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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.


Related in: MedlinePlus

Catalase inhibits Sau killing by Spn. Sau Newman strain was inoculated either alone, with catalase, with wt strain TIGR4, with TIGR4 and catalase or with TIGR4ΔspxB and catalase and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4 (B). 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 only with Sau.
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Figure 5: Catalase inhibits Sau killing by Spn. Sau Newman strain was inoculated either alone, with catalase, with wt strain TIGR4, with TIGR4 and catalase or with TIGR4ΔspxB and catalase and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4 (B). 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 only with Sau.

Mentions: We next incubated Sau and Spn in the presence of bovine liver catalase. In comparison to co-cultures incubated without catalase, incubation of TIGR4 wt with catalase inhibited killing of Sau (Figure 5A). To investigate whether the inhibitory effect of catalase was separate from its enzymatic activity against H2O2, the isogenic TIGR4ΔspxB mutant, which does not produce H2O2, was also incubated with catalase and this treatment was enough to render TIGR4ΔspxB unable to eradicate Sau bacteria (Figure 5A). Whereas, Spn density was similar whether incubated alone or with Sau (Figure 5B), we noticed that in control wells inoculated only with Sau, or Spn, and incubated in the presence of catalase, the bacterial density of both populations, planktonic and biofilms, significantly increased in comparison to wells incubated without the enzyme (Figures 5A,B).


Streptococcus pneumoniae Eradicates Preformed Staphylococcus aureus Biofilms through a Mechanism Requiring Physical Contact
Catalase inhibits Sau killing by Spn. Sau Newman strain was inoculated either alone, with catalase, with wt strain TIGR4, with TIGR4 and catalase or with TIGR4ΔspxB and catalase and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4 (B). 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 only with Sau.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037180&req=5

Figure 5: Catalase inhibits Sau killing by Spn. Sau Newman strain was inoculated either alone, with catalase, with wt strain TIGR4, with TIGR4 and catalase or with TIGR4ΔspxB and catalase and incubated for 4 h at 37°C. Planktonic cells or biofilms were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau counts (A) or blood agar plates with gentamicin to obtain TIGR4 (B). 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 only with Sau.
Mentions: We next incubated Sau and Spn in the presence of bovine liver catalase. In comparison to co-cultures incubated without catalase, incubation of TIGR4 wt with catalase inhibited killing of Sau (Figure 5A). To investigate whether the inhibitory effect of catalase was separate from its enzymatic activity against H2O2, the isogenic TIGR4ΔspxB mutant, which does not produce H2O2, was also incubated with catalase and this treatment was enough to render TIGR4ΔspxB unable to eradicate Sau bacteria (Figure 5A). Whereas, Spn density was similar whether incubated alone or with Sau (Figure 5B), we noticed that in control wells inoculated only with Sau, or Spn, and incubated in the presence of catalase, the bacterial density of both populations, planktonic and biofilms, significantly increased in comparison to wells incubated without the enzyme (Figures 5A,B).

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.


Related in: MedlinePlus