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Specific involvement of pilus type 2a in biofilm formation in group B Streptococcus.

Rinaudo CD, Rosini R, Galeotti CL, Berti F, Necchi F, Reguzzi V, Ghezzo C, Telford JL, Grandi G, Maione D - PLoS ONE (2010)

Bottom Line: Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands.We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype.Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.

ABSTRACT
Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

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Morphological analysis of GBS biofilms by Scanning Electron Microscopy.Representative SEM images of three different GBS high-biofilm former strains (A–F) and of a low-biofilm former strain (G–H). Bacteria were grown on uncoated glass coverslips for 72 h in static conditions. (A and B) Strain CHR-021, serotype Ia with magnification ×500 (A) and ×100 (B). (C and D) Strain ABC020018145, serotype II with magnification ×2000 (C) and ×5000 (D). (E and F) Strain 515, serotype Ia with magnification ×1500 (E) and ×5000 (F). (G and H) Strain CHR-019, serotype III with magnification ×1500 (G) and ×5000 (H).
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pone-0009216-g003: Morphological analysis of GBS biofilms by Scanning Electron Microscopy.Representative SEM images of three different GBS high-biofilm former strains (A–F) and of a low-biofilm former strain (G–H). Bacteria were grown on uncoated glass coverslips for 72 h in static conditions. (A and B) Strain CHR-021, serotype Ia with magnification ×500 (A) and ×100 (B). (C and D) Strain ABC020018145, serotype II with magnification ×2000 (C) and ×5000 (D). (E and F) Strain 515, serotype Ia with magnification ×1500 (E) and ×5000 (F). (G and H) Strain CHR-019, serotype III with magnification ×1500 (G) and ×5000 (H).

Mentions: (A) Determination by crystal violet (CV) assay of biofilm formation of 289 GBS clinical isolates grown in 96-well polystyrene plates in THB supplemented with 1% glucose at 37°C for 18 h. Adherent bacteria were stained with crystal violet (CV) and quantified by measuring the absorbance at 540 nm. The data represent the mean values of three experiments. Standard deviations (not shown) ranged from OD540 values of 0.02 to 0.1. Strains were distributed into 3 groups according to the level of expression of pilus type 2a (PI-2a, Pilus Island 2a), measured by flow cytometry as the Fluorescence Fold Increase (FFI) of cells stained with anti-pilus 2a sera over cells stained with pre-immune sera. Group 1 (High pilus expression: PI-2a >5 FFI) including 171 strains; Group 2 (intermediate or low pilus expression: PI-2a <5 FFI) including 38 strains and Group 3, in which the PI-2a was absent, including 80 strains. Group 1 or Group 2 were also subdivided in strains expressing high levels of pilus type 1 (PI-1>5) and strains carrying only pilus 2a (PI-1 absent) or expressing pilus type 1 below the threshold level (PI-1<5). Group 3 was subdivided into strains expressing either PI-1 or PI-2b at high levels (FFI >5) and strains in which also PI-1 and PI-2b poorly expressed (FFI <5). The horizontal bar intersecting the value of absorbance 0.65 represents the cut-off established to discriminate between high-biofilm forming strains and low-biofilm forming strains. The cut-off was calculated as the value equidistant from the median of absorbance of groups 1, 2 and 3. The percentages are calculated considering the number of strains over or below the cut-off value. Circles indicates strains used for SEM analysis shown in Figure 3. (B) Non parametric multi-comparison statistical analysis. The Kruskal-Wallis test was applied to test the equality of population median among the groups and confirmed that the three groups did not belong to the same population (p-value = 1.742 e−08). The Behrens-Fisher test was applied to distinguish which of the groups were significantly different. Each of the lines connecting two groups is proportional to their comparison p-value and confirms a strong relation between Gr.2 and Gr.3, while the Gr.1 was found to be very different from the others.


Specific involvement of pilus type 2a in biofilm formation in group B Streptococcus.

