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Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4ΔspxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient >0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

No MeSH data available.


Efficient Killing of Sau by Spn requires direct contact. Transwell chambers were installed into 6-well plates and THY was added. TIGR4 was inoculated directly in the Transwell chamber and Sau in the bottom of the well (Sp/Sau), or Sau was inoculated in the Transwell chamber and TIGR4 in the bottom (Sau/Spn). As a control Sau was inoculated alone or with TIGR4 (+Spn). Cultures were incubated for 4 h at 37°C, after which planktonic bacteria (A) or biofilms (B) were harvested from the Transwell chamber, or from the bottom of the well, serially diluted and plated onto salt mannitol agar plates. (C) Planktonic and biofilms were also plated onto BAP plates with gentamicin to obtain Spn counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. *Statistical significance (p < 0.05) in comparison to wells inoculated with Sau.
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Figure 3: Efficient Killing of Sau by Spn requires direct contact. Transwell chambers were installed into 6-well plates and THY was added. TIGR4 was inoculated directly in the Transwell chamber and Sau in the bottom of the well (Sp/Sau), or Sau was inoculated in the Transwell chamber and TIGR4 in the bottom (Sau/Spn). As a control Sau was inoculated alone or with TIGR4 (+Spn). Cultures were incubated for 4 h at 37°C, after which planktonic bacteria (A) or biofilms (B) were harvested from the Transwell chamber, or from the bottom of the well, serially diluted and plated onto salt mannitol agar plates. (C) Planktonic and biofilms were also plated onto BAP plates with gentamicin to obtain Spn counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. *Statistical significance (p < 0.05) in comparison to wells inoculated with Sau.

Mentions: To investigate whether Spn biofilm cells or their supernatants were responsible for the observed phenotype against Sau, strains were inoculated into the same wells, but bacteria were separated using a Transwell system device, which has a membrane with a pore size of 0.4 μm. The Transwell device allows the supernatants to flow throughout the well, but separates bacteria inoculated in the top chamber from those inoculated in the bottom of the well. Neither Sau planktonic cells (Figure 3A), nor biofilms (Figure 3B) were killed when TIGR4 was inoculated in the Transwell device and Sau was inoculated in the bottom of the well (i.e., Spn/Sau). Since the Transwell membrane has a smaller diameter than the bottom of the well, in another set of experiments we inoculated TIGR4 in the bottom of the well and Sau was inoculated directly in the Transwell chamber (i.e., Sau/Spn). Once again, TIGR4 was not able to kill Sau planktonic cells or Sau biofilms within 4 h (Figures 3A,B). TIGR4 planktonic cells and biofilms were similar, whether (1) coincubated with Sau (positive control), (2) inoculated in the Transwell chamber and Sau in the bottom or (3) in the bottom of the well when Sau was inoculated in the Transwell chamber (Figure 3C). Experiments were conducted using Transwell devices with different membrane areas (4.67 and 1.12 cm2) to account for variations in the volume of culture medium obtaining similar results. Altogether, these experiments demonstrated that physical contact is necessary for Spn to kill Sau.


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Efficient Killing of Sau by Spn requires direct contact. Transwell chambers were installed into 6-well plates and THY was added. TIGR4 was inoculated directly in the Transwell chamber and Sau in the bottom of the well (Sp/Sau), or Sau was inoculated in the Transwell chamber and TIGR4 in the bottom (Sau/Spn). As a control Sau was inoculated alone or with TIGR4 (+Spn). Cultures were incubated for 4 h at 37°C, after which planktonic bacteria (A) or biofilms (B) were harvested from the Transwell chamber, or from the bottom of the well, serially diluted and plated onto salt mannitol agar plates. (C) Planktonic and biofilms were also plated onto BAP plates with gentamicin to obtain Spn counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. *Statistical significance (p < 0.05) in comparison to wells inoculated with Sau.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Efficient Killing of Sau by Spn requires direct contact. Transwell chambers were installed into 6-well plates and THY was added. TIGR4 was inoculated directly in the Transwell chamber and Sau in the bottom of the well (Sp/Sau), or Sau was inoculated in the Transwell chamber and TIGR4 in the bottom (Sau/Spn). As a control Sau was inoculated alone or with TIGR4 (+Spn). Cultures were incubated for 4 h at 37°C, after which planktonic bacteria (A) or biofilms (B) were harvested from the Transwell chamber, or from the bottom of the well, serially diluted and plated onto salt mannitol agar plates. (C) Planktonic and biofilms were also plated onto BAP plates with gentamicin to obtain Spn counts. Error bars represent the standard errors of the means calculated using data from at least three independent experiments; the median (cfu/ml) is shown inside bars. *Statistical significance (p < 0.05) in comparison to wells inoculated with Sau.
Mentions: To investigate whether Spn biofilm cells or their supernatants were responsible for the observed phenotype against Sau, strains were inoculated into the same wells, but bacteria were separated using a Transwell system device, which has a membrane with a pore size of 0.4 μm. The Transwell device allows the supernatants to flow throughout the well, but separates bacteria inoculated in the top chamber from those inoculated in the bottom of the well. Neither Sau planktonic cells (Figure 3A), nor biofilms (Figure 3B) were killed when TIGR4 was inoculated in the Transwell device and Sau was inoculated in the bottom of the well (i.e., Spn/Sau). Since the Transwell membrane has a smaller diameter than the bottom of the well, in another set of experiments we inoculated TIGR4 in the bottom of the well and Sau was inoculated directly in the Transwell chamber (i.e., Sau/Spn). Once again, TIGR4 was not able to kill Sau planktonic cells or Sau biofilms within 4 h (Figures 3A,B). TIGR4 planktonic cells and biofilms were similar, whether (1) coincubated with Sau (positive control), (2) inoculated in the Transwell chamber and Sau in the bottom or (3) in the bottom of the well when Sau was inoculated in the Transwell chamber (Figure 3C). Experiments were conducted using Transwell devices with different membrane areas (4.67 and 1.12 cm2) to account for variations in the volume of culture medium obtaining similar results. Altogether, these experiments demonstrated that physical contact is necessary for Spn to kill Sau.

View Article: PubMed Central - PubMed

ABSTRACT

Staphylococcus aureus (Sau) strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA). A previous study found that Streptococcus pneumoniae (Spn) strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1), reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4&Delta;spxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson's coefficient &gt;0.72). Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s) requiring bacterium-bacterium contact, but independent from the production of hydrogen peroxide.

No MeSH data available.