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

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.

Keywords: Staphylococcus aureus; Streptococcus pneumoniae; biofilms; eradication; physical contact.

Figure 1

Spn D39 reduces the population of Sau biofilms. Sau was inoculated alone (Sau) or with Spn strain D39 (Sau+Spn) in abiotic polystyrene plates (A,C) or human pharyngeal cells (B,D). Plates were incubated for 4 or 8 h at 37°C. Planktonic cells were removed, biofilms were harvested, diluted and then plated onto salt mannitol agar plates to obtain Sau biofilm counts (cfu/ml) or blood agar plates with gentamicin to obtain Spn biofilm counts (cfu/ml). Error bars represent the standard errors of the means, calculated using data from at least three independent experiments. ^*statistical significance (p < 0.05) in comparison to wells inoculated only with Sau.

Figure 2

Spn TIGR4 eradicates Sau biofilms produced by strain Newman and MRSA USA300. Sau strain Newman (A,B) or USA300 (C,D) was inoculated alone or with Spn strain TIGR4 and plates were 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 or blood agar plates with gentamicin to obtain TIGR4 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 the bars. ^*statistical significance (p < 0.05) in comparison to wells inoculated only with Sau.

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.

Figure 4

TIGR4ΔspxB mutant eradicate Sau bacteria. Sau Newman strain was inoculated alone or with TIGR4ΔspxB 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 TIGR4ΔspxB counts (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.

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.

Figure 6

Confocal studies of Sau coincubated with Spn strains. (A) Sau, (B) SPJV09 (TIGR4), or (C) SPJV01 (D39), or mixtures of Sau and SPJV09 (D–F) or Sau and SPJV01 (G–I) was inoculated into an eight-well slide and incubated for 4 h at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red). Spn strains were expressing the green fluorescent protein. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm. Gray arrows point out areas where Sau and TIGR4 are located.

Figure 7

Time course study of physical interaction between Sau and Spn strains. Sau (A–D), TIGR4 (E–H), or Sau and TIGR4 (I–P) were inoculated into an eight-well slide and incubated for 1, 2, 3, or 4 h at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). Bacterial DNA was stained by DAPI (blue). Micrographs were taken by confocal microscopy. Panels show representative xy optical sections (~0.4 μm each). Bar at the right panel is valid for its corresponding horizontal panels. Panels (I–L) show the red and green channels while panels (M–P) the red and blue channels. Bars = 10 μm, except were indicated (7 μm).

Figure 8

Colocalization between Sau and Spn. Sau and TIGR4 (A–F) or Sau and D39 (G–L) were inoculated together into an eight-well slide and incubated for 1 h (A–C, G–I) or 2 h (D–F, J–L) at 37°C. Biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). Bacterial DNA was stained by DAPI (blue). Micrographs were taken by confocal microscopy and analyzed using Imaris software. Panels show representative xy optical sections (~0.4 μm each). Bar = 10 μm at right panels and is valid for its corresponding horizontal panels. Vertical panels show specific channels. Arrows point out areas of colocalization between Sau and Spn. (M) Sau colocalized with Spn after 1 h of co-incubation, or free Sau bacteria, were counted in 30 different micrographs. Means were plotted and error bars represent the standard errors. (^*), statistical significance (p < 0.001).

Figure 9

Spn TIGR4 kills preformed Sau biofilms. Sau was inoculated in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Sau biofilms were left uninoculated (Sau) or co-inoculated with Spn strain D39 (Sau/D39) or TIGR4 (Sau/TIGR4) and incubated for 4 h at 37°C. Biofilms (A) or planktonic cells (B) were harvested, serially diluted and plated onto salt mannitol agar plates to obtain bacterial counts. (C) Dilutions were also plated onto blood agar plates with gentamicin to obtain Spn planktonic and biofilm 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 (^*), Sau/D39 (♦) or D39 (#).

Figure 10

Washed TIGR4 bacteria rapidly kill preformed Sau biofilms. (A) Sau was inoculated (Sau) in microtiter plates containing THY and incubated for 4 h, after which planktonic cells were removed and fresh THY medium was added. Another set of wells were inoculated with TIGR4 and incubated for 4 h at 37°C. Planktonic cells, biofilms, or supernatants from this TIGR4 4 h culture were separated as specified in Material and Methods. Preformed Sau biofilms were left uninoculated (Sau), or inoculated with ~1 × 10⁶ cfu/ml of an early-log phase culture of planktonic TIGR4 cells (+Spn), or 4 h cultures of washed bacteria (+Plank/Bio), washed planktonic bacteria (+Plank), washed biofilms (+Bios) or supernatant (+Sup) and incubated for 2 h at 37°C. Cultures were harvested, serially diluted and plated onto salt mannitol agar plates to obtain Sau (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. Statistical significance in comparison to wells inoculated with (^*, p < 0.004) Sau or (♦, p < 0.001) +Plank/Bio. (B–E) Sau was inoculated into an eight-well slide and incubated for 4 h at 37°C. Sau Biofilms were challenged with 4 h cultures of washed TIGR4 bacteria and incubated for 30 min (B), 1 h (C), 1.5 h (D), and 2 h (E). At the end of incubation, biofilms were fixed with 2% PFA and stained with an anti-Sau antibody followed by an Alexa 555-labeled anti-rabbit secondary antibody (red) and then an anti-Spn antibody labeled with Alexa 488 (green). DNA was stained with DAPI. Preparations were analyzed by confocal microscopy. A representative xy optical section is shown. Bar = 20 μm.