Rinaudo CD, Rosini R, Galeotti CL, Berti F, Necchi F, Reguzzi V, Ghezzo C, Telford JL, Grandi G, Maione D - PLoS ONE (2010)

Morphological analysis of GBS biofilms by Scanning Electron Microscopy.Representative SEM images of three different GBS high-biofilm former strains (A–F) and of a low-biofilm former strain (G–H). Bacteria were grown on uncoated glass coverslips for 72 h in static conditions. (A and B) Strain CHR-021, serotype Ia with magnification ×500 (A) and ×100 (B). (C and D) Strain ABC020018145, serotype II with magnification ×2000 (C) and ×5000 (D). (E and F) Strain 515, serotype Ia with magnification ×1500 (E) and ×5000 (F). (G and H) Strain CHR-019, serotype III with magnification ×1500 (G) and ×5000 (H).
© Copyright Policy
Related In: Results  -  Collection

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pone-0009216-g003: Morphological analysis of GBS biofilms by Scanning Electron Microscopy.Representative SEM images of three different GBS high-biofilm former strains (A–F) and of a low-biofilm former strain (G–H). Bacteria were grown on uncoated glass coverslips for 72 h in static conditions. (A and B) Strain CHR-021, serotype Ia with magnification ×500 (A) and ×100 (B). (C and D) Strain ABC020018145, serotype II with magnification ×2000 (C) and ×5000 (D). (E and F) Strain 515, serotype Ia with magnification ×1500 (E) and ×5000 (F). (G and H) Strain CHR-019, serotype III with magnification ×1500 (G) and ×5000 (H).
Mentions: (A) Determination by crystal violet (CV) assay of biofilm formation of 289 GBS clinical isolates grown in 96-well polystyrene plates in THB supplemented with 1% glucose at 37°C for 18 h. Adherent bacteria were stained with crystal violet (CV) and quantified by measuring the absorbance at 540 nm. The data represent the mean values of three experiments. Standard deviations (not shown) ranged from OD540 values of 0.02 to 0.1. Strains were distributed into 3 groups according to the level of expression of pilus type 2a (PI-2a, Pilus Island 2a), measured by flow cytometry as the Fluorescence Fold Increase (FFI) of cells stained with anti-pilus 2a sera over cells stained with pre-immune sera. Group 1 (High pilus expression: PI-2a >5 FFI) including 171 strains; Group 2 (intermediate or low pilus expression: PI-2a <5 FFI) including 38 strains and Group 3, in which the PI-2a was absent, including 80 strains. Group 1 or Group 2 were also subdivided in strains expressing high levels of pilus type 1 (PI-1>5) and strains carrying only pilus 2a (PI-1 absent) or expressing pilus type 1 below the threshold level (PI-1<5). Group 3 was subdivided into strains expressing either PI-1 or PI-2b at high levels (FFI >5) and strains in which also PI-1 and PI-2b poorly expressed (FFI <5). The horizontal bar intersecting the value of absorbance 0.65 represents the cut-off established to discriminate between high-biofilm forming strains and low-biofilm forming strains. The cut-off was calculated as the value equidistant from the median of absorbance of groups 1, 2 and 3. The percentages are calculated considering the number of strains over or below the cut-off value. Circles indicates strains used for SEM analysis shown in Figure 3. (B) Non parametric multi-comparison statistical analysis. The Kruskal-Wallis test was applied to test the equality of population median among the groups and confirmed that the three groups did not belong to the same population (p-value = 1.742 e−08). The Behrens-Fisher test was applied to distinguish which of the groups were significantly different. Each of the lines connecting two groups is proportional to their comparison p-value and confirms a strong relation between Gr.2 and Gr.3, while the Gr.1 was found to be very different from the others.

Bottom Line: Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands.We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype.Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: Novartis Vaccines and Diagnostics, Siena, Italy.

ABSTRACT
Streptococcus agalactiae is the primary colonizer of the anogenital mucosa of up to 30% of healthy women and can infect newborns during delivery and cause severe sepsis and meningitis. Persistent colonization usually involves the formation of biofilm and increasing evidences indicate that in pathogenic streptococci biofilm formation is mediated by pili. Recently, we have characterized pili distribution and conservation in 289 GBS clinical isolates and we have shown that GBS has three pilus types, 1, 2a and 2b encoded by three corresponding pilus islands, and that each strain carries one or two islands. Here we have investigated the capacity of these strains to form biofilms. We have found that most of the biofilm-formers carry pilus 2a, and using insertion and deletion mutants we have confirmed that pilus type 2a, but not pilus types 1 and 2b, confers biofilm-forming phenotype. We also show that deletion of the major ancillary protein of type 2a did not impair biofilm formation while the inactivation of the other ancillary protein and of the backbone protein completely abolished this phenotype. Furthermore, antibodies raised against pilus components inhibited bacterial adherence to solid surfaces, offering new strategies to prevent GBS infection by targeting bacteria during their initial attachment to host epithelial cells.

Show MeSH
Related in: MedlinePlus