bars represent SD. Fig. 2C. COMSTAT assessment of pneumococcal biofilm development after 6 days of culture. Two parameters of surface attached pneumococci are shown: maximum thickness (biofilm towers) (left axis) and biomass (biofilm volume) (right axis). Bars represent an average of 3–5 images taken from duplicate plates in 2 independent experiments (minimum n = 12). Error bars represent standard error of the mean. Fig. 2D. Biofilm forming index (BFI) of the 6 pneumococcal clinical strains (serotype in parentheses) combining statistical analyses from the widely used biofilm assays: CFU/cm2, CV assay and COMSTAT analysis. The index ranks each strain according to biofilm formation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Quantitative assessment of biofilm development by clinical pneumococcal isolates. Fig. 2A. Biofilm development by clinical isolates at days 1, 3 and 6 of culture on polystyrene plates as shown by viable adherent cells (CFUs/cm2). Points represent an average of three duplicate wells per time point in two independent experiments. Error bars represent SD. Fig. 2B. Biofilm development (initial attachment) assayed by crystal violet absorbance comparing 6 clinical pneumococcal isolates on polystyrene over 24 hours. Bars show average triplicate samples of 5 independent experiments. Bars represent SD. Fig. 2C. COMSTAT assessment of pneumococcal biofilm development after 6 days of culture. Two parameters of surface attached pneumococci are shown: maximum thickness (biofilm towers) (left axis) and biomass (biofilm volume) (right axis). Bars represent an average of 3–5 images taken from duplicate plates in 2 independent experiments (minimum n = 12). Error bars represent standard error of the mean. Fig. 2D. Biofilm forming index (BFI) of the 6 pneumococcal clinical strains (serotype in parentheses) combining statistical analyses from the widely used biofilm assays: CFU/cm2, CV assay and COMSTAT analysis. The index ranks each strain according to biofilm formation.
Mentions: To better assess biofilm development quantitatively, CFUs of attached pneumococci were enumerated at 3 time points to assess the kinetics of biofilm development. Viable adherent pneumococci increased over time in all strains. Attached viable pneumococci were present for all clinical isolates at day 1 ranging from 3.1(log10) CFUs/cm2 for BS75 up to 4.9(log10) for BS72 (Fig. 2A). BS75 biofilms contained significantly fewer culturable cells and BS72 biofilms contained a significantly greater number of cells than all of the other 5 strains at day 1, confirming CLSM observation. By day 6, there was a significant difference in the number of adherent cells among the clinical isolates; BS69 and BS75 showed the most viable attached pneumococci with an average CFU/cm2 of 6 and 5.7, respectively. BS72 averaged 5.4 log10 CFUs/cm2, however the number of CFUs/cm2 for this strain was not statistically different from BS68, BS71 or BS73 biofilms, which demonstrated average viable attached pneumococcus on the order of 5.4, 5.0 and 5.5 log10 CFUs/cm2, respectively. Although the kinetics of surface attachment varied among clinical pneumococcal isolates, viable adherent cells increased over 6 days in all strains.

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